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Satellite cells are stem cells with an essential role in skeletal muscle repair. Precise regulation of gene expression is critical for proper satellite cell quiescence, proliferation, differentiation and self-renewal. Nuclear proteins required for gene expression are dependent on the nucleocytoplasmic transport machinery to access to nucleus, however little is known about regulation of nuclear transport in satellite cells. The best characterized nuclear import pathway is classical nuclear import which depends on a classical nuclear localization signal (cNLS) in a cargo protein and the heterodimeric import receptors, karyopherin alpha (KPNA) and beta (KPNB). Multiple KPNA1 paralogs exist and can differ in importing specific cNLS proteins required for cell differentiation and function. We show that transcripts for six Kpna paralogs underwent distinct changes in mouse satellite cells during muscle regeneration accompanied by changes in cNLS proteins in nuclei. Depletion of KPNA1, the most dramatically altered KPNA, caused satellite cells in uninjured muscle to prematurely activate, proliferate and undergo apoptosis leading to satellite cell exhaustion with age. Increased proliferation of satellite cells led to enhanced muscle regeneration at early stages of regeneration. In addition, we observed impaired nuclear localization of two key KPNA1 cargo proteins: p27, a cyclin-dependent kinase inhibitor associated with cell cycle control and lymphoid enhancer factor 1, a critical co-transcription factor for β-catenin. These results indicate that regulated nuclear import of proteins by KPNA1 is critical for satellite cell proliferation and survival and establish classical nuclear import as a novel regulatory mechanism for controlling satellite cell fate. This article is protected by copyright. All rights reserved.
Graphical abstract
Karyopherin alpha 1 (KPNA1), a classical nuclear import receptor, binds to Karyopherin beta (KPNB) to transport proteins such as p27 and LEF1 into the nucleus. After release from KPNA1, p27 acts as a cell cycle inhibitor to maintain satellite cell quiescence and LEF1 stimulates transcription of survivin, an anti-apoptotic protein, to promote satellite cell survival.
Reprogramming of antigen-specific T lymphocytes into induced pluripotent stem cells (iPSCs) and their subsequent re-differentiation has enabled expansion of functional T lymphocytes in vitro, thus opening up new approaches for immunotherapy of cancer and other diseases. In this study, we have established a robust protocol to reprogram human invariant NKT (Vα24+iNKT) cells, which have been shown to act as cellular adjuvants and thus exert anti-tumor activity in mice and humans, and to re-differentiate the iNKT cell-derived iPSCs into functional iNKT cells. These iPSC-derived iNKT cells (iPS-Vα24+iNKT cells) can be activated by ligand-pulsed dendritic cells (DCs) and produce a large amount of interferon-γ upon activation, as much as parental Vα24+iNKT cells, but exhibit even better cytotoxic activity against various tumor cell lines. The iPS-Vα24+iNKT cells possess significant anti-tumor activity in tumor-bearing mice and can activate autologous NK cells upon activation by ligand-pulsed DCs in the NOG mouse model in vivo, further extending their therapeutic potential. This study thus provides a first proof of concept for the clinical application of human iPS-Vα24+iNKT cells for cancer immunotherapy. This article is protected by copyright. All rights reserved.
We developed a protocol to reprogram human Vα24+iNKT cells and to re-differentiate the iNKT cell-derived iPSCs into functional iNKT cells. The iPS-Vα24+iNKT cells activated by murine DC/Gal produce a large amount of interferon-γ upon activation and exhibit even better cytotoxic activity against various tumor cell lines than parental Vα24+iNKT cells. As a preclinical study, we show that the iPS-Vα24+iNKT cells possess significant anti-tumor activity in tumor-bearing NOG mice and adjuvant activity for NK cells in vivo.
Impaired T lymphopoiesis is associated with immunosuppression of the adaptive immune response and plays a role in the morbidity and mortality of patients and animal models of sepsis. Although previous studies examined several intrathymic mechanisms that negatively affect T lymphopoiesis, the extrathymic mechanisms remain poorly understood. Here, we report a dramatic decrease in the percentage of early T lineage progenitors (ETPs) in three models of sepsis in mice (cecal ligation and puncture, lipopolysaccharide continuous injection, and poly I:C continuous injection). However, septic mice did not show a decrease in the number of bone marrow (BM) precursor cells. Instead, the BM progenitors for ETPs expressed reduced mRNA levels of CC chemokine receptor (CCR) 7, CCR9 and P-selectin glycoprotein ligand 1, and exhibited impaired homing capacity in vitro and in vivo. Furthermore, RNA-Seq analysis and real-time PCR showed a marked downregulation of several lymphoid-related genes in hematopoietic stem and progenitor cells. Hematopoietic stem and progenitor cells differentiated into myeloid cells but failed to generate T lymphocytes in vitro and in vivo. Our results indicate that the depletion of ETPs in septic mice might be a consequence of an impaired migration of BM progenitors to the thymus, as well as a defect in lymphoid lineage commitment. This article is protected by copyright. All rights reserved.
A dramatic decrease of early T lineage progenitors (ETPs) in septic mice might be a consequence of an impaired migration of BM progenitors to the thymus, as well as a defect in lymphoid lineage commitment accompanied by enhanced myelopoiesis, suggesting a novel view of how sepsis induces lymphopenia.
Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers—hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1—at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes. Stem Cells 2016
Cardiomyocyte subtype was determined for each human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte from its action potential profile that was recorded optically followed by analysis of pacemaker-specific markers—hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1—by immunostaining in that same cell. This same-single-cell sequential analysis enabled the direct assessment of gene expression by cardiomyocyte subtype.
Human patient-derived induced pluripotent stem cells (hiPSCs) provide unique opportunities for disease modeling and drug development. However, adapting hiPSCs or their differentiated progenies to high throughput assays for phenotyping or drug screening has been challenging. Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and a major genetic cause of autism. FXS is caused by mutational trinucleotide expansion in the FMR1 gene leading to hypermethylation and gene silencing. One potential therapeutic strategy is to reactivate the silenced FMR1 gene, which has been attempted using both candidate chemicals and cell-based screening. However, molecules that effectively reactivate the silenced FMR1 gene are yet to be identified; therefore, a high throughput unbiased screen is needed. Here we demonstrate the creation of a robust FMR1-Nluc reporter hiPSC line by knocking in a Nano luciferase (Nluc) gene into the endogenous human FMR1 gene using the CRISPR/Cas9 genome editing method. We confirmed that luciferase activities faithfully report FMR1 gene expression levels and showed that neural progenitor cells derived from this line could be optimized for high throughput screening. The FMR1-Nluc reporter line is a good resource for drug screening as well as for testing potential genetic reactivation strategies. In addition, our data provide valuable information for the generation of knockin human iPSC reporter lines for disease modeling, drug screening, and mechanistic studies. Stem Cells 2016
We have created robust FMR1-luciferase reporter cell lines using genome editing. The reporter line faithfully reports endogenous FMR1 gene activation therefore will provide a critical resource for drug screening
Since their discovery, human pluripotent stem cells (hPSCs) including embryonic and induced pluripotent stem cells hold great promise in disease modeling and regenerative medicine. Despite intensive research and remarkable progress, it is becoming increasingly acknowledged that their yet incomplete, biological characterisation represents one of the major drawbacks to their successful translation into the clinics. The expression of the transmembrane protein E-Cadherin in hPSCs is well defined to be pivotal to the maintenance of the pluripotent state by mediating intercellular adhesion and intracellular signaling. Next to these canonical functions, were here report for the first time that hPSCs are subject to matrix metalloproteinase-dependent E-Cadherin ectodomain shedding. This generates a ∼80-kD, soluble E-Cadherin fragment which is released into the extracellular space, and which is well described to exert paracrine signaling activity and classified as being oncogenic. Collectively, this finding does not only improve our knowledge on the signaling crosstalk between hPSCs and their cellular environment and the type and nature of the paracrine signals produced by these cells, but also has clear implications for the development of efficient and safe stem cell-based therapies. Stem Cells 2016
E-cadherin ectodomain shedding in hPSCs with implications for basic research and therapy. hESCs and hiPSCs express E-cadherin on the cell surface known to be pivotal to the maintenance of the pluripotent state by mediating intercellular adhesion (1) and intracellular signaling (2). Next to these known, canonical functions of the full-length protein, the E-cadherin ectodomain is proteolytically cleaved upon MMP-mediated processing and released into the extracellular space (termed ectodomain shedding) (3). This E-cadherin fragment of 80 kD (soluble E-cadherin) is well documented to exert paracrine signaling activities by activating receptor tyrosine kinases and downstream signaling cascades leading to enhanced cell survival, proliferation and invasion, and is classified as being oncogenic. Taken together, this observation of E-cadherin ectodomain shedding in hPSCs strongly suggests the involvement of soluble E-cadherin in the signaling crosstalk between stem cells and their therapeutic and/or natural environment including putative roles in hPSC tumorigenicity and embryonic development.
The fundamental tenet that aging is irreversible has been challenged by the development of reprogramming technology that can restore molecular and cellular age by reversing the progression of aging. The use of cells from aged individuals as sources for reprogramming or transplantation creates a major barrier in stem cell therapy with respect to cell quality and quantity. Here, we investigated the molecular features underlying senescence and rejuvenation during aged cell reprogramming and identified novel factors that can overcome age-associated barriers. Enzymes, such as nicotinamide nucleotide transhydrogenase (NNT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), that control mitochondrial NAD+ levels appear to be susceptible to aging. In aged cells, mitochondrial NAD+ levels decrease, accompanied by reduced SIRT3 activity; these changes severely impede cell fate transition. However, in cells collected from aged p16 knockout mice, which exhibit delayed cellular senescence, no changes in NNT or NMNAT3 expression were found. Importantly, restoring mitochondrial NAD+ levels by overexpressing NNT and NMNAT3 enhanced reprogramming efficiency of aged somatic cells and extended the lifespan of human mesenchymal stem cells by delaying replicative senescence. These results demonstrate that maintenance of mitochondrial NAD+ levels is critical for reversing the mechanisms of aging and ensuring that cells collected from aged individuals are of high quality. Stem Cells 2016
Stem cells possess the ability to home in and travel to damaged tissue when injected intravenously. For the cells to exert their therapeutic effect, they must cross the blood vessel wall and enter the surrounding tissues. The mechanism of extravasation injected stem cells employ for exit has yet to be characterized. Using intravital microscopy and a transgenic zebrafish line Tg(fli1a:egpf) with GFP-expressing vasculature, we documented the detailed extravasation processes in vivo for injected stem cells in comparison to white blood cells (WBCs). While WBCs left the blood vessels by the standard diapedesis process, injected cardiac and mesenchymal stem cells underwent a distinct method of extravasation that was markedly different from diapedesis. Here, the vascular wall undergoes an extensive remodeling to allow the cell to exit the lumen, while the injected cell remains distinctively passive in activity. We termed this process Angio-pello-sis, which represents an alternative mechanism of cell extravasation to the prevailing theory of diapedesis. Stem Cells 2016
A stem cell during the middle of the Angiopellosis process. The cell is in the process of extravasating from the inside the lumen of a blood vessel, into the surrounding area. The endothelial cells of the blood vessel are seen remodeling themselves around the cell, as it is removed from the lumen.
Human mesenchymal stem cells (MSCs) hold great promise in cellular therapeutics for skeletal diseases but lack expression of E-selectin ligands that direct homing of blood-borne cells to bone marrow. Previously, we described a method to engineer E-selectin ligands on the MSC surface by exofucosylating cells with fucosyltransferase VI (FTVI) and its donor sugar, GDP-Fucose, enforcing transient surface expression of the potent E-selectin ligand HCELL with resultant enhanced osteotropism of intravenously administered cells. Here, we sought to determine whether E-selectin ligands created via FTVI-exofucosylation are distinct in identity and function to those created by FTVI expressed intracellularly. To this end, we introduced synthetic modified mRNA encoding FTVI (FUT6-modRNA) into human MSCs. FTVI-exofucosylation (i.e., extracellular fucosylation) and FUT6-modRNA transfection (i.e., intracellular fucosylation) produced similar peak increases in cell surface E-selectin ligand levels, and shear-based functional assays showed comparable increases in tethering/rolling on human endothelial cells expressing E-selectin. However, biochemical analyses revealed that intracellular fucosylation induced expression of both intracellular and cell surface E-selectin ligands and also induced a more sustained expression of E-selectin ligands compared to extracellular fucosylation. Notably, live imaging studies to assess homing of human MSC to mouse calvarium revealed more osteotropism following intravenous administration of intracellularly-fucosylated cells compared to extracellularly-fucosylated cells. This study represents the first direct analysis of E-selectin ligand expression programmed on human MSCs by FTVI-mediated intracellular versus extracellular fucosylation. The observed differential biologic effects of FTVI activity in these two contexts may yield new strategies for improving the efficacy of human MSCs in clinical applications. Stem Cells 2016
Two complimentary approaches were used to create E-selectin ligands on human mesenchymal stem cells (MSC) using fucosyltransferase VI (FTVI): extracellularly, by treating cells with purified FTVI enzyme, and intracellularly, by transfecting cells with FTVI-encoding modified mRNA. The extent to which the newly created E-selectin ligands could improve MSC homing to bone was tested using in vivo calvarial imaging. This three-dimensional reconstruction of a xenotransplanted mouse calvarium region shows bone (grey) and blood vessels (red), 24 hours after intravenously co-transplanting fucosylated MSCs (blue) and control MSCs (green). Both fucosylation approaches significantly increased homing of MSCs to the bone marrow, but intracellularly fucosylated (FUT6-mod) MSCs demonstrated increased extravasation into bone marrow parenchyma compared to their exofucosylated counterparts (FTVI-exo). The observed differential biologic effects of FTVI activity in these two contexts may yield new strategies for improving the efficacy of human MSCs in clinical applications. **p < 0.01
CXCR4 is a stem/progenitor cell surface receptor specific for the cytokine stromal cell-derived factor-1 (SDF-1α). There is evidence that bone marrow-derived CXCR4-expressing cells contribute to intimal hyperplasia (IH) by homing to the arterial subintima which is enriched with SDF-1α. We have previously found that transforming growth factor-β (TGFβ) and its signaling protein Smad3 are both upregulated following arterial injury and that TGFβ/Smad3 enhances the expression of CXCR4 in vascular smooth muscle cells (SMCs). It remains unknown, however, whether locally induced CXCR4 expression in SM22 expressing vascular SMCs plays a role in neointima formation. Here, we investigated whether elevated TGFβ/Smad3 signaling leads to the induction of CXCR4 expression locally in the injured arterial wall, thereby contributing to IH. We found prominent CXCR4 upregulation (mRNA, 60-fold; protein, 4-fold) in TGFβ-treated, Smad3-expressing SMCs. Chromatin immunoprecipitation assays revealed a specific association of the transcription factor Smad3 with the CXCR4 promoter. TGFβ/Smad3 treatment also markedly enhanced SDF-1α-induced ERK1/2 phosphorylation as well as SMC migration in a CXCR4-dependent manner. Adenoviral expression of Smad3 in balloon-injured rat carotid arteries increased local CXCR4 levels and enhanced IH, whereas SMC-specific depletion of CXCR4 in the wire-injured mouse femoral arterial wall produced a 60% reduction in IH. Our results provide the first evidence that upregulation of TGFβ/Smad3 in injured arteries induces local SMC CXCR4 expression and cell migration, and consequently IH. The Smad3/CXCR4 pathway may provide a potential target for therapeutic interventions to prevent restenosis. Stem Cells 2016
A schematic model showing transforming growth factor-β/Smad3-stimulated CXCR4 expression in the vascular smooth muscle cell and its signaling activated by the stromal cell-derived factor α ligand.
Human pluripotent stem cells possess remarkable proliferative and developmental capacity and thus have great potential for advancement of cellular therapy, disease modeling, and drug discovery. Twelve years have passed since the first reported isolation of human embryonic stem cell lines (hESC), followed in October 2010 by the first treatment of a patient with hESC-based cellular therapy at the Shepherd Center in Atlanta. Despite seemingly insurmountable challenges and obstacles in the early days, hESC clinical potential reached application in an extraordinarily short time. Eight currently ongoing clinical trials are yielding encouraging results, and these are likely to lead to new trials for other diseases. However, with the discovery of induced pluripotent stem cells (iPSC), disease-specific hESC lines derived from patients undergoing preimplantation genetic diagnosis for single gene disorders fell short of expectations. Lack of ethical controversy made human iPSC (hiPSC) with specific genotypes/phenotypes more appealing than hESC for drug discovery and toxicology-related studies, and in time, lines from HLA-homologous hiPSC banks are likely to take over from hESC in clinical applications. Currently, hESC are indispensable; the results of hESC-based clinical trials will set a gold standard for future iPSC-based cellular therapy. Stem Cells 2016
Ethical issues, the regulatory landscape, and the limited spectrum of diseases were all drawbacks of hESC lines that hiPSC did not have. It is to expect that in foreseeable future hiPSC will take over hESC in both research and clinical applications.
Mesenchymal stem cell (MSC) transplantation has been tested in animal and clinical fracture studies. We have developed a bone-seeking compound, LLP2A-Alendronate (LLP2A-Ale) that augments MSC homing to bone. The purpose of this study was to determine whether treatment with LLP2A-Ale or a combination of LLP2A-Ale and MSCs would accelerate bone healing in a mouse closed fracture model and if the effects are sex dependent. A right mid-femur fracture was induced in two-month-old osterix-mCherry (Osx-mCherry) male and female reporter mice. The mice were subsequently treated with placebo, LLP2A-Ale (500 μg/kg, IV), MSCs derived from wild-type female Osx-mCherry adipose tissue (ADSC, 3 x 105, IV) or ADSC + LLP2A-Ale.
In phosphate buffered saline-treated mice, females had higher systemic and surface-based bone formation than males. However, male mice formed a larger callus and had higher volumetric bone mineral density and bone strength than females. LLP2A-Ale treatment increased exogenous MSC homing to the fracture gaps, enhanced incorporation of these cells into callus formation, and stimulated endochondral bone formation. Additionally, higher engraftment of exogenous MSCs in fracture gaps seemed to contribute to overall fracture healing and improved bone strength. These effects were sex-independent.
There was a sex-difference in the rate of fracture healing. ADSC and LLP2A-Ale combination treatment was superior to on callus formation, which was independent of sex. Increased mobilization of exogenous MSCs to fracture sites accelerated endochondral bone formation and enhanced bone tissue regeneration. Stem Cells 2016
Lack of cell cycle checkpoints and uninterrupted passage through S-phase continuously renew the embryonic stem (ES) cell population and maintain pluripotency. Here, we show that to regulate mitotic progression and pluripotency ES cells must keep the aryl hydrocarbon receptor (AHR), an environmental sensor and transcriptional regulator, in a persistent state of repression. This repression, however, is not always absolute, causing the AHR to fluctuate between reversible states of expression and repression, with a fraction of the cells escaping repression at any one time. Cells that escape AHR repression exhibit reduced levels of the pluripotency factors OCT4 and SOX2 and show an extended mitotic traverse time due to AHR-dependent MID1 repression and the subsequent disruption of the MID1-PP2A-CDC25B-CDK1 signaling pathway that regulates mitosis. Unlike the bulk of the cell population that differentiates into cardiomyocytes upon stimulation, AHR-expressing ES cells restrict cardiogenesis and commit to a neuroglia cell fate. It appears that the untimely expression of the Ahr gene needs to be repressed to maintain ES cell mitotic progression and prevent premature loss of pluripotency. Stem Cells 2016
AHR expression in embryonic stem cells biases cell fate choice. Through downregulation of MID1, AHR delays mitotic progression and promotes premature loss of pluripotency.
The human salivary gland (SG) has an elegant architecture of epithelial acini, connecting ductal branching structures, vascular and neuronal networks that together function to produce and secrete saliva. This review focuses on the translation of cell- and tissue-based research toward therapies for patients suffering from SG hypofunction and related dry mouth syndrome (xerostomia), as a consequence of radiation therapy or systemic disease. We will broadly review the recent literature and discuss the clinical prospects of stem/progenitor cell and tissue-based therapies for SG repair and/or regeneration. Thus far, several strategies have been proposed for the purpose of restoring SG function: (1) transplanting autologous SG-derived epithelial stem/progenitor cells; (2) exploiting non-epithelial cells and/or their bioactive lysates; and (3) tissue engineering approaches using 3D (three-dimensional) biomaterials loaded with SG cells and/or bioactive cues to mimic in vivo SGs. We predict that further scientific improvement in each of these areas will translate to effective therapies toward the repair of damaged glands and the development of miniature SG organoids for the fundamental restoration of saliva secretion. Stem Cells 2016
Considered a feature of the metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), is associated with insulin resistance, type 2 diabetes, obesity and drug toxicity. Its prevalence is estimated at about 30% in western countries mainly due to sedentary life styles and high fat diets. Genome-wide association studies have identified polymorphisms in several genes, for example, PNPLA3, and TM6SF2 which confer susceptibility to NAFLD. Here, we review recent findings in the NAFLD field with a particular focus on published transcriptomics datasets which we subject to a meta-analysis. We reveal a common gene signature correlating with the progression of the disease from steatosis and steatohepatitis and reveal that lipogenic and cholesterol metabolic pathways are main actors in this signature. We propose the use of disease-in-a-dish models based on hepatocyte-like cells derived from patient-specific induced pluripotent stem cells (iPSC). These will enable investigations into the contribution of genetic background in the progression from NALFD to non-alcoholic steatohepatitis. Furthermore, an iPSC-based approach should aid in the elucidation of the function of new biomarkers, thus enabling better diagnostic tests and validation of potential drug targets. Stem Cells 2016
Investigating the progression of nonalcoholic fatty liver disease (NAFLD).
The progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH) still needs to be further elucidated to enable better non-invasive diagnosis and therapies. Here, we review recent findings in this field and propose a multi-omics approach based on liver biopsies, serum samples and induced pluripotent stem cells (iPSC)-differentiated hepatocyte like cell (HLC) models. As a result of a meta-analysis, we reveal several potential biomarkers correlating with the progression of the disease from steatosis and steatohepatitis. The use of disease-in-a-dish models based on hepatocyte-like cells derived from patient-specific induced pluripotent stem cells will enable investigations into the genetic background in the progression from NALFD to NASH. Additionally, an iPSC-based approach will support the assessment of potential biomarkers with the goal of achieving better diagnostic tests and validation of potential drug targets.
It has been proposed that tissue stem cells supply multiple epithelial cells in mature tissues and organs. However, it is unclear whether tissue stem cells generally contribute to cellular turnover in normal healthy organs. Here, we show that liver progenitors distinct from bipotent liver stem/progenitor cells (LPCs) persistently exist in mouse livers and potentially contribute to tissue maintenance. We found that, in addition to LPCs isolated as EpCAM+ cells, liver progenitors were enriched in CD45−TER119−CD31−EpCAM−ICAM-1+ fraction isolated from late-fetal and postnatal livers. ICAM-1+ liver progenitors were abundant by 4 weeks (4W) after birth. Although their number decreased with age, ICAM-1+ liver progenitors existed in livers beyond that stage. We established liver progenitor clones derived from ICAM-1+ cells between 1 and 20W and found that those clones efficiently differentiated into mature hepatocytes (MHs), which secreted albumin, eliminated ammonium ion, stored glycogen, and showed cytochrome P450 activity. Even after long-term culture, those clones kept potential to differentiate to MHs. When ICAM-1+ clones were transplanted into nude mice after retrorsine treatment and 70% partial hepatectomy, donor cells were incorporated into liver plates and expressed hepatocyte nuclear factor 4α, CCAAT/enhancer binding protein α, and carbamoylphosphate synthetase I. Moreover, after short-term treatment with oncostatin M, ICAM-1+ clones could efficiently repopulate the recipient liver tissues. Our results indicate that liver progenitors that can efficiently differentiate to MHs exist in normal adult livers. Those liver progenitors could be an important source of new MHs for tissue maintenance and repair in vivo, and for regenerative medicine ex vivo. Stem Cells 2016
ICAM-1+ hepatocytes can be isolated from healthy adult mouse liver as a distinctive cellular population from mature hepatocytes; they are small, mononucleated hepatocytes and express Cyps at a lower level. ICAM-1+ cells proliferate for long-term in vitro and keep potential to differentiate into functional hepatocytes. When they are used for transplantation, ICAM-1+ cells are integrated to the recipient liver tissue as mature hepatocytes. ICAM-1+ hepatocytes could be a promising cellular source for generating functional hepatocytes ex vivo.
Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016
Two distinct stem cell (SC) compartments in the mouse trachea include basal cells in the surface airway epithelium (SAE) and submucosal glands (SMGs). This research demonstrates that SCs from these two compartments have unique and overlapping properties that respond to dynamic changes in Wnt signaling following injury. Slowly cycling label-retaining (LRC) SCs isolated from the SMGs had a greater proliferative capacity than SAE LRCs and glandular LRCs reside near Wnt-active tubules. Isolated glandular SCs had a greater regenerative capacity to reconstitute a denuded tracheal xenograft than SAE SCs, and only glandular SCs formed gland-like clones in xenografts and organoid cultures.
Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are defined as pluripotent in view of their self-renewal ability and potential to differentiate to cells of all three germ layers. Recent studies have indicated that microRNAs (miRNAs) play an important role in the maintenance of pluripotency and cell cycle regulation. We used a microarray based approach to identify miRNAs that were enriched in hESCs when compared to differentiated cells and at the same time showed significant expression changes between different phases of cell cycle. We identified 34 candidate miRNAs and performed functional studies on one of these, miR-1305, which showed the highest expression change during cell cycle transition. Overexpression of miR-1305 induced differentiation of pluripotent stem cells, increased cell apoptosis and sped up G1/S transition, while its downregulation facilitated the maintenance of pluripotency and increased cell survival. Using target prediction software and luciferase based reporter assays we identified POLR3G as a downstream target by which miR-1305 regulates the fine balance between maintenance of pluripotency and onset of differentiation. Overexpression of POLR3G rescued pluripotent stem cell differentiation induced by miR-1305 overexpression. In contrast, knock-down of POLR3G expression abolished the miR-1305-knockdown mediated enhancement of pluripotency, thus validating its role as miR-1305 target in human pluripotent stem cells. Together our data point to an important role for miR-1305 as a novel regulator of pluripotency, cell survival and cell cycle and uncovers new mechanisms and networks by which these processes are intertwined in human pluripotent stem cells. Stem Cells 2016
Diabetes mellitus is a complex systemic disease characterized by severe morbidity and excess mortality. The burden of its multiorgan complications relies on an imbalance between hyperglycemic cell damage and defective endogenous reparative mechanisms. Inflammation and abnormalities in several hematopoietic components are typically found in diabetes. The discovery that diabetes reduces circulating stem/progenitor cells and impairs their function has opened an entire new field of study where diabetology comes into contact with hematology and regenerative medicine. It is being progressively recognized that such rare circulating cell populations mirror finely regulated processes involved in hematopoiesis, immunosurveillance, and peripheral tissue homeostasis. From a clinical perspective, pauperization of circulating stem cells predicts adverse outcomes and death. Furthermore, studies in murine models and humans have identified the bone marrow (BM) as a previously neglected site of diabetic end-organ damage, characterized by microangiopathy, neuropathy, fat deposition, and inflammation. As a result, diabetes impairs the mobilization of BM stem/progenitor cells, a defect known as mobilopathy or myelokathexis, with negative consequences for physiologic hematopoiesis, immune regulation, and tissue regeneration. A better understanding of the molecular and cellular processes that govern the BM stem cell niche, cell mobilization, and kinetics in peripheral tissues may uncover new therapeutic strategies for patients with diabetes. This concise review summarizes the current knowledge on the interplay between the BM, circulating stem cells, and diabetes, and sets the stages for future developments in the field. Stem Cells 2016
The network of stemness genes and oncogenes in human patient-specific reprogrammed cancer stem cells (CSCs) remains elusive, especially in liver cancer. HepG2-derived induced pluripotent stem cell-like cells (HepG2-iPS-like cells) were generated by introducing Yamanaka factors and the knockdown vector shTP53. They exhibited features of stemness and a higher tumorigenesis after xenograft transplantation compared with HepG2 cells. The cancerous mass of severe combined immunodeficiency (SCID) mice derived from one colony was dissected and cultured to establish reprogrammed HepG2-derived CSC-like cells (designated rG2-DC-1C). A single colony exhibited 42% occurrence of tumors with higher proliferation capacities. rG2-DC-1C showed continuous expression of the OCT4 stemness gene and of representative tumor markers, potentiated chemoresistance characteristics, and invasion activities. The sphere-colony formation ability and the invasion activity of rG2-DC-1C were also higher than those of HepG2 cells. Moreover, the expression of the OCT4 gene and the c-JUN oncogene, but not of c-MYC, was significantly elevated in rG2-DC-1C, whereas no c-JUN expression was observed in HepG2 cells. The positive-feedback regulation via OCT4-mediated transactivation of the c-JUN promoter and the c-JUN-mediated transactivation of the OCT4 promoter were crucial for promoting cancer development and maintaining cancer stemness in rG2-DC-1C. Increased expression of OCT4 and c-JUN was detected in the early stage of human liver cancer. Therefore, the positive feedback regulation of OCT4 and c-JUN, resulting in the continuous expression of oncogenes such as c-JUN, seems to play a critical role in the determination of the cell fate decision from iPS cells to CSCs in liver cancer. Stem Cells 2016
The OCT4/c-JUN positive feedback loop found in reprogrammed HepG2-derived cancer stem cell-like cells (rG2-DC-1C) sheds light on the tumorigenesis of liver cancer. 4F, Yamanaka 4 factors; iPS-like cells, induced pluripotent stem-like cells.
In this study, we identify a novel and essential role for the Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in the regulation of postnatal spermatogenesis. We show that GLIS3 is expressed in gonocytes, spermatogonial stem cells (SSCs) and spermatogonial progenitors (SPCs), but not in differentiated spermatogonia and later stages of spermatogenesis or in somatic cells. Spermatogenesis is greatly impaired in GLIS3 knockout mice. Loss of GLIS3 function causes a moderate reduction in the number of gonocytes, but greatly affects the generation of SSCs/SPCs, and as a consequence the development of spermatocytes. Gene expression profiling demonstrated that the expression of genes associated with undifferentiated spermatogonia was dramatically decreased in GLIS3-deficient mice and that the cytoplasmic-to-nuclear translocation of FOXO1, which marks the gonocyte-to-SSC transition and is necessary for SSC self-renewal, is inhibited. These observations suggest that GLIS3 promotes the gonocyte-to-SSC transition and is a critical regulator of the dynamics of early postnatal spermatogenesis. Stem Cells 2016
Cellular senescence as a result of organismal aging or progeroid diseases leads to stem cell pool exhaustion hindering tissue regeneration and contributing to the progression of age related disorders. Here we discovered that ectopic expression of the pluripotent factor NANOG in senescent or progeroid myogenic progenitors reversed cellular aging and restored completely the ability to generate contractile force. To elicit its effects, NANOG enabled reactivation of the ROCK and Transforming Growth Factor (TGF)-β pathways—both of which were impaired in senescent cells—leading to ACTIN polymerization, MRTF-A translocation into the nucleus and serum response factor (SRF)-dependent myogenic gene expression. Collectively our data reveal that cellular senescence can be reversed and provide a novel strategy to regain the lost function of aged stem cells without reprogramming to the pluripotent state. Stem Cells 2016
NANOG restores the myogenic and contractile capacity of senescent stem cells and progeria derived myofibroblasts. Hair follicle derived Mesenchymal Stem Cells (MSC) were transduced with a tetracycline regulatable vector that carries the NANOG gene. This system allows NANOG expression only when cells are treated with the tetracycline analog, Doxycycline. Cells were serially passaged until they became senescent (late passage, LP MSC). Subsequently Dox was added to the culture medium to induce NANOG expression in LP MSC (LP NANOG MSC) and the myogenic capacity was evaluated and compared to early passage (EP) MSC. Similar experiments were also performed with myofibroblasts derived from patients with an accelerating aging disease (Hutchinson Gilford Progeria Syndrome (HPGS)). (A-B): Western Blot for ACTA2 and SRF. (C-D) SRF dependent transcriptional activity (CArG-box). (E-F) Immunocytochemistry for ACTA2. (G-H): Contractile force using 3D collagen microtissue. (I): Schematic illustration describing the effects of NANOG on senescent cells. GM: Growth Medium, DM: Differentiation Medium. Scale bar: 20 μm for immunocytochemistry and 200 μm for the microtissues, data are presented as mean ± standard deviation, n = 3, *: designates statistical significance as compared to LP, LP DM or HGPS, HGPS DM (p < .05), #: designates statistical significance as compared to LP NANOG GM or HGPS NANOG GM (p < .05).
Numerous studies showed that mesenchymal stem cells derived exosome (MSC-Ex) markedly enhanced tissue regeneration, however, the issue of whether MSC-Ex could control stem cells expansion after a regenerative response to prevent tissue from overcrowding and dysplasia remains to be established. Herein, we found that human umbilical cord MSC (hucMSC)-exosomal14-3-3ζ mediated the binding of YAP and p-LATS by forming a complex to promote the phosphorylation of YAP, which orchestrate exosomal Wnt4 signal in cutaneous regeneration. First, we assessed deep second-degree burn rats treated with hucMSC-Ex and discovered that hucMSC-Ex promoting self-regulation of Wnt/β-catenin signaling at the remodeling phase of cutaneous regeneration. HucMSC-Ex restricted excessive skin cell expansion and collagen deposition at 4 weeks. Under high cell density conditions, hucMSC-Ex inhibited Wnt/β-catenin signaling through induction of YAP phosphorylation. Second, hucMSC-Ex proteomic analysis revealed that 14-3-3 proteins could be transported by exosome. Using gain- and loss-of-function studies, our results showed that hucMSC-exosomal 14-3-3ζ controlled YAP activities and phosphorylation at Ser127 site, and were required for the binding of YAP and p-LATS. Further studies revealed that 14-3-3ζ recruited YAP and p-LATS to form a complex under high cells density status and 14-3-3ζ other than YAP or p-LATS was the key regulatory molecule of this complex. These findings collectively indicate that hucMSC-Ex functions not only as an "accelerator" of the Wnt/β-catenin signal to repair damaged skin tissue but also as a "brake" of the signal by modulating YAP to orchestrate controlled cutaneous regeneration. Stem Cells 2016; 00:000–000
When skin cells were cultured at low cell density condition or Hippo pathway was inactivated, the phosphorylation of LATS was low and hucMSC-Ex delivered 14-3-3ζ did not work in skin cells. HucMSCexosomal Wnt4 promoted transcriptional activity of β-catenin in vitro and stem cells expansion and wound healing in vivo. With the activation of Hippo pathway and the increases of cell density, the expression of p- LATS was accumulated and hucMSC-Ex delivered 14-3-3ζ provided a platform to gather YAP and p-LATS to induce the phosphorylation of YAP. The upregulation of p-YAP reversed the activation of β-catenin induced by hucMSC-exosomal Wnt4 and restricted stem cells expansion to orchestrate controlled cutaneous regeneration.
Activation of mast cells participates in the chronic inflammation associated with cerebral arteries in intracranial aneurysm formation and rupture. Several studies have shown that the anti-inflammatory effect of mesenchymal stem cells (MSCs) is beneficial for the treatment of aneurysms. However, some long-term safety concerns exist regarding stem cell-based therapy for clinical use. We investigated the therapeutic potential of microvesicles (MVs) derived from human MSCs, anuclear membrane bound fragments with reparative properties, in preventing the rupture of intracranial aneurysm in mice, particularly in the effect of MVs on mast cell activation. Intracranial aneurysm was induced in C57BL/6 mice by the combination of systemic hypertension and intrathecal elastase injection. Intravenous administration of MSC-derived MVs on day 6 and day 9 after aneurysm induction significantly reduced the aneurysmal rupture rate, which was associated with reduced number of activated mast cells in the brain. A23187-induced activation of both primary cultures of murine mast cells and a human mast cell line, LAD2, was suppressed by MVs treatment, leading to a decrease in cytokine release and tryptase and chymase activities. Upregulation of prostaglandin E2 (PGE2) production and E-prostanoid 4 (EP4) receptor expression were also observed on mast cells with MVs treatment. Administration of an EP4 antagonist with the MVs eliminated the protective effect of MVs against the aneurysmal rupture in vivo. Human MSC-derived MVs prevented the rupture of intracranial aneurysm, in part due to their anti-inflammatory effect on mast cells, which was mediated by PGE2 production and EP4 activation. Stem Cells 2016; 00:000–000
Effects of intravenous administration of human mesenchymal stem cells-derived microvesicles (MVs) in the rupture of intracranial aneurysms. (A) Schematic diagram of experimental protocol to examine MVs treatment in a mouse model of aneurysm. (B-D) Incidence of aneurysm formation (unruptured and ruptured) (B), aneurysmal rupture rate (C) and symptom-free curve (Kaplan-Meier analysis curve) (D) were examined within 21 days after aneurysm induction in mice treated with vehicle (phosphate buffered saline) or MVs. In (D), the vertical dash lines indicate the time point of MVs or vehicle administration. Mice with aneurysm-free were excluded from graphs in (C) and (D). (E) Representative pictures of normal brain, unruptured and ruptured aneurysms with the whole view and magnified view at aneurysm site. *p < .01 by Fisher exact test; **p < .005 by log-rank test.
Secretory phospholipase A2 Group-IIA (sPLA2-IIA) catalyzes the hydrolysis of the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. sPLA2-IIA is deregulated in various cancers; however, its role in hair follicle stem cell (HFSC) regulation is obscure. Here we report a transgenic mice overexpressing sPLA2-IIA (K14-sPLA2-IIA) showed depletion of HFSC pool. This was accompanied with increased differentiation, loss of ortho-parakeratotic organization and enlargement of sebaceous gland, infundibulum and junctional zone. The colony forming efficiency of keratinocytes was significantly reduced. Microarray profiling of HFSCs revealed enhanced level of epithelial mitogens and transcription factors, c-Jun and FosB that may be involved in proliferation and differentiation. Moreover, K14-sPLA2-IIA keratinocytes showed enhanced activation of EGFR and JNK1/2 that led to c-Jun activation, which co-related with enhanced differentiation. Further, depletion of stem cells in bulge is associated with high levels of chromatin silencing mark, H3K27me3 and low levels of an activator mark, H3K9ac suggestive of alteration in gene expression contributing toward stem cells differentiation. Our results, first time uncovered that overexpression of sPLA2-IIA lead to depletion of HFSCs and differentiation associated with altered histone modification. Thus involvement of sPLA2-IIA in stem cells regulation and disease pathogenesis suggest its prospective clinical implications. Stem Cells 2016
Overexpression of sPLA2-IIA in mice epidermis leads to hyperplasia of both interfollicular epidermis and sebaceous gland, with enlarged size of the junctional zone and dermal papillae. In addition, loss of self-renewal in hair follicle stem cells was due to increased proliferation and differentiation, mediated through increased level of mitogenic signalling followed by enhanced expression of c-Jun, FosB, and Nr4a1.
Gene screenings have identified a number of reprogramming factors that induce pluripotency from somatic cells. However, the screening methods have mostly considered only factors that maintain pluripotency in embryonic stem cells, ignoring a potentially long list of other contributing factors involved. To expand the search, we developed a new screening method that examined 2,008 human genes in the generation of human induced pluripotent stem cells (iPSCs), including not only pluripotent genes but also differentiation-related genes that suppress pluripotency. We found the top 100 genes that increased reprogramming efficiency and discovered they contained many differentiation-related genes and homeobox genes. We selected two, HHEX and HLX, for further analysis. These genes enhanced the appearance of premature reprograming cells in the early phase of human iPSC induction, but had inhibitory effect on the late phase. In addition, when expressed in human iPSCs, HHEX and HLX interfered with the pluripotent state, indicating inverse effects on somatic reprograming and pluripotent maintenance. These results demonstrate that our screening is useful for identifying differentiation-related genes in somatic reprograming. Stem Cells 2016
We used a new screening strategy that investigated the effects of genes from a human cDNA library on reprogramming and discovered two genes, HHEX and HLX, which may offer new understanding on the induction of pluripotency.
Understanding the regulation of cell-cell interactions during the formation of compact myocardial structures is important for achieving true cardiac regeneration through enhancing the integration of stem cell-derived cardiomyocytes into the recipient myocardium. In this study, we found that cellular repressor of E1A-stimulated genes 1 (CREG1) is highly expressed in both embryonic and adult hearts. Gain- and loss-of-function analyses demonstrated that CREG1 is required for differentiation of mouse embryonic stem (ES) cell into cardiomyocytes and the formation of cohesive myocardium-like structures in a cell-autonomous fashion. Furthermore, CREG1 directly interacts with Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Site-directed mutagenesis and rescue of CREG1 knockout ES cells showed that CREG1 binding to Sec8 is required for cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8, and N-cadherin colocalize at intercalated discs in vivo and are enriched at cell-cell junctions in cultured cardiomyocytes. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis. Stem Cells 2016
Schematic model for a role of CREG1 in the formation of intercalated discs between cardiomyocytes. During cardiomyocyte differentiation from embryonic stem cells, CREG1 is upregulated and binds to Sec8 of the exocyst complex. The CREG1-exocyst interaction increases the delivery of N-cadherin from intracellular compartments, such as Golgi and recycling endosomes (ER), to the cell-cell adhesion sites, thereby promoting the formation of intercalated discs.
Acute rejection is commonly encountered for long-term survival in liver transplant (LT) recipients and may impact their long-term survival if rejection is severe or recurrent. The aim of this study is to examine the therapeutic potential of transforming growth factor (TGF-β)-overexpressing mesenchymal stem cells (MSCs) in inducing a local immunosuppression in liver grafts after transplantation. MSCs were transduced with a lentiviral vector expressing the human TGF-β1 gene; TGF-β1-overexpressing MSCs (designated as TGF/MSCs) were then transfused into the liver grafts via the portal vein of a rat LT model of acute rejection. Rejection severity was assessed by clinical and histologic analysis. The immunity suppression effects and mechanism of TGF/MSCs were tested, focusing on their ability to induce generation of regulatory T cells (Tregs) in the liver grafts. Our findings demonstrate that transfusion of TGF/MSCs prevented rejection, reduced mortality, and improved survival of rats after LT. The therapeutic effects were associated with the immunosuppressive effects of MSCs and TGF-β1. Their reciprocal effects on Tregs induction and function resulted in more CD4 + Foxp3 + Helios- induced Tregs, fewer Th17 cells, and improved immunosuppressive effects in local liver grafts. Thus, TGF/MSCs can induce a local immunosuppressive effect in liver grafts after transplantation. The immunomodulatory activity of TGF-β1 modified MSCs may be a gateway to new therapeutic approaches to prevent organ rejection in clinical transplantation. Stem Cells 2016
Bone homeostasis comprises the balance between bone-forming osteoblasts and bone-resorbing osteoclasts (OCs), with an acceleration of osteoclastic bone resorption leading to osteoporosis. OCs can be generated from bone marrow cells (BMCs) under the tightly regulated local bone environment. However, it remained difficult to identify the critical cells responsible for providing an osteoclastogenesis niche. In this study, we used a fluorescence-activated cell sorting technique to determine the cell populations important for forming an appropriate microenvironment for osteoclastogenesis and to verify the associated interactions between osteoclast precursor cells and non-OCs. We isolated and removed a small cell population specific for osteoclastogenesis (CXCR4+ CD45−) from mouse BMCs and cultured the remaining cells with receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage-colony stimulating factor. The resulting cultures showed significantly less large osteoclast formation. Quantitative RT-PCR analysis revealed that these CXCR4+ CD45− cells expressed low levels of RANK and RANKL, but high levels of critical chemokines including stromal cell derived factor 1 (SDF-1), chemokine (C-X-C motif) ligand 7 (CXCL7), and chemokine (C-X3-C motif) ligand 1 (CX3CL1). Furthermore, an SDF-1-specific antibody strongly suppressed OC formation in RAW264.7 cells and antibodies against SDF-1, CXCL7, and CX3CL1 suppressed OC formation in BMCs. These results suggest that isolated CXCR4+ CD45− cells support an appropriate microenvironment for osteoclastogenesis with a direct effect on the cells expressing SDF-1, CXCL7, and CX3CL1 receptors. The regulation of CXCR4+ CD45− cell function might therefore inform therapeutic strategies for diseases involving loss of bone homeostasis. Stem Cells 2016
Recent pre-clinical and clinical studies have suggested that endogenous cardiospheres (eCS) are potentially safe and effective for cardiac regeneration following myocardial infarction (MI). Nevertheless the preparation of autologous eCS requires invasive myocardial biopsy with limited yield. We describe a novel approach to generate induced cardiospheres (iCS) from adult skin fibroblasts via somatic reprogramming. After infection with Sox2, Klf4, and Oct4, iCS were generated from mouse adult skin fibroblasts treated with Gsk3β inhibitor-(2′Z,3′E)- 6-Bromoindirubin-3′-oxime and Oncostatin M. They resembled eCS, but contained a higher percentage of cells expressing Mesp1, Isl1, and Nkx2.5. They were differentiated into functional cardiomyocytes in vitro with similar electrophysiological properties, calcium transient and contractile function to eCS and mouse embryonic stem cell-derived cardiomyocytes. Transplantation of iCS (1 × 106 cells) into mouse myocardium following MI had similar effects to transplantation of eCS but significantly better than saline or fibroblast in improving left ventricular ejection fraction, increasing anterior/septal ventricular wall thickness and capillary density in the infarcted region 4 weeks after transplantation. No tumor formation was observed. iCS generated from adult skin fibroblasts by somatic reprogramming and a cocktail of Gsk3β inhibitor-6-Bromoindirubin-3′-oxime and Oncostatin M may represent a novel source for cell therapy in MI. Stem Cells 2016
We describe a novel approach to generate induced cardiospheres (iCS) from adult skin fibroblasts via somatic reprogramming. After infection with Sox2, Klf4, and Oct4, iCS were generated from adult skin fibroblasts after treatment with a cocktail of cardiotrophic growth factors: cardiotrophic Gsk3β inhibitor-(2′Z,3′E)- 6-Bromoindirubin-3′-oxime and Oncostatin M, through upregulation of β-catenin pathway. The iCSs can be differentiated into functional cardiomyocytes in vitro with similar electrophysiological properties, calcium transient and contractile function to endogenous cardiosphere. Transplantation of iCS into mouse myocardium following myocardial infarction had similar effects to transplantation of cardiosphere in improving left ventricular ejection fraction, increasing anterior/septal ventricular wall thickness, and capillary density in the infarcted region.
Post-translational modification by ubiquitin molecules is a key regulatory process for stem cell fate determination. Ubiquitination and deubiquitination are the major cellular processes used to balance the protein turnover of several transcription factors that regulate stem cell differentiation. Deubiquitinating enzymes (DUBs), which facilitate the processing of ubiquitin, significantly influence stem cell fate choices. Specifically, DUBs play a critical regulatory role during development by directing the production of new specialized cells. This review focuses on the regulatory role of DUBs in various cellular processes, including stem cell pluripotency and differentiation, adult stem cell signaling, cellular reprogramming, spermatogenesis, and oogenesis. Specifically, the identification of interactions of DUBs with core transcription factors has provided new insight into the role of DUBs in regulating stem cell fate determination. Thus, DUBs have emerged as key pharmacologic targets in the search to develop highly specific agents to treat various illnesses. Stem Cells 2016
Regulatory role of deubiquitinating enzymes in stem cell pluripotency and differentiation, cellular reprogramming, and gametogenesis.
Since the discovery of induced pluripotent stem cells there has been intense interest in understanding the mechanisms that allow a somatic cell to be reprogrammed back to a pluripotent state. Several groups have studied the alterations in gene expression that occur as somatic cells modify their genome to that of an embryonic stem cell. Underpinning many of the gene expression changes are modifications to the epigenetic profile of the associated chromatin. We have used a large-scale shRNA screen to identify epigenetic modifiers that act as barriers to reprogramming. We have uncovered an important role for TRIM28 in cells resisting transition between somatic and pluripotent states. TRIM28 achieves this by maintaining the H3K9me3 repressed state and keeping endogenous retroviruses (ERVs) silenced. We propose that knockdown of TRIM28 during reprogramming results in more plastic H3K9me3 domains, dysregulation of genes nearby H3K9me3 marks, and up regulation of ERVs, thus facilitating the transition through reprogramming. Stem Cells 2016
Model of TRIM28 function during reprogramming
A) Ectopic expression of Oct4, Sox2, Klf4 and c-Myc (OSKM) induces the reprogramming of somatic cells into induced pluripotent stem cells at a low efficiency B) Ectopic expression of OSKM along with knock down of TRIM28 increases the expression of genes nearby H3K9me3, and induces the expression of endogenous retroviruses. This decondensed chromatin state increases the reprogramming efficiency.
Few gene targets of Visual System Homeobox 2 (VSX2) have been identified despite its broad and critical role in the maintenance of neural retina (NR) fate during early retinogenesis. We performed VSX2 ChIP-seq and ChIP-PCR assays on early stage optic vesicle-like structures (OVs) derived from human iPS cells (hiPSCs), which highlighted WNT pathway genes as direct regulatory targets of VSX2. Examination of early NR patterning in hiPSC-OVs from a patient with a functional null mutation in VSX2 revealed mis-expression and upregulation of WNT pathway components and retinal pigmented epithelium (RPE) markers in comparison to control hiPSC-OVs. Furthermore, pharmacological inhibition of WNT signaling rescued the early mutant phenotype, whereas augmentation of WNT signaling in control hiPSC-OVs phenocopied the mutant. These findings reveal an important role for VSX2 as a regulator of WNT signaling and suggest that VSX2 may act to maintain NR identity at the expense of RPE in part by direct repression of WNT pathway constituents. Stem Cells 2016
Visual systems homeobox 2 (VSX2) is a key transcription factor involved in neural retinal development. However, surprisingly few gene regulatory targets are known for VSX2 beyond Microphthalmia induced transcription factor (MITF), a protein involved in the development of retinal pigmented epithelium (RPE). We used patient-specific human pluripotent stem cells carrying a mutation in VSX2 that abolishes DNA binding activity (R200Q), combined with pharmacological manipulations and chromatin immunoprecipitation, to demonstrate a novel interaction between VSX2 and WNT signaling genes. In VSX2R200Q cultures, VSX2 binding to WNT signaling genes is abolished and optic vesicle cells aberrantly adopt an RPE fate in lieu of neural retina. This effect that can be partially overcome by inhibition of WNT signaling, or induced in wild type cells through promotion of WNT signaling. This study also presents the first unbiased list of VSX2 targets, thus providing a resource for identification of other genes and mechanisms involved in this biological process.
Osteogenic differentiation and bone formation is suppressed under condition of inflammation induced by proinflammation cytokines. A number of studies indicate miRNAs play a significant role in tumor necrosis factor-α-induced inhibition of bone formation, but whether long non-coding RNAs are also involved in this process remains unknown. In this study, we evaluated the role of MIR31HG in osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo. The results suggested that knockdown of MIR31HG not only significantly promoted osteogenic differentiation, but also dramatically overcame the inflammation-induced inhibition of osteogenesis in hASCs. Mechanistically, we found MIR31HG regulated bone formation and inflammation via interacting with NF-κB. The p65 subunit bound to the MIR31HG promoter and promoted MIR31HG expression. In turn, MIR31HG directly interacted with IκBα and participated in NF-κB activation, which builds a regulatory circuitry with NF-κB. Targeting this MIR31HG–NF-κB regulatory loop may be helpful to improve the osteogenic capacity of hASCs under inflammatory microenvironment in bone tissue engineering. Stem Cells 2016.
Schematic of the regulatory circuitry of MIR31HG and NF-κB. Under TNF-α and IL-17 stimulation, MIR31HG directly binds to IκBα and contributes to IκBα phosphorylation and NF-κB activation. The nuclear translocation of NF-κB in turn binds to MIR31HG promoter, upregulates its expression, and promotes its export to the cytoplasm where it exerts its function.
Glioblastoma multiforme (GBM) is the most common and lethal adult brain tumor. Resistance to standard radiation and chemotherapy is thought to involve survival of GBM cancer stem cells (CSCs). To date, no single marker for identifying GBM CSCs has been able to capture the diversity of CSC populations, justifying the needs for additional CSC markers for better characterization. Employing targeted mass spectrometry, here we present five cell-surface markers HMOX1, SLC16A1, CADM1, SCAMP3, and CLCC1 which were found to be elevated in CSCs relative to healthy neural stem cells (NSCs). Transcriptomic analyses of REMBRANDT and TCGA compendiums also indicated elevated expression of these markers in GBM relative to controls and non-GBM diseases. Two markers SLC16A1 and HMOX1 were found to be expressed among pseudopalisading cells that reside in the hypoxic region of GBM, substantiating the histopathological hallmarks of GBM. In a prospective study (N = 8) we confirmed the surface expression of HMOX1 on freshly isolated primary GBM cells (P0). Employing functional assays that are known to evaluate stemness, we demonstrate that elevated HMOX1 expression is associated with stemness in GBM and can be modulated through TGFβ. siRNA-mediated silencing of HMOX1 impaired GBM invasion—a phenomenon related to poor prognosis. In addition, surgical resection of GBM tumors caused declines (18% ± 5.1SEM) in the level of plasma HMOX1 as measured by ELISA, in 8/10 GBM patients. These findings indicate that HMOX1 is a robust predictor of GBM CSC stemness and pathogenesis. Further understanding of the role of HMOX1 in GBM may uncover novel therapeutic approaches. Stem Cells 2016.
Targeted proteomic analyses revealed elevated expressions of transmembrane proteinsHMOX1 and SLC16A1on the surface of glioblastoma cancer stem cells (CSCs) relative to healthy neural stem cells (NSCs). In the hypoxic region of the tumor, these proteins were found to be expressed among pseudopalisading glioma cells that also express known stem cell factors. From biological assays that are known to evaluate stemness, HMOX1 expression was found to be associated with stemness that could be regulated through TGFβ and PTEN signaling networks. Additionally, siRNA mediated inhibition of HMOX1 expression impaired cancer cell invasion. Together, this study demonstrates the association of HMOX1 expression with cancer stemness in glioblastoma and suggests a possible regulatory mechanism of CSC invasion and survival in the hypoxic region of the tumor.
Stromal support is critical for lung homeostasis and the maintenance of an effective epithelial barrier. Despite this, previous studies have found a positive association between the number of mesenchymal stromal cells (MSCs) isolated from the alveolar compartment and human lung diseases associated with epithelial dysfunction. We hypothesised that bronchoalveolar lavage derived MSCs (BAL-MSCs) are dysfunctional and distinct from resident lung tissue MSCs (LT-MSCs). In this study, we comprehensively interrogated the phenotype and transcriptome of human BAL-MSCs and LT-MSCs. We found that MSCs were rarely recoverable from the alveolar space in healthy humans, but could be readily isolated from lung transplant recipients by bronchoalveolar lavage. BAL-MSCs exhibited a CD90Hi, CD73Hi, CD45Neg, CD105Lo immunophenotype and were bipotent, lacking adipogenic potential. In contrast, MSCs were readily recoverable from healthy human lung tissue and were CD90Hi or Lo, CD73Hi, CD45Neg, CD105Int and had full tri-lineage potential. Transcriptional profiling of the two populations confirmed their status as bona fide MSCs and revealed a high degree of similarity between each other and the archetypal bone-marrow MSC. 105 genes were differentially expressed; 76 of which were increased in BAL-MSCs including genes involved in fibroblast activation, extracellular matrix deposition and tissue remodelling. Finally, we found the fibroblast markers collagen 1A1 and α-smooth muscle actin were increased in BAL-MSCs. Our data suggests that in healthy humans, lung MSCs reside within the tissue, but in disease can differentiate to acquire a profibrotic phenotype and migrate from their in-tissue niche into the alveolar space. Stem Cells 2016.
Human mesenchymal stromal cells were isolated from healthy parenchymal lung tissue (LT-MSCs, n = 24, 4 samples from six patients) and lung allograft bronchoalveolar lavage fluid (BAL-MSCs, n = 12, 1 sample from 12 patients) and gene expression was analysed by microarray. Using Significance Analysis of Microarrays, 105 differentially expressed genes were identified. When visualized as a hierarchical cluster, MSC populations segregate into two distinct groups when samples and probes are clustered using Pearson correlation. Genes that were upregulated in BAL-MSCs include those associated with cellular migration, transforming growth factor-β activation and fibroblastic differentiation. Our findings suggested that BAL-MSCs are a dysregulated population of tissue resident MSCs and may contribute to allograft fibrosis.
As known from model organisms, such as frog, fish, mouse, and chicken, the anterior–posterior patterning of the definitive endoderm (DE) into distinct domains is controlled by a variety of signaling interactions between the DE and its surrounding mesoderm. This includes Wnt/FGFs and BMPs in the posterior half and all-trans-retinoic acid, TGF-β-ligands, Wnt-, and BMP-inhibitors in the anterior half of the DE sheet. However, it is currently unclear how these embryonic tissue interactions can be translated into a defined differentiation protocol for human embryonic stem cells. Activin A has been proposed to direct DE into a SOX2-positive foregut-like cell type. Due to the pleiotropic nature of SOX2 in pluripotency and developing cells of the foregut, we purified DE-cells by magnetic cell sorting and tested the effects of anteriorizing and posteriorizing factors on pure endoderm. We show in contrast to previous studies that the generation of the foregut marked by SOX2/FOXA2 double-positive cells does not depend on activin A/TGF-β-signaling but is mediated by the inhibition of Wnt- and BMP-signaling. Retinoic acid can posteriorize and at the same time dorsalize the foregut toward a PDX1-positive pancreatic duodenal cell type whereas active Wnt/beta-catenin signaling synergistically with FGF-2, BMP-4, and RA induces the formation of CDX2-positive posterior endoderm. Thus, these results provide new insights into the mechanisms behind cell specification of human DE derived from pluripotent stem cells. Stem Cells 2016.
Differentiation of pluripotent stem cells (PSC) via the definitive endoderm (DE) germ layer into the patterned primitive gut tube (PGT). The anterior–posterior axis of the PGT is controlled by differential signaling of Wnt/beta-catenin-, BMP-, FGF-, and RA signaling. BMP inhibition (BMPi) and to a lesser Wnt/beta-catenin inhibition (WNTi) induce a foregut-like SOX2-positive state. Active Wnt/beta-catenin supported by BMPs, FGFs and all-trans retinoic acid (ATRA) induce a hindgut-like state. SOX2 is strongly expressed in PSCs, becomes downregulated during endoderm commitment, and reappears in the foregut domain upon A–P patterning, whereas CDX2 is expressed in the hindgut domain. ATRA is able to posteriorize the foregut.
Adipose tissue-derived stem cells (ASCs) are proposed as an alternative stem cell source to bone marrow-derived cells for immune cell therapy. However, microenvironmental factors may impact the functionality of this population in human adipose tissue (AT). We hypothesized that the fat depot in addition to the donor phenotype controls the immunomodulatory capacity of ASCs. Focusing on obesity and type 2 diabetes (T2D) as metabolic disorders that might affect the immune response of ASCs, we compared the inflammatory response of ASCs from subcutaneous and visceral AT of age-matched donors (lean n = 4, body mass index [BMI] 21.98 ± 1.9; obese n = 4 BMI 33.1 ± 2.1 and T2D n = 4 BMI 35.3 ± 1.5). Obese and particularly T2D-derived ASCs showed increased expression of inflammatory markers, activation of NLRP3 inflammasome and higher migration, invasion and phagocytosis capacities than those derived from lean donors. Remarkably, ASCs derived from obese and T2D subjects exhibited a reduction in typical immunosuppressive activities attributed to stem cells. Accordingly, obese and T2D-ASCs were less effective in suppressing lymphocyte proliferation, activating the M2 macrophage phenotype, and in increasing TGF-β1 secretion, than lean-derived ASCs. Treatment of lean hASCs with interleukin (IL)-1β mimicked the dysfunctional immune behavior of obese and T2D hASCs. Conversely, combined treatment with IL1RA and TGF-β1 reverted the phenotype of obese- and T2D-ASCs. These data indicate that the donor metabolic phenotype compromises the immunomodulatory properties of ASCs. These results are relevant not only for understanding the physiology of ASCs in terms of cell-based therapies but also for their role as key regulators of the immune response. Stem Cells 2016.
Obesity and T2D disturbs the immune properties of ASCs. It is known that obesity increases proliferation of ASCs but reduces their adipogenic differentiation capacity. This study reveals that ASCs from healthy (lean) individuals exhibit typical immunosuppressive activities such as induction of the M2 macrophage phenotype and suppression of T and B cell proliferation. By contrast, ASCs isolated from obese and T2D subjects promote the M1 macrophage phenotype and are less efficient in suppressing lymphocyte proliferation. Thus, obesity and T2D not only disrupt adipose tissue remodeling but also compromise the immunoregulatory properties of ASCs.
Hemogenic endothelial cells (HECs) are considered to be the origin of hematopoietic stem cells (HSCs). HECs have been identified in differentiating mouse embryonic stem cells (ESCs) as VE-cadherin+ cells with both hematopoietic and endothelial potential in single cells. Although the bipotential state of HECs is a key to cell fate decision toward HSCs, the molecular basis of the regulation of the bipotential state has not been well understood. Here, we report that the CD41+ fraction of CD45- CD31+ VE-cadherin+ endothelial cells (ECs) from mouse ESCs encompasses an enriched HEC population. The CD41+ ECs expressed Runx1, Tal1, Etv2, and Sox17, and contained progenitors for both ECs and hematopoietic cells (HCs) at a high frequency. Clonal analyses of cell differentiation confirmed that one out of five HC progenitors in the CD41+ ECs possessed the bipotential state that led also to EC colony formation. A phenotypically identical cell population was found in mouse embryos, although the potential was more biased to hematopoietic fate with rare bipotential progenitors. ESC-derived bipotential HECs were further enriched in the CD41+ CXCR4+ subpopulation. Stimulation with CXCL12 during the generation of VE-cadherin+ CXCR4+ cells attenuated the EC colony-forming ability, thereby resulted in a decrease of bipotential progenitors in the CD41+ CXCR4+ subpopulation. Our results suggest that CXCL12/CXCR4 signaling negatively modulates the bipotential state of HECs independently of the hematopoietic fate. Identification of signaling molecules controlling the bipotential state is crucial to modulate the HEC differentiation and to induce HSCs from ESCs. Stem Cells 2016.
ES cell-derived CD45- VE-cadherin+ CD41+ CXCR4+ cells represent a hemogenic endothelial cell (HEC) population. HECs encompass the differentiation programs for both the endothelial cell (EC) and hematopoietic cell (HC) lineages. A balance between these two programs provides an HEC with a bipotential state that allows the progeny of the cell to differentiate into both ECs and HCs. Fate of the bipotential HECs can be regulated by CXCL12/CXCR4 signaling, which suppresses the EC program independently of the hematopoietic fate.
Hyaluronan-coated surfaces preserve the proliferation and differentiation potential of mesenchymal stem cells by prolonging their G1-phase transit, which maintains cells in a slow-proliferative mode. Mitochondria are known to play a crucial role in stem cell self-renewal and differentiation. In this study, for the first time, the metabolic mechanism underlying the hyaluronan-regulated slow-proliferative maintenance of stem cells was investigated by evaluating mitochondrial functions. Human placenta-derived mesenchymal stem cells (PDMSCs) cultured on hyaluronan-coated surfaces at 0.5, 3.0, 5.0, and 30 µg/cm2 were found to have an average 58% higher mitochondrial mass and an increase in mitochondrial DNA copy number compared to noncoated tissue culture surfaces (control), as well as a threefold increase in the gene expression of the mitochondrial biogenesis-related gene PGC-1α. Increase in mitochondrial biogenesis led to a hyaluronan dose-dependent increase in mitochondrial membrane potential, ATP content, and oxygen consumption rate, with reactive oxygen species levels shown to be at least three times lower compared to the control. Although hyaluronan seemed to favor mitochondrial function, cell entry into a hyaluronan-regulated slow-proliferative mode led to a fivefold reduction in ATP production and coupling efficiency levels. Together, these results suggest that hyaluronan-coated surfaces influence the metabolic proliferative state of stem cells by upregulating mitochondrial biogenesis and function with controlled ATP production. This more efficiently meets the energy requirements of slow-proliferating PDMSCs. A clear understanding of the metabolic mechanism induced by hyaluronan in stem cells will allow future applications that may overcome the current limitations faced in stem cell culture. Stem Cells 2016
Hyaluronan is a key player in the maintenance of slow-proliferative human mesenchymal stem cells through the upregulation of mitochondrial-biogenesis related genes (PGC1-α and mTFA) leading to upregulation of mitochondrial biogenesis (mtDNA, mtMass), mitochondrial function (ATP, OCR, MMP) and mitochondrial respiration with reduced ROS levels and eventual reduced ATP production, as a means of utilizing more efficiently functional mitochondria.
Tumor stromal cells can supply appropriate signals that may develop aggressive phenotypes of carcinoma cells and establish a complex scenario which culminates in metastasis. Recent works proposed that bone marrow-derived mesenchymal stem cells (MSC) are recruited to primary tumors. However, the exact functions of these cells in the tumor microenvironment are not well characterized, as it is reported that MSC can either promote or inhibit tumor progression. In the present study, we aim at investigating the signaling molecules which regulate the interplay between MSC, prostate carcinoma (PCa) cells and two important cellular types constituting the tumor-associated stroma, macrophages and fibroblasts, during their progression toward malignancy. We identified TGF-β1 as a crucial molecule able to attract MSC recruitment both to PCa cells as well as to tumor stroma components. Moreover, PCa- and tumor stroma-secreted TGF-β1 is important to induce MSC transdifferentiation into carcinoma-associated fibroblast (CAF)-like cells. Consequently, the CAF-like phenotype acquired by MSC is central to promote tumor progression related effects. Thus, tumor-educated MSC enhance PCa invasiveness compared to nonactivated MSC. Additionally, differing from normal MSC, CAF-like MSC perform vascular mimicry and recruit monocytes, which can be further polarized to M2 macrophages within the PCa environment. Our findings indicate a prominent role for TGF-β1 in MSC mobilization and activation strengthened by the fact that the blockade of TGF-β1 signaling impairs MSC promotion of PCa progression. Stem Cells 2016
Prostate carcinoma microenvironment recruits and activates mesenchymal stem cells into CAF via TGF-β1. TGF-β 1 produced by prostate carcinoma (PCa) cells and tumor-associated fibroblasts and macrophages is important in bone marrow-derived mesenchymal stem cells (BM-MSC) recruitment to PCa site and further transdifferentiation to the carcinoma-associated fibroblast (CAF)-like phenotype. CAF-like MSC promote PCa cells invasiveness, perform vascular mimicry abilities and recruit monocytes, which can further polarize into M2-like macrophages.
We have previously demonstrated that cryopreservation and thawing lead to altered Mesenchymal stromal cells (MSC) functionalities. Here, we further analyzed MSC's fitness post freeze-thaw. We have observed that thawed MSC can suppress T-cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T-cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous Peripheral Blood Mononuclear Cells (PBMCs) and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO-free cryoprotectants or substitution of Human Serum Albumin (HSA) with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ prelicensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1-methyl Tryptophan, an Indoleamine 2,3-dioxygenase (IDO) inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw-induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T-cell cytolysis and affect MSC's cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use. Stem Cells 2016
Epigenetic reprogramming is a central process during mammalian germline development. Genome-wide DNA demethylation in primordial germ cells (PGCs) is a prerequisite for the erasure of epigenetic memory, preventing the transmission of epimutations to the next generation. Apart from DNA demethylation, germline reprogramming has been shown to entail reprogramming of histone marks and chromatin remodelling. Contrary to other animal models, there is limited information about the epigenetic dynamics during early germ cell development in humans. Here, we provide further characterization of the epigenetic configuration of the early human gonadal PGCs. We show that early gonadal human PGCs are DNA hypomethylated and their chromatin is characterized by low H3K9me2 and high H3K27me3 marks. Similarly to previous observations in mice, human gonadal PGCs undergo dynamic chromatin changes concomitant with the erasure of genomic imprints. Interestingly, and contrary to mouse early germ cells, expression of BLIMP1/PRDM1 persists in through all gestational stages in human gonadal PGCs and is associated with nuclear lysine-specific demethylase-1. Our work provides important additional information regarding the chromatin changes associated with human PGCs development between 6 and 13 weeks of gestation in male and female gonads. Stem Cells 2016
Cardiomyocytes from human pluripotent stem cells (hPSC) are of growing interest as models to understand mechanisms underlying genetic disease, identify potential drug targets and for safety pharmacology as they may predict human relevant effects more accurately and inexpensively than animals or other cell models. Crucial to their optimal use are accurate methods to quantify cardiomyocyte phenotypes accurately and reproducibly. Here, we review current methods for determining biophysical parameters of hPSC-derived cardiomyocytes (hPSC-CMs) that recapitulate disease and drug responses. Even though hPSC-CMs as currently available are immature, various biophysical methods are nevertheless already providing useful insights into the biology of the human heart and its maladies. Advantages and limitations of assays currently available looking toward applications of hPSC-CMs are described with examples of how they have been used to date. This will help guide the choice of biophysical method to characterize healthy cardiomyocytes and their pathologies in vitro. Stem Cells 2016
Measuring pluripotent stem cell-derived cardiomyocyte physiology.
In the mouse brain, neuroblasts generated in the subventricular zone (SVZ) migrate to the olfactory bulb (OB) through the rostral migratory stream (RMS). Although the RMS is not present in the human brain, a migratory pathway that is organized around a ventricular cavity that reaches the OB has been reported. A similar cavity, the lateral ventricle extension (LVE), is found in the adult guinea pig brain. Therefore, we analyzed cytoarchitecture, proliferative activity and precursor cell migration in the SVZ and LVE of 1-, 6- and 12-month-old guinea pigs. In young animals, we used confocal spectral and transmission electron microscopy to identify neuroblasts, astrocytes, and progenitor cells in the SVZ and LVE. Analysis of peroxidase diffusion demonstrated that the LVE was a continuous cavity lined by ependymal cells and surrounded by neuroblasts. Precursor cells were mostly located in the SVZ and migrated from the SVZ to the OB through the LVE. Finally, analysis of 6- and 12-month-old guinea pigs revealed that the LVE was preserved in older animals; however, the number of neurogenic cells was significantly reduced. Consequently, we propose that the guinea pig brain may be used as a new neurogenic model with increased similarity to humans, given that the LVE connects the LV with the OB, as has been described in humans, and that the LVE works a migratory pathway. Stem Cells 2016
Schematic summary of the cellular composition of the subventricular zone and lateral ventricle extension (LVE) of the adult guinea pig brain. In the adult guinea pig brain, a narrow ventricular cavity connects the SVZ with the OB, similar to the LVE previously described in the adult human brain. In the guinea pig brain, ependymal cells line the LV and also LVE; neuroblasts are located in the subependymal area, but mostly along the LVE; proliferating BrdU + cells are mainly found in the SVZ; however, they are also found in LVE. Astrocytes are co-distributed with neuroblasts in the SVZ and surround the neuroblasts in LVE. Finally, in the guinea pig brain the LVE has a reduced proliferative activity; however, it works as a continuous pathway for neuroblasts migration to the OB.
Sfrp2 is overexpressed in stromal cells which maintain hematopoietic stem cells (HSCs) during in vitro culture. We here showed, that coculture of hematopoetic cells with stromal cells with reduced expression of Sfrp2 increases the number lineage-negative Kit+ Sca-1+ (LSK) and progenitor cells in vitro. The LSK cells from these cocultures showed activation of canonical Wnt signaling, higher levels of Ki-67, BrdU incorporation, and the number of γH2A.X positive foci. Total repopulating activity of these cultures was, however, diminished, indicating loss of HSC. To extend these in vitro data, we modelled stress in vivo, i.e., by aging, or 5-FU treatment in Sfrp2−/− mice, or replicative stress in regeneration of HSCs in Sfrp2−/− recipients. In all three in vivo stress situations, we noted an increase of LSK cells, characterized by increased levels of β-catenin and cyclin D1. In the transplantation experiments, the increase in LSK cells in primary recipients was subsequently associated with a progressive loss of HSCs in serial transplantations. Similar to the in vitro coculture stress, in vivo genotoxic stress in 5-FU-treated Sfrp2−/− mice increased cell cycle activity of LSK cells with higher levels of BrdU incorporation, increased expression of Ki-67, and canonical Wnt signaling. Importantly, as noted in vitro, increased cycling of LSKs in vivo was accompanied by a defective γH2A.X-dependent DNA damage response and depolarized localization of acetylated H4K16. Our experiments support the view that Sfrp2 expression in the niche is required to maintain the HSC pool by limiting stress-induced DNA damage and attenuating canonical Wnt-mediated HSC activation. Stem Cells 2016
Hematopoietic stress (coculture, regeneration, genotoxicity, and aging) results in reduced hematopoietic stem cell (HSC) numbers and quality. Here, we show that stromal Sfrp2 dampens the response of HSC to different forms of stress in vitro (cocultures) and in vivo (regeneration, genotoxic insult, and aging). Without Sfrp2 (right hand figure), the niche's ability to limit the stress response is diminished. As a result, HSC activated from quiescence (qHSC) into an activated state (aHSC) show an amplified stress response with an increased DNA damage response. In this Sfrp2-deficient environment, aHSC do not self-renew efficiently, which results in a defective regeneration of the HSC pool. Thus, Sfrp2 secreted by the niche preserves the number of HSCs, a finding which may help to identify new ways to prevent the loss of stem cells under stress conditions.
Osteoclasts form a bone marrow (BM) cavity serving as a hematopoietic niche for the maintenance of hematopoietic stem cells (HSCs). However, the role of osteoclasts in the BM has been controversially reported and remains to be further understood. In the present study, we investigated how osteoclasts affect the modulation of hematopoietic stem/progenitor cells in the BM by administering bisphosphate alendronate (ALN) to B6 mice for 21 consecutive days to inhibit osteoclast activity. ALN treatment caused a reduction in the number of tartrate-resistant acid phosphate (TRAP)-positive osteoclast cells and an increase in bone mineral density, particularly in the trabecular zone, but not in the cortical zone of the BM. Osteoclast inhibition caused by ALN treatment decreased mitochondrial reactive oxygen species (ROS) generation and SA-β-gal activity of CD150+CD48−Lineage-Sca-1+c-Kit+ (LSK) cells, eventually leading to an improvement in the engraftment potential and self-renewal activity of HSCs. Moreover, ALN-treated mice exhibited an enhanced resistance of HSCs in response to the genotoxic stress of 5-fluorouracil, as determined by mitochondrial ROS generation, SA-β-gal activity, and p16INK4a expression in subsets of LSK and CD150+CD48−LSK cells as well as competitive assay. Collectively, our findings indicate that inhibition of osteoclast activity improves the long–term engraftment potential and stress resistance of HSCs. Stem Cells 2016
In most human somatic cells, the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually, DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However, the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped, in contrast to fibroblast cells that enter a state of replicative senescence. Significantly, telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity, we can functionally separate the two unique properties of human pluripotent stem cells, namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation, we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo, and thus sustained telomerase activity. Stem Cells 2016
Inactivation of telomerase results in the functional uncoupling of the two unique properties of human pluripotent stem cells, namely self-renewal and pluripotency. This strategy allows us to create pluripotent stem cells with limited lifespan, which dramatically reduce the tumorigenicity of human pluripotent stem cells for clinical applications.
Melanoma is a highly heterogeneous tumor for which recent evidence supports a model of dynamic stemness. Melanoma cells might temporally acquire tumor-initiating properties or switch from a status of tumor-initiating cells (TICs) to a more differentiated one depending on the tumor context. However, factors driving these functional changes are still unknown. We focused on the role of cyto/chemokines in shaping TICs isolated directly from tumor specimens of two melanoma patients, namely Me14346S and Me15888S. We analyzed the secretion profile of TICs and of their corresponding melanoma differentiated cells and we tested the ability of cyto/chemokines to influence TIC self-renewal and differentiation. We found that TICs, grown in vitro as melanospheres, had a complex secretory profile as compared to their differentiated counterparts. Some factors, such as CCL-2 and IL-8, also produced by adherent melanoma cells and melanocytes did not influence TIC properties. Conversely, IL-6, released by differentiated cells, reduced TIC self-renewal and induced TIC differentiation while IL-10, produced by Me15888S, strongly promoted TIC self-renewal through paracrine/autocrine actions. Complete neutralization of IL-10 activity by gene silencing and antibody-mediated blocking of the IL-10Rα was required to sensitize Me15888S to IL-6-induced differentiation. For the first time these results show that functional heterogeneity of melanoma could be directly influenced by inflammatory and suppressive soluble factors, with IL-6 favoring TIC differentiation, and IL-10 supporting TIC self-renewal. Thus, understanding the tumor microenvironment (TME) role in modulating melanoma TIC phenotype is fundamental to identifying novel therapeutic targets to achieve long-lasting regression of metastatic melanoma. Stem Cells 2016
Melanoma is a heterogeneous tumor that shows a model of dynamic stemness (1). Melanoma cells temporally acquire tumor-initiating properties or switch from a status of tumor-initiating cells (TICs) to a more differentiated one depending on the tumor context (2). TICs produce IL-10, while the differentiated cells release IL-6. The self-produced or exogenous IL-10 sustains self-renew and expansion of TICs (3) while IL-6 promotes their differentiation (4). The functional heterogeneity of melanoma can be influenced by inflammatory and suppressive factors released also by cells constituting the tumor microenviroment. Our in vitro and in vivo experiments suggest that anti-IL-10 antibodies (αIL10 mAb) or anti-IL-6 receptor α (tocilizumab) might represent drugs for novel therapeutic strategies in melanoma patients (5).
Recent studies have shown that Sca-1+ (stem cell antigen-1) stem/progenitor cells within blood vessel walls may contribute to neointima formation, but the mechanism behind their recruitment has not been explored. In this work Sca-1+ progenitor cells were cultivated from mouse vein graft tissue and found to exhibit increased migration when cocultured with smooth muscle cells (SMCs) or when treated with SMC-derived conditioned medium. This migration was associated with elevated levels of chemokines, CCL2 (chemokine (C-C motif) ligand 2) and CXCL1 (chemokine (C-X-C motif) ligand 1), and their corresponding receptors on Sca-1+ progenitors, CCR2 (chemokine (C-C motif) receptor 2) and CXCR2 (chemokine (C-X-C motif) receptor 2), which were also upregulated following SMC conditioned medium treatment. Knockdown of either receptor in Sca-1+ progenitors significantly inhibited cell migration. The GTPases Cdc42 and Rac1 were activated by both CCL2 and CXCL1 stimulation and p38 phosphorylation was increased. However, only Rac1 inhibition significantly reduced migration and p38 phosphorylation. After Sca-1+ progenitors labeled with green fluorescent protein (GFP) were applied to the adventitial side of wire-injured mouse femoral arteries, a large proportion of GFP-Sca-1+-cells were observed in neointimal lesions, and a marked increase in neointimal lesion formation was seen 1 week post-operation. Interestingly, Sca-1+ progenitor migration from the adventitia to the neointima was abrogated and neointima formation diminished in a wire injury model using CCL2−/− mice. These findings suggest vascular stem/progenitor cell migration from the adventitia to the neointima can be induced by SMC release of chemokines which act via CCR2/Rac1/p38 and CXCR2/Rac1/p38 signaling pathways. Stem Cells 2016
Schematic illustration of the role of CCL2 and CXCL1 released from SMCs in enhancing VPCs chemotaxis. SMCs release CCL2 and CXCL1 into the medium. When VPCs are treated with this SMC conditioned medium, these chemokines bind to their corresponding receptors CCR2 and CXCR2 on the VPCs. The GTPase Rac1 become activated and then p38 is phosphorylated via Rac1, finally leading to increased VPC migration. SMC conditioned medium also induces expression of cytoskeleton related proteins axillin, vinculin and phosphorylated FAK, which may also activate the Rac1 signaling pathway.
Mesenchymal stem cells (MSCs) have been proven to be therapeutically effective against atopic dermatitis (AD) in preclinical studies. However, the safety and efficacy of MSCs against AD have not yet been investigated in a clinical study. To establish the safety and efficacy of human umbilical cord blood-derived MSCs (hUCB-MSCs) in AD, 34 adult patients with moderate-to-severe AD were enrolled in two phase trials with a follow-up for 1 month and 3 months, respectively. Patients were randomly allocated to receive low dose (2.5 × 107) or high dose (5.0 × 107) of hUCB-MSCs subcutaneously. An Eczema Area and Severity Index (EASI) score, Investigator's Global Assessment (IGA) score, Severity Scoring for Atopic Dermatitis (SCORAD) score, adverse effect assessments, and serum biomarker levels were evaluated as end points. A single treatment of hUCB-MSCs resulted in dose-dependent improvements in AD manifestation. Fifty-five percent of patients in high dose hUCB-MSC-treated group showed a 50% reduction in the EASI score. The IGA score and SCORAD score decreased by 33% and 50%, respectively, in high dose-treated group. Particularly, the administration of high dose hUCB-MSCs reduced the pruritus score by 58%. The serum IgE levels and number of blood eosinophils were downregulated by the treatment. No serious adverse events occurred, and none of the patients discontinued the trial due to adverse events. This is the first report to demonstrate a marked improvement of AD features with cell therapeutics. These data suggest that the infusion of hUCB-MSCs might be an effective therapy for patients with moderate-to-severe AD. Stem Cells 2016
Prenatal folic acid (FA) supplementation prevents neural tube defects. Folate receptor alpha (FRα) is critical for embryonic development, including neural crest (NC) development. Previously we showed that FRα translocates to the nucleus in response to FA, where it acts as a transcription factor. In this study, we examined if FA through interaction with FRα regulates stem cell characteristics of cranial neural crest cells (CNCCs)-critical for normal development. We hypothesized that FRα upregulates coding genes and simultaneously downregulates non-coding miRNA which targets coding genes in CNCCs. Quantitative RT-PCR and chromatin immunoprecipitation showed that FRα upregulates Oct4, Sox2, and Klf4 by binding to their cis-regulator elements-5′ enhancer/promoters defined by H3K27Ac and p300 occupancy. FA via FRα downregulates miRNAs, miR-138 and miR-let-7, which target Oct4 and Trim71 (an Oct4 downstream effector), respectively. Co-immunoprecipitation data suggests that FRα interacts with the Drosha-DGCR8 complex to affect pre-miRNA processing. Transfecting anti-miR-138 or anti-miR-let-7 into non-proliferating neural crest cells (NCCs) derived from Splotch (Sp−/−), restored their proliferation potential. In summary, these results suggest a novel pleiotropic role of FRα: (a) direct activation of Oct4, Sox2, and Klf4 genes; and (b) repression of biogenesis of miRNAs that target these genes or their effector molecules. Stem Cells 2016
Hypothetical model of FRα function in NCCs. In response to FA, FRα translocates to the nucleus where it binds the cis-regulatory elements of Oct4, Sox2, and Klf4, activating transcription; this in turn activates downstream targets such as Trim71 and downregulates levels of miR-138 and miR-let-7, which target Oct4 and Trim71 respectively thus removing the inhibitory effects on Oct4 and Trim71 expression.
Pluripotent stem cells, both human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC), provide an important resource to produce specialized cells such as osteogenic cells for therapeutic applications such as repair or replacement of injured, diseased or damaged bone. hESCs and iPSCs can also be used to better define basic cellular and genetic mechanisms that regulate the earliest stages of human bone development. However, current strategies to mediate osteogenic differentiation of hESC and iPSC are typically limited by the use of xenogeneic components such as fetal bovine serum (FBS) that make defining specific agents that mediate human osteogenesis difficult. Runt-related transcription factor 2 (RUNX2) is a key regulator required for osteogenic differentiation. Here, we used a RUNX2-YFP reporter system to characterize the novel ability of fibrinogen to mediate human osteogenic development from hESC and iPSC in defined (serum-free) conditions. These studies demonstrate that fibrinogen mediates significant osteo-induction potential. Specifically, fibrinogen binds to the surface integrin (α9β1) to mediate RUNX2 gene expression through the SMAD1/5/8 signaling pathway. Additional studies characterize the fibrinogen-induced hESC/iPSC-derived osteogenic cells to demonstrate these osteogenic cells retain the capacity to express typical mature osteoblastic markers. Together, these studies define a novel fibrinogen-α9β1-SMAD1/5/8-RUNX2 signaling axis can efficiently induce osteogenic differentiation from hESCs and iPSCs. Stem Cells 2016
Fibrinogen interaction with surface integrin (α9β1) mediates RUNX2 expression through SMAD1/5/8 signaling pathway and leads to osteogenic differentiation of human pluripotent stem cells.
With ongoing clinical trials, human embryonic stem cells (hESCs) have shown substantial potential for regenerative medicine. However, due to the mismatch of human leukocyte antigens (HLAs) between hESC-derived allografts and recipients, immunosuppressant regimens must be used to prevent immune rejection of the grafts. Considerable efforts have been devoted to overcoming this hurdle via the derivation and banking of human nuclear transfer ESCs, parthenogenetic ESCs, and induced pluripotent stem cells. However, ethical and safety concerns remain, hindering the application of these types of pluripotent cells. Other approaches have recently been explored to generate universally compatible hESCs through the silencing or deletion of HLAs or genes essential for HLA expression, including β-2-microglobulin and class-II MHC transactivator, as well as the induction of immunosuppression via the ectopic expression of non-classical HLAs (e.g., HLA-E and -G), cytotoxic T lymphocyte antigen 4 fused with immunoglobulin, and programmed death ligand-1. In this review, we introduce developments in this line of research and discuss strategies to reduce the tumorigenic concerns regarding hESCs, especially after they acquire the capability to escape immune surveillance. Stem Cells 2016
Immune cells in a recipient, including dendritic cells, effector T cells etc., recognize and attack foreign cells from a donor via class-I and -II human leukocyte antigens (HLAs), while a host's natural killer (NK) cells kill foreign cells without HLAs. Universally compatible human embryonic stem cells (UC-hESCs) can be generated via genetic manipulations, for example, repression or deletion of the heavy chain or light chain (B2M) of class-I HLA and CIITA to abolish immune response, mediated by class-I and -II HLAs, respectively, and ectopic expression of immunosuppressive molecules such as HLA-G, HLA-E, CTLA4 fused with immunoglobulin, and programmed death ligand-1 to inhibit NK or T cell-mediated killing. Thus, UC-hESCs and their progeny can escape immune recognition and elimination by the host.
The AKT pathway plays an important role in various aspects of stem cell biology. However, the consequences of constitutive activation of AKT in mesenchymal stromal cells (MSCs) on the fate of hematopoietic stem cells (HSCs) were unknown. Here, we show that bone marrow-derived MSCs expressing a constitutively active AKT1 expand HSCs, but severely affect their functionality. Conversely, stromal cells with silenced AKT1 limit HSC proliferation, but boost their functionality. These effects were related to differential modulation of several important regulatory genes, in both, the cocultured HSCs and in the stromal cells themselves. The detrimental effect of stromal cells with constitutively activated AKT1 involved dynamin-dependent endocytosis, whereas the salutary effect of stromal cells devoid of AKT1 was mediated via GAP junctions. Constitutive activation of AKT1 led to deregulated formation of GAP junctions in the stromal cells, which consequently exhibited strikingly increased intercellular transfer of molecular cargo to the HSCs. Conversely, stromal cells with silenced AKT1 exhibited normal intercellular arrangement of GAP junctions at appositional membrane areas, and did not show aberrant intercellular transfer. Micro-vesicles isolated from conditioned media of the stromal cells not only mimicked the effect of these cells, but also showed stronger effects. This is perhaps the first report demonstrating that AKT1 signaling prevailing in the MSCs regulates HSC functionality through various intercellular communication mechanisms. These findings could have important implications in the use of MSCs in regenerative medicine. Stem Cells 2016
The data presented in this manuscript demonstrate that the AKT1 signaling prevailing in the mesenchymal stromal cells (MSCs) regulates functionality of hematopoietic stem cells (HSCs) via intercellular communication pathways. MSCs modified with a constitutively activated AKT1 expand phenotypically defined HSCs, but severely affect their functionality. On the other hand, MSCs with silenced AKT1 limit the expansion of HSCs, but boost their functionality. These effects are mediated through distinctly different intercellular communication pathways – the negative effects of MSCs having activated AKT1 are mediated via Dynamin-dependent endocytosis, whereas the salutary effects of MSCs having silenced AKT1 involve GAP junctions. These findings could also have implications in the use of MSCs in regenerative medicine.
Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and regeneration. Though of prime interest, their potentially protective role on neutrophil-induced tissue damage, associated with high morbidity and mortality, has not been explored in sufficient detail. Here we report the therapeutic skill of MSCs to suppress unrestrained neutrophil activation and to attenuate severe tissue damage in a murine immune-complex mediated vasculitis model of unbalanced neutrophil activation. MSC-mediated neutrophil suppression was due to intercellular adhesion molecule 1-dependent engulfment of neutrophils by MSCs, decreasing overall neutrophil numbers. Similar to MSCs in their endogenous niche of murine and human vasculitis, therapeutically injected MSCs via upregulation of the extracellular superoxide dismutase (SOD3), reduced superoxide anion concentrations and consequently prevented neutrophil death, neutrophil extracellular trap formation and spillage of matrix degrading neutrophil elastase, gelatinase and myeloperoxidase. SOD3-silenced MSCs did not exert tissue protective effects. Thus, MSCs hold substantial therapeutic promise to counteract tissue damage in conditions with unrestrained neutrophil activation. Stem Cells 2016
Scheme of adaptive mesenchymal stem cell (MSC) responses to unrestrained neutrophil activation. Immune complexes inside and outside the vessel lead to unrestrained neutrophil activation with enhanced generation and release of . which can stimulate neutrophil extracellular trap formation with expulsion of chromatin decorated with granules and highly concentrated myeloperoxidase, neutrophil elastase, and matrix metalloproteinase-9, neutrophil death with spillage of their toxic cargo leading to tissue damage and tissue break down. Further damage to macromolecules occur by . and derivatives thereof like hydrogen peroxide (H2O2), peroxynitrate (ONNO.) and hydroxyl radicals (HO.). MSCs are able to mount an adaptive response to unrestrained neutrophil activation, with the release of . detoxifying extracellular superoxide dismutase and intercellular adhesion molecule 1/CD18 dependent engulfment of dying neutrophils thus preventing the spillage of their toxic cargo. By employing independent strategies MSCs effectively protect from tissue damage due to unrestrained neutrophil activation.
Mechanisms determining intrinsic differentiation bias inherent to human pluripotent stem cells (hPSCs) toward cardiogenic fate remain elusive. We evaluated the interplay between ErbB4 and Epidemal growth factor receptor (EGFR or ErbB1) in determining cardiac differentiation in vitro as these receptor tyrosine kinases are key to heart and brain development in vivo. Our results demonstrate that during cardiac differentiation, cell fate biases exist in hPSCs due to cardiac/neuroectoderm divergence post cardiac mesoderm stage. Stage-specific up-regulation of EGFR in concert with persistent Wnt3a signaling post cardiac mesoderm favors commitment toward neural progenitor cells (NPCs). Inhibition of EGFR abrogates these effects with enhanced (>twofold) cardiac differentiation efficiencies by increasing proliferation of Nkx2-5 expressing cardiac progenitors while reducing proliferation of Sox2 expressing NPCs. Forced overexpression of ErbB4 rescued cardiac commitment by augmenting Wnt11 signaling. Convergence between EGFR/ErbB4 and canonical/noncanonical Wnt signaling determines cardiogenic fate in hPSCs. Stem Cells 2016
The size of neural stem cell (NSC) pool at birth determines the starting point of adult neurogenesis. Aberrant neurogenesis is associated with major mental illness, in which ULK4 is proposed as a rare risk factor. Little is known about factors regulating the NSC pool, or function of the ULK4. Here, we showed that Ulk4tm1a/tm1a mice displayed a dramatically reduced NSC pool at birth. Ulk4 was expressed in a cell cycle-dependent manner and peaked in G2/M phases. Targeted disruption of the Ulk4 perturbed mid-neurogenesis and significantly reduced cerebral cortex in postnatal mice. Pathway analyses of dysregulated genes in Ulk4tm1a/tm1a mice revealed Ulk4 as a key regulator of cell cycle and NSC proliferation, partially through regulation of the Wnt signaling. In addition, we identified hemizygous deletion of ULK4 gene in 1.2/1,000 patients with pleiotropic symptoms including severe language delay and learning difficulties. ULK4, therefore, may significantly contribute to neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Stem Cells 2016
Ulk4 regulates neural stem cell pool by tuning cell cycle and Wnt signaling pathway. Ulk4 is highly expressed in the G2/M phases and Ulk4 disruption alters expression levels of cell cycle-related molecules including Eml1, Pura and Apc. Ulk4 also modulates Wnt signaling pathway and Ulk4 gene deficiency decreases expression of Fzd, Apc and Lef1 which are known to positively influence neural stem cells, as well as elevated Wnt target of Vim which negatively regulates neurogenesis. Ulk4 is highly co-expressed in the SVZ of newborn mice with the proliferating marker Ki67 and Ulk4 mutation dramatically reduces the size of neural stem cell pool.
Human mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into osteoblasts, chondrocytes, and adipocytes. The importance of epigenetic regulation for osteogenic differentiation of MSCs is widely accepted. However, the molecular mechanisms are poorly understood. Here, we show that histone H3K9 acetyltransferase PCAF plays a critical role in osteogenic differentiation of MSCs. Knockdown of PCAF significantly reduced the bone formation both in vitro and in vivo. Mechanistically, PCAF controls BMP signaling genes expression by increasing H3K9 acetylation. Most importantly, PCAF expression is significantly decreased in bone sections of ovariectomized or aged mice. Histone modification enzyme is chemically modifiable; therefore, PCAF may represent a novel therapeutic target for stem cell-mediated regenerative medicine and the treatment of osteoporosis. Stem Cells 2016
Osteogenic differentiation induced PCAF expression through SMADs; PCAF promoted activation of BMP2, BMP4, BMPR1B and RUNX2 by increasing H3K9ac, which led to osteogenic differentiation.
Cancer stem-like cells (SLC) resist conventional therapies, necessitating searches for SLC-specific targets. We established that cyclo-oxygenase(COX)-2 expression promotes human breast cancer progression by activation of the prostaglandin(PG)E-2 receptor EP4. Present study revealed that COX-2 induces SLCs by EP4-mediated NOTCH/WNT signaling. Ectopic COX-2 over-expression in MCF-7 and SKBR-3 cell lines resulted in: increased migration/invasion/proliferation, epithelial-mesenchymal transition (EMT), elevated SLCs (spheroid formation), increased ALDH activity and colocalization of COX-2 and SLC markers (ALDH1A, CD44, β-Catenin, NANOG, OCT3/4, SOX-2) in spheroids. These changes were reversed with COX-2-inhibitor or EP4-antagonist (EP4A), indicating dependence on COX-2/EP4 activities. COX-2 over-expression or EP4-agonist treatments of COX-2-low cells caused up-regulation of NOTCH/WNT genes, blocked with PI3K/AKT inhibitors. NOTCH/WNT inhibitors also blocked COX-2/EP4 induced SLC induction. Microarray analysis showed up-regulation of numerous SLC-regulatory and EMT-associated genes. MCF-7-COX-2 cells showed increased mammary tumorigenicity and spontaneous multiorgan metastases in NOD/SCID/IL-2Rγ-null mice for successive generations with limiting cell inocula. These tumors showed up-regulation of VEGF-A/C/D, Vimentin and phospho-AKT, down-regulation of E-Cadherin and enrichment of SLC marker positive and spheroid forming cells. MCF-7-COX-2 cells also showed increased lung colonization in NOD/SCID/GUSB-null mice, an effect reversed with EP4-knockdown or EP4A treatment of the MCF-7-COX-2 cells. COX-2/EP4/ALDH1A mRNA expression in human breast cancer tissues were highly correlated with one other, more marked in progressive stage of disease. In situ immunostaining of human breast tumor tissues revealed co-localization of SLC markers with COX-2, supporting COX-2 inducing SLCs. High COX-2/EP4 mRNA expression was linked with reduced survival. Thus, EP4 represents a novel SLC-ablative target in human breast cancer. Stem Cells 2016
COX-2 upregulation or overexpression in human breast cancer cells increase EP4 expression, cell migration, invasion, proliferation, EMT and stem like cells (SLC) in human breast cancer. COX-2 overexpression activates NOTCH and WNT pathways and spheroids formation. COX-2 high cells promote tumor growth and metastassis in NOD/SCID mice. Serial transplantation of COX-2 high tumors promotes tumor growth and metastasis. Treatments with COX-2 inhibitor (I) or EP4 antagonist (A) or EP4 knock down (KD) in COX-2 high cells reduce above all phenotypes including lung metastasis. NOTCH and WNT inhibitors could abrogate COX-2 induced SLC functions. PI3K/AKT inhibition could reduce EP4 mediated SLC functions and NOTCH/WNT expression, establishing COX-2 induces SLC via EP4/PI3K/AKT/NOTCH/WNT pathway. In human breast tumor tissues COX-2 and EP4 expression increases with tumor progression, SLC marker co-localizes in COX-2 high cells and both COX-2/EP4 expression is associated with poor patient survival.
The healing of a corneal epithelial defect is essential for preventing infectious corneal ulcers and subsequent blindness. We previously demonstrated that mesenchymal stem cells (MSCs) in the corneal stroma, through a paracrine mechanism, yield a more favorable therapeutic benefit for corneal wound re-epithelialization than do MSCs in the corneal epithelium. In this study, MSCs were grown on a matrix with the rigidity of the physiological human vitreous (1 kPa), corneal epithelium (8 kPa), or corneal stroma (25 kPa) for investigating the role of corneal tissue rigidity in MSC functions regarding re-epithelialization promotion. MSC growth on a 25-kPa dish significantly promoted the wound healing of human corneal epithelial (HCE-T) cells. Among growth factors contributing to corneal epithelial wound healing, corneal stromal rigidity selectively enhanced transforming growth factor-beta (TGF-β) secretion from MSCs. Inhibitors of TGF-β pan receptor, TGF-β receptor 1, and Smad2 dose dependently abrogated MSC-mediated HCE-T wound healing. Furthermore, MSCs growth on a matrix with corneal stromal rigidity enhanced the ability of themselves to promote corneal re-epithelialization by activating matrix metalloproteinase (MMP) expression and integrin β1 production in HCE-T cells through TGF-β signaling pathway activation. Smad2 activation resulted in the upregulation of MMP-2 and −13 expression in HCE-T cells, whereas integrin β1 production favored a Smad2-independent TGF-β pathway. Altogether, we conclude that corneal stromal rigidity is a critical factor for MSC-induced promotion of corneal re-epithelialization. The activation of the TGF-β signaling pathway, which maintains the balance between integrin and MMP expression, in HCE-T cells is the major pathway responsible for MSC-mediated wound healing. Stem Cells 2016
Mesenchymal stem cells (MSCs) grown on a matrix with rigidity of corneal stroma (25 kPa) promoted corneal wound healing. A 25 kPa matrix rigidity stimulated MSCs to secret transforming growth factor β (TGF-β), and which activated the TGF-β signaling pathway in the corneal epithelial cells. TGF-β in corneal epithelial cells not only upregulated the expression of metalloproteinase (MMP)-2, -13 via Smad2/3 phosphorylation, but also enhanced integrin-β1 expression through a Smad2/3 independent manner.
Studies to evaluate the therapeutic potential of stem cells in humans would benefit from more realistic animal models. In veterinary medicine, companion animals naturally develop many diseases that resemble human conditions, therefore, representing a novel source of preclinical models. To understand how companion animal disease models are being studied for this purpose, we reviewed the literature between 2008 and 2015 for reports on stem cell therapies in dogs and cats, excluding laboratory animals, induced disease models, cancer, and case reports. Disease models included osteoarthritis, intervertebral disc degeneration, dilated cardiomyopathy, inflammatory bowel diseases, Crohn's fistulas, meningoencephalomyelitis (multiple sclerosis-like), keratoconjunctivitis sicca (Sjogren's syndrome-like), atopic dermatitis, and chronic (end-stage) kidney disease. Stem cells evaluated in these studies included mesenchymal stem-stromal cells (MSC, 17/19 trials), olfactory ensheathing cells (OEC, 1 trial), or neural lineage cells derived from bone marrow MSC (1 trial), and 16/19 studies were performed in dogs. The MSC studies (13/17) used adipose tissue-derived MSC from either allogeneic (8/13) or autologous (5/13) sources. The majority of studies were open label, uncontrolled studies. Endpoints and protocols were feasible, and the stem cell therapies were reportedly safe and elicited beneficial patient responses in all but two of the trials. In conclusion, companion animals with naturally occurring diseases analogous to human conditions can be recruited into clinical trials and provide realistic insight into feasibility, safety, and biologic activity of novel stem cell therapies. However, improvements in the rigor of manufacturing, study design, and regulatory compliance will be needed to better utilize these models. Stem Cells 2016;34:1709–1729
Companion animal diseases (reviewed in Table 1) have the potential to serve as realistic models of human disease, as they more closely approximate the natural history, symptoms, pathology, biomarkers, therapeutic responses, tolerance to therapies, and survival characteristics of analogous human conditions. Stem cell trials in companion animal disease models are, therefore, of interest as translational systems in regenerative medicine. We reviewed the study design, manufacturing, endpoints, safety, and efficacy data from stem cell trials in dogs and cats between the years of 2008-2015 (n = 19) (Table 2). Most clinical trials were open label design involving MSC (see Cells Evaluated), informing safety, route of administration, feasibility of protocols with modest power to evaluate efficacy. Overall safety was excellent, and patients showed responses that exceeded expectations based on baseline, historical, or interventional (placebo) controls. Companion animal disease models have potential to inform hypotheses concerning stem cell trials in humans. Improved rigor in study design and manufacturing will unmask the full potential of this approach to benefit humans and animals.
Interaction between hematopoietic stem/progenitor cells (HSPCs) with their niche is critical for HSPC function. The interaction also plays an important role in the multistep process of leukemogenesis. Rac1 GTPase has been found to be highly expressed and activated in leukemia patients. Here, by forced expression of constitutively active form of Rac1 (Rac1-V12) in HSPCs, we demonstrate that active Rac1 promotes interaction of HSPC with niche. We then established an active Rac1 associated acute myeloid leukemia (AML) model by expression of Rac1-V12 cooperated with AML1-ETO9a (AE9a) in mouse HSPCs. Compared with AE9a alone, Rac1-V12 cooperated with AE9a (AER) drives an AML with a short latency, demonstrating that activation of Rac1 GTPase in mice promotes AML development. The mechanism of this AML promotion is by a better homing and lodging of leukemia cells in niche, which further enhancing their colony formation, quiescence and preventing leukemia cells from apoptosis. Further study showed that an inhibitor targeting activated Rac1 can increase the efficacy of chemotherapeutic agents to leukemia cells. This study provides evidence that activation of Rac1 promotes leukemia development through enhancing leukemia cells' homing and retention in niche, and suggests that inhibition of Rac1 GTPase could be an effective way of eliminating AML cells. Stem Cells 2016;34:1730–1741
By establishing an active Rac1 associated acute myeloid leukemia (AML) model via expression of Rac1-V12 cooperated with AML1-ETO9a (AE9a) in mouse hematopoietic stem/progenitor cells, we show that compared with AE9a alone, Rac1-V12 cooperated with AE9a (AER) drives an AML with a short latency, demonstrating that activation of Rac1 GTPase in mice promotes AML development. The mechanism of this AML promotion is by a better homing and lodging of leukemia cells in niche, which further enhancing their colony formation, quiescence and preventing leukemia cells from apoptosis. Further study shows that an inhibitor targeting activated Rac1 can increase the efficacy of chemotherapeutic agents to leukemia cells.
Polysialic acid (PSA) is a carbohydrate polymer of repeating α-2,8 sialic acid residues that decorates multiple targets, including neural cell adhesion molecule (NCAM). PST and STX encode the two enzymes responsible for PSA modification of target proteins in mammalian cells, but despite widespread polysialylation in embryonic development, the majority of studies have focused strictly on the role of PSA in neurogenesis. Using human pluripotent stem cells (hPSCs), we have revisited the developmental role of PST and STX and show that early progenitors of the three embryonic germ layers are polysialylated on their cell surface. Changes in polysialylation can be attributed to lineage-specific expression of polysialyltransferase genes; PST is elevated in endoderm and mesoderm, while STX is elevated in ectoderm. In hPSCs, PST and STX genes are epigenetically marked by overlapping domains of H3K27 and H3K4 trimethylation, indicating that they are held in a "developmentally-primed" state. Activation of PST transcription during early mesendoderm differentiation is under control of the T-Goosecoid transcription factor network, a key regulatory axis required for early cell fate decisions in the vertebrate embryo. This establishes polysialyltransferase genes as part of a developmental program associated with germ layer establishment. Finally, we show by shRNA knockdown and CRISPR-Cas9 genome editing that PST-dependent cell surface polysialylation is essential for endoderm specification. This is the first report to demonstrate a role for a glycosyltransferase in hPSC lineage specification. Stem Cells 2016;34:1742–1752
Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)—known as pluripotent stem cells (PSC)—is influenced by culture conditions, including the substrate on which they are grown. However, details of the molecular mechanisms interconnecting the substrate and self-renewal of these cells remain unclear. We describe a signaling pathway in hPSCs linking self-renewal and expression of pluripotency transcription factors to integrin α6β1 and inactivation of focal adhesion kinase (FAK). Disruption of this pathway results in hPSC differentiation. In hPSCs, α6β1 is the dominant integrin and FAK is not phosphorylated at Y397, and thus, it is inactive. During differentiation, integrin α6 levels diminish and Y397 FAK is phosphorylated and activated. During reprogramming of fibroblasts into iPSCs, integrin α6 is upregulated and FAK is inactivated. Knockdown of integrin α6 and activation of β1 integrin lead to FAK phosphorylation and reduction of Nanog, Oct4, and Sox2, suggesting that integrin α6 functions in inactivation of integrin β1 and FAK signaling and prevention of hPSC differentiation. The N-terminal domain of FAK, where Y397 is localized, is in the nuclei of hPSCs interacting with Oct4 and Sox2, and this immunolocalization is regulated by Oct4. hPSCs remodel the extracellular microenvironment and deposit laminin α5, the primary ligand of integrin α6β1. Knockdown of laminin α5 resulted in reduction of integrin α6 expression, phosphorylation of FAK and decreased Oct4. In conclusion, hPSCs promote the expression of integrin α6β1, and nuclear localization and inactivation of FAK to supports stem cell self-renewal. Stem Cells 2016;34:1753–1764
Human embryonic stem cells (hESCs) have an abbreviated G1 phase of the cell cycle that allows rapid proliferation and maintenance of pluripotency. Lengthening of G1 corresponds to loss of pluripotency during differentiation. However, precise mechanisms that link alterations in the cell cycle and early differentiation remain to be defined. We investigated initial stages of mesendodermal lineage commitment in hESCs, and observed a cell cycle pause. Transcriptome profiling identified several genes with known roles in regulation of the G2/M transition that were differentially expressed early during lineage commitment. WEE1 kinase, which blocks entry into mitosis by phosphorylating CDK1 at Y15, was the most highly expressed of these genes. Inhibition of CDK1 phosphorylation by a specific inhibitor of WEE1 restored cell cycle progression by preventing the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. Our findings identify a novel G2 cell cycle pause that is required for endodermal differentiation and provide important new mechanistic insights into early events of lineage commitment. Stem Cells 2016;34:1765–1775
Early after induction of differentiation to certain lineages there is an increase in nuclear WEE1 that phosphorylates the CDK1/cyclin complex at Y15, inhibiting progression into mitosis and forcing cells to pause in G2. This pause is seen in mesendoderm, mesoderm, and endoderm differentiation but is only required for endoderm differentiation.
The transition of embryonic stem cell (ESC) pluripotency to differentiation is accompanied by an expansion of mRNA and proteomic diversity. Post-transcriptional regulation of ESCs is critically governed by cell type-specific splicing. However, little is known about the splicing factors and the molecular mechanisms directing ESC early lineage differentiation. Our study identifies RNA binding motif protein 24 (Rbm24) as a key splicing regulator that plays an essential role in controlling post-transcriptional networks during ESC transition into cardiac differentiation. Using an inducible mouse ESC line in which gene expression could be temporally regulated, we demonstrated that forced expression of Rbm24 in ESCs dramatically induced a switch to cardiac specification. Genome-wide RNA sequencing analysis identified more than 200 Rbm24-regulated alternative splicing events (AS) which occurred in genes essential for the ESC pluripotency or differentiation. Remarkably, AS genes regulated by Rbm24 composed of transcriptional factors, cytoskeleton proteins, and ATPase gene family members which are critical components required for cardiac development and functionality. Furthermore, we show that Rbm24 regulates ESC differentiation by promoting alternative splicing of pluripotency genes. Among the Rbm24-regulated events, Tpm1, an actin filament family gene, was identified to possess ESC/tissue specific isoforms. We demonstrated that these isoforms were functionally distinct and that their exon AS switch was essential for ESC differentiation. Our results suggest that ESC's switching into the differentiation state can be initiated by a tissue-specific splicing regulator, Rbm24. This finding offers a global view on how an RNA binding protein influences ESC lineage differentiation by a splicing-mediated regulatory mechanism. Stem Cells 2016;34:1776–1789
Rbm24 controls ESC lineage differentiation by a splicing-mediated regulatory mechanism. (a) Forced expression of Rbm24 promotes cardiac lineage specification from ESCs. (b) Rbm24 is a major splicing factor regulating alternative splicing events important in ESC cardiac differentiation. (c) Rbm24 decreased the expression of ESC-specific exon 5 and increased the expression of tissue-specific exon 6 of Tpm1; Knockdown of ESC-specific the Tpm1 isoform markedly affected the maintenance of ESC pluripotency. Results suggest that Rbm24 could reduce ESC pluripotency by switching splicing of the pluripotency transcripts toward tissue-specific isoforms.
Presomitic mesoderm (PSM) cells are the precursors of the somites, which flank both sides of the neural tube and give rise to the musculo-skeletal system shaping the vertebrate body. WNT and FGF signaling control the formation of both the PSM and the somites and show a graded distribution with highest levels in the posterior PSM. We have used reporters for the mesoderm/PSM control genes T, Tbx6, and Msgn1 to investigate the differentiation of mouse ESCs from the naïve state via EpiSCs to PSM cells. Here we show that the activation of WNT signaling by CHIR99021 (CH) in combination with FGF ligand induces embryo-like PSM at high efficiency. By varying the FGF ligand concentration, the state of PSM cells formed can be altered. High FGF concentration supports posterior PSM formation, whereas low FGF generates anterior/differentiating PSM, in line with in vivo data. Furthermore, the level of Msgn1 expression depends on the FGF ligand concentration. We also show that Activin/Nodal signaling inhibits CH-mediated PSM induction in EpiSCs, without affecting T-expression. Inversely, Activin/Nodal inhibition enhances PSM induction by WNT/high FGF signaling. The ability to generate PSM cells of either posterior or anterior PSM identity with high efficiency in vitro will promote the investigation of the gene regulatory networks controlling the formation of nascent PSM cells and their switch to differentiating/somitic paraxial mesoderm. Stem Cells 2016;34:1790–1800
There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering-based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation toward the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the antichondrogenic regulator microRNA-221 (miR-221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF-β. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR-221 silencing was sufficient to drive hMSCs toward chondrogenic differentiation in the absence of TGF-β. In vivo, the potential of miR-221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR-221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate-only). Notably, miR-221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR-221 has a prochondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. Stem Cells 2016;34:1801–1811
Cellular transplantation strategies utilizing intraspinal injection of mesenchymal progenitor cells (MPCs) have been reported as beneficial for spinal cord injuries. However, intraspinal injection is not only technically challenging, but requires invasive surgical procedures for patients. Therefore, we investigated the feasibility and potential benefits of noninvasive intravenous injection of MPCs in two models of cervical spinal cord injury, unilateral C5 contusion and complete unilateral C5 hemisection. MPCs isolated from green fluorescence protein (GFP)-luciferase transgenic mice compact bone (1 × 106 cells), or vehicle Hank's Buffered Saline Solution (HBSS), were intravenously injected via the tail vein at D1, D3, D7, D10, or D14. Transplanted MPCs were tracked via bioluminescence imaging. Live in vivo imaging data showed that intravenously injected MPCs accumulate in the lungs, confirmed by postmortem bioluminescence signal—irrespective of the time of injection or injury model. The results showed a rapid, positive modulation of the inflammatory response providing protection to the injured spinal cord tissue. Histological processing of the lungs showed GFP+ cells evenly distributed around the alveoli. We propose that injected cells can act as cellular target decoys to an immune system primed by injury, thereby lessening the inflammatory response at the injury site. We also propose that intravenous injected MPCs modulate the immune system via the lungs through secreted immune mediators or contact interaction with peripheral organs. In conclusion, the timing of intravenous injection of MPCs is key to the success for improving function and tissue preservation following cervical spinal cord injury. Stem Cells 2016;34:1812–1825
Mesenchymal progenitor cells intravenously injected after cervical spinal cord injury are found in the lungs. Luc-GFP+ mesenchymal progenitor cells were intravenously injected at D1, D3, D7, D10 or D14 after C5 unilateral contusion or hemisection spinal cord injury. (A) Bioluminescence imaging showed that intravenous injected cells traveled to the lung region regardless of injury type or time of injection. (B, C) A subset of mice receiving C5 unilateral contusion spinal cord injury with mesenchymal progenitor cells intravenously injected at D1 was sacrificed at 6 hours and GFP+ cells were found in the lungs of these mice. Scale bar = 50 μm.
First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826–1835
Mesenchymal stem cell (MSC) immunosuppressive functions make them attractive candidates for anti-inflammatory therapy in allergic asthma. However, the mechanisms by which they ensure therapeutic effects remain to be elucidated. In an acute mouse model of house dust mite (Der f)-induced asthma, one i.v. MSC injection was sufficient to normalize and stabilize lung function in Der f-sensitized mice as compared to control mice. MSC injection decreased in vivo airway responsiveness and decreased ex vivo carbachol-induced bronchial contraction, maintaining bronchial expression of the inhibitory type 2 muscarinic receptor. To evaluate in vivo MSC survival, MSCs were labeled with PKH26 fluorescent marker prior to i.v. injection, and 1 to 10 days later total lungs were digested to obtain single-cell suspensions. 91.5 ± 2.3% and 86.6 ± 6.3% of the recovered PKH26+ lung cells expressed specific macrophage markers in control and Der f mice, respectively, suggesting that macrophages had phagocyted in vivo the injected MSCs. Interestingly, only PKH26+ macrophages expressed M2 phenotype, while the innate PKH26− macrophages expressed M1 phenotype. Finally, the remaining 0.5% PKH26+ MSCs expressed 10- to 100-fold more COX-2 than before injection, suggesting in vivo MSC phenotype modification. Together, the results of this study indicate that MSCs attenuate asthma by being phagocyted by lung macrophages, which in turn acquire a M2 suppressive phenotype. Stem Cells 2016;34:1836–1845
Lung macrophages have been cell-sorted from lung cell suspensions, 24 hours after intravenous injection of PKH26 + mesenchymal stem cells (MSCs). PKH26 staining is characterized by incorporation of red fluorescent molecules into the cell membrane and induces intense and reproducible fluorescence. Lung macrophages show PKH26 + (red) labeling, suggesting that they had in vivo phagocyted the PKH26 + MSCs.
Our previous study showed that the therapeutic effects of mesenchymal stem cells (MSCs) transplantation were improved by enhancing migration. MicroRNA-211 (miR-211) can modulate the migratory properties of some cell types by mechanisms that are not fully understood. This study was designed to investigate a possible role for miR-211 in MSC migration, and whether genetic manipulation of miR-211 in MSCs could be used to enhance its beneficial effects of cell transplantation. Transwell assays confirmed that MSCs migration of was significantly impaired by miR-211 knockdown but enhanced by miR-211 overexpression. MiR-211 overexpressing MSCs also exhibited significantly increased cell engraftment in the peri-infarct areas of female rat hearts 2 days after intravenous transplantation of male MSCs as shown by GFP tracking and SYR gene quantification. This conferred a significant decrease in infarct size and improved cardiac performance. By using a loss or gain of gene function approach, we demonstrated that miR-211 targeted STAT5A to modulate MSCs migration, possibly by interacting with MAPK signaling. Furthermore, the beneficial effects of miR-211 overexpression in MSCs were abolished by simultaneous overexpression of STAT5A whereas the negative effects of miR-211 silencing on MSC migration were rescued by simultaneous downregulation of STAT5A. Finally, using ChIP-PCR and luciferase assays, we provide novel evidence that STAT3 can directly bind to promoter elements that activate miR-211 expression. STAT3/miR-211/STAT5A signaling plays a key role in MSCs migration. Intravenous infusion of genetically modified miR-211 overexpressing MSCs conveys enhanced protection from adverse post-MI remodeling compared with unmodified MSCs. Stem Cells 2016;34:1846–1858
Previous studies indicate that Kras is dispensable for fetal liver hematopoiesis, but its role in adult hematopoiesis remains unclear. Here, we generated a Kras conditional knockout allele to address this question. Deletion of Kras in adult bone marrow (BM) is mediated by Vav-Cre or inducible Mx1-Cre. We find that loss of Kras leads to greatly reduced thrombopoietin (TPO) signaling in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), while stem cell factor-evoked ERK1/2 activation is not affected. The compromised TPO signaling is associated with reduced long term- and intermediate-term HSC compartments and a bias toward myeloid differentiation in MPPs. Although granulocyte macrophage colony-stimulating factor (GM-CSF)-evoked ERK1/2 activation is only moderately decreased in Kras-/- myeloid progenitors, it is blunted in neutrophils and neutrophil survival is significantly reduced in vitro. At 9-12 months old, Kras conditional knockout mice develop profound hematopoietic defects, including splenomegaly, an expanded neutrophil compartment, and reduced B cell number. In a serial transplantation assay, the reconstitution potential of Kras-/- BM cells is greatly compromised, which is attributable to defects in the self-renewal of Kras-/- HSCs and defects in differentiated hematopoietic cells. Our results demonstrate that Kras is a major regulator of TPO and GM-CSF signaling in specific populations of hematopoietic cells and its function is required for adult hematopoiesis. Stem Cells 2016;34:1859–1871
Histone-modifying enzymes dynamically regulate the chromatin status and have been implicated in the fate specification of stem cells, including neural stem cells (NSCs), which differentiate into three major cell types: neurons, astrocytes, and oligodendrocytes. Lysine-specific demethylase 1 (LSD1, also known as KDM1A) catalyzes the demethylation of H3K4me1/2 and H3K9me1/2, and it was recently suggested that functional disruption of LSD1 links to various human diseases. However, the mechanism by which LSD1 regulates human neural development remains unclear. Here, we present evidence that specific inhibition of LSD1 suppresses the neurogenesis of cultured human fetal NSCs (hfNSCs) isolated from the human fetal neocortex. Notably, we found that LSD1 directly associates with the promoter of the HEYL gene, and controls the demethylation of H3K4me2, which is accompanied by repression of HEYL expression during hfNSC neuronal differentiation. Furthermore, we also showed that HEYL expression is sufficient to inhibit the neuronal differentiation of hfNSCs. This mechanism seems to be primate-specific because mouse NSCs do not exhibit the LSD1 inhibitor-induced upregulation of Heyl. Our findings suggest that LSD1 plays an important role in primate neurogenesis and may contribute to the characterization of an evolved primate brain. Stem Cells 2016;34:1872–1882
LSD1 mediates neuronal differentiation of human fetal neural stem cells (hfNSCs) by controlling the expression of HEYL (A): The cells treated with or without LSD1 inhibitor were stained with antibody against βIII-tubulin. ** p < .01. (B): Schematic representation of the proximal 5′ region (∼ -1kb) of the human HEYL locus and designed primer sets (Primer1-3). ChIP assays for hfNSC after induction of differentiation were performed, followed by real-time PCR with the proper primer sets in the HEYL promoter. *, p < .05; **, p < .01; ***, p < .005. (C): The cells overexpressing HEYL were stained with antibody against βIII-tubulin. ****, p < .001. (D): Scheme for neuronal differentiation through histone demethylation of LSD1 in hfNSC. At the induction of neuronal differentiation, LSD1 associates with a site close to the RBP-J binding region, demethylates H3K4 methylation on the HEYL promoter, and represses its expression. Because of the repression of HEYL expression by LSD1, hfNSCs are induced to differentiate into excitatory and inhibitory neurons. On the other hand, LSD1 inhibitor treatment blocks the effect of LSD1 on the HEYL promoter resulting in high levels of HEYL, which may repress the expression of genes such as CTIP2 and DLX5 directly or indirectly, and prevent neuronal differentiation.
IκB kinase β (IKKβ), a central coordinator of inflammation through activation of nuclear factor-κB, has been identified as a potential therapeutic target for the treatment of obesity-associated metabolic dysfunctions. In this study, we evaluated an antisense oligonucleotide (ASO) inhibitor of IKKβ and found that IKKβ ASO ameliorated diet-induced metabolic dysfunctions in mice. Interestingly, IKKβ ASO also inhibited adipocyte differentiation and reduced adiposity in high-fat (HF)-fed mice, indicating an important role of IKKβ signaling in the regulation of adipocyte differentiation. Indeed, CRISPR/Cas9-mediated genomic deletion of IKKβ in 3T3-L1 preadipocytes blocked these cells differentiating into adipocytes. To further elucidate the role of adipose progenitor IKKβ signaling in diet-induced obesity, we generated mice that selectively lack IKKβ in the white adipose lineage and confirmed the essential role of IKKβ in mediating adipocyte differentiation in vivo. Deficiency of IKKβ decreased HF-elicited adipogenesis in addition to reducing inflammation and protected mice from diet-induced obesity and insulin resistance. Further, pharmacological inhibition of IKKβ also blocked human adipose stem cell differentiation. Our findings establish IKKβ as a pivotal regulator of adipogenesis and suggest that overnutrition-mediated IKKβ activation serves as an initial signal that triggers adipose progenitor cell differentiation in response to HF feeding. Inhibition of IKKβ with antisense therapy may represent as a novel therapeutic approach to combat obesity and metabolic dysfunctions. Stem Cells 2016;34:1883–1895
Working model of the role of IκB kinase β (IKKβ) signaling in adipocyte lineage cells in obesity. (A): Under normal feeding condition, the IKKβ/NF-κB signaling is maintained at basal activity. (B): Under high-fat feeding condition, overnutrition (e.g., excess free fatty acids) activates IKKβ, leading to increased adipogenesis and inflammation. (C): In the current study, pharmacological inhibition or genetic deletion of IKKβ in adipocyte lineage cells impairs adipogenesis and protects mice from high-fat diet-induced obesity and metabolic dysfunctions
The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ (HG). Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here, we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary HG marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary HG activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. Stem Cells 2016;34:1896–1908
Mesenchymal stromal cells (MSCs) support hematopoiesis and exert immunoregulatory activities. Here, we analyzed the functional outcome of the interactions between MSCs and monocytes/macrophages. We showed that MSCs supported the survival of monocytes that underwent differentiation into macrophages, in the presence of macrophage colony-stimulating factor. However, MSCs skewed their polarization toward a peculiar M2-like functional phenotype (MMSC), through a prostaglandin E2-dependent mechanism. MMSC were characterized by high expression of scavenger receptors, increased phagocytic capacity, and high production of interleukin (IL)-10 and transforming growth factor-β. These cytokines contributed to the immunoregulatory properties of MMSC, which differed from those of typical IL-4-induced macrophages (M2). In particular, interacting with activated natural killer (NK) cells, MMSC inhibited both the expression of activating molecules such as NKp44, CD69, and CD25 and the production of IFNγ, while M2 affected only IFNγ production. Moreover, MMSC inhibited the proliferation of CD8+ T cells in response to allogeneic stimuli and induced the expansion of regulatory T cells (Tregs). Toll-like receptor engagement reverted the phenotypic and functional features of MMSC to those of M1 immunostimulatory/proinflammatory macrophages. Overall our data show that MSCs induce the generation of a novel type of alternatively activated macrophages capable of suppressing both innate and adaptive immune responses. These findings may help to better understand the role of MSCs in healthy tissues and inflammatory diseases including cancer, and provide clues for novel therapeutic approaches. Stem Cells 2016;34:1909–1921
Mesenchymal stromal cells (MSC) induce the generation of a novel subset of alternatively activated macrophages (MMSC), via the release of PGE2. MMSC are characterized by high expression of CD14, CD16, CD206, HLA-II, CD11b, CD163, and CD209 and secrete high amounts of IL-1β, IL-10, and TGF-β. These cytokines contribute to the immunoregulatory properties of MMSC. Interacting with autologous activated NK cells, MMSC inhibit both the expression of activating molecules such as NKp44, CD69, and CD25 and the production of IFNγ. Moreover, MMSC inhibit the proliferation of CD8+ T cells and induce the expansion of regulatory T cells (Treg). LPS revert the phenotypic and functional features of MMSC to those of M1 immunostimulatory/proinflammatory macrophages.
Shortly after the discovery of endothelial progenitor cells (EPCs) in 1997, many clinical trials were conducted using EPCs as a cellular based therapy with the goal of restoring damaged organ function by inducing growth of new blood vessels (angiogenesis). Results were disappointing, largely because the cellular and molecular mechanisms of EPC-induced angiogenesis were not clearly understood. Following injection, EPCs must migrate to the target tissue and engraft prior to induction of angiogenesis. In this study EPC migration was investigated in response to tumor necrosis factor α (TNFα), a pro-inflammatory cytokine, to test the hypothesis that organ damage observed in ischemic diseases induces an inflammatory signal that is important for EPC homing. In this study, EPC migration and incorporation were modeled in vitro using a coculture assay where TNFα treated EPCs were tracked while migrating toward vessel-like structures. It was found that TNFα treatment of EPCs increased migration and incorporation into vessel-like structures. Using a combination of genomic and proteomic approaches, NF-kB mediated upregulation of CADM1 was identified as a mechanism of TNFα induced migration. Inhibition of NF-kB or CADM1 significantly decreased migration of EPCs in vitro suggesting a role for TNFα signaling in EPC homing during tissue repair. Stem Cells 2016;34:1922–1933
Mesenchymal stem cells (MSCs) are multipotent stromal cells residing in the bone marrow. MSCs have the potential to differentiate to adipocytes, chondrocytes, and other types of cells. In this study, we investigated the molecular mechanism that controls MSC cell fate decisions for differentiation. We found that Vav1, a guanine nucleotide exchange factor for Rho GTPase, was highly expressed in MSCs. Interestingly, loss of Vav1 in MSCs led to spontaneous adipogenic but impaired chondrogenic differentiation, and accordingly Vav1 null mice displayed an increase in fat content and a decrease in cartilage. Conversely, ectopic expression of Vav1 in MSCs reversed this phenotype, and led to enhanced MSC differentiation into chondrocyte but retarded adipogenesis. Mechanistically, loss of Vav1 reduced the level of Sirt1, which was responsible for an increase of acetylated PPARγ. As acetylation activates PPARγ, it increased C/EBPα expression and promoted adipogenesis. On the other hand, loss of Vav1 resulted in an increase of acetylated Sox9, a target of Sirt1. As acetylation represses Sox9 activity, it led to a dramatic reduction of collagen 2α1, a key regulator in chondrocyte differentiation. Finally, we found that Vav1 regulates Sirt1 in MSCs through Creb. Together this study reveals a novel function of Vav1 in regulating MSC cell fate decisions for differentiation through Sirt1. Sirt1 deacetylates PPARγ and Sox9, two key mediators that control adipocyte and chondrocyte differentiation. The acetylation status of PPARγ and Sox9 has opposite effects on its activity, thereby controlling cell fate decision. Stem Cells 2016;34:1934–1946
Effective and specific therapeutic approaches are still needed for treating acute lung injury caused by severe pneumonia. Adipose-derived stem cells (ADSCs) are well-characterized adult stem cells that have antibacterial and anti-inflammatory effects. In this study, we evaluated the therapeutic effect of ADSCs on Staphylococcus aureus-induced acute lung injury in mice. Our results showed that intratracheal injection of ADSCs could attenuate the severity of lung inflammation, and reduce the bacterial load as well as mortality among infected mice. Our experiments also revealed that the secretion of regenerating islet-derived IIIγ (RegIIIγ) is responsible for the protective effect of ADSCs. Moreover, the expression of RegIIIγ requires TLR2, MyD88, and JAK2/STAT3 activation. In conclusion, ADSCs exhibit a direct antimicrobial activity that is mediated primarily by the TLR2-MyD88-JAK2/STAT3-dependent secretion of RegIIIγ. Stem Cells 2016;34:1947–1956
Recent studies have demonstrated the therapeutic potential of mesenchymal stem cells (MSCs) for the treatment of acute inflammatory injury and bacterial pneumonia, but their therapeutic applications in mycobacterial infections have not been investigated. In this study, we demonstrated the use of MSCs as a novel therapeutic strategy against Mycobacterium abscessus (M. abscessus), which is the most drug-resistant and difficult-to-treat mycobacterial pathogen. The systemic intravenous injection of MSCs not only improved mouse survival but also enhanced bacterial clearance in the lungs and spleen. Additionally, MSCs enhanced IFN-γ, TNF-α, IL-6, MCP-1, nitric oxide (NO) and PGE2 production and facilitated CD4+/CD8+ T cell, CD11bhigh macrophage, and monocyte recruitment in the lungs of M. abscessus-infected mice. To precisely elucidate the functions of MSCs in M. abscessus infection, an in vitro macrophage infection system was used. MSCs caused markedly increased NO production via NF-κB activation in M. abscessus-infected macrophages cultured in the presence of IFN-γ. Inhibiting NO or NF-κB signaling using specific inhibitors reduced the antimycobacterial activity of MSCs. Furthermore, the cellular crosstalk between TNF-α released from IFN-γ-stimulated M. abscessus-infected macrophages and PGE2 produced by MSCs was necessary for the mycobacterial-killing activity of the macrophages. Finally, the importance of increased NO production in response to MSC administration was confirmed in the mouse M. abscessus infection model. Our results suggest that MSCs may offer a novel therapeutic strategy for treating this drug-resistant mycobacterial infection by enhancing the bacterial-killing power of macrophages. Stem Cells 2016;34:1957–1970
Effects of aminoguanidine on MSC-induced M. abs-R clearance in mice. (A) Schematic diagram of the experimental design. (B) C57BL/6J mice were infected with an aerosolized strain of M. abs-R (1.6 × 107 CFUs/mouse), and MSCs (2.5 × 105 cells/mouse) were administered by intravenous injection at one day post-infection. Aminoguanidine was administered at a dose of 0.25% ad libitum in the drinking water each day, starting one day post-infection. The data are presented as the means ± SEM of two experiments (**, p < .01 and ***, p < .001). (C) The effects of MSCs on iNOS expression in the lungs of M. abs-R-infected mice. Immunohistochemistry was performed as described in "Materials and Methods" (scale bar = 100 μm). (E) C57BL/6J mice were infected with an aerosolized strain of M. abs-R (1.6 × 107 CFUs/mouse), and Cox-2−/− and iNOS−/− MSCs (2.5 × 105 cells/mouse) were administered by intravenous injection at one day post-infection. After 10 day after infection, bacterial CFUs were measured at lung and spleen as described in "Materials and Methods." The results are expressed as the mean estimated CFU ± SEM from the lung samples of five to six mice in two independent experiments. (D) Representative immunohistochemistry staining of iNOS and F4/80 (scale bar = 10 μm).
Transplantation of mesenchymal stem cells (MSCs) into injured or diseased tissue—for the in situ delivery of a wide variety of MSC-secreted therapeutic proteins—is an emerging approach for the modulation of the clinical course of several diseases and traumata. From an emergency point-of-view, allogeneic MSCs have numerous advantages over patient-specific autologous MSCs since "off-the-shelf" cell preparations could be readily available for instant therapeutic intervention following acute injury. Although we confirmed the in vitro immunomodulatory capacity of allogeneic MSCs on antigen-presenting cells with standard coculture experiments, allogeneic MSC grafts were irrevocably rejected by the host's immune system upon either intramuscular or intracerebral transplantation. In an attempt to modulate MSC allograft rejection in vivo, we transduced MSCs with an interleukin-13 (IL13)-expressing lentiviral vector. Our data clearly indicate that prolonged survival of IL13-expressing allogeneic MSC grafts in muscle tissue coincided with the induction of an alternatively activated macrophage phenotype in vivo and a reduced number of alloantigen-reactive IFNγ- and/or IL2-producing CD8+ T cells compared to nonmodified allografts. Similarly, intracerebral IL13-expressing MSC allografts also exhibited prolonged survival and induction of an alternatively activated macrophage phenotype, although a peripheral T cell component was absent. In summary, this study demonstrates that both innate and adaptive immune responses are effectively modulated in vivo by locally secreted IL13, ultimately resulting in prolonged MSC allograft survival in both muscle and brain tissue. Stem Cells 2016;34:1971–1984
Upon transplantation of IL13-engineered MSCs into allogeneic mice, local IL13 production modulated both innate and adaptive immune responses, ultimately resulting in prolonged MSC allograft survival. (A): FVB mouse-derived MSC-Luc/eGFP and MSC-Luc/eGFP/IL13 implants in the muscle or brain of C57BL/6 mice. Expression of Arg1, a marker of alternative M2a activation, was restricted to F4/80+ macrophages infiltrating IL13-producing MSC grafts. (B): T cells from naive, FVB MSC-transplanted or FVB MSC-IL13-transplanted C57BL/6 mice were analyzed by flow cytometry, with or without prior FVB MSC stimulation. IL13-expressing MSC allografts induced significantly fewer alloreactive CD8+ T cells than wildtype MSC allografts. *p<0.05; ***p<0.001 (C+D): Quantitative analysis of in vivo bioluminescence imaging of intramuscular (panel C) and intracerebral (panel D) FVB MSC-Luc/eGFP or FVB MSC-Luc/eGFP/IL13 allografts in C57BL/6 mice. Background radiance is demarcated by the gray area. IL13-expressing MSC allografts display a prolonged survival in both muscle and brain tissue of immune competent mice. **p<0.01 Abbreviations: MSC, mesenchymal stem cell; IL13, interleukin-13; Luc, firefly luciferase; eGFP, enhanced green fluorescent protein; Arg1, arginase-1.
The embryonic stem cell cycle (ESCC) and let-7 families of miRNAs function antagonistically in the switch between mouse embryonic stem cell self-renewal and somatic differentiation. Here, we report that the human ESCC miRNA miR-372 and let-7 act antagonistically in germline differentiation from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). hESC and iPSC-derived primordial germ cell-like cells (PGCLCs) expressed high levels of miR-372 and conversely, somatic cells expressed high levels of let-7. Manipulation of miRNA levels by introduction of miRNA mimics or knockdown with miRNA sponges demonstrated that miR-372 promotes whereas let-7 antagonizes PGCLC differentiation. Knockdown of the individual miR-372 targets SMARCC1, MECP2, CDKN1, RBL2, RHOC, and TGFBR2 increased PGCLC production, whereas knockdown of the let-7 targets CMYC and NMYC suppressed PGCLC differentiation. These findings uncover a miR-372/let-7 axis regulating human primordial germ cell (PGC) specification. Stem Cells 2016;34:1985–1991
Let-7 and miR-294 antagonistically regulate somatic versus germline fates through their influence on multiple cellular pathways.