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Neural crest‐like stem cells for tissue regeneration

Abstract Neural crest stem cells (NCSCs) are a transient population of cells that arise during early vertebrate development and harbor stem cell properties, such as self‐renewal and multipotency. These cells form at the interface of non‐neuronal ectoderm and neural tube and undergo extensive migration whereupon they contribute to a diverse array of cell and tissue derivatives, ranging from craniofacial tissues to cells of the peripheral nervous system. Neural crest‐like stem cells (NCLSCs) can be derived from pluripotent stem cells, placental tissues, adult tissues, and somatic cell reprogramming. NCLSCs have a differentiation capability similar to NCSCs, and possess great potential for regenerative medicine applications. In this review, we present recent developments on the various approaches to derive NCLSCs and the therapeutic application of these cells for tissue regeneration.

these cells to migrate extensively within the developing embryo whereupon they experience various environmental cues. Accumulative evidence indicates that some cells lose plasticity and confer a fate during or even before migration, although a subpopulation of migratory cells appear to retain their multipotency. [5][6][7][8] After settling in their final sites of differentiation, these cells proceed to give rise to wide array of cell types and tissues, including bone, cartilage, melanocytes, fibroblasts, and smooth muscle cells (SMCs), in addition to neurons and glial cells. [2][3][4] As NCSCs are responsible for generating a diverse population of cells and tissues, dysregulation of neural crest (NC) development, cell migration, and differentiation can lead to a broad spectrum of human congenital disorders, including cardiovascular defects, melanoma, craniofacial defects, and neuroblastoma, collectively known as neurocristopathies. 9,10 Therefore, NCSCs can be used to generate a variety of specific cell types for disease modeling. Furthermore, the multipotency of NCSC differentiation makes NCSCs a valuable cell source for tissue regeneration.
In the past, NCSC isolation was limited to embryonic tissues.
NCSCs generally express markers such as Sox10, Slug, Snail, Twist, AP-2α, p75 NTR , and HNK1. Sox10 is critical for neurogenesis and maintenance of multipotency, [11][12][13] whereas Sox17 functions in the development of definitive endoderm and vasculogenesis. 14,15 Sox9, [16][17][18] Slug, 19,20 Snail, [21][22][23] and Twist 24,25 are all found closely related to NC and neural tube development. AP-2α is required for NC induction and expressed among actively migrating NCSCs. [26][27][28] p75 NTR and HNK1 can be utilized to identify migratory NCSCs, albeit HNK1 only labels a small proportion of migrating human NCSCs. 28,29 Recently, the development of pluripotent stem cells (PSCs), especially induced pluripotent stem cells (iPSCs) has enabled the derivation of human neural crest-like stem cells (NCLSCs), providing an opportunity to not only better understand human development, but also an unlimited cell source for patientspecific disease modeling and therapy. In addition, significant progress has been made in identifying and isolating NCLSCs from fetal, perinatal, and adult tissues that can potentially serve as viable cell sources for tissue regeneration. In this review, we will present recent findings on available sources of NCLSCs, and discuss their potential application for tissue engineering and regenerative medicine ( Figure 1).
Generally speaking, NCSCs are a cell population in NC-derived tissues during embryonic development, whereas all other sources of NCSCs, including those derived from PSCs in vitro and those isolated from placental and adult tissues, should be defined as NCLSCs. However, previous studies have interchangeably utilized NCSC to describe NCLSC derived from PSCs and adult tissues, and some NCLSCs have been named differently based on their tissue origin. Furthermore, there is also an argument that some adult NCLSCs are remnants of NCSCs arising during the development. To clearly distinguish all of these NCSCs and NCLSCs, single cell RNA sequencing, in addition to lineage tracing, needs to be performed to classify the cells based on genetic profile, which has not been done in a vast majority of previous studies. Therefore, when presenting results from specific studies, we generally use NCSC or NCLSC as we define it, but sometimes utilize the term as defined by the authors of the study to accommodate their hypothesis and viewpoint and to avoid confusion and controversy.
1.1 | Embryonic, fetal, and placental tissue-derived NCLSCs for tissue regeneration NCSCs have been identified and isolated from various embryonic and fetal tissues, 30 including the trunk neural tube, sciatic nerve, dorsal root ganglia (DRG), gut, heart, and branchial arches. These NCSCs can self-renew and differentiate in vitro and in vivo into distinct NC derivatives, 30 such as neurons, glia, myofibroblasts, osteocytes, and SMCs. Recently, a new method to culture premigratory NCSCs as "crestospheres" was developed, which enabled the maintenance of these cells in vitro for an extended period of time without the loss of their stem-cell characteristics. 31,32 Early studies using postmigratory NCSCs isolated from distinct regions within the embryo revealed that spatially distinct NCSCs displayed cell intrinsic differences that regulated their developmental potential in vivo, 33,34 suggesting that for tissue regeneration strategies, postmigratory NCSCs should be isolated from locations that match where these cells will be utilized therapeutically.
The boundary cap located at the dorsal root entry zone has also served as a source of embryonic NCSCs, 35 which have shown potential as a therapy for pancreatic and neurodegenerative disorders.
Boundary cap NCSCs (bNCSCs) cotransplanted with mouse or human pancreatic islets were found to enhance beta cell proliferation and improve islet graft reinnervation and revascularization in diabetic mice, possibly restoring neural-islet interactions that consequently improved islet engraftment and function after transplantation. [36][37][38] Furthermore, in an in vitro coculture system, bNCSCs partially prevented the cell death of human insulin producing cells in response to pro-inflammatory cytokines, suggesting a potential mechanism by which bNCSCs offer a protective effect that improves islet transplantation and thus, in combination with pancreatic islets, may serve as attractive cell source to treat patients with type 1 diabetes. 39  system. 40,41 These bNCSCs not only improved the survival of motor neuron precursors following transplantation into the spinal cord of adult mice, 42 but also prevented the loss of spinal cord neurons and glial activation in acutely injured spinal cord slice cultures through the secretion of brain-derived neurotrophic factor (BDNF), 43 suggesting these cells exhibit neuroprotective, anti-apoptotic, glia-inhibitory, and neurotrophic effects that may aid in neuroregenerative therapies.
Extraembryonic placental tissues, including early or later gestation chorionic villus tissues, are a unique cell source, yielding robust placental mesenchymal stem/stromal cells (PMSCs) well-suited for autologous or allogeneic cell therapy and tissue engineering. Interestingly, PMSCs express NCSC transcription factor markers including Sox9, Sox10, Sox17, Slug, Snail, and Twist, 44,45 suggesting PMSCs may be NC-derived and a type of NCLSC. The uniformity of expression of these transcription factors in PMSC cultures may reflect the developmental origins of these cells and could serve as predictors of some related functional properties. PMSCs were found to also express stem cell-related intracellular structural proteins Nestin, neurofilament medium, and S100β that are often associated with neural lineage phenotypes. Methods have been established to expand PMSCs from various placental tissues. [44][45][46][47][48] The in vitro characteristics of PMSCs are also analogous to those of MSCs isolated from various source tissues 49 in terms of surface marker expression and multipotency. [44][45][46][47][48] It has been shown that PMSCs display notable immunomodulatory capabilities, 50 Enteric neural crest cell (ENCC) is another type of NCLSC that has been utilized for the treatment of enteric neuropathies. ENCCs have been isolated from embryonic and postnatal murine intestine 66,67 and more recently, from fetal and postnatal human gut. [68][69][70][71] In murine studies, ENCCs were capable of colonizing the gut upon migrating and differentiating into enteric neurons and glia. Furthermore, these cells formed neural networks and were able to functionally integrate within the host bowel in vivo without any long-term safety issues. 72 Despite this, NCLSCs from adult tissues possess stem cell-like qualities, which make them great potential candidates for tissue regeneration. 97 Most of the studies carried out, thus far, using adult tissuederived NCLSCs have primarily focused on blood vessel, nerve, and bone regeneration.
Vascular stem cells (VSCs) play an important role in vascular remodeling and regeneration. 98 NCLSCs, as a type of VSCs, were found in the media and adventitia layers. 99 These Sox10 + NCLSCs not only differentiated into SMCs in the neointima, but also contributed to chondrogenic and osteogenic cell types in the atherosclerotic lesion, providing a novel perspective on the development of vascular diseases.
NCLSCs were also identified in the perivascular cells around microvessels throughout the body. 100 possibly through the secretion of various trophic factors that were distinct from those of BM-MSCs. 106 In addition to trophic support, it has been suggested that NCLSCs may promote tissue repair through the modulation of the immune system, 104,108 suggesting these cells may alter pro-and anti-inflammatory factors that could therefore improve tissue regeneration. The potential mechanisms by which transplanted NCLSCs regulate peripheral nerve repair require further elucidation.
Additionally, the application of adult tissue-derived NCLSCs transplanted alone or with other support cells in preclinical large animal studies will provide further insights into clinical outcomes and the potential of this new therapy for PNI.
Spinal cord injury (SCI) has many distinct factorial aspects including primary mechanical damage, reactive gliosis, secondary cell apoptosis, and the inability of axons to regenerate. 109 Indeed, in several case studies and preliminary clinical trials, the transplantation of DPSCs has proven to be a safe and effective therapeutic strategy, 127 indicating that DPSCs are a promising cell source for tissue regeneration and the treatment of various diseases.

| PSC-derived NCLSCs for tissue regeneration
PSCs, including embryonic stem cells (ESCs) and iPSCs, offer the advantage that they can proliferate extensively and give rise to cells from all three germ layers, thus making them powerful and instrumental for regenerative cell therapy, disease modeling, and drug discovery. [132][133][134][135] Utilizing existing knowledge of NCSC specification during development, researchers have created protocols to generate NCLSCs from mouse and human PSCs in vitro. 30 Although initial protocols relied on deriving NCLSCs through a neural progenitor cell population or coculture with stromal cells, direct and specific NCSC induction protocols have been developed that are based on using small molecule activators of Wnt signaling and inhibitors of BMP and Activin/ Nodal signaling. [136][137][138] This direct induction approach was further modified and refined into a completely defined system utilizing inhibition of transforming growth factor beta and glycogen synthase kinase 3 to generate NCLSCs, thereby improving the potential translation of these cells for clinical application. 139 Moreover, inclusion of additional factors, such as BMP-4, RA, and FGF-2, and modulation of Wnt signal 140 has enabled researchers to induce NCLSCs with distinct regional identity (eg, cranial, 141 trunk, 142 and vagal 138,143,144 NCLSCs), and as a result provides an approach to not only study regional identity in vitro, but also the possibility to generate NC derivatives that may closely resemble the in vivo counterparts and be utilized for tissue regeneration. As these NCLSCs can be differentiated in vitro into various cell types, 30  NCLSCs did not undergo chondrogenesis in vivo nor did they effectively repair osteochondral defects. 158 On the other hand, mouse iPSC-NCLSC-MSCs transplanted into calvarial defects differentiated into osteoblasts, produced no tumors, and contributed to bone regeneration. 159 These studies highlight how these derived cells may behave differently in vivo. However, a direct comparison cannot be made as the cells used in these studies were from different species and distinct defect models were implemented. Further investigation of craniofacial bone repair using MSCs from human iPSC-derived NCLSCs would reveal whether these cells could be a potential cell source for clinical application.
Melanocytes, which produce melanin and play a critical role in skin homeostasis and protection, have also been derived from ESCs and iPSCs after proceeding through a NC stage. 138,[161][162][163] Notably, human ESC-derived melanocytes were able to localize to the appropriate layer after being introduced into a human skin xenograft model.
Engraftment of these skin reconstructs into SCID mice revealed that these cells survived and were functional for over 4 weeks. 161 Furthermore, these melanocytes were capable of producing melanin and functionally integrated into a reconstituted pluristratified epidermis in vitro. 162 Altogether, this in vitro system provides an opportunity to study melanocyte developmental biology and may serve as a potential cellular therapy for hypopigmentation disorders.
Corneal scarring or blindness resulting from damage to the corneal stroma or corneal endothelial (CE) dysfunction is currently being treated with surgical corneal transplantation. 164  CECs. 167 Yet, whether these cells would integrate and aid in tissue regeneration in vivo remains unknown. Preliminary studies using CECs generated from human ESCs that proceeded through a periocular mesenchymal phase, rather than an NCSC stage, have shown promising results in improving CE dysfunction in rabbit models, 170 suggesting the possibility that keratocytes and CECs generated from ESC/iPSCderived NCLSCs may yield similar beneficial effects. Although further investigation into the functionality of these cells in vivo is still required, these NCLSC-derived corneal cells may serve a promising alternative for corneal repair.
Mouse iPSC-derived NCLSCs can also undergo differentiation into odontoblast-like cells upon cotransfection of Pax9 and BMP4 expression plasmids. 171 Interestingly, transplantation of control and transfected NCLSCs did not give rise to teratomas after they were subcutaneously injected into mice, suggesting these cells were not tumorigenic and, thus, a safe cell source for tooth regeneration.
Another study also showed that mouse iPSC-derived NCLSCs could differentiate into odontogenic mesenchymal cells. 172 Culturing these NCLSCs with conditioned medium from mouse dental epithelium further promoted their differentiation into odontoblasts. However, whether PSC-derived NCLSCs can be differentiated into other dental cell types (eg, dental pulp and dental follicle cells) and aid in dental tissue regeneration has yet to be determined.
In addition to tissue regeneration, numerous studies have demonstrated the potential of using iPSCs from patients affected by neurocristopathies for modeling human disease in vitro. Various diseases have been studied, thus far, including CHARGE syndrome, 173,174 Ewing sarcomas, 175 familial dysautonomia, 176,177 pigmentation disorders (eg, Hermansky-Pudlak and Chediak-Higashi syndromes), 138 HSCR disease, 143,178 Bardet-Biedl syndrome, 179 Treacher Collins syndrome, 180 and cardiovascular malformations, such as bicuspid aortic valves. 181 Findings from these studies have not only provided demonstration of disease-related phenotypes, but also evidence on the origin of certain neurocristopathies, which appear to arise from defects in NCSCs, rather than MSCs. 174,175,181 Additionally, this technology has served as a platform to identify potential drug candidates that are capable of restoring impaired function and possibly serve as therapeutic agents. 143,176,182 Apart from deriving NCLSCs from PSCs through directed differentiation, it has also been shown the NCLSCs can be generated using direct reprogramming, a process that bypasses the iPSC stage during the conversion of somatic cells into distantly related cell types. 183,184 In contrast to directed differentiation, which can take up to several weeks, 137 reprogramming can yield NCLSCs in 10 to 14 days. 177,185 Therefore, this approach provides a rapid method of obtaining NCLSCs that can be potentially administered clinically. To date, NCLSCs have been derived from human and mouse fibroblasts, 177,[186][187][188] keratinocytes, 189,190 and melanocytes 191 via the introduction of transcription factors, such as SOX10 and FOXD3, specific growth factors, or forced expression of Notch1 signaling, respectively. It has been reported that the derived NCLSCs are functional in vivo when they were investigated for neural repair in a zebrafish model 186 and migration capability in a chick embryo model system 177,191 ; however, more detailed and comprehensive analysis of in vivo functional outcomes is still necessary. Furthermore, this reprogramming approach has been applied to generate NCLSCs from fibroblasts isolated from patients with familial autonomic dystrophy, enabling the production of patientspecific cells, which holds great promise for personalized medicine and disease modeling. 177 Further elucidation on whether reprogrammedderived NCLSCs can be used for the various aforementioned tissue regeneration applications will greatly expand their therapeutic utility and clinical applicability. shown that iPSC-derived NCLSCs exhibit a nonimmunogenic phenotype, rather than an immunosuppressive one, as demonstrated by low levels of immune-related antigens in a noninflammatory environment and no induction of T-cell proliferation or pro-inflammatory cytokine production. 195 In contrast, another study proposed that PSC-derived NCLSCs exhibited immunosuppressive properties. 196 More work on the immunogenic properties of these cells will aid to provide more clarity on the issue. Overall, careful consideration of these concerns will ensure these cells are clinically safe and effective upon transplantation.

| CONCLUSIONS AND FUTURE DIRECTIONS
To facilitate the therapeutic applications of NCLSCs in the clinical setting, current NCLSC isolation, expansion, and differentiation protocols may require further optimization to consider good manufacturing practices. 197 Furthermore, during the development of favorable scaleup procedures to achieve large numbers for cell transplantation, certain aspects, such as passage number, should be taken into account as increased cell passaging can potentially diminish the therapeutic effects of these cells 198 and result in chromosomal defects that may lead to tumorigenesis. 199 In stem cell-based therapies, often times materials are used in combination with stem cells. 200,201 These materials can provide a scaffold that not only promotes cell survivability and the regeneration process, but also greatly influences cell fate and function in vivo. Future developments of next-generation biomaterials that are biocompatible and able to further improve NCSC expansion and differentiation in vitro and in vivo will be highly desirable for regenerative therapies.
Although NCSC and NCLSC appear to share common characteristics, such as multipotency, NC marker expression (eg, Sox10, p75 NTR , AP-2, and Nestin) and pluripotent gene expression, and a molecular signature representative of EPI-NCSC and embryonic NCSC has been defined, 202 there also are some apparent differences. For instance, long SAGEtranscriptome profiling revealed that human NCSCs exhibited a unique NC molecular signature, expressed pluripotent genes (Nanog, POU5F1. and Sox2), and were found to have global molecular profile similar to ESCs. 203 On the other hand, mouse epidermal NCLSCs only partially share the gene expression pattern of PSCs in that they express Myc, Klf4, and Sox2 but significantly lower levels of Nanog and Oct-4 when compared with mouse ESCs, 204 an attribute that can potentially reduce their tumorigenicity potential. A recent study performed transcriptome profiling at different time points during the induction of cranial neural crest cells (cNCCs) from mouse iPSCs and found that cNCCs exhibited gene expression profiles that were only partially similar to those previously reported. 205 Interestingly, they observed that these cNCCs did not express certain NC specifier genes (eg, FoxD3, Gbx2, Msx1, Dlx3, Zic2, and Zic3) during the derivation process and other markers, such as Sox10, were only expressed at day 14, indicating that cNCCs take longer to acquire a migratory phenotype in vitro in comparison to mouse embryos in vivo. 205 These findings suggest that the molecular network that gov-

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

AUTHOR CONTRIBUTIONS
J.S., X.D., A.W.: wrote and edited the manuscript; S.L.: conceived overall review content and edited the manuscript.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.