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Induced pluripotent stem cells as a cellular model for studying Down Syndrome.

Down Syndrome (DS), or Trisomy 21 Syndrome, is one of the most common genetic diseases. It is a chromosomal abnormality caused by a duplication of chromosome 21. DS patients show the presence of a third copy (or a partial third copy) of chromosome 21 (trisomy), as result of meiotic errors. These patients suffer of many health problems, such as intellectual disability, congenital heart disease, duodenal stenosis, Alzheimer’s disease, leukemia, immune system deficiencies, muscle hypotonia and motor disorders. About one in 1000 babies born each year are affected by DS. Alterations in the dosage of genes located on chromosome 21 (also called HSA21) are responsible for the DS phenotype. However, the molecular pathogenic mechanisms of DS triggering are still not understood; newest evidences suggest the involvement of epigenetic mechanisms. For obvious ethical reasons, studies performed on DS patients, as well as on human trisomic tissues are limited. Some authors have proposed mouse models of this syndrome. However, not all the features of the syndrome are represented. Stem cells are considered the future of molecular and regenerative medicine. Several types of stem cells could provide a valid approach to offer a potential treatment for some untreatable human diseases. Stem cells also represent a valid system to develop new cell-based drugs and/or a model to study molecular disease pathways. Among stem cell types, patient-derived induced pluripotent stem (iPS) cells offer some advantages for cell and tissue replacement, engineering and studying: self-renewal capacity, pluripotency and ease of accessibility to donor tissues. These cells can be reprogrammed into completely different cellular types. They are derived from adult somatic cells via reprogramming with ectopic expression of four transcription factors (Oct3/4, Sox2, c-Myc and Klf4; or, Oct3/4, Sox2, Nanog, and Lin28). By reprogramming cells from DS patients, it is possible to obtain new tissue with the same genetic background, offering a valuable tool for studying this genetic disease and to design customized patient-specific stem cell therapies.

as a nuchal translucency screening test. When the child has abnormalities, more fluid than usual tends to collect in this neck tissue. The results of ultrasound and blood tests combined with the mother's age can produce an estimate of the risk of having a baby with DS.
The integrated screening test includes the features of the first trimester combined test with a maternal blood test to measure the levels of four pregnancy-associated substances: alpha fetoprotein, estriol, HCG and inhibin A. This test can achieve the same sensitivity of detection as the first trimester combined test, but with a lower false-positive rate. The cell-free fetal DNA test checks for fetal DNA circulating in the mother's blood. If this screening test indicates a high risk of DS, diagnostic tests should be used.
There are also several diagnostic tests that are able to identify DS. These include: -Amniocentesis. It uses the amniotic fluid to analyze the chromosomes of the fetus. This test is performed after 15 weeks of pregnancy. The test is associated with a slight risk of miscarriage.
-Chorionic villus sampling (CVS). In CVS, cells are taken from the placenta and used to analyze the fetal chromosomes. This test is performed after 10 weeks of pregnancy and it has a higher risk of miscarriage than amniocentesis. -Cordocentesis. The fetal blood is taken from a vein in the umbilical cord and examined for chromosomal defects. This test is performed between 18 and 22 weeks of pregnancy. It has a higher risk of miscarriage than amniocentesis and chorionic villus sampling, so it is only recommended when results of other tests are unclear and the desired information cannot be obtained by any other method. Individuals with DS have the particular phenotypic features that include intellectual disability, short stature, motor disorders, and characteristic facies [8] . In addition to these features, these patients have specific major congenital malformations, such as atrioventricular septal defect of the heart, duodenal stenosis or atresia, and imperforate anus of the gastrointestinal tract.
People with DS have a higher risk of developing Alzheimer's disease, acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) [9,10] . The incidence of ALL is 20-fold higher in children with DS than in the general population, and the incidence of AML is between 46-to 83-fold higher, with a particular susceptibility to acute megakaryoblastic leukemia [11] .
Both the phenotypes and the pathological conditions associated with DS are due to alterations in the dosage of genes located on chromosome HSA21. However, the molecular pathogenic mechanisms of DS induction are still not understood. Newest evidences suggest the involvement of epigenetic mechanisms. Indeed, in response to changes in gene dosage, trisomy could disrupt normal epigenetic marks across the genome. Distinct DNA methylation pattern on chromosome 21 seems to be associated with accelerated cognitive aging in DS patients [12] .
The quality of life of people with DS is compromised by multiple health problems affecting them. The patients are treated by "early intervention programs" to promote development and independence individually, as recommended by the US National DS Society (NDSS). Some of the therapies used in the "early intervention programs" are: Physical therapy includes exercises to help the child develop good posture, increasing muscle strength, and improving balance.
Speech-language therapy can help DS children by improving their communication skills by using language more effectively.
Occupational therapy helps finding ways to adjust everyday tasks and conditions to match a person's needs and abilities.
Emotional and behavioral therapies. Children with DS may become frustrated because of difficulty communicating, may develop compulsive behaviors, Attention Deficit Hyperactivity Disorder, and other mental health issues. Therapists identify the cause of these behaviors to develop a strategy for avoiding or preventing these situations and teach better or more positive ways to respond to situations [13] .
Sometimes in addition to these non-drug therapies, patients with DS assume amino acid supplements or drugs that affect their brain activity.

Down Syndrome animal model
For obvious ethical reasons, studies performed on DS patients, as well as on human trisomic tissues are limited. Some authors have proposed mouse models of this syndrome. The mouse model more used to study the DS is Ts65Dn mouse. It bears segmental trisomy for a distal region of the mouse chromosome Mmu16 which contains approximately 55% of Hsa21 conserved genes [14] . This model is additionally trisomic for approximately 50 genes that are nonorthologous to Hsa21 [15] . Ts65Dn mouse, although it is an invaluable tool for the study DS, provides only a limited representation of human T21 syndrome modelling. Indeed, this mouse model has limitations including the lack of numerous Hsa21 orthologous genes and the addition of some Mmu17 genes that are non-trisomic in humans. These differences in genes may confound results of therapeutic interventions. Since these males are sterile, mice must be generated from Ts65Dn dams.

These limitations could be overcome studying stem cells in vitro.
Among stem cell types, patient-derived induced pluripotent stem (iPS) cells can be used as in vitro human disease models [16,17] .
These cells can be reprogrammed into completely different cellular types, differentiation into various cellular types can provide new insights into disease pathophysiology that are not possible with murine models [18,19] . The next paragraph will explain in detail the advantages of using this type of stem cells.

Induced Pluripotent Stem (iPS) Cells
Stem cells are considered the future of molecular and regenerative medicine [20] . Several types of stem cells could provide a valid approach to offer a potential treatment for some untreatable human diseases [21] . Stem cells also represent a valid system to develop new cell-based drugs and/or a model to study molecular disease pathways. Among stem cell types, patient-derived induced pluripotent stem (iPS) cells offer some advantages for cell and tissue replacement, engineering and studying self-renewal capacity, pluripotency, and ease of accessibility to donor tissues [22] . These cells, discovered by the Nobel Prize winner S. Yamanaka, can be reprogrammed into completely different cellular types [23] . They are derived from adult somatic cells via reprogramming with ectopic expression of four transcription reprogramming factors (Oct3/4, Sox2, c-Myc and Klf4; or, Oct3/4, Sox2, Nanog, and Lin28, otherwise known as Yamanaka's cocktail). The expression of the Yamanaka's cocktail transcription factors lead to the suppression of the genes responsible for differentiation, together with the expression of genes and epigenetic changes which sustain pluripotency, in this way reverting the cells to a pluripotent state [24] .
Yamanaka's research team was the first to generate iPS cells from mouse embryonic and adult fibroblasts by retrovirus-mediated transfection of four transcription factors under embryonic stem cell (ESC) culture conditions [24] . In this work, they paved the way to overcome the problems related to the use of ESCs for cell transplantation. Indeed, the use of human embryos is accompanied by several ethical issues, as well as scientific problems, such as tissue rejection following transplantation in patients, in vivo teratoma formation and uncontrollable cell proliferation of undifferentiated ESCs [25,26] . The generation of pluripotent cells directly from the patients' own cells could resolve these ESC limitations. Yamanaka's group successfully demonstrated efficacious reprogramming of differentiated human somatic cells into a pluripotent state. Human somatic cell-derived iPS cells are comparable to human ESCs in many aspects including morphology, proliferation, cell markers, gene expression, in vitro differentiation capacity and teratoma formation [27] . The technology for iPS cells generation is simple and reproducible, thus providing hope for potential human disease treatments. However, therapeutic potential of iPS cells remains to be further elucidated. The work of Rudolf Jaenisch's group demonstrated that autologous iPS cells were effective in a humanized sickle cell anemia mouse model [28] , opening the way for a possible human transfer of the technology. Beyond cell transplantation, patientderived iPS cells are also useful for modeling diseases. Derivation of patient-specific cells could be a potential cure for many diseases. Indeed, several diseases could be modeled by using iPS cells: from neurological disorders (i.e. Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal muscular atrophy, amyotrophic lateral sclerosis, Duchenne muscular dystrophy, schizophrenia, and autism spectrum disorders) to kidney diseases, metabolic diseases, blood diseases, diabetes and cardiovascular diseases [29][30][31][32][33][34] . In the near future, modeling diseases with iPS cells could overcome or reduce the need for animal disease models, since iPS cell-models represent more accurately the physiology of human cells as compared to animal cells [23] . This point is of particular interest in DS research, whereas animal models could be inadequate. It has been demonstrated that in a chromosome rodent model, in which the mice were engineered with the murine chromosome segment orthologous to the DS critical region on HSA21, the human cranial abnormalities commonly associated with trisomy 21 were not seen [35] . This result was probably due to the different chromosomal distribution of genetic segments between human and mouse. Indeed, the orthologous segments to human chromosome 21 are located on mouse chromosomes 10 and 17.

Modeling Down Syndrome with Patient-Specific iPS cells
By reprogramming cells from DS patients, it is possible to obtain new tissue with the same genetic background, offering a valuable tool for studying this genetic disease and to design potential customized patient-specific stem cell therapies. For an efficient DS model, it is necessary that the new cell system is able to reproduce the features of the disease.
In 2008, Park et al., described derivation of human iPS cell lines from two young male DS patients [36] . Donor cells were fibroblasts transduced with the four (OCT4, SOX2, KLF4, and c-MYC) reprogramming factors cocktail, via the Maloney murine leukemia virus (MMLV)-derived retroviruses pMXs. Noteworthy, the authors performed analysis of the iPS cell karyotype, showing the characteristic trisomy 21 anomaly. Morphology, pluripotency and differentiation capacity of iPS cells were confirmed. In 2013, Weick et al., reprogrammed fibroblasts from two DS subjects to iPS cells [37] . One of the fibroblast lines had low level mosaicism for trisomy 21. They also differentiated iPS cells into neurons expressing dorsal forebrain neurons markers. These DS iPS cell -derived neurons showed significant synaptic deficits, such as reduced synaptic activity attributable to developmental abnormalities consistent with cognitive deficits in DS patients.
By using lentiviral vectors, dermal fibroblasts from DS patients of various karyotypes (trisomy 21 and translocation) have been reprogrammed into iPS cells. The iPS cells maintained the same karyotypes as the parental human dermal fibroblasts [38] .
Hematopoietic disturbances associated with DS have also been modeled using iPS cells. Induced PS cells were differentiated into hematopoietic cells, showing that trisomy 21 is accompanied by an abnormal expansion of hematopoietic progenitor cells [39] . Hematopoietic defects have also been detected by Chou et al. [40] . Blood progenitor cells generated from DS iPS cells enhanced erythropoiesis and reduced myelopoiesis, even if proliferation rate was normal.
DS iPS cells from fetal fibroblasts of monozygotic twins discordant for trisomy 21 show remarkable differences. The DS vs. normal twin's iPS cells show differences in proliferation, neurogenesis and neuronal differentiation [41] .
Astrocytes also take a role in DS pathogenesis. DS patient fibroblasts were reprogrammed into iPS cells and subsequently differentiated to astroglia and neurons [42] . Derived astroglia cells showed cellular dysfunction and oxidative stress. They negatively interacted with derived neurons in regard to the regulation of neurite outgrowth, neuronal ion channel maturation, synaptic activity formation and non-cell-autonomous toxic effects on neurons [42] . Very importantly, once transplanted in mouse neonatal brain, DS iPS cell-derived astroglia contributed to the impaired brain function [42] .
Through these techniques, it has been confirmed that iPS cells generated from DS somatic cells possess the genetic and phenotypic characteristics of the disease. In addition, differentiation of DSderived iPS cells into neural cells still retain cellular dysfunctions, providing evidence that iPS cell system is an efficient model to study the syndrome and to enable discovery of the underlying causes of and pharmaceutical therapeutic treatments for this disorder.

Using Patient-Specific iPS cells for treating Down Syndrome Gene therapy
In DS research, removal of the trisomy condition is a priority goal. Induced PSC generation from DS patients allows experiments of gene therapy correction. In an elegant research experiment, DS fibroblast-derived iPS cells were engineered by TKNEO fusion gene (a hybrid gene combined by human thymidine kinase (tk) gene and the coding region of the bacterial neomycin-resistance gene (neo)) [43] . Briefly, derived cells showed trisomy removal, providing insights for a future clinical application.
Chromosome therapy has been proposed in the work of Jiang et al. [44] . Through a zinc finger nuclease (ZFN)-driven targeted addition, they inserted the human X-inactivation gene, XIST (which produces a non-coding RNA from the inactive X chromosome) into the trisomic 21chromosome of DS patient-derived iPS cells to enhance epigenetic chromosome-wide changes. XIST induced longrange silencing in iPS cells, and XIST-iPS cell differentiated neural precursor cells showed improved cell proliferation and neurogenesis. This strategy highlights that X-chromosome inactivation via iPS cells could be an useful strategy to correct chromosomal dosage imbalance [45] .

Cell-based therapy
Induced PS cells derived from amniotic fluid stem cells (AFSCs) represent an attractive potential cell system in DS modeling [28] . These stem cells are very interesting for cell-based applications, as they show low expression of major histocompatibility complex antigens, high pluripotency, low immunogenicity and anti-inflammatory properties [46] . DS AFSC-derived iPS cells retain their cytogenetic signatures and are able to differentiate into specialized hematopoietic and neural precursor cells [47,48] .Very interestingly, in their work, Lu's group used amniotic stem cells as a source to generate human DS iPS cells [49] . Reprogramming was achieved by co-expressing Yamanaka factors through lentiviral delivery. DS iPS cells maintained the abnormal chromosomal karyotype signature and were then differentiated into neuronal progenitor cells (NPCs). These differentiated cells showed amyloid precursor protein (APP) gene over-expression. In addition, DS iPS-NPCs also displayed overexpression of microRNAs miR-155 and miR-802, which in turn trigger methyl-CpG-binding protein 2 (MeCP2) degradation with consequent neurogenesis and neuron maturation impairment.
AFSC-iPS cell system is also a valuable tool for drug screening. DS AFSC iPS cell-derived neural cells treated with Nbutylidenephthalide, a major phthalide ingredient of Angelica sinensis, showed a reduction in secreted Aβ40 deposits in the total and hyperphosphorylated Tau levels [50] . This study indicates that DS AFSC-iPS cell derived neural cells can be utilized as both an ideal cellular model of DS with early-onset dementia and as highthroughput screening model of candidate drugs.
What would be the best donor cell type choice for reprogramming? The answer should be based on the use of iPS cells: disease modeling to study the basic mechanism of the disease, drug screening, or cell therapy [51] . Due to easy, well-validated cell culture procedures and high proliferation rate, fibroblasts are the most used cell type for the generation of iPS cells. Apart the above mentioned AFSCs, fetal stromal cells and mononuclear cells have been also efficaciously used for reprogramming [52] .
Optimal cell culture conditions need to be further improved and standardized. Several improvements in such conditions have been proposed. Dietary supplements (i.e. vitamin C and anti-oxidants) added in the reprogramming media are able to improve iPS cell state [53,54] . Serum-and feeder-free defined culture conditions have been also implemented [53] . Of course, while looking for new cell culture improvements, proliferation rate, pluripotency and selfrenewal capacity should not be lost.

Limitations
Using retroviruses, transgenes, or the oncogene c-Myc for cell reprogramming could be a limitation for human applications of iPS cells [55][56][57] . Viral vectors may integrate into the host genome and create single-base (i.e. insertions or deletions) mutations. The following processes should be avoided in order to ensure genomic stability: mutations during reprogramming process, epigenetic reactions, abnormalities in the X chromosome, and copy number variations. Several other methods have been proposed to overcome the use of integrating viral vectors. These methods are integration free systems: plasmid DNA, recombinant proteins, and synthetic mRNA [58] . Transfection of two expression plasmids (one containing the complementary DNAs of Oct3/4, Sox2, and Klf4 and the other containing the c-Myc cDNA) into embryonic fibroblasts results in iPS cells generation without plasmid integration, which produced teratomas when transplanted [59] . The expression of the miR302/367 microRNA-cluster is able to give an efficient reprogramming of mouse and human somatic cells to iPS cells, without the use of exogenous transcription factors [60] . In addition, whereas miR302/367-iPS cells show similar characteristics to transcription factors-iPS cells, in order to produce pluripotency and teratoma formation, miRNAbased reprogramming is two orders of magnitude more efficient than standard Yamanaka's factors-based approach. The microRNA-induced PS cells have also a reduced risk of mutations and tumorigenesis [61] .
Briggs at al., generated non-viral iPS cells from DS fibroblasts by episomal reprogramming [62] . These DS iPS cells are able to model in vitro the phenotype of the disorder. Their differentiation toward neural phenotype indicated increased neural cell death due to oxidative stress. Other limitations to be considered are: -Age of the somatic donor cells. Pluripotency is achieved more easily in young tissue (i.e. embryonic tissue) rather than adult tissue [51] .
-Differentiation status of somatic donor cells. Differentiation state could influence reprogramming process. This fact is noteworthy to be considered, as some iPS cells are able to retain the "epigenetic memory" of their primitive tissue [63] . It has been demonstrated that hematopoietic stem cells are more efficiently reprogrammed than differentiated B and T cells [64] . Recently, it has been reported that MUC1, a growth factor receptor of human stem cells acting to maintain the naïve stem cell state [65,66] , is able to change the DNA methylation pattern in cancer cells [67,68] , also contributing to inflammation state [69] .
Novel genome editing technologies could help correct mutations that have arisen during reprogramming process. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (CRISPR/Cas9) system is an efficient tool to efficaciously edit and correct gene mutations in iPS cells [70] .

Conclusions
By reprogramming cells from DS patients, it is possible to obtain new tissue with the same genetic background, offering a valuable tool for studying this genetic disease and to design customized patient-specific stem cell therapies. However, several limitations still need to be overcome and further experimental validations are required before translation into therapy.