Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells

Pluripotency of embryonic stem cells (ESCs) is maintained by the balancing of several signaling pathways, such as Wnt, BMP, and FGF, and differentiation of ESCs into a specific lineage is induced by the disruption of this balance. Sulfated glycans are considered to play important roles in lineage choice of ESC differentiation by regulating several signalings. We examined whether reduction of sulfation by treatment with the chemical inhibitor chlorate can affect differentiation of ESCs. Chlorate treatment inhibited mesodermal differentiation of mouse ESCs, and then induced ectodermal differentiation and accelerated further neural differentiation. This could be explained by the finding that several signaling pathways involved in the induction of mesodermal differentiation (Wnt, BMP, and FGF) or inhibition of neural differentiation (Wnt and BMP) were inhibited in chlorate-treated embryoid bodies, presumably due to reduced sulfation on heparan sulfate and chondroitin sulfate. Furthermore, neural differentiation of human induced pluripotent stem cells (hiPSCs) was also accelerated by chlorate treatment. We propose that chlorate could be used to induce efficient neural differentiation of hiPSCs instead of specific signaling inhibitors, such as Noggin.     

Cardiac differentiation of human pluripotent stem cells

Due to the extremely limited proliferative capacity of adult cardiomyocytes, human embryonic (pluripotent) stem cell derived cardiomyocytes (hESC-CMs) are currently almost the only reliable source of human heart cells which are suited to large-scale production. These cells have the potential for wide-scale application in drug discovery, heart disease research and cell-based heart repair. Embryonic atrial-, ventricular- and nodal-like cardiomyocytes can be obtained from differentiated human embryonic stem cells (hESCs). In recent years, several highly efficient cardiac differentiation protocols have been developed. Significant progress has also been made on understanding cardiac subtype specification, which is the key to reducing the heterogeneity of hESC-CMs, a major obstacle to the utilization of these cells in medical research and future cell-based replacement therapies. Herein we review recent progress in cardiac differentiation of hESCs and cardiac subtype specification, and discuss potential applications in drug screening and cell-based heart regeneration.     

T lineage differentiation from induced pluripotent stem cells

Induced pluripotent stem (iPS) cells have potential to differentiate into T lymphocytes, however, the actual ability of iPS cells to develop into T lineages is not clear. In this study, we co-cultured iPS cells on OP9 cells expressing the Notch ligand Delta-like 1 (DL1), the iPS cells differentiated into T lymphocytes. In addition, in vitro stimulation of iPS cell-derived T lymphocytes resulted in secretion of IL-2 and IFN-γ. Moreover, adoptive transfer of iPS cell-derived T lymphocytes into Rag-deficient mice reconstituted their T cell pools. These results indicate that iPS cells are able to follow the normal program of T cell differentiation.     

Induction of oligodendrocyte differentiation from adult human fibroblast-derived induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) prepared from somatic cells might become a novel therapeutic tool in regenerative medicine, especially for the central nervous system (CNS). In this study, we attempted to induce O4-positive (O4⁺) oligodendrocytes from adult human fibroblast-derived iPSCs in vitro. We used two adult human iPSC cell lines, 201B7 and 253G1. 201B7 was induced by four-gene transduction (oct4, sox2, klf4, c-myc), and 253G1 was induced by three-gene transduction (oct4, sox2, klf4). We treated these cells with two in vitro oligodendrocyte-directed differentiation protocols that were optimized for human embryonic stem cells. One protocol used platelet-derived growth factor as the major mitogen for oligodendrocyte lineage cells, and the other protocol used epidermal growth factor (EGF) as the mitogen. Although the differentiation efficiency was low (less than 0.01%), we could induce O4⁺ oligodendrocytes from 253G1 cells using the EGF-dependent differentiation protocol. This is the first report of the in vitro induction of oligodendrocytes differentiation from human iPSCs.     

Glycomics of human embryonic stem cells and human induced pluripotent stem cells

Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.     

Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells

Human induced pluripotent stem (hiPS) cells have potential uses for drug discovery and cell therapy, including generation of pancreatic β-cells for diabetes research and treatment. In this study, we developed a simple protocol for generating insulin-producing cells from hiPS cells. Treatment with activin A and a GSK3β inhibitor enhanced efficient endodermal differentiation, and then combined treatment with retinoic acid, a bone morphogenic protein inhibitor, and a transforming growth factor-β (TGF-β) inhibitor induced efficient differentiation of pancreatic progenitor cells from definitive endoderm. Expression of the pancreatic progenitor markers PDX1 and NGN3 was significantly increased at this step and most cells were positive for anti-PDX1 antibody. Moreover, several compounds, including forskolin, dexamethasone, and a TGF-β inhibitor, were found to induce the differentiation of insulin-producing cells from pancreatic progenitor cells. By combined treatment with these compounds, more than 10% of the cells became insulin positive. The differentiated cells secreted human c-peptide in response to various insulin secretagogues. In addition, all five hiPS cell lines that we examined showed efficient differentiation into insulin-producing cells with this protocol.     

Thalidomide induces apoptosis during early mesodermal differentiation of human induced pluripotent stem cells

Thalidomide was once administered to pregnant women as a mild sedative; however, it was subsequently shown to be strongly teratogenic. Recently, there has been renewed interest in thalidomide because of its curative effects against intractable diseases. However, the teratogenicity of thalidomide is manifested in various ways and is still not fully understood. In the present study, we evaluated the effects of thalidomide on early mesodermal differentiation by examining the differentiation of human induced pluripotent stem cells (hiPSCs). The most common symptom of thalidomide teratogenicity is limb abnormality, which led us to hypothesize that thalidomide prevents early mesodermal differentiation. Therefore, mesodermal differentiation of hiPSCs was induced over a 6-d period. To induce early mesoderm differentiation, 1 d after seeding, the cells were incubated with the small molecule compound CHIR99021 for 3 d. Thalidomide exposure was initiated at the same time as CHIR99021 treatment. After 5 d of thalidomide exposure, the hiPSCs began expressing a mesodermal marker; however, the number of viable cells decreased significantly as compared to that of control cells. We observed that the proportion of apoptotic and dead cells increased on day 2; however, the proportion of dead cells on day 5 had decreased, suggesting that the cells were damaged by thalidomide during early mesodermal differentiation (days 0–2). Our findings may help elucidate the mechanism underlying thalidomide teratogenicity and bring us closer to the safe use of this drug.     

Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks

Efficient derivation of human cerebral neocortical neural stem cells (NSCs) and functional neurons from pluripotent stem cells (PSCs) facilitates functional studies of human cerebral cortex development, disease modeling and drug discovery. Here we provide a detailed protocol for directing the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to all classes of cortical projection neurons. We demonstrate an 80-d, three-stage process that recapitulates cortical development, in which human PSCs (hPSCs) first differentiate to cortical stem and progenitor cells that then generate cortical projection neurons in a stereotypical temporal order before maturing to actively fire action potentials, undergo synaptogenesis and form neural circuits in vitro. Methods to characterize cortical neuron identity and synapse formation are described.     

Immediate expression of Cdh2 is essential for efficient neural differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) exhibit reduced efficiency and higher variability in neural differentiation compared to embryonic stem cells (ESCs). In this study, we showed that mouse iPSCs failed to efficiently give rise to neuronal cells using conventional methods previously established for driving mouse ESC differentiation. We reported a novel approach which remarkably increases neural differentiation of mouse iPSCs. This novel approach initiated embryoid body (EB) formation directly from the whole cell clones isolated from the top of feeder cells. Compared to conventional neural induction methods such as single cell suspension or monolayer culture, the cell clone-derived EB method led to a pronounced increase in directed generation of various types of neural cells including neural stem cells, motoneurons and dopaminergic neurons in response to different inducers. Through gene expression microarray analysis, we identified 14 genes that were highly expressed in the cell clone-derived EBs. Among them, we found that Cdh2, also known as N-cadherin, played important roles in controlling the neural differentiation efficiency of mouse iPSCs. Forced expression of Cdh2 in iPSCs substantially enhanced the differentiation efficiency while knocking-down of Cdh2 by shRNA blocked the neural differentiation. Our results revealed a critical role of Cdh2 in the process of efficient neural differentiation of mouse iPS cells.     

miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells

The use of pluripotent stem cells in regenerative medicine and disease modeling is complicated by the variation in differentiation properties between lines. In this study, we characterized 13 human embryonic stem cell (hESC) and 26 human induced pluripotent stem cell (hiPSC) lines to identify markers that predict neural differentiation behavior. At a general level, markers previously known to distinguish mouse ESCs from epiblast stem cells (EPI-SCs) correlated with neural differentiation behavior. More specifically, quantitative analysis of miR-371-3 expression prospectively identified hESC and hiPSC lines with differential neurogenic differentiation propensity and in vivo dopamine neuron engraftment potential. Transient KLF4 transduction increased miR-371-3 expression and altered neurogenic behavior and pluripotency marker expression. Conversely, suppression of miR-371-3 expression in KLF4-transduced cells rescued neural differentiation propensity. miR-371-3 expression level therefore appears to have both a predictive and a functional role in determining human pluripotent stem cell neurogenic differentiation behavior.     

Directed differentiation of induced pluripotent stem cells towards T lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies (1). CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines (2-7). However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases (8-10). However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic (11-13), HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture (14-16). Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.     

Biomaterial aided differentiation and maturation of induced pluripotent stem cells

Engineering/reprogramming differentiated adult somatic cells to gain the ability to differentiate into any type of cell lineage are called as induced pluripotent stem cells (iPSCs). Offering unlimited self-renewal and differentiation potential, these iPSC are aspired to meet the growing demands in the field of regenerative medicine, tissue engineering, disease modeling, nanotechnology, and drug discovery. Biomaterial fabrication with the rapid evolution of technology increased their versatility and utility in regenerative medicine and tissue engineering, revolutionizing the stem cell biology research with the property to guide the process of proliferation, differentiation, and morphogenesis. Combining traditional culture platforms of iPSC with biomaterials aids to overcome the limitations associated with derivation, proliferation, and maturation, thereby could improve the clinical translation of iPSC. The present review discusses in brief about the reprogramming techniques for the derivation iPSC and details on several biomaterial guided differentiation of iPSC to different cell types with specific relevance to tissue engineering/regenerative medicine.     

Stability of neural differentiation in human adipose derived stem cells by two induction protocols

There are some evidences for suggesting that adipose derived stem cells (ADSCs) can be differentiated to the fate of neural cell type. ADSCs can be expanded rapidly in vitro and can be obtained by a less invasive method. In this study, we attempted to compare the stability of neural differentiation in human ADSCs by using two induction protocols.Isolated ADSCs were induced into neural-like cells using diverse effects of two specific procedures. For protocol 1, ADSCs were induced by chemical induction. In protocol 2, ADSCs were treated for sphere formation. Then, the singled cells were cultured in neurobasal media supplemented with special components. Differentiated ADSCs were evaluated for Nestin, MAP2 and GFAP expression by immunocytochemistry and semi quantitative RT-PCR techniques. Moreover, MTT assay was employed to detect cell viability and proliferation.Immunocytochemical analysis of both protocols demonstrated that ADSCs had large expression of the neural-specific markers. In RT-PCR, protocol 1 showed the highest percentage of MAP2 expression, but with time passing, the neural like state was reversible. Protocol 2 found with express of Nestin at week 1, however MAP2 and GFAP expression increased after 3 weeks. The neural-like cells produced by protocol 1 led to the further cell death.Comparative analysis showed that neural-like cell differentiation of ADSCs in chemical induction protocol was rapid but transitory, while it was approximately steady in neurosphere formation protocol.     

Genomic integrity of human induced pluripotent stem cells:Reprogramming,differentiation and applications

Ten years after the initial generation of induced pluripotent stem cells(hiPSCs)from human tissues, their potential is no longer questioned, with over 15000 publications listed on PubMed, covering various fields of research; including disease modeling, cell therapy strategies, pharmacology/toxicology screening and 3 D organoid systems. However, despite evidences that the presence of mutations in hiPSCs should be a concern, publications addressing genomic integrity of these cells represent less than 1% of the literature. After a first overview of the mutation types currently reported in hiPSCs, including karyotype abnormalities, copy number variations, single point mutation as well as uniparental disomy, this review will discuss the impact of reprogramming parameters such as starting cell type and reprogramming method on the maintenance of the cellular genomic integrity. Then, a specific focus will be placed on culture conditions and subsequent differentiation protocols and how their may also trigger genomic aberrations within the cell population of interest.Finally, in a last section, the impact of genomic alterations on the possible usages of hiPSCs and their derivatives will also be exemplified and discussed. We will also discuss which techniques or combination of techniques should be used to screen for genomic abnormalities with a particular focus on the necessary quality controls and the potential alternatives.