Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell‐based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC‐derived mesenchymal‐like progenitor population. We found the direct plating of undifferentiated iPSCs into high‐density micromass cultures in the presence of BMP‐2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP‐2 treatment of iPSC‐derived MSC‐like (iPSC–MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage‐specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell‐based approaches to repair joint cartilage damage. J. Cell. Biochem. 114: 480–490, 2013. © 2012 Wiley Periodicals, Inc.     

Synthetic sulfonyl-hydrazone-1 positively regulates cardiomyogenic microRNA expression and cardiomyocyte differentiation of induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) are obtained from adult cells through overexpression of pluripotency factors. iPSCs share many features with embryonic stem cells (ESCs), circumventing ethical issues, and, noteworthy, match donor's genotype. iPSCs represent therefore a valuable tool for regenerative medicine. Cardiac differentiation of ESCs can be enhanced via microRNAs (miRNAs) and small chemical compounds, which probably act as chromatin remodelers. Cardiomyogenic potential of iPSCs is currently intensely investigated for cell therapy or in vitro drug screening and disease modeling. However, influences of small compounds on iPSC‐related cardiomyogenesis have not yet been investigated in details. Here, we compared the effects of two small molecules, bis‐peroxo‐vanadium (bpV) and sulfonyl‐hydrazone‐1 (SHZ) at varying concentrations, during cardiac differentiation of murine iPSCs. SHZ (5 µM) enhanced specific marker expression and cardiomyocyte yield, without loss of cell viability. In contrast, bpV showed negligible effects on cardiac differentiation rate and appeared to induce Casp3‐dependent apoptosis in differentiating iPSCs. Furthermore, SHZ‐treated iPSCs were able to increase beating foci rate and upregulate early and late cardiomyogenic miRNA expression (miR‐1, miR‐133a, and miR‐208a). Thus, our results demonstrate that small chemical compounds, such as SHZ, can constitute a novel and clinically feasible strategy to improve iPSC‐derived cardiac differentiation. J. Cell. Biochem. 112: 2006–2014, 2011. © 2011 Wiley‐Liss, Inc.     

The tumourigenicity of iPS cells and their differentiated derivates

Induced pluripotent stem cell (iPSC) provides a promising seeding cell for regenerative medicine. However, iPSC has the potential to form teratomas after transplantation. Therefore, it is necessary to evaluate the tumorigenic risks of iPSC and all its differentiated derivates prior to use in a clinical setting. Here, murine iPSCs were transduced with dual reporter gene consisting of monomeric red fluorescent protein (mRFP) and firefly luciferase (Fluc). Undifferentiated iPSCs, iPSC derivates from induced differentiation (iPSC‐derivates), iPSC‐derivated cardiomyocyte (iPSC‐CMs) were subcutaneously injected into the back of nude mice. Non‐invasive bioluminescence imaging (BLI) was longitudinally performed at day 1, 7, 14 and 28 after transplantation to track the survival and proliferation of transplanted cells. At day 28, mice were killed and grafts were explanted to detect teratoma formation. The results demonstrated that transplanted iPSCs, iPSC‐derivates and iPSC‐CMs survived in receipts. Both iPSCs and iPSC‐derivates proliferated dramatically after transplantation, while only slight increase in BLI signals was observed in iPSC‐CM transplanted mice. At day 28, teratomas were detected in both iPSCs and iPSC‐derivates transplanted mice, but not in iPSC‐CM transplanted ones. In vitro study showed the long‐term existence of pluripotent cells during iPSC differentiation. Furthermore, when these cells were passaged in feeder layers as undifferentiated iPSCs, they would recover iPSC‐like colonies, indicating the cause for differentiated iPSC's tumourigenicity. Our study indicates that exclusion of tumorigenic cells by screening in addition to lineage‐specific differentiation is necessary prior to therapeutic use of iPSCs.     

Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+)/CXCR4(+)/VE-cadherin(-) (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+) cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+) cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.     

Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells

Human induced pluripotent stem cells (HiPSCs) appear to be highly similar to human embryonic stem cells (HESCs). Using two genetic lineage-tracing systems, we demonstrate the generation of iPSC lines from human pancreatic islet beta cells. These reprogrammed cells acquired markers of pluripotent cells and differentiated into the three embryonic germ layers. However, the beta cell-derived iPSCs (BiPSCs) maintained open chromatin structure at key beta-cell genes, together with?a unique DNA methylation signature that distinguishes them from other PSCs. BiPSCs also demonstrated an increased ability to differentiate into insulin-producing cells both in?vitro and in?vivo, compared with ESCs and isogenic non-beta iPSCs. Our results suggest that the epigenetic memory may predispose?BiPSCs to differentiate more readily into insulin producing cells. These findings demonstrate that HiPSC phenotype may be influenced by their cells of origin, and suggest that their skewed differentiation potential may be advantageous for cell replacement therapy.     

Derivation of murine induced pluripotent stem cells (iPS) and assessment of their differentiation toward osteogenic lineage

Induced pluripotent stem cells (iPSCs) have generated hope and excitement because of the potential they possess for generating patient‐specific embryonic‐like stem cells (ESCs). Although many hurdles remain to be solved before the cells can be applied clinically; studies directed toward understanding factors that control differentiation of the cells toward various cell lineages are prerequisites for their future application. In the present study, we generated murine iPSC and assessed their differentiation toward osteogenic lineage. Murine tail tip fibroblasts were reprogrammed into embryonic‐like state by transduction with defined factors (Oct3/4, Sox2, c‐Myc, and klf4) carried in a retroviral vector. The reprogrammed cells expressed ESC markers, gave rise to three germ layers as demonstrated by teratoma formation and immunofluorescence staining. These data confirmed that the reprogrammed cells exhibited ESC‐like state. Treatment of iPSCs‐derived embryoid bodies (EBs) with transforming growth factor beta 1 (TGF‐β1) in the presence of retinoic acid enhanced generation of MSC‐like cells. The MSCs‐like cells expressed putative makers associated with MSCs; the cells deposited calcium in vitro when cultured in osteogenic medium. Interestingly MSCs‐like cells generated from iPSC directed EBs by treatment with retinoic acid and TGF‐β1 deposited more calcium in vitro than cells derived without TGF‐β1 treatment. Taken together, the data demonstrate that iPSC give rise to MSCs‐like state and that the cells have potential to differentiate toward osteoblasts. In addition, brief treatment of iPSC‐derived EBs with TGF‐β1 may be an approach for directing iPSC toward MSC‐like state. J. Cell. Biochem. 109: 643–652, 2010. © 2009 Wiley‐Liss, Inc.     

iPSC technology-Powerful hand for disease modeling and therapeutic screen

Cardiovascular and neurodegenerative diseases are major health threats in many developed countries. Recently, target tissues derived from human embryonic stem (hES) cells and induced pluripotent stem cells (iPSCs), such as cardiomyocytes (CMs) or neurons, have been actively mobilized for drug screening. Knowledge of drug toxicity and efficacy obtained using stem cell-derived tissues could parallel that obtained from human trials. Furthermore, iPSC disease models could be advantageous in the development of personalized medicine in various parts of disease sectors. To obtain the maximum benefit from iPSCs in disease modeling, researchers are now focusing on aging, maturation, and metabolism to recapitulate the pathological features seen in patients. Compared to pediatric disease modeling, adult-onset disease modeling with iPSCs requires proper maturation for full manifestation of pathological features. Herein, the success of iPSC technology, focusing on patient-specific drug treatment, maturation-based disease modeling, and alternative approaches to compensate for the current limitations of patient iPSC modeling, will be further discussed. [BMB Reports 2015; 48(5): 256-265]     

Genetically Matched Human iPS Cells Reveal that Propensity for Cartilage and Bone Differentiation Differs with Clones,not Cell Type of Origin

Background

For regenerative therapy using induced pluripotent stem cell (iPSC) technology, cell type of origin to be reprogrammed should be chosen based on accessibility and reprogramming efficiency. Some studies report that iPSCs exhibited a preference for differentiation into their original cell lineages, while others did not. Therefore, the type of cell which is most appropriate as a source for iPSCs needs to be clarified.

Methodology/Principal Findings

Genetically matched human iPSCs from different origins were generated using bone marrow stromal cells (BMSCs) and dermal fibroblasts (DFs) of the same donor, and global gene expression profile, DNA methylation status, and differentiation properties into the chondrogenic and osteogenic lineage of each clone were analyzed. Although genome-wide profiling of DNA methylation suggested tissue memory in iPSCs, genes expressed differentially in BMSCs and DFs were equally silenced in our bona fide iPSCs. After cell-autonomous and induced differentiation, each iPSC clone exhibited various differentiation properties, which did not correlate with cell-of-origin.

Conclusions/Significance

The reprogramming process may remove the difference between DFs and BMSCs at least for chondrogenic and osteogenic differentiation. Qualified and genetically matched human iPSC clone sets established in this study are valuable resources for further basic study of clonal differences.     

Hepatic differentiation of mouse iPS cells and analysis of liver engraftment potential of multistage iPS progeny

Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies.     

Derivation of Induced Pluripotent Stem Cells from Human Peripheral Blood T Lymphocytes

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models, genomic health analyses, and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small, clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs (“TiPS”) retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology, pluripotency-associated marker expression and capacity to generate neurons, cardiomyocytes, and hematopoietic progenitor cells. Additionally, they retain their characteristic T-cell receptor (TCR) gene rearrangements, a property which could be exploited for iPSC clone tracking and T-cell development studies. Reprogramming T-cells procured in a minimally invasive manner can be used to characterize and expand donor specific iPSCs, and control their differentiation into specific lineages.     

iPS cells: a game changer for future medicine

The induced pluripotent stem cell (iPSC) technology is instrumental in advancing the fields of disease modeling and cell transplantation. We herein discuss the various issues regarding disease modeling and cell transplantation presented in previous reports, and also describe new iPSC‐based medicine including iPSC clinical trials. In such trials, iPSCs from patients can be used to predict drug responders/non‐responders by analyzing the efficacy of the drug on iPSC‐derived cells. They could also be used to stratify patients after actual clinical trials, including those with sporadic diseases, based on the drug responsiveness of each patient in the clinical trials. iPSC‐derived cells can be used for the identification of response markers, leading to increased success rates in such trials. Since iPSCs can be used in micromedicine for drug discovery, and in macromedicine for actual clinical trials, their use would tightly connect both micro‐ and macromedicine. The use of iPSCs in disease modeling, cell transplantation, and clinical trials could therefore lead to significant changes in the future of medicine.     

Inducing pluripotency in vitro: recent advances and highlights in induced pluripotent stem cells generation and pluripotency reprogramming

Induced pluripotent stem cells (iPSCs) are considered patient‐specific counterparts of embryonic stem cells as they originate from somatic cells after forced expression of pluripotency reprogramming factors Oct4, Sox2, Klf4 and c‐Myc. iPSCs offer unprecedented opportunity for personalized cell therapies in regenerative medicine. In recent years, iPSC technology has undergone substantial improvement to overcome slow and inefficient reprogramming protocols, and to ensure clinical‐grade iPSCs and their functional derivatives. Recent developments in iPSC technology include better reprogramming methods employing novel delivery systems such as non‐integrating viral and non‐viral vectors, and characterization of alternative reprogramming factors. Concurrently, small chemical molecules (inhibitors of specific signalling or epigenetic regulators) have become crucial to iPSC reprogramming; they have the ability to replace putative reprogramming factors and boost reprogramming processes. Moreover, common dietary supplements, such as vitamin C and antioxidants, when introduced into reprogramming media, have been found to improve genomic and epigenomic profiles of iPSCs. In this article, we review the most recent advances in the iPSC field and potent application of iPSCs, in terms of cell therapy and tissue engineering.     

人诱导多能干细胞与人胚胎干细胞分化过程中Oct4/Nanog基因表达的比较

目的：比较通过慢病毒方法获得的人诱导多能性干细胞（iPSCs）与人胚胎干细胞（hESCs）分化过程中全能性基因Oct4、Nanog的表达变化。方法：收集分化不同时间点的拟胚体（EBs）,检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果：iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷（SCID）小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论：通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。     

ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell‐derived neurons

Induced pluripotent stem cells (iPSCs) hold promise to revolutionize studies of intracellular transport in live human neurons and to shed new light on the role of dysfunctional transport in neurodegenerative disorders. Here, we describe an approach for live imaging of axonal and dendritic transport in iPSC‐derived cortical neurons. We use transfection and transient expression of genetically‐encoded fluorescent markers to characterize the motility of Rab‐positive vesicles, including early, late and recycling endosomes, as well as autophagosomes and mitochondria in iPSC‐derived neurons. Comparing transport parameters of these organelles with data from primary rat hippocampal neurons, we uncover remarkable similarities. In addition, we generated lysosomal‐associated membrane protein 1 (LAMP1)‐enhanced green fluorescent protein (EGFP) knock‐in iPSCs and show that knock‐in neurons can be used to study the transport of endogenously labeled vesicles, as a parallel approach to the transient overexpression of fluorescently labeled organelle markers.     

Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells

We compared bona fide human induced pluripotent stem cells (iPSCs) derived from umbilical cord blood (CB) cells and neonatal keratinocytes (K). As a consequence of both incomplete erasure of tissue-specific methylation and aberrant de novo methylation, CB-iPSCs and K-iPSCs were distinct in genome-wide DNA methylation profiles and differentiation potential. Extended passage of some iPSC clones in culture did not improve their epigenetic resemblance to embryonic stem cells, implying that some human iPSCs retain a residual 'epigenetic memory' of their tissue of origin.     

A functionally characterized test set of human induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.