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Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers in vitro and in vivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines., Competing Interests: Declaration of interests This study was also funded by Anicom Specialty Medical Institute. T.A. and K.W. are employees of Anicom Specialty Medical Institute. M.I. and M.N. are the inventors of the patent application (JP 6770224 B2, JP 7174958 B2, US 10544431 B1, EP 3246406 B1, TW I 696700 B, CN 107109400 B, SG 11201705820U, KR 6102459458 B1)., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the development of various in vitro differentiation protocols for the efficient derivation of specific cell types, human induced pluripotent stem cell (hiPSC) lines have varing ability to differentiate into specific lineages. Therefore, surrogate markers for accurately predicting the differentiation propensity of hiPSC lines may facilitate cell-based therapeutic product development and manufacture. We attempted to identify marker genes that could predict the differentiation propensity of hiPSCs into neural stem/progenitor cells (NS/PCs). Using Spearman's rank correlation coefficients, we investigated genes in the undifferentiated state, the expression levels of which were significantly correlated with the neuronal differentiation propensity of several hiPSC lines. Among genes significantly correlated with NS/PC differentiation (P < 0.01), we identified ROR2 as a novel predictive marker. ROR2 expression in hiPSCs was negatively correlated with NS/PC differentiation tendency, regardless of the differentiation method, whereas its knockdown enhanced differentiation. ROR2 regulates NS/PC differentiation, suggesting that ROR2 is functionally essential for NS/PC differentiation. Selecting cell lines with relatively low ROR2 expression facilitated identification of hiPSCs that can differentiate into NS/PCs. Cells with ROR2 knockdown showed increased efficiency of differentiation into forebrain GABAergic neurons compared to controls. These findings suggest that ROR2 is a surrogate marker for selecting hiPSC lines appropriate for NS/PC and GABAergic neuronal differentiations., (© 2024. The Author(s).)

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by steroidogenic enzymes containing monogenetic defects. Most steroidogenic enzymes are cytochrome P450 groups that can be categorized as microsomal P450s, including 21-hydroxylase and 17α-hydroxylase/17,20 lyase, and mitochondrial P450s, including 11β-hydroxylase. It has been shown that ectopic administration of Cyp21a1 ameliorates steroid metabolism in 21-hydroxylase-deficient mice. However, the effectiveness of this approach for mitochondrial P450 has not yet been evaluated. In this study, primary fibroblasts from patients with 21-hydroxylase deficiency (CYP21A2D) ( n  = 4), 17α-hydroxylase/17,20 lyase deficiency (CYP17A1D) ( n  = 1), and 11β-hydroxylase deficiency (CYP11B1D) ( n  = 1) were infected with adeno-associated virus type 2 (AAV2) vectors. Steroidogenic enzymatic activity was not detected in the AAV2-infected CYP11B1D fibroblasts. Induced pluripotent stem cells (iPSCs) of CYP11B1D were established and differentiated into adrenocortical cells by induction of the NR5A1 gene. Adrenocortical cells established from iPSCs of CYP11B1D (CYP11B1D-iPSCs) were infected with an AAV type 9 (AAV9) vector containing CYP11B1 and exhibited 11β-hydroxylase activity. For an in vivo evaluation, we knocked out Cyp11b1 in mice by using the CRISPR/Cas9 method. Direct injection of Cyp11b1 -containing AAV9 vectors into the adrenal gland of Cyp11b1 -deficient mice significantly reduced serum 11-deoxycorticosterone/corticosterone ratios at 4 weeks after injection and the effect was prolonged for up to 12 months. This study indicated that CYP11B1D could be ameliorated by gene induction in the adrenal glands, which suggests that a defective-enzyme-dependent therapeutic strategy for CAH would be required. Defects in microsomal P450, including CYP21A2D and CYP17A1D, can be treated with extra-adrenal gene induction. However, defects in mitochondrial P450, as represented by CYP11B1D, may require adrenal gene induction.

Background: An extra copy of chromosome 21 in humans can alter cellular phenotypes as well as immune and metabolic systems. Down syndrome is associated with many health-related problems and age-related disorders including dermatological abnormalities. However, few studies have focused on the impact of trisomy 21 (T21) on epidermal stem cells and progenitor cell dysfunction. Here, we investigated the differences in keratinocytic characteristics between Down syndrome and euploid cells by differentiating cells from trisomy 21-induced pluripotent stem cells (T21-iPSCs) and autonomous rescued disomy 21-iPSCs (D21-iPSCs)., Methods: Our protocol for keratinocytic differentiation of T21-iPSCs and D21-iPSCs was employed. For propagation of T21- and D21-iPSC-derived keratinocytes and cell sheet formation, the culture medium supplemented with Rho kinase inhibitor on mouse feeder cells was introduced as growth rate decreased. Before passaging, selection of a keratinocytic population with differential dispase reactivity was performed. Three-dimensional (3D) air-liquid interface was performed in order to evaluate the ability of iPSC-derived keratinocytes to differentiate and form stratified squamous epithelium., Results: Trisomy-rescued disomy 21-iPSCs were capable of epidermal differentiation and expressed keratinocytic markers such as KRT14 and TP63 upon differentiation compared to trisomy 21-iPSCs. The lifespan of iPSC-derived keratinocytes could successfully be extended on mouse feeder cells in media containing Rho kinase inhibitor, to more than 34 population doublings over a period of 160 days. Dispase-based purification of disomy iPSC-derived keratinocytes contributed epidermal sheet formation. The trisomy-rescued disomy 21-iPSC-derived keratinocytes with an expanded lifespan generated 3D skin in combination with a dermal fibroblast component., Conclusions: Keratinocytes derived from autonomous trisomy-rescued iPSC have the ability of stratification for manufacturing 3D skin with restoration of keratinocytic functions., (© 2021. The Author(s).)

The use of human induced pluripotent stem cells (iPSCs), used as an alternative to human embryonic stem cells (ESCs), is a potential solution to challenges, such as immune rejection, and does not involve the ethical issues concerning the use of ESCs in regenerative medicine, thereby enabling developments in biological research. However, comparative analyses from previous studies have not indicated any specific feature that distinguishes iPSCs from ESCs. Therefore, in this study, we established a linear classification-based learning model to distinguish among ESCs, iPSCs, embryonal carcinoma cells (ECCs), and somatic cells on the basis of their DNA methylation profiles. The highest accuracy achieved by the learned models in identifying the cell type was 94.23%. In addition, the epigenetic signature of iPSCs, which is distinct from that of ESCs, was identified by component analysis of the learned models. The iPSC-specific regions with methylation fluctuations were abundant on chromosomes 7, 8, 12, and 22. The method developed in this study can be utilized with comprehensive data and widely applied to many aspects of molecular biology research.

Introduction: Early development of the human placenta remains poorly understood due to the lack of proper model systems. Previous reports have demonstrated that human induced pluripotent stem cells (hiPSCs) treated with bone morphogenetic protein 4 (BMP4) can differentiate into extraembryonic tissues as useful models of the early stage of trophoblast (TB) differentiation. In our previous study, we optimized the culture conditions of hiPSC-derived TB lineages, but the differentiated cells were heterogeneous., Methods: In order to characterize the hiPSC-derived TB lineage cells, four types of hiPSCs were treated with 50 ng/mL of BMP4 for 10 days. Subsequently, cells that were positive for the pan-TB marker keratin 7(KRT7) were purified from the differentiated cells using flow cytometry and identified with a DNA microarray., Results: Comparisons of our microarray data with the human transcriptome in a previous large-scale analysis showed that the gene expression patterns of KRT7+ cells were similar to the placenta. In total, 259 upregulated genes were commonly expressed in all four KRT7+ groups, including well-known TB markers. Among these upregulated genes, several with poorly investigated expression patterns and functions were confirmed as expressed in the primary placenta. While only XAGE2 and KCNQ2 were expressed in TB layers, XAGE2 was expressed throughout pregnancy and KCNQ2 was expressed only in cytotrophoblasts of the first trimester placenta., Conclusion: BMP4-treated KRT7+ cells were in the course of the human placental development. In addition, this approach allowed the identification of new genes that might be involved in placentation. However, further studies are needed to confirm their functions., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Down syndrome is the most frequent chromosomal abnormality among live-born infants. All Down syndrome patients have mental retardation and are prone to develop early onset Alzheimer's disease. However, it has not yet been elucidated whether there is a correlation between the phenotype of Down syndrome and the extra chromosome 21. In this study, we continuously cultivated induced pluripotent stem cells (iPSCs) with chromosome 21 trisomy for more than 70 weeks, and serendipitously obtained revertant cells with normal chromosome 21 diploids from the trisomic cells during long-term cultivation. Repeated experiments revealed that this trisomy rescue was not due to mosaicism of chromosome 21 diploid cells and occurred at an extremely high frequency. We herewith report the spontaneous correction from chromosome 21 trisomy to disomy without genetic manipulation, chemical treatment or exposure to irradiation. The revertant diploid cells will possibly serve a reference for drug screening and a raw material of regenerative medicinal products for cell-based therapy.

During reprogramming into human induced pluripotent stem cells (iPSCs), several stem cell marker genes are induced, such as OCT-4, NANOG, SALL4, and TERT. OCT-4, NANOG, and SALL4 gene expression can be regulated by DNA methylation. Their promoters become hypomethylated in iPSCs during reprogramming, leading to their induced expression. However, epigenetic regulation of the TERT gene remains unclear. In this study, we focused on epigenetic regulation of the human TERT gene and identified a differentially methylated region (DMR) at a distal region in the TERT promoter between human iPSCs and their parental somatic cells. Interestingly, the TERT-DMR was highly methylated in iPSCs, but low-level methylation was observed in their parental somatic cells. Region-specific, methylated-promoter assays showed that the methylated TERT-DMR up-regulated the promoter activity in iPSCs. In addition, Lamin B1 accumulated at the TERT-DMR in iPSCs, but not in their parent somatic cells. These results suggested that the TERT transcription was enhanced by DNA methylation at the TERT-DMR via binding to nuclear lamina during reprogramming. Our findings shed light on a new functional aspect of DNA methylation in gene expression.

Human induced pluripotent stem cells (hiPSCs) are potentially useful in both clinical applications and basic biological research. hiPSCs can differentiate into extra-embryonic cells in the presence of BMP4. However, the differentiation potential of hiPSCs can be affected by culture conditions or genetic variation. In this study, we investigated the effect of various BMP4 concentrations on the expression states of trophoblast markers and the optimal conditions for trophoblast induction. A high-fidelity gene expression assay using hiPSC lines showed that the expression levels of various trophoblast marker genes, such as KRT7, GCM1, CGB, and HLA-G, were upregulated by BMP4 in a dose-dependent manner in all types of hiPSCs used in this study. Treatment with high doses of BMP4 for prolonged periods increased the ratio of cells with trophoblast markers irrespective of the presence of bFGF. We found that the expression states of major pluripotency- and differentiation-related protein-coding genes in BMP4-treated cells depended on culture conditions rather than donor cell types. However, miRNA expression states were affected by donor cell types rather than BMP4 dose. Furthermore, the effect of the presence of bFGF on differentiation potential of KRT7-positive cells differed among iPSC types. Mechanistically, chromatin states around KRT7 promoter regions were comparable among the iPSC types used in this study, indicating that hiPSC chromatin state at these regions is not a parameter for cytotrophoblast differentiation potential. In conclusion, the optimal conditions for trophoblast differentiation from hiPSCs differ according to parental cell line.

Myelomeningocele (MMC) is a congenital disease without genetic abnormalities. Neurological symptoms are irreversibly impaired after birth, and no effective treatment has been reported to date. Only surgical repairs have been reported so far. In this study, we performed antenatal treatment of MMC with an artificial skin using induced pluripotent stem cells (iPSCs) generated from a patient with Down syndrome (AF-T21-iPSCs) and twin-twin transfusion syndrome (AF-TTTS-iPSCs) to a rat model. We manufactured three-dimensional skin with epidermis generated from keratinocytes derived from AF-T21-iPSCs and AF-TTTS-iPSCs and dermis of human fibroblasts and collagen type I. For generation of epidermis, we developed a protocol using Y-27632 and epidermal growth factor. The artificial skin was successfully covered over MMC defect sites during pregnancy, implying a possible antenatal surgical treatment with iPSC technology., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Functional intestines are composed of cell types from all 3 primary germ layers and are generated through a highly orchestrated and serial developmental process. Directed differentiation of human pluripotent stem cells (hPSCs) has been shown to yield gut-specific cell types; however, these structures do not reproduce critical functional interactions between cell types of different germ layers. Here, we developed a simple protocol for the generation of mature functional intestinal organoids from hPSCs under xenogeneic-free conditions. The stem cell-derived gut organoids produced here were found to contain distinct types of intestinal cells, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, that were derived from all 3 germ layers; moreover, they demonstrated intestinal functions, including peptide absorption, and showed innervated bowel movements in response to stimulation with histamine and anticholinergic drugs. Importantly, the gut organoids obtained using this xenogeneic-free system could be stably maintained in culture for prolonged periods and were successfully engrafted in vivo. Our xenogeneic-free approach for generating gut organoids from hPSCs provides a platform for studying human intestinal diseases and for pharmacological testing., Competing Interests: The authors have declared that no conflict of interest exists.

Cancer stem cells (CSCs) retain the capacity to propagate themselves through self-renewal and to produce heterogeneous lineages of cancer cells constituting the tumor. Novel drugs that target CSCs can potentially eliminate the tumor initiating cell population therefore resulting in complete cure of the cancer. We recently established a CSC-like model using induced pluripotent stem cell (iPSC) technology to reprogram and partially differentiate human mammary epithelial MCF-10A cells. Using the induced CSC-like (iCSCL) model, we developed a phenotypic drug assay system to identify agents that inhibit the stemness and self-renewal properties of CSCs. The selectivity of the agents was assessed using three distinct assays characterized by cell viability, cellular stemness and tumor sphere formation. Using this approach, we found that withaferin A (WA), an Ayurvedic medicine constituent, was a potent inhibitor of CSC stemness leading to cellular senescence primarily via the induction of p21Cip1 expression. Moreover, WA exhibited strong anti-tumorigenic activity against the iCSCL. These results indicate that our iCSCL model provides an innovative high throughput platform for a simple, easy, and cost-effective method to search for novel CSC-targeting drugs. Furthermore, our current study identified WA as a putative drug candidate for abrogating the stemness and tumor initiating ability of CSCs.

Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes., (©AlphaMed Press.)

Cancer stem cells (CSCs), a small and elusive population of undifferentiated cancer cells within tumors that drive tumor growth and recurrence, are believed to resemble normal stem cells. Although surrogate markers have been identified and compelling CSC theoretical models abound, actual proof for the existence of CSCs can only be had retrospectively. Hence, great store has come to be placed in isolating CSCs from cancers for in-depth analysis. On the other hand, although induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, concern exists over the inadvertent co-transplantation of partially or undifferentiated stem cells with tumorigenic capacity. Here we demonstrate that the introduction of defined reprogramming factors (OCT4, SOX2, Klf4 and c-Myc) into MCF-10A nontumorigenic mammary epithelial cells, followed by partial differentiation, transforms the bulk of cells into tumorigenic CD44(+)/CD24(low) cells with CSC properties, termed here as induced CSC-like-10A or iCSCL-10A cells. These reprogrammed cells display a malignant phenotype in culture and form tumors of multiple lineages when injected into immunocompromised mice. Compared with other transformed cell lines, cultured iCSCL-10A cells exhibit increased resistance to the chemotherapeutic compounds, Taxol and Actinomycin D, but higher susceptibility to the CSC-selective agent Salinomycin and the Pin1 inhibitor Juglone. Restored expression of the cyclin-dependent kinase inhibitor p16INK4a abrogated the CSC properties of iCSCL-10A cells, by inducing cellular senescence. This study provides some insight into the potential oncogenicity that may arise via cellular reprogramming, and could represent a valuable in vitro model for studying the phenotypic traits of CSCs per se.

Human induced pluripotent stem (iPS) cells were differentiated into the endoderm using activin A and were then treated with fibroblast growth factor 2 (FGF2) for differentiation into intestinal stem cell-like cells. These immature cells were then differentiated into enterocyte-like cells using epidermal growth factor (EGF) in 2% fetal bovine serum (FBS). At the early stage of differentiation, mRNA expression of caudal type homeobox 2 (CDX2), a major transcription factor related to intestinal development and differentiation, and leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), an intestinal stem cell marker, was markedly increased by treatment with FGF2. When cells were cultured in medium containing EGF and a low concentration of FBS, mRNAs of specific markers of intestinal epithelial cells, including sucrase-isomaltase, the intestinal oligopeptide transporter SLC15A1/peptide transporter 1 (PEPT1), and the major metabolizing enzyme CYP3A4, were expressed. In addition, sucrase-isomaltase protein expression and uptake of β-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA), a fluorescence-labeled substrate of the oligopeptide transporter, were detected. These results demonstrate a simple and direct method for differentiating human iPS cells into functional enterocyte-like cells.

The use of human induced pluripotent stem (iPS) cells would be of great value for a variety of applications involving drug development studies. Several reports have been published on the differentiation of human iPS cells into hepatocyte-like cells; however, the cells were insufficient for application in drug metabolism studies. In this study, we aimed to establish effective methods for differentiation of human iPS cells into hepatocytes. Two human iPS cell lines were differentiated by addition of activin A, dimethyl sulfoxide, hepatocyte growth factor, oncostatin M, and dexamethasone. The differentiated cells expressed hepatocyte markers and drug-metabolizing enzymes, revealing that the human iPS cells were differentiated into hepatocyte-like cells. Expression of CYP3A4 and UGT1A1 mRNAs increased with treatment with typical inducers of the enzymes, and the response of the cells against the inducers was similar to that of human hepatocytes. Furthermore, the drug-metabolizing activity of CYP3A4, as monitored by testosterone 6β-hydroxylase activity, was elevated by these inducers. In conclusion, we established methods for differentiation of hepatocyte-like cells expressing drug metabolizing activity from human iPS cells. The hepatocyte-like cells derived from human iPS cells will be useful for drug metabolism studies.

Embryonic stem (ES) cells recapitulate normal developmental processes and serve as an attractive source for routine access to a large number of cells for research and therapies. We previously reported that ES cells cultured on M15 cells, or a synthesized basement membrane (sBM) substratum, efficiently differentiated into an endodermal fate and subsequently adopted fates of various digestive organs, such as the pancreas and liver. Here, we established a novel hepatic differentiation procedure using the synthetic nanofiber (sNF) as a cell culture scaffold. We first compared endoderm induction and hepatic differentiation between murine ES cells grown on sNF and several other substrata. The functional assays for hepatocytes reveal that the ES cells grown on sNF were directed into hepatic differentiation. To clarify the mechanisms for the promotion of ES cell differentiation in the sNF system, we focused on the function of Rac1, which is a Rho family member protein known to regulate the actin cytoskeleton. We observed the activation of Rac1 in undifferentiated and differentiated ES cells cultured on sNF plates, but not in those cultured on normal plastic plates. We also show that inhibition of Rac1 blocked the potentiating effects of sNF on endoderm and hepatic differentiation throughout the whole differentiation stages. Taken together, our results suggest that morphological changes result in cellular differentiation controlled by Rac1 activation, and that motility is not only the consequence, but is also able to trigger differentiation. In conclusion, we believe that sNF is a promising material that might contribute to tissue engineering and drug delivery.

Here we attempted to clarify telomere metabolism in parental cells and their derived clonal human induced pluripotent stem cells (iPSCs) at different passages using quantitative fluorescence in situ hybridization (Q-FISH). Our methodology involved estimation of the individual telomere lengths of chromosomal arms in individual cells within each clone in relation to telomere fluorescence units (TFUs) determined by Q-FISH. TFUs were very variable within the same metaphase spread and within the same cell. TFUs of the established iPSCs derived from human amnion (hAM933 iPSCs), expressed as mean values of the median TFUs of 20 karyotypes, were significantly longer than those of the parental cells, although the telomere extension rates varied quite significantly among the clones. Twenty metaphase spreads from hAM933 iPSCs demonstrated no chromosomal instability. The iPSCs established from fetal lung fibroblasts (MRC-5) did not exhibit telomere shortening and chromosomal instability as the number of passages increased. However, the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased, and one (5%) of 20 metaphase spreads showed chromosomal abnormalities including X trisomy at an early stage and all 20 showed abnormalities including X and 12 trisomies at the late stage., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Glycogen storage disease type Ib (GSDIb) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), which leads to neutrophil dysfunction. However, the underlying causes of these dysfunctions and their relationship with glucose homeostasis are unclear. Induced pluripotent stem cells (iPSCs) hold a great promise for advances in developmental biology, cell-based therapy and modeling of human disease. Here, we examined the use of iPSCs as a model for GSDIb. In this study, one 2-year-old patient was genetically screened and diagnosed with GSDIb. We established iPSCs and differentiated these cells into hepatocytes and neutrophils, which comprise the main pathological components of GSDIb. Cells that differentiated into hepatocytes exhibited characteristic albumin secretion and indocyanine green uptake. Moreover, iPSC-derived cells generated from patients with GSDIb metabolic abnormalities recapitulated key pathological features of the diseases affecting the patients from whom they were derived, such as glycogen, lactate, pyruvate and lipid accumulation. Cells that were differentiated into neutrophils also showed the GSDIb pathology. In addition to the expression of neutrophil markers, we showed increased superoxide anion production, increased annexin V binding and activation of caspase-3 and caspase-9, consistent with the GSDIb patient's neutrophils. These results indicate valuable tools for the analysis of this pathology and the development of future treatments., (© 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.)

Background: hiPSCs are generated through epigenetic reprogramming of somatic tissue. Genomic imprinting is an epigenetic phenomenon through which monoallelic gene expression is regulated in a parent-of-origin-specific manner. Reprogramming relies on the successful erasure of marks of differentiation while maintaining those required for genomic imprinting. Loss of imprinting (LOI), which occurs in many types of malignant tumors, would hinder the clinical application of hiPSCs., Results: We examined the imprinting status, expression levels and DNA methylation status of eight imprinted genes in five independently generated hiPSCs. We found a low frequency of LOI in some lines. Where LOI was identified in an early passage cell line, we found that this was maintained through subsequent passages of the cells. Just as normal imprints are maintained in long-term culture, this work suggests that abnormal imprints are also stable in culture., Conclusions: Analysis of genomic imprints in hiPSCs is a necessary safety step in regenerative medicine, with relevance both to the differentiation potential of these stem cells and also their potential tumorigenic properties.

Despite the fact that cells in vivo are largely affected by the spatial heterogeneity in their surroundings, in vitro experimental procedures for stem cell differentiation have been relying on spatially uniform culture environments so far. Here, we present a method to form spatiotemporally non-uniform culture environments for stem cell differentiation using a membrane-based microfluidic device. By adopting a porous membrane with relatively large pores, patterned delivery of soluble factors is maintained stably over a period of time long enough for cell differentiation. We report that spatial patterns of mouse induced pluripotent stem cells (miPSCs) differentiation can be controlled by the present method. Furthermore, it is shown that the cell fate decision of miPSCs is determined by time-dependent switching of the delivery pattern. The present technique could be of relevance to the detailed analyses of the characteristics of stem cell differentiation in time and space, opening up a new insight into regenerative biology.

Induced pluripotent stem (iPS) cells, obtained from reprogramming somatic cells by ectopic expression of a defined set of transcription factors or chemicals, are expected to be used as differentiated cells for drug screening or evaluations of drug toxicity and cell replacement therapies. As pluripotent stem cells, iPS cells are similar to embryonic stem (ES) cells in morphology and marker expression. Several types of iPS cells have been generated using combinations of reprogramming molecules and/or small chemical compounds from different types of tissues. A comprehensive approach, such as global gene or microRNA expression analysis and whole genomic DNA methylation profiling, has demonstrated that iPS cells are similar to their embryonic counterparts. Considering the substantial variation among iPS cell lines reported to date, the safety and therapeutic implications of these differences should be thoroughly evaluated before they are used in cell therapies. Here, we review recent research defining the concept of standardization for iPS cells, their ability to differentiate and the identity of the differentiated cells.

Human-induced pluripotent stem cells (hiPSCs) are expected to become a powerful tool for regenerative medicine. Their efficacy in the use of clinical purposes is currently under intensive verification. It was reported that hiPSC-derived hemangioblasts had severely limited expansion capability due to an induction of early senescence: hiPSC-derived vascular endothelial cells (VECs) senesced after one passage and hiPSC-derived hematopoietic progenitor cells (HPCs) showed substantially decreased colony-forming activities. Here we show that early senescence is not an inevitable fate of hiPSC-derived cells. Applying our unique feeder-free culture methods for the differentiations of human embryonic stem cells (hESCs), we successfully generated VECs and HPCs from three lines of hiPSCs that were established by using a retrovirus vector system. All hiPS-derived VECs could be subcultured by 2:1∼3:1 dilutions up to 10∼20 passages, after which the cells underwent senescence. Among the three lines of hiPSCs, two lines generated HPCs that bore comparable granulocyte colony-forming units to those of hESCs. Moreover, one line effectively reproduced HPCs within the sac-like structures, the fields of in vitro hematopoiesis, as in the case of hESCs. Surprisingly, release of neutrophils into culture supernatant persisted even longer (∼60 days) than the case of hESCs (∼40 days). Thus, the problem of early senescence can be overcome by selecting appropriate lines of hiPSCs and applying proper differentiation methods to them.

Induced pluripotent stem cells (iPSCs) can now be produced from various somatic cell (SC) lines by ectopic expression of the four transcription factors. Although the procedure has been demonstrated to induce global change in gene and microRNA expressions and even epigenetic modification, it remains largely unknown how this transcription factor-induced reprogramming affects the total glycan repertoire expressed on the cells. Here we performed a comprehensive glycan analysis using 114 types of human iPSCs generated from five different SCs and compared their glycomes with those of human embryonic stem cells (ESCs; nine cell types) using a high density lectin microarray. In unsupervised cluster analysis of the results obtained by lectin microarray, both undifferentiated iPSCs and ESCs were clustered as one large group. However, they were clearly separated from the group of differentiated SCs, whereas all of the four SCs had apparently distinct glycome profiles from one another, demonstrating that SCs with originally distinct glycan profiles have acquired those similar to ESCs upon induction of pluripotency. Thirty-eight lectins discriminating between SCs and iPSCs/ESCs were statistically selected, and characteristic features of the pluripotent state were then obtained at the level of the cellular glycome. The expression profiles of relevant glycosyltransferase genes agreed well with the results obtained by lectin microarray. Among the 38 lectins, rBC2LCN was found to detect only undifferentiated iPSCs/ESCs and not differentiated SCs. Hence, the high density lectin microarray has proved to be valid for not only comprehensive analysis of glycans but also diagnosis of stem cells under the concept of the cellular glycome.

Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs). Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell) and five human embryonic stem cell (ESC) lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the "convergence" of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs., Competing Interests: The authors have declared that no competing interests exist.

The prominent characteristics of pluripotent stem cells are their unique capacity to self-renew and pluripotency. Although pluripotent stem cell proliferation is maintained by specific intracellular phosphorylation signaling events, it has not been well characterized how the resulting phosphorylated proteins are subsequently regulated. We here report that the peptidylprolyl isomerase Pin1 is indispensable for the self-renewal and maintenance of pluripotent stem cells via the regulation of phosphorylated Oct4 and other substrates. Pin1 expression was found to be up-regulated upon the induction of induced pluripotent stem (iPS) cells, and the forced expression of Pin1 with defined reprogramming factors was observed to further enhance the frequency of iPS cell generation. The inhibition of Pin1 activity significantly suppressed colony formation and induced the aberrant differentiation of human iPS cells as well as murine ES cells. We further found that Pin1 interacts with the phosphorylated Ser(12)-Pro motif of Oct4 and that this in turn facilitates the stability and transcriptional activity functions of Oct4. Our current findings thus uncover an atypical role for Pin1 as a putative regulator of the induction and maintenance of pluripotency via the control of phosphorylation signaling. These data suggest that the manipulation of Pin1 function could be a potential strategy for the stable induction and proliferation of human iPS cells.

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.

Background: Human induced pluripotent stem (iPS) cells are currently used as powerful resources in regenerative medicine. During very early developmental stages, DNA methylation decreases to an overall low level at the blastocyst stage, from which embryonic stem cells are derived. Therefore, pluripotent stem cells, such as ES and iPS cells, are considered to have hypo-methylated status compared to differentiated cells. However, epigenetic mechanisms of "stemness" remain unknown in iPS cells derived from extra-embryonic and embryonic cells., Methodology/principal Findings: We examined genome-wide DNA methylation (24,949 CpG sites covering 1,3862 genes, mostly selected from promoter regions) with six human iPS cell lines derived from human amniotic cells and fetal lung fibroblasts as well as two human ES cell lines, and eight human differentiated cell lines using Illumina's Infinium HumanMethylation27. A considerable fraction (807 sites) exhibited a distinct difference in the methylation level between the iPS/ES cells and differentiated cells, with 87.6% hyper-methylation seen in iPS/ES cells. However, a limited fraction of CpG sites with hypo-methylation was found in promoters of genes encoding transcription factors. Thus, a group of genes becomes active through a decrease of methylation in their promoters. Twenty-three genes including SOX15, SALL4, TDGF1, PPP1R16B and SOX10 as well as POU5F1 were defined as genes with hypo-methylated SS-DMR (Stem cell-Specific Differentially Methylated Region) and highly expression in iPS/ES cells., Conclusions/significance: We show that DNA methylation profile of human amniotic iPS cells as well as fibroblast iPS cells, and defined the SS-DMRs. Knowledge of epigenetic information across iPS cells derived from different cell types can be used as a signature for "stemness" and may allow us to screen for optimum iPS/ES cells and to validate and monitor iPS/ES cell derivatives for human therapeutic applications.

The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.