Long-term culture of Japanese human embryonic stem cells in feeder-free conditions

Human pluripotent embryonic stem cells (hESCs) have great promise for research into human developmental biology, development of cell therapies for the treatment of diseases, toxicology, and drug discovery. Traditionally, undifferentiated hESCs are maintained on mouse embryonic fibroblasts (MEFs), which impede the clinical applications of hESCs. Here we have examined the long-term stability of the Japanese hESC line (KhES-1) in feeder-free culture. KhES-1 cells were cultured with MEF conditioned medium (CM) and different doses of basic fibroblast growth factor (bFGF) in six-well-plates of which the surface was coated with Matrigel. KhES-1 cells were maintained for at least 40 passages. In this culture system, the cells maintained stable proliferation rates and steadily expressed Oct-4, Nanog, and alkaline phosphatase. In addition, KhES-1 cells maintained without direct feeder contact formed embryonic bodies with expression of markers from the three germ layers. Here we demonstrated that Japanese human embryonic stem cells KhES-1 were cultured long term in a feeder-free method, while retaining pluripotency in vitro.     

人卵泡壁层颗粒细胞源性非整合诱导多能干细胞的制备

目的探索将卵泡液中壁层颗粒细胞诱导为非整合的诱导多能干细胞(iPS细胞),并检测其颗粒细胞方向的分化能力。方法在取卵的操作过程中,收集废弃的壁层颗粒细胞,在颗粒细胞培养至第4天时,加入iPS仙台病毒,经过20余天的维持,挑取iPS细胞克隆,并扩增培养。对其多能基因表达情况、外源基因整合情况、自然分化能力、颗粒细胞定向分化能力进行鉴定,并与皮肤细胞来源的iPSC系进行平行分化能力比较。结果成功建立了壁层颗粒细胞来源的iPS细胞系,其通过了类似胚胎干细胞的多能性检测及体外三胚层的分化能力检测,尤其在定向分化为颗粒细胞时,分化出了大量FOXL2、CYP19A1和FSHR阳性的细胞,经ELISA试剂盒检测,发现该分化细胞可以分泌AMH并且能够将雄激素转化成雌激素;且颗粒细胞源iPS系较皮肤细胞源的iPS系在颗粒细胞方向的分化效率更高。结论提供了一种从人颗粒细胞建立iPSC的方法,并验证了其颗粒细胞方向分化的优势。该系统不仅可以用于建立生殖不孕疾病的iPSC库,还为颗粒细胞功能障碍不孕的患者提供了一种细胞治疗的新思路。     

Comparative analysis of endoderm formation efficiency between mouse ES cells and iPS cells

Definitive endoderm (DE) derived from stem cells holds potential to differentiate into hepatocytes. Stem cell therapy using those cells has potential for a treatment of liver disease. To date, various ways of inducing hepatocytes from embryonic stem (ES) cells have been reported by researchers. However, it has not been proved enough that induced pluripotent stem (iPS) cells behave in the same manner as ES cells in endoderm differentiation. The purpose of this study was to establish an efficient method to induce DE from iPS cells, through comparatively analyzing the efficacy of endoderm formation from mouse ES cells. Furthermore, the efficiency of a serum-free medium in the differentiation into DE was investigated. Mouse ES cells and iPS cells were floated in culture medium for 2 or 5 days and embryoid bodies (EB) were formed. Subsequently, DE was induced with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF). RT-PCR and real-time PCR analyses were carried out at each step to determine the gene expression of EB markers. The difference in cellular proliferation between serum-containing and serum-free media was examined by an MTS assay in EB and DE induction. iPS cells showed the paralleled mRNA expression to ES cells in each step of differentiation into EB, but the levels of expression of Sox17 and Foxa2 were relatively higher in ES cell-derived DE, whereas Cxcr4 expression was higher in iPS cell-derived DE. The utilization of serum-free medium for iPS cells showed significantly favorable cellular proliferation during EB formation and subsequent DE induction. Forming EB for 5 days and subsequently DE induction with activin A and bFGF with serum-free medium was an appropriate protocol in iPS cells. This may represent an important step for generating hepatocytes from iPS cells for the development of cell therapy.     

诱导的多潜能干细胞类胚体分化过程中残留未分化细胞的研究

目的：探讨诱导的多潜能干细胞（induced pluri potent stem cells,iPS cells）通过类胚体长期分化后残留未分化细胞的特性。方法：小鼠iPS细胞株,体外类胚体分化20天后消化打散,重新给予i PS细胞常规培养液培养。观察扩增的残留细胞形态;流式细胞仪和免疫荧光染色检测和观察残留细胞表面标志物及体外再次分化能力。将残留细胞扩增后注射入裸鼠背部皮下,6周后注射部位取材进行大体和组织学检查。结果：分化20天的类胚体中存在残留未分化的细胞,呈克隆样生长,高度表达SSEA-1、CD-9和OCT-4等多潜能性标志。残留细胞能反复传代,并可在体外再次分化和残留。残留细胞注射部位形成畸胎瘤,瘤体组织中存在成熟的内胚层、中胚层和外胚层组织。结论：iPS细胞分化为类胚体后残留部分未分化细胞,残留细胞在体内、外可再次分化,并能在体外分化中再次残留。     

Regenerative cells for transplantation in hepatic failure

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have an enormous potential; however, their potential clinical application is being arrested due to various limitations such as teratoma formation followed by tumorigenesis, emergent usage, and the quality control of cells, as well as safety issues regarding long-term culture are also delaying their clinical application. In addition, human ES cells have two crucial issues: immunogenicity and ethical issues associated with their clinical application. The efficient generation of human iPS cells requires gene transfer, yet the mechanism underlying pluripotent stem cell induction has not yet been fully elucidated. Otherwise, although human adult regenerative cells including mesenchymal stem cells have a limited capacity for differentiation, they are nevertheless promising candidates for tissue regeneration in a clinical setting. This review highlights the use of regenerative cells for transplantation in hepatic failure.     

Hepatic differentiation of mouse iPS cells in vitro

Induced pluripotent stem (iPS) cells are pluripotent and are able to unlimitedly proliferate in vitro. This technical breakthrough in creating iPS cells from somatic cells has noteworthy implications for overcoming the immunological rejection and the ethical issues associated with the derivation of embryonic stem cells from embryos. In the current work, we present an efficient hepatic differentiation of mouse iPS cells in vitro. iPS cells were cultured free floating to induce the formation of embryoid bodies (EB) for 5 days. EB were transferred to a gelatin-coated plate and treated with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF) for 3 days to induce definitive endoderm. Cells were further cultured for 8 days with 100 ng/ml hepatocyte growth factor (HGF) to generate hepatocytes. Characterization was performed by RT-PCR assay. Functional analysis for albumin secretion and ammonia removal was also carried out. iPS cell-derived hepatocyte-like cells (iPS-Heps) were obtained at the end of the differentiation program. Expression levels of a gestational hepatocyte gene and lineage-specific hepatic genes intensified in iPS-Heps. The production of albumin increased in a time-dependent manner. iPS-Heps were capable of metabolizing ammonia. We present here instant hepatic differentiation of mouse iPS cells using combined 3-day treatments of activin A and bFGF with subsequent 8-day HGF. Our study will be an important step to generate hepatocytes from human iPS cells as a new source for liver-targeted cell therapies.     

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.     

Strategies for replacing myocytes with induced pluripotent stem in clinical protocols

Induced pluripotent stem (iPS) cells can be established via forced epigenetic reprogramming of various somatic cells and selection based on given criteria. Human iPS cells are preferred for clinical applications because they present fewer ethical concerns than embryonic stem cells. However, technical issues of stability and safety must be fully validated before clinical use. Moreover, heart regenerative therapies would require approximately 10(9) cardiomyocytes per person, necessitating the following technical considerations: (1) genetically and epigenetically stable scaling up of the iPS cells, (2) efficient and reproducible differentiation, and (3) highly reliable cell purification. Furthermore, (4) strategies for efficient iPS transplantation, rapid maturation, and functional integration of grafted cardiomyocytes into the host myocardium are critical unmet technologies. Herein, we discuss current progress and unmet problems for the realization of heart regenerative therapy.     

Kidney organogenesis and regeneration: a new era in the treatment of chronic renal failure?

The recent development of a strategy to establish human inducible pluripotent stem (iPS) cells has created a second surge in the field of regenerative research, which had been slowed by restrictions on the use of pluripotent embryonic stem cells. Research on regenerative nephrology offers hope for patients on dialysis. However, due to its anatomic complexity, the kidney is the most difficult organ for the application of regenerative medicine. Very recently, the establishment of a functional whole kidney has been attempted using various stem cells, which may lead to clinical applications. We review recent progress in the field of regenerative nephrology, focusing on the de novo establishment of a whole kidney.     

Introduction to induced pluripotent stem cells: advancing the potential for personalized medicine

Induced pluripotent stem (iPS) cell technology has enormous potential to advance medical therapy by personalizing regenerative medicine and creating novel human disease models for research and therapeutic testing. Before this technology is broadly used in the clinic, we must realistically evaluate its disease modeling and therapeutic potential. Recent advances including the use of iPS cells to successfully model spinal muscular atrophy in vitro, as well as new techniques in generating iPS cells with recombinant proteins have accelerated the prospects of iPS cells for clinical use in regenerative therapy. This review explores the development and limitations of iPS cell technology, presents a critical comparison of iPS cells and embryonic stem cells, and discusses potential clinical applications and future research directions.     

Highly efficient generation of definitive endoderm lineage from human induced pluripotent stem cells

Background

Although hepatocytes can be an option for liver transplantation, the shortage of donor organs continues to worsen. Since the development of induced pluripotent stem (iPS) cell technology, it is eagerly anticipated to produce functional elements from pluripotent stem cells. These functional cells differentiated from iPS cells could be used for transplantation, drug screening, and in vitro toxicology.

Methods

Human iPS cells are maintained on Mitomycin C-treated mouse embryonic fibroblast layers in DMEM-Ham F12-based medium supplemented with Knockout Serum Replacement, nonessential amino acids, 2-mercaptoethanol, and Glutamax. Differentiation of human iPS cells into a definitive endodermal lineage was induced with PRMI 1640 medium supplemented with B27 and 100 ng/mL human activin A. Two B27 supplements were examined with and without insulin. Furthermore, the PI3 kinase inhibitor LY294002 was used to examine the effect of inhibiting insulin signaling.

Results and Discussion

We established efficient induction of definitive endodermal differentiation from iPS cells. Quantitative analysis revealed efficient (93.03 ± 2.74%) differentiation of human iPS cells into definitive endoderm cells using B27 minus insulin. This protocol may contribute as a fundamental technique to promote human iPS studies to develop cellular sources for transplantation.     

From embryonic stem cells to induced pluripotent stem cells—Ready for clinical therapy?

Embryonic stem cells and induced pluripotent stem cells have increasingly important roles in many different fields of research and medicine. Major areas of impact include improved in vitro disease models, drug screening, and the development of cell‐based clinical therapies. Here, we review the generation and uses of embryonic stem cells compared to induced pluripotent stem cells and discuss their advantages and limitations. We also evaluate the feasibility of clinical therapies and the future prospects for induced pluripotent cell‐based treatments.     

Derivation of high-purity definitive endoderm from human parthenogenetic stem cells using an in vitro analog of the primitive streak

Human parthenogenetic stem cells (hpSCs) are pluripotent stem cells with enormous potential as cell sources for cell-based therapies: hpSCs may have histocompatibilty advantages over human embryonic stem cells (hESCs) and derivation of hpSCs does not require viable blastocyst destruction. For translation of all pluripotent stem cell-based therapies, derivation of differentiated cell products that are not contaminated with undifferentiated cells is a major technical roadblock. We report here a novel method to derive high-purity definitive endoderm (DE) from hpSCs, based on reproducing features of the normal human embryonic microenvironment. The method mimics the developmental process of transition through a primitive streak, using a differentiation device that incorporates a three-dimensional extracellular matrix (ECM) combined with a porous membrane. Treatment of undifferentiated hpSCs above the membrane results an epithelial-to-mesenchymal transition (EMT); thus, responsive cells acquire the ability to migrate through the membrane into the ECM, where they differentiate into DE. Importantly, the resultant DE is highly purified, and is not contaminated by undifferentiated cells, as assessed by OCT4 expression using immunocytochemistry and flow cytometry. The functional properties of the DE are also preserved by the process: DE differentiated in the device can generate a highly enriched population of hepatocyte-like cells (HLCs) characterized by expression of hepatic lineage markers, indocyanine green clearance, glycogen storage, cytochrome P450 activity, and engraftment in the liver after transplantation into immunodeficient mice. The method is broadly applicable and we obtained purified DE using hESCs, as well as several hpSC lines. The novel method described here represents a significant step toward the efficient generation of high-purity cells derived from DE, including hepatocytes and pancreatic endocrine cells, for use in regenerative medicine and drug discovery, as well as a platform for studying cell fate specification and behavior during development.     

Differential role of natural killer group 2D in recognition and cytotoxicity of hepatocyte‐like cells derived from embryonic stem cells and induced pluripotent stem cells

Stem cell–based approaches have the potential to address the organ shortage in transplantation. Whereas both embryonic stem cells and induced pluripotent stem cells have been utilized as cellular sources for differentiation and lineage specification, their relative ability to be recognized by immune effector cells is unclear. We determined the expression of immune recognition molecules on hepatocyte‐like cells (HLC) generated from murine embryonic stem cells and induced pluripotent stem cells, compared to adult hepatocytes, and we evaluated the impact on recognition by natural killer (NK) cells. We report that HLC lack MHC class I expression, and that embryonic stem cell–derived HLC have higher expression of the NK cell activating ligands Rae1, H60, and Mult1 than induced pluripotent stem cell–derived HLC and adult hepatocytes. Moreover, the lack of MHC class I renders embryonic stem cell–derived HLC, and induced pluripotent stem cell–derived HLC, susceptible to killing by syngeneic and allogeneic NK cells. Both embryonic stem cell–derived HLC, and induced pluripotent stem cell–derived HLC, are killed by NK cells at higher levels than adult hepatocytes. Finally, we demonstrate that the NK cell activation receptor, NKG2D, plays a key role in NK cell cytotoxicity of embryonic stem cell–derived HLC, but not induced pluripotent stem cell–derived HLC.     

人诱导性多能干细胞的无饲养层培养及鉴定

目的：探索人诱导性多能干细胞（induced pluripotent stem cells,iPSCs）的无饲养层培养方法,并对此方法培养的iPSCs进行鉴定。方法将人iPSCs接种于玻璃粘连蛋白（Vitronectin XF）包被的培养皿上培养,采用EDTA消化传代。倒置显微镜下观察iPSCs的生长状态;碱性磷酸酶（ALP）染色鉴定;采用PCR和免疫荧光检测iPSCs多能性基因SSEA?1、Nanog、Sox2的表达情况。结果倒置显微镜下可见iPSCs呈典型的克隆状生长,克隆呈圆形或椭圆形,边界清晰、整齐;ALP染色结果阳性;PCR结果显示人iPSCs强表达多能性基因SSEA?1、Nanog、Sox2;免疫荧光结果显示多能干细胞特异性指标SSEA?1、Nanog、Sox2均呈阳性。结论无饲养层培养体系培养人iPSCs,细胞能稳定增殖,保持自我更新潜能及多能性。     

Docosahexaenoic acid promotes dopaminergic differentiation in induced pluripotent stem cells and inhibits teratoma formation in rats with Parkinson-like pathology

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic (DA) neurons in the midbrain. Induced pluripotent stem (iPS) cells have shown potential for differentiation and may become a resource of functional neurons for the treatment of PD. However, teratoma formation is a major concern for transplantation-based therapies. This study examined whether functional neurons could be efficiently generated from iPS cells using a five-step induction procedure combined with docosahexaenoic acid (DHA) treatment. We demonstrated that DHA, a ligand for the RXR/Nurr1 heterodimer, significantly activated expression of the Nurr1 gene and the Nurr1-related pathway in iPS cells. DHA treatment facilitated iPS differentiation into tyrosine hydroxylase (TH)-positive neurons in vitro and in vivo and functionally increased dopamine release in transplanted grafts in PD-like animals. Furthermore, DHA dramatically upregulated the endogenous expression levels of neuroprotective genes (Bcl-2, Bcl-xl, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor) and protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis in iPS-derived neuronal precursor cells. DHA-treated iPS cells significantly improved the behavior of 6-hydroxydopamine (6-OHDA)-treated PD-like rats compared to control or eicosapentaenoic acid-treated group. Importantly, the in vivo experiment suggests that DHA induces the differentiation of functional dopaminergic precursors and improves the abnormal behavior of 6-OHDA-treated PD-like rats by 4 months after transplantation. Furthermore, we found that DHA treatment in iPS cell-grafted rats significantly downregulated the mRNA expression of embryonic stem cell-specific genes (Oct-4 and c-Myc) in the graft and effectively blocked teratoma formation. Importantly, 3 Tesla-magnetic resonance imaging and ex vivo green fluorescence protein imaging revealed that no teratomas were present in transplanted grafts of DHA-treated iPS-derived DA neurons 4 months after implantation. Therefore, our data suggest that DHA plays a crucial role in iPS differentiation into functional DA neurons and that this approach could provide a novel therapeutic approach for PD treatment.