Generation of functional endothelial-like cells from adult mouse germline-derived pluripotent stem cells

Functional endothelial cells and their progenitors are required for vascular development, adequate vascular function, vascular repair and for cell-based therapies of ischemic diseases. Currently, cell therapy is limited by the low abundance of patient-derived cells and by the functional impairment of autologous endothelial progenitor cells (EPCs). In the present study, murine germline-derived pluripotent stem (gPS) cells were evaluated as a potential source for functional endothelial-like cells.     

Epigenetic alteration of imprinted genes during neural differentiation of germline-derived pluripotent stem cells

Spermatogonial stem cells (SSCs), which are unipotent stem cells in the testes that give rise to sperm, can be converted into germline-derived pluripotent stem (gPS) by self-induction. The androgenetic imprinting pattern of SSCs is maintained even after their reprogramming into gPS cells. In this study, we used an in vitro neural differentiation model to investigate whether the imprinting patterns are maintained or altered during differentiation. The androgenetic patterns of H19, Snrpn, and Mest were maintained even after differentiation of gPS cells into NSCs (gPS-NSCs), whereas the fully unmethylated status of Ndn in SSCs was altered to somatic patterns in gPS cells and gPS-NSCs. Thus, our study demonstrates epigenetic alteration of genomic imprinting during the induction of pluripotency in SSCs and neural differentiation, suggesting that gPS-NSCs can be a useful model to study the roles of imprinted genes in brain development and human neurodevelopmental disorders.     

Coloring skin with pluripotent stem cells

Comment on: Nissan X, et al. Proc Natl Acad Sci USA 2011; 108:14861-6.     

In vitro organogenesis from pluripotent stem cells

Pluripotent stem cells (PSCs) have the ability to spontaneously generate structured tissues in vitro reminiscent of embryonic tissue development. Recently, complex organoids such as cortical tissues, cerebral brain organoids, optical cups, intestinal tissues, and liver buds have been generated from PSCs derived from healthy individuals and patients with genetic diseases, providing powerful tools to understand morphogenesis and disease pathology. This article highlights recent advances in the state-of-art generation of organoids from PSCs, possible signaling pathways and mechanisms involved in organogenesis, and the understanding of extracellular microenvironment. Challenges involved in the organoid generation such as increasing organoid size, enhancing the tissue complexity, and improving functional maturation are also discussed.     

In vitro organogenesis from pluripotent stem cells

Pluripotent stem cells (PSCs) have the ability to spontaneously generate structured tissues in vitro reminiscent of embryonic tissue development. Recently, complex organoids such as cortical tissues, cerebral brain organoids, optical cups, intestinal tissues, and liver buds have been generated from PSCs derived from healthy individuals and patients with genetic diseases, providing powerful tools to understand morphogenesis and disease pathology. This article highlights recent advances in the state-of-art generation of organoids from PSCs, possible signaling pathways and mechanisms involved in organogenesis, and the understanding of extracellular microenvironment. Challenges involved in the organoid generation such as increasing organoid size, enhancing the tissue complexity, and improving functional maturation are also discussed.     

Dissecting nephron morphogenesis using kidney organoids from human pluripotent stem cells

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Generating human intestinal tissue from pluripotent stem cells in vitro

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1-3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro.     

Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. For the application of iPSCs to forms of autologous cell therapy, suitable animal models are required. Among species that could potentially be used for this purpose, nonhuman primates are particularly important, and among these the marmoset offers significant advantages. In order to demonstrate the feasibility of the application of iPSC technology to this species, here we derived lines of marmoset iPSCs. Using retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc, we derived clones that fulfil critical criteria for successful reprogramming: they exhibit typical iPSC morphology; they are alkaline phosphatase positive; they express high levels of NANOG, OCT4 and SOX2 mRNAs, while the corresponding vector genes are silenced; they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice they produce teratomas that have derivatives of all three germ layers (endoderm, α-fetoprotein; ectoderm, βIII-tubulin; mesoderm, smooth muscle actin). Starting with a population of 4 × 10⁵ newborn marmoset skin fibroblasts, we obtained ～ 100 colonies with iPSC-like morphology. Of these, 30 were expanded sufficiently to be cryopreserved, and, of those, 8 were characterized in more detail. These experiments provide proof of principle that iPSC technology can be adapted for use in the marmoset, as a future model of autologous cell therapy.     

Avian pluripotent stem cells

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.     

Induced pluripotent stem cells

Induced pluripotent stem cells (iPS) result from a reprogramming of somatic cells via transduction with viral vectors expressing the Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28 genes, which are essential for the establishment and maintenance of the pluripotent state. In properties, iPS are almost fully similar to embryonic stem cells (ESC). To date, iPS have been obtained from various differentiated cells of mice and humans. Along with ESC, iPSs are highly promising for research and medicine.     

MicroRNAs与多潜能干细胞的研究进展

microRNAs(miRNAs)是一类内源性非编码小RNA,通过调控基因表达来参与生命过程中的一系列重要进程。越来越多的证据表明,miRNAs参与了几乎所有生物代谢过程,其胚胎干细胞的自我更新与分化和在多能干细胞(iPSCs)中的诱导调节作用也日益受到关注。该文介绍了miRNAs的生成、检测方法以及miRNAs对胚胎干细胞(ESCs)及诱导多能性干细胞的调控作用,并对miRNAs的应用前景进行了展望。     

体细胞诱导为多能干细胞的最新进展

2007年11-12月,Cell、Science和Nature发表一系列体外诱导人类体细胞转变为多能干细胞的论文。来自日本和美国的研究小组利用慢病毒载体分别将Oct-4、Sox2、C-Myc、Klf4和Oct-4、Sox2、Nanog、Lin28两套基因转入人成纤维细胞,均获得类似ES细胞的克隆。小鼠诱导性多能干细胞已初步用于镰刀细胞性贫血的基因治疗。短短一年半,诱导性多能干细胞的研究和关注度呈现了爆炸式成长;体细胞重编程、去分化、多能干细胞来源等一系列热点问题再次成为大众瞩目的中心。     

Epithelial stem cells

New molecular markers for epidermal stem cells have enabled their isolation both in vitro and from the epidermis lying between hair follicles. Micro-dissection experiments have localised a second population of stem cells within hair follicles. Epidermal stem cells have a patterned distribution in vivo. The patterning can be reconstituted in vitro, showing that it is generated by interactions between keratinocytes and that the differentiation of epidermal stem cells is regulated by signals from other keratinocytes. Recent evidence from transgenic mice suggests that stem cell behaviour in the gut may be regulated by similar cell-cell interactions in vivo. Candidate genes for mediating these interactions are the homologues of Drosophila cell fate patterning genes such as Notch and Wingless and the Cadherin family of cell-cell adhesion molecules. The roles of stem cells and of mutations of the Patched gene in epithelial carcinogenesis are discussed.     

多能干细胞体外分化为类神经管模型的研究进展

近年来多能干细胞(PSCs)的体外培养与分化技术发展迅速,并广泛应用于再生医学和发育生物学等领域。PSCs能够在体外神经诱导的条件下分化为类神经管模型,这为探索体内早期神经发育与中枢神经系统发育疾病的形成机制提供了全新的实验平台。本文总结了近年来应用小鼠和人PSCs建立体外类神经管模型的研究进展,其中体外模型主要包括在不同培养体系下诱导获得的二维(2D)与三维(3D)类神经管模型,并针对早期类神经管模型在神经系统发育性疾病机制研究中的前景和挑战作进一步探讨,同时为疾病预防和治疗提供新的思路。     

Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.     

Ovarian pluripotent/multipotent stem cells and in vitro oogenesis in mammals

There has been a long persisting dilemma about potential ovarian stem cells in adult mammalian ovaries, including human, and now there is steadily increasing experimental evidence on their existence. After some previous indirect evidence about the presence of stem cells in adult mouse ovaries, an important breakthrough was made by Zou and his co-workers who successfully established long-persisting pluripotent/multipotent ovarian stem cell lines in neonatal and adult mice, and were followed by some other important studies in mouse and human. Moreover, oocyte-like cells can be developed in vitro from pluripotent stem cells of different origins (embryonic stem cells, induced pluripotent stem cells, fetal skin stem cells, pancreatic stem cells). The aim of this article is to elucidate the fast growing new knowledge on the ovarian stem cells and potential in vitro oogenesis in mammals.