Potential applications of germline cell-derived pluripotent stem cells in organ regeneration

Impressive progress has been made since the turn of the century in the field of stem cells. Different types of stem cells have now been isolated from different types of tissues. Pluripotent stem cells are the most promising cell source for organ regeneration. One such cell type is the germline cell-derived pluripotent cell, which is derived from adult spermatogonial stem cells. The germline cell-derived pluripotent stem cells have been obtained from both human and mouse and, importantly, are adult stem cells with embryonic stem cell-like properties that do not require specific manipulations for pluripotency acquisition, hence bypassing problems related to induced pluripotent stem cells and embryonic stem cells. The germline cell-derived pluripotent stem cells have been induced to differentiate into cells deriving from the three germ layers and shown to be functional in vitro. This review will discuss the plasticity of the germline cell-derived pluripotent stem cells and their potential applications in human organ regeneration, with special emphasis on liver regeneration. Potential problems related to their use are also highlighted.     

Generation of rabbit pluripotent stem cell lines

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.     

Physical passaging of embryoid bodies generated from human pluripotent stem cells

Spherical three-dimensional cell aggregates called embryoid bodies (EBs), have been widely used in in vitro differentiation protocols for human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Recent studies highlight the new devices and techniques for hEB formation and expansion, but are not involved in the passaging or subculture process. Here, we provide evidence that a simple periodic passaging markedly improved hEB culture condition and thus allowed the size-controlled, mass production of human embryoid bodies (hEBs) derived from both hESCs and hiPSCs. hEBs maintained in prolonged suspension culture without passaging (>2 weeks) showed a progressive decrease in the cell growth and proliferation and increase in the apoptosis compared to 7-day-old hEBs. However, when serially passaged in suspension, hEB cell populations were significantly increased in number while maintaining the normal rates of cell proliferation and apoptosis and the differentiation potential. Uniform-sized hEBs produced by manual passaging using a 1∶4 split ratio have been successfully maintained for over 20 continuous passages. The passaging culture method of hEBs, which is simple, readily expandable, and reproducible, could be a powerful tool for improving a robust and scalable in vitro differentiation system of human pluripotent stem cells.     

Human pluripotent stem cells: a progress report

The derivation of diploid human pluripotent stem cell lines from either human blastocysts or embryonic gonads in 1998 attracted a great deal of interest because of the widespread potential applications of these cells in research and in regenerative medicine. Since the initial reports, there has been some progress in the characterisation of blastocyst-derived stem cells, and some technical advances in their manipulation. Conditions for differentiation in vitro of pluripotent stem cells from either blastocysts or gonads have been defined. In some studies, committed progenitor cell populations have been isolated from mixed cultures of differentiating ES cells.     

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.     

Clonal expansion of human pluripotent stem cells on gelatin-coated surface

The research of human pluripotent stem cells is important for providing the molecular basis for their future application to regenerative medicine. To date, they are usually cultured on feeder cells and passaged by partial dissociation with either enzymatic or mechanical methods, which are problematic for the research using them in the convenience and reproducibility. Here we established a new culture system that allows handling as easily as culturing feeder-free mouse ES cells. This newly developed culture system is based on the combinatorial use of ROCK inhibitor and soluble fibronectin, which enables us to expand human pluripotent stem cells from single cell dissociation on gelatin-coated surface without any feeder cells. In this new culture system, these human pluripotent stem cells can stably grow, even if in clonal density with keeping expression of stem cell markers. These cells also have abilities to differentiate into three germ layers in vivo and in vitro. Furthermore, no chromosomal abnormalities are found even after sequential passage. Therefore this system will dramatically simplify genetic engineering of these human pluripotent stem cells or defining process of their signal pathway.     

Somatic cell reprogramming for regenerative medicine: SCNT vs. iPS cells

Reprogramming of somatic cells to a pluripotent state holds huge potentials for regenerative medicine. However, a debate over which method is better, somatic cell nuclear transfer (SCNT) or induced pluripotent stem (iPS) cells, still persists. Both approaches have the potential to generate patient-specific pluripotent stem cells for replacement therapy. Yet, although SCNT has been successfully applied in various vertebrates, no human pluripotent stem cells have been generated by SCNT due to technical, legal and ethical difficulties. On the other hand, human iPS cell lines have been reported from both healthy and diseased individuals. A recent study reported the generation of triploid human pluripotent stem cells by transferring somatic nuclei into oocytes, a variant form of SCNT. In this essay, we discuss this progress and the potentials of these two reprogramming approaches for regenerative medicine.     

Tracking the progression of the human inner cell mass during embryonic stem cell derivation

The different pluripotent states of mouse embryonic stem cells (ESCs) in vitro have been shown to correspond to stages of mouse embryonic development. For human cells, little is known about the events that precede the generation of ESCs or whether they correlate with in vivo developmental stages. Here we investigate the cellular and molecular changes that occur during the transition from the human inner cell mass (ICM) to ESCs in vitro. We demonstrate that human ESCs originate from a post-ICM intermediate (PICMI), a transient epiblast-like structure that has undergone X-inactivation in female cells and is both necessary and sufficient for ESC derivation. The PICMI is the result of progressive and defined ICM organization in vitro and has a distinct state of cell signaling. The PICMI can be cryopreserved without compromising ESC derivation capacity. As a closer progenitor of ESCs than the ICM, the PICMI provides insight into the pluripotent state of human stem cells.     

High-efficient generation of induced pluripotent stem cells from human astrocytes

The reprogramming of human somatic cells to induced pluripotent stem (hiPS) cells enables the possibility of generating patient-specific autologous cells for regenerative medicine. A number of human somatic cell types have been reported to generate hiPS cells, including fibroblasts, keratinocytes and peripheral blood cells, with variable reprogramming efficiencies and kinetics. Here, we show that human astrocytes can also be reprogrammed into hiPS (ASThiPS) cells, with similar efficiencies to keratinocytes, which are currently reported to have one of the highest somatic reprogramming efficiencies. ASThiPS lines were indistinguishable from human embryonic stem (ES) cells based on the expression of pluripotent markers and the ability to differentiate into the three embryonic germ layers in vitro by embryoid body generation and in vivo by teratoma formation after injection into immunodeficient mice. Our data demonstrates that a human differentiated neural cell type can be reprogrammed to pluripotency and is consistent with the universality of the somatic reprogramming procedure.     

X chromosome inactivation in human and mouse pluripotent stem cells

Since the groundbreaking hypothesis of X chromosome inactivation (XCI) proposed by Mary Lyon over 50 years ago, a great amount of knowledge has been gained regarding this essential dosage compensation mechanism in female cells. For the mammalian system, most of the mechanistic studies of XCI have so far been investigated in the mouse model system, but recently, a number of interesting XCI studies have been extended to human pluripotent stem cells, including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Emerging data indicate that XCI in hESCs and hiPSCs is much more complicated than that of their mouse counterparts. XCI in human pluripotent stem cells is not as stable and is subject to environmental influences and epigenetic regulation in vitro. This mini-review highlights the key differences in XCI between mouse and human stem cells with a greater emphasis placed on the understanding of the epigenetic regulation of XCI in human stem cells.     

Progenitor Cells for Regenerative Medicine and Consequences of ART and Cloning-Associated Epimutations

The “holy grail” of regenerative medicine is the identification of an undifferentiated progenitor cell that is pluripotent, patient specific, and ethically unambiguous. Such a progenitor cell must also be able to differentiate into functional, transplantable tissue, while avoiding the risks of immune rejection. With reports detailing aberrant genomic imprinting associated with assisted reproductive technologies (ART) and reproductive cloning, the idea that human embryonic stem cells (hESCs) derived from surplus in vitro fertilized embryos or nuclear transfer ESCs (ntESCs) harvested from cloned embryos may harbor dangerous epigenetic errors has gained attention. Various progenitor cell sources have been proposed for human therapy, from hESCs to ntESCs, and from adult stem cells to induced pluripotent stem cells (iPS and piPS cells). This review highlights the advantages and disadvantages of each of these technologies, with particular emphasis on epigenetic stability.     

Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral‐nerve injury: An advantageous alternative to ES and iPS cells

The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral‐, plasmid‐ or transposon‐mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection. To address these fundamental problems, our group is developing hair follicle pluripotent stem (hfPS) cells. Our previous studies have shown that mouse hfPS cells can differentiate to neurons, glial cells in vitro, and other cell types, and can promote nerve and spinal cord regeneration in vivo. hfPS cells are located above the hair follicle bulge in what we have termed the hfPS cell area (hfPSA) and are nestin positive and keratin 15 (K‐15) negative. Human hfPS cells can also differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In the present study, human hfPS cells were transplanted in the severed sciatic nerve of the mouse where they differentiated into glial fibrillary‐acidic‐protein (GFAP)‐positive Schwann cells and promoted the recovery of pre‐existing axons, leading to nerve generation. The regenerated nerve recovered function and, upon electrical stimulation, contracted the gastrocnemius muscle. The hfPS cells can be readily isolated from the human scalp, thereby providing an accessible, autologous and safe source of stem cells for regenerative medicine that have important advantages over ES or iPS cells. J. Cell. Biochem. 107: 1016–1020, 2009. © 2009 Wiley‐Liss, Inc.     

Liver engraftment potential of hepatic cells derived from patient-specific induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) are potential renewable sources of hepatocytes for drug development and cell therapy. Differentiation of human iPSCs into different developmental stages of hepatic cells has been achieved and improved during the last several years. We have recently demonstrated the liver engraftment and regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo. Here we describe the in vitro and in vivo activities of hepatic cells derived from patientspecific iPSCs, including multiple lines established from either inherited or acquired liver diseases, and discuss basic and clinical applications of these cells for disease modeling, drug screening and discovery, gene therapy and cell replacement therapy.Key words: induced pluripotent stem cells (iPSCs), hepatic differentiation, liver ngraftment, disease modeling, drug testing, alpha-1 antitrypsin, liver cirrhosis, hepatocellular carcinoma, cell therapy     

Safeguarding clinical translation of pluripotent stem cells with suicide genes

The generation of human induced pluripotent stem cells (hiPSCs) opens a new avenue in regenerative medicine. However, transplantation of hiPSC-derived cells carries a risk of tumor formation by residual pluripotent stem cells. Numerous adaptive strategies have been developed to prevent or minimize adverse events and control the in vivo behavior of transplanted stem cells and their progeny. Among them, the application of suicide gene modifications, which is conceptually similar to cancer gene therapy, is considered an ideal means to control wayward stem cell progeny in vivo. In this review, the choices of vectors, promoters, and genes for use in suicide gene approaches for improving the safety of hiPSCs-based cell therapy are introduced and possible new strategies for improvements are discussed. Safety-enhancing strategies that can selectively ablate undifferentiated cells without inducing virus infection or insertional mutations may greatly aid in translating human pluripotent stem cells into cell therapies in the future.     

诱导型多能干细胞iPSc研究现状及其移植治疗帕金森综合症应用前景

诱导多能干细胞(i PS细胞)在小鼠和人上的成功获取,使干细胞领域的研究进入了一个崭新的时代。干细胞研究是再生医学的重要组成部分,研究干细胞的最终目的是应用干细胞治疗疾病,其在疾病模型建立、药物筛选、细胞移植等方面具有极大的应用潜力。i PSCs是由体细胞诱导分化而成的"多能细胞",具备和胚胎干细胞类似的功能,既解决了ESCs的伦理障碍,又为ESCs的获得提供了一条全新的途径,具有重要的理论和应用价值。i PS细胞不仅打破了道德理论的束缚,而且在再生医学、组织工程和药物发现及评价等方面具有积极的价值。神经系统遗传性疾病发病率居各系统遗传病之首,但其发病的分子机制仍不完全清楚,运用体细胞重编程技术建立的疾病特异性诱导多能干细胞模型将有助于揭示神经系统遗传性疾病的发病机理。近几年i PS细胞最新研究成果表明,利用疾病患者i PS细胞模型已逐渐应用于帕金森氏病、老年性痴呆症、脊髓侧索硬化症、脊髓肌肉萎缩症及舞蹈症等5种常见神经性退行性疾病发病机理的研究。本文主要对i PSc的发展历程,避免病毒基因干扰诱导i PS细胞进行的优化,以及干细胞尤其是i PS细胞移植治疗帕金森病等神经系统疾病的现状及应用前景进行系统阐述与论证。