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.     

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.     

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.     

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.     

A molecular view on pluripotent stem cells

Pluripotent stem cells are undifferentiated cells that are capable of differentiating to all three embryonic germ layers and their differentiated derivatives. They are transiently found during embryogenesis, in preimplantation embryos and fetal gonads, or as established cell lines. These unique cell types are distinguished by their wide developmental potential and by their ability to be propagated in culture indefinitely, without loosing their undifferentiated phenotype. This short review intends to give a general overview on the pluripotent nature of embryo-derived stem cells with a focus on human embryonic stem cells.     

Generation of Induced Pluripotent Stem Cell Lines from Tibetan Miniature Pig

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model.     

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     

Making gametes from alternate sources of stem cells: past,present and future

Infertile couples including cancer survivors stand to benefit from gametes differentiated from embryonic or induced pluripotent stem (ES/iPS) cells. It remains challenging to convert human ES/iPS cells into primordial germ-like cells (PGCLCs) en route to obtaining gametes. Considerable success was achieved in 2016 to obtain fertile offspring starting with mouse ES/iPS cells, however the specification of human ES/iPS cells into PGCLCs in vitro is still not achieved. Human ES cells will not yield patient-specific gametes unless and until hES cells are derived by somatic cell nuclear transfer (therapeutic cloning) whereas iPS cells retain the residual epigenetic memory of the somatic cells from which they are derived and also harbor genomic and mitochondrial DNA mutations. Thus, they may not be ideal starting material to produce autologus gametes, especially for aged couples. Pluripotent, very small embryonic-like stem cells (VSELs) have been reported in adult tissues including gonads, are relatively quiescent in nature, survive oncotherapy and can be detected in aged, non-functional gonads. Being developmentally equivalent to PGCs (natural precursors to gametes), VSELs spontaneously differentiate into gametes in vitro. It is also being understood that gonadal stem cells niche is compromised by oncotherapy and with age. Improving the gonadal somatic niche could regenerate non-functional gonads from endogenous VSELs to restore fertility. Niche cells (Sertoli/mesenchymal cells) can be directly transplanted and restore gonadal function by providing paracrine support to endogenous VSELs. This strategy has been successful in several mice studies already and resulted in live birth in a woman with pre-mature ovarian failure.     

胚胎干细胞定向分化为心肌细胞研究进展

胚胎干细胞在体外可分化为 3个胚层的所有组织细胞。诱导人类胚胎干细胞定向分化为心肌细胞可为心肌梗死、心肌坏死等重大心脏疾病患者实施细胞治疗 ,也可作为种子细胞 ,用于构建供器官移植用的人造心脏 ;进一步可研究心肌细胞发育分化的分子机理及更直观的用于体外筛选人类心血管药物等。对人类胚胎干细胞及其定向分化为心肌细胞分子机理的研究进展及其所面临的问题作一综述。     

Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.     

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.     

多能干细胞诱导分化及体细胞转分化为心肌细胞的研究进展

心血管疾病是威胁人类健康的重大疾病,而心肌细胞数量逐渐减少,甚至衰竭是其核心病变。心肌细胞补偿性替代治疗是未来用于治疗这类疾病的重要手段,因此,心肌细胞的来源和有效治疗将成为关键。目前,心肌细胞构建的主要方法有多能干细胞诱导分化成心肌祖细胞或心肌细胞、心源性心肌祖细胞,以及体细胞重编程等。其中,多能干细胞向心肌细胞分化是最常用的方法;而体细胞转分化技术相较于传统的诱导多潜能干细胞衍生心肌细胞缩短了时间窗,为潜在的心血管疾病治疗提供了另一种思路。随着获取心肌细胞效率及其质量的提升,未来心血管疾病的治疗将有望获得重大突破。     

Teratoma generation in the testis capsule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines(1,2,3). Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells(4). With the generation of human ESC lines(5,6), the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)(7,8,9). Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard(7). In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers(10). In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly(11). Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth.     

In vitro strategies for human gametes production from stem cells

Embryonic stem cells (ESC) are self-renewal and pluripotent cells that are able to differentiate in vitro into several cell types in favourable conditions. Technical protocols for in vitro gametes production have been developed in mice and human species. The functionality of such differentiated cells is not always analysed and an early meiotic arrest is a current observation. These kinds of experimentations have also been tested from human induced pluripotent stem cells (IPSC). However, differentiation ends shortly at the primordial germ cell stage.