Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations.     

Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells

Pluripotency of embryonic stem cells (ESCs) is maintained by the balancing of several signaling pathways, such as Wnt, BMP, and FGF, and differentiation of ESCs into a specific lineage is induced by the disruption of this balance. Sulfated glycans are considered to play important roles in lineage choice of ESC differentiation by regulating several signalings. We examined whether reduction of sulfation by treatment with the chemical inhibitor chlorate can affect differentiation of ESCs. Chlorate treatment inhibited mesodermal differentiation of mouse ESCs, and then induced ectodermal differentiation and accelerated further neural differentiation. This could be explained by the finding that several signaling pathways involved in the induction of mesodermal differentiation (Wnt, BMP, and FGF) or inhibition of neural differentiation (Wnt and BMP) were inhibited in chlorate-treated embryoid bodies, presumably due to reduced sulfation on heparan sulfate and chondroitin sulfate. Furthermore, neural differentiation of human induced pluripotent stem cells (hiPSCs) was also accelerated by chlorate treatment. We propose that chlorate could be used to induce efficient neural differentiation of hiPSCs instead of specific signaling inhibitors, such as Noggin.     

Biobanking of human pluripotent stem cells in China

In recent years, significant progress has been made internationally in the development of human pluripotent stem cell (hPSC)‐derived products for serious and widespread disorders. Biobanking of the cellular starting materials is a crucial component in the delivery of safe and regulatory compliant cell therapies. In China, key players in these developments have been the recently launched National Stem Cell Resource Center (NSCRC) and its partner organizations in Guangzhou and Shanghai who together, have more than 600 hPSC lines formally recorded in the Chinese Ministry of Science and Technology''s stem cell registry. In addition, 47 of these hPSCs have also been registered with the hPSCreg project which means they are independently certified for use in European Commission funded research projects. The NSCRC are currently using their own cell lines to manufacture eight different cell types qualified for clinical use, that are being used in nine clinical studies for different indications. The Institute of Zoology at the Chinese Academy of Sciences (IOZ‐CAS) has worked with NSCRC to establish Chinese and international standards in stem cell research. IOZ‐CAS was also a founding partner in the International Stem Cell Banking Initiative which brings together key stem cell banks to agree minimum standards for the provision of pluripotent stem cells for research and clinical use. Here, we describe recent developments in China in the establishment of hPSCs for use in the manufacture of cell therapies and the significant national and international coordination which has now been established to promote the translation of Chinese hPSC‐based products into clinical use according to national and international standards.     

线粒体与多潜能干细胞功能

线粒体是细胞内重要的细胞器,主要功能是通过氧化磷酸化为细胞生命活动提供能量。近年来,研究表明,在多潜能干细胞（Pluripotent stem cells, PSCs）中线粒体表现出独有的特征,即在多能性状态下,PSCs主要依靠糖酵解提供能量,其分化期间线粒体氧化磷酸化代谢能力逐渐增强。相反,体细胞重编程为多潜能干细胞期间,线粒体氧化磷酸化向糖酵解途径的转变是其成功重编程必需的代谢过程。另外,线粒体通过生物合成和形态结构的动态重塑维持了PSCs多能性、诱导分化及诱导多能干细胞（Induced pluripotent stem cells, iPSCs）的重编程。因此,本文综述了PSCs线粒体形态结构及其在调控PSCs多能性、合成代谢、氧化还原状态的平衡、分化及重新编程中的作用,为深入了解线粒体调控PSCs功能的作用提供理论基础。     

Generation of skeletal muscle cells from embryonic and induced pluripotent stem cells as an in vitro model and for therapy of muscular dystrophies

Muscular dystrophies (MDs) are a heterogeneous group of inherited disorders characterized by progressive muscle wasting and weakness likely associated with exhaustion of muscle regeneration potential. At present, no cures or efficacious treatments are available for these diseases, but cell transplantation could be a potential therapeutic strategy. Transplantation of myoblasts using satellite cells or other myogenic cell populations has been attempted to promote muscle regeneration, based on the hypothesis that the donor cells repopulate the muscle and contribute to its regeneration. Embryonic stem cells (ESCs) and more recently induced pluripotent stem cells (iPSCs) could generate an unlimited source of differentiated cell types, including myogenic cells. Here we review the literature regarding the generation of myogenic cells considering the main techniques employed to date to elicit efficient differentiation of human and murine ESCs or iPSCs into skeletal muscle. We also critically analyse the possibility of using these cellular populations as an alternative source of myogenic cells for cell therapy of MDs.     

Application of induced pluripotent stem cell and embryonic stem cell technology to the study of male infertility

Stem cells (SCs) are classes of undifferentiated biological cells existing only at the embryonic, fetal, and adult stages that can divide to produce specialized cell types during fetal development and remain in our bodies throughout life. The progression of regenerative and reproductive medicine owes the advancement of respective in vitro and in vivo biological science on the stem cell nature under appropriate conditions. The SCs are promising therapeutic tools to treat currently of infertility because of wide sources and high potency to differentiate. Nevertheless, no effective remedies are available to deal with severe infertility due to congenital or gonadotoxic stem cell deficiency in prepubertal childhood. Some recent solutions have been developed to address the severe fertility problems, including in vitro formation of germ cells from stem cells, induction of pluripotency from somatic cells, and production of patient‐specific pluripotent stem cells. There is a possibility of fertility restoration using the in vitro formation of germ cells from somatic cells. Accordingly, the present review aimed at studying the literature published on the medical application of stem cells in reproductive concerns.     

The cell cycle inhibitor p27Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle,Brachyury and Twist

The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27^Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27^Kip1 in hESC lead to a G₁ phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27^Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27^Kip1 protein occupies the Twist1 gene promoter and manipulation of p27^Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27^Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27^Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC.     

FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells

Human embryonic stem cell (hESC) lines are traditionally derived and maintained on mouse embryonic fibroblasts (MEF) which are xenogeneic and enter senescence rapidly. In view of the clinical implications of hESCs, the use of human fibroblast as feeders has been suggested as a plausible alternative. However, use of fibroblast cells from varying sources leads to culture variations along with the need to add FGF2 in cultures to sustain ES cell pluripotency. In this study we report the derivation of FGF2 expressing germ layer derived fibroblast cells (GLDF) from hESC lines. These feeders could support the pluripotency, karyotypes and proliferation of hESCs with or without FGF2 in prolonged cultures as efficiently as that on MEF. GLDF cells were derived from embryoid bodies and characterized for expression of fibroblast markers by RT-PCR, Immunofluorescence and by flow cytometry for CD marker expression. The expression and secretion of FGF2 was confirmed by RT-PCR, Western blot, and ELISA. The hESC lines cultured on MEF and GLDF were analyzed for various stemness markers. These feeder cells with fibroblast cells like properties maintained the properties of hESCs in prolonged culture over 30 passages. Proliferation and pluripotency of hESCs on GLDF was comparable to that on mouse feeders. Further we discovered that these GLDF cells could secrete FGF2 and maintained pluripotency of hESC cultures even in the absence of supplemental FGF2. To our knowledge, this is the first study reporting a novel hESC culture system which does not warrant FGF2 supplementation, thereby reducing the cost of hESC cultures.     

The Janus face of pluripotent stem cells--connection between pluripotency and tumourigenicity

Pluripotent stem cells have gained special attraction because of their almost unlimited proliferation and differentiation capacity in vitro. These properties substantiate the potential of pluripotent stem cells in basic research and regenerative medicine. Here three types of in vitro cultured pluripotent stem cells (embryonic carcinoma, embryonic stem and induced pluripotent stem cells) are compared in their historical context with respect to their different origin and properties. It became evident that tumourigenicity is an inherent property of pluripotent cells based on p53 down-regulation, expression of tumour-related genes and high telomerase activity that allow unlimited proliferation. In addition, culture-adapted genetic and epigenetic changes may induce tumourigenicity of pluripotent cells. The use of stem cells in regenerative medicine, however, requires non-malignant cell types and strategies that circumvent stages of malignancy.Reprogramming strategies of adult somatic cells that avoid the tumourigenic state of pluripotency may offer alternatives for future biomedical application.     

The cell cycle inhibitor p27Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle,Brachyury and Twist

The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27^Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27^Kip1 in hESC lead to a G₁ phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27^Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27^Kip1 protein occupies the Twist1 gene promoter and manipulation of p27^Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27^Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27^Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC.     

Expression of the 49 human ATP binding cassette (ABC) genes in pluripotent embryonic stem cells and in early- and late-stage multipotent mesenchymal stem cells

The 49-member human ATP binding cassette (ABC) gene family encodes 44 membrane transporters for lipids, ions, peptides or xenobiotics, four translation factors without transport activity, as they lack transmembrane domains, and one pseudogene. To understand the roles of ABC genes in pluripotency and multipotency, we performed a sensitive qRT-PCR analysis of their expression in embryonic stem cells (hESCs), bone marrow-derived mesenchymal stem cells (hMSCs) and hESC-derived hMSCs (hES-MSCs). We confirm that hES-MSCs represent an intermediate developmental stage between hESCs and hMSCs. We observed that 44 ABCs were significantly expressed in hESCs, 37 in hES-MSCs and 35 in hMSCs. These variations are mainly due to plasma membrane transporters with low but significant gene expression: 18 are expressed in hESCs compared with 16 in hES-MSCs and 8 in hMSCs, suggesting important roles in pluripotency. Several of these ABCs shared similar substrates but differ regarding gene regulation. ABCA13 and ABCB4, similarly to ABCB1, could be new markers to select primitive hMSCs with specific plasma membrane transporter^low phenotypes. ABC proteins performing basal intracellular functions, including translation factors and mitochondrial heme transporters, showed the highest constant gene expression among the three populations. Peptide transporters in the endoplasmic reticulum, Golgi and lysosome were well expressed in hESCs and slightly upregulated in hMSCs, which play important roles during the development of stem cell niches in bone marrow or meningeal tissue. These results will be useful to study specific cell cycle regulation of pluripotent stem cells or ABC dysregulation in complex pathologies, such as cancers or neurological disorders.     

诱导多能干细胞研究进展

人类诱导多能干细胞（induced pluripotent stem cells,iPS细胞）的建立被公认为目前最重要的科技进展之一。iPS细胞在动物疾病模型上的成功治疗,病患特异性iPS细胞的研究及iPS细胞的定向分化研究将有可能使人们避开治疗性克隆的伦理和技术障碍,给人类疾病的干细胞治疗带来光明的前景。本文从iPS细胞的诱导策略和方法,来源细胞及筛选、重编程机制的研究现状、应用前景以及研究中存在的问题等方面对其作一综述和讨论。     

Immunological considerations for embryonic and induced pluripotent stem cell banking

Recent advances in stem cell technology have generated enthusiasm for their potential to study and treat a diverse range of human disease. Pluripotent human stem cells for therapeutic use may, in principle, be obtained from two sources: embryonic stem cells (hESCs), which are capable of extensive self-renewal and expansion and have the potential to differentiate into any somatic tissue, and induced pluripotent stem cells (iPSCs), which are derived from differentiated tissue such as adult skin fibroblasts and appear to have the same properties and potential, but their generation is not dependent upon a source of embryos. The likelihood that clinical transplantation of hESC- or iPSC-derived tissues from an unrelated (allogeneic) donor that express foreign human leucocyte antigens (HLA) may undergo immunological rejection requires the formulation of strategies to attenuate the host immune response to transplanted tissue. In clinical practice, individualized iPSC tissue derived from the intended recipient offers the possibility of personalized stem cell therapy in which graft rejection would not occur, but the logistics of achieving this on a large scale are problematic owing to relatively inefficient reprogramming techniques and high costs. The creation of stem cell banks comprising HLA-typed hESCs and iPSCs is a strategy that is proposed to overcome the immunological barrier by providing HLA-matched (histocompatible) tissue for the target population. Estimates have shown that a stem cell bank containing around 10 highly selected cell lines with conserved homozygous HLA haplotypes would provide matched tissue for the majority of the UK population. These simulations have practical, financial, political and ethical implications for the establishment and design of stem cell banks incorporating cell lines with HLA types that are compatible with different ethnic populations throughout the world.     

The prospect of pluripotent stem cell-based therapy

Human embryonic stem cells (hESC) are able to maintain pluripotency in culture, to proliferate indefinitely and to differentiate into all somatic cell types. Due to these unique properties, hESC may become an exceptional source of tissues for transplantation and have a great potential for the therapy of incurable diseases. Here, we review new developments in the area of embryonic stem cells and discuss major challenges — standardization of protocols for cell derivation and cultivation, identification of specific molecular markers, development of new approaches for directed differentiation, etc. — which remain to be settled, prior to safe and successful clinical application of stem cells. We appraise several potential approaches in hESC-based therapy including derivation of autologous cells via therapeutic cloning (1), generation of immune tolerance to allogenic donor cells via hematopoetic chimerism (2), and development of the banks of hESC lines compatible with the main antigens and exhibiting equivalent pluripotency (3). In addition, we discuss briefly induced pluripotent cells, which are derived via genetic modification of autologous somatic cells and are analogous to ESC. Our analysis demonstrates that uncontrollable differentiation in vivo and teratogenic potential of hESC are critical limitations of their application in clinical practice. Therefore, the major approach in hESC therapy is derivation of a specific differentiated progeny, which has lower proliferative potential and immune privilege, yet poses fewer risks for organism. The review demonstrates that cell therapy is far more complex and resource-consuming process as compared with drug-based medicine and consequently pluripotent stem cell biology and technology still requires further investigation and development before these cells can be used in clinical practice.