人诱导多能干细胞与人胚胎干细胞分化过程中Oct4/Nanog 基因 表达的比较

目的:比较通过慢病毒方法获得的人诱导多能性干细胞(iPSCs)与人胚胎干细胞(hESCs)分化过程中全能性基因Oct4、Nanog的表达变化。方法:收集分化不同时间点的拟胚体(EBs),检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果:iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷(SCID)小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论:通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。     

人诱导多能干细胞与人胚胎干细胞分化过程中Oct4/Nanog基因表达的比较

目的：比较通过慢病毒方法获得的人诱导多能性干细胞（iPSCs）与人胚胎干细胞（hESCs）分化过程中全能性基因Oct4、Nanog的表达变化。方法：收集分化不同时间点的拟胚体（EBs）,检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果：iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷（SCID）小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论：通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。     

Real microgravity condition promoted regeneration capacity of induced pluripotent stem cells during the TZ‐1 space mission

Induced pluripotent stem cells (iPSCs) are reprogrammed somatic cells that gained self‐renewal and differentiation capacity similar to embryonic stem cells. Taking the precious opportunity of the TianZhou‐1 spacecraft mission, we studied the effect of space microgravity (µg) on the self‐renewal capacity of iPSCs. Murine iPSCs carrying pluripotency reporter Oct4‐GFP were used. The Oct4‐EGFP‐iPSCs clones were loaded into the bioreactor and exposed to μg in outer space for 14 days. The control experiment was performed in identical device but on the ground in earth gravity (1 g). iPSCs clones were compact and highly expressed Oct4 before launch. In μg condition, cells in iPSC clones spread out more rapidly than those in ground 1 g condition during the first 3 days after launch. However, in 1 g condition, as the cell density increases, the Oct4‐GFP signal dropped significantly during the following 3 days. Interestingly, in μg condition, iPSCs originated from the spread‐out clones during the first 3 days appeared to cluster together and reform colonies that activated strong Oct4 expression. On the other hand, iPSC clones in 1 g condition were not able to recover Oct4 expression after overgrown. Our study for the first time performed real‐time imaging on the proliferation process of iPSCs in space and found that in μg condition, cell behaviour appeared to be more dynamic than on the ground.     

Cytoplast containing reprogramming-related factors from human embryonic stem cells arrested at metaphase

Generating pluripotent stem cells directly from a patient's somatic cells is one of the major methods to avoid rejection in future regenerative medicine. It is reported that human embryonic stem cells (hESCs) are able to reprogram the nuclei of fully differentiated human somatic cells, apparently conferring on them a pluripotent state. However, the ability of the cytoplasts from enucleated hESCs to reprogram somatic cells causes much controversy. Here we detect the location of pluripotency-related factors such as Oct4/Nanog/Sox2 in the hESCs at division and non-division stage and obtain the cytoplasts of hESCs by centrifugation. We demonstrate for the first time that the cytoplast from hESCs arrested at the division phase of cell the cycle contains the reprogramming factors and this kind of cytoplast can be obtained through gradient centrifugation. These give us direct proof of the possibility of reprogramming somatic cell using cytoplast of hESCs and make this a possible method for getting patient-specific pluripotent cells without extrinsic DNA introduction.     

Induced pluripotency and direct reprogramming: a new window for treatment of neurodegenerative diseases

Human embryonic stem cells (hESCs) are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages, including neural stem (NS) cells. Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered, and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro. However, the limitations of hESCs resource along with the religious and ethical concerns impede the progress of ESCs application. Therefore, the induced pluripotent stem cells (iPSCs) via somatic cell reprogramming have opened up another new territory for regenerative medicine. iPSCs now can be derived from a number of lineages of cells, and are able to differentiate into certain cell types, including neurons. Patient-specific iPSCs are being used in human neurodegenerative disease modeling and drug screening. Furthermore, with the development of somatic direct reprogramming or lineage reprogramming technique, a more effective approach for regenerative medicine could become a complement for iPSCs.     

Generation of induced pluripotent stem cells using elastin like polypeptides as a non-viral gene delivery system

Induced pluripotent stem cells (iPSCs) have been generated from various somatic cells using different approaches; however, a major restriction of reprogramming methods is the use of viral vectors, which have the risk of causing genome-integration of viral DNA. Here, without a viral vector, we generated iPSCs from mouse fibroblasts using an elastin-like polypeptide (ELP)-based transfection method. Our findings support the possible use of ELPs for delivery of the reprogramming genes in to somatic cells for generation of iPSCs. Results of gel retardation assay demonstrated efficient complexation of ELPs with a plasmid containing the four Yamanaka stem cell factors, Oct-4, Klf4, c-myc, and Sox2. After transfection, the iPSCs showed embryonic stem cell-like characteristics, including expression of endogenous pluripotency genes, differentiation into three germ layer lineages, and formation of teratomas in vivo. Our results demonstrate that ELP-based gene delivery may provide a safe method for use in generation of virus-free and exogenous DNA-free iPSCs, which will be crucial for future applications in stem cell-based therapies.     

Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

Background

Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored.

Methodology/Principal Findings

We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation.

Conclusions/Significance

Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance.     

Inducing pluripotency in vitro: recent advances and highlights in induced pluripotent stem cells generation and pluripotency reprogramming

Induced pluripotent stem cells (iPSCs) are considered patient‐specific counterparts of embryonic stem cells as they originate from somatic cells after forced expression of pluripotency reprogramming factors Oct4, Sox2, Klf4 and c‐Myc. iPSCs offer unprecedented opportunity for personalized cell therapies in regenerative medicine. In recent years, iPSC technology has undergone substantial improvement to overcome slow and inefficient reprogramming protocols, and to ensure clinical‐grade iPSCs and their functional derivatives. Recent developments in iPSC technology include better reprogramming methods employing novel delivery systems such as non‐integrating viral and non‐viral vectors, and characterization of alternative reprogramming factors. Concurrently, small chemical molecules (inhibitors of specific signalling or epigenetic regulators) have become crucial to iPSC reprogramming; they have the ability to replace putative reprogramming factors and boost reprogramming processes. Moreover, common dietary supplements, such as vitamin C and antioxidants, when introduced into reprogramming media, have been found to improve genomic and epigenomic profiles of iPSCs. In this article, we review the most recent advances in the iPSC field and potent application of iPSCs, in terms of cell therapy and tissue engineering.     

Anatomical templates for tissue (re)generation and beyond

Induced pluripotent stem cells (iPSCs) represent a valuable alternative to stem cells in regenerative medicine overcoming their ethical limitations, like embryo disruption. Takahashi and Yamanaka in 2006 reprogrammed, for the first time, mouse fibroblasts into iPSCs through the retroviral delivery of four reprogramming factors: Oct3/4, Sox2, c-Myc, and Klf4. Since then, several studies started reporting the derivation of iPSC lines from animals other than rodents for translational and veterinary medicine. Here, we review the potential of using these cells for further intriguing applications, such as “cellular agriculture.” iPSCs, indeed, can be a source of in vitro, skeletal muscle tissue, namely “cultured meat,” a product that improves animal welfare and encourages the consumption of healthier meat along with environmental preservation. Also, we report the potential of using iPSCs, obtained from endangered species, for therapeutic treatments for captive animals and for assisted reproductive technologies as well. This review offers a unique opportunity to explore the whole spectrum of iPSC applications from regenerative translational and veterinary medicine to the production of artificial meat and the preservation of currently endangered species.     

Generation of Induced Pluripotent Stem Cells with High Efficiency from Human Umbilical Cord Blood Mononuclear Cells

Human induced pluripotent stem cells （iPSCs） hold great promise for regenerative med- icine. Generating iPSCs from immunologically immature newborn umbilical cord blood mononu- clear cells （UCBMCs） is of great significance. Here we report generation of human iPSCs with great efficiency from UCBMCs using a dox-inducible lentiviral system carrying four Yamanaka factors. We generated these cells by optimizing the existing iPSC induction protocol. The UCBMC-derived iPSCs （UCB-iPSCs） have characteristics that are identical to pluripotent human embryonic stem cells （hESCs）. This study highlights the use of UCBMCs to generate highly functional human iPSCs that could accelerate the development of cell-based regenerative therapy for patients suffering from various diseases.