Induced pluripotent stem cell generation from bovine somatic cells indicates unmet needs for pluripotency sustenance

Mechanisms that direct reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs), albeit incomplete in understanding, are highly conserved across all mammalian species studied. Equally, proof of principle that iPSCs can be derived from domestic cattle has been reported in several publications. In our efforts to derive and study bovine iPSCs, we encountered inadequacy of methods to generate, sustain, and characterize these cells. Our results suggest that iPSC protocols optimized for mouse and human somatic cells do not effectively translate to bovine somatic cells, which show some refractoriness to reprogramming that also affects sustenance. Moreover, methods that enhance reprogramming efficiency in mouse and human cells had no effect on improving bovine cell reprogramming. Although use of retroviral vectors coding for bovine OCT4, SOX2, KLF4, cMYC, and NANOG appeared to produce consistent iPSC‐like cells from both fibroblasts and cells from the Wharton's jelly, these colonies could not be sustained. Use of bovine genes could successfully reprogram both mouse and human cells. These findings indicated either incomplete reprogramming and/or discordant/inadequate culture conditions for bovine pluripotent stem cells. Therefore, additional studies that advance core knowledge of bovine pluripotency are necessary before any anticipated iPSC‐driven bovine technologies can be realized.     

Molecular signature and colony morphology affect in vitro pluripotency of porcine induced pluripotent stem cells

Overall efficiency of cell reprogramming for porcine fibroblasts into induced pluripotent stem cells (iPSCs) is currently poor, and few cell lines have been established. This study examined gene expression during early phase of cellular reprogramming in the relationship to the iPSC colony morphology and in vitro pluripotent characteristics. Fibroblasts were reprogrammed with OCT4, SOX2, KLF4 and c-MYC. Two different colony morphologies referred to either compact (n = 10) or loose (n = 10) colonies were further examined for proliferative activity, gene expression and in vitro pluripotency. A total of 1,697 iPSC-like colonies (2.34%) were observed after gene transduction. The compact colonies contained with tightly packed cells with a distinct-clear border between the colony and feeder cells, while loose colonies demonstrated irregular colony boundary. For quantitative expression of genes responsible for early phase cell reprogramming, the Dppa2 and EpCAM were significantly upregulated while NR0B1 was downregulated in compact colonies compared with loose phenotype (p < .05). Higher proportion of compact iPSC phenotype (5 of 10, 50%) could be maintained in undifferentiated state for more than 50 passages compared unfavourably with loose morphology (3 of 10, 30%). All iPS cell lines obtained from these two types of colony morphologies expressed pluripotent genes and proteins (OCT4, NANOG and E-cadherin). In addition, they could aggregate and form three-dimensional structure of embryoid bodies. However, only compact iPSC colonies differentiated into three germ layers. Molecular signature of early phase of cell reprogramming coupled with primary colony morphology reflected the in vitro pluripotency of porcine iPSCs. These findings can be simply applied for pre-screening selection of the porcine iPSC cell line.     

Nanog基因的生物学功能研究进展

胚胎干细胞具有无限增殖能力和多向分化潜能决定了它在医学及生物学基础研究中具有巨大的应用潜力。探索维持胚胎干细胞特性的分子机制成为胚胎干细胞的生物学研究中的热点。研究发现与维持胚胎干细胞多能性相关的基因有Oct4、Nanog、Sox2等，其中Nanog是2003年5月末发现的一个基因，它对维持胚胎干细胞多能性起关键性作用，能够独立于L1F/Stats维持ICM和ES细胞的多能性。几年来，Nanog的生物学功能及其与Oct4、Sox2等多能性维持基因之间的相互作用关系已有较为深入的研究。作者在综述Nanog基因的表达特征和功能的基础上，重点探讨Nanog基因表达调控以及Oct4、Sox2等多能性维持基因之间的相互作用关系，并展望其应用前景。     

Generation of induced pluripotent stem cells from Asian patients with chronic neurodegenerative diseases

Induced pluripotent stem (iPS) cells derived from disease patients are an invaluable resource for biomedical research and may provide a source for replacement therapies. In this study, we have generated iPS cells from Asian patients with chronic degenerative diseases of the nervous system, including spinal muscular atrophy (SMA), Parkinson disease (PD) and amyotrophic lateral sclerosis (ALS) by transduction with four factors (KLF4, SOX2, OCT4 and c-MYC). All of the iPS cells showed pluripotency similar to that of human embryonic stem cells (hESCs) and were able to differentiate into various somatic cell types in vitro and in vivo. Furthermore, the iPS cells also can be committed to differentiate into neural cells, the cell type that is affected in chronic degenerative diseases. Therefore, the patient-specific iPS cells we generated offer a cellular model in which to investigate disease mechanisms, discover and test novel drugs and develop new therapies for chronic neurodegenerative diseases.     

Na?ve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use.     

Derivation of Canine Induced Pluripotent Stem Cells

Dogs and humans have many inherited genetic diseases in common and conditions that are increasingly prevalent in humans also occur naturally in dogs. The use of dogs for the experimental and clinical testing of stem cell and regenerative medicine products would benefit canine health and welfare and provide relevant animal models for the translation of therapies to the human field. Induced pluripotent stem cells (iPSCs) have the capacity to turn into all cells of the body and therefore have the potential to provide cells for therapeutic use and for disease modelling. The objective of this study was to derive and characterize iPSCs from karyotypically abnormal adult canine cells. Aneuploid adipose‐derived mesenchymal stromal cells (AdMSCs) from an adult female Weimeraner were re‐programmed into iPSCs via overexpression of four human pluripotency factors (Oct 4, Sox2, Klf4 and c‐myc) using retroviral vectors. The iPSCs showed similarity to human ESCs with regard to morphology, pluripotency marker expression and the ability to differentiate into derivatives of all three germ layers in vitro (endoderm, ectoderm and mesoderm). The iPSCs also demonstrated silencing of the viral transgenes and re‐activation of the silent X chromosome, suggesting full reprogramming had occurred. The levels of aneuploidy observed in the AdMSCs were maintained in the iPSCs. This finding demonstrates the potential for generating canine induced pluripotent stem cells for use as disease models in addition to regenerative medicine and pharmaceutical testing.     

Generation of Human β-thalassemia Induced Pluripotent Stem Cells from Amniotic Fluid Cells Using a Single Excisable Lentiviral Stem Cell Cassette

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may have a wide range of applications in cell and gene therapy. However, the safety issues and the low efficiency associated with generating human iPSCs have limited their usage in clinical settings. The cell type used to create iPSCs can significantly influence the reprogramming efficiency and kinetics. Here, we show that amniotic fluid cells from the prenatal diagnosis of a β-thalassemia patient can be efficiently reprogrammed using a doxycycline (DOX)-inducible humanized version of the single lentiviral "stem cell cassette" vector flanked by loxP sites, which can be excised with Cre recombinase. We also demonstrated that the patient-derived iPSCs can be characterized based on the expression of pluripotency markers, and they can be differentiated into various somatic cell types in vitro and in vivo. Moreover, microarray analysis demonstrates a high correlation coefficient between human β-thalassemia iPS cells and human embryonic stem (hES) cells but a low correlation coefficient between human β-thalassemia amniotic fluid cells and human β-thalassemia iPS cells. Our data suggest that amniotic fluid cells may be an ideal human somatic cell resource for rapid and efficient generation of patient-specific iPS cells.     

绵羊胚胎成纤维细胞饲养层用于培养人诱导性多能干细胞的效果研究

试验在体外分离培养1月龄绵羊胚胎成纤维细胞（sheep embryonic fibroblast，SEF），经丝裂霉素C处理后探讨其作为诱导性多能干细胞（induced pluripotent stem cells，iPSC）体外培养饲养层的可行性。试验以SNL饲养层细胞为对照，人iPSC（hiPSC）为培养对象，通过形态学观察、碱性磷酸酶（AP）染色、以及实时荧光定量PCR和免疫细胞化学对hiPSC标志基因mRNA和蛋白表达的检测，比较了SEF细胞和SNL细胞作为干细胞饲养层的效果。结果表明，在试验期内，与SNL饲养层体外培养的hiPSC相似，SEF饲养层体外培养的hiPSC在形态上呈集落样生长，增殖速度快；AP染色呈蓝紫色，能够维持未分化状态；能正常表达多能性标志基因。两种饲养层细胞培养的hiPSC多能性标志基因c-Myc、Klf4、OCT4和SOX2 mRNA的表达以及OCT4、SOX2、SSEA4和TRA-1-60蛋白的表达并无显著差异（P>0.05）。本研究结果初步表明SEF可作为体外培养iPSC的饲养层细胞，为进一步建立可表达促生长因子的基因修饰SEF细胞系奠定了基础。     

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot^TM reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.     

鸡体细胞诱导重编程早期的糖代谢变化研究

为研究鸡体细胞诱导重编程早期的糖代谢方式的变化,试验采用OCT4、SOX2、NANOG和LIN28A(OSNL)四因子诱导体系将鸡胚成纤维细胞(Chicken embryo fibroblasts,CEF)重编程为诱导多能干细胞(Induced pluripotent stem cells,iPS),并利用碱性磷酸酶染色、阶段特异性胚胎抗原1(Stage-specific embyronic antigen-1,SSEA-1)免疫荧光染色、体外诱导分化及多能性基因表达检测等对iPS进行鉴定。通过检测重编程过程中糖代谢相关基因表达及酶活性的变化,并对葡萄糖摄取量、乳酸产生量及线粒体膜电位检测等研究鸡体细胞诱导重编程早期的糖代谢变化。结果显示,鸡CEF诱导重编程形成的iPS呈碱性磷酸酶染色阳性,表达SSEA-1蛋白,体外分化形成类胚体且表达多能性标记基因。同时重编程过程中氧化磷酸化基因表达下调而糖酵解相关基因表达上调,糖酵解关键酶活性均增强,且iPS的葡萄糖吸收量及乳酸产生量增加,而线粒体膜电位则下降。结果表明,OSNL四因子体系将鸡CEF诱导重编程形成iPS的过程中,细胞的主要糖代谢方式从氧化磷酸化转变为糖酵解,而糖酵解的激活可能会进一步促进iPS的形成。     

Sox2基因真核表达载体的构建及转Sox2基因绵羊骨髓间充质干细胞系的建立

Sox2是多能干细胞的主要标记之一,有研究发现高表达Sox2基因的神经干细胞作为供体细胞进行核移植时具有较高的重编程能力。本研究旨在通过对绵羊骨髓间充质干细胞(bonemarrowmesenchymal stem cells,BMSC) Sox2基因进行外源性增强表达,以期提高其重编程能力,从而改善动物体细胞克隆效率。试验提取绵羊胎儿生殖腺组织RNA,以其为模板克隆Sox2基因cDNA序列,装入真核表达载体pEGFP-N1,构建出pEGFP-N1-Sox2表达载体。经脂质体转染将重组质粒转染入绵羊BMSC,经G418与荧光标记双筛选后挑选单克隆并扩增培养。测序鉴定表明,克隆得到绵羊Sox2基因CDS区全长,重组质粒构建成功;荧光检测表明,成功建立表达Sox2基因的绵羊BMSC系。本研究得到了高表达Sox2基因的绵羊BMSC系,为提高体细胞克隆过程中的重编程效率提供了新思路。     

Livestock induced pluripotent stem cells

Chimeric animals generated from livestock-induced pluripotent stem cells (iPSCs) have opened the door of opportunity to genetically manipulate species for the production of biomedical models, improving traits of agricultural importance and potentially providing a system to test novel iPSC therapies. The potential of pluripotent stem cells in livestock has long been recognized, with many attempts being chronicled to isolate, culture and characterize pluripotent cells from embryos. However, in most cases, livestock stem cells derived from embryonic sources have failed to reach a pluripotent state marked by the inability to form chimeric animals. The in-depth understanding of core pluripotency factors and the realization of how these factors can be harnessed to reprogram adult cells into an induced pluripotent state has changed the paradigm of livestock stem cells. In this review, we will examine the advancements in iPSC technology in mammalian and avian livestock species.     

Construction of Eukaryotic Expression Vector of Sox2 Gene and Establishment of Sheep Bone Marrow Mesenchymal Stem Cell Lines Transfected with Sox2 Gene

Sox2 is one important marker of pluripotent stem cells, a study found that neural stem cells with high expression of Sox2 as donor cells showed higher reprogramming ability in nuclear transplantation.In this study, through enhancing exogenous Sox 2 gene expression of sheep bone marrow mesenchymal stem cells, in order to raise their reprogramming ability, and improve the efficiency of somatic cell cloning in animal.Total RNA was extracted from sheep testicular tissue, and with this template, Sox2 cDNA sequence was amplified and inserted into the eukaryotic expression vector pEGFP-N1 to build a recombinant vector pEGFP-N1-Sox2.The vector was transfected into the sheep bone marrow mesenchymal stem cells by liposome method, and through G418 and fluorescence screening to obtain and amplify monoclone.DNA sequencing showed that sheep Sox 2 gene CDS sequence was obtained, and recombinant plasmid was successfully constructed.Identification of fluorescence confirmed that stable sheep bone marrow mesenchymal stem cell lines transfected with Sox2 were established.This study obtained the sheep bone marrow mesenchymal stem cell lines with high expression of Sox2, and provided a new idea for raising reprogramming efficiency in the process of somatic cell cloning.