Pluripotency of somatic cell hybrids between nullipotent and pluripotent embryonal carcinoma cells

Embryonal carcinoma (EC) cells are stem cells of teratocarcinomas. Many lines of EC cells can differentiate into a wide spectrum of cell types and are termed pluripotent. Some EC cell lines, however, cannot differentiate and are termed nullipotent. Nullipotent and pluripotent cells are both developmentally uncommitted but differ in their potential for differentiation. As a first step in analyzing this difference we asked whether pluripotent × nullipotent somatic cell hybrids could differentiate. The pluripotent EC cell line PSA-1 was fused with a ouabainresistant, hypoxanthine phosphoribosyltransferase-deficient subclone of the nullipotent embryonal carcinoma cell line F9. The developmental potential of nine independent hybrid clones was assayed in vivo. The tumors derived from these hybrid cell lines contained the same spectrum of differentiated tissue types found in tumors produced by the pluripotent parent. Control nullipotent × nullipotent somatic cell hybrids produced tumors containing only embryonal carcinoma tissue. The pluripotent phenotype therefore behaves in a dominant fashion in these somatic cell hybrids. One explanation for these results is that nullipotent F9 cells lack a critical function required for differentiation in vivo, and this function is supplied to the hybrids by the pluripotent parent.     

Ethics and Embryonic Stem Cell Research

Ethical controversy in stem cell research arises because current methods to produce embryonic stem cell lines require the destruction of living human embryos. For this reason, there is increasing interest in developing alternative, non-embryonic sources of pluripotent stem cells. This effort is especially important in the US due to the prevailing policy against federal funding of embryo-destructive research. Altered nuclear transfer (ANT) is one of several potential methods to develop alternative sources of pluripotent stem cells. This approach employs the technique of somatic cell nuclear transfer, but the somatic cell nucleus or egg cytoplasm (or both) are first altered before the somatic cell nucleus is transferred into the oocyte. This alteration precludes the coordinated organization and developmental potential that is necessary for the resulting biological entity to be an embryo, but it still allows the entity to generate pluripotent stem cells. Proof-of-principle for one variant of ANT has been established in mice by silencing the functional expression of the gene Cdx2 in the somatic cell nucleus prior to its transfer into an enucleated egg. From the resulting non-embryonic laboratory construct, fully functional pluripotent stem cells were procured. Other more recent studies have suggested the possibility of achieving the same results by preemptively silencing maternally derived Cdx2 messenger RNA in the egg before the act of nuclear transfer. The procedure would produce the equivalent of a tissue culture of pluripotent stem cells. In contrast to the use of embryos 'left over' from clinical in vitro fertilization, ANT could produce pluripotent stem cell lines with an unlimited range of specifically selected and controlled genotypes. Such flexibility would greatly facilitate the study of disease, drug development, and toxicology testing, and may allow the production of therapeutically useful pluripotent stem cells that are immune-compatible. If developed to the point of scientific reliability, ANT would be a valuable research tool for the study of other aspects of cell development and differentiation, including gene expression patterns, imprinting, and cell-cell signaling. ANT would also help to clarify definitions and boundaries that distinguish true organisms from 'biological artifacts' and, thereby, provide moral precedent to guide future progress in developmental biology.     

Four new human germ cell tumor cell lines

Four new human tumor cell lines, NCC-EC-1, NCC-EC-2, NCC-EC-3, and NCC-IT, were derived from different germ cell tumors established in vitro. On the basis of morphologic studies of cell cultures and nude mice xenografts, it was concluded that NCC-EC-1, and NCC-EC-2 cell lines are equivalent to developmentally nullipotent embryonal carcinoma; cell line NCC-EC-3 showed trophoblastic differentiation, whereas the NCC-IT cell line was composed of developmentally pluripotent cells capable of somatic and extraembryonic differentiation. Nude mouse-xenografts of NCC-IT contained foci of embryonal carcinoma, yolk sac tumor, immature somatic tissues, and trophoblastic giant cells indicating that this cell line is indeed developmentally pluripotent. We conclude that human embryonal carcinoma cell lines may be developmentally nullipotent, show restricted capacity for differentiation, or be developmentally pluripotent.     

Human embryonic stem cell lines derived from discarded embryos

Human pluripotent embryonic stem (ES) cells have important potential in regenerative medicine and as models for human preimplantation development; however, debate continues over whether embryos should be destroyed to produce human ES cells. We have derived four ES cell lines on mouse embryonic fibroblast cells in medium supplemented with basic fibroblast growth factor, human recombinant leukemia inhibitory factor, and fetal bovine serum. The source of these cell lines was poor-quality embryos that in the course of routine clinical practice would have been discarded. After continuous proliferation in vitro for more than 12 months, these ES cell lines maintained their developmental potential to form trophoblast and somatic cells, including cardiac muscle and neuronal tissue.     

De novo reestablishment of gap junctional intercellular communications during reprogramming to pluripotency and differentiation

Gap junctional intercellular communication (GJIC) has been described in embryonic stem cells (ESCs) and various somatic cells. GJIC has been implicated in the regulation of cell proliferation, self-renewal, and differentiation. Recently, a new type of pluripotent stem cells was generated by direct reprogramming of somatic cells. Here, for the first time, we show that during reprogramming events GJIC is re-established upon reaching complete reprogramming. The opposite process of cell differentiation from the pluripotent state leads to the disruption of GJIC between pluripotent and differentiated cell subsets. However, GJIC is subsequently re-established de novo within each differentiated cell type in vitro, forming communication compartments within a histotype. Our results provide the important evidence that reestablisment of functional gap junctions to the level similar to human ESCs is an additional physiological characteristic of somatic cell reprogramming to the pluripotent state and differentiation to the specific cell type.     

Dppa2 and Dppa4 are closely linked SAP motif genes restricted to pluripotent cells and the germ line

Despite the enormous medical potential of ESCs, the molecular mechanisms conferring the ability to differentiate into all cell types of the embryo remain elusive. We used an in silico approach to identify genes expressed exclusively in mouse preimplantation embryos and pluripotent cell lines. Two of these genes were developmental pluripotency-associated gene 2 (Dppa2) and Dppa4, which we show are closely linked genes encoding putative nuclear SAP domain proteins expressed in human and mouse pluripotent stem cells and germ cell tumor-derived embryonal carcinoma cells. In the mouse, these genes are transcribed in germinal vesicle-stage oocytes and throughout the cleavage stages of embryogenesis. They then become restricted to the pluripotent inner cell mass of blastocysts and are subsequently downregulated. After gastrulation, Dppa2 and Dppa4 are expressed only in the developing germ line, showing that these genes mark cells of the pluripotent cycle. In the germ line, both genes are downregulated as the germ cells commit to the oogenic pathway or soon after commitment to the spermatogenic pathway. We have observed similar germ line expression profiles for other pluripotent markers, and these results are consistent with the hypothesis that pluripotent markers must be downregulated during fetal germ line development, a process that may be required to facilitate appropriate germ line differentiation. The study of expression and function of pluripotent markers such as Dppa2 and Dppa4 is likely to unveil new aspects of the regulation of pluripotency and germ line development in mammals.     

Reprogramming somatic gene activity by fusion with pluripotent cells

Fertilized eggs and early blastomeres, that have the potential to develop to fetuses when placed into a uterus, are totipotent. Those cells in the embryo, that can give rise to all cell types of an organism, but not to an organism itself, are pluripotent. Embryonic stem (ES), embryonic carcinoma (EC), and embryonic germ (EG) cells are powerful in vitro artifacts derived from different embryonic stages and are pluripotent. Totipotent and pluripotent cells have the potential to greatly benefit biological research and medicine. One powerful feature is that the genetic program of somatic cells can be converted into that of totipotent or pluripotent cells, as shown by nuclear transfer or cell fusion experiments. During reprogramming by cell fusion various features of pluripotent cells are acquired. These include the typical morphology of the respective pluripotent fusion partner, a specific epigenetic state, a specific gene profile, inactivation of tissue-specific genes expressed in the somatic fusion partner, and the developmental as well as differentiation potential of pluripotent cells. In this review, we will discuss what is known about the reprogramming process mediated by cell fusion and the potential use of fusion-induced reprogramming for therapeutic applications.     

Generation of Induced Pluripotent Stem Cells From Buffalo (Bubalus bubalis) Fetal Fibroblasts with Buffalo Defined Factors

Ectopically, expression of defined factors could reprogram mammalian somatic cells into induced pluripotent stem cells (iPSCs), which initiates a new strategy to obtain pluripotent stem cell lines. Attempts have been made to generate buffalo pluripotent stem cells by culturing primary germ cells or inner cell mass, but the efficiency is extremely low. Here, we report a successful method to reprogram buffalo fetal fibroblasts (BFFs) into pluripotent stem cells [buffalo induced pluripotent stem cell (biPSCs)] by transduction of buffalo defined factors (Oct4, Sox2, Klf4, and c-Myc) using retroviral vectors. The established biPSCs displayed typical morphological characteristics of pluripotent stem cells, normal karyotype, positive staining of alkaline phosphatase, and expressed pluripotent markers including Oct4, Sox2, Nanog, Lin28, E-Cadherin, SSEA-1, SSEA-4, TRA-1-81, STAT3, and FOXD3. They could form embryoid bodies (EBs) in vitro and teratomas after injecting into the nude BALB/C mice, and 3 germ layers were identified in the EBs and teratomas. Methylation assay revealed that the promoters of Oct4 and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs, while the promoters of Sox2 and E-Cadherin were hypomethylated in both BFFs and biPSCs. Further, inhibiting p53 expression by coexpression of SV40 large T antigen and buffalo defined factors in BFFs or treating BFFs with p53 inhibitor pifithrin-a (PFT) could increase the efficiency of biPSCs generation up to 3-fold, and nuclear transfer embryos reconstructed with biPSCs could develop to blastocysts. These results indicate that BFFs can be reprogrammed into biPSCs by buffalo defined factors, and the generation efficiency of biPSCs can be increased by inhibition of p53 expression. These efforts will provide a feasible approach for investigating buffalo stem cell signal pathways, establishing buffalo stem cell lines, and producing genetic modification buffaloes in the future.     

Analysis of Esg1 expression in pluripotent cells and the germline reveals similarities with Oct4 and Sox2 and differences between human pluripotent cell lines

Establishment of pluripotent epiblast cells is a critical event during early mammalian development because all somatic lineages and the primordial germ cells (PGCs) are derived from them. The epiblast and PGCs are in turn the precursors of pluripotent embryonic stem cells and embryonic germ cells, respectively. Although PGCs are specialized cells, they express several key pluripotency-related genes, such as Oct4 and Sox2. We have analyzed Esg1 expression in mouse and human cells and shown that in the mouse the gene is specifically expressed in preimplantation embryos, stem cells, and the germline. Moreover, Esg1 coexpresses with Oct4 and Sox2, confirming its identity as a marker of the pluripotent cycle. Esg1 is also expressed with Oct4 and Sox2 by human embryonic stem cells and in germ cell carcinoma tissue but not by all human embryonal carcinoma cell lines. These data suggest that together with Oct4 and Sox2, Esg1 plays a conserved role in the pluripotent pathway of mouse and human stem and germ cells.     

重编程诱导多功能干细胞的研究进展

Induced pluripotent stem cells (iPSCs) have been first induced from mouse fibroblasts since 2006, and the research on iPSCs has made great progress in the following years. iPS cell lines were established from different somatic cells through DNA, RNA, protein, and small molecule compounds and various methods of transduction, making the induction of iPSCs more secure and effective, and more attractive prospect of clinical application. In this review, different somatic cell reprogramming, different levels of reprogramming, different transduction pathways, and prospect of application are discussed.     

人诱导性多潜能干细胞建立心肌细胞疾病模型的研究进展

诱导性多潜能干细胞是干细胞研究领域里程碑式的发现,近10年发展迅速。通过体细胞获得诱导性多潜能干细胞,再进一步分化成心肌细胞,可建立不同的心肌细胞疾病模型,并用于疾病的机制研究、药物筛选和基因治疗等。本文对已成功构建的人诱导多潜能干细胞来源的心肌细胞疾病模型进行了介绍,并指出了其面临的问题及展望。     

转录因子调节的体细胞重编程分子和表观机制

诱导性多能干细胞(induced pluripotent stem cells,iPS)是通过转录因子诱导的体细胞重编程建立的,它与胚胎干细胞具有共同的发育多潜能性及在细胞替代治疗上潜在的应用前景。然而,转录因子调节的重编程过程是一个复杂而漫长的过程,涉及大量、未知的分子及表观事件,而且产生的iPS细胞发育潜能差异较大。对其重编程机制的揭示,将有助于获得更高效、安全的iPS细胞,为最终实现应用于人类疾病的治疗提供理论依据。本文关注了近几年转录因子调节的重编程分子机制的研究,阐述了重编程过程中转录与表观的变化。     

Pluripotential reprogramming of the somatic genome in hybrid cells occurs with the first cell cycle

The fusion of pluripotent embryonic cells with somatic cells results in reprogramming of the somatic cell genome. Oct4-green fluorescent protein (GFP) transgenes that do not contain the proximal enhancer (PE) region are widely used to visualize reprogramming of the somatic to the pluripotent cell state. The temporal onset of Oct4-GFP activation has been found to occur 40-48 hours postfusion. We asked whether activation of the transgene actually reflects activation of the endogenous Oct4 gene. In the current study, we show that activation of an Oct4-GFP transgene that contains the PE region occurs within 22 hours of fusion. In addition, demethylation of the Oct4-GFP transgene and that of the endogenous Oct4 and Nanog genes was found to occur within 24 hours of fusion. As this timing corresponds with the timing of cell cycle completion in embryonic stem cells and fusion hybrids (approximately 22 hours), we postulate that pluripotential reprogramming of the somatic cell genome begins during the first cell cycle after the fusion of a somatic cell with a pluripotent cell and has been completed by day 2 postfusion.