Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells

BackgroundCell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.MethodsFused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.ResultsColonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.ConclusionsOur results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.     

Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression. These small, non-coding RNAs are believed to regulate more than a third of all protein coding genes, and they have been implicated in the control of virtually all biological processes, including the biology of stem cells. The essential roles of miRNAs in the control of pluripotent stem cells were clearly established by the finding that embryonic stem (ES) cells lacking proteins required for miRNA biogenesis exhibit defects in proliferation and differentiation. Subsequently, the function of numerous miRNAs has been shown to control the fate of ES cells and to directly influence critical gene regulatory networks controlled by pluripotency factors Sox2, Oct4, and Nanog. Moreover, a growing list of tissue-specific miRNAs, which are silenced or not processed fully in ES cells, has been found to promote differentiation upon their expression and proper processing. The importance of miRNAs for ES cells is further indicated by the exciting discovery that specific miRNA mimics or miRNA inhibitors promote the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Although some progress has been made during the past two years in our understanding of the contribution of specific miRNAs during reprogramming, further progress is needed since it is highly likely that miRNAs play even wider roles in the generation of iPS cells than currently appreciated. This review examines recent developments related to the roles of miRNAs in the biology of pluripotent stem cells. In addition, we posit that more than a dozen additional miRNAs are excellent candidates for influencing the generation of iPS cells as well as for providing new insights into the process of reprogramming.     

Reprogramming mediated by stem cell fusion

Advances in mammalian cloning prove that somatic nuclei can be reprogrammed to a state of totipotency by transfer into oocytes. An alternative approach to reprogram the somatic genome involves the creation of hybrids between somatic cells and other cells that contain reprogramming activities. Potential fusion partners with reprogramming activities include embryonic stem cells, embryonic germ cells, embryonal carcinoma cells, and even differentiated cells. Recent advances in fusion-mediated reprogramming are discussed from the standpoints of the developmental potency of hybrid cells, genetic and epigenetic correlates of reprogramming, and other aspects involved in the reprogramming process. In addition, the utility of fusion-mediated reprogramming for future cell-based therapies is discussed.     

细胞融合致体细胞重编程机制研究进展

已分化的体细胞能够通过重编程转化回多能干细胞,在细胞移植、疾病细胞模型的制备以及药物筛选等领域具有重要意义。通过干细胞和体细胞的细胞融合,可使体细胞重编程。细胞融合致体细胞重编程速度快、效率高,是一种研究重编程机制的重要手段。对细胞融合致体细胞重编程的机制作一综述。     

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.     

Genomic integrity of human induced pluripotent stem cells:Reprogramming,differentiation and applications

Ten years after the initial generation of induced pluripotent stem cells(hiPSCs)from human tissues, their potential is no longer questioned, with over 15000 publications listed on PubMed, covering various fields of research; including disease modeling, cell therapy strategies, pharmacology/toxicology screening and 3 D organoid systems. However, despite evidences that the presence of mutations in hiPSCs should be a concern, publications addressing genomic integrity of these cells represent less than 1% of the literature. After a first overview of the mutation types currently reported in hiPSCs, including karyotype abnormalities, copy number variations, single point mutation as well as uniparental disomy, this review will discuss the impact of reprogramming parameters such as starting cell type and reprogramming method on the maintenance of the cellular genomic integrity. Then, a specific focus will be placed on culture conditions and subsequent differentiation protocols and how their may also trigger genomic aberrations within the cell population of interest.Finally, in a last section, the impact of genomic alterations on the possible usages of hiPSCs and their derivatives will also be exemplified and discussed. We will also discuss which techniques or combination of techniques should be used to screen for genomic abnormalities with a particular focus on the necessary quality controls and the potential alternatives.     

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.     

A spin-label study on fusion of red blood cells induced by hemagglutinating virus of Japan.

Fusion of red blood cells (RBC) induced by hemagglutinating virus of Japan (HVJ) has been studied using a phosphatidylcholine spin label. The spin label was readily incorporated and diffused into the lipid bilayer portion of the viral envelope. The exchange broadening in the electron spin resonance (ESR) spectrum of densely labeled virus disappeared rapidly when the virus was mixed with RBC at 37 degrees. The spectrum gradually approached that of the host cell spin labeled with the phosphatidylcholine label. The results directly indicate transfer and intermixing of phospholipid molecules between the viral envelope and RBC membrane. The transfer reaction was strongly dependent on temperature. No transfer was observed at lower temperatures where the virus adsorbed to the cell and caused aggregation but no hemolysis and fusion. The transfer rate remained negligibly small until 19 degrees and increased rapidly between 25 and 30 degrees. The virus-induced hemolysis showed similar temperature dependence. The transfer rate was greatly reduced under inhibitory conditions of fusion: glutaraldehyde treatment of RBC, trypsin treatment of HVJ, or the presence of concanavalin A. Only slight transfer was observed from fusion-inactive influenza virus to RBC. The transfer was greatly enhanced by the help of HVJ. The close parallelism suggests that the transfer and intermixing are necessary steps to the cell fusion. The transfer rate was dependent on fluidity of the host cell membrane and independent of the viral dose. The virus-induced transfer of phospholipid molecules between RBC's was also detected by the spin label. Its temperature dependence was quite similar to that for the virus-to-cell transfer. The intercellular transfer was nearly proportional to the viral dose.     

Cellular reprogramming by single-cell fusion with mouse embryonic stem cells in pig

Fusion of differentiated somatic cells with pluripotent stem cells can be used for cellular reprogramming, but the efficiency to obtain hybrid cells is extremely low. Here, we explored a novel cell fusion system, termed single-cell fusion, the efficiency was significantly improved verified by fusion of mouse embryonic stem cells (mESCs), comparing to traditional polyethylene glycol fusion. Then, we employed the optimized system to perform cell fusion of porcine embryonic fibroblasts (PEFs) and porcine pluripotent stem cells (pPSCs) with mESCs. The hybrid cells showed both red and green fluorescence and expressed species-specific genes of mouse and pig to evidence that the fusion was successful. The hybrid cells displayed characteristics similar with mESCs, including colony morphology, alkaline phosphatase positive and formation of embryoid body, and the expressions of core pluripotent factors OCT4, NANOG, and SOX2 of the pig were induced in the mESC/PEF hybrid cells. The results indicate PEFs and pPSCs could be reprogrammed by mESCs via the single-cell fusion. Taking advantage of the hybrid cells to investigate the signaling pathways depended on the pluripotency of pig, we suggest the transforming growth factor-β signaling pathways may play important roles. In summary, the single-cell fusion is highly efficient, and we believe in the future it will be widely used in the application and fundamental research.     

组蛋白去乙酰化酶抑制剂调控干细胞分化与体细胞重编程的研究进展

表观遗传调控,如组蛋白乙酰化修饰,是决定干细胞分化方向的重要机制。组蛋白去乙酰化酶抑制剂(HDACi)通过影响不同亚类的组蛋白去乙酰化酶(HDAC)活性,提高组蛋白乙酰化水平,调控基因表达,从而影响胚胎干细胞自我更新,以及沿神经元、心肌和造血等细胞谱系的定向分化。HDACi类小分子化合物在体细胞重编程中也有广泛的应用,可替代致癌因子c-Myc和Klf4,促进体细胞克隆。研究显示,HDACi的效应与药物剂量、细胞类型和细胞分化状态密切相关。本文主要阐述了HDACi在干细胞分化和体细胞重编程中的应用进展,并对所涉及的分子通路进行讨论,有助于揭示干细胞定向分化的关键分子机制,优化干细胞定向分化诱导策略,对干细胞诱导分化具有重要的理论和实用价值。     

Promotion of haematopoietic activity in embryonic stem cells by the aorta-gonad-mesonephros microenvironment

We investigated whether the in vitro differentiation of ES cells into haematopoietic progenitors could be enhanced by exposure to the aorta-gonadal-mesonephros (AGM) microenvironment that is involved in the generation of haematopoietic stem cells (HSC) during embryonic development. We established a co-culture system that combines the requirements for primary organ culture and differentiating ES cells and showed that exposure of differentiating ES cells to the primary AGM region results in a significant increase in the number of ES-derived haematopoietic progenitors. Co-culture of ES cells on the AM20-1B4 stromal cell line derived from the AGM region also increases haematopoietic activity. We conclude that factors promoting the haematopoietic activity of differentiating ES cells present in primary AGM explants are partially retained in the AM20.1B4 stromal cell line and that these factors are likely to be different to those required for adult HSC maintenance.     

Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system

One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo.     

A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.     

The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism

Mouse pluripotent embryonic stem (ES) cells, once reintroduced into a mouse blastocyst, can contribute to the formation of all tissues, including the germline, of an organism referred to as a chimaeric. However, the reasons why this contribution often appears erratic are poorly understood. We have tested the notion that the chromosome make-up may be important in contributing both to somatic cell chimaerism and to germ line transmission. We found that the percentage of chimaerism of ES cell-embryo chimaeras, the absolute number of chimaeras and the ratio of chimaeras to total pups born all correlate closely with the percentage of euploid metaphases in the ES cell clones injected into the murine blastocyst. The majority of the ES cell clones that we tested, which were obtained from different gene targeting knockout experiments and harboured 50 to 100% euploid metaphases, did transmit to the germline; in contrast, none of the ES cell clones with more than 50% of chromosomally abnormal metaphases transmitted to the germline. Euploid ES cell clones cultured in vitro for more than 20 passages rapidly became severely aneuploid, and again this correlated closely with the percentage of chimaerism and with the number of ES cell--embryo chimaeras obtained per number of blastocysts injected. At the same time, the ability of these clones to contribute to the germline was lost when the proportion of euploid cells dropped below 50%. This study suggests that aneuploidy, rather than loss of totipotency, in ES cells, is the major cause of failure in obtaining contributions to all tissues of the adult chimaera, including the germline. Because euploidy is predictive of germline transmission, karyotype analysis is crucial and time/cost saving in any gene-targeting experiment     

Pluripotential competence of cells associated with Nanog activity

Nanog is a novel pluripotential cell-specific gene that plays a crucial role in maintaining the undifferentiated state of early postimplantation embryos and embryonic stem (ES) cells. We have explored the expression pattern and function of Nanog and a Nanog-homologue, Nanog-ps1.Nanog-ps1 was mapped on Chromosome 7 and shown to be a pseudogene. Immunocytochemical analysis in vivo showed that the NANOG protein was absent in unfertilized oocytes, and was detected in cells of morula-stage embryos, the inner cell mass of blastocysts and the epiblast of E6.5 and E7.5 embryos, but not in primordial germ cells of early postimplantation embryos. In monkey and human ES cells, NANOG expression was restricted to undifferentiated cells. Furthermore, reactivation of the somatic cell-derived Nanog was tightly linked with nuclear reprogramming induced by cell hybridization with ES cells and by nuclear transplantation into enucleated oocytes. Notably, mouse Nanog (+/-) ES cells, which produced approximately half the amount of NANOG produced by wild-type ES cells, readily differentiated to multi-lineage cells in culture medium including LIF. The labile undifferentiated state was fully rescued by constitutive expression of exogenous Nanog. Thus, the activity of Nanog is tightly correlated with an undifferentiated state of cells even in nuclear reprogrammed somatic cells. Nanog may function as a key regulator for sustaining pluripotency in a dose-dependent manner.