遗传修饰小鼠胚胎干细胞种系嵌合体小鼠的研制

利用显微注射的方法,分别将三株不同类型的经过遗传修饰的中靶ES细胞注射到C57BL/6J小鼠的囊胚中,通过胚胎移植将注射后的囊胚引入受体小鼠子宫中,分别获得了不同整合度的嵌合体小鼠,将高碳合度小鼠与C57BL/6J小鼠杂交,对这些仔鼠进行PCR及Southern鉴定的结果表明,三株修饰后的ES细胞均能整合入生殖系,得到了棕褐色子代鼠,表明获得了种系嵌合体小鼠。     

Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF)

The isolation of pluripotent murine embryonic stem (ES) cells has previously been achieved by coculturing the ES cells with fibroblast feeder cells. In this report we demonstrate that ES cell lines can be isolated from murine 129/Sv He blastocysts in the absence of feeder cells in culture medium supplemented with recombinant leukemia inhibitory factor (LIF). Three of the ES cell lines (MBL-1, MBL-2, and MBL-3) were isolated by directly explanting blastocysts, whilst two ES cell lines (MBL-4 and MBL-5) were isolated from blastocysts pretreated by immunosurgery. Three of the ES cell lines contained the Y chromosome (MBL-1, MBL-2, and MBL-5) with a high proportion of the cells displaying a normal diploid karyotype with a modal chromosome number of 40. All of the ES cell lines tested expressed the stem cell markers ECMA-7 and alkaline phosphatase, which were lost on removal of LIF when the ES cells differentiated into a variety of cell types. The full developmental potential of the ES cells was determined by injecting cells from two of the independently derived ES cell lines, MBL-1 and MBL-5, into C57BL/6J blastocysts. A high proportion of the pups born were chimeric as judged by coat pigmentation. Subsequent breeding established that the ES cells had contributed to the germ line. These results demonstrate that feeder cells are not essential for the isolation of pluripotent ES cell lines.     

Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state

Mouse embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family. In other mammals, this is not possible with LIF alone. Chicken ES-like cells (blastodermal cells) have only been cultured with mouse LIF because chicken LIF was not available. However the culture system is imperfect and chicken ES-like cells equivalent to mouse ES cells were not observed. In the present study, we cloned the cDNA-encoding chicken LIF using mRNA subtraction and RACE methodology. The chicken LIF cDNA encodes a protein with approximately 40% sequence identity to mouse LIF. It has 211 amino acids including a putative N-terminal signal peptide of 24 residues. Chicken blastodermal cells were cultured in the presence of bacterially expressed chicken LIF or mouse LIF. The expression of alkaline phosphatase and embryonal carcinoma cell monoclonal antibody-1 and stage-specific embryonic antigen-1 and the activation of STAT3 were examined, all of which are indices of the undifferentiated state. Exposure in the blastodermal cells to recombinant chicken LIF but not to mouse LIF maintained the expression of these various markers. After 9 days of incubation, the blastodermal cells formed cystic embryoid bodies in the presence of mouse LIF but not in the presence of recombinant chicken LIF. We conclude that chicken LIF is able to maintain chicken ES cell cultures in the undifferentiated state.     

High-grade transgenic somatic chimeras from chicken embryonic stem cells

Male and female embryonic stem (ES) cell lines were derived from the area pellucidae of Stage X (EG&K) chicken embryos. These ES cell lines were grown in culture for extended periods of time and the majority of the cells retained a diploid karyotype. When reintroduced into Stage VI-X (EG&K) recipient embryos, the cES cells were able to contribute to all somatic tissues. By combining irradiation of the recipient embryo with exposure of the cES cells to the embryonic environment in diapause, a high frequency and extent of chimerism was obtained. High-grade chimeras, indistinguishable from the donor phenotype by feather pigmentation, were produced. A transgene encoding GFP was incorporated into the genome of cES cells under control of the ubiquitous promoter CX and GFP was widely expressed in somatic tissues. Although cES cells made extensive contributions to the somatic tissues, contribution to the germline was not observed.     

Differentiation of embryonic stem cells into cardiomyocytes induced by leukemia inhibitory factor (LIF)

An experimental model of mouse embryonic stem cell (ESC) differentiation into cells with contractile activity (similar to that of cardiomyocytes) without embryoid body formation has been obtained. The main factor inducing ESC differentiation along the cardiomyocyte pathway is recombinant cytokine LIF added in the course of long-term culturing. The contractile cells respond positively to treatment with isoproterenol, a cardioactive drug, which is evidence for the presence in these cells of β-adrenoreceptors characteristic of terminally differentiated mammalian cardiomyocytes.     

Differentiation of embryonic stem cells into cardiomyocytes with the help of cytokine LIF (leukemia inhibitory factor)

An experimental model of differentiated mouse embryonic stem cells with retractive activity similar to that of cardiomyocytes without preliminary formation of embryoid bodies was obtained. The basic factor that induced in vitro embryonic cell differentiation into cardial type is the recombinant cytokine LIF under prolonged cultivation. The positive reaction of the cells with retractive activity to isoproterenol indicates the presence of the beta-adrenergic receptor activity characteristic only for terminal differentiated mammalian cardiomyocytes.     

CD9 is associated with leukemia inhibitory factor-mediated maintenance of embryonic stem cells

Mouse embryonic stem (ES) cells can proliferate indefinitely in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), or differentiate into all three germ layers upon removal of this factor. To determine cellular factors associated with self-renewal of undifferentiated ES cells, we used polymerase chain reaction-assisted cDNA subtraction to screen genes that are expressed in undifferentiated ES cells and down-regulated after incubating these cells in a differentiation medium without LIF for 48 h. The mRNA expression of a tetraspanin transmembrane protein, CD9, was high in undifferentiated ES cells and decreased shortly after cell differentiation. An immunohistochemical analysis confirmed that plasma membrane-associated CD9 was expressed in undifferentiated ES cells but low in the differentiated cells. Addition of LIF to differentiating ES cells reinduced mRNA expression of CD9, and CD9 expression was accompanied with a reappearance of undifferentiated ES cells. Furthermore, activation of STAT3 induced the expression of CD9, indicating the LIF/STAT3 pathway is critical for maintaining CD9 expression. Finally, addition of anti-CD9 antibody blocked ES cell colony formation and reduced cell viability. These results indicate that CD9 may play a role in LIF-mediated maintenance of undifferentiated ES cells.     

Formation of human prostate tissue from embryonic stem cells

Rodent models and immortalized or genetically modified cell lines are frequently used-but have limited utility-for studying human prostate development and maturation. Using rodent mesenchyme to establish reciprocal mesenchymal-epithelial cell interactions with human embryonic stem cells (hESCs), we generated human prostate tissue expressing prostate-specific antigen (PSA) within 8-12 weeks. This human prostate model shows species-conserved signalling mechanisms that could extend to integumental, gastrointestinal and genital tissues.     

Dodecaploid H1 embryonic stem cells abolished pluripotency in L15F10 medium both with and without leukemia inhibitory factor

Two lines of dodecaploid H1 embryonic stem cells, 12H1 and 12H1(?) cells (mouse-originated cells), were established through polyploidization of two hexaploid H1 cells, 6H1 and 6H1(?) cells, which were cultured in L15F10 (7:3) medium with and without leukemia inhibitory factor (LIF), respectively. The G₁, S, and G₂/M phase fractions of 12H1 and 12H1(?) cells were almost the same as those of 6H1 and 6H1(?) cells, respectively, but the doubling time of cell proliferation was prolonged, suggesting that cell death occurred in 12H1 and 12H1(?)cells. The cell volumes of 12H1 and 12H1(?) cells were about double those of 6H1 and 6H1(?) cells, respectively. 12H1 and 12H1(?) cells showed near-negative activity of alkaline phosphatase and no ability to form teratocarcinomas in mouse abdomen, suggesting that 12H1 and 12H1(?) cells lost pluripotency. The DNA contents of 12H1 and 12H1(?) cells decayed in long-term culturing, suggesting that 12H1 and 12H1(?) cells were DNA-unstable. Possible explanations for the lost pluripotency and for the DNA decay in 12H1 and 12H1(?) cells are presented.     

Gene expression in human neural stem cells: effects of leukemia inhibitory factor

Human neural precursor cells grown in culture provide a source of tissue for drug screening, developmental studies and cell therapy. However, mechanisms underlying their growth and differentiation are poorly understood. We show that epidermal growth factor (EGF) responsive precursors derived from the developing human cortex undergo senescence after 30-40 population doublings. Leukemia inhibitory factor (LIF) increased overall expansion rates, prevented senescence and allowed the growth of a long-term self renewing neural stem cell (ltNSCctx) for up to 110 population doublings. We established basal gene expression in ltNSCctx using Affymetrix oligonucleotide microarrays that delineated specific members of important growth factor and signaling families consistently expressed across three separate lines. Following LIF withdrawal, 200 genes showed significant decreases. Protein analysis confirmed LIF-regulated expression of glial fibrillary acidic protein, CD44, and major histocompatibility complex I. This study provides the first molecular profile of human ltNSCctx cultures capable of long-term self renewal, and reveals specific sets of genes that are directly or indirectly regulated by LIF.     

Development of embryonic stem cells in recombinant kidneys

Embryonic stem cells (ESC) are self-renewing and can generate all cell types during normal development. Previous studies have begun to explore fates of ESCs and their mesodermal derivatives after injection into explanted intact metanephric kidneys and neonatal kidneys maturing in vivo. Here, we exploited a recently described recombinant organ culture model, mixing fluorescent quantum dot labeled mouse exogenous cells with host metanephric cells. We compared abilities of undifferentiated ESCs with ESC-derived mesodermal or non-mesodermal cells to contribute to tissue compartments within recombinant, chimeric metanephroi. ESC-derived mesodermal cells downregulated Oct4, a marker of undifferentiated cells, and, as assessed by locations of quantum dots, contributed to Wilms’ tumor 1-expressing forming nephrons, synaptopodin-expressing glomeruli, and organic ion-transporting tubular epithelia. Similar results were observed when labeled native metanephric cells were recombined with host cells. In striking contrast, non-mesodermal ESC-derived cells strongly inhibited growth of embryonic kidneys, while undifferentiated ESCs predominantly formed Oct4 expressing colonies between forming nephrons and glomeruli. These findings clarify the conclusion that ESC-derived mesodermal cells have functional nephrogenic potential, supporting the idea that they could potentially replace damaged epithelia in diseased kidneys. On the other hand, undifferentiated ESCs and non-mesodermal precursors derived from ESCs would appear to be less suitable materials for use in kidney cell therapies.     

Effective production of microinjectable blastocysts for germ-line transmission of embryonic stem cells.

Blastocysts of C57BL/6 mice obtained either 2.5 or 3.5 days post-coitum (dpc) were examined for efficient microinjection after overnight in vitro culture. Incidences of zona-free embryos were much higher at 3.5 dpc after natural mating (1.05/mouse) and superovulation (2.83/mouse) than at 2.5 dpc after natural mating (0.05/mouse) and superovulation (0.01/mouse). By testing germ-line competency of gene-targeted J1 embryonic stem cells, superovulation and/or in vitro culture should be recommended for producing microinjectable blastocysts for production of high germ-line chimeras.     

Production of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos

The production of mouse chimeras is a common step in the establishment of genetically modified animal strains. Chimeras also provide a powerful experimental tool for following cell behavior during both prenatal and postnatal development. This protocol outlines a simple and economical technique for the production of large numbers of mouse chimeras using traditional diploid morula<-->diploid embryonic stem (ES) cell aggregations. Additional steps are included to describe the procedures necessary to produce specialized tetraploid chimeras using tetraploid morula<-->diploid ES cell aggregations. This increasingly popular form of chimera produces embryos of nearly complete ES cell derivation that can be used to speed transgenic production or ask developmental questions. Using this protocol, mouse chimeras can be generated and transferred to pseudopregnant surrogate mothers in a 5-d period.     

Comparison of aggregation and injection techniques in producing chimeras with embryonic stem cells in mice

We analyzed embryonic stem cell lines for their capacity to produce aggregation chimeras with diploid or developmentally compromised tetraploid embryos. Descendants of embryonic stem cells which contributed to midgestation fetuses at high levels were capable of supporting fetal development also with tetraploid partners. Different numbers of embryonic stem cells were introduced into diploid and tetraploid morulae as well as into blastocysts by microinjection. There were no differences in the frequency of embryonic stem cell-containing fetuses when comparing aggregation or injection into morulae versus blastocysts. However, the distribution pattern of embryonic stem cell derivatives in chimeric fetuses suggested that pre-compaction embryos are more suitable for generating fetuses with high embryonic stem cell contribution. Injection of embryonic stem cells into tetraploid embryos showed that completely embryonic stem cell-derived fetuses can also be produced by this technique. Totally embryonic stem cell derived fetuses were observed in each group, when embryonic stem cells were injected into diploid embryos. However, the rate of chimeras and chimerism was lower when 1 or 3 embryonic stem cells were used versus 8 or 15 cells. This suggests that the number of embryonic stem cells introduced might play a role in the colonization ability.     

调控胚胎干细胞生长的转录因子

胚胎干细胞的生长是由一个极其复杂的网络系统调控的,本文简要叙述了Oct-4、Nanog、Sox2三个转录因子对胚胎干细胞生长的调控作用,为将来更好的开发利用胚胎干细胞资源奠定理论基础.