Formation of germ-line chimeras from embryonic stem cells maintained with recombinant leukemia inhibitory factor

Murine embryonic stem (ES) cells can be maintained as stem cells in vitro only in the presence of feeder cells or a soluble factor produced by a number of cell lines. We have previously demonstrated that leukemia inhibitory factor (LIF) is the molecule which prevents ES cell differentiation in culture. In this report we demonstrate that recombinant LIF can substitute for feeder cells in maintaining the full developmental potential of ES cells. The totipotent D3 ES cell line, previously isolated and maintained on growth-arrested primary embryo fibroblasts, was transferred to media supplemented with 1000 U/ml (10 ng/ml) recombinant LIF. In the presence of LIF the ES cells were maintained for over 2 months as undifferentiated cells in the absence of any feeder cells. When injected into blastocysts the ES cells which had been maintained in LIF-supplemented media efficiently formed germ-line chimeras.     

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.     

CD9 correlates with cancer stem cell potentials in human B-acute lymphoblastic leukemia cells

Cancer stem cell (CSC) theory suggests that only a small subpopulation of cells having stem cell-like potentials can initiate tumor development. While recent data on acute lymphoblastic leukemia (ALL) are conflicting, some studies have demonstrated the existence of such cells following CD34-targeted isolation of primary samples. Although CD34 is a useful marker for the isolation of CSCs in leukemias, the identification of other specific markers besides CD34 has been relatively unsuccessful. To identify new markers, we first performed extensive analysis of surface markers on several B-ALL cell lines. Our data demonstrated that every B-ALL cell line tested did not express CD34 but certain lines contained cell populations with marked heterogeneity in marker expression. Moreover, the CD9⁺ cell population possessed stem cell characteristics within the clone, as demonstrated by in vitro and transplantation experiments. These results suggest that CD9 is a useful positive-selection marker for the identification of CSCs in B-ALL.     

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.     

5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells

Background  

5-Hydroxymethylcytosine (5hmC) was recently found to be abundantly present in certain cell types, including embryonic stem cells. There is growing evidence that TET proteins, which convert 5-methylcytosine (5mC) to 5hmC, play important biological roles. To further understand the function of 5hmC, an analysis of the genome-wide localization of this mark is required.     

bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling

Background

Human pluripotent stem cells (PSCs) open new windows for basic research and regenerative medicine due to their remarkable properties, i.e. their ability to self-renew indefinitely and being pluripotent. There are different, conflicting data related to the role of basic fibroblast growth factor (bFGF) in intracellular signal transduction and the regulation of pluripotency of PSCs. Here, we investigated the effect of bFGF and its downstream pathways in pluripotent vs. differentiated human induced (hi) PSCs.

Methods

bFGF downstream signaling pathways were investigated in long-term culture of hiPSCs from pluripotent to differentiated state (withdrawing bFGF) using immunoblotting, immunocytochemistry and qPCR. Subcellular distribution of signaling components were investigated by simple fractionation and immunoblotting upon bFGF stimulation. Finally, RAS activity and RAS isoforms were studied using RAS assays both after short- and long-term culture in response to bFGF stimulation.

Results

Our results revealed that hiPSCs were differentiated into the ectoderm lineage upon withdrawing bFGF as an essential pluripotency mediator. Pluripotency markers OCT4, SOX2 and NANOG were downregulated, following a drastic decrease in MAPK pathway activity levels. Notably, a remarkable increase in phosphorylation levels of p38 and JAK/STAT3 was observed in differentiated hiPSCs, while the PI3K/AKT and JNK pathways remained active during differentiation. Our data further indicate that among the RAS paralogs, NRAS predominantly activates the MAPK pathway in hiPSCs.

Conclusion

Collectively, the MAPK pathway appears to be the prime signaling pathway downstream of bFGF for maintaining pluripotency in hiPSCs and among the MAPK pathways, the activity of NRAS-RAF-MEK-ERK is decreased during differentiation, whereas p38 is activated and JNK remains constant.

     

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.     

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.     

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.     

CD74 is a regulator of hematopoietic stem cell maintenance

Hematopoietic stem and progenitor cells (HSPCs) are a small population of undifferentiated cells that have the capacity for self-renewal and differentiate into all blood cell lineages. These cells are the most useful cells for clinical transplantations and for regenerative medicine. So far, it has not been possible to expand adult hematopoietic stem cells (HSCs) without losing their self-renewal properties. CD74 is a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF), and its mRNA is known to be expressed in HSCs. Here, we demonstrate that mice lacking CD74 exhibit an accumulation of HSCs in the bone marrow (BM) due to their increased potential to repopulate and compete for BM niches. Our results suggest that CD74 regulates the maintenance of the HSCs and CD18 expression. Its absence leads to induced survival of these cells and accumulation of quiescent and proliferating cells. Furthermore, in in vitro experiments, blocking of CD74 elevated the numbers of HSPCs. Thus, we suggest that blocking CD74 could lead to improved clinical insight into BM transplant protocols, enabling improved engraftment.

Hematopoietic stem and progenitor cells (HSPCs) can self-renew and differentiate into all blood cell lineages, making them useful for clinical transplantations and regenerative medicine. This study shows that blocking the MIF receptor CD74 increases the accumulation of HSPCs and could improve the efficacy of bone marrow transplantation protocols.     

CD146 expression on mesenchymal stem cells is associated with their vascular smooth muscle commitment

Bone marrow mesenchymal stem cells (MSCs) are plastic adherent cells that can differentiate into various tissue lineages, including osteoblasts, adipocytes and chondrocytes. However, this progenitor property is not shared by all cells within the MSC population. In addition, MSCs vary in their proliferation capacity and expression of markers. Because of heterogeneity of CD146 expression in the MSC population, we compared CD146^−/Low and CD146^High cells under clonal conditions and after sorting of the non-clonal cell population to determine whether this expression is associated with specific functions. CD146^−/Low and CD146^High bone marrow MSCs did not differ in colony-forming unit-fibroblast number, osteogenic, adipogenic and chondrogenic differentiation or in vitro haematopoietic-supportive activity. However, CD146^−/Low clones proliferated slightly but significantly faster than did CD146^High clones. In addition, a strong expression of CD146 molecule was associated with a commitment to a vascular smooth muscle cell (VSMC) lineage characterized by a strong up-regulation of calponin-1 and SM22α expression and an ability to contract collagen matrix. Thus, within a bone marrow MSC population, certain subpopulations characterized by high expression of CD146, are committed towards a VSMC lineage.     

Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture

Fibroblast feeder cells play an important role in supporting the derivation and long term culture of undifferentiated, pluripotent human embryonic stem cells (hESCs). The feeder cells secrete various growth factors and extracellular matrix (ECM) proteins into extracellular milieu. However, the roles of the feeder cell-secreted factors are largely unclear. Animal feeder cells and use of animal serum also make current feeder cell culture conditions unsuitable for derivation of clinical grade hESCs. We established xeno-free feeder cell lines using human serum (HS) and studied their function in hESC culture. While human foreskin fibroblast (hFF) feeder cells were clearly hESC supportive, none of the established xeno-free human dermal fibroblast (hDF) feeder cells were able to maintain undifferentiated hESC growth. The two fibroblast types were compared for their ECM protein synthesis, integrin receptor expression profiles and key growth factor secretion. We show that hESC supportive feeder cells produce laminin-511 and express laminin-binding integrins α3ß1, α6ß1 and α7ß1. These results indicate specific laminin isoforms and integrins in maintenance of hESC pluripotency in feeder-dependent cultures. In addition, several genes with a known or possible role for hESC pluripotency were differentially expressed in distinct feeder cells.     

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.     

Modified hyaluronan hydrogels support the maintenance of mouse embryonic stem cells and human induced pluripotent stem cells

These studies provide evidence for the ability of a commercially available, defined, hyaluronan-gelatin hydrogel, HyStem-C?, to maintain both mouse embryonic stem cells (mESCs) and human induced pluripotent stem cells (hiPSCs) in culture while retaining their growth and pluripotent characteristics. Growth curve and doubling time analysis show that mESCs and hiPSCs grow at similar rates on HyStem-C? hydrogels and mouse embryonic fibroblasts and Matrigel?, respectively. Immunocytochemistry, flow cytometry, gene expression and karyotyping reveal that both human and murine pluripotent cells retain a high level of pluripotency on the hydrogels after multiple passages. The addition of fibronectin to HyStem-C? enabled the attachment of hiPSCs in a xeno-free, fully defined medium.