Relationship between the ability to support differentiation of osteoclast-like cells and adipogenesis in murine stromal cells derived from bone marrow

In vitro osteoclast differentiation is supported by stromal cells. In order to isolate a stromal cell line that can support osteoclast differentiation, 22 cell lines were cloned from mouse bone marrow. One of these clones, TMS-14, is a line of preadipocytes that supports osteoclast-like cell formation without any bone resorbing factors; and another, TMS-12, is a line of preosteoblasts that supports osteoclast-like cell formation with bone resorbing factors such as prostaglandin E(2)(PGE(2)). The difference of these two lines for osteoclast formation was not related with their abilities of PGE(2)production, but with the expression of osteoclast differentiation factor (ODF, also called OPGL, RANKL, and TRANCE), which detected with RT-PCR, in both cell lines. In TMS-14 cells, ODF mRNA was detected with or without PGE(2). In TMS-12 cells, ODF expression was detected in the PGE(2)-treated cells alone. When TMS-14 cells were induced to undergo adipogenic differentiation in response to treatment with thiazolidinedione, a ligand and activator of peroxisome proliferator-activated receptor gamma (PPARgamma), the ability of TMS-14 cells to support osteoclast-like cell formation was prevented in the presence or absence of 1,25(OH)(2)D(3). The gene expression of ODF in TMS-14 cells was also inhibited by treatment with thiazolidinedione. These results suggest that adipogenesis in bone marrow cells is related to the ability to support osteoclast differentiation. This is the first report of a cloned stromal cell line that can support osteoclastogenesis without the treatment with any osteotropic factors. Furthermore, this murine clonal preadipose cell line may be useful for studying senescence-dependent osteoporosis.     

Establishment and characterization of stromal cell lines that support differentiation of murine hematopoietic blast cells into osteoclast-like cells

Summary This study aimed to establish and characterize a new stromal cell line that supports the proliferation of hematopoietic blast cells and their differentiation into osteoclast-like cells. Cells isolated from the calvaria of neonatal Balb/c mice were subcultured every 2 to 4 days at 1.2×10⁴ cells/cm². After 18 passages the cells had become immortalized and were designated as MCHT-1. MCHT-1 cells were found to support the proliferation of hematopoietic blast cells and their differentiation into osteoclast-like cells when these two cells were co-cultured in the presence of 1α,25(OH)₂D₃ and dexamethasone. However, because the MCHT-1 cells showed heterogeneity, cloning was performed and each clone was characterized. All the clones obtained supported the proliferation of hematopoietic blast cells and their differentiation into osteoclast-like cells irrespective of their obvious differences in growth capacities and cytochemical characteristics. However, the time-course of the appearance of osteoclast-like cells differed among clones. The supportive effect of these clonal stromal cells on differentiation of hematopoietic blast cells into osteoclast-like cells was completely dependent on the presence of 1α,25(OH)₂D₃ and dexamethasone. These clonal MCHT-1 cells are expected to be useful for precise analysis of the proliferation and differentiation of osteoclasts.     

Vascular cell adhesion molecule-1 expression and hematopoietic supportive capacity of immortalized murine stromal cell lines derived from fetal liver and adult bone marrow

Ontogeny-specific differences in hematopoietic behavior may be influenced by unique adhesive interactions between hematopoietic cells and the microenvironment, such as that mediated by vascular cell adhesion molecule-1 (VCAM-1, CD 106). Although VCAM-1 is variably expressed during vertebrate development, we hypothesized that VCAM-1 expression might be linked to the enhanced capacity of the fetal liver microenvironment to support hematopoiesis. To test this we used immortalized murine stromal cell lines derived from midgestation fetal liver and adult bone marrow to compare the functional expression of VCAM-1. Molecular analysis of VCAM-1 expression was performed on stromal cell lines using Northern blot analysis, immunoprecipitation studies, and solid-phase enzyme-linked immunosorbent assay. Hematopoietic studies were performed by coculturing fetal liver cells with stromal cell lines, and the functional readout was determined by high-proliferative potential colony-forming cell (HPP-CFC) adherence assays. In contrast to our initial hypothesis, we observed greater expression of VCAM-1 messenger ribonucleic acid and protein on an adult marrow stromal cell line. In functional studies, anti-VCAM-1 antibody inhibited the binding of nearly half of the HPP-CFCs to adult marrow stroma but had a minimal effect on their binding to fetal liver stroma, despite the greater adherence of HPP-CFCs to fetal stroma. We conclude that VCAM-1 influences the hematopoietic supportive capacity of immortalized murine stroma derived from adult bone marrow. Our studies suggest that cellular interactions other than those mediated by VCAM-1 are involved in the increased adhesive capacity of immortalized murine stroma derived from fetal liver.     

Effects of different Sertoli cell types on the maintenance of adult spermatogonial stem cells in vitro

Spermatongonial stem cells (SSCs) are unique testis cells that are able to proliferate, differentiate, and transmit genetic information to the next generation. However, the effect of different Sertoli cell types on the expression of specific SSC genes is not yet well understood. In this study, we compare the in vitro effect of adult Sertoli cells, embryonic Sertoli cells, and TM4 (a Sertoli cell line) as feeder layers on the expression of SSC genes. SSCs were isolated from the testis of adult male mice and purified by differential plating. Following enrichment, SSCs were cultivated for 1 and 2 wk in the presence of various feeders. The expression of SSC-specific genes (Mvh, ZBTB, and c-kit) was evaluated by real-time polymerase chain reaction. Our results revealed that expression of the specific SSC genes was significantly higher in the embryonic Sertoli cells after 1 and 2 wk compared to the adult Sertoli cells and the TM4 group. Our finding suggest that co-culturing of SSCs with embryonic Sertoli cells is helpful for in vitro cultivation of SSCs and might improve the self-renewal of these stem cells.     

Quantitative and qualitative in vitro analysis of the stem cell potential of hematopoietic cells purified from murine skeletal muscle

The murine skeletal muscle contains hematopoietic stem cells, but this potential has so far not been studied quantitatively or qualitatively in vitro. To quantify the hematopoietic stem cell potential, we have used highly purified SP/CD45^＋ cells in long-term culture initiating cell （LTC-IC） assays. The SP/CD45^＋ cell population purified from murine muscle was found to have significant stem cell activity with an LTC-IC frequency of 1/640. Single-cell-sorted SP/CD45^＋ cells from muscle exhibited robust proliferative activity in vitro at day 16 （380-fold amplification）, especially after culture with OP-9 layers that also support embryonic stem cells. Amplified cell populations originating from single cells exhibited multilineage differentiation ability with evidence of myeloid, lymphoid and NK cell markers. Thus, our results demonstrate that hematopoietic stem cells that can be quantified by LTC-IC assays exist in the murine skeletal muscle and show also for the first time, at the single-cell level, that these cells exhibit multilineage differentiation ability and major proliferative potential.     

Comparison of adult stem cells derived from multiple stem cell niches

Objectives

Adult stem cells (ASCs) have great potential for tissue regeneration; however, comparative studies of ASCs from different niches are required to understand the characteristics of each population for their potential therapeutic uses.

Results

We compared the proliferation, stem cell marker expression, and differentiation potential of ASCs from bone marrow, skin dermis, and adipose tissue. ASCs from bone marrow and skin dermis showed 50–100 % increased proliferation in comparison to the ASCs from adipose tissues. Furthermore, ASCs from each stem cell niche showed differential expression of stem cell marker genes, and preferentially differentiated into cell types of their tissue of origin.

Conclusion

Different characters of each ASC might be major factors for their effective use for therapeutics and tissue regeneration.

     

Derivation of hematopoietic stem cells from murine embryonic stem cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny. Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability. Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types. The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol. HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders. However, many patients that might benefit from HSC therapy lack access to suitable donors. ESC could provide an alternative source of HSC for these patients. The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC. In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation. EBs are then dissociated and infected with retroviral HoxB4. Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days. During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100 s of millions of cells. These cells can then be used to rescue and reconstitute lethally irradiated mice.     

Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues

Mesenchymal stem cells (MSCs), which evoke only minimal immune reactivity, may have anti-inflammatory and immunomodulatory effects. In this study, we conducted a comparative analysis of the immunomodulatory properties of MSCs derived from adult human tissues including bone marrow (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Wharton’s jelly (WJ). Using a multiple cytokine detection assay, we showed that there were no significant differences in levels of secreted factors from non-stimulated MSCs. We compared the immunosuppressive effect of BM-MSCs, AT-MSCs, CB-MSCs, and WJ-MSCs on phytohemagglutinin-induced T-cell proliferation. AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed mitogen-induced T-cell proliferation as effectively as did BM-MSCs. Levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with each type of MSCs. In addition, the expression of hepatocyte growth factor, IL-10, transforming growth factor-β₁, cyclooxygenase (COX)-1, and COX-2 were unchanged in MSCs treated with IFN-γ and/or TNF-α, while indoleamine 2,3-dioxygenase (IDO) expression increased. IFN-γ and/or TNF-α produced by activated T-cells were correlated with induction of IDO expression by MSCs, which, in turn, suppressed T-cell proliferation. These findings suggest that MSCs derived from AT, CB, or WJ could be substituted for BM-MSCs for treatment of allogeneic conflicts.     

Human bone marrow stromal cell lines from myeloma and rheumatoid arthritis that can support murine pre-B cell growth.

In order to elucidate the pathologic significance of the bone marrow (BM) microenvironment in multiple myeloma (MM) and rheumatoid arthritis (RA), we established patient- or healthy donor (HD)-derived BM stromal cell lines by transfecting the plasmid for expression of SV40 large T Ag and examined their ability to support the stromal cell-dependent growth of a pre-B cell line, DW34. The means of recovered cell numbers of DW34 co-cultured with MM- and RA-derived BM stromal cell lines ranged from 6- to 10-fold more than those with HD-derived ones. Their enhanced ability to support DW34 cell growth was not caused by cytokines, including IL-6, IL-7, and c-kit ligand, although exogenous IL-7 could augment the growth-supporting ability. DW34 cell growth on the stromal cell lines was abolished by inhibiting cell-to-cell interaction with a membrane filter. FACS analysis revealed that the stromal cell lines did not express LFA-1 alpha, beta, NCAM, or ELAM-1. Both patient and HD BM stromal cell lines variably expressed ICAM-1, VCAM-1, and CD44. However, surface expression levels of these molecules did not correlate with the ability of the stromal cell lines to support DW34 cell growth. Taken together, these results suggested that BM microenvironment might play important roles in the pathogenesis of MM and RA.     

In vitro inhibition of murine hematopoietic progenitors and stromal cells by vinorelbine

Hematopoietic progenitor colony assays were used to establish the effects of the vinca alkaloid vinorelbine (VRB) on murine bone marrow. The in vitro growth of colony-forming units–granulocyte/macrophage (CFU-GM), burst forming units–erythroid (BFU-E) and colony-forming units–mix (CFU-mix) was dose-dependently inhibited by VRB. The highest dose assayed (0.02 μg/ml) suppressed all of the different progenitor cells by 100%. A comparison of the dose–response curves showed that CFU-GM, BFU-E, and CFU-mix exhibited similar patterns of sensitivity to the cytotoxic action of VRB. Long-term bone marrow cultures have provided a valuable in vitro model for studying the role of the microenvironment of bone marrow. Cellularity of stromal layers was reduced with increasing doses of VRB. The appearence of these layers was altered minimally with the lowest dose used; a gradual loss of cellularity was seen in cultures exposed to 0.05 and 0.075 μg/ml; and a marked loss at the dose of 0.1 μg/ml. Our results show that VRB has an important effect on hematopoietic progenitors at the highest dose tested, while the stromal cells were not affected at a similar dose (0.025 μg/ml), suggesting that the stroma is more resistant to this drug. This revised version was published online in July 2006 with corrections to the Cover Date.     

人胎儿造血组织来源间充质干细胞基本生物学特性的比较

目的:分离、纯化人胎儿造血组织来源的胎儿骨髓间充质干细胞(MSCs derived from human fetal bone marrow,hfBM-MSCs)和胎儿肝脏间充质干细胞(MSCs derived from human fetal liver,hfL-MSCs),比较其基本生物学特性.方法:取材12-20周流产胎儿,冲洗骨髓腔得到全骨髓细胞悬液;通过二步离心联合羟乙基淀粉沉淀法富集肝非实质细胞,接种、培养、纯化MSCs.绘制MSCs生长曲线.检测MSCs表面标志、细胞周期.诱导MSCs向脂肪细胞分化并鉴定.结果:hfBM-MSCs与hfL-MSCs均为平行排列的纤维状细胞,但前者较细长,后者稍宽大;hfBM-MSCs与hfL-MSCs的原代培养时间、原代培养细胞数有显著性差异:而第二代细胞数无显著性差异;二者生长曲线相似,但hfBM-MSCs的增殖速度较快;细胞周期分析显示G0-G1期细胞在hfBM-MSCs与hfL-MSCs分别占96.1%和91.2%.流式检测结果显示HLA-DR、CD40和CD80在hfBM-MSCs与hfL-MSCs均极低表达,而后者更低;hfBM-MSCs与hfL-MSCs均可成脂分化,但前者更容易向脂肪样细胞分化.结论:在相同时间内,从胎儿骨髓中可以获得更多的MSCs;hfBM-MSCs具有较大的增殖潜能、增殖速度以及分化能力,而hfL-MSCs的免疫原性更弱.     

In vitro development of murine T cells from prethymic and preliver embryonic yolk sac hematopoietic stem cells.

Mature T cells are derived from prethymic stem cells, which arise at one or more extrathymic sites and enter and differentiate in the thymus. The nature of these prethymic stem cells is a critical factor for the formation of the T-cell repertoire. Although the bone marrow of adult mice can provide such stem cells, their origin during murine embryogenesis is still undetermined. Among potential sites for these progenitor cells are the fetal liver and the embryonic yolk sac. Our studies focus on the yolk sac, both because the yolk sac appears earlier than any other proposed site, and because the mammalian yolk sac is the first site of hematopoiesis. Although it has been shown that the yolk sac in midgestation contains stem cells that can enter the thymic rudiment and differentiate toward T-cell lineage, our aim was to analyze the developmental potential of cells in the yolk sac from earlier stages, prior to the formation of the liver and any other internal organ. We show here that the yolk sac from 8- and 9-day embryos (2-9 and 13-19 somites, respectively) can reconstitute alymphoid congenic fetal thymuses and acquire mature T-cell-specific characteristics. Specifically, thymocytes derived from the early embryonic yolk sac can progress to the expression of mature T lymphocyte markers including CD3/T-cell receptor (TCR), CD4 and CD8. In contrast, we have been unable to document the presence of stem cells within the embryo itself at these early stages. These results support the hypothesis that the stem cells capable of populating the thymic rudiment originate in the yolk sac, and that their presence as early as at the 2- to 9-somite stage may indicate that prethymic stem cells found elsewhere in the embryo at later times may have been derived by migration from this extra-embryonic site. Our experimental design does not exclude the possibility of multiple origins of prethymic stem cells of which the yolk sac may provide the first wave of stem cells in addition to other later waves of cells.     

Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.     

Human mesenchymal and murine stromal cells support human lympho-myeloid progenitor expansion but not maintenance of multipotent haematopoietic stem and progenitor cells

A major goal in haematopoietic stem cell (HSC) research is to define conditions for the expansion of HSCs or multipotent progenitor cells (MPPs). Since human HSCs/MPPs cannot be isolated, NOD/SCID repopulating cell (SRC) assays emerged as the standard for the quantification of very primitive haematopoietic cell. However, in addition to HSCs/MPPs, lympho-myeloid primed progenitors (LMPPs) were recently found to contain SRC activities, challenging this assay as clear HSC/MPP readout. Because our revised model of human haematopoiesis predicts that HSCs/MPPs can be identified as CD133⁺CD34⁺ cells containing erythroid potentials, we investigated the potential of human mesenchymal and conventional murine stromal cells to support expansion of HSCs/MPPs. Even though all stromal cells supported expansion of CD133⁺CD34⁺ progenitors with long-term myeloid and long-term lymphoid potentials, erythroid potentials were exclusively found within erythro-myeloid CD133^lowCD34⁺ cell fractions. Thus, our data demonstrate that against the prevailing assumption co-cultures on human mesenchymal and murine stromal cells neither promote expansion nor maintenance of HSCs and MPPs.     

Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and, (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes (alpha-globin, betaH-1 globin, beta-major globin, epsilon -globin, and zeta-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, epsilon-globin, gamma-globin, betaH1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of secondary EB origin cultured in media containing dexamethasone showed a down-regulation of GATA-3 and an up-regulation of GATA-1, Flk-1, and Epo-R in comparison to the two other cytokines and growth factor combinations containing media. The secondary differentiation also showed an enhanced production of erythrocytic precursors in dexamethasone containing media in comparison to that in the control media. Our results indicate that dexamethasone can prove to be an effective agent which can be employed to enhance differentiation towards erythrocytic progenitors from ES cells.