兔骨髓间充质细胞分化为肌细胞的实验研究

目的　探讨兔骨髓间充质细胞 (BMSCs)体外分化为肌细胞的可能性。方法　穿刺法抽取成年新西兰兔双侧股骨骨髓 ,分离培养BMSCs ,5 -溴脱氧尿苷 (bromod eoxyuridine)标记 ,再经过 5一氮杂胞苷 (5 azacytidine)诱导或与乳鼠心肌细胞共培养 ,通过免疫细胞化学染色及透射电镜检查 ,研究其分化情况。结果　体外培养的BMSCs无论是在 5 aza诱导还是在与心肌细胞共培养的条件下 ,均有部分细胞表达横纹肌肌动蛋白 (α actin)。电镜检查显示分化后的细胞具有原始肌小节样结构。结论　成年兔BMSCs在 5 aza诱导或与心肌细胞共培养的条件下均可向肌细胞方向分化     

Production of hematopoietic stem cell-chemotactic factor by bone marrow stromal cells

Chemotactic factors produced by stromal cells in the bone marrow are characterized. Two kinds of factors produced by stromal cell lines are identified using blind-well Boyden's Chambers; one is a neutrophil- chemotactic factor and the other a hematopoietic stem cell (HSC)- chemotactic factor. The latter attracts blastic cells in a low-density fraction, which are Thy1lo, wheat germ agglutinin (WGA)hi, H-2Khi, Ly-1- , Ly-2-, L3T4-, Ly5-, and slg-. The molecular weight of this HSC- chemotactic factor is estimated to be more than 200 kD. Putative cytokines and growth factors, such as granulocyte colony-stimulating factor (CSF), macrophage CSF, granulocyte-macrophage CSF, stem cell factor (SCF), interleukin (IL)-6, and IL-3, do not possess HSC- chemotactic activity. These findings strongly suggest that bone marrow stromal cells produce a new factor that attracts HSCs.     

Formation of hematopoietic territories and bone by transplanted human bone marrow stromal cells requires a critical cell density

OBJECTIVE: Bone marrow stromal cells (BMSCs) include multipotent cells with the ability to form mature bone organs upon in vivo transplantation. Hematopoiesis in these bone organs has been ascribed to the action of skeletal stem cells, which are capable of differentiating towards bone and hematopoiesis-supporting stroma. Yet, the creation of hematopoietic territories may be in part a natural consequence of the formation of a sufficiently mature and large bone microenvironment. Here, we describe, for the first time, a relationship between BMSC numbers and the extent of bone/hematopoiesis formation in heterotopic transplants. METHODS: Human BMSCs were transplanted along with hydroxyapatite/tricalcium phosphate, utilizing a spectrum of dosages, into immunotolerant mice; the transplants were followed for up to 29 months. RESULTS: The extent of bone and hematopoiesis formation increased with increasing BMSC numbers; however, the relationship was sigmoid in character, and a threshold number of BMSCs was necessary for extensive bone formation or any hematopoiesis. Hematopoiesis only occurred in conjunction with extensive bone formation, and no hematopoiesis occurred where bone formation was poor. Consistent with our earlier studies of long-term BMSC transplantation, the transplants underwent a change in bone morphology but not bone content after 8 weeks. CONCLUSION: Our results have provided evidence that the formation of both hematopoiesis and a mature bone organ is as much a consequence of a sufficiently high local density of bone marrow stromal cells as it is the product of skeletal stem cell action.     

Fibronectin-mediated attachment of hematopoietic cells to stromal elements in continuous bone marrow culture

Hematopoiesis in continuous bone marrow culture is dependent upon close-range interaction between hematopoietic cells and marrow-derived adherent cells. The latter have been shown to include a significant proportion of fibroblastic elements, synthesizing the attachment protein fibronectin. The role of fibronectin in continuous bone marrow culture was therefore investigated. Continuous marrow cultures were stained by the peroxidase-anti-peroxidase technique, using an affinity-purified anti-fibronectin antibody. The stained cultures were then examined by transmission electron microscopy for the presence of fibronectin at sites of cellular attachment. Fibronectin was detected on the substratum attachment surfaces of marrow-derived adherent cells and at sites of interaction between stromal elements in the adherent layer. Fibronectin was also detected at many attachment sites between marrow-derived adherent cells and developing granulocytes and monocytes. The possible significance of these observations is discussed in relation to the hematopoietic microenvironment.     

Monocytes stimulate fibroblastoid bone marrow stromal cells to produce multilineage hematopoietic growth factors

In previous studies we have found that monocytes produce soluble factors that stimulate human umbilical vein endothelial cells to produce granulocyte-macrophage colony-stimulating activity (CSA), burst- promoting activity (BPA), and megakaryocyte colony-stimulating activity (Meg-CSA) as well as factors that stimulate T lymphocytes and neonatal fibroblasts to produce CSA. To test the hypothesis that monocytes would similarly stimulate the production of hematopoietic growth factors by autologous bone marrow stromal cells, multiply-passaged adherent fibroblastoid cells derived from the bone marrow of normal volunteers were exposed to conditioned media prepared by incubating autologous peripheral blood monocytes in complete medium for three days. When conditioned media from stromal cells incubated in monocyte-conditioned medium were compared with those of stromal cells cultured in the absence of monocyte-conditioned medium, BPA was increased fourfold and CSA was increased more than 30-fold. We conclude that mononuclear phagocytes recruit stromal cells of the marrow to produce multilineage growth factors in vitro. We suggest that these monocyte-derived recruiting activities may play an important role in orchestration of hematopoietic growth factor production by cells of the marrow microenvironment.     

Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates

In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.     

Ultraviolet B light inactivates bone marrow T lymphocytes but spares hematopoietic precursor cells

Bone marrow cells from ten normal donors were exposed to ultraviolet (UV)C or UVB light for total exposures of 0.1 to 100 mJ/cm2, and assayed for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and phytohemagglutinin (PHA)- stimulated proliferative responses. After exposure to UVC CFU-GM, BFU-E and PHA responses showed a UV dose-dependent sharp decrease to levels less than 1% of controls with 0.5, 2.0, and 10 mJ/cm2, respectively. With UVB, PHA responses were most sensitive, declining to less than 1% at 5 mJ/cm2. BFU-E decreased to less than 1% of control with 15 mJ/cm2 UVB. CFU-GM, at UVB doses of 0.1 to 2.0 mJ/cm2, increased to 125% to 130% of control and decreased to less than 1% only at exposures greater than 20 mJ/cm2. Thus, these studies show that UVB, but not UVC light, can be used to inactivate bone marrow T lymphocytes selectively while sparing hematopoietic precursor cells. The data suggest that UVB irradiation can be used for T-lymphocyte purging for allogeneic marrow transplantation.     

The effect of bone marrow transplantation on the osteoblastic differentiation of human bone marrow stromal cells.

Osteoporosis is a serious and relatively common complication of transplantation procedures. However, little is known about the exact mechanism or severity of osteoporosis complicated by bone marrow transplantation (BMT). We conducted both ex vivo and clinical studies to identify the mechanism and extent of bone loss after BMT. In a prospective clinical study, we intended to identify the changes in bone turnover markers and bone mineral density (BMD) after BMT. During a 1-yr follow-up, BMD was measured before BMT and 1 yr after BMT in 67 patients undergoing BMT. Biochemical markers of bone formation and resorption were measured in all patients at short-term intervals during the yearlong follow-up. In ex vivo study, we cultured human bone marrow cells of normal controls and BMT recipients in osteogenic medium and compared their osteogenic potential. Using a DNA fingerprinting method, we also investigated the origin of bone marrow stromal cells that were harvested 3-4 wk after BMT. In a clinical study of 67 patients undergoing BMT, the mean serum carboxy-terminal cross-linked telopeptide of type I collagen increased progressively until 4 wk after BMT. Thereafter, it began to decrease and reached basal values after 1 yr. Serum osteocalcin decreased progressively until 3 wk after BMT and reached basal values after 3 months. One year after BMT, lumbar spine BMD had decreased by 3.3% (P < 0.05), and total proximal femoral BMD had decreased by 8.9% (P < 0.001). For the ex vivo study, bone marrow was obtained from healthy donors (n = 7) and transplant recipients (n = 7). Then, mononuclear cells including marrow stromal cells were isolated and cultured to osteoblastic lineage. Alkaline phosphatase activities of each group were measured by the time course of secondary culture, and the mineralizing potentials were compared between the two groups. Cells cultured in our system showed characteristics of osteoblast-like cells differentiated from marrow stromal cells. They were initially in a fibroblastic-like spindle shape and became cuboidal with the formation of nodules that were later confluent. The cells were stained to both alkaline phosphatase histochemistry and Von Kossa histochemistry, demonstrating that these cells were of osteoblastic lineage differentiating from marrow stromal cells. The mean time required for the near-confluence in the primary culture was 15 and 22.9 d in healthy donors and transplant recipients, respectively (P = 0.003). Alkaline phosphatase activity was significantly lower in the bone marrow recipients than in the healthy donors at d 7 and 10 of the secondary cultures. The period at which peak activity of alkaline phosphatase was reached was also delayed in the osteoblasts derived from BMT recipient bone marrow compared with those of healthy donors. Using Von Kossa histochemistry, much more mineralization was observed in osteoblasts of healthy donors than those of BMT recipients. After BMT, although the peripheral mononuclear cells in the recipients were of donor origin, the bone marrow stromal cells were of recipient origin according to the PCR analysis using YNZ 22 mini-satellite probe. In conclusion, the differentiation of bone marrow stromal cells into osteoblasts was impaired after BMT, and this might contribute to post-BMT bone loss.     

Clonal precursor of bone,cartilage, and hematopoietic niche stromal cells

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.     

Circulating hematopoietic stem cells do not efficiently home to bone marrow during homeostasis

OBJECTIVE: Hematopoietic stem cells (HSC), normally resident in bone marrow, can be detected in the murine and human circulation. It is thought that HSC move in and out of bone marrow daily and that returning HSC are generally equivalent to their bone marrow counterparts in phenotype and function. However, large numbers of mononuclear blood cells are required to rescue animals from lethal irradiation, indicating either that the prevalence of circulating HSC is low, or they are inherently deficient in their repopulating ability. Accordingly, recent data suggest that circulating HSC may be unable to stably engraft WBM under homeostatic conditions. The purpose of this study was to explore these dynamics in detail using parabiosis and bone marrow transplantation. MATERIALS AND METHODS: The WBM and skeletal muscle HSC stem cell compartments of parabiosed CD45 congenic mice were analyzed functionally (via bone marrow transplantation) and phenotypically (via flow cytometry) for circulating stem cells at specific time points postparabiosis and after surgical separation. RESULTS: Surprisingly, we find that stem cells trafficking out of bone marrow and into the circulation do not stably return to bone marrow, although long-lived lymphoid precursors do stably re-engraft. Circulating HSC do, however, take up residence in skeletal muscle, wherein they account for HSC activity. CONCLUSION: Circulating HSC are not in flux with the bone marrow HSC and can persist in peripheral tissues.     

In vitro functions of stromal cells from human and mouse bone marrow

Human fibroblastoid cell strains obtained from primary bone marrow cultures and continuous stromal cell lines recently derived from mouse bone marrow were studied. The incidence of fibroblastoid precursors (CFU-F) varied considerably in human bone marrow samples, and no differences could be detected between marrows from a group of myelodysplastic patients (age range 70-82 years) and groups of age-matched controls or younger individuals. A lack of direct correlation between initial clonogenicity and ultimate capacity of fibroblastoid cells to grow in continuous culture was observed in both the normal and the myelodysplastic groups. Despite the apparently normal clonogenicity of CFU-F in patients with myelodysplastic syndromes, some of these marrows failed to grow when subcultured. Normal fibroblastoid cells at 10(4) per culture exhibited myelopoietic activities when cocultured with fresh bone marrow cells. At higher concentrations, these cells inhibited myeloid colony formation. Fibroblastoid cells from only one out of four myelodysplastic patients examined exhibited comparable inhibitory activity. The specificity of the inhibitor(s) was demonstrated by the lack of effect of fibroblastoid cells from normal human bone marrow on the clonogenicity of mouse erythroleukemia cells. Moreover, human foreskin fibroblasts were devoid of such inhibitory activity. These functions of cultured stromal cells may correlate with some of their activities in the bone marrow microenvironment.     

Neuropilin-1 is expressed on bone marrow stromal cells: a novel interaction with hematopoietic cells?

In adult bone marrow, hematopoietic stem cells are found in close association with distinctive stromal cell elements. This association is necessary for maintenance of hematopoiesis, but the precise mechanisms underlying the cross-talk between stromal cells and hematopoietic stem cells are poorly understood. In this study, we used a bone marrow stromal cell line (MS-5) that is able to support human long-term hematopoiesis. This hematopoietic-promoting activity cannot be related to expression of known cytokines and is abolished by addition of hydrocortisone. Using a gene trap strategy that selects genes encoding transmembrane or secreted proteins expressed by MS-5 cells, we obtained several insertions that produced fusion proteins. In one clone, fusion protein activity was downregulated in the presence of hydrocortisone, and we show that insertion of the trap vector has occurred into the neuropilin-1 gene. Neuropilin-1 is expressed in MS-5 cells, in other hematopoietic-supporting cell lines, and in primary stromal cells but not in primitive hematopoietic cells. We show that neuropilin-1 acts as a functional cell-surface receptor in MS-5 cells. Two neuropilin-1 ligands, semaphorin III and VEGF 165, can bind to these cells, and the addition of VEGF 165 to MS-5 cells increases expression of 2 cytokines known to regulate early hematopoiesis, Tpo and Flt3-L. Finally, we show that stromal cells and immature hematopoietic cells express different neuropilin-1 ligands. We propose that neuropilin-1 may act as a novel receptor on stromal cells by mediating interactions between stroma and primitive hematopoietic cells.     

Identification of the hematopoietic growth factors elaborated by bone marrow stromal cells using antibody neutralization analysis

These studies were undertaken to characterize the subclasses of hematopoietic growth factors produced by stromal cells in long-term murine bone marrow cultures. Exposure of these cultures to extremely high doses of irradiation (500 Gy), followed by endotoxin stimulation, permitted detection and characterization of various growth factor activities in the unconcentrated conditioned medium. To determine the nature of these activities, neutralization studies were performed using antisera against the following subclasses of purified colony-stimulating factors (CSFs): purified L-cell CSF-1, recombinant granulocyte-macrophage CSF (rGM-CSF), and recombinant interleukin 3 (rIL3). The antiserum against CSF-1 consistently abrogated 100% of the CSF bioactivity in irradiated stromal cell-conditioned medium (CM) but was only capable of neutralizing 62%-91% of the bioactivity in endotoxin-stimulated, irradiated stromal cell-CM. Antisera against rGM-CSF and rIL3 demonstrated variable effects. When the antisera were used in combinations, only the mixture of anti-CSF-1 + anti-GM-CSF resulted in 100% neutralization of the activities in endotoxin-stimulated, irradiated stromal cell-CM. This CM stimulated the IL3/GM-CSF-responsive cell line FDC-P1 but not the IL3-responsive (GM-CSF-unresponsive) cell line 32D cl-23. The FDC-P1 growth-promoting activity was inhibited only by the antiserum against GM-CSF and not by antiserum against IL3. These experiments indicate that stromal cells from long-term bone marrow cultures can produce and release CSF-1 and GM-CSF while the production of IL3 in this system, if there is any, could not be demonstrated.     

BCNU-induced increase in sulfated glycosaminoglycan production by human bone marrow stromal cells

The etiology of alkylator-induced leukemia is obscure, but may be due in part to alternations in the bone marrow stromal microenvironment. Marrow extracellular matrix, including collagen, glycosaminoglycans/proteoglycans, and glycoproteins, may play a crucial role in the control of normal and abnormal hematopoiesis. Twenty-four hours after seeding, confluent human bone marrow stromal cell cultures were exposed for 3 h to 15 micrograms/ml of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), an alkylating agent with leukemogenic potential. On the eighth day of culture, [35S]sulfate was added and radiolabeled glycosaminoglycan(s) (GAGs) was harvested 24 h later. BCNU treatment resulted in a 104% increase of the radiolabel incorporation into cetylpyridinium chloride precipitable GAG. In addition, spectrophotometric measurement of total GAG in treated cells revealed a similar GAG increase. However, BCNU treatment did not alter compartmental GAG distribution or GAG species. Our results demonstrate a profound quantitative change in the production of important extracellular matrix components by bone marrow stromal cells after exposure to a nitrosourea. This increase may be a factor in microenvironmental alterations leading to bone marrow toxicity following alkylator exposure.     

Human osteoblasts support human hematopoietic progenitor cells in vitro bone marrow cultures

Hematopoietic stem cell differentiation occurs in direct proximity to osteoblasts within the bone marrow cavity. Despite this striking affiliation, surprisingly little is known about the precise cellular and molecular impact of osteoblasts on the bone marrow microenvironment. Recently, we showed that human osteoblasts produce a variety of cytokine mRNAs including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-6. We examined here the ability of osteoblasts to support the development of hematopoietic colonies from progenitors as well the ability to maintain long-term culture-initiating cells (LTC-IC) in vitro. Examination of the hematopoietic cells recovered after 2 weeks of culture showed that osteoblasts support the maintenance of immature hematopoietic phenotypes. In methylcellulose assays, osteoblasts stimulate the development of hematopoietic colonies to a level at least 10-fold over controls from progenitor cells. Using limiting dilutional bone marrow cultures, we observed an activity produced by osteoblasts resulting in an threefold to fourfold expansion of human LTC-IC and progenitor cells in vitro. Thus, the presence of hematopoietic stem cells in close proximity to endosteal surfaces in vivo may be due in part to a requirement for osteoblast-derived products.