Characterization of engraftable hematopoietic stem cells in murine long-term bone marrow cultures

OBJECTIVE: Long-term bone marrow cultures (LTBMC) are a potential source of hematopoietic stem cells (HSC) for transplantation. Previous reports indicate that feeding LTBMCs induces hematopoietic progenitor cycling, and other studies link HSC cycle phase with engraftability. Our study was initiated to further characterize LTBMC engraftability and determine if a cycle phase-related engraftment defect affects HSC from LTBMCs. MATERIALS AND METHODS: Competitive repopulation of lethally irradiated BALB/c females was used to examine engraftability of LTBMCs under "fed" or "unfed" conditions at 3 to 5 weeks culture. Tritiated thymidine suicide was used to determine the cycle status of HPP-CFC and CFU-S from LTBMCs. RESULTS: Total cell number in LTBMCs decreases from input. Quantitatively, both fed and unfed 3-, 4-, or 5-week cultures compete strongly with fresh marrow for 2 and 8 weeks, but not 6 months, after transplantation. Short-term engraftable HSCs expand between 3 and 5 weeks of culture. Clonal assays indicate no peak in S-phase of CFU-S at 24 and 48 hours after feeding, and fluctuation in both content and cycle status of HPP-CFC after feeding. CONCLUSIONS: Our LTBMCs engraft in all conditions, and the level of engraftment capability does not correlate with cell-cycle phase of CFU-S or HPP-CFC, or with time from feeding. Although the total cell number decreases from input, the proportion of short- and intermediate-term engrafting HSC in whole LTBMCs approximates that of fresh marrow and expands from 3 to 5 weeks in culture, whereas long-term engraftable HSCs are decreased in culture.     

Molecular and cell biologic aspects of erythropoiesis in long-term bone marrow cultures

In long-term marrow cultures, proliferation and differentiation of hemopoietic stem cells occurs for several months. Normally, only the most primitive erythroid progenitor cells are produced (the BFU-E). Following treatment with anemic mouse serum (AMS) or normal mouse serum plus erythropoietin, the BFU-E mature into CFU-E, which then go to produce mature nonnucleated red cells. This development is associated with the production of adult type hemoglobin. Furthermore, erythropoiesis and granulopoiesis occur in association with discrete cellular elements of the adherent cell layer in the long-term culture. Following treatment with AMS, erythropoiesis is enhanced while granulopoiesis is depressed, with no apparent competition at the stem cell or progenitor cell level.     

Studies on the role of serum in long-term bone marrow cell cultures

The effect of fetal calf serum (FCS) concentration on hemopoiesis in long-term bone marrow cell cultures (supplemented with 10(-6) M hydrocortisone) was investigated. The duration of hemopoiesis was directly related to the concentration of serum in the cultures. At each concentration, marked differences were seen between different batches of FCS. The longevity of cultures established with a suboptimum concentration of serum was increased by the addition of HEPES buffer. Removal of all non-adherent cells and growth medium for the weekly feedings increased the overall production of hemopoietic cells when compared to the standard feeding procedure of replacing one-half of the growth medium.     

The regulation of hemopoiesis in long-term bone marrow cultures. II. Stimulation and inhibition of stem cell proliferation

The isolation of a DNA synthesis inhibitor (NBME fraction IV) and stimulator (RBME fraction III) specific for the hemopoietic stem cell (CFU-s) from freshly isolated normal adult and regenerating murine bone marrow, respectively, has been well documented. We have utilized long- term liquid bone marrow cultures in a further analysis of the role of these factors in the regulation of CFU-s proliferation. Our results show that shortly after feeding, at a time when the cultured CFU-s are actively proliferating, high levels of the hemopoietic stem cell proliferation stimulator fraction III can be isolated from the culture medium. In contrast, the presence of essentially noncycling CFU-s found in cultures fed 8-10 days previously correlates with high levels of the hemopoietic stem cell inhibitor fraction IV. These results suggest that a certain balance between these factors determines CFU-s proliferation in the long-term cultures. In support of this, DNA synthesis in actively cycling CFU-s in the long-term cultures is inhibited for at least 3 days by the addition of excess NBME fraction IV (inhibitor). Furthermore, DNA synthesis in noncycling cultured CFU-s is stimulated for at least 5 days by the addition of RBME fraction III (stimulator).     

Resident progenitors and bone marrow stem cells in myocardial renewal and repair

Although cardiac transplantation is still the treatment of choice for end-stage heart disease, the side effects derived from the use of immunosuppressants and the limited availability of donors have prompted the search for alternative therapeutic strategies. Among other possibilities, cell transplantation approaches have recently emerged as new alternatives to stimulate myocardial regeneration. These approaches are mainly based on the increasing number of reports documenting the plasticity of stem cells of various origins, particularly the ability of several types of embryonic and adult stem cells to give rise to cardiomyocytes. Unprecedented in the field of 'translational research' and based on the urgent need for alternative therapies, the promising results obtained with animal models have been quickly transferred to the clinical arena, where numerous small pilot studies using different cell types are already ongoing and/or have reported promising results. Nevertheless, the lack of randomization, the variability and small size of the treated cohorts and the use of mixed populations of cells have often clouded the significance and prevented a mechanistic interpretation of the results. Here, we briefly review the use of bone-marrow-derived and cardiac-derived stem/progenitor cells in myocardial regeneration studies and discuss their significance for the future of the field of myocardial regeneration.     

Use of scid mice to identify and quantitate lymphoid-restricted stem cells in long-term bone marrow cultures

Mice homozygous for an autosomal recessive scid (severe combined immune deficiency) mutation on chromosome 16 exhibit a defect that specifically impairs lymphoid differentiation but not myelopoiesis. Consequently such mice are deficient in both humoral and cell-mediated immune functions. Despite their defect, scid mice survive under pathogen-free conditions and are fertile. The mutation does not impair the hematopoietic microenvironment necessary for lymphoid differentiation, since these mice can be cured with grafts of normal bone marrow (BM) or cells from long-term BM cultures (LTBMC); however, reconstitution requires sublethal (400 cGy) irradiation of recipients. Engraftment with cells from LTBMC gave near-normal levels of colony-forming B cells (CFU-B) in spleen and BM of the recipients by 6 weeks postgrafting. Since LTBMC are devoid of all mature B and pre-B cells but contain stem cells that restore lymphoid function in scid mice, we used a limiting-dilution assay to characterize and enumerate the number of stem cells in LTBMC capable of restoring lymphoid function. Curing was determined by the CFU-B-cell assay, since CFU-B are not detectable in normal scid mice. The results indicate that fewer cells from LTBMC than from fresh BM are required to obtain lymphoid reconstitution. As few as 10(3) LTBMC cells can repopulate significant B- and T-cell function in scid recipients. From these results we conclude that scid mice can be used as recipients to quantify lymphoid-restricted stem cells and that there is a functional separation of lymphoid- and myeloid-restricted stem cells in LTBMC with an enrichment for lymphoid-restricted stem cells in these cultures.     

Regulation of megakaryopoiesis in long-term murine bone marrow cultures

Megakaryocytes and their precursor cells were sustained in mouse bone marrow suspension cultures for over 4-6 wk. Megakaryocyte precursor cells were detected by their capacity to form colonies of megakaryocytes in semisolid agar cultures. Colony formation was dependent on the presence of medium conditioned by a myelomonocytic leukemic cell line (WEHI-3CM). Megakaryocytes from the liquid and semisolid cultures were identified by cytoplasmic acetylcholine esterase and by ultrastructural analysis. The suspension medium from the bone marrow liquid cultures which sustained megakaryopoiesis was not directly acitive in stimulating megakaryocyte colony formation in the semisolid agar cultures, but potentiated the number of colonies detected when WEHI-3CM was present. Bone marrow-conditioned medium increased the sensitivity of megakaryocyte progenitor cells to the stimulus in WEHI-3CM. Addition of the activities present in the two sources produced a quantitative assay for the detection of mouse megakaryocyte progenitor cells. These studies showed: (1) that no inductive regulator of in vitro clones of megakaryocytes was present in the supernatants from the long-term marrow cultures and, (2) that at least two factors were necessary for the induction of megakaryocyte progenitors to proliferate and differentiate in semisolid cultures in vitro.     

Haemopoiesis in long-term bone marrow cultures. A review.

Bone marrow-derived adherent cell layers, containing endothelial cells, fat cells and macrophages will support prolonged haemopoiesis in vitro. Evidence suggests that the adherent layer is acting as an in vitro haemopoietic inductive environment for stem cell proliferation and differentiation into the variety of committed progenitor cells of the granulocytic, erythroid, megakaryocytic and lymphoid lineages. Using this system we have analysed the factors controlling proliferation of stem cells (CFU-S), differentiation and maturation of granulocyte/macrophage precursor cells (CFU-C), leukaemic transformation by chemicals and viruses, the role of environment versus stem cells in the aetiology of haemopoietic aplasias and the possible usefulness of long-term cultures for bone marrow transplantation.     

Proteoglycans in human long-term bone marrow cultures: biochemical and ultrastructural analyses

Proteoglycans within the extracellular matrix of human bone marrow have been implicated in the process of hematopoiesis, but little is known about the structure and composition of these macromolecules in this tissue. Hematopoietically active human long-term bone marrow cultures were incubated with medium containing 35S-sulfate and 3H-glucosamine as labeling precursors. Proteoglycans present in the medium and cell layer were extracted with 4 mol/L guanidine HCI and purified by diethylaminoethyl (DEAE)-Sephacel ion exchange and molecular sieve chromatography. Both culture compartments contain a large chondroitin sulfate proteoglycan (MI, CI) that eluted in the void volume of a Sepharose CL-4B column and contained glycosaminoglycan chains of molecular weight (mol wt) approximately 38,000. A second population of sulfate-labeled material was identified as a broad heterogenous peak (MII, CII) that was included on Sepharose CL-4B at Kav = 0.31. This material when chromatographed on Sepharose CL-6B could be further separated into a void peak (MIIa, CIIa) and an included peak eluting at Kav = 0.39 (MIIb, CIIb). The void peaks (MIIa, CIIa) were susceptible to chondroitinase ABC digestion (99%) but slightly less susceptible to chondroitinase AC digestion (90%). Papain digestion of these peaks revealed them to be proteoglycans with glycosaminoglycan chains of mol wt approximately 38,000. The included peaks on Sepharose CL-6B (MIIb, CIIb) from both medium and cell layer compartments resisted digestion with papain, indicating the presence of glycosaminoglycan chains of mol wt approximately 38,000 either free or attached to a small peptide. Although this material was susceptible to chondroitinase ABC (98%), it was considerably less susceptible to chondrotinase AC (approximately 60%), indicating that it contained dermatan sulfate. A small amount of heparan sulfate proteoglycan was also identified but constituted only approximately 10% of the total sulfated proteoglycan extracted from these cultures. Additionally, approximately 40% of the incorporated 3H- activity radioactivity was present as hyaluronic acid. Electron microscopy revealed a layer of adherent cells covered by a mat containing ruthenium red-positive granules that were connected by thin filaments. The extracellular matrix layer above the adherent cells contained a mixture of hematopoietic cells. Chondroitinase ABC treatment of the cultures completely removed the ruthenium red-positive granules overlying the cells and resulted in a loss of approximately 70% of the 35S-sulfate-labeled material from the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Persistence of murine myeloid leukaemic cells in long-term bone marrow cultures

Patterns of growth in long-term bone marrow culture were compared for a number of murine myeloid leukaemias. A leukaemic pattern of haematopoiesis was maintained in culture for a period of at least 15 weeks for one of the lines. However in the other five murine myeloid leukaemic lines studies, the leukaemic cells appeared not to survive in culture and normal haematopoiesis was established. Transplantation of cells from these established cultures at weeks 7 or 11 into syngeneic recipients revealed that leukaemic cells were present. Thus leukaemic cells seem to persist in long-term bone marrow cultures even in morphologically normal cultures.     

The radiation response and recovery of bone marrow stroma with particular reference to long-term bone marrow cultures

There is evidence for long-term haematopoietic dysfunction in some patients treated with radiotherapy. Although the underlying mechanisms are unclear, both stem cell and environmental defects have been implicated. In the present article we review the evidence concerning the role of stromal cells. According to the endpoints used, a wide range of radiosensitivities for the stroma have been reported. Long-term bone marrow cultures provide a system in which both functional and regenerative aspects of the stroma can be studied. A dose of 5 Gy applied prior to the establishment of long-term bone marrow cultures decreases both the formation of a confluent adherent stromal layer and its capacity to support haematopoiesis. In contrast, in its fully established phase, the adherent layer displays a high radioresistance due to the low proliferative stress applied to its stromal populations. A dose of 10 Gy given to a fully established adherent layer does not prevent haematopoietic engraftment and sustained haematopoiesis. At doses above 100 Gy a macrophage-like and epithelioid cell-type become dominant, which preserve their ability of producing growth regulatory molecules at doses as high as 500 Gy. These data suggest that the main effect on the stroma is a delayed expression of irradiation damage due to the slow rate of turnover of stromal cells. So far, there is little evidence for persistent deficiencies in the functional roles of stromal cell populations.     

Cloned stromal cell lines derived from human Whitlock/Witte-type long-term bone marrow cultures.

We report a human bone marrow culture technique that initially parallels the murine Whitlock/Witte culture system. As in the murine system, B cells predominate over other cell types, and all differentiation stages from pre-B to plasma cell are observed. Although these human long-term cultures pass through stages resembling phases I to III of murine Whitlock/Witte cultures, no outgrowth of nonadherent cells was seen after cultures had reached the "crisis" phase unless Epstein-Barr virus (EBV)-transformants appeared. The stromal cells persisted well beyond crisis, but they could not be maintained and passaged as cell lines, limiting their use in molecular analysis. Transfection of these stromal cells with plasmid DNA containing the simian virus 40 (SV40) early region yielded 124 cloned cell lines. Analysis of these lines showed that all expressed SV40 large T antigen, but they retained most phenotypic markers found on non-transformed stromal cells. When adherent and T-cell-depleted bone marrow cells were cultured on either nontransformed stromal layers or transformed cell lines they proliferated actively and soon yielded predominantly lymphoid nonadherent populations. Moreover, prolonged survival of acute lymphoblastic leukemia cells of pre-B phenotype was regularly achieved on both normal and transformed adherent cell layers. Although the liquid culture system favored lymphocytes, transformed stroma supported colony formation by both human and murine hemopoietic progenitors when marrow was added in agar medium. This was not explained by colony-stimulating factor (CSF) production, because striking heterogeneity in the levels of granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF) secretion by the lines was noted. Some lines that did not produce detectable CSF demonstrated good support of fresh bone marrow growth and acute lymphoblastic leukemia (ALL) cell survival. The heterogeneity of these cell lines and their capacity to support hemopoiesis suggest that they will be useful in studying the molecular basis of in vitro lymphohemopoiesis in man.     

Resistance of the stromal cell in murine long-term bone marrow cultures to damage by ionizing radiation

The ability of bone marrow stromal cells to survive and function after exposure to ionizing radiation remains controversial. Therefore, we used the murine long-term bone marrow culture system to analyze the effects of single doses of ionizing radiation (9-500 Gy) on the function of a preexisting, nearly confluent stroma that was supportive of hematopoiesis. Hematopoiesis ceased promptly in all the irradiated cultures and did not recover unless fresh marrow cells were inoculated. Radiation doses less than or equal to 100 Gy caused no obvious morphologic change in the cells. Total RNA, total protein, and collagen synthesis declined by 35%-60% within two days after even 9 Gy; but radiation doses up to 100 Gy caused minimal or no additional decline. Although RNA synthesis recovered nearly to normal within three weeks after radiation doses less than 100 Gy, total protein and collagen synthesis remained suppressed. Normal adherent layers irradiated with 9-50 Gy supported long-term hematopoiesis by fresh Sl/Sld marrow cells, although Sl/Sld marrow did not demonstrate sustained hematopoiesis when cultured in plain culture dishes or over normal stroma irradiated with 200 Gy. Thus, bone marrow stromal cells in long-term cultures did not show evidence of substantial cell death over at least the six-week period studied after irradiation with as much as 100 Gy, and they maintained hematopoietic supportive functions when irradiated with up to at least 50 Gy.     

骨髓干细胞移植治疗肝硬化研究进展

肝硬化是一种常见的,由不同病因引起的肝脏慢性、进行性、弥漫性病变,引起肝硬化的病因在国内以病毒性肝炎多见,目前对肝硬化的治疗主要是保护肝功能及对症支持疗法,疗效欠佳。终末期肝病最后考虑肝移植,但供肝来源越来越少,肝移植费用昂贵,手术并发症,且术后出现的移植排斥等问题困扰患者终生,近来兴起的骨髓干细胞移植治疗肝硬化具有操作简单、可重复进行、免疫源性弱、治疗费用低等优点,     

骨髓间充质干细胞的免疫学特性

目的 研究骨髓间充质干细胞(MSC)的免疫学特性,为临床应用提供实验依据.方法 培养鉴定MSC,检测其生长动力学、细胞周期.检测单纯和200 U/ml干扰素(IFN)γ干预后MSC的程序性死亡配体(PDL)-1、CD54、CD40、CD80、CD86、主要组织相容性复合物(MHC)-Ⅰ、MHC-Ⅱ的表达情况,并以此为调节细胞进行混合淋巴细胞反应.封闭PDL-1、CD54,观察它们在MSC免疫调节中的作用.检测培养上清液中IFN γ、白细胞介素(IL)-2,IL-4、IL-10的水平.将MSC移植于体内观察肝脏归巢和诱导微嵌合体情况.结果 分离、培养的MSC纯度较高;生长曲线显示第1、2天为潜伏期,第3、4、5天为对数生长期,第6、7天为平台期.细胞周期显示G0/G1期为76.0%±2.0%,S期为13.0%±2.0%,(G2+M)期为10.0%±1.7%.IFN γ上调CD54、PDL-1、MHC-Ⅰ、MHC-Ⅱ的表达,CD40、CD80、CD86为阴性表达.MSC呈剂量依赖性地抑制T淋巴细胞增殖,IFN γ增强其抑制作用.封闭PDL-1、CD54后能减弱MSC对T淋巴细胞增殖的抑制作用.培养上清液中IFN γ、IL-10水平较高,IL-4较低,IL-2未检测到.MSC在肝实质内有多处定居,并能诱导肝移植术后微嵌合体形成.结论 IFN γ能增强MSC对淋巴细胞增殖的抑制作用,PDL-1和CD54可能发挥关键作用;MSC移植后能在体内存活并诱导微嵌合体形成.     

Role of colony-stimulating factor in myelopoiesis in murine long-term bone marrow cultures

Weekly medium change or midweek feeding of long-term bone marrow cultures (LTMBCs) results in a significant increase in total myeloid cell production. Proliferative myeloid cells peak 48 hours after feeding, and nonproliferative myeloid cells reach maximum levels at 72 hours. This increase in myelopoiesis is invariably preceded by a significant elevation in biologically and immunologically measurable colony-stimulating factor (CSF) in the supernatants of LTBMC. The level peaks 24 hours after medium change, then gradually returns to basal values. The decrease in CSF relates to its consumption by generating myeloid precursors because no fluctuation in the levels occur in cultures without active myelopoiesis. No significant inhibitors or promoters of CSF were detected. When highly purified L cell CSF, CSF in lung-conditioned medium, or CSF concentrated from LTBMC supernatant is added to cultures, an identical increase in myelopoiesis occurs. Anti- CSF antiserum, added to culture at the time of medium change, totally neutralizes supernatant CSF levels but does not affect myelopoiesis. These findings suggest a potential regulatory role for CSF in myelopoiesis in LTBMC. CSF appears to function within the microenvironment through a mechanism involving cell:cell interactions or by causing the production of other substances that stimulate myelopoiesis. Because exogenous CSF stimulates myelopoiesis, it is likely that it too can react either directly or through microenvironmental cells to stimulate primitive myeloid cells to divide.     

Basic fibroblast growth factor stimulates myelopoiesis in long-term human bone marrow cultures

We previously showed that basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow (BM) stromal cells and significantly delays their senescence. In the present study, we demonstrated that low concentrations of bFGF (0.2 to 2 ng/mL) enhance myelopoiesis in long-term human BM culture. Addition of bFGF to long-term BM cultures resulted in an increase in (a) the number of nonadherent cells (sixfold), particularly those of the neutrophil granulocyte series; (b) the number of nonadherent granulocyte colony-stimulating factor (G-CSF)- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive progenitor cells; (c) the number of adherent foci of hematopoietic cells (10-fold); and (d) the number of progenitor cells in the adherent stromal cell layer. These effects were not noted with higher concentrations of bFGF (20 ng/mL). Thus, low concentrations of bFGF effectively augment myelopoiesis in human long-term BM cultures, and bFGF may therefore be a regulator of the hematopoietic system in vitro and in vivo.     

Granulopoietic differentiation in long-term bone marrow cultures from children with congenital neutropenia

The capacity of granulopoietic precursor cells (CFU-GM) to differentiate in vitro was evaluated in five children with congenital neutropenia using short-term colony assays and long-term marrow cultures. In all five children, methylcellulose assays revealed normal numbers of CFU-GM, which displayed an appropriate response to various sources of GM-CSF and differentiated up to the polymorphonuclear leukocyte state (PMN). In contrast, neutrophil PMN were not observed in long-term bone marrow cultures from three patients, despite a normal production of CFU-GM, myeloblasts, and promyelocytes during the 5-6 week culture period. Thus, in these patients, the characteristic "block" in granulocytic maturation observed in vivo was reproduced in vitro in long-term cultures. Granulocytic differentiation proceeded normally in long-term cultures from the two other patients, thus indicating heterogeneity in the expression of the defect. These results might indicate abnormal interactions between stromal and hematopoietic cells in long-term marrow cultures from some patients with congenital neutropenia. Furthermore, our results showed some correlation between the granulocytic defect in vitro and the clinical outcome in vivo.     

Diethyldithiocarbamate induction of cytokine release in human long-term bone marrow cultures.

Diethyldithiocarbamate (DDTC) is a biochemical modulating agent that protects murine bone marrow progenitor cells from the cytotoxicity of a variety of cancer chemotherapeutic agents. However, the mechanism of this protection is not well understood. Long-term human bone marrow cultures (LTBMC) were established and at day 17 treated with 30 mumol/L DDTC for 1 hour, after which DDTC was removed and replaced with complete medium. Conditioned medium was then collected 6, 12, 24, and 48 hours later and analyzed for the presence of cytokines. A time-dependent increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) (12-fold), granulocyte-CSF (G-CSF) (66-fold), interleukin (IL)-6, (three-fold), IL-1 beta (161-fold), and tumor necrosis factor (TNF)-alpha (25-fold) was observed. The maximum increase for the factors other than TNF-alpha was at 24 to 48 hours posttreatment. However, TNF-alpha peaked as early as 6 hours post-DDTC. When conditioned medium from these cultures was tested in a granulocyte-macrophage progenitor cell (GM-CFC) assay, an increase in colony formation was observed that correlated with the increased levels of cytokines in the medium. The specificity of this effect was confirmed by the fact that the closely related congener bis(hydroxyethyl)dithiocarbamate was devoid of colony-stimulating activity. The addition of antibodies for TNF-alpha and/or IL-1 alpha following DDTC treatment did not inhibit the release of GM-CSF, G-CSF, or IL-6 from the LTBMC. These results suggest that DDTC accelerates bone marrow recovery following myelotoxic drug treatment via increased production of cytokines that are known to be essential for hematopoiesis.