The role of cells sensitive to anti-mouse brain serum in the regulation of CFU-S proliferation

CFU-S differentiation and regeneration kinetics in the spleen and femur was studied after treatment of bone marrow cells with RAMB serum. The effect of thymocytes on the rate of CFU-S regeneration was also investigated. It was found that CFU-S regeneration in the spleen was similar in RAMBS-treated and intact cell populations on days 4-14 after transplantation. On the contrary, the rate of CFU-S regeneration in the femur was slower in RAMBS-treated than in intact bone marrow cells. However, the growth rate in the femur could be restored to the normal level by the administration of freshly isolated syngeneic thymocytes to mice pre-injected with RAMBS-treated CFU-S population. The treatment of bone marrow suspension with RAMB serum did not affect the differentiation of spleen colonies. It is suggested that RAMBS eliminates cell population regulating CFU-S proliferation, without affecting its differentiation.     

Cloning stem cells in the bone marrow of irradiated mice]

Different amount of intact or irradiated bone marrow from syngenous donors was administered to mice irradiated with a lethal dose. There was revealed a linear dependence of the number of the 8-9-day colonies grown in the bone marrow of the femur on the amount of the administered cells, and an exponential dependence on the irradiation dose. Regularity of the stem cell cloning in the bone marrow was analogous to such in the spleen. Radiosensitivity of the colony-forming units (CFU) differed depending on the site (the spleen, the bone marrow) of their colony formation. The CFU settling in the marrow proved to be more radioresistant (D(0) equalled 160-200 P) in comparison with the CFU settling in the spleen (D(0) constituted 80-100 P). It is supposed that a different radiosensitivity of the CFU was caused by the presence of heterogenic population of the stem cells and also by specific peculiarities of the organ (the spleen, the bone marrow) in which the colonies formed.     

Effect of thymus cells on the radiosensitivity and differentiation trends of hematopoietic stem cells]

A stimulating influence of thymus cells on the capability of irradiated (from 100 to 500 r) bone marrow of mice of producing colonies in spleen of syngenous recipient has been proven. The intensification of colony formation involves an increased radioresistance of stem cells. It is supposed that radioresistant thymus cells have a stimulating effect. Thymus cells exert their influence not only to the rate of survival of stem cells proliferating in the bone marrow of femur, but also increase their erythropoetic potention.     

Factors that control the differentiation of stem cells. I. The change in direction of hematopoietic stem cell differentiation under the effect of differentiating T-lymphocytes]

A mixed transplantation of bone marrow cells, and lymph nodes or thymic cells of mice CBA strain into lethally irradiated hybrid recipients (CBAXC57B1)F1 is accompanied with changes in the differentiation pattern from a mainly erythroid to a mainly granuloid way. Thymectomy of either donor of bone marrow cells or recipients, or both, destroys the stem cell differentiation in the direction of granulopoieseis. Intact syngeneic lymphocytes normalize differentiation of the stem cells, but in the presence of tissue antigens these provide for the stem cell differentiation mainly in the direction of granulopoiesis. The differentiation of stem haemopoietic cells is accomplished under the thymic and lymphocyte control. T-differentiating lymphocytes (Td) are the lymphocytes controlling the stem cell differentiation.     

Two subpopulations of stem cells for T cell lineage

An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells, the generation of donor-derived T cells being observed in two out of 14 recipients transferred with as few as 1.5 X 10(4) cells. The stem cell activity of spleen cells was estimated to be about 1% of that of bone marrow cells, and no activity was found in thymus cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. Spleen cells showed a markedly high level of activity 7 days after the reconstitution, followed by a decline, whereas the activity of bone marrow cells was very low on day 7 and increased crosswise. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells. Such patterns of compartmentalization of stem cells in the spleen and bone marrow of irradiated recipients completely conform to the general scheme of the relationship between restricted stem cells and less mature stem cells, including pluripotent stem cells, which became evident in other systems such as in the differentiation of spleen colony-forming cells or of stem cells for B cell lineage.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Cramp过表达对小鼠骨髓造血干细胞功能的影响

目的研究Cramp蛋白过表达对小鼠骨髓造血干细胞自我更新和分化能力的影响。方法应用流式细胞仪分析Cramp过表达转基因小鼠及同龄野生型小鼠的骨髓、脾脏、胸腺等组织器官中各种细胞的比例;分选骨髓造血干细胞,体外培养,观察其克隆形成能力。结果与野生型小鼠相比,Cramp过表达转基因小鼠的骨髓、脾脏、胸腺等组织器官中各种细胞的比例、骨髓造血干细胞的克隆形成能力等均无明显变化。结论本研究中,Cramp过表达转基因小鼠骨髓造血干细胞的分化能力、克隆形成能力无明显变化。     

Hemopoietic colony studies. VI. Increased eosinophil-containing colonies obtained by antigen pre-treatment of irradiated mice reconstituted with bone marrow cells

The cellular response to an intraperitoneal injection of antigen (tetanus toxoid) was studied in reconstituted animals in order to determine the mechanism of control of eosinophil granulocytopoiesis. Antigen treatment of the marrow cell donors did not consistently increase the number of spleen and bone marrow colonies in recipient animals or change the percentage of eosinophil or other hemopoietic colony types. Antigen pre-treatment of the irradiated recipients increased the percentage of eosinophil-containing colonies in the spleen and femoral bone marrow without significantly changing the total number of either spleen or marrow colonies. Antigen treatment of both the bone marrow cell donor and recipient produced a further increase in the percentage of eosinophil-containing colonies in the marrow cavity, but not in the spleen. Antigen treatment of the irradiated recipient increased the number of eosinophilic cells (but not the total number of cells) in both the peritoneal cavity and the bone marrow. Antigen treatment of both the marrow donor and recipient produced a further increase in the number of eosinophilic cells in the peritoneal cavity, but not in a single femur. Since antigen treatment of the marrow recipient, or recipient and donor, but not of the marrow donor alone, results in increased eosinophilic cell and colony numbers, the effect of antigen appears to be mediated through some host factor(s), perhaps the eosinophilic hemopoietic inducing microenvironment (HIM), rather than directly on the hemopoietic stem cells.     

DISTRIBUTION AND MULTIPLICATION OF COLONY FORMING UNITS FROM BONE MARROW AND SPLEEN AFTER INJECTION IN IRRADIATED MICE

The distribution and proliferation of CFUs from bone marrow and spleen cell suspensions were followed after injection in lethally irradiated isogeneic mice. It was found that a larger proportion of the injected bone marrow CFUs than of the spleen derived CFUs could be recovered from the recipient's spleen and femur. This consistently higher recovery points to the conclusion that a larger fraction of bone marrow-derived CFUs than of spleen-derived CFUs is capable of producing daughter CFUs, most likely due to a commitment to early differentiation of many spleen CFUs.     

Colony-forming units of the bone marrow and spleen of immunodeficient mice after stimulation of the cells with thymalin

It has been previously demonstrated by the authors that histological characteristics of colony-forming units (CFUs) in normal mice prove a certain shift in their differentiation in erythroid direction comparing to the bone marrow CFUs. Thymectomy of mature animals is accompanied with weakening growth of granular colonies at cloning of the bone marrow CFUs and with loss of stability in direction of splenic CFUs differentiation. Polypeptide preparation of the thymus--thymalin stimulates growth of the granulocytic colonies from the splenic CFUs in thymectomized mice both in in vivo and in vitro experiments. Differentiation of the bone marrow CFUs is normalized under the effect of thymalin in in vivo experiment only. The data obtained confirm the suggestion made by R. V. Petrov on existence of T-cell clone, enhancing CFUs differentiation in granulocytic direction. Activation of this clone in the spleen is revealed at thymectomy and stimulation of the cells with thymalin both in in vivo and in vitro experiments. Thus, affirmations are obtained on differences of clonic T-cell regulation of the CFUs differentiation in the bone marrow and in the spleen.     

In vivo studies on the regeneration kinetics of enriched populations of haemopoietic spleen colony-forming cells from normal bone marrow

Functional properties of mouse haemopoietic spleen colony-forming cells, enriched 40- to 80-fold, from normal bone marrow were studied. It was found that: (1) the number of partially purified CFU-s (colony forming unit-spleen) required to rescue lethally irradiated mice was similar to the number of normal unfractionated bone marrow CFU-s giving the same level of protection; (2) the homing of partially purified CFU-s was similar to that of CFU-s from unfractionated bone marrow; (3) the regeneration of CFU-s in spleen was similar for enriched and unfractionated cell populations between 4 and 11 days after transplantation. In contrast, the rate of regeneration of CFU-s in femur was slower with enriched progenitor cells than with unfractionated bone marrow. The growth rate in femur, however, could be restored to normal by injecting freshly isolated syngeneic thymocytes with the enriched CFU-s population. The results indicate that the partially purified CFU-s are by themselves functionally normal and show that the rate of CFU-s repopulation in bone marrow can be affected by cell types other than spleen colony-forming cells.     

M-CSF诱导人骨髓间充质干细胞分化为肝样细胞的分子机制

本研究主要目的是明确M-CSF诱导骨髓间充质干细胞分化为肝样细胞的分子机制,为临床中的肝移植和治疗肝病提供新思路。对取自于本院骨科治疗的患者的股骨骨髓间充质干细胞进行提取、分离、传代培养及鉴定。流式细胞仪检测BMSCs的表面表型。为了诱导BMSCs的肝分化,本研究将BMSCs加入到培养基中。骨髓间充质干细胞诱导21 d后,BMSCs表达了肝细胞特异性标志物a-蛋白(AFP)和细胞角蛋白18(CK18),通过免疫荧光染色证实了分化与为分化的BMSCs表达的差异性。分化的BMSCs还显示了肝细胞的体外功能特征,包括白蛋白产生、尿素分泌和糖原储存。本研究结果表明,BMSCs在M-CSF诱导下可分化为功能性肝细胞样细胞,可作为肝病治疗的细胞来源。     

调控软骨形成的信号通路及相关因子在骨髓间充质干细胞骨向分化中的作用*

骨髓间充质干细胞是一类具有自我复制和多向分化潜能的成体干细胞,可以通过定向诱导分化为成骨细胞、软骨细胞、脂肪细胞等,是目前骨再生医学和细胞治疗研究最多的理想种子细胞。在骨缺损的修复过程中,骨髓间充质干细胞内成软骨相关基因表达升高进而分化为软骨细胞,后期随着成骨细胞和破骨细胞的形成及血管长入,软骨基质逐步降解并被骨基质所替换。软骨细胞参与了骨缺损前期的修复过程,调控软骨形成的信号通路及相关因子不仅调控骨髓间充质干细胞成软骨细胞分化,同时在成骨细胞分化过程中也发挥着重要的作用。对调控软骨形成的信号通路及相关因子在骨髓间充质干细胞骨向分化中的调控作用和研究现状进行了总结,以期为临床寻找更好的治疗骨缺损的方法提供理论依据和研究方向。     

Infection of bone marrow cells in vitro with FLV: Effects on stem cell proliferation,differentiation and leukemogenic capacity

Long-term cultures of proliferating hematopoietic stem cells derived from bone marrow permit the study of the interaction between murine leukemia virus (MuLV) infection and the proliferation and differentiation of stem cells. We have used this system to analyze the replication of different biological variants of MuLV in bone marrow cells; the effect of MuLV infection upon pluripotent stem cell (CFU-S) proliferation; and the effect of MuLV on differentiation of CFU-S along different hematopoietic pathways. Two MuLV variants were studied in detail: the Moloney strain of lymphatic leukemia virus (Mol-MuLV) and the erythroleukemic Friend virus complex (FLV) consisting of the lymphoid leukemia helper virus and the defective spleen focus-forming virus (SFFV). Mol-MuLV and its sarcoma virus pseudotype, MSV(Mol-MuLV), replicate efficiently in the bone marrow cultures; however, CFU-S are lost more readily than in uninfected cultures, and the cultures are soon represented by a majority population of mononuclear macrophages. On the other hand, infection with FLV produces a prolonged survival of the spleen colony-forming cells, CFU-S, and CFU-C (the committed granulocytic precursor cells). Production of erythroleukemogenic SFFV is maintained in these cultures for more than 40 weeks. No erythroblastic differentiation was observed in vitro, however, neither erythroblast precursor cells (CFU-E) nor hemoglobin-producing cells could be detected. This suggests that the target cell for FLV is an earlier precursor cell.     

Effect of T-cell mitogens and T-lymphocyte deficiency on splenic colony formation

A study was made of the influence of T-cell mitogens (Con A and PHA) on the colony formation and differentiation of hemopoietic stem cells from normal and thymectomized mice, as well as of the relationship between the colony formation and the dose of injected thymocytes. The incubation of bone marrow cells with Con A and PHA was shown to inhibit the growth of spleen colonies. This inhibition is reduced by thymocytes within the dose intervals of 0.25-2.0 X 10(7) cells/mouse. Administration of these agents serially has led to the potentiation of inhibition effect and to the inability of thymocytes to reverse it. Con A and PHA exert no effect on the differentiation of stem cells. Incubation of the bone marrow cells from thymectomized mice with Con A is much less effective in the depression of colony formation, if compared with the treatment by intact bone marrow preparations. A reversed picture was observed using antiserum to mouse brain (RAMBS). It is proposed that regulation of stem cells is governed by different subpopulations of thymocytes.     

Self-maintenance of migrating hematopoietic stem cells]

Decreased self-maintenance ability of the migrating stem cells (CFU) from the peripheral blood or ectopic focus of hemopoiesis in comparison to the settled bone marrow CFU, as measured by the spleen colony method or by means of chromosomal markers, has been studied. The competence for myeloid and lymphoid differentiation was essentially the same for migrating and settled stem cells.     

Different effect of testosterone on polypotential stem hematopoietic stem cells and immunocompetent B-lymphocytes]

A research was made to study the dynamics of the proliferative, colony-forming and migration capacity of stem hemopoietic cells in (CBA X C57Bl) F1 hybrid mice under the influence of testosterone propionate, 10 mg/100 g, as well as the migration of immunocompetent B lymphocytes from the bone marrow to the spleen and the accumlation of their progeny, antibody-producing cells, in the spleen. The immunodepressive effect of testosterone was manifested by a decrease in the migration of B cells and the number of antibody-producing cells in the spleen. On the contrary, testosterone had a stimulating effect on the functional activity of stem hemopoietic cells, increasing their proliferation and migration. Under conditions of the suppressed erythropoietic differentiation of multipotent stem hemopoietic cells the injection of testosterone resulted in an increase in the number of antibody-producing cells in the spleen. This suggests that the stimulation of erythropoiesis and immunosuppression, induced by testosterone, are interconnected and determined by the direct action of the hormone on the cellular cycle of the stem cells, as well as by their prevailing differentiation towards the erythroid series, resulting in the decrease of their differentiation into B cells.     

Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction.

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.     

An experimental analysis of factors affecting the localization of embryonic bone marrow

Summary The present investigations have been concerned with factors which determine and influence the localization and development of hemopoietic bone marrow in the embryo mouse and the adult. These studies, which have employed organ cultures and the transplantation of mouse embryo femur and tail rudiments, indicate that the surrounding mesenchyme is required for the normal development of the cartilage rudiment and its ossification, and for the formation and colonization of the marrow cavity. It was suggested that hemopoiesis results from the colonization of the “prepared” marrow cavity by stem cells arising from sources external to the rudiment. The addition of erythropoietin and L-thyroxine produced distinct erythropoietic differentiation in the normally myelocytic embryonic marrow cavity. The significance of the microenvironment present in developing bone rudiments and the initiation of hemopoiesis in stem cells was discussed. A hypothesis was developed to explain marrow localization in adults based on the colonization of bone rudiments which are developing their marrow sites at a time when the blood contains large numbers of colony-forming units.