Genetic control of allogenic inhibition of hematopoietic stem cells]

Adult mice of C57BL/6, CBA (CBA X C57BL/6) F1, (CBA X C57BL/6) F2, F1 X CBA and F1 X C57BL/6 strains were lethally irradiated and reconstituted with a constant dose of 3-10(5) C57BL/6 bone marrow cells. At the 9th day after the bone marrow transplantation the colony count was performed in spleen of irradiated recipients. In the spleen of F1, CBA and C57BL/6 mice were registered low (0--8, intermediate (6--18) and high (22-40) numbers of colonies respectively. The segregation ratios in F2 progeny were close to 2 (low): 1(intermediate): 1(high). The segregation ratios in backcross (F1 X CBA) were close to 1(low): 1(intermediate)numbers of colonies. Backcrosses (F1 X C57BL/6) were distributed to low and high numbers of colonies with the ratio 1:1. The number of spleen colonies of males and females was the same in all segregating progeny. The results of hybrid analysis suggest that a single pair of allelic genes is involved in genetic control of allogenic inhibition, and that the resistance (manifestation of inhibition) to C57BL/6 stem cells is conferred by the dominant allele.     

Influence of the maternal effect on allogenic inhibition of hematopoietic stem cells]

Bone marrow cells (0,5-10(6)) of female mice of CBA or C57BL strains were injected intravenously to lethally irradiated CBA, C57BL/6, (femaleCBA X maleC57BL/6)F1 and (femaleC57BL/6 X maleCBA)F1 mice. Spleen of recipients as assayed for colony count on the 9th day after bone marrow transplantation by the method of Till and McCullouch. Stem cells of CBA mice demonstrated failure of allogenic inhibition in (CBA X C57BL/6)F1 hybrid mice and formed the same number of colonies as in the spleen of syngenic recipients. The level of allogenic inhibition of CBA stem cells transplanted to (C57BL/6 X X CBA)F1 hybrid mice was 50%. Bone marrow cells of C57BL/6 mice formed colonies in spleen of (CBA X C57BL/6)F1 mice at least in 20 times less than in syngenic combination. In the transplantation of bone marrow from C57BL/6 mice to (C57BL/6 X CBA)F1 hybrid mice the allogenic inhibition was less pronounced (77-85%) as compared with the transfer of cells to (CBA X C57BL/6)F1 hybrid mice (95%). The sex of a recipient did not influence the number of formed colonies. The different level of allogenic inhibition of parental stem cells can not be explained by the effect of linkage with sex as the female of reciprocal hybrid mice have identical structure of sex chromosomes (X(CBA)XC57BL/6). The data obtained indicate that the maternal effect affects allogenic inhibition of stem cells in parent--F1 system. It is possible that the maternal influence may be determined by cytoplasmic factors of inheritance which affect the expressivity of recessive genes Hh, controlling the inheritance of specific haematopoietic cell antigens.     

Effect of different temperature fractions of RNA on allogenic inhibition of hematopoietic stem cells of mouse bone marrow and embryonal liver]

In the system of non-syngeneic transfer of stem hemopoietic cells, the preliminary incubation of the cells of bone marrow or embryonic liver of the C57BL mice with different temperature RNA fractions isolated from the spleen of (CBAXC57BL) F1 was shown to lead to the complete or partial restoration of the colony forming ability of the donor cells. The 63 degrees RNA fraction was shown to have the greatest restorative effect.     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

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.     

胚胎干细胞向造血细胞分化研究

胚胎干（embryonic stem,ES）细胞是来源于囊胚的内细胞团（inner cell mass,ICM）,具有发育的全能性或多能性,能嵌合到早期胚胎,在体内可以参与各种组织发育甚至包括生殖细胞;在体外分化培养条件下,可以顺序分化出各种组织细胞,与体内完整胚胎发育过程相符合,而且可以通过调节ES细胞某些基因的表达而调节其分化。因此,ES细胞是研究哺乳动物早期胚胎发育、细胞分化及其关键基因鉴定的理想模型。另外,胚胎生殖脊（embryonic germ,EG）细胞系也具有同样的生物学特性,它是由早期胚胎的原始生殖脊（primordial germ,PG）细胞建株而来。最近研究显示：ES细胞在体外不但可以分化为所有造血细胞系,而且还可以分化为具有长期增殖能力的造血干细胞。作者就胚胎干细胞向造血细胞和造血干细胞分化及其诱导因子和调控基因的表达作一综述。     

胚胎干细胞向造血干/祖细胞定向诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ES细胞)是指由胚胎内细胞团(inner cell mass,ICM)细胞经体外抑制培养而筛选得到的细胞,具有无限增殖潜能,在体外可以向造血细胞分化,有可能为造血干细胞移植和血细胞输注开辟新的来源．此外,ES细胞向造血干/祖细胞的定向诱导分化也为阐明哺乳动物造血发育的细胞和分子机制提供了良好的体外模型．对ES细胞向造血干/祖细胞定向分化的研究进展进行了综述．     

Repair of the radiation damage in hematopoietic stem cells from the mouse embryonic liver. Kinetic aspects]

The method of fractionated irradiation was used to study kinetic aspects of repair of sublethal radiation damages in precursor cells from mouse embryonal liver that form in vivo colonies on 8th and 11th days. It was shown that 11-day CFUs had a lesser ability to repair sublethal radiation damages than 8-day ones at different time-intervals between radiation fractions (from 2 to 6 h). These two CFUs sub-populations differed also in the repair kinetics.     

Effect of long-term cytostatic therapy on the hematopoietic stem cells]

CFU-C and diffusion chamber studies were performed in patients with teratocarcinoma, who underwent long term chemotherapy. No significant decline of bone marrow CFU-C or diffusion chamber cell recovery was found during twelve months of cytotoxic treatment. In contrast to the results in these patients the CFU-C-content of the remission marrow in leukemic patients showed a significant decrease in relation to the duration of remission and chemotherapy.     

Effect of tissue macrophage destruction products on hematopoietic stem cells]

Products of mouse peritoneal macrophage destruction (PMD) obtained by aseptic freezing-thawing of the cells, repeated thrice, were found to elicit in syngeneic mice injected with PMD intraperitoneally an increase of CFUs count in the hemopoietic bone marrow tissue and the spleen, as demonstrated by the Till and McCullooch technique. This proved to be a true increase since the transplatned stem cell fraction sorbed by the recipient's spleen was relatively lower in donor mice given PMD than in the control. Although PMD caused an increase of both erythropoietic (E) and granulocytopoietic-monocytic (G) colonies number, the E/G ratio was decreased; one of the mechanisms of the described effect could be the influence of PMD on the hemopoiesis-inducing microenvironment, as the same effects were obtained in mice injected repeatedly with PMD prior to the transplantation of bone marrow tissue of normal donors. Other possible mechanisms of these effects were analyzed, with consideration to the fact that in experiments with preincubation of bone marrow tissue with PMD prior to injection to the lethally irradiated mice no direct stimulating influence of PMD on the stem cell could be revealed.     

Effect of polyinosinic-polycytidylic acid on the colony-forming ability of hematopoietic stem cells in conditions of allogeneic inhibition]

It was found that the colony-forming capacity of parental bone marrow transplant (C57BL/6) was partially restored in the (CBA X C57BL/6) hybrid recipient irradiated with 800 rad when poly I -- poly C preparation was injected. The effect of poly I -- poly C injection on the colony formation was equivalent to addition of the thymus cells syngeneic with the marrow. In either case the number of splenic colonies was more than double that in the control. On the other hand, it was found that in a completely syngeneic system the number of splenic colonies was not influenced by the thymus cells and poly I -- poly C preparation. Poly I -- poly C doses ranging from 50 to 100 mug and thymus cell doses ranging from 4-10(6) to 8-10(6) did not increase the efficiency of the colony formation with a stable bone marrow dose transplant.     

Effect of the lining of the thymus gland on hematopoietic stem cell kinetics in a suspension of embryonic mouse liver]

A study was made of the kinetics of hemopoietic stem cells in the organic culture of a suspension of mouse embryonic liver cultivated directly on the filters and on the preliminarily grown embryonic thymic feeder. It was shown that stem hemopoietic cells were retained in the course of one month of cultivation in the embryonic liver suspensions; during the first two weeks hemopoiesis in the suspension practically failed to differ from such in the fragments of the same age and cultivation period, whereas during the subsequent periods hemopoiesis proved to be more active in the suspension cultivated on thymic feeder. A possible participation of the thymus in the hemopoiesis is discussed.     

Effect of various factors on the differentiation of hematopoietic stem cells]

The treatment in vitro of bone marrow cells in mice by phytohemagglutinin, concanavaline, or antilimphocytic globulin resulted in the suppression of exogenous hemopoietic colonies in the spleen of lethally irradiated (830r) syngenic recipients, whereas lipopolysaccharide, tuberculin, anti-theta serum or nati-gamma-globulin serum exerted no influence on the colony-forming function of hemopoietic stem cells. The morphological analysis of the ratio and cell composition of hemopoietic colonies has revealed no marked differences between the experimental and control groups. The suppression of hemopoietic stem cells by mitogens might be due both to their direct effect and indirect one, possibly, through a humoral factor.     

Role of B-lymphocytes in inactivating allogenic stem cells]

Antisera to membrane antigens of B lumphocytes eliminated the capacity of lymphocytes to inactivate allogenous stem cells by 60%; however, lymphocytes from the lymph nodes of B mice possessed no inactivating capacity. T-lymphocytes were the main criteria inactivating allogenous stem cells. Cooperating with T-lymphocytes, B-lymphocytes probably contributed to inactivation of precursor cells realized by T-lymphocytes. However, the presence of B-lymphocytes in the killer cells population was not a determinant, since T-lymphocytes were capable of inactivating allogenous stem cells without any participation of B-lymphocytes.     

Temporary characteristics of the process of stem cell inactivation by allogenic lymphocytes]

The time during which transplated lymphocytes block proliferation and differentiation of non-syngeic stem cells has been determined by retrasplantation of immuno-competent cells from one lethally irradiated recipient to another one. It was established that process of inactivation of CFU by allogeneic lymphocytes proceeds itwo stages. At the first stage, the colonization of recipient's tissues takes place. The colonization of tissues and processes of early recognition are completed during the first hours after transplantation of cell mixtures. At the second stage, the processes of redistribution of injected cells occur and a complete inactivation of stem cells take place. These events are completed in bone marrow and spleen 4-5 days after transplantation of cells mixture, possibly with the participation of lymphocytes sensibilized with the target-cells.     

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.