Factors controlling the recirculation of hematopoietic stem cells. IV. The effect of thymosin on the migration and differentiation of hematopoietic stem cells

Thymosine, a thymus hormone, restores the thymectomy induced deterioration of the routine pathways of migration and differentiation ofhemopoietic stem cells in mice. Administration of thymosine together with bone marrow cells from thymectomized mice to irradiated recipients also restores the level of migration and differentiation of hemopoietic stem cells. The inducing effect of thymosine on the maturation of T-lymphocyte precursors, which in their turn restore the usual rate of migration and differentiation of hemopoietic stem cells, has been suggested.     

Proliferation and differentiation of hematopoietic stem cells in hypokinesia]

Using the method of exogenous cloning in vivo of the hemopoietic stem cells of the bone marrow and spleen in the femur and the spleen of mice it was shown that during hypokinesia the kinetics of the stem cells differed in both organs (the spleen and the bone marrow). Differentiation of transplanted stem cells from different sources was unchanged in the spleen, but stem cells of the bone marrow seeding in the femur changed the character of their differentiation in the direction of increase of the erythopoietic function, whereas stem cells of the spleen failed to alter the direction of differentiation.     

Factors affecting the proliferation and differentiation of clonogenic hematopoietic stem cells in vitro

The characteristics of hematopoietic factors modulating the growth of clonogenic pluripotent stem cells (PSC) in culture (i.e., CFU-GEMM) are examined. The chemical and biologic properties of "burst-promoting activity" (BPA) are compared with those of the lymphokine interleukin-3 (Il-3). The preponderance of the data available suggests that the active moiety of each of these growth factors may be chemically identical since both BPA and purified Il-3 are capable of supporting the growth of PSC in vitro. The effects of hemin on the growth of CFU-GEMM are also examined experimentally. The results show conclusively that, in the absence of exogenous (BPA), hemin is also capable of supporting the growth of CFU-GEMM in culture and to a level that is consistently higher than that of BPA alone. It is therefore suggested that hemin is capable of augmenting the growth of GEMM colonies in vitro in conjunction with BPA/Il-3. However, the in situ role of each of these "candidate" regulators of hemopoiesis remains largely unknown.     

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.     

The effect of carnosine on hematopoietic stem cell activity in irradiated animals]

Carnosine given to adult animals together with potable water one day prior to gamma-irradiation or injected in a single intraperitoneal dose one hour after irradiation enhances colony formation by haemopoietic stem cells migrating from the bone marrow to the spleen. In young animals with a high colony-forming activity carnosine either decreases or does not influence at all the efficiency of colony production.     

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.     

The effect of serotonin on the hematopoietic stem cells of the bone marrow]

Haemopoietic stem cells content and proliferative activity were studied in the bone marrow of female F1 (CBA x C57Bl6) mice after single (50 mg/kg) and chronic (0.5 mg/kg daily for 7 days) serotonin (S) injections. It is shown that 9-day and 12-day COEs contents in the bone marrow of experimental mice has been increasing for 24 h after single S injection. After chronic S injections twofold increase of 12-day COEs is observed without any increasing of 9-day COEs. Total myelokaryocyte number is increased too. The study of proliferative status by in vitro incubation of bone marrow cells with ARA-C has shown that the numbers of 9-day and 12-day COEs in S-phase have increased both after single and chronic S injections. Possible mechanisms of stimulating effect of S on bone marrow stem cells are discussed.     

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.     

The effect of cyclophosphamide on hematopoietic stem cells

The effects of cyclophosphamide on hematopoietic stem cells (HSC) and on hematopoiesis was studied. Administration of 5 mg of cyclophosphamide to mice resulted in a rapid decrease in the number of HSC in the leg and spleen. The exocolonizing potential of HSC in the spleen recovered and reached supranormal levels in 7 days. That of HSC in the legs recovered to pretreatment values after 15 days. The endocolonizing potential of HSC in the leg, however, recovered in less than 4 days. The peripheral blood counts all dropped to a fraction of normal except for the platelet count, which was affected to a minimal degree. We suggest that cyclophosphamide irreparably damages most of the body's HSC. Those remaining in the leg then proliferate rapidly and repopulate other sites. HSC cannot, however, accumulate in the leg for more than a week after cyclophosphamide administration. Erythropoiesis does not recover until after this occurs. Platelets are least affected because of their resistance to the effects of the drug and because of the ability of megakaryocytes to increase their output of platelets without requiring differentiation of HSC into their compartment.     

The phenotypic characteristics of T-lymphocytes that interact with hematopoietic stem cells: the expression of the antigens Lyt-1, Lyt-2 and L3T4]

The role of T-lymphocytes, bearing antigens Lyt-1, Lyt-2 and L3T4, in regulation of the functional activity of blood-forming precursor cells of syngen and non-syngen origin was investigated. The treatment of cells of murine lymphatic nidi with monoclonal antibodies to the above antigens and with the complement did not abolish the capacity of T-lymphocytes of controlling proliferation and differentiation of syngen and allogen blood-forming precursor cells. The subpopulation characteristics of lymphocytes interacting with the stem cells is discussed.     

间充质干细胞在造血干细胞移植中的应用

间充质干细胞(MSCs)是一种具有自我更新和多向分化潜能的成体干细胞,存在于骨髓、脂肪组织、脐血及多种胎儿组织.它可分泌多种细胞因子及生长因子,促进造血干细胞(HSC)的增殖与分化.MSCs还具有免疫调节、抗炎和组织修复作用,可减轻移植物抗宿主病(GVHD)及其他移植相关并发症.     

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.     

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.     

Changing modified regions in the genome in hematopoietic stem cell differentiation

In the process of hematopoietic stem cell (CD133⁺ cell) differentiation, a drastic change in gene expression occurs which must be regulated by epigenetic mechanisms. One strategy for CD133⁺ cell differentiation analysis is to identify genomic DNA regions that have been modified in the process of differentiation. However, it is difficult to obtain large amounts of genomic DNA from uniform CD133⁺ cells. Based on this situation, we screened genomic DNA regions where modifications change during the process of differentiation in human CD133⁺ cells using differential methylation site scanning (DMSS), which is a method of identifying differentially methylated regions of the genome from a small number of cells. As a result, we cloned three DNA fragments which corresponded to centrosomal protein 68 kDA (Cep68), TRIO and F-actin binding protein (TRIOBP), and AMP-activated protein kinase beta (AMPKb).     

The effect of autoblood in a hyposmotic state on the colony-forming activity of hematopoietic stem cells]

It has been shown that hypoosmotic autoblood injected sub- or intracutaneously stimulates the colony-forming activity of haemopoietic stem cells in mice. Autoblood injected to animals immediately after their irradiation stimulates haemopoiesis even after a single dose. When mice are injected with autoblood prior to irradiation, the time between the first injection and the day of irradiation is critical for manifestation of the immunomodulating effect. Autoblood infusions immediately before, the day before, or two days before irradiation markedly deteriorate the clinical status of experimental animals and cause death in some of them. It is suggested that stimulation of haemopoiesis is associated with the appearance in the blood stream of a population of radiosensitive cells, apparently T-cell precursors.     

Genetic control of quiescence in hematopoietic stem cells

Cellular quiescence is a reversible cell cycle arrest that is poised to re-enter the cell cycle in response to a combination of cell-intrinsic factors and environmental cues. In hematopoietic stem cells, a coordinated balance between quiescence and differentiating proliferation ensures longevity and prevents both genetic damage and stem cell exhaustion. However, little is known about how all these processes are integrated at the molecular level. We will briefly review the environmental and intrinsic control of stem cell quiescence and discuss a new model that involves a protein-to-protein interaction between G0S2 and the phospho-nucleoprotein nucleolin in the cytosol.     

The differentiation of T-lymphocytes. III. The behaviour of subpopulations of mouse thymus cells in short-term cell culture.

Seven human cultured lymphoblastoid cell lines (CLL) were divided into two major groups based on studies of their cell cycle characteristics and surface Ig. CLL I (lines CL, MW, HH and TM) had generation times ranging from 25–40 hr, S phase times of 10–12 hr, G₂ + M times of 6–8 hr, and demonstrated sharp differences between the percentage of SIg(+) cells in different phases of the cell cycle. Line TM was particularly discordant with the highest percentage of SIg(+) cells in G₂ + M. CLL II (lines PS, JR and HT) demonstrated generation times ranging from 18–21 hr, S phase times of 7–10 hr and G₂ + M phase times of 2 hr. In this second group, two of the three CLLs had no differences between cells taken from different points of the cell cycle. DNA synthesis and cell density could not be correlated with either of the above major parameters, i.e. cell cycle times or SIg expression. The results suggest that human CLLs fall into subgroups in which specific patterns of cellular and immune functions may predominate.     

Differential effect of activin on mouse embryonic stem cell differentiation in insulin-secreting cells under nestin-positive selection and spontaneous differentiation protocols

Parallel to the importance of the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function like primary islets. To increase the efficiency of endocrine pancreatic-like cell differentiation from mouse embryonic stem cells (ESCs), we applied activin-B to nestin-positive selection (protocol 1) and spontaneous differentiation (protocol 2) in different groups including: [A] activin-B, or [B] basic fibroblast growth factor (bFGF), and/or [C] activin-B+bFGF. The differentiated cells expressed most pancreatic-related genes. The number of insulin- and C peptide-positive cells, as well as dithizone-positive clusters in group A of protocol 1 was higher than in the other groups. Significant insulin concentrations in protocol 1 were produced when glucose was added to the medium, in comparison with protocol 2. Moreover, insulin release was increased significantly in group A of protocol 1 even with lower glucose. In conclusion, Addition of activin-B in a nestin-positive selection protocol increased the insulin-secreting cells in comparison with the same protocol with bFGF and/or spontaneous differentiation in presence of bFGF and/or activin-B alone. However, improvements of the current method are required to generate a sufficient source of true beta-cells for the treatment of diabetes mellitus.     

A genetic determinant that specifically regulates the frequency of hematopoietic stem cells.

The regulation of hematopoietic stem cell (HSC) homeostasis is not well understood. We screened for genetic polymorphisms that were linked to differences between mouse strains in the numbers of long-term reconstituting HSCs or restricted progenitors in the bone marrow. AKR/J mice had significantly higher frequencies and numbers of both HSCs and restricted progenitors in their bone marrow than C57BL/Ka-Thy-1.1 mice. The C57BL/Ka-Thy-1.1 alleles were partially dominant. A locus on chromosome 17, including the H-2 complex, was significantly linked to the frequency of long-term self-renewing HSCs but showed no evidence of linkage to the frequency of restricted progenitors. Conversely, a chromosome 1 locus exhibited suggestive linkage to restricted progenitor frequencies but was not linked to HSC frequency. This demonstrates that there are distinct genetic determinants of the frequencies of HSCs and restricted progenitors in vivo. The AKR/J chromosome 17 locus was not sufficient to increase HSC frequencies when bred onto a C57BL background. This suggests that to affect HSC frequencies, the product(s) of this locus likely depend on interactions with unlinked modifying loci.