Some observations on the growth requirements of multipotent stem cells under defined culture conditions.
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Biomedical subjects
F C Monette
Publications and source records attributed to F C Monette.
The role of interleukin-3 and heme in the induction of erythropoiesis.
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Molecularly characterized factors governing the growth of murine multipotent stem cells in serum-depleted marrow cultures.
The growth requirements of normal murine marrow-derived multipotent stem cells (CFU-GEMM) in a simple clonal cell culture system substantially devoid of exogenous serum proteins was assessed. The ability of murine interleukin-3 (Il-3), recombinant human erythropoietin (rEPO), and a crystalline preparation of the protoporphyrin hemin to support colony growth in "serum-free" cultures was examined by titration. The results suggest that both Il-3 and hemin are limiting for multipotential colony growth in "serum-free" cultures, but that EPO is not. In addition, the 'sensitivity' of CFU-GEMM to each growth factor appeared to increase in the "serum-free" environment as evidenced by a "shift-to-the-left" in all the titration curves. Nearly half of the GEMM colonies grew to full maturity in the absence of exogenous EPO. Given the optimal concentration of each growth factor, high colony growth was consistently observed in the "serum-free" cultures, with a range from 65% to 119% of the serum control level. It is therefore concluded that supplementation of murine marrow cultures with Il-3 and hemin alone may provide the necessary setting for studying the factors which modulate the growth of multipotent stem cells in a serum-free environment.
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Growth of murine multipotent stem cells in a simple "serum-free" culture system: role of interleukin-3, erythropoietin, and hemin.
The growth requirements of normal murine marrow-derived multipotent stem cells (CFU-GEMM) in a simple clonal cell culture system substantially devoid of exogenous serum proteins was assessed. The ability of murine interleukin-3 (Il-3), recombinant human erythropoietin (rEPO), and a crystalline preparation of the protoporphyrin hemin to support colony growth in "serum-free" cultures was examined by titration. The results suggest that both Il-3 and hemin are limiting for multipotential colony growth in "serum-free" cultures, but that EPO is not. In addition, the "sensitivity" of CFU-GEMM to each growth factor appeared to increase in the "serum-free" environment as evidenced by a "shift-to-the-left" in all the titration curves. Nearly half of the GEMM colonies grew to full maturity in the absence of exogenous EPO. Given the optimal concentration of each growth factor, high colony growth was consistently observed in the "serum-free" cultures, with a range from 65% to 119% of the serum control level. It is therefore concluded that supplementation of murine marrow cultures with Il-3 and hemin alone may provide the necessary setting for studying the factors that modulate the growth of multipotent stem cells in a serum-free environment.
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Hemin acts synergistically with interleukin-3 to promote the growth of multipotent stem cells (CFU-GEMM) in "serum-free" cultures of normal murine bone marrow.
The role of hemin (iron protoporphyrin 9) in the enhancement of interleukin-3 (IL-3)-stimulated multipotent stem cell colony formation was assessed in both serum-containing as well as in "serum-free" marrow culture systems. A greater than 7-fold enhancement in colony number was observed when cultures were supplemented with both IL-3 and hemin compared with either factor alone. In addition, this effect was observed over a wide concentration range. Hemin by itself failed to promote CFU-GEMM in the "serum-free" marrow culture system. The results suggest that hemin acts synergistically with IL-3 to promote the growth of CFU-GEMM in a dose-dependent manner.
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Sensitivity of murine multipotential stem cell colony (CFU-GEMM) growth to interleukin-3, erythropoietin, and hemin.
The in vitro growth of murine marrow-derived CFU-GEMM in response to partially purified preparations of interleukin-3 and erythropoietin was assessed in the presence of hemin. Although CFU-GEMM exhibited a near-absolute requirement for interleukin-3, some colony growth was observed in the absence of exogenous erythropoietin. Erythropoietin was nevertheless capable of further stimulating CFU-GEMM growth in the presence of optimal concentrations of interleukin-3 and hemin. Optimal concentrations of all three factors allowed at least a 60% reduction in the serum concentration without an effect on colony numbers or their detection efficiency. Furthermore, nearly half the colonies continued to grow without the addition of serum, suggesting that hemin supplementation, along with interleukin-3 and erythropoietin, may provide the basis for a relatively simple serum-free culture system for murine CFU-GEMM.
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Effect of rabbit antimouse brain serum on marrow cells capable of curing W/Wv mice.
The W/Wv mouse has a recessively inherited defect in hematopoietic stem cells (HSC) but can be cured of its hematopoietic abnormalities by infusion of marrow from a co-isogeneic, +/+ mouse. The "curative" cell for the W/Wv is thought to be a subcompartment of the HSC that is capable of forming hematopoietic spleen colonies (CFU-S) in irradiated mice. The curative HSC must have a very high proliferative potential and it is known that HSC with variable degrees of proliferative potential are found within the CFU-S compartment. Rabbit antimouse brain serum (RAMBS) was used to treat +/+ marrow and its effect upon CFU-S and upon curative cells was compared with the effect of normal rabbit serum (NRS) or of sham treatment. CFU-S were reduced to 70%-79% of control by NRS and to 8%-9% by RAMBS. Curative cells for the W/Wv were not detectably reduced by NRS; they were reduced by RAMBS, but to only approximately 20%-30% of control. Thus, it appeared to a certain degree that RAMBS spared HSC with a high proliferative potential when compared with its effect on the entire CFU-S compartment.
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Antibodies against pluripotent stem cells: their use in studying stem cell function.
The biologic characteristics and specificity of rabbit anti-mouse brain (RAMB) serum for pluripotent hemopoietic stem cells (CFU-s) is reviewed. The application of RAMB serum to the functional analysis of stem cell differentiation and self renewal characteristics is discussed. Preliminary data are presented which suggest the existence of two stem cell subcompartments. The majority of stem cells express membrane determinants that are detected by RAMB serum. A minor (5%-10%) stem cell subpopulation lacks the stem cell antigen and exhibits a greater self-renewal capacity than those cells expressing the antigen.
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The relationship between stem cell seeding efficiency and position in cell cycle.
The seeding efficiency of colony-forming cells from normal, regenerating and velocity-sedimented cycling and non-cycling narrow preparations was compared. Colony-forming cells in cycle were found to exhibit a 50% reduction in splenic seeding when compared to normal marrow or sedimented non-cycling cells. The results of this study indicate that the spleen colony assay underestimates the total number of colony-forming cells by a fraction which is directly related to the number of cells in cycle.
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Characterization of the anti-stem cell activity of anti-mouse brain serum.
The anti-stem cell activity of a high-titer rabbit anti-mouse brain serum preparation has been further characterized. Following absorption with bone marrow and erythrocytes the antiserum had dose-dependent cytotoxicity against pluripotent stem cells. Rigorous absorptions with bone marrow, spleen, liver, erythrocytes, and thymus failed to remove the anti-stem cell activity of the serum. Adult brain, the immunogen, but not neonatal brain, removed a substantial amount of the activity against stem cells. Maximal cytotoxicity occurred both with and without complement and was maximal following only a 4 degrees C incubation of cells with serum. The anti-stem cell activity was present in the serum globulin fraction. No increase in the frequency of microcolonies or, with longer growth periods, in splenic macrocolonies was observed, suggesting that CFU-s were completely inactivated by exposure to the antiserum. Injected antiserum also reduced CFU-s in vivo.
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Hydroxyurea-induced erythroid differentiation.
Hydroxyurea, a cytotoxic agent which destroys cells in DNA synthesis, has been shown to evoke the differentiation of a small number of hemopoietic precursor cells in the erythroid series of erythropoietically suppressed hypertransfused mice. This effect does not appear to be mediated by erythropoietin (EP) since the simultaneous injection of anti-EP did not alter this response.
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