Murine Pluripotent Stem Cells Research Articles

CD4 Is Expressed on Murine Pluripotent Hematopoietic Stem Cells

Abstract We show here for the first time that pluripotent hematopoietic stem cells express the CD4 antigen. CD4+ cells isolated from mouse marrow repopulated all hematopoietic lineages in both the long-term repopulation assay and the competitive repopulation assay. This finding indicates that the CD4+ population contains primitive stem cells with extensive repopulation capacity. Interestingly, the CD4- population had significant life-sparing activity, even though this population was depleted of long-term repopulating stem cells when compared with CD4+ cells. The majority of the cells that respond to the stroma in Whitlock- Witte cultures with B-cell differentiation were recovered in the CD4- population. Thus, this bone marrow (BM)-derived B-cell precursor lacks CD4, which is in contrast to myeloid precursors and thymus-derived lymphoid precursors that reportedly express CD4. We show further that the CD4 molecule expressed on BM cells is similar in molecular weight and epitope makeup to the CD4 antigen found on thymocytes. Detection of CD4 on BM cells is dependent on using high concentrations of antibodies. Thus, it is not surprising that expression of CD4 on pluripotent stem cells has been missed previously. Taken together, our data suggest that the CD4 molecule may play an important role in lineage definition in early hematopoietic differentiation.

IL-6 Production by Retrovirus Packaging Cells and Cultured Bone Marrow Cells

Retrovirus integration into the host cell genome occurs most efficiently in replicating cells. In agreement with this notion, it was observed that the efficiency with which hemopoietic stem cells (HSC) can be transduced is greatly enhanced when the hemopoietic growth factor (HGF) interleukin 3 (IL-3) is added to co-cultures of bone marrow cells with retrovirus-producing cells. The HGF IL-6, which enhances the IL-3-induced formation of blast cell colonies in vitro, is also believed to improve the transduction of HSC. Because IL-6 can be produced by a number of different cell types, we investigated whether IL-6 was present in the culture supernatant of retrovirus packaging cells and bone marrow cells. We found that the six retrovirus packaging cells tested produced large amounts of IL-6. Bone marrow cells cultured with IL-1 alpha and IL-3 also make IL-6, and, following co-cultivation of both cell types, the concentration of IL-6 in the medium is even up to 10-fold higher than the sum of the concentrations obtained when both are cultured separately. Considering that IL-6 is produced in large amounts during co-cultivations, we believe that its effect on the transduction of HSC cannot be measured by adding extra growth factor to the co-culture medium.

A Low‐Molecular‐Weight Factor Extracted from Human Placenta Inhibits the Entry into Cell Cycle of Murine Pluripotent Stem Cells

A Low‐Molecular‐Weight Factor Extracted from Human Placenta Inhibits the Entry into Cell Cycle of Murine Pluripotent Stem Cells

Mock Retroviral Infection Alters the Developmental Potential of Murine Bone Marrow Stem Cells

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.

Pluripotent Embryonal Stem Cell Lines Can Be Established from Disaggregated Mouse Morulae: (mouse embryonal stem (ES) cells/disaggregated morulae/in vitro development/teratoma).

Mouse pluripotent embryonal stem (ES) cell lines hitherto have been conventionally isolated from the 'inner cell mass' of mouse blastocysts. In this report, I describe a new and simplified method for establishing pluripotent cell lines from mouse morulae of the 16- to 20-cell stage, which were disaggregated by the use of EDTA. From 17 cell lines established in such a way, 7 were characterized with respect to their differentiation potential: (i) When injected into syngeneic mice, the cells gave rise to solid, fully differentiated teratomas representing derivatives of all three germ layers. (ii) When cultured in suspension in vitro, the cells were able to differentiate into complex organized 'embryoid bodies' analogous to mouse early postimplantation embryos. These results strongly imply that embryonal stem cell lines isolated from mouse morulae are highly homologous to conventionally isolated ES cells. In addition, my results indicate that murine pluripotent embryonal stem (ES) cell lines can be derived with more ease and higher efficiency from disaggregated morulae than from the 'inner cell mass' of blastocysts.

Tumor-promoting phorbol esters support the in vitro proliferation of murine pluripotent hematopoietic stem cells.

The effect of tumor-promoting phorbol esters on the in vitro proliferation of mouse pluripotent hematopoietic stem cells (CFU-S) was examined using a short-term in vitro culture system and an 11-d spleen colony assay. Phorbol myristate acetate (PMA, 10(-7) M), but not the inert compound phorbol, supported the in vitro survival of day 11 CFU-S for 72 h in a manner similar to IL 3. PMA also enhanced the effect of IL 3 on the in vitro survival of day 11 CFU-S and as little as 1 h of exposure to PMA was sufficient for this purpose. The effect of PMA on CFU-S survival in vitro was not mediated by prostaglandins, did not require an established adherent cell population, and was observed at a concentration of 10(-9) M. PMA alone did not enhance the in vitro survival of day 11 CFU-S at very low concentrations of FCS but was still able to potentiate the effect of IL 3 on these cells. PMA also enhanced the in vitro survival of day 11 CFU-S from mice treated with 5-fluorouracil or from marrow cells exposed to merocyanine 540 and light. The interaction of PMA with day 11 CFU-S was not inhibited by a neutralizing antiserum to IL 3 but was inhibited by the protein kinase inhibitor H-7. Together, the data indicate that tumor-promoting phorbol esters interact with pluripotent hematopoietic stem cells. Like IL 3, their effect appears to be permissive and involves stem cells with marrow repopulating ability.

Effects of aldehyde dehydrogenase inhibitors on the ex vivo sensitivity of murine late spleen colony-forming cells (day-12 CFU-S) and hematopoietic repopulating cells to mafosfamide (ASTA Z 7557)

The effects of inhibitors of aldehyde dehydrogenase activity on the sensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorine anticancer agents, e.g. mafosfamide, were examined using two different assay procedures. In the first part of the investigation, the ex vivo sensitivity of murine day-12 spleen colony-forming cells (CFU-S) to mafosfamide was determined in the absence and presence of known inhibitors of aldehyde dehydrogenase activity, viz. diethyldithiocarbamate and cyanamide. These results were compared to those generated for day-8 CFU-S. Day-12 CFU-S were less sensitive to mafosfamide, and to phosphoramide mustard, although the difference in sensitivity to the latter was less marked. Diethyldithiocarbamate and cyanamide each potentiated the cytotoxic action of mafosfamide toward both day-12 and day-8 CFU-S; they did not potentiate the cytotoxic action of phosphoramide mustard toward these cells. Since cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophosphamide/aldophosphamide, the major transport form of mafosfamide, to the relatively nontoxic acid, carboxyphosphamide, the results suggest that intracellular aldehyde dehydrogenase activity is a determinant of the sensitivity of day-12 CFU-S, as well as of day-8 CFU-S, to mafosfamide and other oxazaphosphorines, e.g. cyclophosphamide. In the second part of this investigation, a murine syngeneic bone marrow transplantation model was used to determine the ex vivo sensitivity of murine hematopoietic repopulating cells to mafosfamide in the absence and presence of diethyldithiocarbamate. Specifically, the ability of treated marrow grafts to repopulate the hematopoietic system, and thereby save recipients from the otherwise lethal effect of total body irradiation, was determined. Diethyldithiocarbamate potentiated the cytotoxic action of mafosfamide, but not that of phosphoramide mustard, toward hematopoietic repopulating cells. These observations support our previous contention that aldehyde dehydrogenase activity is an operative determinant with regard to the sensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorines.

Direct morphological and functional examination of murine pluripotent hemopoietic stem cells.

Pluripotent hemopoietic stem cells (PHSC) were isolated from adult mouse bone marrow by a combination of equilibrium density centrifugation and light-activated cell sorting for WGA-positive and H-2k antigen-positive cells. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 days and 6.6 colonies per 100 cells at 12 days after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony forming unit-spleen) equalled 135 (range, 90-230; n = 15). Enrichment for the cell type that provides radioprotection was equal to 180 +/- 70, indicating that PHSC and CFU-S are identical. Evidence is provided that the spleen seeding efficiency (the f-factor) of these cells was 0.10 and, therefore, that the average purity of the sorted PHSC was 65% (range in 15 experiments, 35-110%). The sorted cells were all in the G0 or G1 phase of the cycle. They appeared to be undifferentiated blasts by morphological criteria, the majority of the cells being similar in structure to the PHSC previously identified in less purified concentrates. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. Thirty percent of the sorted cells could be induced to form progeny in vitro. This procedure facilitates direct examination of the first events of hemopoietic regulation.

Aldehyde dehydrogenase activity as the basis for the relative insensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorines

The ex vivo sensitivity of murine pluripotent hematopoietic stem cells (CFU-S) and myeloid progenitor cells (CFU-GM) to 4-hydroperoxycyclophosphamide, ASTA Z 7557, phosphoramide mustard, acrolein, melphalan, and cis-platinum was determined in the absence and presence of known (disulfiram, diethyldithiocarbamate, cyanamide) or suspected [ethylphenyl(2-formylethyl)phosphinate] inhibitors of aldehyde dehydrogenase activity. As compared to CFU-GM, CFU-S were less sensitive to the oxazaphosphorine agents, 4-hydroperoxycyclophamide and ASTA Z 7557. The two cell populations were approximately equisensitive to acrolein as well as to the non-oxazaphosphorine cross-linking agents, phospharamide mustard, melphalan and cis-platinum. All four inhibitors pf aldehyde dehydrogenase activity potentiated the cytotoxic action of the oxazaphosphorines toward CFU-S; they did not potentiate the cytotoxic action of acrolein or the non-oxazaphosphorines toward these cells. The inhibitors did not potentiate the cytotoxic action of the oxazaphosphorines, non-oxazaphosphorines, or acrolein toward CFU-GM. Pyridoxal, a substrate for aldehyde oxidase, did not potentiate the cytotoxic action of oxazaphosphorines toward CFU-S. Cellular NAD-linked aldehyde dehydrogenase are known to catalyze the oxidation of the major transport form of cyclophosphamide, 4-hydroxycyclophosphamide/aldophosphamide, to an inactive metabolite, carboxyphosphamide. Our observations suggest that (1) aldehyde dehydrogenase activity is an important determinant of the sensitivity of a cell population to the oxazaphosphorines, (2) CFU-GM lack the relevant aldehyde dehydrogenase activity, and (3) the phenotypic basis for the relative insensitivity of CFU-S to oxazaphosphorines is the aldehyde dehydrogenase activity contained by these cells.

Interleukin 3 promotes the in vitro proliferation of murine pluripotent hematopoietic stem cells.

Medium conditioned by activated T lymphocytes stimulates the in vitro proliferation of pluripotent hematopoietic stem cells (spleen colony-forming units [CFU-S]) but the factors involved have not been identified. Because the lymphokine interleukin 3 (IL-3) enhances in vitro colony formation by committed hematopoietic progenitor cells, we examined the effect of IL-3 on the in vitro proliferation of CFU-S using an 11-d spleen colony assay. When mouse marrow cells were placed in liquid culture, CFU-S content declined progressively and by 96 h only 13% of the CFU-S remained. By contrast, after 96 h in the presence of 20 U/ml of IL-3, the number of CFU-S were the same as that in the initial inoculum. Although the number of CFU-S eventually declined, they could still be recovered after 264 h of culture. In the absence of IL-3, the number of CFU-S synthesizing DNA was negligible; in its presence, greater than 20% of the CFU-S were in cycle. IL-3 stimulated CFU-S proliferation at a concentration of 0.2 U/ml. The dose-response curve was similar to that observed for other biologic effects of the lymphokine, and as little as 1 h of exposure to IL-3 enhanced the survival of CFU-S in vitro. Treatment of marrow cells with anti-Thy 1.2 antibody and complement before exposure to IL-3 did not inhibit spleen colony formation, but treatment of the cells with anti-Thy 1.2 antibody and complement after exposure to IL-3 reduced CFU-S recovery after 96 h of culture by 45%. The cell composition of day 11 spleen colonies formed by IL-3-treated marrow cells was similar to that of colonies formed by untreated marrow cells. Finally, day 11 CFU-S persisting in the marrow of mice treated with 5-fluorouracil required IL-3 for proliferation in vitro. Taken together, these data indicate that IL-3 promotes the proliferation of CFU-S in vitro, increases the number of CFU-S synthesizing DNA, but does not alter their commitment program, and the target cell population includes CFU-S with self-renewal and marrow-repopulating ability.

Human Spleen Cell Generation of Factors Stimulating Human Pluripotent Stem Cell, Erythroid, and Myeloid Progenitor Cell Growth

Mitogen-stimulated murine spleen cells produce humoral substances capable of supporting murine hematopoiesis and pluripotent stem cell proliferation in vitro. Thus, we evaluated conditioned media generated by human spleen cells (SCM) in the presence or absence of mitogens for factors stimulatory for human pluripotent (CFU-GEMM), erythroid (BFU-E), and myeloid (CFU-GM) precursors. Two and one half percent to 10% SCM stimulated proliferation of all three types of precursor cells from nonadherent buoyant human marrow target cells. Mitogen-stimulated SCM augmented CFU-GM (175% to 225%), whereas CFU-GEMM and BFU-E growth was essentially unchanged. Cell separation procedures used to determine which cells provided these microenvironmental stimuli indicated that nonadherent mononuclear spleen cells provided the bulk of the CSF-GM, whereas adherent cells (95% nonspecific esterase + monocyte-macrophages) and nonadherent cells provided similar proportions of CSF-mix and erythroid burst-promoting activity (BPA). The nonadherent cells generating high levels of CSF-mix, BPA, and CSF-GM were predominantly Leu-1-negative, ie, non-T, cells. In the presence or absence of mitogens, SCM was a more potent source (1.3- to 3.8-fold) than peripheral leukocyte CM of the growth factors for the three progenitor cell types. Specific in situ cytochemical stains for analyzing morphology of myeloid colonies demonstrated that SCM stimulated the proliferation of the same types and proportions of colonies as human placental CM, suggesting that these CMs may contain similar CSF-GMs. These data show the contribution of spleen cell subsets to the generation of hematopoietic growth factors and the responsiveness of these cells to various mitogenic stimuli.

Human spleen cell generation of factors stimulating human pluripotent stem cell, erythroid, and myeloid progenitor cell growth

Mitogen-stimulated murine spleen cells produce humoral substances capable of supporting murine hematopoiesis and pluripotent stem cell proliferation in vitro. Thus, we evaluated conditioned media generated by human spleen cells (SCM) in the presence or absence of mitogens for factors stimulatory for human pluripotent (CFU-GEMM), erythroid (BFU- E), and myeloid (CFU-GM) precursors. Two and one half percent to 10% SCM stimulated proliferation of all three types of precursor cells from nonadherent buoyant human marrow target cells. Mitogen-stimulated SCM augmented CFU-GM (175% to 225%), whereas CFU-GEMM and BFU-E growth was essentially unchanged. Cell separation procedures used to determine which cells provided these microenvironmental stimuli indicated that nonadherent mononuclear spleen cells provided the bulk of the CSF-GM, whereas adherent cells (95% nonspecific esterase + monocyte- macrophages) and nonadherent cells provided similar proportions of CSF- mix and erythroid burst-promoting activity (BPA). The nonadherent cells generating high levels of CSF-mix, BPA, and CSF-GM were predominantly Leu-1-negative, ie, non-T, cells. In the presence or absence of mitogens, SCM was a more potent source (1.3- to 3.8-fold) than peripheral leukocyte CM of the growth factors for the three progenitor cell types. Specific in situ cytochemical stains for analyzing morphology of myeloid colonies demonstrated that SCM stimulated the proliferation of the same types and proportions of colonies as human placental CM, suggesting that these CMs may contain similar CSF-GMs. These data show the contribution of spleen cell subsets to the generation of hematopoietic growth factors and the responsiveness of these cells to various mitogenic stimuli.

Isolation of murine pluripotent hemopoietic stem cells.

A method described to purify pluripotent hemopoietic stem cells ( PHSC ) from adult mouse bone marrow. The method consists of three separation steps. First, bone marrow cells are centrifuged in a discontinuous metrizamide gradient and simultaneously labeled with wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC). Second, the low density cells are analyzed by a fluorescence-activated cell sorter (FACS) and the WGA-positive cells with medium forward and low perpendicular light scatter intensities are sorted. The WGA-FITC is removed from the cells by incubation with N-acetyl-D-glucosamine. Finally, the sorted cells are incubated with anti-H-2K-biotin and avidin-FITC and sorted a second time to enrich cells with high H-2K density. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 d and 6.6 colonies per 100 cells at 12 d after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony-forming unit/spleen) was 135 (range, 90--230; n = 15) and was similar to that for the cell type that provides radioprotection (180 +/- 70), indicating that these functional properties were copurified. Indirect evidence suggests that the spleen-seeding efficiency (f factor) of these cells is 0.10 and, therefore, the average purity of the sorted PHSC was 65% (range in 15 experiments, 35--110%). The sorted cells were all in the G1 or G0 phase of the cell cycle. They appeared to be undifferentiated blasts by morphological criteria. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. 32% of the sorted cells could be induced to form myeloid progeny in vitro. This procedure should be useful for direct studies on the regulation of hemopoietic cell differentiation.

An antigenic difference between cells forming early and late haematopoietic spleen colonies (CFU-S).

Murine pluripotent haematopoietic stem cells have generally been assayed by their ability to form macroscopic colonies of haematopoietic cells in the spleens of heavily irradiated recipient mice (colony-forming unit-spleen or CFU-S assay). However, recent evidence suggests that there are distinct subpopulations of CFU-S. Most spleen colonies present 7-8 days after injection consist of differentiated erythroid cells, contain no primitive myeloid or erythroid precursor cells and disappear from the spleen within 3 days, whereas the majority of colonies present at 10-12 days contain primitive precursors, are multilineal and cannot be detected at 7-8 days. Furthermore, many 10-day-old spleen colonies do not contain cells capable of forming spleen colonies in secondary irradiated recipients (an index of the self-renewal capacity of stem cells) whereas at day 14 almost all colonies contain these CFU-S. These studies suggest that only when colonies are scored at or later than 11-12 days is the spleen colony technique adequate for the assay of multipotential stem cells. We now report an antigenic difference between subpopulations of CFU-S forming early (day 8) and late (day 14) spleen colonies which has been used to purify multipotential haematopoietic stem cells from murine bone marrow.

Dose-dependent responses of murine pluripotent stem cells and myeloid and erythroid progenitor cells following administration of the immunomodulating agent glucan

The hemopoletic effects produced by six different doses of a commercially available glucan preparation were Investigated, C3H/HeN mice were Intravenously injected with either 0.1, 0.4, 0.8, 1.2, 1.6, or 2.0 mg of glucan Five days later, total nucleated cellularity, pluripotent stem cefls (CFU-s), grunulocyte-macrophage colony-forming cells (GM-CFC), macrophage collonyfarming cells (M-CFC), and erythroid burst- and colony-forming cells (EFU-e and CFU-e) were assayed in the bone marrow and spleen. Neither femoral cellularity nor GM-CFC content was alterede by any of the glucan doses tested. By contrast, bone marrow CFU-s content increased and CFU-e and BFU-e contents decreased with Increasing glucan doses. In the spleen, all aspects of hemopoiesils (i.e., cellularity, CFU-s, GM-CFC, M-CFC, and CFU-e) increased after glucan administration, The degree of splenic enhancement was directly correlated with the dose of glucan administered.

Temporal response of murine pluripotent stem cells and myeloid and erythroid progenitor cells to low-dose glucan treatment.

B6D2F1 female mice were intravenously administered 0.4 mg of glucan. 1, 5, 11, and 17 days later, the total nucleated cellularity (TNC) and the numbers of pluripotent hemopoietic stem cells (CFU-s), granulocyte-macrophage progenitor cells (GM-CFC), and erythroid colony-forming (CFU-e) and burst-forming (BFU-e) cells were assayed in the bone marrow and spleen. Bone marrow TNC was not altered, but splenic TNC increased approximately twofold on day 5 and remained increased on days 11 and 17 after glucan treatment. The concentrations of bone marrow and splenic CFU-s and GM-CFC both significantly increased (p less than 0.01) by 5 days after glucan administration; however, they returned to control levels by day 17. Splenic CFU-e concentration increased on days 5, 11, and 17, whereas splenic BFU-e concentration increased only on day 11 after treatment. By contrast, bone marrow CFU-e and BFU-e concentrations were either unaffected or slightly decreased by glucan treatment. When peripheral blood was assayed for CFU-s and GM-CFC, no detectable increase in the concentrations of these progenitors was noted at any time after glucan treatment. The relevance of these effects of low-dose (0.4 mg) glucan treatment is discussed with respect to previously reported effects of higher-dose (e.g., 4.0 mg) glucan treatment.

Proliferation inhibition of murine pluripotent haemopoietic stem cells by interferon or poly I:C.

The effect of mouse serum interferon (IF) in vitro and an inducer in vivo on the proliferation of a pluripotent stem cell population with high turnover rate was studied. Proliferation rate was characterized by the number of CFUs in the S phase of the cell cycle. Increased proliferation of bone marrow stem cell populations was produced either by irradiating the donor mice with 3.36 Gy (336 rad) 60Co-gamma rays 7 days before the experiment or by incubating normal bone marrow cells with 10(-11) M concentration of isoproterenol. IF considerably reduced the number of CFUs in S phase in both cases without reducing the CFUs content of the samples. Injection of IF inducer (4 mg/kg poly I:C) into regenerating mice also inhibited the proliferation of CFUs without decreasing the femoral CFUs level. Regeneration kinetics of CFUs from irradiated poly I:C-treated mice ran parallel with that of irradiated untreated animals but showed a characteristic delay corresponding to approximately one CFUs doubling. A transient, non-cytotoxic proliferation inhibitory effect of IF or IF inducer is, therefore, proposed.

The effect of lithium on murine hematopoiesis in a liquid culture system.

Lithium carbonate has been shown to increase granulocyte production. We studied the effect of lithium on murine hematopoiesis in a liquid culture system providing for the prolonged growth of stem cells and their progeny. After one week of incubation, lithium, at a supernatant concentration of 1 mmol per liter, increased murine pluripotent stem cells (CFU-S, or colony-forming units in spleen) to 232 per cent of control values (P less than 0.001), granulocyte-monocyte progenitor cells (CFU-C, or colony-forming units in culture) to 218 per cent of control values (P less than 0.0001), granulocytes to 125 per cent of control values (P less than 0.01), and megakaryocytes to 246 per cent of control values (P less than 0.001). These increases were associated with transient elevations in colony-stimulatory activity. Prolonged exposure to lithium (three to 12 weeks) was associated with a dose-dependent progressive depletion of stem cells and their progeny. Lithium enhancement of granulopoiesis may be explained by primary stimulation of the pluripotent stem cell. Prolonged proliferative stress induced by lithium when the stem-cell reserve is limited may be associated with diminished replicative potential of the stem cells and rapid depletion of cells.

Stem cell migration induced by erythropoietin or haemolytic anaemia: the effects of actinomycin and endotoxin contamination of erythropoietin preparations.

The injection of erythropoietin or the induction of anaemia with phenylhydrazine leads to changes in murine pluripotent and granulocyte-macrophage stem cells indicating migration from marrow to spleen. In order to evaluate the interrelationship between erythroid differentiation and stem cell migration we have selectively suppressed erythroid differentiation with actinomycin D. Anaemia or EP injection resulted in stem cell changes consistent with migration; actinomycin blocked these changes in anaemic but not EP injected mice while blocking erythropoiesis in both groups. The erythropoietin contained from 0.01 to 1000 microgram/ml of endotoxin as defined by the limulus test; it decreased marrow erythropoiesis and stimulated marrow granulopoiesis. Adsorption of the erythropoietin preparation with limulus lysate removed endotoxin without decreasing erythropoietin activity. Adsorbed erythropoietin stimulated erythropoiesis and not granulopoiesis, and stem cell changes induced by its administration were largely blocked by actinomycin, suggesting that endotoxin in the non-adsorbed erythropoietin caused the actinomycin resistant stem cell changes. The observation that actinomycin blocks both erythroid differentiation and stem cell migration suggests that these two physiologic events are closely linked. The effects of injected erythropoietin on murine haemopoietic stem cells may, to a significant extent, be secondary to the presence of endotoxin in the erythropoietin preparations.