Lymphoid differentiation of the hematopoietic stem cell that reconstitutes total erythropoiesis of a genetically anemic W/Wv mouse

We investigated whether the stem cell that reconstitutes total erythropoiesis of a WBB6F1-W/Wv mouse differentiates into lymphoid lineage. The electrophoretic pattern of hemoglobin was used as a marker of the reconstitution; 3-phosphoglycerate kinase (PGK), an X chromosome-linked enzyme was used as a tool for estimating clonality. We injected 10(5) bone marrow cells of 5-FU treated C57BL/6-Pgk-1b/Pgk-1a female mice, in which each stem cell had either A-type PGK or B-type PGK due to random inactivation of one of two X chromosomes, into genetically anemic (WB x C57BL/6)F1-W/Wv (hereafter WBB6F1-W/Wv) mice that contained only B-type PGK. The recipient WBB6F1-W/Wv mice, in which erythropoiesis was reconstituted with donor cells for a long term, were killed and the PGK patterns of bone marrows, thymus, lymph nodes, and Peyer's patches were examined. A considerable amount of A-type PGK was detected in the lymphoid organs of the WBB6F1-W/Wv mice in which erythrocytes showed only A-type PGK when killed. In contrast, A-type PGK was scarcely detectable in the lymphoid organs of the WBB6F1-W/Wv mice in which erythrocytes showed only B-type PGK when killed. The present results suggest that the hematopoietic stem cells estimated by the erythropoiesis reconstituting assay differentiate into lymphoid lineage and that the long-term erythropoiesis reconstitution assay is useful for detecting the true primitive hematopoietic stem cells.     

Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.     

In vivo expansion of purified hematopoietic stem cells transplanted in nonablated W/Wv mice

We have evaluated the in vivo amplification potential of purified murine hematopoietic stem cells, identified as Wheat Germ Agglutinin+ (WGA+), 15-1.1(-) , Rhodamine 123 Dull (Rho-dull) cells, by serial transplantation into stem cell defective nonmyeloablated W/Wv mice. C57BL Rho-dull cells (250/ 500 cells/mouse) permanently engrafted nonablated W/Wv mice as defined by the presence of > 95% red and > 20% white donor-derived circulating cells for at least 1.5 years following transplantation. At this time, approximately 61% of Rho-dull cells and all the Rho-bright progenitor and colony forming cells of the engrafted mice were found to be donor-derived by c-Kit genotyping and by their response to stem cell factor (SCF). Retransplantation of 250-1000 Rho-dull cells from primary into secondary W/Wv recipients generated C57BL hematopoiesis in 40%-64% of animals revealing the presence of donor derived hematopoietic stem cells (HSC) in the bone marrow of the primary recipients. One and half years after transplantation, the bone marrow of the secondary engrafted animals contained C57BL Rho-dull cells approximately = 51% by genotype), which were capable of reconstituting tertiary W/Wv recipients. In this respect, 25% of tertiary mice expressed C57BL hematopoiesis when transplanted with 250-1000 Rhodull cells purified from secondary W/Wv recipients. On the basis of the number of Rho-dull cells purified from a single mouse, we calculate that approximately 7.3x10(4) Rho-dull cells, which are genotypically and functionally defined as C57BL long-term repopulating stem cells, were generated in the marrow of reconstituted primary W/Wv recipients transplanted 1.5 years earlier with 250-500 C57BL Rho-dull cells. We conclude that murine HSC have extensive amplification capacity in nonmyeloablated animals.     

Pluripotent hematopoietic stem cells of low and high density can repopulate W/Wv mice.

We have studied several features of pluripotent hematopoietic stem cells (PHSC) and day-12 spleen colony-forming units (CFU-S12) in murine bone marrow. C57BL/6J marrow cell suspensions were separated by elutriation and fractions were obtained at flow rates (FR) of 25 ml/min, 29/30 ml/min, 35 ml/min, and with the rotor off. All four fractions contained PHSC that could repopulate W/Wv mice, but significant numbers of CFU-S12 were found only in the three higher FR fractions. Cells in the FR29/30 fraction were shown to have almost three-fold more repopulating activity than fresh marrow in a competitive repopulation assay. The PHSC in fractions separated by elutriation were enriched by depleting cells expressing specific lineage markers with monoclonal antibodies and magnetic immunobeads. As few as 10(4) lineage negative (lin-) cells from FR35 or 10(5) lin--cells from FR25 conferred long-term multilineage repopulation in W/Wv mice, as demonstrated by Southern blot analysis of DNA from recipient thymus and bone marrow. We conclude that PHSC are heterogeneous for cell size and density and that the highest concentration of PHSC resides in the subset of intermediate density present in the FR29/30 fraction.     

Different repopulation profile between erythroid and nonerythroid progenitor cells in genetically anemic W/Wv mice after bone marrow transplantation

Repopulation kinetics of erythrocytes and neutrophils and replacement of hematopoietic progenitors were studied in genetically anemic (WB x C57BL/6)F1-W/Wv (WBB6F1-W/Wv) hosts after bone marrow transplantation from C57BL/6-bgJ/bgJ or C57BL/6-bgJ/bgJ;Pgk-1a/Y mice. Electrophoretic pattern of hemoglobin was used as a marker of donor-type erythrocytes, giant granules of bgJ/bgJ mice as a marker of donor-type neutrophils, and A-type phosphoglycerate kinase-1 (PGK-1) as a marker of hematopoietic colonies produced by donor-derived progenitor cells. Repopulation of donor-type erythrocytes was significantly faster than that of donor-type neutrophils. Moreover, the extent of replacement was greater for erythroid progenitor cells than for nonerythroid progenitor cells. When nonirradiated WBB6F1-W/Wv mice with B-type PGK-1 received 10(5) bone marrow cells from C57BL/6-bgJ/bgJ;Pgk-1a donors, only approximately 20% replacement of erythroid progenitor cells gave rise to total reconstitution of erythrocytes. The present result suggests that normal multipotential stem cells may preferentially differentiate into erythroid lineage cells in anemic WBB6F1-W/Wv hosts and that normal erythroid progenitor cells may suppress the differentiation of erythroid progenitors of WBB6F1-W/Wv hosts.     

Clonal contributions of small numbers of retrovirally marked hematopoietic stem cells engrafted in unirradiated neonatal W/Wv mice.

Mice were repopulated with small numbers of retrovirally marked hematopoietic cells operationally definable as totipotent hematopoietic stem cells, without engraftment of cells at later stages of hematopoiesis, in order to facilitate analysis of stem cell clonal histories. This result depended upon the use of unirradiated W/Wv newborn recipients. Before transplantation, viral integration markers were introduced during cocultivation of fetal liver or bone marrow cells with helper cell lines exporting defective recombinant murine retroviruses of the HHAM series. Omission of selection in culture [although the vector contained the bacterial neomycin-resistance (neo) gene] also limited the proportion of stem cells that were virally labeled. Under these conditions, engraftment was restricted to a small population of marked and unmarked normal donor stem cells, due to their competitive advantage over the corresponding defective cells of the mutant hosts. A relatively simple and coherent pattern emerged, of one or a few virally marked clones, in contrast to previous studies. In order to establish the totipotent hematopoietic stem cell identity of the engrafted cells, tissues were sampled for viral and inbred-strain markers for periods close to one year after transplantation. The virally labeled clones were characterized as stem cell clones by their extensive self-renewal and by formation of the wide range of myeloid and lymphoid lineages tested. Results clearly documented concurrent contributions of cohorts of stem cells to hematopoiesis. A given stem cell can increase or decrease its proliferative activity, become completely inactive or lost, or become active after a long latent period. The contribution of a single clone present in a particular lineage was usually between 5% and 20%.     

Purified murine hematopoietic stem cells function longer on nonirradiated W41/Wv than on +/+ irradiated stroma

Mouse bone marrow (BM) was separated into low-density, lineage- negative, wheat germ agglutinin-positive (WGA+), Rhodamine-123 bright (Rhbright) or dim (Rhdim) cells to obtain populations that were highly enriched for committed progenitors (Rhbright cells) or for more primitive stem cells (Rhdim). When 2,500 Rhbright or Rhdim cells were seeded onto 6-week-old irradiated (20 Gy) long-term BM cultures (LTBMC), the nonadherent cell production from Rhbright cells was transient and ended after 5 weeks. Production from Rhdim cells did not begin until week 3, peaked at week 5, and ended at week 8, when the irradiated stroma seemed to fail. Termination of cell production from Rhdim cells did not occur in nonirradiated LTBMC from W41/Wv mice. During peak nonadherent cell production, 25% to 30% of the cells in the nonirradiated LTBMC from W41/Wv mice had donor cell markers. Two approaches were tested to try to enhance the proportion or number of donor cells. Addition of Origen-HGF at the time of seeding Rhdim cells caused a nonspecific increase in both host and donor cell production, but a specific increase in production of donor cells was obtained by seeding the cultures at 2 weeks rather than 6 weeks. Limiting dilution of Rhdim cells gave the same frequency of wells producing cells on both irradiated +/+ and nonirradiated W41/Wv or W/Wv cultures.     

Interleukin 3 can compensate for the deficiency in erythroid burst-forming cells in anemic mice of genotype W/Wv

Bone marrow cells from mice with the W/Wv mutation have about one-third the erythroid burst-forming cells (BFU-E) found with wild-type littermates. The mutant cells are also less responsive in vitro to exogenous erythropoietin (epo) than are the wild-type cells. Addition of interleukin 3 (IL-3) to the culture medium largely corrects the deficit in burst formation and in responsiveness to epo. When mutant cells are incubated for 2 days in the absence of epo but with IL-3 present, burst formation is the same as with wild-type cells, suggesting that IL-3 acts on maintenance in vitro and/or proliferation of primitive precursor cells of both mutant and wild-type mice.     

Latent deficiency of the hematopoietic microenvironment of aged mice as revealed in W/Wv mice given +/+ cells

The macrocytic anemia of W/Wv mice can be cured by injection of +/+ bone marrow cells (BMC) from WBB6F1 mice. However, it has been observed that some W/Wv recipients appear to "lose" their cure with time, an effect that does not appear to be related to the age of the BMC donor. The present study was undertaken to determine the effect of recipient age on W/Wv responses to BMC injection. The effect of aging on erythroid parameters was similar in untreated W/Wv mice and +/+ controls. In both genotypes, hematocrit (HCT) and red blood cell count (RBC) decreased, and the modal red blood cell size (peak) increased between 13 and 150 weeks of age. As anticipated, mean HCT and RBC values were lower and peak values higher in W/Wv mice compared to +/+ controls at every age. However, the rate of decrease in HCT and RBC with age was the same for both genotypes, suggesting that the age effect and W gene effect were independent. Peak values increased slightly more with age for W/Wv than for +/+ controls. When female W/Wv mice in three age groups (23.5, 70, and 91.5 weeks old) were injected with 5 x 10(5) BMC from 20-week-old +/+ female donors and HCT, RBC, and peak were determined monthly, improvement was seen in most W/Wv recipients. However, in the older mice this improvement was slower and often was not sustained; 100% of the youngest recipients, 80% of the middle-aged, and only 30% of the older groups were cured after 3 months. Taken together, these data suggest a latent deficiency of the aging hematopoietic microenvironment that is revealed in W/Wv mice by the stress of continuing erythroid demand on the limited number of normal donor BMC.     

Temporal replacement of donor erythrocytes and leukocytes in nonanemic W44J/W44J and severely anemic W/Wv mice

The dominant white spotting, W, locus in the mouse encodes Kit, a receptor molecule with cytosolic tyrosine kinase activity. Mutations in Kit deplete hematopoietic cells by an as yet unknown mechanism, but one that presumably affects the early progenitors of all cell lineages. To examine cell lineage-specific changes caused by different W mutations, we injected genetically marked normal marrow cells into mutant mice and monitored repopulation kinetics. In the present report, we compare repopulation of the various peripheral blood cells in nonanemic W44J/W44J and severely anemic W/Wv mice administered increasing increments of donor cells. At all doses of cells tested, donor erythrocyte repopulation precedes leukocyte repopulation regardless of the recipient phenotype. There is, in fact, little difference in the rate or extent of nonerythroid repopulation in W44J/W44J mice injected with between 6 x 10(6) and 2 x 10(7) donor cells. The fact that donor cells rapidly replace erythrocytes, even in the nonanemic W44J/W44J host, while other cell lineages become donor type more slowly provides further evidence that mutations at the W locus are especially damaging to erythrocyte progenitors. We suggest that host nonerythroid hematopoietic cells compete with normal cells, probably at the level of early progenitors rather than at the level of the totipotent hematopoietic stem cell. The fact that successively higher doses of donor cells do not markedly alter nonerythroid repopulation kinetics implies that it may be possible to maximize autologous therapeutic marrow transplantation.     

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.     

Development of an anti-porcine CD34 monoclonal antibody that identifies hematopoietic stem cells

OBJECTIVES: The isolation of porcine hematopoietic stem cells (HSC) would be an important step toward development of porcine-to-human chimerism for induction of tolerance in clinical xenotransplantation. CD34 is a common marker of HSC and has not been developed as a marker in pigs. In this study we have generated and characterized a monoclonal antibody (mAb) that identifies porcine CD34 on a subset of porcine bone marrow (BM) stem/progenitor cells. METHODS: The porcine CD34 gene was cloned and a recombinant protein produced. An anti-porcine CD34 mAb was produced that could detect both the recombinant protein and a subset of porcine BM cells. The CD34(+) cells were phenotyped by lineage and HSC associated markers. Furthermore, the CD34(+) cells were analyzed by colony-forming unit (CFU) assay. RESULTS: Two splice variants of the porcine CD34 gene were cloned and a recombinant protein produced for mAb production. The mAb developed can detect both the recombinant protein and the native CD34 protein on a range of pig tissues, including BM. This subset of BM cells was negative for hematopoietic lineage makers, including CD3, CD14, and CD21 and positive for other known porcine HSC markers, including CD90, CD172a, histocompatibility complex (MHC) class I, and MHC class II. Moreover, the CD34(+) BM cells were enriched for multilineage progenitor cells as determined by CFU assay. CONCLUSIONS: Similar to human and mouse CD34, pig CD34 detects a subset of BM progenitor cells. This mAb will now provide a means for isolating porcine CD34(+) cells to be further analyzed for HSC activity and to assess their potential to develop pig-to-human chimeras to induce xenograft tolerance.     

Phorbol 12-myristate 13-acetate-induced development of functionally active mast cells in W/Wv but not Sl/Sld genetically mast cell-deficient mice

The normal skin and other tissues of adult genetically mast cell-deficient WBB6F1-W/Wv or WCB6F1-Sl/Sld mice contain less than 1.0% the number of mast cells present in the corresponding tissues of the congenic normal (+/+) mice. We previously reported that mature dermal mast cells developed locally in the skin of W/Wv, but not Sl/Sld, mice at sites of chronic idiopathic dermatitis. We now report that the repeated application of phorbol 12-myristate 13-acetate (PMA) to the ear skin of either W/Wv or +/+ mice induces both dermatitis and a striking and dose-dependent increase in the number of dermal mast cells. The number of dermal mast cells at sites treated for 6 weeks with 5 micrograms PMA, three times per week, was 39 +/- 7/mm2 and 305 +/- 34/mm2 for W/Wv and +/+ mice, respectively; the corresponding values for vehicle-treated skin were 1.5 +/- 1.0/mm2 and 145 +/- 8/mm2, respectively. The PMA-induced dermal mast cells in W/Wv mice appeared mature by morphology, stained with the heparin-binding fluorescent dye, berberine sulfate, and were competent to express IgE-dependent passive cutaneous anaphylaxis responses. The development of mast cells was a local, not systemic, effect of PMA treatment. PMA treatment also induced dermatitis in both WCB6F1-Sl/Sld and +/+ mice, but was associated with increased numbers of dermal mast cells only in the WCB6F1(-)+/+ mice. PMA treatment had no detectable effect on the ability of bone marrow-derived cultured mast cells to survive in the skin of Sl/Sld mice. These findings establish a convenient model system for analyzing factors associated with the development of endogenous populations of mast cells in genetically mast cell-deficient W/Wv mice.     

Expression of lineage markers by CD34+ hematopoietic stem cells of adult mice

OBJECTIVE: By using a murine transplantation model, we studied the relationship between CD34 expression and expression of CD4 and Mac-1 by hematopoietic stem cells of normal adult mice. MATERIALS AND METHODS: Cells from Ly-5.1 C57BL/6 mice were used as test cells and lethally irradiated Ly-5.2 mice were used as recipient mice. Peripheral blood was obtained 6 months posttransplantation to analyze engraftment of donor-derived cells. RESULTS: First, we determined that CD34- long-term reconstituting cells are CD4-, while some CD34+ stem cells express CD4. We then studied Mac-1 expression. Mac-1(-) and Mac-1(low) populations of both CD34- and CD34+ cells were capable of long-term reconstitution. Mac-1(high) population of CD34+ cells but not of CD34- cells also engrafted. CONCLUSIONS: These results strongly indicate that depletion of Mac-1(+) and CD4(+) cells in stem cell purification may inadvertently discard significant populations of CD34+ stem cells. Since positive selection based on CD34 expression is the current practice for purification of human stem cells, our studies may possess implications in the purification of human hematopoietic stem cells.     

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45(+)/CD34(-) cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow-derived CD34(+)/CD38(-) stem/progenitor cells. The human CD45(+) cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45(+) cell were recovered from the marrow of bnx mice than from the marrow of human stem cell-engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45(+)/CD34(-) cells recovered from the bnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34(+) progeny following retransplantation. These studies demonstrate that the human CD34(+) population can act as a reservoir for generation of CD34(-) cells. In the current studies we demonstrate that human CD34(+)/CD38(-) cells can generate CD45(+)/CD34(-) progeny in a long-term xenograft model and that those CD45(+)/CD34(-) cells can regenerate CD34(+) progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.     

Effect of human recombinant granulocyte colony-stimulating factor on hematopoietic injury in mice induced by 5-fluorouracil

Intravenous (IV) administration of 5-fluorouracil (5-FU) caused severe hematopoietic injury in mice. Daily administration of purified human recombinant granulocyte colony-stimulating factor (rG-CSF) accelerated recovery from neutropenia in these injured mice. Granulocyte-macrophage progenitors (CFU-GM) in spleen were markedly increased by rG-CSF, while rG-CSF was less effective on the regeneration of CFU-GM in femoral marrow. The accelerated recovery from neutropenia in the mice might be mainly due to the stimulation of granulopoiesis in spleen by rG-CSF.     

Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation.

Production of tissue mast cells was evaluated in genetically anemic mice of W/Wv genotype and was found to be abnormal. In the skin of adult W/Wv mice the number of mast cells/cm was less than 1% of the number observed in the congeneic +/+ mice. No mast cells were detectable in other tissues of the W/Wv mice. After transplantation of bone marrow cells from +/+ mice the number of mast cells in the skin, stomach, caecum, and mesentery of the W/Wv mice increased to levels similar to those of the +/+ mice. These results show that the W/Wv mouse is a useful tool for the investigations concerning the physiologic roles and the origin of mast cells.     

The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence- activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38- , HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.