Persistence of human multilineage, self-renewing lymphohematopoietic stem cells in chimeric sheep

We have previously reported the ability of uncharacterized human bone marrow (BM) cells to engraft into preimmune fetal sheep, thereby creating sheep-human chimera suitable for in vivo examination of the properties of human hematopoietic stem cells (HSC). Adult human bone marrow CD34+ HLA-DR- cells have been extensively characterized in vitro and have been demonstrated to contain a number of primitive hematopoietic progenitor cells (PHPC). However, the capacity of such highly purified populations of human marrow CD34+ HLA-DR- cells to undergo in vivo self-renewal and multipotential lymphohematopoietic differentiation has not been previously demonstrated. To achieve that, human CD34+ HLA-DR- cells were transplanted in utero into immunoincompetent fetal sheep to investigate the BM-populating potential of these cells. Long-term chimerism, sustained human hematopoiesis, and expression of human cells belonging to all human blood cell lineages were demonstrated in two animals for more than 7 months' posttransplantation. Chimeric BM contained erythroid, granulocytic/monocytic, and megakaryocytic hematopoietic progenitor cells, as well as the primitive high proliferative potential colony- forming cell (HPP-CFC). Under a variety of in vitro experimental conditions, chimeric BM cells gave rise to human T cells expressing T- lymphocyte-specific markers, human natural killer (NK) cells, and human IgG-producing B cells. In vivo expansion and possibly self-renewal of transplanted PHPC was confirmed by the detection in chimeric BM 130 days' posttransplantation of CD34+ HLA-DR- cells, the phenotype of human cells constituting the stem-cell graft. These studies demonstrate not only the BM-populating capacity, multipotential differentiation, and most likely self-renewal capabilities of human CD34+ HLA-DR- cells, but also that this BM population contains human HSC. Furthermore, it appears that this animal model of xenogeneic stem-cell transplantation is extremely useful for in vivo examination of human hematopoiesis and the behavioral and functional characteristics of human HSC.     

Hematopoietic capability of CD34+ cord blood cells: a comparison with CD34+ adult bone marrow cells

The characteristics of hematopoietic progenitor and stem cell (HPC/HSC) populations in mammals vary according to their ontogenic stage. In humans, HPC/HSCs from umbilical cord blood (CB) are increasingly used as an alternative to HPC/HSCs from adult bone marrow (BM) for the treatment of various hematologic disorders. How the hematopoietic activity of progenitor and stem cells in CB differs from that in adult BM remains unclear, however. We compared CD34+ cells, a hematopoietic cell population, in CB with those in adult BM using phenotypic subpopulations analyzed by flow cytometry, the colony-forming activity in methylcellulose clonal cultures, and the repopulating ability of these cells in NOD/Shi-scid (NOD/SCID) mice. Although the proportion of CD34+ cells was higher in adult BM than in CB mononuclear cells, the more immature subpopulations, CD34+ CD33- and CD34+ CD38- cells, were present in higher proportions in CD34+ CB cells. Clonal culture assay showed that more multipotential progenitors were present in CD34+ CB cells. When transplanted into NOD/SCID mice. CD34+ adult BM cells could not reconstitute human hematopoiesis in recipient BM, but CD34+ CB cells achieved a high level of engraftment, indicating that CD34+ CB cells possess a greater repopulating ability. These results demonstrated that human hematopoiesis changes with development from fetus to adult. Furthermore, CD34+ CB cells contained a greater number of primitive hematopoietic cells, including HSCs, than did adult BM, suggesting the usefulness of CD34+ CB cells not only as a graft for therapeutic HSC transplantation but also as a target cell population for ex vivo expansion of transplantable HSCs and for gene transfer in gene therapy.     

Engraftment of human hematopoietic precursor cells with secondary transfer potential in SCID-hu mice

Human fetal bone fragments implanted subcutaneously in immunodeficient (SCID) mice maintain active human hematopoiesis. In this study, we show that this human hematopoietic microenvironment supports the engraftment and differentiation of HLA-mismatched, CD34+ primitive hematopoietic progenitor cells isolated from fetal and adult human bone marrow (BM). The BM CD34+ cells were depleted of CD2, CD14, CD15, CD16, glycophorin A, and CD19 lineage-committed cells (CD34+Lin-). Donor cell engraftment was manifested by the presence of B (CD19+) and myeloid (CD33+) cells of donor HLA phenotype. Successful engraftment was observed as early as 4 weeks after fetal BM donor cell injection and sustained for at least 12 weeks, with engraftment success rates of 100% (11/11 grafts) and 92% (11/12 grafts) at 8 and 12 weeks, respectively. Mixed BM chimerism of donor and endogenous cells was consistently observed in SCID-hu bones successfully engrafted with HLA-mismatched CD34+Lin- donor cells. Preconditioning of the SCID-hu bone with a single dose of sublethal (350 rad) whole body irradiation (WBI) immediately before cell injection enhanced the repopulation of the bone grafts with donor cells and, in some instances, resulted in complete repopulation. After WBI, as few as 500 fetal bone marrow CD34+Lin- cells injected in the human bone grafts resulted in donor-derived hematopoiesis. Donor progenitor cells recovered from the SCID-hu bone grafts 8 weeks postinjection had the capacity to repopulate secondary groups of HLA-disparate fetal human bones in SCID-hu mice with B and myeloid cells as well as CD34+ cells in some recipients. In addition, these cells repopulated fetal human thymus fragments in SCID mice with donor thymocytes including immature CD4+CD8+ and mature CD4+CD8- as well as CD4-CD8+ subsets. These results indicate that the fetal human bone implants of SCID-hu mice can support the maintenance of a cell population that has both multilineage potential and repopulating potential for periods of time as long as 16 weeks. The SCID-hu bone model consistently supported the engraftment of both fetal and adult CD34+Lin- cells without the administration of exogenous human cytokines to these animals. This model is currently being used to permit the isolation and characterization of candidate human hematopoietic stem cells (HSCs) and provide important information critical for human HSC therapy in humans.     

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.     

Disparate regulation of human fetal erythropoiesis by the microenvironments of the liver and bone marrow

The liver and the bone marrow (BM) are the major organs that support hematopoiesis in the human fetus. Although both tissues contain the spectrum of hematopoietic cells, erythropoiesis dominates the liver. Previous studies suggested that a unique responsiveness of fetal burst-forming units erythroid (BFU-E) to erythropoietin (EPO) obviates the need for cytokines with burst-promoting activity (BPA) in fetal erythropoiesis. This potential regulatory mechanism whereby fetal erythropoiesis is enhanced was further investigated. Fluorescence-activated cell sorting was used to isolate liver and BM progenitors based on their levels of CD34 and CD38 expression. The most mature population of CD34+ lineage (Lin-) cells was also the most prevalent of the three subpopulations and contained BFU-E responsive to EPO alone under serum-deprived conditions. Kit ligand (KL) also strongly synergized with EPO in stimulating the growth of these BFU-E. An intermediate subset of CD34++CD38+Lin- cells contained erythroid progenitors responsive to EPO alone, but also displayed synergism between EPO and KL, granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin (IL)-3, demonstrating that erythroid progenitors that respond to cytokines with BPA do exist in fetal tissues as in the adult BM. Candidate stem cells (CD34++CD38-Lin- cells) did not respond to EPO. Synergisms among KL, GM-CSF, and IL-3, and to a lesser extent granulocyte colony-stimulating factor (G-CSF) and FLK-2/FLT-3 ligand (FL), supported the growth of primitive multipotent progenitors that became responsive to EPO. These data define the limits of EPO activity in fetal erythropoiesis to cells that express CD38 and demonstrate the potential for various cytokine interactions to be involved in regulating fetal erythropoiesis. Furthermore, a comparison of the responses of liver and BM erythroid progenitors revealed similarity in their responses to cytokines but a difference in the frequency of BFU-E among the three subpopulations examined. A higher frequency of BFU-E among the intermediate and late progenitor subsets in the liver indicates that regulatory factors acting on stem cells and their immediate progeny are partially responsible for the high content of erythropoiesis in the liver. These data implicate a critical role for the microenvironments of the liver and BM in regulating the disparate levels of erythropoiesis in these tissues.     

Changes in the growth properties of CD34+, CD38- bone marrow progenitors during human fetal development

We have previously described the isolation of separate populations of CD34+, CD38- stromal and hematopoietic progenitors cells within fetal bone marrow. The CD34+, CD38-, CD50+, HLA-DR+ population contained the majority of primitive hematopoietic progenitor cells, whereas stromal progenitors were contained within the CD34+, CD38-, CD50-, HLA-DR- population. In this study, we compared the frequencies and total numbers of clonogenic CD34+, CD38- stromal and hematopoietic cells as a function of fetal gestational age using single-cell fluorescent- activated cell sorting (FACS). At 14 weeks of gestation, 1/500 fetal bone marrow mononuclear cells were primitive hematopoietic CD34+, CD38- , HLA-DR+ progenitor cells, whereas 1/1,000 were stromal progenitors with the CD34+, CD38-, HLA-DR- phenotype. During fetal ontogeny there was a continuous, age-dependent decrease in the frequency of stromal progenitors, such that, at 24 weeks of gestation, only 1/100,000 of bone marrow cells had the CD34+, CD38-, HLA-DR- phenotype and were clonogenic stromal cells when isolated by FACS. In contrast, 1/250 bone marrow cells in a 24-week fetus had the CD34+, CD38-, HLA-DR+ phenotype and were clonogenic hematopoietic progenitors. The decrease in the frequency of stromal progenitors was a function of both a decreased frequency of cells with the CD34+, CD38-, HLA-DR- phenotype and a decrease in the growth potential of individual with this phenotype. The total numbers of mononuclear cells and the total numbers of hematopoietic progenitors in two fetal femurs increased in parallel, 100-fold, between 14 and 24 weeks of gestation. In contrast, the total numbers of clonogenic CD34+, CD38-, HLA-DR- stromal progenitor cells remained constant during this period. Although adult bone marrow samples contained stromal progenitor cells at a frequency of approximately 1/7,000 mononuclear cells, clonogenic stromal cells with the CD34+, CD38-, HLA-DR- phenotype could not be isolated by single- cell FACS from these samples. Thus, there are significant differences between the frequencies and biologic characteristics of stromal and hematopoietic stem cells during fetal and postnatal ontogeny.     

Reversible expression of CD34 by adult human bone marrow long-term engrafting hematopoietic stem cells

OBJECTIVE: We previously reported that CD34(-) population of bone marrow (BM) cells from adult humans contains cells capable of engraftment and multilineage differentiation. We also reported on the reversibility of CD34 expression by murine hematopoietic stem cells. Based on long-term observations in primary, secondary, and tertiary sheep recipients, we now present definitive evidence for the long-term engrafting capability of human BM CD34(-) cells, and the reversibility of CD34 expression by human BM hematopoietic stem cells (HSC) in vivo. MATERIALS AND METHODS: We used serial transplantations into primary, secondary, and tertiary preimmune fetal sheep recipients to evaluate and compare the long-term engraftment and differentiation of adult human bone marrow-derived CD34(-) and CD34(+) cells in vivo. RESULTS: In primary hosts CD34(-) or CD34(+) cells produced multilineage human cell activity that persisted for 31 months. To confirm the long-term engrafting characteristics of CD34(-) cells and determine whether CD34 expression on human HSC is reversible, we transplanted human CD34(-) and CD34(+) cells obtained from primary hosts into secondary sheep recipients. Multilineage engraftment occurred in all secondary hosts, and in tertiary hosts transplanted with CD34(-) or CD34(+) cells obtained from BM of secondary recipients. CONCLUSION: These results demonstrate that human BM CD34(-) cells are capable of long-term multilineage engraftment in vivo. The finding that both CD34(-) and CD34(+) cells from primary/secondary groups engraft secondary/tertiary hosts indicates that CD34 expression on human HSC is reversible, a process that does not impair HSC function in vivo.     

Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion

To examine potential mechanisms by which hematopoiesis may be regulated by endothelial cells within the bone marrow (BM) microenvironment, we have devised a technique for the in vitro study of the interaction of human BM microvascular endothelial cells (BMEC) with hematopoietic cells. Microvessels isolated by collagenase digestion of spicules obtained from filtered BM aspirate were plated on gelatin-coated plastic dishes, and colonies of endothelial cells grown from microvessel explants were further purified by Ulex europaeus lectin affinity separation. BMEC monolayers isolated by this technique grew in typical cobblestone fashion, stained positively with antibody to factor VIII/von Willebrand factor, and incorporated acetylated LDL. Immunohistochemical studies showed that BM microvessels and BMEC monolayers express CD34, PECAM, and thrombospondin. Incubation of resting BMEC with BM mononuclear hematopoietic cells resulted in the selective adhesion of relatively large numbers of CD34+ progenitor cells and megakaryocytes. The binding of purified BM-derived CD34+ progenitor cells to BMEC was dependent on divalent cations and was partially blocked by antibodies to CD34. IL-1 beta treatment of BMEC monolayers resulted in an increase of CD34+ progenitor cell adhesion by mechanisms independent of CD34 or divalent cations. BMEC exhibit specific affinity for CD34+ progenitor cells and megakaryocytes, suggesting that the BM microvasculature may play a role in regulating the trafficking, proliferation, and differentiation of lineage specific hematopoietic elements, and possibly of pluripotent stem cells within the CD34+ population.     

CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.     

The Clinical Implications of Mixed Lymphocyte Reaction with Leukemic Cells

CD34 has been widely used as a stem and progenitor cell marker, and clinical CD34+ stem cell transplantation (CD34+ SCT) has been performed for tumor purging and for prevention of graft-versus-host disease. Recently, CD34-negative hematopoietic stem cells (CD34 HSCs) were identified in mice and humans. Xenogeneic chimera engraftment assays with immunodeficient mice or preimmune fetal sheep resulted in identification of human CD34- HSCs in cord blood, bone marrow, and granulocyte colony-stimulating factor-mobilized peripheral blood, although no significant clonogenic activity was detected in vitro. These characteristics of CD34- HSCs make the assessment of clinical samples difficult. The generation of CD34+ HSCs from CD34 cells in vitro may be a surrogate assay for detecting CD34- HSC activity. This approach was used in recipients of CD34+ SCT and revealed the absence of a CD34 precursor population. The identification of a positive marker in CD34- HSCs and the development of a simpler and more efficient in vivo assay for CD34- HSCs may allow the diagnostic evaluation of human CD34- HSCs in various clinical procedures and diseases.     

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis

Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.     

Characterization and functional analysis of adult human bone marrow cell subsets in relation to B-lymphoid development

To study the frontiers between pluripotent stem cells and committed progenitors and to further define the B-cell pathway in adult bone marrow (BM), CD34+ subpopulations and CD34- B-lineage cells were analyzed by multiparameter flow cytometry, studied by light and electron microscopy, and in short-term and long-term cultures (LTC). While the total CD34+ cells represent 4.9% +/- 0.8 of BM mononuclear cells within the lymphoid-blast window, 73.8 +/- 3.5%, 14.4 +/- 1.8% and 8.8 +/- 2.9% of them were CD34+ CD10- CD19-, CD34+ CD10+ CD19+, and CD34+ CD10+ CD19-, respectively. CD34+ CD10+ CD19+ cells represent a smal homogeneous TdT4 c micro-blast population. Although expressing CD38 and high level of HLA-DR antigens, like myeloid committed progenitors, they did not generate LTC, myeloid, and T lymphoid colonies suggesting that the CD34+ CD10+ CD19+ population represents exclusively B lymphoid committed progenitors. By contrast, all myeloid progenitors and LTC-initiating cells were found in the CD34+ CD10- CD19- cell fraction. This fraction appeared more heterogeneous and contained CD38- HLA-DRlow small cells, larger blasts, and promonocyte-like cells exhibiting small peroxidase-positive granules. Interestingly, CD10 was also present on CD34+ CD19- cells. This population mainly coexpressed CD33 and gave rise to macrophagic colonies.     

Generation of T cells from cytokine-mobilized peripheral blood and adult bone marrow CD34+ cells

The present study compared the T-cell progenitor content of CD34+ lineage (Lin)- cells isolated from normal adult bone marrow (ABM) and mobilized peripheral blood (MPB). Both cell populations were found to differentiate into T cells when injected into human fetal thymi implanted into severe combined immunodeficient mice. Cytokine-MPB cells were less efficient than ABM cells in engrafting in the fetal human thymus, although both gave rise to thymocytes with identical phenotypes based on the analysis of CD1a, CD3, CD4, and CD8 expression. Thymocytes derived from adult CD34+ Lin- cells were capable of fully differentiating into mature CD3+ T cells expressing either the T-cell receptor (TCR) gamma delta or the TCR alpha beta (the later associated with CD4 or CD8), showing that the T-cell progenies of adult CD34+ cells were polyclonal and functional. Our data indicate that human MPB CD34+ cells are qualitatively identical to their BM counterparts, and demonstrate the existence of T-lymphoid progenitor cell activity in MPB.     

The generation of human natural killer cells from CD34+/DR- primitive progenitors in long-term bone marrow culture

We have adapted the stroma-dependent long-term bone marrow culture (LTBMC) system to study the development of human natural killer cells (NK) from the CD34+/HLA-DR- (CD34+/DR-) BM mononuclear cell (BMMNC) population. The CD34+/DR- population does not express any known antigens associated with myeloid or lymphoid lineage and has been shown by us and others to contain primitive hematopoietic progenitors capable of both self-renewal and differentiation to myeloid lineage. CD34+/DR- cells obtained from normal human BM by fluorescence-activated cell sorting were plated on allogeneic, irradiated BM stromal layers. After 5 weeks of culture in the presence of media containing recombinant interleukin-2 and human serum, 147- +/- 21-fold expansion of cells with the morphologic appearance of large granular lymphocytes was observed. Cultured cells (84.8% +/- 1.5%) expressed the characteristic CD56+/CD3- phenotype of NK. A proportion of CD56+/CD3- cells expressed other markers of lymphoid lineage that have been associated with mature NK, including CD2 (7.8% +/- 1.2%), CD7 (19.5% +/- 2.8), CD8 (3.1% +/- 1.0%), and CD16 (4.5% +/- 1.3%). The cultured cells did not express other antigens associated with T-lymphocyte (CD3, CD5, T-cell receptor [TCR] alpha/beta and TCR gamma/delta), B-lymphocyte (CD19), myeloid (MY8, CD33, and CD71), or monocytoid (CD14 and CD15) lineage and did not express the CD34 antigen associated with hematopoietic progenitors present on the starting population. This NK population was cytotoxic against both K562 (E:T 20:1; 79% +/- 1.9%) and Raji (E:T 20:1; 38% +/- 5.7%) target cell lines. The NK progenitor frequency in the CD34+/DR- cell population determined by limiting dilution of CD34+DR- on stromal layers followed by a functional chromium release assay against K562 targets was 1:169 +/- 50 CD34+/DR- cells. The data suggest that human LTBMC developed to study myeloid differentiation can be modified to study the origin and development of the NK and possibly other lymphoid lineages. Modified cultures show that cells with morphologic, phenotypic, and functional characteristics of NK can be derived from a population of BMMNC with the phenotype of primitive hematopoietic progenitors and without phenotypic evidence of lymphoid- or myeloid- lineage commitment. Further studies will address the cell of origin and the ontogeny of human NK and other lymphoid lineages.     

CD34+CD38dim cells in the human thymus can differentiate into T, natural killer, and dendritic cells but are distinct from pluripotent stem cells

Recently we reported that the human thymus contains a minute population of CD34+CD38dim cells that do not express the T-cell lineage markers CD2 and CD5. The phenotype of this population resembled that of CD34+CD38dim cells present in fetal liver, umbilical cord blood, and bone marrow known to be highly enriched for pluripotent hematopoietic stem cells. In this report we tested the hypothesis that the CD34+CD38dim thymocytes constitute the most primitive hematopoietic cells in the thymus using a combination of phenotypic and functional analyses. It was found that in contrast to CD34+CD38dim cells from fetal liver and bone marrow, CD34+CD38dim cells from the thymus express high levels of CD45RA and are negative for Thy-1. These data indicate that the CD34+CD38dim thymocytes are distinct from pluripotent stem cells. CD34+CD38dim thymocytes differentiate into T cells when cocultured with mouse fetal thymic organs. In addition, individual cells in this population can differentiate either to natural killer cells in the presence of stem cell factor (SCF), interleukin-7 (IL-7), and IL-2 or to dendritic cells in the presence of SCF, granulocyte- macrophage colony-stimulating factor, and tumor necrosis factor alpha(TNFalpha), indicating that CD34+CD38dim thymocytes contain multi- potential hematopoietic progenitors. To establish which CD34+ fetal liver subpopulation contains the cells that migrate to the thymus, we investigated the T-cell-developing potential of CD34+CD38dim and CD34+CD38+ fetal liver cells and found that the capacity of CD34+ fetal liver cells to differentiate into T cells is restricted to those cells that are CD38dim. Collectively, these findings indicate that cells from the CD34+CD38dim fetal liver cell population migrate to the thymus before upregulating CD38 and ommitting to the T-cell lineage.     

Peripheral blood harvest of unaffected CD34+ CD38- hematopoietic precursors in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) arises from somatic mutation of a bone marrow progenitor that disrupts glycosylinositol phospholipid (GPI) anchoring of cell surface proteins. We recently characterized the expression of GPI-anchored decay acclerating factor (DAF) and CD59 during hematopoietic development in PNH marrow. We found that, although a subset of early hematopoietic precursors identified by the CD34+CD38- phenotype exhibits normal DAF and CD59 expression, DAF and CD59 are absent on the majority of CD34+CD38- cells. Pluripotent CD34+CD38- hematopoietic stem cells normally circulate in the peripheral blood and can be collected by apheresis, cryopreserved, and later used for reconstitution of hematopoiesis. In this study, we examined the phenotypes of CD34+ cells that are released into the blood of PNH patients. Analyses of apheresis samples from three affected individuals showed discrete populations of circulating DAF+CD59+CD34+ and DAF-CD59- CD34+ cells. Variable proportions of CD34+CD38- cells were present within the peripheral blood CD34+ cells of each patient, but in all three cases the DAF+CD59+CD34+CD38- cell subset subset. Because CD34+ cells lacking CD38 antigen are highly enriched for self-renewing hematopoietic stem cells, these findings indicate that apheresis samples can serve as a source of unaffected stem cells for autologous marrow transplantation of PNH patients.     

In vitro evidence for the presence of hematopoietic stem cells in the adult human liver

Previous studies have identified novel lymphoid phenotypes in the adult human liver and provided evidence to suggest that lymphoid differentiation can occur locally in this organ. The aim of this study was to examine the adult human liver for the presence of hematopoietic stem cells that may provide the necessary precursor population for local hematopoietic and lymphoid differentiation. Hepatic mononuclear cells (HMNC) were extracted from normal adult liver biopsy specimens using a combination of mechanical disruption and enzymatic digestion. The stem cell marker CD34 was found on 0.81% to 2.35% of isolated HMNCs by flow cytometry. CD34(+) HMNCs were positively selected using magnetically labeled beads, and the enriched population was further examined for surface markers characteristically expressed by immature hematopoietic cells and early progenitors. CD45 was expressed by 49% (+/-23%) of CD34(+) HMNCs, indicating their hematopoietic origin. CD38, one of the first markers to be expressed by developing progenitor cells was found on 50% (+/-22%) of CD34(+) HMNCs indicating the presence of both pluripotent stem cells and committed precursors. The majority (90%) of CD34(+) HMNCs coexpressed the activation marker human leukocyte antigen DR, consistent with actively cycling cells. Functional maturation of these hepatic progenitors was shown by the detection of multilineage hematopoietic colony formation after tissue culture. Erythroid (BFU-E), granulocyte-monocyte (CFU-GM), and mixed colonies (CFU-GEMM) were detected after culture of unseparated HMNCs and the enriched CD34(+) HMNC population; 14.3 +/- 13.2 (mean +/- SD) BFU-E, 3.1 +/- 3.1 CFU-GM, and 0.4 +/- 0.9 CFU-GEMM per 1 x 10(5) unseparated HMNCs and 16.0 +/- 9.5 BFU-E and 1.7 +/- 0.9 CFU-GM were identified per 2.4 x 10(3) CD34(+) HMNCs plated. The detection of surface markers characteristic of immature hematopoietic cells and colony formation in tissue culture provides evidence for the presence of hematopoietic stem cells and early progenitor cells in the adult human liver. This would suggest that the adult human liver continues to contribute to hematopoiesis and may be an important site for the differentiation of lymphohematopoietic cells involved in disease states, such as autoimmune hepatitis and graft rejection after liver transplantation.     

Characterization of CD34+ subsets derived from bone marrow, umbilical cord blood and mobilized peripheral blood after stem cell factor and interleukin 3 stimulation

We characterized CD34+ cells purified from bone marrow (BM), mobilized peripheral blood (PB) and cord blood (CB) and we tried to establish correlations between the cell cycle kinetics of the CD34+CD38- and CD34+CD38+ subpopulations, their sensitivity to SCF and IL-3 and their expression of receptors for these two CSFs. At day 0, significantly fewer immature CD34+CD38- cells from CB and mobilized PB are in S + G2M phases of the cell cycle (respectively 2.0 +/- 0.4 and 0.9 +/- 0.3%) than their BM counterpart (5.6 +/- 1.2%). A 48-h incubation with SCF + IL-3 allows a significant increase in the percentage of cycling CD34+CD38- cells in CB (19.2 +/- 2.2%, P < 0.0002) and PB (14.1 +/- 5.5%, P < 0.05) while the proliferative potential of BM CD34+CD38- progenitors remains constant (8.6 +/- 1.0%, NS). CD123 (IL-3 receptor) expression is similar in the three sources of hematopoietic cells at day 0 and after 48-h culture. CD117 (SCF receptor) expression, although very heterogeneous according to the subpopulations and the sources of progenitors evaluated, seems not to correlate with the difference of progenitor cell sensitivity to SCF nor with their proliferative capacity. Considering the importance of the c-kit/SCF complex in the adhesion of stem cells to the microenvironment, several observations are relevant. The density of CD117 antigen expression (expressed in terms of mean equivalent soluble fluorescence, MESF) is significantly lower on fresh PB cells than on their BM (P < 0.017) and CB (P < 0.004) counterparts, particularly in the immature CD34+CD38- population (560 +/- 131, 2121 +/- 416 and 1192 +/- 129 MESF respectively); moreover, when PB and BM CD34+CD38- cells are stimulated for 48 h with SCF + IL-3, the CD117 expression decreases by 1.5- and 1.66-fold, respectively. This reduction could modify the functional capacities of ex vivo PB and BM manipulated immature progenitor cells.     

Proliferation and differentiation of myelodysplastic CD34+ cells: phenotypic subpopulations of marrow CD34+ cells

In a search for a mechanism to explain the impaired growth of progenitor cells in patients with myelodysplastic syndromes (MDS), marrow CD34+ cells were purified up to 94.9% +/- 4.2% for normal individuals and 88.1% +/- 17.6% for MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). Phenotypic subpopulations of these CD34+ cells were analyzed for CD38, HLA-DR, CD33, CD13, CD14, CD41 and CD3 plus CD19, in association with proliferative and differentiative capacities. The 15 studies performed included 12 MDS patients. Coexpression rate of CD13 significantly increased in the MDS CD34+ cell population with a value of 91.4% +/- 11.6% and ranging from 60.3% to 100%, and exceeded 99% in four studies, whereas that of normal CD34+ cells was 49.9% +/- 15.8%, ranging from 28.2% to 70.1% (P < .001). Coexpression rate of CD38, HLA-DR, CD33, CD14, and CD3 plus CD19 in MDS CD34+ cells did not significantly differ from that of normal CD34+ cells. The total number of colonies and clusters grown from 100 normal marrow CD34+ cells was 40.4 +/- 8.6, the range being from 27.2 to 50.3; this varied in MDS marrow CD34+ cells with a value of 34.0 +/- 28.7, the range being 0 to 95.9. The lineage of colonies and clusters promoted by MDS marrow CD34+ cells was predominantly committed to nonerythroid with impaired differentiation in 13 of 15 studies (87%). CD13 is first expressed during hematopoiesis by colony-forming unit granulocyte-macrophage and is absent in erythroid progenitors. Therefore, this study provides direct evidence for the lineage commitment of MDS CD34+ cells to nonerythroid with impaired differentiation and explains the mechanism of nil or low colony expression of MDS progenitor cells to erythroid lineage.     

Immunophenotypic and functional characterization of CD33(+)CD34(+) cells in human cord blood of preterm neonates

OBJECTIVE: To characterize CD33(+)CD34(+) cells, a major population in human cord blood (CB) CD34(+) cells of preterm neonates. MATERIALS: The proportion of CD33(+) cells was analyzed on CB CD34(+) cells from preterm and full-term neonates. CD33(+)CD34(+) cells were purified by cell sorting and analyzed on their clonogenic activity, proliferative activity in short-time liquid suspension culture, and GATA-2 mRNA expression by RT-PCR and Southern blot. RESULTS: The absolute numbers and proportion of CD34(+) cells in mononuclear cells inversely correlated with gestational age. CD33 was expressed on a majority of CB CD34(+) cells of preterm neonates but on only a minor population of them in full-term neonates. In addition, CD33 was dominantly expressed on CD38(-)CD34(+) cells or CD117(low)CD34(+) cells in CB of preterm neonates. CD33(+)CD34(+) cells of preterm cord blood had high proliferative and reproducible potentials compared with CD33(-)CD34(+) cells. CD33(+)CD34(+) cells as well as CD33(-)CD34(+) cells from preterm CB highly expressed GATA-2, in contrast to those from BM. CONCLUSIONS: These results suggest that CD33(+)CD34(+) cells, which are a major population in CB CD34(+) cells of preterm neonates, do not simply represent relatively mature myeloid lineage hematopoietic progenitor cells as those in adult BM CD34(+) cells, and may contain hematopoietic stem cells or primitive progenitor cells as in fetal liver.