Proliferative responses to interleukin-3 and granulocyte colony-stimulating factor distinguish a minor subpopulation of CD34-positive marrow progenitors that do not express CD33 and a novel antigen, 7B9.

Human hematopoietic colony-forming cells (CFC) express the CD34 antigen (CD34+) as well as differentiation antigens such as CD33 and HLA-DR. CD34+ cells that do not express these latter differentiation antigens have been shown to contain few CFC in direct culture, but generate increasing numbers of CFC when cultured over a marrow stromal cell layer in the long-term culture system. In this study we determined if CD34+ cells with low or absent expression of CD33 and a novel antigen, 7B9 (CD34+CD33-7B9-), could be distinguished from CD34+ cells expressing these antigens (CD34+CD33+7B9+) based on their proliferative responses to interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) in a short-term liquid culture system. These two populations were separated by fluorescence-activated cell sorting, cultured with IL-3 (10 ng/mL), G-CSF (100 ng/mL), or IL-3 and G-CSF, and 3H-thymidine uptake was measured. CD34+CD33-7B9- cells proliferated in the presence of IL-3, but not G-CSF. However, a synergistic response to the combination of IL-3 and G-CSF was seen in most experiments. In contrast, CD34+CD33+7B9+ cells proliferated in the presence of either IL-3 or G-CSF but did not display an additive or synergistic response to the combination of IL-3 and G-CSF. In colony-forming assays performed before and after liquid culture, the CD34+CD33-7B9- cells in two experiments contained 0.3% and 2.2% of all sorted marrow CFC before liquid culture and generated 40-fold and ninefold increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), respectively, after liquid culture with IL-3 and G-CSF. In contrast, the CD34+CD33+7B9+ cells contained 99.7% and 97.8% of all sorted marrow CFC before liquid culture and had no change or a threefold increase in the number of CFU-GM, respectively, after liquid culture with IL-3 and G-CSF. Single-cell liquid cultures containing IL-3 and G-CSF with cells that were either CD34+CD33-7B9- and depleted of mature lymphoid cells (CD34+lin-) or were CD34+lin+ showed that a higher proportion of wells containing a CD34+lin- cell gave rise to one or more CFC (8.7%) than did wells containing a CD34+lin+ cell (2.9%), with the responding cells in the former population giving rise to an average of 2.9 +/- 0.6 CFC and in the latter population, 2.0 +/- 1.0 CFC.(ABSTRACT TRUNCATED AT 400 WORDS)     

CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells

Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane ("stroma- noncontact" culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma- noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA- DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.     

Further investigations on the expression of HLA-DR, CD33 and CD13 surface antigens in purified bone marrow and peripheral blood CD34± haematopoietic progenitor cells

Summary. We evaluated the HLA-DR, CD33 and CD13 antigen expression on CD34± haematopoietic progenitor cells (HPC) isolated from the bone marrow (BM) and peripheral blood (PB) of normal donors. The majority of both BM and PB CD34± HPC expressed CD13 and HLA-DR. The coexpression of CD34 and CD33 was found in a minor CD34± subset. After 7 d of culture in the presence of interleukin-3 and granulocyte-macrophage colony-stimulating factor, CD33 expression was detected in about 50% of HPC. At this point CD34 antigen expression was lost and CD13 and HLA-DR expression was partially lost. After 14 d of culture, the majority of HPC were CD33±. HPC maintained the capacity to generate colony forming unit granulocyte-macrophage but they lost the capacity to generate burst forming unit-erythroid. A correlation was found between the percentage of CD34±/HLA-DR± cells and the total number of colony forming cells in unfractionated samples from BM and PB of patients with malignancies. These studies demonstrate that, in normal conditions, only a minor subset of CD34± cells coexpress CD33 antigen either in BM or in PB and CD33 antigen is a lineage marker which is coexpressed with HLA-DR and CD13 on a progenitor committed to the granulocytic-macrophagic lineage.     

Tumor necrosis factor-alpha strongly potentiates interleukin-3 and granulocyte-macrophage colony-stimulating factor-induced proliferation of human CD34+ hematopoietic progenitor cells.

Previous studies have shown that tumor necrosis factors (TNFs) inhibit the proliferative effects of crude or purified colony-stimulating factors (CSFs) on low density human bone marrow cell fractions. In the present study we investigated the effects of TNF alpha on the growth of highly purified CD34+ human hematopoietic progenitor cells (HPC) in response to recombinant CSFs. In short-term liquid cultures (5 to 8 days), TNF alpha strongly potentiates interleukin-3 (IL-3) and granulocyte-macrophage-CSF (GM-CSF)-induced growth of CD34+ HPC, while it has no proliferative effect per se. Within 8 days, the number of viable cells obtained in TNF alpha-supplemented cultures is threefold higher than in cultures carried out with IL-3 or GM-CSF alone. Secondary liquid cultures showed that the potentiating effect of TNF alpha on IL-3-induced proliferation of CD34+ HPC does not result from an IL-3-dependent generation of TNF alpha responsive cells. Limiting dilution analysis indicates that TNF alpha increases both the frequency of IL-3 responding cells and the average size of the IL-3-dependent clones. The potentiating effect of TNF alpha on IL-3- and GM-CSF-dependent growth of CD34+ HPC is also observed in day 7 colony assays. Under these short-term culture conditions, TNF alpha does not appear to accelerate cell maturation as a precursor morphology is retained. Finally, TNF alpha inhibits the relatively weak growth-promoting effect of granulocyte-CSF (G-CSF), which acts on a more committed subpopulation of CD34+ HPC different from that recruited by IL-3 and GM-CSF. TNF beta displays the same modulatory effects as TNF alpha. Thus, TNFs appear to enhance the early stages of myelopoiesis.     

IL-6 precludes the differentiation induced by IL-3 on expansion of CD34+ cells from cord blood

BACKGROUND AND OBJECTIVES: Ex vivo expansion of hematopoietic progenitor cells (HPC) from umbilical cord blood (UCB) is an interesting strategy to obtain a sufficient number of transplantable cells for adults. To define the optimal culture conditions allowing the generation of HPC that retain their proliferative capacity without loss of long-term culture-initiating cells (LTC-IC), the effect of different cytokine combinations on the expansion of CD34+ cells from UCB was assessed. DESIGN AND METHODS: CD34+ cells were cultured in serum-free culture medium with four cytokine combinations: stem cell factor plus thrombopoietin plus flk2/flt3 ligand (STF), STF plus interleukin-3 (IL-3), STF plus interleukin-6 (IL-6) and STF plus IL-6 plus IL-3. After a 1-week culture, the number of CD34+ and CD133+ cells, colony forming units (CFU), LTC-IC and telomerase activity were determined. RESULTS: The addition of IL-6 or IL-3 to the combination of STF significantly enhanced the expansion of CD34+, CD133+ cells and CFU. All cytokine combinations tested induced a slight increase in LTC-IC number except that composed by STF plus IL-3. The greatest induction of telomerase activity was observed with the combination of STF plus IL-3 or plus IL-3 plus IL-6. Decay of the activity along time was observed when the combination of STF plus IL-3 was used, and this effect was reverted by the addition of IL-6. INTERPRETATION AND CONCLUSIONS: Our results demonstrate that the inclusion of IL-6 in a serum-free short-term culture has a beneficial effect on HPC expansion from UCB, and precludes the negative effects induced by IL-3 on LTC-IC expansion and telomerase activity.     

Lymphokine requirement for the generation of natural killer cells from CD34+ hematopoietic progenitor cells

We have established a cell culture system without stromal cells that allows the CD34+ hematopoietic progenitor cells (HPC) to differentiate into natural killer (NK) cells. CD34+Lin (CD3, CD16, CD56)- cells were purified using fluorescence-activated cell sorting from normal adult bone marrow (BM) and cultured for 28 days in medium supplemented with interleukin-2 (IL-2) and stem cell factor (SCF). NK (CD3-CD16-CD56+) cells were generated in a dose-dependent manner in response to SCF. NK cells originated from CD34+CD33+Lin- cells, but they were barely detectable in cultures of CD34+CD33-Lin- cells. However, on addition of IL-3, an induced differentiation of NK cells from CD34+CD33-Lin- cells was observed, although at a lower frequency. Supplementing of the cell cultures with SCF alone or both SCF and IL-3 for the first 7 days followed by IL-2 for the next 21 days is essential for production of NK cells from CD34+CD33+Lin- cells and from CD34+CD33-Lin- cells, respectively. These data provide direct evidence that NK cells arise from CD34+HPC and show the minimum lymphokine requirement for their differentiation.     

CD133-enriched CD34(-) (CD33/CD38/CD71)(-) cord blood cells acquire CD34 prior to cell division and hematopoietic activity is exclusively associated with CD34 expression

We compared the cell division behavior of CD34(-) and CD34(+) (CD33/CD38/CD71)-negative (Lin(-)) CD133(+) cord blood cells stimulated with the cytokines Flt3-ligand, stem cell factor, and thrombopoietin. Within a 4-day time frame, Lin(-)CD34(-) CD133(+) (CD34(-)) cells underwent more cell divisions in serum-free culture than their Lin(-)CD34(+) CD133(+) (CD34(+)) counterparts. The majority of CD34(-) cells acquired expression of CD34 in vitro, including most undivided cells. Moreover, hematopoietic activity from both CD34(-) and CD34(+) cells was exclusively retained within the cell fraction expressing CD34 after 4 days in culture. Most strikingly, in cultures from Lin(-)CD34(-) cells hematopoietic activity was associated with the fraction of divided cells, whereas in cultures of CD34(+) cells, hematopoietic activity associated with the undivided cell fraction. Therefore, clonogenic CD34(+) cells either do not divide or lose their clonogenic capacity upon cell division in vitro, while CD34(-) cells divide and retain this capacity under the same specific conditions. In conclusion, we demonstrate that CD133-enriched Lin(-)CD34(-) cord blood cells acquire CD34 prior to cell division and that long-term hematopoietic activity is associated exclusively with expression of CD34.     

CD34 cell subsets and long-term culture colony-forming cells evaluated on both autologous and normal bone marrow stroma predict long-term hematopoietic engraftment in patients undergoing autologous peripheral blood stem cell transplantation

OBJECTIVE: The aim of this study was to evaluate which CD34(+) cell subset contained in leukapheresis products could be regarded as the most predictive of long-term hematopoietic recovery after autologous peripheral blood stem cell transplantation (auto-PBSCT). MATERIALS AND METHODS: Based on data from 34 patients with hematologic malignancies, doses of CD34(+) cells and CD34(+) cell subsets, defined by the expression of HLA-DR, CD38, CD117 (c-kit/R), CD123 (alpha subunit of IL-3/R), CD133 (AC133), and CD90 (Thy-1) antigens, were correlated with the number of short-term (i.e., colony-forming cells [CFC]) and long-term culture CFC (LTC-CFC) (generated at week 5 of culture) and with the kinetics of hematopoietic engraftment following auto-PBSCT. The capacity of autologous stroma (AS), normal human bone marrow stroma, and M2-10B4 murine cell line to sustain CD34(+) cell growth was comparatively evaluated in the LTC assay. RESULTS: Our data demonstrated that some of the most primitive progenitor subsets (CD34(+)CD117(-)HLA-DR(-), and CD34(+)CD38(+)HLA-DR(-)) showed the strongest correlation with LTC-CFC numbers generated within the AS, whereas no significant correlation was noted using normal bone marrow stroma. Multivariate analysis showed that the only CD34 cell subset independently associated with long-term (3 to 6 months) platelet engraftment after auto-bone marrow transplantation was the CD34(+)CD117(-)HLA-DR(-) phenotype; long-term erythrocyte engraftment was correlated with CD34(+)CD38(+)HLA-DR(-) cell content. The latter further influenced platelet engraftment in the first 3 months after auto-PBSCT. The most predictive parameters for neutrophil engraftment were CD34(+)CD38(+)HLA-DR(-) cell subtype and the total LTC-CFC quantity infused. CONCLUSIONS: These data further support the hypothesis that the type of stromal feeders influences the frequency of LTC-CFC, possibly because they differ in their ability to interact with distinct subsets of hematopoietic stem cells. Furthermore, as the use of AS in LTC assay can mimic in vitro the human bone marrow microenvironment, it can be speculated that this culture system could be a useful means to study the kinetics of recovery of bone marrow stroma following chemotherapy and PBSCT. From these results, it can be concluded that some CD34(+) cell subsets appear to be more reliable predictors of long-term hematopoietic recovery rates than total CD34(+) cell quantity.     

A comparative study of the phenotype and proliferative capacity of peripheral blood (PB) CD34+ cells mobilized by four different protocols and those of steady-phase PB and bone marrow CD34+ cells

Peripheral blood (PB) CD34+ cells from four commonly used mobilization protocols were studied to compare their phenotype and proliferative capacity with steady-state PB or bone marrow (BM) CD34+ cells. Mobilized PB CD34+ cells were collected during hematopoietic recovery after myelosuppressive chemotherapy with or without granulocyte- macrophage colony-stimulating factor (GM-CSF) or granulocyte colony- stimulating factor (G-CSF) or during G-CSF administration alone. The expression of activation and lineage-associated markers and c-kit gene product were studied by flow cytometry. Proliferative capacity was measured by generation of nascent myeloid progenitor cells (granulocyte- macrophage colony-stimulating factor; CFU-GM) and nucleated cells in a stroma-free liquid culture stimulated by a combination of six hematopoietic growth factors (interleukin-1 (IL-1), IL-3, IL-6, GM-CSF, G-CSF, and stem cell factor). G-CSF-mobilized CD34+ cells have the highest percentage of CD38- cells (P < .0081), but otherwise, CD34+ cells from different mobilization protocols were similar to one another in their phenotype and proliferative capacity. The spectrum of primitive and mature myeloid progenitors in mobilized PB CD34+ cells was similar to their steady-state counterparts, but the percentages of CD34+ cells expressing CD10 or CD19 were lower (P < .0028). Although steady-state PB and chemotherapy-mobilized CD34+ cells generated fewer CFU-GM at day 21 than G-CSF-mobilized and steady-state BM CD34+ cells (P < .0449), the generation of nucleated cells and CFU-GM were otherwise comparable. The presence of increased or comparable numbers of hematopoietic progenitors within PB collections with equivalent proliferative capacity to BM CD34+ cells is not unexpected given the rapid and complete hematopoietic reconstitution observed with mobilized PB. However, all four types of mobilized PB CD34+ cells are different from steady-state BM CD34+ cells in that they express less c-kit (P < .0002) and CD71 (P < .04) and retain less rhodamine 123 (P < .0001). These observations are novel and suggest that different mobilization protocols may act via similar pathways involving the down-regulation of c-kit and may be independent of cell-cycle status.     

Interferon-gamma enhances factor-dependent myeloid proliferation of human CD34+ hematopoietic progenitor cells.

Numerous studies have shown that interferon-gamma (IFN gamma) inhibits the proliferative effects of colony-stimulating factors (CSFs) on human bone marrow cells. In the present study we investigated the effects of IFN gamma and other described inhibitory factors on the proliferation of highly purified CD34+ human hematopoietic progenitor cells (HPC) in response to recombinant CSFs. While transforming growth factor-beta (TGF beta) and IFN alpha were highly inhibitory, IFN gamma strongly potentiated interleukin-3 (IL-3) and, to a lesser extent, granulocyte-macrophage-CSF (GM-CSF) induced growth of CD34+ HPC. IFN gamma had no significant proliferative effect per se, and did not affect granulocyte-CSF (G-CSF)-dependent cell proliferation. Within 10 days the number of viable cells generated in the presence of IL-3 + IFN gamma was two times higher than in the presence of IL-3 alone. Limiting dilution analysis showed that IFN gamma acts directly on its target cell to increase the frequency of IL-3-responding cells without affecting the average size of the IL-3-dependent clones. Enhanced frequency of IL-3- and GM-CSF-responding cells was also observed in colony assays where the addition of IFN gamma increased by twofold to threefold the number of granulocyte colony-forming units (CFU-G), macrophage CFUs (CFU-M), granulocyte-macrophage CFUs (CFU-GM), and mixed erythroid (E-MIX). In contrast, IFN gamma did not affect the generation of erythroid burst-forming units (BFU-e) in such cultures. In longer-term culture, the combination of IFN gamma and IL-3 did not alter the lineage distribution of the cells when compared with IL-3 alone. However, after 15 days, when mature cells were present in the cultures, IFN gamma displayed cell concentration-related growth-inhibitory effects. Thus, IFN gamma appears to stimulate the early stage of myelopoiesis by enhancing the frequency of growth factor-responding cells but, unlike tumor necrosis factor alpha (TNF alpha), does not alter cell differentiation.     

Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- progenitor cells.

Multiparameter flow cytometry was applied on normal human bone marrow (BM) cells to study the lineage commitment of progenitor cells ie, CD34+ cells. Lineage commitment of the CD34+ cells into the erythroid lineage was assessed by the coexpression of high levels of the CD71 antigen, the myeloid lineage by coexpression of the CD33 antigen and the B-lymphoid lineage by the CD10 antigen. Three color immunofluorescence experiments showed that all CD34+ BM cells that expressed the CD71, CD33, and CD10 antigens, concurrently stained brightly with anti-CD38 monoclonal antibodies (MoAbs). In addition, the CD38 antigen was brightly expressed on early T lymphocytes in human thymus, characterized by CD34, CD5, and CD7 expression. Only 1% of the CD34+ cells, 0.01% of nucleated cells in normal BM, did not express the CD38 antigen. The CD34+, CD38- cell population lacked differentiation markers and were homogeneous primitive blast cells by morphology. In contrast the CD34+, CD38 bright cell populations were heterogeneous in morphology and contained myeloblasts and erythroblasts, as well as lymphoblasts. These features are in agreement with properties expected from putative pluripotent hematopoietic stem cells; indeed, the CD34 antigen density decreased concurrently with increasing CD38 antigen density suggesting an upregulation of the CD38 antigen on differentiation of the CD34+ cells. Further evidence for a strong enrichment of early hematopoietic precursors in the CD34+, CD38- cell fraction was obtained from culture experiments in which CD34+ cell fractions with increasing density of the CD38 antigen were sorted singularly and assayed for blast colony formation. On day 14 of incubation, interleukin-3 (IL-3), IL-6, and GM-CSF, G-CSF, and erythropoietin (Epo) were added in each well. Twenty-five percent of the single sorted cells that expressed CD34 but lacked CD38 antigen gave rise to primitive colonies 28 to 34 days after cell sorting. The ability to form primitive colonies decreased rapidly with increasing density of the CD38 antigen. During 120 days of culture, up to five sequential generations of colonies were obtained after replating of the first-generation primitive colonies. This study provides direct evidence for the existence of a single class of progenitors with extensive proliferative capacity in human BM and provides an experimental approach for their purification, manipulation, and further characterization.     

Thymic repopulation by CD34(+) human cord blood cells after expansion in stroma-free culture

Thymic repopulation by transplanted hematopoietic progenitor cells (HPC) is likely to be important for long-term immune reconstitution and for successful gene therapy of diseases affecting the T-cell lineage. However, the T-cell progenitor potential of HPC, cultured in vitro for cell number expansion and gene transfer remains largely unknown. Here, we cultured highly purified human umbilical cord blood (CB) CD34(+)CD38(-) or CD34(+)CD38(+) cells for up to 5 weeks in stroma-free cultures supplemented with various combinations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2 ligand (FL), interleukin-3 (IL-3), and IL-6 and investigated thymus-repopulating ability of expanded cells in vitro and in vivo. After up to 5 weeks of culture in IL-3 + SCF + IL-6 or TPO + FL + SCF supplemented medium, the progeny of CD34(+)CD38(-) CB cells generated T cells and natural killer cells in the thymus. Limiting dilution experiments demonstrated increase in the number of T-cell progenitors during culture. After 3 weeks of culture, gene marked CD34(+)CD38(-) CB cells injected in the human thymus fragment transplanted in severe combined immunodeficient (SCID) mice (SCID-hu) generated thymocytes expressing the retroviral encoded marker gene GFP in vivo. Thus, our results show that the progeny of CD34(+)CD38(-) CB cells cultured for extensive periods, harbor thymus-repopulating cells that retain T-cell progenitor potential after expansion and gene transfer.     

CD34(+) cord blood cells expressing cutaneous lymphocyte-associated antigen are enriched in granulocyte-macrophage progenitors and support extensive amplification of dendritic cell progenitors

OBJECTIVE: We evaluated the frequency of hematopoietic progenitor cells (HPC) in CD34(+)CLA(+) (cutaneous lymphocyte-associated antigen) and CD34(+)CLA(-) cord blood cells, and followed cellular growth and HPC production during cultures in Flt3 ligand, thrombopoietin, and stem cell factor (FTS). MATERIALS AND METHODS: Immunomagnetic bead-purified CD34(+) cells were sorted into CD34(+)CLA(+) or CD34(+)CLA(-) cells. HPC frequency was assessed by clonal assays in methylcellulose either ex vivo or after, 7, 14, or 21 days of culture with FTS. Dendritic cell (DC) progenitors were evaluated after induction of FTS-amplified cells into DC using secondary cultures containing granulocyte-macrophage colony-stimulating factor and interleukin-4. RESULTS: Ex vivo, granulocyte-macrophage progenitors were more frequent and erythroid progenitors were less frequent in the CLA(+) fraction. In FTS culture, CD34(+)CLA(+) cells produced greater absolute numbers of CD34(+) cells, granulocyte-macrophage-, erythroid-, and DC (including Langerhans cell-related) progenitors compared to CD34(+)CLA(-) cells. In CD34(+)CLA(+) cultures, CLA(+) cells steadily decreased with time, and CD34(+)CLA(-) cells appeared. In CD34(+)CLA(-) cultures, CLA(+) cells were generated, increased up to day 7, and decreased thereafter. CLA was expressed only on CD34(-) cells in these cultures. Ex vivo, CD34(+)CLA(+) cells could be subdivided further into CD38(low) and CD38(high) cells. Cord blood and growth factor-mobilized CD34(+) cells contained more CLA(+)CD38(low) cells than nonmobilized peripheral blood CD34(+) cells and proliferated more extensively with FTS than the latter cells. CONCLUSIONS: CD34(+)CLA(+) cells contain a rather immature progenitor capable of high proliferation and extensive amplification of HPC in vitro. This progenitor may be localized in the CD34(+)CLA(+)CD38(low) fraction. In addition, cultures of CD34(+)CLA(+) cells from cord blood produced CD34(+)CLA(-) cells, suggesting that these cells may derive directly from CD34(+)CLA(+) cells in vivo.     

Efficient Ex Vivo Generation of Human Dendritic Cells from Mobilized CD34p+ Peripheral Blood Progenitors

We tried to efficiently generate human dendritic cells (DCs) from CD34+ peripheral blood hematopoietic progenitor cells mobilized by high-dose chemotherapy and subsequent administration of granulocyte colony-stimulating factor, using a liquid suspension culture system. Among various combinations, the combination of c-kit ligand, flt-3 ligand, c-mpl ligand (TPO), and interleukin (IL)-4 most potently generated the number of CD1a+CD14- DCs in cultures containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The delayed addition of IL-4 on day 6 of culture gave rise to an additional increase in the yield of CD1a+CD14-DCs that were characterized by the expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. The majority of the sorted CD1a-CD14+ cells derived from 6-day culture of CD34+ cells gave rise to CD1a+CD14- DCs and CD1a-CD14+ macrophages on day 12 of culture in the presence and absence of IL-4, respectively. These findings suggest that IL-4 promotes the differentiation of CD1a- CD14+ cells derived from mobilized CD34+ peripheral blood hematopoietic progenitors to CD1a+ CD14- DCs. The majority of these DCs expressed CD68 but not the Langerhans-associated granule antigen, a finding that suggests they emerge through the monocyte differentiation pathway. The addition of TPO and IL-4 to cultures did not affect the potential of DCs to stimulate the primary allogeneic T-cell response. These findings demonstrated that the combination of c-kit ligand plus flt-3 ligand plus TPO with GM-CSF plus TNF-alpha, followed by IL-4, is useful for ex vivo generation of human DCs from mobilized CD34+ peripheral blood progenitors.     

Ex vivo generation of CD34(+) cells from CD34(-) hematopoietic cells

The human Lin(-)CD34(-) cell population contains a newly defined class of hematopoietic stem cells that reconstitute hematopoiesis in xenogeneic transplantation systems. We therefore developed a culture condition in which these cells were maintained and then acquired CD34 expression and the ability to produce colony-forming cells (CFC) and SCID-repopulating cells (SRCs). A murine bone marrow stromal cell line, HESS-5, supports the survival and proliferation of Lin(-)CD34(-) cells in the presence of fetal calf serum and human cytokines thrombopoietin, Flk-2/Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin-3, and interleukin-6. Although Lin(-)CD34(-) cells do not initially form any hematopoietic colonies in methylcellulose, they do acquire the colony-forming ability during 7 days of culture, which coincides with their conversion to a CD34(+) phenotype. From 2.2% to 12.1% of the cells became positive for CD34 after culture. The long-term multilineage repopulating ability of these cultured cells was also confirmed by transplantation into irradiated NOD/SCID mice. These results represent the first in vitro demonstration of the precursor of CD34(+) cells in the human CD34(-) cell population. Furthermore, the in vitro system we reported here is expected to open the way to the precise characterization and ex vivo manipulation of Lin(-)CD34(-) hematopoietic stem cells.     

Functional differences between CD38- and DR- subfractions of CD34+ bone marrow cells

Several studies have previously demonstrated enrichment in primitive progenitor cells in subfractions of CD34+ bone marrow (BM) cells not expressing CD38 or HLA-DR (DR) antigens. However, no studies have directly compared these two cell populations with regard to their content of primitive and more committed progenitor cells. Flow cytometric analysis of immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+CD38- and CD34+DR- progenitor subpopulations in that only 12% to 14% of total CD34+DR- and CD34+CD38- cells were double negative (CD34+CD38-DR-). Although the number of committed myeloid progenitor cells (colony-forming units granulocyte- macrophage) was reduced in both subpopulations, only CD34+CD38- cells were significantly depleted in committed erythroid progenitor cells (burst-forming units-erythroid). In single-cell assay, CD34+CD38- cells showed consistently poorer response to single as opposed to multiple hematopoietic growth factors as compared with unfractionated CD34+ cells, indicating that the CD34+CD38- subset is relatively enriched in primitive hematopoietic progenitor cells. Furthermore, CD34+CD38- and CD34+DR- cells, respectively, formed 3.2-fold and 1.6-fold more high proliferative potential colony-forming cell (HPP-CFC) colonies than did unfractionated CD34+ cells. Finally, CD34+CD38-DR- cells were depleted in HPP-CFCs as compared with CD34+CD38+DR+ cells. The results of the present study suggest that both the CD38- and DR- subfractions of CD34+ bone marrow cells are enriched in primitive hematopoietic progenitor cells, with the CD34+CD38- subpopulation being more highly enriched than CD34+DR- cells.     

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.     

Extensive generation of human cord blood CD34(+) stem cells from Lin(-)CD34(-) cells in a long-term in vitro system

Human CD34(-) hematopoietic stem cells (HSCs) have been identified as potential precursors of CD34(+) HSCs by using xenogeneic transplantation systems. However, the properties of CD34(+) cells generated from CD34(-) cells have not been precisely analyzed due to the lack of an in vitro system in which CD34(+) cells are continuously produced from CD34(-) cells. We conducted this study to determine whether CD34(+) cells generated in vitro from CD34(-) cells have long-term multilineage reconstitution abilities. Lin(-)CD34(-) population isolated from human cord blood was cultured in the presence of murine bone marrow stroma cell line, HESS-5, and human cytokines, thrombopoietin, Flk2/Flt3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin 3 (IL-3), and IL-6. They were analyzed weekly for their surface markers expressions, colony-forming cells, long-term culture initiating cells (LTC-IC), and SCID repopulating cells (SRC) abilities up to 30 days of culture.In this culture system, more than 10(7) CD34(+) cells can be continuously generated from 10(4) CD34(-) cells over 30 days. These CD34(+) cells produce colony-forming units, LTC-IC, and SRC with multi-lineage differentiation, all of which are characteristic features of hematopoietic stem/progenitor cells.These findings suggest that CD34(-) HSCs have extensive potential for the generation of CD34(+) HSCs in vitro. This system provides a novel and potentially useful procedure to generate CD34(+) cells for clinical transplantation and gene therapy.     

Alloantigen presenting function of normal human CD34+ hematopoietic cells

The identification of the CD34 molecule, expressed almost exclusively on human hematopoietic stem cells and committed progenitors, and the development of CD34-specific monoclonal antibodies have made procurement of relatively pure populations of CD34+ marrow cells for autologous transplantation feasible. Characterization of the immunogenicity of CD34+ marrow cells may facilitate the design of successful strategies to use these cells for allogeneic transplantation. CD34+ marrow cells from normal volunteers were enriched to greater than 98% purity by immunoaffinity chromatography on column followed by fluorescence-activated cell sorting. Purified CD34+ cells were tested for expression of HLA-DR and other accessory molecules, and function in hematopoietic colony growth and mixed leukocyte culture (MLC) assays. Greater than 95% CD34+ cells were positive for HLA-DR and 74% +/- 10% were highly positive for CD18, the common beta-chain of a leukointegrin family. CD34+/CD18- cells were small, agranular lymphocytes which contained the majority of precursors for colony-forming cells detected in long-term cultures. They produced almost no stimulation of purified T cells from HLA-DR-incompatible individuals in bulk MLC or in limiting dilution assay. In contrast, CD34+/CD18+ cells were large, were enriched for cells forming mixed colonies in short- but not long-term assays, and were capable of stimulating allogeneic T cells. CD86, a natural ligand for the T-cell activation molecule CD28, was coexpressed with CD18 in 6% +/- 3% of CD34+ cells. CD34+/CD86+ cells, but not CD34+/CD86- cells, exhibited strong alloantigen presenting function. Thus, pluripotent hematopoietic activity and alloantigen presenting function are attributes of distinct subsets of CD34+ marrow cells. CD34+/CD18- or CD34+/CD86- cells may be more effective than either the whole CD34+ population or unseparated marrow in engrafting allogeneic recipients and may also facilitate induction of tolerance.     

Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor.

We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.