Human bone marrow progenitor cells stimulated by cord blood

Agar cultures of human bone marrow cells stimulated by cord plasma or irradiated cord blood feeder layers demonstrated the presence of a multilineage hemopoietic growth factor in cord blood. When bone marrow cultures stimulated with giant cell tumor-conditioned medium (GCT-CM) were supplemented with this cord blood-derived growth factor, total colony numbers increased by more than 50% after day 23 and persisted in culture for approximately 40 days. Marrow cultures stimulated by the cord blood-derived growth factor formed colonies of neutrophils, monocyte-macrophages, eosinophils, mast cells, and a few colonies containing a mixed cell population. The results suggest that, while GCT-CM contains granulocyte-monocyte colony-stimulating factor (GM-CSF), cord blood contains a high concentration of a multilineage hemopoietic growth factor, which may be multi-CSF.     

人巨细胞病毒对粒-单及红系祖细胞体外增殖的影响

目的：探讨人巨细胞病毒(HCMV)对脐血粒—单系祖细胞(CFU—GM)、红系爆式祖细胞(BFU—E)及红系祖细胞(CFU—E)体外增殖的抑制作用。方法：采用造血祖细胞体外半固体培养技术，研究HCMV—ADl69株对脐血CFU—GM、BFU—E及CFU—E集落生长的影响；用聚合酶链反应(PCR)技术检测集落细胞中HCMV—ADl69 DNA。结果：①HCMV—ADl69对CFU—GM、BFU—E及CFU—E均有抑制作用。②其抑制作用与HCMV浓度有关，高浓度(1：10，1：100)感染组与对照组比较集落产率明显下降(P＜0.01)，集落维持的时间明显缩短(P＜0.01)，而低浓度组(1：1000)无此作用。③经PCR检测发现病毒感染组CFU—GM、BFU—E及CFU—E集落细胞中有HCMV—ADl69 DNA存在。结论：①HCMV—ADl69可抑制CFU—GM、BFU—E及CFU—E集落的生长。②造血祖细胞是HCMV—ADl69的宿主细胞之一，HCMV—ADl69能直接感染造血祖细胞。HCMV感染患儿出现的粒细胞减少和贫血可能与此有关。     

人类巨细胞病毒感染对脐血造血祖细胞体外增殖的影响

目的:探讨人类巨细胞病毒(HCMV)感染对脐血造血祖细胞(CFU-Gm、CFU-E、BFU-E、CFU-Mix及CFU-Mk)体外增殖的抑制作用及其机制。方法:采用造血祖细胞体外半固体培养技术,培养、观察HCMV-AD169株对脐血CFU-Gm、CFU-E、BFU-E、CFU-Mix及CFU-Mk集落数、抑制率、集落峰值时间和集落维持时间;并用聚合酶链反应(PCR)技术检测集落细胞内HCMV-DNA。结果:①HCMV-AD169株的不同感染滴度[103、104及105空斑形成单位(PFU)/ml]均能明显抑制CFU-Gm、CFU-E、BFU-E、CFU-Mix及CFU-Mk集落的形成,集落数随病毒感染滴度的增高而明显减少;②与对照组比较集落的抑制率分别是a.105PFU感染组依次为40.9%、47.9%、53.3%、55.2%、42.1%(均P<0.01);b.104PFU感染组32.7%、30.2%、36.1%、35.1%、29.7%(均P<0.01);c.103PFU感染组8.0%、6.9%、8.2%、20.3%、14.5%(CFU-Gm、CFU-E、BFU-E与对照组比较差异无统计学意义,余均P<0.01),显示抑制程度与病毒感染滴度呈剂量依赖关系;③集落维持时间感染组较对照组明显缩短(P<0.05);④PCR检测感染组发现CFU-Gm、CFU-E、CFU-Mix及CFU-Mk集落细胞内均有HCMV-AD169DNA存在。结论:HCMV-AD169能直接感染CFU-Gm、CFU-E、BFU-E、CFU-Mix及CFU-Mk造血祖细胞,并抑制造血祖细胞的增殖,此可能与HCMV感染患儿出现粒细胞减少、血小板减少和贫血等造血功能紊乱有关。     

Kinetics of myeloid progenitor cells in human micro long-term bone marrow cultures

Hemopoiesis was analyzed in a miniaturized long-term culture of human bone marrow cells by quantifying the production of granulocyte-macrophage progenitor cells. As in the conventional long-term culture system, hemopoiesis was dependent on the presence of a marrow-derived adherent layer. Adipocytes proved to be essential for long-term proliferation. Optimal growth conditions were maintained by incubation in McCoy's medium supplemented with hydrocortisone, fetal calf serum, and horse serum. When calculated back to the volume of conventional cultures, the numbers and kinetics of nucleated cells and granulocyte-macrophage colony-forming cells were comparable in both culture systems. The microsystem is therefore suitable for performing multiple analyses on small samples of cells.     

Preferential suppression of myelopoiesis in normal human bone marrow cells after in vitro challenge with human cytomegalovirus.

The pathogenic effects of human cytomegalovirus (CMV) infection in vitro on hematopoiesis were investigated. Normal human bone marrow cells from both seronegative and seropositive donors were challenged with CMV (Towne or wild-type strain) and tested for their responsiveness to the recombinant hematopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF), respectively. Regardless of the serostatus of the donor, infection with CMV resulted in a significant decrease in the proliferation and colony formation of hematopoietic progenitor cells in response to both growth factors, with more pronounced suppression in response to G-CSF being observed. Evaluation of the colony composition revealed a profound decrease in colonies of the granulocytic (CFU-G), or granulocyte-macrophage (CFU-GM) lineages, while suppression of multipotential (CFU-GEMM) and erythroid (BFU-E) colony-forming cells occurred after infection with wild-type but not the laboratory strain of CMV. Although no evidence of productive virus infection could be seen in colony-forming cells, in situ hybridization studies and immunohistochemical staining revealed the presence of CMV-specific mRNA and immediate-early antigens, demonstrating that a small proportion of cells were abortively infected. These studies demonstrate that CMV can infect bone marrow progenitor cells and interfere with normal hematopoiesis in vitro, which may help to explain the hematologic defects seen during acute infections with CMV in vivo.     

5-Azacytidine-treated human mesenchymal stem/progenitor cells derived from umbilical cord, cord blood and bone marrow do not generate cardiomyocytes in vitro at high frequencies

Background and Objectives   Mesenchymal stem/progenitor cells (MSCs) are multipotent progenitors that differentiate into such lineages as bone, fat, cartilage and stromal cells that support haemopoiesis. Bone marrow MSCs can also contribute to cardiac repair, although the mechanism for this is unclear. Here, we examine the potential of MSCs from different sources to generate cardiomyocytes in vitro , as a means for predicting their therapeutic potential after myocardial infarction.
Materials and Methods   Mesenchymal stem/progenitor cells were isolated from the perivascular tissue and Wharton's jelly of the umbilical cord and from cord blood. Their immunophenotype and differentiation potential to generate osteoblasts, chondrocytes, adipocytes and cardiomyoxcytes in vitro was compared with those of bone marrow MSCs.
Results   Mesenchymal stem/progenitor cells isolated from umbilical cord and cord blood were phenotypically similar to bone marrow MSCs, the exception being in the expression of CD106, which was absent on umbilical cord MSCs, and CD146 that was highly expressed in cord blood MSCs. They have variable abilities to give rise to osteoblasts, chondrocytes and adipocytes, with bone marrow MSCs being the most robust. While a small proportion (~0·07%) of bone marrow MSCs could generate cardiomyocyte-like cells in vitro, those from umbilical cord and cord blood did not express cardiac markers either spontaneously or after treatment with 5-azacytidine.
Conclusion   Although MSCs may be useful for such clinical applications as bone or cartilage repair, the results presented here indicate that such cells do not generate cardiomyocytes frequently enough for cardiac repair. Their efficacy in heart repair is likely to be due to paracrine mechanisms.     

Biology of umbilical cord blood progenitors in bone marrow niches

Within the bone marrow (BM), hematopoietic progenitor cells (HPCs) are localized in poorly oxygenated niches where they interact with the surrounding osteoblasts (OBs) through VLA4/VCAM-1 engagement, and are exposed to interleukin-6 (IL-6), stem cell factor (SCF), and chemokines such as CXCL12 (OB factors). Umbilical cord (UC) is more highly oxygenated that the BM microenvironment. When UC-HPCs are exposed to the 2% to 3% O(2) concentration found in the bone endosteum, their survival is significantly decreased. However, engagement of VLA-4 integrins on UCB-derived CD34(+) cells reduced cell death in 2% to 3% O(2) conditions, which was associated with an increase in phospho-Ser473 AKT and an increase in phospho-Ser9 GSK3b. Consistent with the role of GSK3b in destabilizing beta-catenin, there was more cytoplasmic beta-catenin in UC-HPCs exposed to 2% to 3% O(2) on fibronectin, compared with suspension culture. UC-HPCs cultured at 2% to 3% O(2) with OB factors showed an increase in nuclear beta-catenin and persistence of a small pool of CD34(+)38(-) HPCs. CFU assays followed by surface phenotyping of the plated colonies showed improved maintenance of mixed lineage colonies with both erythroid and megakaryocytic precursors. These studies provide a biologic perspective for how UC-derived HPCs adapt to the bone endosteum, which is low in oxygen and densely populated by osteoblasts.     

In vitro suppression of bone marrow progenitor cell differentiation by human herpesvirus 6 infection.

Suppression of marrow function may be one of the most serious effects of human herpesvirus 6 (HHV-6) infection in marrow transplant patients. In this study, normal bone marrow mononuclear cells were infected in vitro with HHV-6, and a methylcellulose-based colony formation assay was used to evaluate the impact of the infection on marrow cell differentiation and proliferation. Results demonstrated that the outgrowth of colony-forming units of granulocyte and macrophage lineages (cfu-GM) was decreased by approximately 43%, that growth of cfu of granulocyte, erythrocyte, macrophage, and megakaryocyte lineages (cfu-GEMM) was inhibited by an average of 71%, and that the erythroid burst-forming unit (bfu-E) was decreased by approximately 73%. Further, outgrowth of the marrow stromal layer was reduced 74%. Direct infection of bone marrow monocytes was observed, although cell-free virus could not be detected in infected culture supernatants. Addition of a neutralizing monoclonal antibody specific for interferon-alpha to the infected cultures resulted in an almost complete reversal of the viral suppressive effects.     

Interleukin-6 is a cofactor for the growth of myeloid cells from human bone marrow aspirates but does not affect the clonogenicity of myeloma cells in vitro

Several groups have claimed that IL-6 is a growth factor for human myeloma cells in vitro. Bone marrow aspirates from 30 patients at different stages of treatment with VAMP/high dose melphalan, were examined for myeloma colony formation (MY-CFUc) using a clonogenic assay in vitro. Myeloma cells from 16/30 patients produced MY-CFUc in our assay system, which uses heavily irradiated HL60 cells as an underlay in soft agar. These heavily irradiated cells were shown to be essential for the inhibition of granulocyte-macrophage colonies (GM-CFUc). The addition of recombinant human IL-6 (10 ng/plate) reduced the number of bone marrow samples which produced MY-CFUc from 16 to six. Furthermore, the addition of antibody to IL-6 (1 microgram/plate) failed to inhibit MY-CFUc from 6/7 samples. Conditioned medium from human peripheral blood mononuclear cells (PBMC-CM) contains approximately 2 ng/ml IL-6 and can be used to stimulate the growth and maintenance of the B9 murine IL-6 dependent hybridoma cell line. Recombinant human IL-6 supported the growth of B9 cells in a clonogenic assay and growth was inhibited by anti-IL-6 in the presence of rhIL-6 or PBMC-CM. Mononuclear cells from a second group of myeloma patients were cultured in soft agar in a mixture of PBMC-CM and fresh growth medium. Nine of the 10 samples produced myeloid colonies which consisted of granulocytes, monocytes and macrophages and the number of colonies was reduced by at least 50% in 6/8 samples when anti-IL-6 was added to the cultures. In no instance were MY-CFUc produced. Also, conditioned medium from the bladder carcinoma cell line 5637, which is used routinely as a source of granulocyte-macrophage colony stimulating factor (GM-CSF), contains approximately 4 ng/ml IL-6. Although rhIL-6 failed to stimulate GM-CFUc in the absence of other growth factors, addition of anti-IL-6 to cultures containing a suboptimal amount of 5637-CM reduced the number of colonies by 50%. These data provide evidence that IL-6 is a cofactor for the growth of myeloid precursors but does not affect the proliferation of human myeloma cells in vitro.     

Plastic-adherent progenitor cells in human bone marrow

Human bone marrow contains plastic-adherent hemopoietic progenitor cells whose plating efficiency is increased by brief (2 h) exposure to methylprednisolone (MP). When subsequently covered with methylcellulose medium, they form colonies of monoblastoid cells. Colony size, but not number, and mature cell production are increased by erythropoietin (epo) and granulocyte-macrophage colony-stimulating factor (GM-CSF). However, colonies do not grow under serum-free conditions. The resistance of plastic-adherent progenitors to treatment with 5-fluorouracil (5FU), their growth pattern, and their capacity to produce granulocytic and erythroid colonies on replating, suggest that they may be similar to the primitive, 5FU-resistant, plastic-adherent progenitor cells (HPP-CFC) in murine marrow.     

Inhibition of human bone marrow and myeloid progenitors by interleukin 2-activated lymphocytes

We have shown that unstimulated and interleukin 2 (IL-2)-activated peripheral blood lymphocytes from both normal donors and cancer patients in remission significantly inhibited the proliferation and granulocyte-macrophage colony formation (granulocyte-macrophage colony-forming cells, GM-CFC) of autologous and allogeneic bone marrow (BM). The inhibition was mediated primarily by CD5+ T cells, although lower levels of inhibition were also displayed by CD56+, CD5- lymphocytes, most of which were CD16-. The CD5+ lymphocytes were also the major effectors responsible for lysis of BM. Inhibition of BM proliferation and GM-CFC colony formation was not dependent on proliferation of the effector cells or cell-to-cell contact, because it was also mediated by a soluble factor produced by IL-2-activated lymphocytes. The relevance of these findings to future approaches to leukemia treatment is discussed.     

Comparison of in vitro drug-sensitivity of human granulocyte-macrophage progenitors from two different origins: umbilical cord blood and bone marrow

Predictive in vitro hematotoxicity assays using human cells will provide estimation of tolerable level and aid considerably the development of agents with greater therapeutic activity and less toxicity. Human hematopoietic cells can be derived from three sources: human bone marrow by sternal or femoral aspiration, mobilized peripheral blood, or umbilical cord blood samples collected from placentas after deliveries. Because of the difficulties to have a continuous supply of bone marrow cells from normal human donors and the related ethical problems, we performed a study to compare the sensitivity of human bone marrow cells (h-BMC) and human cord blood cells (h-CBC) to chemicals in order to confirm if h-CBC can readily replace bone marrow cells in checking the sensitivity of GM-CFU progenitors to drugs as preliminarily reported in literature. Our results showed that the prediction of IC50 values in human model is quite similar by using h-BMC or h-CBC. On the contrary, the type of medium influenced in a significant way the ICs determination of some drugs.     

Human osteoblasts support human hematopoietic progenitor cells in vitro bone marrow cultures

Hematopoietic stem cell differentiation occurs in direct proximity to osteoblasts within the bone marrow cavity. Despite this striking affiliation, surprisingly little is known about the precise cellular and molecular impact of osteoblasts on the bone marrow microenvironment. Recently, we showed that human osteoblasts produce a variety of cytokine mRNAs including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-6. We examined here the ability of osteoblasts to support the development of hematopoietic colonies from progenitors as well the ability to maintain long-term culture-initiating cells (LTC-IC) in vitro. Examination of the hematopoietic cells recovered after 2 weeks of culture showed that osteoblasts support the maintenance of immature hematopoietic phenotypes. In methylcellulose assays, osteoblasts stimulate the development of hematopoietic colonies to a level at least 10-fold over controls from progenitor cells. Using limiting dilutional bone marrow cultures, we observed an activity produced by osteoblasts resulting in an threefold to fourfold expansion of human LTC-IC and progenitor cells in vitro. Thus, the presence of hematopoietic stem cells in close proximity to endosteal surfaces in vivo may be due in part to a requirement for osteoblast-derived products.     

Direct contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long-term in vitro hematopoiesis.

Long-term bone marrow cultures support both differentiation and conservation of primitive human hematopoietic progenitors in the absence of exogenous cytokines. It is believed that hematopoiesis in such cultures requires direct contact between hematopoietic progenitors and stroma. In the present study, we demonstrate that primitive progenitors physically separated from the stromal layer by a 0.45-microns microporous membrane continue to generate differentiated progenitors for at least 8 weeks. Moreover, primitive progenitors are conserved to a greater extent under these conditions, as when cultured in direct contact with the stroma. However, excessive production of granulocyte-macrophage progenitors occurs when primitive progenitors are not allowed to interact directly with the stroma. Thus, direct contact between hematopoietic and stromal cells is not required for either differentiation or conservation of primitive hematopoietic progenitors but is essential for the regulated production of mature blood elements. These findings can now be used to define the role of diffusible factors and cell-cell or cell-extracellular matrix adhesion events in the regulation of conservation, proliferation, and differentiation of primitive human hematopoietic progenitors in vitro.     

Different haematopoietic growth factors have different capacity in overcoming the in vitro interferon gamma-induced suppression of bone marrow progenitor cells

Interferon gamma (IFN gamma) inhibits haematopoiesis in vitro and an in vivo role in bone marrow suppression has been implied from clinical studies. We investigated the capacity of three recombinant (r), human (h), haematopoietic growth factors to overcome the in vitro IFN gamma inhibition of bone marrow progenitor cells in a methylcellulose culture system. Granulocyte macrophage-colony stimulating factor (GM-CSF) partially reversed IFN gamma-induced suppression of granulocyte-macrophage colony formation, by increasing colony forming units-granulocyte macrophage (CFU-GM) in a proportion ranging from 54-101%. Interleukin-3 (IL-3) and granulocyte-colony stimulating factor (G-CSF) were much less effective. For erythropoiesis, IL-3 was much more effective and partially reversed IFN gamma-mediated inhibition by increasing burst forming units-erythroid (BFU-E) in a proportion ranging from 52-138%. GM-CSF and G-CSF had no significant effect on IFN gamma-induced suppression of BFU-E. In conclusion, haematopoietic growth factors have different capacity to overcome IFN gamma-induced suppression of marrow progenitor cells in vitro. The findings may have therapeutic implications, as combinations of growth factors may be more effective in treating bone marrow failure syndromes.     

Proliferation of myeloid progenitor cells in human long-term bone marrow cultures is stimulated by interleukin-1 beta

To study the effect of interleukin-1 (IL-1) beta on the proliferation of hematopoietic progenitor cells (HPC) in long-term bone marrow cultures (LTBMC), stromal cell layers were established from normal human bone marrow. Autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells were reinoculated on the stromal layers in fresh culture medium, with or without the addition of human IL-1 beta (30 U/mL). Once a week, half of the culture supernatant was replaced with fresh culture medium with or without IL-1, and all nonadherent cells were returned to the flasks. At weekly intervals during a period of 5 weeks, one culture was sacrificed to determine the total number of cells and hematopoietic progenitor cells, present in the adherent and the nonadherent cell fractions. In IL-1-stimulated cultures, the number of cells recovered during a period of 5 weeks exceeded the number of cells in unstimulated control cultures by 1.5 times. This difference was attributed to a twofold increase in the number of adherent cells. The number of HPC recovered from IL-1- stimulated cultures was not different from that recovered from controls. The levels of colony-stimulating activity (CSA) in supernatants from IL-1-stimulated cultures were significantly higher than those in supernatants from control cultures. These results indicate that IL-1 enhances the recovery of cells in LTBMC by stimulating the proliferation of HPC with the concurrent release of CSA from stromal cells, without diminishing the number of HPC.     

Adult bone marrow is a rich source of human mesenchymal 'stem' cells but umbilical cord and mobilized adult blood are not

In postnatal life, mesenchymal stem cells (MSC) self-replicate, proliferate and differentiate into mesenchymal tissues, including bone, fat, tendon, muscle and bone marrow (BM) stroma. Possible clinical applications for MSC in stem cell transplantation have been proposed. We have evaluated the frequency, phenotype and differentiation potential of MSC in adult BM, cord blood (CB) and peripheral blood stem cell collections (PBSC). During culture, BM MSC proliferated to confluence in 10-14 d, maintaining a stable non-haemopoietic phenotype, HLA class-1+, CD29+, CD44+, CD90+, CD45-, CD34- and CD14 through subsequent passages. Using the colony forming unit fibroblasts assay, the estimated frequency of MSC in the BM nucleated cell population was 1 in 3.4 x 10(4) cells. Both adipogenic and osteogenic differentiation of BM MSC was demonstrated. In contrast, CB and PBSC mononuclear cells cultured in MSC conditions for two passages produced a population of adherent, non-confluent fibroblast-like cells with a haemopoietic phenotype, CD45+, CD14+, CD34-, CD44-, CD90- and CD29-. In paired experiments, cultured BM MSC and mature BM stroma were seeded with CB cells enriched for CD34+. Similar numbers of colony-forming units of granulocytes-macrophages were produced by MSC-based and standard stroma cultures over 10 weeks. We conclude that adult BM is a reliable source of functional cultured MSC, but CB and PBSC are not.     

Enrichment of myeloid progenitor cells from normal human bone marrow using an immune-rosette technique

In this study we have developed methods for purification of myeloid progenitor cells (CFU-Cs) from normal human bone marrow cells. Bone marrow aspirates were obtained from volunteers, and mononuclear cells (MNCs) were separated by Ficoll-Hypaque gradient centrifugation. T- and B-lymphocytes, monocytes, mature granulocytes, and erythroid precursors were eliminated by an immune-rosette technique using a panel of murine monoclonal antibodies and immunoglobulin (Ig)-coated sheep red blood cells (SRBCs). MNCs were treated with OKT3, B1, M3, Mo5, and EP1 monoclonal antibodies, which are reactive with T cells, B cells, monocytes, granulocytes, and erythroid precursors, respectively. Antibody-treated MNCs were incubated with SRBCs that had been coated with goat antirabbit IgG F(ab')2 and rabbit antimouse Ig for immune rosetting. Rosetted cells were then separated from nonrosetted cells in Ficoll-Hypaque. Nonrosetted cells were, in the second step, treated with an OKIa1 monoclonal antibody and again separated into an Ia+ and Ia- cell fraction by the same manner; 39% +/- 19.2% (mean +/- 1 SD, range 16.3%-75.4%) of CFU-Cs (colonies plus clusters) were recovered in the OKT3-, B1-, M3-, Mo5-, EP1- cell fraction, and the number of CFU-Cs grown in semisolid agar was 149.6 +/- 73.0 (64.0-309.0)/10(4) plated cells in this purified fraction, representing an enrichment of 14.2 +/- 6.4 (6.0-27.3)-fold when compared with unseparated marrow cell fractions. CFU-Cs were enriched 17.7 +/- 8.6 (6.1-28.3)-fold in the Ia+ cell fraction. These purified myeloid precursors would be of value for in-depth studies of the interactions between hematopoietic progenitor cells and regulatory factors that influence their proliferation and differentiation and also of drug metabolism and determinants of cytotoxicity.     

Direct comparison by limiting dilution analysis of long-term culture- initiating cells in human bone marrow, umbilical cord blood, and blood stem cells

Limiting-dilution analysis of long-term culture-initiating cells (LTCIC) is a quantitative method of estimating hematopoietic stem cell activity in clinical samples. We compared the numbers of LTCIC in bone marrow (BM), umbilical cord blood, and blood progenitor cells (obtained from patients with solid tumors at leukapheresis after mobilization with induction chemotherapy and filgrastim administration), using a two- stage long-term culture system and a limiting-dilution technique, scoring cobblestone areas of greater than 15 hematopoietic cells weekly for up to 8 weeks. Samples were obtained from 30 normal BMs, 20 human umbilical cords, and 32 leukapheresis products. Direct comparison of LTCIC in the three sources showed that the median proportions of cells generating hematopoietic foci from unfractionated mononuclear cells at 5 and 8 weeks, respectively, were 1:13,314 and 1:33,949 for BM, 1:12,506 and 1:34,546 for umbilical cord blood, and 1:10,302 and 1:12,891 for leukapheresis product. The estimated proportions of LTCIC from unfractionated mononuclear cells and CD34+ cells were similar in experiments with leukapheresis product. Leukapheresis product was superior to umbilical cord blood and cord blood to BM at 5 and 8 weeks of culture (P = .01). In two-stage long-term cultures, more colonies per flask and CD34+ cells were found in assays of leukapheresis product than in BM or umbilical cord blood cultures (P = .0005). Results obtained by this simplified limiting-dilution analysis correlated well with standard long-term cultures and can be used as a measure of the stem cell population. These data suggest that the incidence of putative stem cells in leukapheresis product and umbilical cord blood are at least comparable with that of BM.