Phenotype of Peripheral Blood Cells Mobilized by Granulocyte Colony-Stimulating Factor in Patients with Chronic Heart Failure

Administration of granulocyte CSF preparation to patients with chronic heart failure produced a hemostimulating effect and increased the content of leukocytes and neutrophils in the peripheral blood. Granulocyte CSF induced mobilization of bone marrow progenitor cells into the peripheral blood. The content of hemopoietic CD34(+)progenitor cells, which attained 0.42% (0.25-0.64) by the end of mobilization, inversely correlated with the number of myocardial infarctions. Administration of granulocyte CSF not only led to mobilization of bone marrow hemopoietic cells, but also increased the pool of endothelial progenitor cells in the peripheral blood: the content of CD34(+)/CD133(+)and CD34(+)/KDR(+)attained 0.02% (0.013-0.075) and 0.1% (0.05-0.20), respectively. Peripheral blood is an available source of progenitor cells, while mononuclear cells after administration of granulocyte CSF can produce a reparative effect on ischemic myocardium.     

脐血与成人骨髓淋巴细胞及其亚群比较

比较脐血与骨髓T淋巴细胞、B淋巴细胞和NK细胞亚群的分布特点及血清sIL-2R和IgG水平的不同,探讨脐血移植后免疫重建延迟、GVHD及GVL效应的发生机制。应用流式细胞仪分别检测脐血及骨髓CD3+、CD3+CD5+、CD3+CD45RA+、CD3+CD45RO+、CD3+CD25+、CD19+、CD19+CD10+、CD19+CD40+、CD19+CD23+、CD3-CD16+CD56-及CD3-CD16+CD56+细胞的含量。应用双抗夹心酶联免疫吸附法(ELISA)及速率免疫散射比浊法分别检测脐血及骨髓血清中sIL-2R及IgG水平。CD3+T细胞数在脐血及骨髓中无显著差异(P>0.05)。脐血中CD3+CD45RA+、CD19+、CD19+CD10+及CD3-CD16+CD56-细胞数显著高于骨髓(P<0.05)。脐血中CD3+CD5+、CD3+CD45RO+、CD3+CD25+、CD19+CD40+、CD19+CD23+及CD3-CD16+CD56+细胞数显著低于骨髓(P<0.05)。脐血血清中sIL-2R及IgG水平显著低于骨髓(P<0.05)。脐血中成熟T细胞、成熟B细胞及成熟NK细胞亚群的细胞数显著低于骨髓,这可能是脐血移植后免疫重建延迟、GVHD发生率低及GVL效应低的原因之一。     

骨髓基质细胞的造血支持作用等生物学特性的研究

目的研究人骨髓基质细胞体外长期培养的生物学特性和造血支持功能。方法①采用静置贴壁细胞培养法,体外长期培养胎儿、儿童和成人的骨髓基质细胞。②采用免疫细胞化学染色法和流式细胞仪检测法,分析细胞的表型。③将不同发育阶段的骨髓基质细胞体外培养,并扩增脐血造血干细胞。结果①建立了成纤维肌样细胞系,可传至10代,维持6个月,同时还培养出内皮细胞和巨噬细胞。②儿童骨髓基质肌样细胞的染色特征为波形纤维蛋白(viementin)呈阳性,第VIII因子呈阴性;儿童骨髓基质细胞的表型为CD33     

Characterization of ''adult-type'' mast cells derived from human bone marrow CD34+ cells cultured in the presence of stem cell factor and interleukin-6. Interleukin-4 is not required for constitutive expression of CD54, FcεRIα and chymase, and CD13 expression is reduced during differentiation

BACKGROUND: In vitro-derived human mast cells exhibit different properties, depending in part on the source of progenitor cells. Most investigations have used fetal liver, cord blood or peripheral blood. Few have used adult bone marrow. OBJECTIVE: Human mast cells derived in vitro from the CD34(+) progenitors in bone marrow and cord blood that had been cultured with recombinant human stem cell factor (rhSCF) and recombinant human interleukin-6 (rhIL-6) were compared. METHODS AND RESULTS: After 12 weeks of culture, nearly all of the cells were mast cells, and nearly all of these had cytoplasmic granules containing both tryptase and chymase (MCTC type), stained metachromatically with acidic toluidine blue, and expressed CD117 on the cell surface. Both tryptase protein and mRNA were detected by two weeks of culture. Chymase mRNA and protein were detected at 4 weeks but not at 2 weeks of culture. By 12 weeks, chymase content per cell, measured by ELISA, was significantly higher (P < 0.05) in human bone marrow-derived mast cells (HBMMC) (5.6 +/- 0.9 pg) than in cord blood-derived mast cells (CBMC) (2.4 +/- 0.9 pg), whereas histamine and tryptase levels were not significantly different. Of the cluster designations tested, CD29, CD49d, CD51 and CD61 were strongly expressed on HBMMC. CD54 and Fc epsilon RI alpha also were expressed constitutively. Approximately half of CD34-sorted cells at day 0 were CD13(+) and this diminished as mast cell maturation occurred. Electron microscopy revealed that 12-week-old HBMMC had many secretory granules that contained spherical electron dense cores surrounded by electron lucent space, consistent with previous reports of immature MCTC cells developing in vivo. CONCLUSIONS: CD34(+) progenitors of human bone marrow are a rich source of mast cell progenitors capable of expressing granule and surface markers of mature mast cells in the presence of rhSCF and rhIL-6.     

Developmental differences in megakaryocyte maturation are determined by the microenvironment

Historically, physicians have attributed delayed platelet engraftment following umbilical cord blood transplant to decreased numbers of stem cells in cord blood compared with adult bone marrow. However, recent studies suggest that delayed platelet engraftment may be caused by an intrinsic inability of neonatal stem cells to produce mature, polyploid megakaryocytes. We tested this hypothesis by transplanting adult bone marrow and newborn liver hematopoietic stem and progenitor cells from transgenic mice expressing green fluorescent protein into myeloablated wild-type recipients and comparing the size and ploidy levels of megakaryocytes that developed in adult transplant recipients. Transplanted stem and progenitor cells, regardless of their source, gave rise to megakaryocytes that were larger than normal adult megakaryocytes as early as 7 days post-transplant. However, megakaryocytes that developed after transplant of neonatal stem and progenitor cells were significantly smaller than those derived from adult stem and progenitor cells. Furthermore, megakaryocytes derived from neonatal cells had lower ploidy values than megakaryocytes derived from adult cells at 18 days post-transplant, when ploidy could first be reliably measured in the bone marrow. These differences in size and ploidy disappeared by 1 month post-transplant. The largest megakaryocytes developed in the spleen. These results suggest that, in the mouse, the microenvironment is responsible for some of the maturational differences in size and ploidy between neonatal and adult megakaryocytes. Furthermore, neonatal and adult megakaryocyte progenitors also have cell-intrinsic differences in the way they engraft and respond to thrombocytopenic stress. These differences may contribute to the delay in platelet engraftment that frequently complicates cord blood transplants.     

An in vitro model of allogeneic stimulation of cord blood: induction of Fas independent apoptosis.

Cord blood is increasingly used in transplantation as it is a readily available source of progenitor cells and is reputed to generate less severe graft-versus-host disease (GVHD) than adult bone marrow. We have compared apoptosis of cord blood lymphocytes (CB) and adult lymphocytes (PBMC) after stimulation via HLA class I, HLA class II or CD3 in order to reproduce in vitro some of the stimuli occurring after allotransplantation. CB spontaneously apoptose more than PBMC ex vivo, stimulation via HLA class I dramatically increased CB apoptosis without altering viability of PBMC. Expression of Fas was markedly lower on CB than on PBMC and this difference was maintained even after activation. Fas ligand was expressed in CB and in PBMC. CB were activated via either HLA class I or class II molecules although proliferation was not observed. Only phorbol ester pre-activation allowed Fas to subsequently induce a death signal. Proliferation of PBMC via CD3 led to enhanced Fas signals. CB therefore differ from PBMC with regard to both spontaneous and activation induced apoptosis and either allo- or CD3 mediated stimulation. Finally, the apoptosis of CB via HLA-class I could have an important role in the moderation of graft-versus-host disease.     

The role of blood stem cells in hematopoietic cell renewal

It has been the purpose of this keynote address to review available evidence for the notion that the stem and progenitor cells circulating in the peripheral blood play a decisive role in the homeostasis of blood cell formation distributed throughout dozens of bone marrow units in the skeleton. Furthermore, if this notion is correct, one could speculate that the quantity and quality of stem and progenitor cells in the blood should reflect the functional state of the hematopoietic stem cell system throughout the skeletal bone marrow and provide a new tool for the evaluation of alteration in blood cell production. On this basis, the following questions are considered: A) What do we know about the quality and quantity of blood stem cells in steady state conditions? B) In what way do blood stem cells respond to perturbations of the "steady state" of blood cell formation? C) Which role do blood stem cells play during hemopoietic development assuming that the establishment of bone marrow hemopoiesis requires the "seeding" of blood stem cells into an appropriate cellular environment? D) What is the role of blood stem cells in hemopoietic regeneration after partial body irradiation with a small volume of marrow (and hence stem cells) protected? and E) What are the mechanisms and/or kinetics of hemopoietic recovery if stem cells introduced into the circulation were collected from exogenous (autologous or allogeneic) sources? In this review presentation, experimental work of our group and of other members of the scientific community is summarized. It becomes obvious that blood stem and progenitor cells play a key role in hematopoietic homeostasis. Furthermore, their physiology and pathophysiology deserve rigorous experimental studies in order to develop a novel tool in the diagnosis and prognosis of neoplastic and non-neoplastic disorders of blood cell formation.     

Absolute values of dendritic cell subsets in bone marrow,cord blood,and peripheral blood enumerated by a novel method

Dendritic cells (DCs) are pivotal in inducing immunity or alternatively downregulating immune reactivity. In humans, the opposing phenotypic subsets of CD11c(+)/CD123(-) "myeloid" DCs and CD123(+)/CD11c(-) "lymphoid" DCs have been proposed to orchestrate these immune responses. In this study we determined the absolute numbers of both subsets in three resting hematopoietic tissues by employing a novel flow cytometry method, eliminating processing steps and calculations based on mononuclear cell percentages. Internal bead standards along with the cells of interest were simultaneously acquired directly from unmanipulated whole blood specimens. We found significant differences (p < 0.001) between the mean absolute numbers of CD123(+)/CD11c(-) lymphoid DCs among the three sources, with the fewest present in peripheral blood (8.2/ micro l), about 50% more in cord blood (12.2/ micro l), and fivefold more in bone marrow (40.2/ micro l). Cord blood and bone marrow CD11c(+)/CD123(-) myeloid DC counts did not differ from each other (23.5/ micro l and 28.9/ micro l, respectively) but peripheral blood contained significantly fewer (15.5/ micro l, p = 0.006). CD11c(+)/CD123(-) DCs had significantly higher surface expression of HLA-DR (p < 0.001) in all three sources. To test for association with the DC subsets, T, B, and natural killer (NK) lymphocytes were also enumerated. In bone marrow only, significant correlations were found between the size of the CD123(+)/CD11c(-) lymphoid DC pool and NK cells (p = 0.0029) and B cells (p = 0.0033). These data support the hypothesis that a common CD123(+)/CD11c(-) DC, NK cell, and B cell progenitor is resident in marrow, and this cell may be identical to the common lymphoid progenitor previously described in mice and/or the human CD34(+)/Lin(-)/CD10(+) progenitor.     

人骨骼肌源性血管内皮细胞对造血干/祖细胞的体外支持

背景:人骨骼肌源性血管内皮细胞位于血管壁,共表达肌肉干细胞和血管内皮细胞的标记(CD56+CD34+CD144+CD45-).研究显示,人肌血管内皮细胞与间充质干细胞存在相似性,表达间充质干细胞表面标记物,具有多向分化潜能.目的:建立人肌血管内皮细胞作为滋养层与人脐血CD34+细胞体外培养体系,以培养前后CD34+细胞...     

Seven week culture of functional human mast cells from buffy coat preparations

Functional, mature human mast cells have been generated by in vitro differentiation of CD133(+)/CD34(+) progenitor cells isolated from e.g. cord blood, peripheral blood, bone marrow or fetal liver. However, the protocols published so far require long term cultivation, i.e. up to 15 weeks for mast cell differentiation, which makes such approaches not only laborious but also costly. Here, we have developed a protocol for generating functional human mast cells from peripheral blood already within 7 weeks. Human CD133(+) progenitors were isolated from buffy coat preparations of peripheral blood and cultured in the presence of stem cell factor (SCF) and IL-6 for 7 weeks. IL-3 was added to the culture medium during the first 3 weeks, and fetal calf serum (FCS) added during the last week. In vitro differentiated CD133(+) cells exhibited multiple characteristics of mature mast cells. Thus, cells contained tryptase and expressed functional levels of FcepsilonRI. Anti-IgE stimulation induced significant release of histamine and PGD(2) and also of chemokines including MCP-1, IL-8, MIP-1alpha, and MIP-1beta. The fact that our in vitro differentiated mast cells are derived from a generally available source of progenitor cells makes this novel protocol widely applicable to any patient group, irrespective of age. Moreover, this progenitor source is more readily available than e.g. bone marrow or cord blood-derived progenitors. Consequently, our protocol has great potential in studies on mast cell biology and mast cell pathology, and e.g. on evaluation of drug effects.     

大鼠骨髓和外周血早晚期内皮祖细胞的分离培养和鉴定

背景：旨在从大鼠外周血及骨髓中提取内皮祖细胞,培养晚期内皮祖细胞,为干细胞移植治疗或通过内皮祖细胞联合基因治疗使内皮祖细胞高表达血管新生诱导因子,达到促进缺血性脑血管病血管新生的目的。 目的：从大鼠骨髓及外周血中分离出内皮祖细胞并对其进行鉴定。 方法：使用密度梯度离心及贴壁筛选法从大鼠骨髓和外周血中分离获得单个核细胞,进行诱导培养,观察并记录贴壁细胞的生物学特征;选取内皮祖细胞特异性表面标志CD133、CD34、KDR对原代细胞进行免疫荧光检测,利用流式细胞学技术检测KDR、CD34表达,并通过吞噬功能实验进一步鉴定培养细胞。 结果与结论：大鼠骨髓和外周血能够分离获得早晚期内皮祖细胞;贴壁细胞免疫荧光检测CD34、CD133、KDR表达阳性;流式细胞学检测CD34、KDR表达阳性;贴壁细胞能够吞噬ac-LDL,结合UEA-1。实验成功从大鼠骨髓及外周血中分离出内皮祖细胞;并获得了增殖活性强的晚期内皮祖细胞,找到了更好的成血管干细胞的种子来源。     

Colocalization analysis of sialomucins CD34 and CD164

Flow cytometric protocols are employed to identify and characterize hemopoietic stem/progenitor populations before transplantation. Cell surface antigens, including CD34, are employed in this process and widely used in harvest protocols, which largely ignores the potential functional role of such antigens. Transmembrane glycoprotein sialomucins, including CD34 and CD164, have been implicated in cell-to-cell interactions and activation. CD164, also expressed on early hemopoietic populations, was reported to have a possible function facilitating CD34(+) cells to adhere to bone marrow stroma. In this study, we employed high-definition laser-scanning confocal microscopy to investigate CD34 and CD164 surface co-localization patterns on bone marrow and cord blood cells and to compare the expression patterns using a three-dimensional computer-generated method developed in house. Differential interference microscopy analysis revealed bone marrow membrane activity was higher than the corresponding cord blood counterpart, perhaps indicating the marrow microenvironmental nature. Fluorescence analysis of CD34 and CD164 antigens showed both were expressed first in a halo-like pattern and second in antigen-dense pockets. Three-dimensional computer analyses further revealed that this pocketing corresponded to dense crest-like surface structures appearing to rise from the point of adherence on the slide. Further, it was found that CD34 and CD164 display strong colocalization patterns on cells expressing both antigens. The dual nature of the CD34 and CD164 antigens discovered here lends further evidence to the previous literature implicating a strong functional link between these two sialomucins, which should be considered in the transplantation arena and in the function of such sialomucins as negative regulators of cell proliferation.     

Cord blood transplant: strategy of alternative donor search

Unrelated cord blood transplantation has been used to treat patients with malignant and non-malignant hematopoietic disorders for whom an HLA-compatible hematopoietic stem cell donor is not available. The establishment of cord blood banks worldwide, the increased number of cord blood units frozen, and the shorter time to find a donor, have made it possible to use this source of hematopoietic stem cells to treat more than 2,500 patients. The Eurocord registry was established to study the clinical results of cord blood transplantation and to compare the outcomes of unrelated transplants using either cord blood or bone marrow. Briefly, we have found, in two distinct retrospective analyses of children or adults with acute leukemia given either an unrelated cord blood or bone marrow transplant, that leukemia-free survival and relapse were similar in both types of graft (with adjustment for confounding clinical factors). Cord blood recipients experienced a decreased incidence of acute graft-versus-host disease and delayed hematopoietic recovery compared to bone marrow recipients. To improve the delayed hematopoietic recovery after cord blood transplantation, certain approaches have been investigated such as ex vivo expansion of cord blood cells, double cord blood transplantation and reduced intensity conditioning regimen. We have also attempted to establish some guidelines for cord blood-donor choice based on cord blood cell dose and number of HLA disparities that have been found to be associated with hematopoietic recovery. In conclusion, an unrelated hematopoietic stem cell donor should be simultaneously searched for in cord blood banks and in bone marrow donor registries for patients lacking an HLA-identical sibling hematopoietic stem cell donor. The option of performing cord blood transplants should be based on urgency of the transplant, cord blood cell dose and number of HLA disparities.     

Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers.

The stromal cell population in bone marrow has been the focus of much attention since it has been shown that this cell population can be expanded and differentiated into cells with the phenotype of bone, cartilage, muscle, stroma, neural, and fat cells. We evaluated umbilical cord blood (UCB) for the presence of these cells. From the mononuclear fraction of UCB, we demonstrated the presence of a subset of cells that have been maintained in continuous culture for more than 6 months (>10 passages). These adherent cell populations express adhesion molecules CD13+, CD29+, and CD44+, but not antigens of hematopoietic differentiation. Exposure of these cells to osteogenic agents resulted in an increase in expression of alkaline phosphatase and the appearance of hydroxyapatite nodules by Von Kossa staining. Incubation with adipogenic agents resulted in morphological change and staining with Oil Red O. In addition, when exposed to basic fibroblast growth factor and human epidermal growth factor the cells underwent changes consistent with cells of neural origin. These changes were demonstrated by a combination of immunofluorescent labeling and Western immunoblots for neural-specific markers. Thus, similar to what has been previously reported with bone marrow, cord blood contains a population of cells that can be expanded in culture and are able to express the phenotype of multiple lineages. Cord blood multilineage cells are slower to establish in culture, have a lower precursor frequency and a lower level of bone antigen expression, and lack constitutive expression of neural antigens when compared to bone marrow, suggesting a more primitive population. Cord blood may prove to be a new source of cells for cellular therapeutics for stromal, bone, and, potentially, neural repair.     

mPEG遮蔽供者淋巴细胞减轻移植物抗宿主病的研究

目的 :探讨向SCID小鼠移植甲氧基聚乙二醇 (mPEG)修饰的人脐血单个核细胞时 ,因供体淋巴细胞表面抗原被遮蔽降低了移植物抗宿主病 (GVHD)而不影响其干祖细胞造血功能重建。方法 :(1)用流式细胞仪检测修饰前后人脐血单个核细胞中CD4 、CD8 T细胞及CD4 /CD8 T细胞比率的变化。 (2 )观察遮蔽前后 ,人脐血干祖细胞在体外培养形成CFU GM的差异。 (3)将修饰前后的单个核细胞移植到SCID小鼠体内 ,观察GVHD出现的时间和小鼠活存状况。 (4)移植后 7wk左右测定小鼠骨髓中人源CD4 5 细胞的含量。结果 :(1)mPEG几乎可完全遮蔽T细胞表面的CD4和CD8抗原。 (2 )修饰前、后的干祖细胞 ,体外培养形成CFU GM的数量没有明显差异。 (3)将修饰的人脐血单个核细胞移植到小鼠体内 ,GVHD出现的时间晚于未修饰组 ,提高了小鼠的活存率。 (4)移植后 4 7d ,活杀小鼠的骨髓细胞中 ,可检测到人的CD4 5 细胞。结论 :用mPEG遮蔽供体T细胞表面的CD4、CD8抗原 ,最终减轻了宿主对移植物的免疫应答 ,而干祖细胞增殖、分化的功能未受到明显影响。     

A method for enriching myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells from human cord blood by accessory cell depletion.

Human cord blood provides a convenient alternative to bone marrow as a rich source of hemopoietic progenitor cells. This study reports a simple means for enriching a cord blood progenitor cell population by accessory cell depletion. Two methods of monocyte depletion were tested. A Cytodex 3 microcarrier system using collagen coated dextran beads was compared to the more commonly used method of plastic plate adhesion. The method of plastic plate adhesion gave a significantly higher cell recovery. T cell depletion using a recently characterized rat monoclonal antibody which fixes human complement was also investigated. A combined method of monocyte depletion by plate adhesion and T cell depletion resulted in the removal of > 96% of monocytes and > 98% of T cells. This led to a significant enrichment of myeloid (CFU-GM) and erythroid (BFU-E) colony growth. Such enriched progenitor cell populations provide a useful starting population for any study on hemopoiesis.     

Replication and cytopathology of human parvovirus B19 in human umbilical cord blood erythroid progenitor cells.

Human parvovirus B19 productively infected erythroid progenitor (EP) cells from umbilical cord blood, in vitro as shown by an increase of viral DNA in supernatant fluid assayed by dot blot hybridization and liquid scintillation counting. Progeny virus was released into the supernatant fluid of CD34+ EP cells which had been purified by immunomagnetic separation. This supernatant fluid was infectious for bone marrow cells. Erythroid bursts infected with virus showed characteristic cytopathic effect by electron microscopy consisting of cytoplasmic vacuolization, marginated chromatin, and nuclear inclusions of lattice or crystalline arrays. Cultures of umbilical cord blood EP cells may be useful for the propagation of parvovirus B19 serological testing reagents and the study of virus-host cell interactions.     

Possible Role of Interleukin-10 in Autoantibody Production and in the Fate of Human Cord Blood CD5+ B Lymphocytes

Mononuclear cell subsets in 25 human umbilical cord blood samples and 10 healthy adults were studied. We found a decreased percentage of CD3+ cells, CD8+ cells and gammadelta T cells in cord blood compared with blood from healthy adults. The CD16+56+ and CD19+CD5+ phenotypes were overexpressed in cord blood. We then measured spontaneous gene expression and the production of interleukin-10 in mononuclear cells from cord blood and adult subjects. Although we found no difference between cord blood cells and those from healthy adults, a tendency towards spontaneous interleukin-10 production was observed in cord blood. Phorbol 12-myristate 13-acetate stimulation revealed that, among lymphocytes, cord blood B cells are the main cellular source of interleukin-10. Finally, we found no evidence of augmented spontaneous apoptosis but an increased bcl-2 gene expression in non-T cells from cord blood. Interleukin-10 might protect CD19+CD5+ B cells from apoptosis by inducing bcl-2 and promoting autoantibody production in this B-cell subpopulation.     

Reactions of the blood system and stem cells in bleomycin-induced model of lung fibrosis

On the model of toxic diffuse pulmonary fibrosis induced by intratracheal administration of bleomycin, we studied reactions of the blood system, content of stem cells, committed hemopoietic and stromal progenitor cells in the bone marrow, spleen and peripheral blood of C57Bl/6 mice. It was shown that the development of diffuse pulmonary fibrosis was accompanied by hyperplasia of bone marrow hemopoiesis and leukocytosis in the peripheral blood. Activation of the erythroid and granulocytic hemopoietic stems was related to stimulation of hemopoietic stem cells (polypotent cells, granulocyte/erythroid/macrophage/megakaryocyte precursor cells) and committed erythroid and myeloid progenitor cells in the bone marrow. At the same time, the number of stromal precursors increased. Bleomycin increased the count of hemopoietic stem cells the peripheral blood and spleen and reduced the content of mesenchymal stem cells in the spleen and bone marrow.     

The role of c-Mpl ligands in the expansion of cord blood hematopoietic progenitors

The major limitations to the widespread use of high-dose chemotherapy or radiotherapy followed by autologous or allogeneic transplantation are the scarcity of stem cell donors and the depletion of the autologous stem cell reservoir. Cord blood is a readily available source of stem cells, which, however, might be limited in number. For this reason, up to now, cord blood transplantation has been restricted to children. Therefore, a major goal for experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. Two systems have been described to identify in vitro these progenitor cell populations in both mice and humans: A) long-term culture-initiating cells (LTC-IC), so named because of their ability to support the growth of hemopoietic colonies (colony-forming cell [CFC]) for five to six weeks when cocultured on stromal layers, and B) the generation of hematopoietic progenitors CFC from stroma-free liquid cultures for extended periods of time, which is another indirect evidence for the presence of primitive stem cells. The two systems detect largely overlapping but not identical cell populations of progenitor cells; thus, the identification of the growth factor requirements for the maintenance and amplification of both systems is relevant. The studies presented here demonstrate that CD34+ cord blood cells can be grown in stroma-free liquid cultures for extremely prolonged periods of time (up to six months). During such a period, hemopoietic precursors and committed progenitors belonging to all of the hematopoietic lineages are continuously and massively generated. Such a massive expansion is sustained by an increasingly larger expansion of primitive stem cells (CFU-BI and LTC-IC). The presence of both FL and thrombopoietin (TPO) was necessary and sufficient to support this phenomenon. The addition of KL +/- interleukin 6 (IL-6) does not appear to substantially modify the extent of LTC-IC expansion. FL and TPO appear to be two unique growth factors that preferentially support the self-renewal of primitive stem cells; the additional presence of KL and IL-6 seems to enhance the proliferative potential of at least a subpopulation of daughter stem cells which can undergo at least three differentiation pathways.