Tunica Interna Endothelial Cell Kinase Expression and Hematopoietic and Angiogenic Potentials in Cord Blood CD34+ Cells

Tunica interna endothelial cell kinase (TEK) is expressed in both hematopoietic and endothelial cells and plays a crucial role in hematopoiesis and angiogenesis in mouse development. In humans, however, little is known about the hematopoietic and angiogenic potentials of TEK-expressing cells in umbilical cord blood (CB) cells, which originate during the human fetal period. We therefore compared the hematopoietic and angiogenic abilities of CB CD34+TEK+ and CD34+TEK- cells by using a clonogenic assay and xenotransplantation into immunodeficient NOD/SCID mice. The results showed that colony-forming cells and cells capable of repopulating in NOD/SCID mice were present in both CD34+TEK+ and CD34+TEK- cells and that the hematopoietic activities of the cell types were similar. In contrast, the potential to differentiate into endothelial cells in vivo was greater in the CD34+TEK+ cells. All NOD/SCID mice engrafted with CD34+TEK+ cells had human CD31-expressing and VE-cadherin-expressing endothelial cells in the vessels of the ischemic muscles and/ or human endothelial cells expressing CD31, kinase-insert domain-containing receptor, and endothelial nitric oxide synthase in liver sinusoidal cells, whereas such endothelial cells were detected in only 3 of the 7 recipients engrafted with CD34+TEK- cells. This result has important implications in cell therapy using CB cells for treating hematopoietic disorders and vascular diseases.     

Effects of anti-CD33 blocked ricin immunotoxin on the capacity of CD34+ human marrow cells to establish in vitro hematopoiesis in long-term marrow cultures.

Human marrow cells that express the CD34 antigen but lack CD33 are able to initiate sustained, multilineage in vitro hematopoiesis in long-term Dexter cultures and are believed to include the primitive stem cells responsible for effecting long-term hematopoietic reconstitution in vivo following marrow transplantation. In studies described in this report we investigated the effects of a novel anti-CD33 immunotoxin on the clonogenic potential of normal human CD34+ marrow cells and on the ability of these cells to initiate hematopoiesis in two-stage Dexter cultures (long-term marrow cultures, LTMC). This immunotoxin (anti-CD33-bR), shown previously to kill both clonogenic myelogenous leukemia cells and normal mature myeloid progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM), consists of an anti-CD33 monoclonal antibody conjugated to purified ricin that has been modified by blocking the carbohydrate binding domains of the ricin B-chain to eliminate nonspecific binding. For our studies, normal CD34+ human marrow cells were isolated from the light-density (less than 1.070 g/ml) cells of aspirated marrow by positive selection with immunomagnetic beads linked to the monoclonal antibody K6.1. These cell isolates were highly enriched with both multipotential and lineage-restricted clonogenic, hematopoietic progenitors (mixed lineage colony-forming units, CFU-Mix; CFU-GM; and erythroid burst-forming units, BFU-E) which constituted greater than or equal to 20% of the cells. Recovery of clonogenic progenitors from these CD34+ cell preparations, following treatment with anti-CD33-bR (10 nM), was reduced by greater than or equal to 85% for CFU-GM and 20%-40% for CFU-Mix and BFU-E. However, the capacity of these cells to initiate hematopoietic LTMC was preserved. Indeed, the production of high proliferative potential (HPP) CFU-GM, BFU-E, and CFU-Mix in cultures seeded with 10(5) anti-CD33-bR-treated CD34+ marrow cells was substantially greater than that observed in LTMC seeded with equivalent numbers of untreated CD34+ cells. Moreover, concentrations of long-term culture initiating cells in CD34+ cell isolates, quantified by a limiting dilution technique, were found to be increased following anti-CD33-bR treatment. These findings support the potential usefulness of anti-CD33-bR for in vitro marrow purging or in vivo treatment to eliminate CD33+ leukemic clones, while sparing normal CD34+/CD33- stem cells that support normal hematopoiesis and hematopoietic reconstitution in vivo.     

Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development

We elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac-like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis.     

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 defects in mice lacking the sialomucin CD34

Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34- null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34- null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.     

Erythropoiesis from acetyl LDL incorporating endothelial cells at the preliver stage

Erythropoiesis is characterized by 2 waves of production during mouse embryogenesis: a primitive one originating from the yolk sac (YS) and a definitive one produced from both the YS and the embryo proper. How the latter wave is generated remains unclear. To investigate our hypothesis that endothelial cells (ECs) could generate erythroid cells, we designed a method to label ECs at 10 days after coitus. This labeling method associates 2 techniques: an intracardiac inoculation that allows molecules to be delivered into the bloodstream followed by a whole-embryo culture period. DiI-conjugated acetylated low-density lipoproteins (Ac-LDL-DiI) were used to specifically tag ECs from the inside. One hour after inoculation, DiI staining was found along the entire endothelial tree. Fluorescence-activated cell sorter (FACS) analysis revealed that DiI+ cells were CD31+, CD34+, and CD45-, an antigen makeup characteristic of the endothelial lineage. Twelve hours after inoculation, 43% of DiI+ circulating cells belonged to the erythroid lineage. These cells expressed Ter119 and displayed an adult globin chain arrangement; thus they belonged to the definitive lineage as confirmed in erythroid colony formation. The remaining cells likely represent committed white blood cells or multipotent progenitors, as revealed by a mixed-colony formation. Beyond the 29-somite stage, the proportion of DiI+ erythroid cells gradually decreased. These results demonstrate the generation of hematopoietic cells from an endothelial intermediate, using in vivo tracing. We provide evidence for a release of these cells into the circulation and hypothesize that these cells are able to colonize the fetal liver and generate definitive erythrocytes in vivo.     

Granulocytic and monocytic differentiation of CD34hi cells is associated with distinct changes in the expression of the PU.1- regulated molecules, CD64 and macrophage colony-stimulating factor receptor

The present study investigated the possibility that macrophage colony- stimulating factor (M-CSF) responsiveness of hematopoietic progenitor cells is regulated at the level of receptor expression and that M-CSF receptor (M-CSFR) may be used as an early marker of monocyte lineage commitment. Immunofluorescence measurements with an anti-M-CSFR antibody showed that 44% +/- 5% of CD34hi cells expressed the receptor. The M-CSFR was present on progenitor cells that were positive for the granulo-monocytic marker CD64, but not on primitive, erythroid, or lymphoid progenitors. The CD34hiCD64+ population could be divided into subsets of M-CSFRhi and M-CSFRlo cells. In addition, a subset of CD34hiCD64-M-CSFRhi cells was found. CD34+ cells that were positive for M-CSFR, CD64, or both gave rise exclusively to granulo-monocytic cells, and 65% of the granulomonocytic colony-forming cells in the CD34+ population were recovered from these cells. Approximately 70% of the colony-forming cells (CFCs) derived from CD34hiM-CSFRhi cells were macrophage colony-forming units (CFU-M), whereas 91% of the CFCs in the CD34hiCD64+M-CSFRlo population were granulocyte colony-forming units (CFU-G). The M-CSFRhi cells with the highest frequency of colony- forming and bipotent cells and largest average colony size were found in the CD64- subset, indicating that M-CSFR appears earlier than CD64 during monocyte development. After 60 hours in culture, a subset of the CD34hiM-CSFRhi cells had downmodulated M-CSFR (29% to 38%). This population gave rise almost exclusively to granulocytes, whereas the cells that remained M-CSFRhi gave rise exclusively to monocytes. In all experiments, the M-CSFRhi population responded to M-CSF, whereas minimal responses were observed among M-CSFRlo cells. These results suggest that M-CSF target specificity among human hematopoietic progenitor cells is determined by lineage-specific regulation of the M- CSFR and show that M-CSFR is a useful marker to discriminate between monocytic and granulocytic progenitor cells.     

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

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

CD34+ endothelial cell lines derived from murine yolk sac induce the proliferation and differentiation of yolk sac CD34+ hematopoietic progenitors

Embryonic hematopoiesis is initiated in part in the blood islands of the yolk sac. Previous confocal microscopic analysis has shown that the CD34 antigen, a mucin-like cell surface glycoprotein that is expressed by hematopoietic progenitors and all endothelial cells of the adult and embryo, is also found on a subset of luminal hematopoietic-like cells in the yolk sac blood islands as well as on the vascular endothelium lining these early hematopoietic locations. We show here that, as in all other hematopoietic sites thus far examined, immunoaffinity- purified CD34+ nonadherent cells from murine yolk sacs contain the vast majority of erythroid and myeloid progenitor cell colony forming activity. To examine the developmental interactions between these CD34+ hematopoietic progenitor cells of the yolk sac and the CD34+ yolk sac endothelium, we have immunaffinity-purified adherent endothelial cells from day 10.5 yolk sacs using CD34 antiserum and produced cell lines by transformation with a retrovirus expressing the polyoma middle T antigen. Analysis of these cell lines for CD34, von Willebrand's factor, FLK 1 and FLT 1 expression, and capillary growth in Matrigel indicates that they appear to be endothelial cells, consistent with their original phenotype in vivo. Coculture of yolk sac CD34+ hematopoietic cells on these endothelial cell lines results in up to a 60-fold increase in total hematopoietic cell number after approximately 8 days. Analysis of these expanded hematopoietic cells showed that the majority were of the monocyte/macrophage lineage. In addition, examination of the cultures showed the rapid formation of numerous cobblestone areas, a previously described morphologic entity thought to be representative of early pluripotential stem cells. Scrutiny of the ability of these endothelial cell lines to expand committed progenitor cells showed up to a sixfold increase in erythroid and myeloid colony- forming cells after 3 to 6 days in culture, consistent with the notion that these embryonic endothelial cells mediate the expansion of these precursor cells. Polymerase chain reaction analyses showed that most of the cell lines produce FLK-2/FLT-3 ligand, stem cell factor, macrophage colony-stimulating factor, leukemia-inhibitory factor, and interleukin- 6 (IL-6), whereas there is a generally low or not measurable production of granulocyte colony-stimulating factor, granulocyte-macrophage colony- stimulating factor, IL-1, IL-3, transforming growth factor beta-1, erythropoietin, or thrombopoietin. The output of mature hematopoietic cells from these cocultures can be modified to include an erythroid population by the addition of exogenous erythropoietin. These data suggest that endothelial cell lines derived form the yolk sac provide an appropriate hematopoietic environment for the expansion and differentiation of yolk sac progenitor cells into at least the myeloid and erythroid lineages.     

PECAM-1 is expressed on hematopoietic stem cells throughout ontogeny and identifies a population of erythroid progenitors

Platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31) is an adhesion molecule expressed on endothelial cells and subsets of leukocytes. Analysis of phenotypically defined hematopoietic stem cells (HSCs) from the yolk sac, fetal liver, and adult bone marrow demonstrates CD31 expression on these cells throughout development. CD31+ c-kit+ cells, but not CD31- c-kit+ cells, isolated from day-9.5 yolk sac give rise to multilineage hematopoiesis in vivo. Further evaluation of the CD31+ lineage marker-negative fraction of adult bone marrow reveals functionally distinct cell subsets. Transplantation of CD31+ Lin- c-kit- cells fails to protect lethally irradiated recipients, while CD31+ Lin- c-kit+ Sca-1- cells (CD31+ Sca-1-) provide radioprotection in the absence of long-term donor-derived hematopoiesis. Although donor-derived leukocytes were not detected in CD31+ Sca-1- recipients, donor-derived erythroid cells were transiently produced during the initial phases of bone marrow recovery. These results demonstrate CD31 expression on hematopoietic stem cells throughout ontogeny and identify a population of CD31+ short-term erythroid progenitors cells that confer protection from lethal doses of radiation.     

Searching for common stem cells of the hepatic and hematopoietic systems in the human fetal liver: CD34+ cytokeratin 7/8+ cells express markers for stellate cells

BACKGROUND/AIMS: The hematopoietic and hepatic systems are intertwined in the liver during fetal life. Cells expressing the hematopoietic stem cell marker CD34 and cytokeratin 7/8 (CK7/8) are hypothesized to be common stem cells for the hematopoietic and hepatic systems. Our aim was to determine if human fetal liver cells expressing CD34 and CK7/8 represent a common stem cell for both the hematopoietic and hepatic systems. METHODS: CD34+CK7/8+ cells from midgestation livers were analyzed for the expression of various markers by flow cytometry and isolated based on their expression of CD34, nerve growth factor receptor (NGFR) and lack of CD45 expression. CD34+CD38- hematopoietic stem cells were also isolated and cultured in the presence of various hepatopoietins. RESULTS: CD34+CK7/8+ cells comprised 3.4-8.5% of the erythrocyte-depleted liver. CD34+CK7/8+ cells had unique light-scatter properties compared to hematopoietic precursors and did not express most markers associated with hematopoietic cells. They did stain with CD13, CD59, NGFR, desmin and alpha-smooth muscle actin. In culture, these cells had a stellate appearance. Cultured hematopoietic stem cells failed to generate hepatocytes. CONCLUSIONS: CD34+CK7/8+ cells are not common stem cells but rather appear to be hepatic stellate cells. A link between the hematopoietic and hepatic systems during fetal life requires further investigation.     

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

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

Study of early hematopoietic precursors in human cord blood.

Human cord blood is a source of transplantable stem cells. These stem cells express the antigen CD34, are resistant to treatment with 4-hydroperoxycyclophosphamide (CD34+/4-HCres), and do not give rise to colonies when plated in clonogenic assays. We studied the number of CD34+ cells present in cord blood and developed a two-step assay for early precursors (pre-colony-forming units, pre-CFU) capable of giving rise to committed progenitors. In this assay CD34+/4-HCres cord blood cells were cultured in suspension with different growth factors. After 7 days in suspension the remaining cells were plated in clonogenic assays, for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and mixed lineage colony-forming units (CFU-MIX), in the presence of pure factors or a combination of recombinant human (rh) interleukin 3 (IL-3) and medium conditioned by the PU34 primate cell line. Pre-CFU for all precursors were identified. These pre-CFU developed into committed progenitors in response to rhIL-3. The combinations of rhIL-3 plus rh interleukin 1 (IL-1) or rhIL-3 plus rh interleukin 6 (IL-6) did not enhance recovery of progenitors. The developing CFU-GM were responsive to rh granulocyte-macrophage colony-stimulating factor (GM-CSF) and rh granulocyte colony-stimulating factor (G-CSF) but much less so to rhIL-3. BFU-E and CFU-MIX developed in suspension but could only be detected when cells were replated in the presence of a combination of rhIL-3 and PU34 but not rhIL-3 alone. This assay may be useful in evaluating the number of early hematopoietic precursors present in cord blood samples and in defining growth factor combinations that could enhance hematopoietic recovery after cord blood stem cell transplants.     

Hemangioblastic characteristics of fetal bone marrow-derived Flk1(+)CD31(-)CD34(-) cells

OBJECTIVE: To investigate whether Flk1(+)CD31(-)CD34(-) cells isolated from fetal bone marrow (BM) have characteristics of hemangioblasts, i.e., progenitors of endothelial and hematopoietic cells. MATERIALS AND METHODS: Mononuclear cells from fetal BM were negatively sorted by CD45, GlyA, and CD34 micromagnetic beads, then cultured to form cell colonies. A single colony was harvested. Culture-expanded cells were seeded on ECM gel or semisolid media supplemented with endothelial and hematopoietic growth factors, respectively. Immunochemistry staining and RT-PCR were performed for cell characterization. RESULTS: 99% of cells from the single colony maintained Flk1(+) and CD31/CD34(-) during passaging. On ECM gel, Flk1(+)CD31(-)CD34(-) cells could grow into vascular structure that was positive for CD31 and vWF. There were round CD34(+) cells around the vascular structure. When angiogenesis inhibitor suramin was added before tube formation, formation of vascular structure was blocked. Additionally, Flk1(+)CD31(-)CD34(-) cells cultured on hematopoietic condition could differentiate into hematopoietic cells which expressed GATA-1, 2, and gamma, beta globin gene. After being replated in methylcellulose medium, they formed typical erythroid colonies. CONCLUSIONS: Flk1(+)CD31(-)CD34(-) cells derived from fetal BM could differentiate into endothelial and hematopoietic cells. The results suggested that these Flk1(+)CD31(-)CD34(-) cells after embryo stage bear characteristics of hemangioblast and may have potential application for the hematopoietic and vascular diseases.     

Expression of Fas/CD95 and Bcl-2 by primitive hematopoietic progenitors freshly isolated from human fetal liver

The cell-surface expression and the functional status of the CD95/Fas antigen on primitive hematopoietic progenitors (PHPs) freshly isolated from human fetal liver (FL) were studied. PHPs were phenotypically defined as CD34++ CD38 -/+ cells. The most immature subfractions of PHPs, CD34++CD38- and CD34+2CD38+ FL cells, expressed CD95, whereas the more mature CD34++CD38++ and CD34+CD38++2 FL cells displayed low CD95 expression. Combinations of cytokines, such as kit ligand (KL) + interleukin-3 or KL + granulocyte-macrophage colony-stimulating factor (GM-CSF) upregulated the expression of CD95 on PHPs upon in vitro culture. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) further increased the CD95 expression induced by KL+GM-CSF. The hematopoietic potential of sorted CD34++lineage (lin)- CD95+ versus CD34++ lin-CD95-FL cells was compared by colony-forming unit-culture (CFU-C) assays performed in serum-deprived medium. Lin+ cells were composed of erythrocytes, monocytes, T cells, B cells, and natural killer cells. Our results indicated that both CD95- and CD95+ subsets contained pluripotent progenitors, generating myeloid and erythroid progenitors. The functional status of CD95 and the effects of TNF-alpha and IFN-gamma, cytokines known to induce CD95-mediated apoptosis, were analyzed by incubation of PHPs in the presence of anti-CD95 monoclonal antibodies (MoAbs). The effect of anti-CD95 MoAbs was measured by viable cell counting, flow cytometry, and CFU-C assays. A decrease of CFU-C numbers was observed in the presence of anti-CD95 MoAbs and TNF- alpha and/or IFN-gamma. However, whereas growth factor deprivation induced apoptosis of PHPs, cross-linking of CD95 did not lead to apoptosis of PHPs measured by flow cytometry and viable cell counting. The correlation of increased intracytoplasmic levels of bcl-2 with high levels of cell-surface CD34 and the presence of CD95 on fresh FL cells suggests that bcl-2 may be involved in protecting against CD95-mediated apoptosis of FL PHPs.     

Clinical paroxysmal nocturnal hemoglobinuria is the result of expansion of glycosyl-phosphatidyl-inositol-anchored protein-deficient clone in the condition of Deficient Hematopoiesis

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired, clonal hematopoietic stem cell disorder in which PIG-A, gene essential for the biosynthesis of the glycosyl-phosphatidyl-inositol (GPI) anchor, is somatically mutated. Absence of GPI-linked proteins from the surface of blood cells is characteristic of the PIG-A mutant (PNH) clone and is also accountable fo certain manifestations, such as intravascular hemolysis. It is unclear how the PNH clone expands and comes to dominate hematopoiesis. In this study, CD34+ cells--committed progenitors (colony-forming cells) representing immature hematopoietic stem cells--and reticulocytes representing the differentiated erythroid cells were quantitated in peripheral blood of patients with PNH. Compared with normal controls (n = 29), CD34+ cell levels were significantly lower in PNH patients who did not have preexisting aplastic anemia (AA) (n = 12) (2.47+/-1.23 versus 4.68+/-1.05 x 106/L, mean +/- standard error; P = .022). PNH patients with precedent aplastic anemia (AA+/PNH) showed markedly low CD34+ cell levels compared with normal control subjects (0.6+/-0.29 versus 4.68+/-1.05 x 10(6)/L; P = .0001). In addition, colony-forming cells from PNH patients were significantly decreased compared with those from normal volunteers (erythroid burst-forming units, 2.8+/-1.2 versu 25.6+/-6.2/5 x 10(5) mononuclear cells; P = .0006; and granulocyte/macrophage colony-forming units, 1.2+/-0.5 versus 13.3+/-3.0/ 5 x 10(5) mononuclear cells; P = .0006). These findings occur in both aplastic and hemolytic types of PNH, suggesting hematopoietic failure in PNH. On the contrary, the numbers of reticulocytes and the reticulocyte production index of PNH patients were significantly higher than those of normal persons and comparable to those from patients with autoimmune hemolytic anemia, indicating accelerating erythropoiesis in PNH. The degree of reticulocytosis correlated well with the proportion of CD59- (PNH) reticulocytes. All of the findings suggest that in the condition of deficient hematopoiesis, the PNH clone arising from the mutated hematopoietic stem cell expands and maintains a substantial proportion of the patient's hematopoiesis.     

Novel Method for Efficient Production of Multipotential Hematopoietic Progenitors from Human Embryonic Stem Cells

We propose a novel method for the efficient production of hematopoietic progenitors from human embryonic stem cells (hESC) via coculture with murine fetal liver-derived stromal cells, in which embryonic hematopoiesis dramatically expands at midgestation. We generated various hematopoietic progenitors in coculture, and this hematopoietic activity was concentrated in cobblestone-like cells derived from differentiated hESC. The cobblestone-like cells mostly expressed CD34 and retained an endothelial cell potential. They also contained hematopoietic colony-forming cells, especially erythroid and multilineage colony-forming cells at high frequency. The multipotential hematopoietic progenitors abundant among the cobblestone-like cells produced almost all types of mature blood cells, including adult-type alpha-globin-expressing erythrocytes and tryptase/chymase double-positive mast cells. These progenitors showed neither the immature properties of ESC nor the potential to differentiate into endoderm and ectoderm at a clonal level. The coculture system developed for hESC can provide a novel source of hematopoietic and blood cells for applications in cellular therapy and drug screening.     

CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells.

CD109 is a monomeric cell surface glycoprotein of 170 kD that is expressed on endothelial cells, activated but not resting T-lymphocytes, activated but not resting platelets, leukemic megakaryoblasts, and a subpopulation of bone marrow CD34+ cells. Observing an apparent association between CD109 expression and the megakaryocyte lineage (MK), we sought to determine whether CD109 was expressed on MK progenitors. In fetal bone marrow (FBM), a rich source of MK progenitors, CD109 is expressed on a mean of 11% of CD34- cells. Fluorescence activated cell sorting (FACS) of FBM CD34+ cells into CD109+ and CD109- fractions revealed that the CD34+CD109+ subset contained virtually all assayable MK progenitors, including the colony-forming unit-MK (CFU-MK) and the more primitive burst-forming unit-MK (BFU-MK). The CD34+CD109+ subset also contained all the assayable burst-forming units-erythroid (BFU-E), 90% of the colony-forming units-granulocyte/macrophage (CFU-GM), and all of the more primitive mixed lineage colony-forming units (CFU-mix). In contrast, phenotypic analysis of the CD34+CD109- cells in FBM, adult bone marrow (ABM) and cytokine-mobilized peripheral blood (MPB) demonstrated that this subset comprises lymphoid-committed progenitors, predominantly of the B-cell lineage. CD109 was expressed on the brightest CD34 cells identifiable not only in FBM, but also in ABM and MPB indicating that the most primitive, candidate hematopoietic stem cells (HSC) might also be contained in the CD109+ subset. In long-term marrow cultures of FBM CD34+ cells, all assayable cobblestone area forming cell (CAFC) activity was contained within the CD109+ cell subset. Further phenotypic analysis of the CD34+CD109+ fraction in ABM indicated that this subset included candidate HSCs that stain poorly with CD38, but express Thy-1 (CD90) and AC133 antigens, and efflux the mitochondrial dye Rhodamine 123 (Rho123). When selected CD34+ cells were sorted for CD109 expression and Rho123 staining, virtually all CAFC activity was found in the CD109+ fraction that stained most poorly with Rho123. CD34+ cells were also sorted into Thy-1 CD109+ and Thy-1 CD109+ fractions and virtually all the CAFC activity was found in the Thy-1+CD109+ subset. In contrast, the Thy-1-CD109+ fraction contained most of the short-term colony-forming cell (CFC) activity. CD109, therefore, is an antigen expressed on a subset of CD34+ cells that includes pluripotent HSCs as well as all classes of MK and myelo-erythroid progenitors. In combination with Thy-1, CD109 can be used to identify and separate myelo-erythroid and all classes of MK progenitors from candidate HSCs.     

All-trans retinoic acid prevents apoptosis of human marrow CD34+ cells deprived of haematopoietic growth factors

The regulation of apoptosis plays a key role in haematopoiesis. It has been demonstrated that haematopoietic progenitor cells progressively undergo apoptotic cell death in the absence of appropriate growth factors. We studied the effects of pharmacological doses of all-transretinoic acid (ATRA) on the apoptosis of human adult marrow CD34+ progenitor cells cultured for 7 d in a serum-free medium. We quantified CD34+ cells, clonogenic progenitors and 5 week colony-forming cells (CFC) before and after ATRA exposure. Moreover, we defined the apoptotic status of the CD34+ cell fraction by analysis of phosphatidylserine externalization (using annexin V), the relative membrane permeability to 7-aminoactinomycin D (7AAD) and the mitochondrial membrane potential [using 3,3'-dihexyloxacarbocyanine iodide, DiOC6(3)]. In the drastic experimental conditions used, a decrease in viable CD34+ cells, granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E) and 5 week CFC were observed. Exposure to ATRA partially prevented the decrease in viable CD34+, without a concomitant effect on the clonogenic and more immature progenitors. ATRA-treated CD34+ cells displayed changes in apoptotic status compared with control cultures, particularly in lower annexin V-binding. These results were confirmed using 7AAD and DiOC6(3). Our results demonstrate that ATRA exerts a protective effect on CD34+ cells exposed to such apoptotic stress.