Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3784-3793 ◽  
Author(s):  
Ryan Reca ◽  
Dimitrios Mastellos ◽  
Marcin Majka ◽  
Leah Marquez ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Calcium Flux ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Wide Range ◽  
Normal Human ◽  
Anaphylatoxin C3a

Abstract Complement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34+ cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell–derived factor 1 (SDF-1)–dependent chemotaxis of human CD34+ cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1–dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)–mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1+ cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF–CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

Platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 3143-3149 ◽  
Author(s):  
Anna Janowska-Wieczorek ◽  
Marcin Majka ◽  
Jacek Kijowski ◽  
Monika Baj-Krzyworzeka ◽  
Ryan Reca ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Clinical Implications ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Platelet Binding ◽  
Mobilized Peripheral Blood

Abstract Because human CD34+ and murine Sca-1+hematopoietic stem–progenitor cells (HSPCs) express platelet-binding sialomucin P-selectin (CD162) and integrin Mac-1 (CD11b–CD18) antigen, it was inferred that these cells might interact with platelets. As a result of this interaction, microparticles derived from platelets (PMPs) may transfer many platelet antigens (CD41, CD61, CD62, CXCR4, PAR-1) to the surfaces of HSPCs. To determine the biologic significance of the presence of PMPs on human CD34+ and murine Sca-1+ cells, their expressions on mobilized peripheral blood (mPB) and on nonmobilized PB- and bone marrow (BM)–derived CD34+ cells were compared. In addition, the effects of PMPs on the proliferation of CD34+ and Sca-1+ cells and on adhesion of HSPCs to endothelium and immobilized SDF-1 were studied. Finally, the hematopoietic reconstitution of lethally irradiated mice receiving transplanted BM mononuclear cells covered or not covered with PMPs was examined. It was found that PMPs are more numerous on mPB than on BM CD34+cells, do not affect the clonogenicity of human and murine HSPCs, and increase adhesion of these cells to endothelium and immobilized SDF-1. Moreover, murine BM cells covered with PMPs engrafted lethally irradiated mice significantly faster than those not covered, indicating that PMPs play an important role in the homing of HSPCs. This could explain why in a clinical setting human mPB HSPCs (densely covered with PMPs) engraft more rapidly than BM HSPCs (covered with fewer PMPs). These findings indicate a new role for PMPs in stem cell transplantation and may have clinical implications for the optimization of transplantations.

Neuropeptides Orexin A and B Are Funktionally Aktive in CD34+ Hematopoietic Stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4593-4593
Author(s):  
Ron-Patrick Cadeddu ◽  
Akos G. Czibere ◽  
Sebastian Büst ◽  
Johannes C Fischer ◽  
Ulrich Steidl ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Calcium Influx ◽  
Receptor Stimulation ◽  
Orexin A ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Orexin Receptors ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Abstract 4593 Orexin receptors are involved in the regulation of sleep-wake-rhythm, food intake and energy homeostasis and it was still recently believed that their expression is restricted to the nervous system. But, during the last years orexin receptors have been detected in an increasing number of peripheral tissues. We have earlier found orexin receptor 1 and 2 expression on human CD34+ hematopoietic stem and progenitor cells. Still, the sources of their physiological ligands, the peptides orexin A and B, seemed so far to be restricted to the central nerve system. Ca2+-dependent signaling and activation of mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (ERK1/2) pathways are considered as main downstream signaling pathways of the orexin receptors. In this study, we investigated the signaling and functional role of orexin receptors in CD34+ hematopoietic stem and progenitor cells. Using confocal fluorescence microscopy and flow cytometry we found that stimulation of purified CD34+ cells with orexin A and B led to an increase of the intracellular calcium concentration due to both calcium influx and calcium release from intracellular stores. Of interest, incubation with orexin reduces the SDF-1β-induced calcium influx. Furthermore orexin receptor stimulation led to a decrease of the intracellular cAMP concentration. Following orexin receptor stimulation with orexin A and B, we observed an initial increase of ERK1/2 phosphorylation up to 30 minutes upon incubation with orexin followed by a decrease at several time points up to 8 hours in comparison to the unstimulated control. To investigate a potential impact on the functional properties of human CD34+ cells we performed proliferation and apoptosis assays, migration and adhesion assays as well as colony forming and long-term culture assays. Remarkably, stimulation with orexin A and B led to a significant higher proportion of early pluripotent hematopoietic progenitor (CFU-GEMM) colonies and a significant reduction of erythroid precursors. A more immature phenotype of orexin-stimulated CD34+ cells is also reflected by array-based gene expression profiling. Long-term culture assays revealed a significant higher frequency of LTC-IC indicating also a more immature phenotype of orexin-stimulated cells. In line, orexin receptor stimulation led to a significant increase of the proportion of Lin-, CD34+, CD38- HSC in the G0-phase of the cell cycle. Furthermore, stimulation with orexin A and B increased the number of apoptotic cells in the Lin-, CD34+, CD38- HSC fraction and the total hematopoietic stem and progenitor population determined by flowcytometric analysis of intracellular cleaved caspase 3 content. The adhesive capacity of CD34+ cells to fibronectin and collagen coated dishes and the migratory capacity was significantly decreased upon orexin receptor stimulation. Concurrent incubation with the selective Gi-protein inhibitor pertussis toxin abrogated these effects. Given the functional impact of the orexin system on CD34+ cells, we asked if orexins are secreted locally in the bone marrow or autocrine by CD34+ cells or if they are humorally transported to the bone marrow cavity. Using FACS analysis, immunfluorescent staining and western blotting we could detect prepro-Orexin in CD34+ cells and using ELISA orexin was found in the serum obtained by bone marrow biopsies and peripheral blood. Taken together, the phenotype of orexin-stimulated hematopoietic stem and progenitor cells suggest a mobilizing effect of the orexin receptor stimulation as well as an increased repopulation capacity which might be of relevance in clinical stem cell mobilization and transplantation and is currently verified in murine models. Disclosures: No relevant conflicts of interest to declare.

R4 Rgs Subfamily Proteins Negatively Regulates SDF-1/CXCR4 Signaling in CD34+ Hematopoietic Stem and Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5048-5048
Author(s):  
Kam Tong Leung ◽  
Yorky Tsin Sik Wong ◽  
Karen Li ◽  
Kathy Yuen Yee Chan ◽  
Xiao-Bing Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Rgs Proteins ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract RGS family proteins are known to negatively regulate G-protein-coupled receptor signaling through their GTPase-accelerating activity. In several types of hematopoietic cells (e.g., B lymphocytes and megakaryocytes), responses to stromal cell-derived factor-1 (SDF-1) are subjected to regulation by R4 subfamily RGS proteins. However, their expression patterns and functional roles in hematopoietic stem and progenitor cells (HSC) are poorly characterized. Here, we showed that human CD34+ HSC derived from cord blood (CB, n = 10) expressed 7 out of 10 R4 RGS proteins at mRNA level (RGS1-3, 5, 13, 16 and 18), whereas expressions of RGS4, 8 and 21 were undetectable. Exposure of CB CD34+ cells to SDF-1 significantly increased RGS1, 2, 13 and 16 expressions and decreased RGS3 and 18 expressions (P ≤ 0.0402, n = 5). Expressions of RGS1, 13 and 16 were significantly higher in bone marrow (BM, n = 10) CD34+ cells when compared to mobilized peripheral blood (MPB, n = 5) CD34+ cells (P ≤ 0.0160), while RGS3 and 18 expressions were lower in BM CD34+ cells (P ≤ 0.0471), suggesting a SDF-1- and niche-dependent regulation of RGS expressions. To investigate the potential involvement of RGS proteins in SDF-1-mediated homing-related functions, we introduced RGS overexpression constructs into CB CD34+ cells by lentiviral transduction. With >80% transduction efficiency, we showed that overexpression of RGS1, 13 and 16 but not RGS2 significantly inhibited migration of CD34+ cells to a SDF-1 gradient (P ≤ 0.0391, n = 4-5). Similarly, RGS1, 13 and 16 overexpression suppressed SDF-1-induced Akt phosphorylation (n = 2), but none of them affected SDF-1-mediated actin polymerization (n = 3). In the NOD/SCID mouse xenotransplantation model, preliminary results showed that bone marrow homing was impaired in RGS1- (16.3% reduction), RGS13- (12.7% reduction) or RGS16-overexpressing CD34+ cells (33.7% reduction). Taken together, we provided the first evidence that expressions of R4 RGS proteins are regulated by the SDF-1/CXCR4 axis in human CD34+ HSC. We also presented evidence that specific R4 RGS proteins (RGS1, 13 and 16) negatively regulate in vitro SDF-1-mediated responses and in vivo homing of CD34+ cells, suggesting that RGS proteins may serve as a feedback mechanism to regulate SDF-1/CXCR4 signaling. Strategies to inhibit RGS signaling could thus be a potential method for enhancing efficiency of HSC homing and long-term engraftment, which is particularly important in the setting of CB transplantation. Disclosures No relevant conflicts of interest to declare.

scholarly journals R4 Rgs Subfamily Proteins Suppress Engraftment and Regulate SDF-1/CXCR4 Signaling in Human CD34+ Hematopoietic Stem/Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1927-1927
Author(s):  
Kathy Chan ◽  
Tsin Sik Wong ◽  
Karen Li ◽  
Xiao-Bing Zhang ◽  
Ronald Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Actin Polymerization ◽  
Conflicts Of Interest ◽  
Calcium Flux ◽  
Rgs Proteins ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Stat3 Phosphorylation ◽  
Cd34 Cells

Members of the Regulators of G-protein Signaling (RGS) are GTPase-accelerating proteins and have been implicated in SDF-1-directed trafficking of mature hematopoietic cells. However, their roles in hematopoietic stem and progenitor cells (HSPC) remain largely unknown. In this study, we investigated the expression, functions and mechanism of R4 RGS subfamily members on migration and engraftment of human HSPC. Our results demonstrated that cord blood (CB), bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells expressed specific RGS mRNAs, of which RGS1, RGS2, RGS13 and RGS16 were significantly upregulated by SDF-1 (1.6-1.9 fold, n=5, P<0.05). In the presence of AMD3100, a CXCR4 inhibitor, the stimulating effects of SDF-1 on RGS expression were completely abolished (n=6). SDF-1-directed functions (chemotaxis, trans-matrigel migration and calcium flux) and signaling (phosphorylation of Akt, ERK and Stat3) were significantly inhibited in RGS1, RGS13 and RGS16-overexpressing CD34+ cells (n=4-6, P<0.05) but not in RGS2-overexpressing cells, whereas actin polymerization, adhesion and colony formation were unaffected by these RGS members. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1, RGS13 or RGS16 in CD34+ cells did not impact their short-term homing but substantially compromised their long-term engraftment efficiency in bone marrow and spleens of recipient mice by 91.4%, 83.7% and 71.2%, respectively (n= 8-9; P<0.05). Genome-wide expression microarray and qPCR validation identified 32 common differentially expressed genes (1 upregulated and 31 downregulated) in RGS1, RGS13 or RGS16-overexpressing CD34+ cells. Network analysis revealed the potential mechanisms of RGS1, RGS13 and RGS16 downstream of SDF1/CXCR4 and Gαi protein, leading to compromised Akt, ERK and Stat3 phosphorylation and negative regulation of stem cell functions (CCNA1, SPP1, LPAR5, IL1RL1, HPSE), complement activation (C3AR1, C5AR2, C5AR1), proteolysis (TIMP3, MMP14) and cell migration (THBS1, F2RL2, PROS1, CCL1). Our results highlight the unprecedented functions of R4 RGS proteins in HSPC migration and engraftment, and provide the foundation of future design of RGS-targeting strategies to enhance the efficiency of clinical HSPC transplantation. Disclosures No relevant conflicts of interest to declare.

Superiority of 3-D Hematopoietic Stem Cell (HSC) & Stroma Cell Co-Cultures in Supporting the Proliferation of Transplantable HSC.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4260-4260
Author(s):  
Shai Meretzki ◽  
Avinoam Kadouri ◽  
Dov Zipori ◽  
Ora Berger ◽  
Jacob M. Rowe
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Cell Cultures ◽  
High Density ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Stroma Cell ◽  
Cd34 Cells ◽  
Wide Range

Abstract Stem cells are intimately associated in-vivo with discrete niches within the 3-dimensional (3-D) bone marrow micro-environment collectively provide molecular signals that mediate their differentiation and self-renewal. A recently developed Plug-Flow Bioreactor System (PluriXTM), which closely mimics the physiological bone marrow environment by combining 3-D stromal cultures with a continuous flow system, is capable of supporting long term maintenance/expansion of transplantable human HSC. The stroma cells, growing inside the PluriXTM bioreactor on porrosive carriers enable the propagation of high density (>100-fold input) 3-D stroma cell cultures for prolonged periods of time. Within 40 days, the number of primary human stroma cells seeded in the bioreactor at a density of 5*104 cells/ml reached 1.5*106 cells/ml and the stroma cell lines reached a density of 5*107–1*108 cells/ml. Three-dimensional static stroma HSC co-cultures were found to be superior to 2-D co-cultures in supporting the maintenance of CB HSC and early hematopoietic progenitors. When cord blood CD34+ cells were seeded on 2-D & 3-D stroma cell static cultures, the 3-D stroma cultures proved superior in supporting the growth of CD34+, CD34+38− and CD34+38−CXCR4+ cells. The 3-D primary human stroma static cultures supported these cells 4.7, 22, 7.4 fold better than the 2-D cultures, respectively. The CD34+ cells seeded on the high-density 3-D stroma cell cultures within the PluriXTM bioreactor demonstrated significant superiority to 2-D and 3-D stroma cell static cultures and 2-D stroma cultures in supporting the growth and expansion of HSC and earlier hematopoietic progenitors. These findings verify that a system based on 3-D stroma co-cultures is preferable for ex-vivo expansion of human HSC’s. The PluriXTM system offers wide-range potential for expansion of stem cells from cord blood and production of specific blood products.

Platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 3143-3149
Author(s):  
Anna Janowska-Wieczorek ◽  
Marcin Majka ◽  
Jacek Kijowski ◽  
Monika Baj-Krzyworzeka ◽  
Ryan Reca ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Clinical Implications ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Platelet Binding ◽  
Mobilized Peripheral Blood

Because human CD34+ and murine Sca-1+hematopoietic stem–progenitor cells (HSPCs) express platelet-binding sialomucin P-selectin (CD162) and integrin Mac-1 (CD11b–CD18) antigen, it was inferred that these cells might interact with platelets. As a result of this interaction, microparticles derived from platelets (PMPs) may transfer many platelet antigens (CD41, CD61, CD62, CXCR4, PAR-1) to the surfaces of HSPCs. To determine the biologic significance of the presence of PMPs on human CD34+ and murine Sca-1+ cells, their expressions on mobilized peripheral blood (mPB) and on nonmobilized PB- and bone marrow (BM)–derived CD34+ cells were compared. In addition, the effects of PMPs on the proliferation of CD34+ and Sca-1+ cells and on adhesion of HSPCs to endothelium and immobilized SDF-1 were studied. Finally, the hematopoietic reconstitution of lethally irradiated mice receiving transplanted BM mononuclear cells covered or not covered with PMPs was examined. It was found that PMPs are more numerous on mPB than on BM CD34+cells, do not affect the clonogenicity of human and murine HSPCs, and increase adhesion of these cells to endothelium and immobilized SDF-1. Moreover, murine BM cells covered with PMPs engrafted lethally irradiated mice significantly faster than those not covered, indicating that PMPs play an important role in the homing of HSPCs. This could explain why in a clinical setting human mPB HSPCs (densely covered with PMPs) engraft more rapidly than BM HSPCs (covered with fewer PMPs). These findings indicate a new role for PMPs in stem cell transplantation and may have clinical implications for the optimization of transplantations.

Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow–derived and circulating human CD34+ hematopoietic stem cells

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2037-2044 ◽  
Author(s):  
Ulrich Steidl ◽  
Ralf Kronenwett ◽  
Ulrich-Peter Rohr ◽  
Roland Fenk ◽  
Slawomir Kliszewski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Dna Synthesis ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract CD34+ hematopoietic stem cells are used clinically to support cytotoxic therapy, and recent studies raised hope that they could even serve as a cellular source for nonhematopoietic tissue engineering. Here, we examined in 18 volunteers the gene expressions of 1185 genes in highly enriched bone marrow CD34+(BM-CD34+) or granulocyte–colony-stimulating factor–mobilized peripheral blood CD34+(PB-CD34+) cells by means of cDNA array technology to identify molecular causes underlying the functional differences between circulating and sedentary hematopoietic stem and progenitor cells. In total, 65 genes were significantly differentially expressed. Greater cell cycle and DNA synthesis activity of BM-CD34+ than PB-CD34+ cells were reflected by the 2- to 5-fold higher expression of 9 genes involved in cell cycle progression, 11 genes regulating DNA synthesis, and cell cycle–initiating transcription factor E2F-1. Conversely, 9 other transcription factors, including the differentiation blocking GATA2 and N-myc, were expressed 2 to 3 times higher in PB-CD34+ cells than in BM-CD34+cells. Expression of 5 apoptosis driving genes was also 2 to 3 times greater in PB-CD34+ cells, reflecting a higher apoptotic activity. In summary, our study provides a gene expression profile of primary human CD34+ hematopoietic cells of the blood and marrow. Our data molecularly confirm and explain the finding that CD34+ cells residing in the bone marrow cycle more rapidly, whereas circulating CD34+ cells consist of a higher number of quiescent stem and progenitor cells. Moreover, our data provide novel molecular insight into stem cell physiology.

scholarly journals Enhanced Megakaryocyte and Erythroid Development From Normal Human CD34+ Cells: Consequence of Enforced Expression of SCL

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3756-3765 ◽  
Author(s):  
Ngaire J. Elwood ◽  
Helen Zogos ◽  
Daniel S. Pereira ◽  
John E. Dick ◽  
C. Glenn Begley
Keyword(s):  
Bhlh Transcription Factor ◽  
Cell Compartment ◽  
Cell Cycle Time ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Proliferative Effect ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Normal Human ◽  
Erythroid Development

Abstract The product of the SCL gene is a basic helix-loop-helix (bHLH) transcription factor that is essential for the development of hematopoietic stem cells in both the embryo and the adult. However, once the stem cell compartment is established, the function of SCL in subsequent differentiation and commitment events within normal hematopoietic cells remains undefined. The aim of the current study was to investigate this role using purified normal human hematopoietic CD34+ cells. An SCL retrovirus was used to transduce CD34+ cells isolated from human bone marrow, peripheral blood, and umbilical cord blood. Enforced expression of SCL increased by a median of twofold the number of erythroid colonies, with an increase in both colony size and the rate of hemoglobinization. Unexpectedly, enforced expression in CD34+ cells also significantly increased the number of megakaryocyte colonies, but with no impact on the size of colonies. There was no consistent effect on the number nor size of granulocyte-macrophage (GM) colonies. The proliferative effect of enforced SCL expression on erythroid cells was attributed to a shortened cell cycle time; the self-renewal capacity of erythroid or GM progenitors was unchanged, as was survival of cells within colonies. These results demonstrate a role for SCL in determining erythroid and megakaryocyte differentiation from normal human hematopoietic CD34+ cells.

Action of interleukin-3, G-CSF, and GM-CSF on highly enriched human hematopoietic progenitor cells: synergistic interaction of GM-CSF plus G-CSF

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 110-114 ◽  
Author(s):  
I McNiece ◽  
R Andrews ◽  
M Stewart ◽  
S Clark ◽  
T Boone ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Synergistic Interaction ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Normal Human ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

Abstract Purified preparations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and interleukin 3 (IL-3 or multi-CSF) alone and in combination, have been compared for their stimulatory effects on human granulocyte-macrophage colony forming cells (GM-CFC). In cultures of unseparated normal human bone marrow, the combinations of G-CSF plus IL-3 and GM-CSF plus IL-3 stimulated additive numbers of GM colonies, while GM-CSF plus G-CSF stimulated greater than additive numbers of GM colonies, compared with the sum of the colony formation obtained with each factor alone. Cultures of unseparated bone marrow, harvested from patients four to six days after administration of 5-fluorouracil (5-FU), resulted in additive GM colony formation with GM-CSF plus G-CSF, GM-CSF plus IL-3, and G-CSF plus IL-3. In order to address the possibility of secondary factor involvement in the synergistic interaction of GM-CSF and G-CSF, CD33+/CD34+ colony forming cells were separated from normal and post FU marrow by two color fluorescence activated cell sorting. In cultures of CD33+/CD34+ cells the combination of GM-CSF plus G-CSF stimulated a synergistic increase in GM colonies while GM-CSF plus IL-3 stimulated additive numbers of colonies. These results suggest that GM-CSF, G-CSF, and IL-3 stimulate distinct populations of GM-CFC. Furthermore GM-CSF and G-CSF interact synergistically and this action is a direct effect on progenitor cells not stimulated by GM-CSF or G-CSF alone.

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