A Specialized Polarized Membrane Domain Found On Normal but Not Leukemic Hematopoietic Progenitor Cells Is Required for Homing to the Bone Marrow Microenvironment.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 82-82
Author(s):  
Andre Larochelle ◽  
Jennifer Gillette ◽  
Amy Cantilena ◽  
Alexandra Cerf ◽  
Jennifer Lippincott-Schwartz ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Bone Marrow Microenvironment ◽  
Bone Marrow Niche ◽  
Membrane Domains ◽  
Membrane Domain ◽  
Cd34 Cells ◽  
G0 Phase

Abstract Abstract 82 Hematopoietic stem/progenitor cells (HSPCs) reside in a bone marrow niche, where adhesive interactions with osteoblasts provide essential cues for their proliferation and survival. In co-cultures of osteoblasts with primary human normal CD34+ cells, CD34+CD38- cells, or the KG1a progenitor cell line, we previously showed, using live cell imaging approaches, that HSPCs made prolonged contact with the osteoblast surface via a specialized membrane domain enriched in prominin 1 (CD133), the very late antigen-4 (VLA-4), the phosphatidylethanolamine (PE) analogue rhodamine-PE, and the tetraspanins CD63 and CD81. At the contact site, portions of the specialized domain of the CD34+ cells containing these molecules were taken up by osteoblasts and internalized into signaling endosomes within the osteoblasts. This caused the osteoblasts to downregulate Smad signaling and to increase their production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow (Gillette J. et al, Nat. Cell Biol. 11(3): 303–311, 2009). We have now evaluated the functional significance of these specialized membrane domains for in vivo homing of normal HSPCs to the bone marrow microenvironment. G-CSF mobilized human peripheral blood CD34+ cells from two normal donors were treated for 30 minutes with the cholesterol sequestration agent methyl-β-cyclodextrin (MβCD). This treatment resulted in disruption of the CD34+ cell membrane domains but had no effect on cell viability, proliferation or colony forming capacity in vitro. However, in two independent experiments, we observed a three-fold decrease in homing of MβCD-treated CD34+ cells to the bone marrow of NOD/SCID IL2rψcnull mice 16 hours after transplantation as compared to mock-treated CD34+ cells (p=0.0002). In contrast to homing studies, long-term human cell engraftment determined by CD45 cell surface expression 2 months after transplantation in two independent experiments was not significantly different in mice transplanted with MβCD-treated CD34+ cells compared to mock-treated CD34+ cells (p=0.13). Rapid repolarization of the membrane domain after transplantation may have resulted in engraftment of MβCD-treated CD34+ cells at levels similar to those observed with mock-treated CD34+ cells. Given the known homing/engraftment defect of actively cycling HSPC, we compared membrane domains on quiescent and cycling CD34+ cells from two normal donors. At baseline, 55–68% of CD34+ cells were in the G0 phase of the cell cycle as measured by Hoechst/Pyronin Y staining and specialized membrane domains were detected on a similar percentage of CD34+ cells for both donors. After culture for 4 days in the presence of stimulatory cytokines (SCF, TPO and Flt3), less than 10% of the CD34+ cells remained in G0 and, similarly, less than 10% of the cells analyzed by microscopy had a specialized membrane domain. After 4 days, cells were continued in culture for 2 days under non-stimulatory conditions (SCF alone). Under these conditions, for one donor, the percentage of cells in G0 increased from 9% to 18% and the percentage of cells with membrane domains increased similarly from 6% to 11%. For the other donor, the percentage of cells in G0 and with membrane domains both remained unchanged after 2 days in non-stimulatory culture. Surprisingly, the polarized membrane domains detected on normal CD34+ cells were not found on peripheral blood blast cells from patients diagnosed with relapsed AML (n=2 patients) or CML (n=1 patient). Accordingly, in a preliminary experiment, no difference in homing was observed 16 hours after transplantation of MβCD- or mock-treated peripheral blood cells from an AML patient in NOD/SCID IL2rψcnull mice. Additional homing and engraftment studies using cells from leukemic patients are ongoing. In combination, these findings indicate that specialized membrane domains are found on normal but not leukemic HSPCs and that these domains are required for homing to the bone marrow microenvironment. Disruption of these domains in actively cycling progenitor cells may provide an explanation for the previously demonstrated homing/engraftment defect of cycling cells compared to quiescent cells in the G0 phase of the cell cycle. Disclosures: No relevant conflicts of interest to declare.

Upregulation of CXCR4 Is Essential for the Migration and Survival of Quiescent CML Progenitor Cells in the Bone Marrow Microenvironment after Imatinib Treatment.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1004-1004
Author(s):  
Yoko Tabe ◽  
Linhua Jin ◽  
Sergej Konoplev ◽  
Lu Hongbo ◽  
Shinya Kimura ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Blast Crisis ◽  
Cxcr4 Expression ◽  
Bone Marrow Microenvironment ◽  
Imatinib Treatment ◽  
Protective Effects

Abstract Chronic myeloid leukemia (CML) is driven by constitutively activated Bcr-Abl tyrosine kinase, which causes the defective adhesion of CML cells to bone marrow (BM) stroma. The overexpression of p210Bcr-Abl was reported to down-regulate CXCR4 expression, resulting in cell migration defects in CML. We proposed that the tyrosine kinase inhibitors imatinib or INNO-406 may restore CXCR4 expression and cause the migration of CML cells to BM microenvironment niches which results in acquisition of stroma-mediated chemoresistance of CML progenitor cells. In KBM5 and K562 cells, imatinib or INNO-406 increased CXCR4 expression and migration. Imatinib induced a G0/1 cell cycle block in CML cells, which was further enhanced in a bone marrow-derived stromal cell (MSC) co-culture system. Quiescent KBM5 cells migrating to stromal cells were resistant to Imatinib-induced cell death. These anti-apoptotic effects were abrogated by inhibition of CXCR4 (AMD3465) or Integrin-linked kinase (QLT0267). BM CD34+ cells from newly diagnosed (n=15) or blast crisis CML patients (n=5) expressed markedly lower CXCR4 levels compared with normal CD34+ BM progenitor cells (n=12) (p<0.05), and was associated with deficient migration of CD34+ CML cells. A longitudinal analysis of peripheral blood samples from CML patients in blast crisis treated with imatinib (n=5) revealed induction of CXCR4 in peripheral blood CD34+ progenitor cells within the first few days after initiation of treatment, which coincided with decreased WBC and absolute blast counts. After prolonged (3–10 day) exposure to imatinib, however, CXCR4 levels of CD34+ PB CML cells decreased, frequently below the starting levels. CXCR4 immunostaining on blasts/immature cells in BM biopsy sections from patients demonstrated that 5 of 7 patient samples were CXCR4-negative prior to imatinib therapy; 4 of these 5 became CXCR4 positive after imatinib treatment. While patients in blast-phase had short-lived hematologic responses to imatinib, Ph+ clone remained predominant in the bone marrow, and all patients ultimately relapsed with recurrence of blasts in the BM. Altogether, these findings suggest that the up-regulation of CXCR4 by imatinib promotes migration of CML cells to BM stroma, causing G0/1 cell-cycle arrest and enhancing the survival of quiescent CML progenitor cells within bone marrow microenvironment. This provides the rationale for interfering with the protective effects of BM stroma cells by inhibiting CXCR4 and/or integrin signaling, which could be of benefit in eradicating residual quiescent CML cells.

scholarly journals Orderly Process of Sequential Cytokine Stimulation Is Required for Activation and Maximal Proliferation of Primitive Human Bone Marrow CD34+ Hematopoietic Progenitor Cells Residing in G0

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 658-668 ◽  
Author(s):  
Amy C. Ladd ◽  
Robert Pyatt ◽  
Andre Gothot ◽  
Susan Rice ◽  
Jon McMahel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Phase I ◽  
Progenitor Cells ◽  
Phase Ii ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Cytokine Stimulation ◽  
Phase Iv

Abstract Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+ ). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with &lt;20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0 /G1 , unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation.

ALL Cells Mobilized by AMD3100 Are More Proliferative, and Remain In the Circulation Longer Than Normal HSC, Enhancing the Action of Vincristine

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2137-2137 ◽  
Author(s):  
Linda J. Bendall ◽  
Robert Welschinger ◽  
Florian Liedtke ◽  
Carole Ford ◽  
Aileen Dela Pena ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Bone Marrow Microenvironment ◽  
Hematopoietic Progenitors ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Cell Mobilization ◽  
Cycle Dependent

Abstract Abstract 2137 The chemokine CXCL12, and its receptor CXCR4, play an essential role in homing and engraftment of normal hematopoietic cells in the bone marrow, with the CXCR4 antagonist AMD3100 inducing the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. We have previously demonstrated that AMD3100 similarly induces the mobilization of acute lymphoblastic leukemia (ALL) cells into the peripheral blood. The bone marrow microenvironment is thought to provide a protective niche for ALL cells, contributing to chemo-resistance. As a result, compounds that disrupt leukemic cell interactions with the bone marrow microenvironment are of interest as chemo-sensitizing agents. However, the mobilization of normal hematopoietic stem and progenitor cells may also increase bone marrow toxicity. To better evaluate how such mobilizing agents affect normal hematopoietic progenitors and ALL cells, the temporal response of ALL cells to the CXCR4 antagonist AMD3100 was compared to that of normal hematopoietic progenitor cells using a NOD/SCID xenograft model of ALL and BALB/c mice respectively. ALL cells from all 7 pre-B ALL xenografts were mobilized into the peripheral blood by AMD3100. Mobilization was apparent 1 hour and maximal 3 hours after drug administration, similar to that observed for normal hematopoietic progenitors. However, ALL cells remained in the circulation for longer than normal hematopoietic progenitors. The number of ALL cells in the circulation remained significantly elevated in 6 of 7 xenografts examined, 6 hours post AMD3100 administration, a time point by which circulating normal hematopoietic progenitor levels had returned to baseline. No correlation between the expression of the chemokine receptor CXCR4 or the adhesion molecules VLA-4, VLA-5 or CD44, and the extent or duration of ALL cell mobilization was detected. In contrast, the overall motility of the ALL cells in chemotaxis assays was predictive of the extent of ALL cell mobilization. This was not due to CXCL12-specific chemotaxis because the association was lost when correction for background motility was undertaken. In addition, AMD3100 increased the proportion of actively cells ALL cells in the peripheral blood. This did not appear to be due to selective mobilization of cycling cells but reflected the more proliferative nature of bone marrow as compared to peripheral blood ALL cells. This is in contrast to the selective mobilization of quiescent normal hematopoietic stem and progenitor cells by AMD3100. Consistent with these findings, the addition of AMD3100 to the cell cycle dependent drug vincristine, increased the efficacy of this agent in NOD/SCID mice engrafted with ALL. Overall, this suggests that ALL cells will be more sensitive to effects of agents that disrupt interactions with the bone marrow microenvironment than normal progenitors, and that combining agents that disrupt ALL retention in the bone marrow may increase the therapeutic effect of cell cycle dependent chemotherapeutic agents. Disclosures: Bendall: Genzyme: Honoraria.

scholarly journals Transendothelial Migration of CD34+ and Mature Hematopoietic Cells: An In Vitro Study Using a Human Bone Marrow Endothelial Cell Line

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 72-80 ◽  
Author(s):  
Robert Möhle ◽  
Malcolm A.S. Moore ◽  
Ralph L. Nachman ◽  
Shahin Rafii
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Transendothelial Migration ◽  
Plating Efficiency ◽  
Cd34 Cells ◽  
Upper Chamber

To study the role of bone marrow endothelial cells (BMEC) in the regulation of hematopoietic cell trafficking, we have designed an in vitro model of transendothelial migration of hematopoietic progenitor cells and their progeny. For these studies, we have taken advantage of a human BMEC-derived cell line (BMEC-1), which proliferates independent of growth factors, is contact inhibited, and expresses adhesion molecules similar to BMEC in vivo. BMEC-1 monolayers were grown to confluency on 3 μm microporous membrane inserts and placed in 6-well tissue culture plates. Granulocyte-colony stimulating factor (G-CSF )–mobilized peripheral blood CD34+ cells were added to the BMEC-1 monolayer in the upper chamber of the 6-well plate. After 24 hours of coincubation, the majority of CD34+ cells remained nonadherent in the upper chamber, while 1.6 ± 0.3% of the progenitor cells had transmigrated. Transmigrated CD34 cells expressed a higher level of CD38 compared with nonmigrating CD34+ cells and may therefore represent predominantly committed progenitor cells. Accordingly, the total plating efficiency of the transmigrated CD34+ cells for lineage-committed progenitors was higher (14.0 ± 0.1 v 7.8% ± 1.5%). In particular, the plating efficiency of transmigrated cells for erythroid progenitors was 27-fold greater compared with nonmigrating cells (8.0% ± 0.8% v 0.3% ± 0.1%) and 5.5-fold compared with unprocessed CD34+ cells (2.2% ± 0.4%). While no difference in the expression of the β1-integrin very late activation antigen (VLA)-4 and β2-integrin lymphocyte function-associated antigen (LFA)-1 was found, L-selectin expression on transmigrated CD34+ cells was lost, suggesting that shedding had occurred during migration. The number of transmigrated cells was reduced by blocking antibodies to LFA-1, while L-selectin and VLA-4 antibodies had no inhibitory effect. Continuous coculture of the remaining CD34+ cells in the upper chamber of the transwell inserts resulted in proliferation and differentiation into myeloid and megakaryocytic cells. While the majority of cells in the upper chamber comprised proliferating myeloid precursors such as promyelocytes and myelocytes, only mature monocytes and granulocytes were detected in the lower chamber. In conclusion, BMEC-1 cells support transmigration of hematopoietic progenitors and mature hematopoietic cells. Therefore, this model may be used to study mechanisms involved in mobilization and homing of CD34+ cells during peripheral blood progenitor cell transplantation and trafficking of mature hematopoietic cells.

scholarly journals Orderly Process of Sequential Cytokine Stimulation Is Required for Activation and Maximal Proliferation of Primitive Human Bone Marrow CD34+ Hematopoietic Progenitor Cells Residing in G0

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 658-668 ◽  
Author(s):  
Amy C. Ladd ◽  
Robert Pyatt ◽  
Andre Gothot ◽  
Susan Rice ◽  
Jon McMahel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Phase I ◽  
Progenitor Cells ◽  
Phase Ii ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Cytokine Stimulation ◽  
Phase Iv

Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+ ). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with <20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0 /G1 , unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation.

Phenotypic Examination of Side Population (SP) Cells in Highly Enriched Human CD34+ Cells Obtained from Peripheral Blood Progenitor Cells (PBPC) and Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4140-4140
Author(s):  
Dag Josefsen ◽  
Leiv S. Rusten ◽  
Trond Stokke ◽  
Lise Forfang ◽  
Erlend B. Smeland ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Cell Population ◽  
Peripheral Blood ◽  
Side Population ◽  
Hoechst 33342 ◽  
Hematopoietic Stem ◽  
Immature Cell ◽  
Cd38 Expression ◽  
Cd34 Cells

Abstract CD34+ cells isolated from bone marrow include hematopoietic stem cells (HSC) as well as more lineage committed hematopoietic progenitor cells (HPC), demonstrating that CD34+ cells are a relatively heterogeneous cell population. Highly enriched CD34+ cells isolated from peripheral blood (PBPC) after mobilization shows a more immature profile with less expression of lineage restricted markers indicating that CD34+ cells from PBPC are a more homogenous immature cell population than CD34+ cells obtained from bone marrow. By using Hoechst 33342-dye efflux assay, which identifies a population of immature HPC, termed side population (SP) cells we have examined the phenotypical profile of SP+CD34+ cells obtained from bone marrow and SP+CD34+ cells isolated from PBPC. Highly enriched CD34+ cells were isolated from PBPC obtained from patients with Hodgkin lymphoma, and bone marrow was obtained from healthy volunteer donors by iliac crest aspiration after informed consent. To identify the SP+ cells, enriched CD34+ cells were stained with Hoechst 33342 dye. Using flowcytometric techniques (FACStar+, FACSDiva, Becton Dickinson, San Jose, CA) we were able to visualize the dye efflux in SP+ cells. SP+ cells were functionally confirmed using Verapamil staining. The frequenzy of LTC-IC was markedly increased in SP+CD34+ cells compared to SP−CD34+ cells (n=5), in line with previous reports. The percentage of SP+CD34+ cells varied from 0,4 to 18% of the total CD34+ cell population obtained from PBPC (n= 16), whereas the level of SP+CD34+ cells obtained from bone marrow varied between 4–7% of the total CD34+ cell population (n=4). Expression of lineage committed markers, including CD10, CD15 and CD19 was less then 10% of the whole CD34+ cell population obtained from PBPC, whereas we found a higher level of expression of these markers in CD34+ cells isolated from bone marrow. However, when we examined the SP+CD34+ cells from either PBPC or bone marrow, we observed that the phenotypical profile of these cells were similar with almost no expression of lineage markers. Thus, the more lineage-committed cells in the CD34+ cell population obtained from bone marrow seems to be restricted to the SP−CD34+ cell fraction. Examination of CD90 and CD133 expression revealed a higher level in the SP+ CD34+ cell fractions compared to the SP− fractions. Furthermore, we investigated the level of CD38 expression. Previous studies have demonstrated that lack of CD38 expression in CD34+ cells identifies a more immature cell population. Surprisingly, we observed that 30–40% of SP+CD34+ cells obtained from bone marrow were CD38 negative, whereas the level of SP+CD34+CD38− cells from PBPC was 2–5%, which is similar to the level of CD38− cells in the CD34+ cell population isolated from both PBPC and bone marrow. Currently, we are exploring the frequency of LTC-IC in SP+CD34+CD38− cells from bone marrow, and we are also planning cell sorting of these cells for functional analyses. In conclusion, we find that the level of CD38 negative cells in SP+CD34+ subpopulation of CD34+ bone marrow cells are higher than what observed in SP+CD34+ and SP−CD34+ from PBPC as well as in SP−CD34+ from bone marrow. Our ongoing studies will clarify if these results define SP+CD34+CD38− cells from bone marrow as a source of highly enriched primitive HPC.

The Ets Transcription Factor, GABP, Is Required by Myeloid Progenitors for Normal Myeloid Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 256-256
Author(s):  
Zhongfa Yang ◽  
Karen Drumea ◽  
Junling Wang ◽  
James Cormier ◽  
Alan G. Rosmarin
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Transcriptional Repressor ◽  
Myeloid Cell ◽  
Myeloid Differentiation ◽  
Ets Domain

Abstract Abstract 256 GABP transcription factor regulates genes that are required for innate immunity. GABP is an obligate tetrameric protein complex that contains two molecules of GABPa, which binds to DNA through its ets domain, and two molecules of GABPb, which contains a transcription activation domain. GABP is an essential component of a multiprotein enhanceosome that is required for retinoic acid-dependent myeloid gene transcription. Disruption in mouse embryo fibroblasts of Gabpa, the mouse gene that encodes mouse Gabpa, causes profound cell cycle arrest at the G1-S boundary, due to reduced expression of DNA Polymerase a and Thymidylate Synthase, which are required for DNA synthesis, and of Skp2, a ubiquitin ligase that controls degradation of the cyclin-dependent kinase inhibitors (CDKIs), p21 and p27. Thus, GABP is a key regulator of the cell cycle. In order to define the role of GABP in myeloid differentiation, we generated mice in which exons that encode the Gabpa ets domain are flanked by loxP recombination sites, and bred these floxed mice to mice that bear the Mx1-Cre transgene. Their progeny were treated with pI-C and Gabpa was efficiently deleted in hematopoietic cells of these Gabpa−/− mice. As controls for all experiments, mice that bear Mx1-Cre but which lack the floxed Gabpa allele were also injected with pI-C. Within days, the peripheral blood white blood cell count fell in the Gabpa−/− mice compared to the controls; half of the Gabpa−/− mice died within two weeks. Gabpa−/− mice exhibited a striking loss of Gr1+, CD11b+ cells in the peripheral blood, spleen, and bone marrow. Myeloid cells of Gabpa−/− mice were immature, morphologically dysplastic, and demonstrated aberrant patterns of myeloid gene expression. Bone marrow from Gabpa−/− mice formed reduced numbers of in vitro myeloid colonies in the presence of G-CSF, M-CSF, or GM-CSF; cells isolated from in vitro colonies from Gabpa−/− mice exhibited a strong bias toward macrophage-like morphology. Multicolor flow cytometry revealed a loss of granulocyte-monocyte committed progenitor cells (GMPs) in the bone marrow of Gabpa−/− mice, and these progenitors expressed aberrant patterns of key transcription factors. Especially notable in Gabpa−/− GMPs was reduced expression of Gfi-1, a transcriptional repressor that is mutated in some congenital neutropenic syndromes, and whose genetic disruption causes abnormalities in granulocyte development. We used chromatin immunoprecipitation (ChIP) to identify ets sites in the Gfi-1 promoter that are bound by GABP in vivo. We conclude that GABP is required for proliferation or survival of committed myeloid progenitor cells and for normal maturation of granulocytes. We hypothesize that defects in myeloid cell proliferation and differentiation associated with Gabpa disruption are caused, at least in part, by its regulation of the Gfi-1 transcriptional repressor. Furthermore, we propose that the regulation of Gfi-1 by GABP constitutes a key regulatory pathway in myeloid cell development. Disclosures: No relevant conflicts of interest to declare.

Involvement of the Retinoblastoma Protein in Monocytic and Neutrophilic Lineage Commitment of Human Bone Marrow Progenitor Cells

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 1971-1978 ◽  
Author(s):  
Gösta Bergh ◽  
Mats Ehinger ◽  
Inge Olsson ◽  
Sten Eirik W. Jacobsen ◽  
Urban Gullberg
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Differentiation ◽  
Progenitor Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Lineage Commitment ◽  
Flt3 Ligand ◽  
Monocytic Differentiation ◽  
Cd34 Cells

The retinoblastoma gene product (pRb) is involved in both cell cycle regulation and cell differentiation. pRb may have dual functions during cell differentiation: partly by promoting a cell cycle brake at G1 and also by interacting with tissue-specific transcription factors. We recently showed that pRb mediates differentiation of leukemic cell lines involving mechanisms other than the induction of G1 arrest. In the present study, we investigated the role of pRb in differentiation of human bone marrow progenitor cells. Human bone marrow cells were cultured in a colony-forming unit–granulocyte-macrophage (CFU-GM) assay. The addition of antisense RB oligonucleotides (-RB), but not the addition of sense orientated oligonucleotides (SO) or scrambled oligonucleotides (SCR), reduced the number of colonies staining for nonspecific esterase without affecting the clonogenic growth. Monocytic differentiation of CD34+ cells supported by FLT3-ligand and interleukin-3 (IL-3) was correlated to high levels of hypophosphorylated pRb, whereas neutrophilic differentiation, supported by granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF), was correlated to low levels. The addition of -RB to liquid cultures of CD34+ cells, supported with FLT3-ligand and IL-3, inhibited monocytic differentiation. This was judged by morphology, the expression of CD14, and staining for esterase. Moreover, the inhibition of monocytic differentiation of CD34+ cells mediated by -RB, which is capable of reducing pRb expression, was counterbalanced by an enhanced neutrophilic differentiation response, as judged by morphology and the expression of lactoferrin. CD34+ cells incubated with oligo buffer, -RB, SO, or SCR showed similar growth rates. Taken together, these data suggest that pRb plays a critical role in the monocytic and neutrophilic lineage commitment of human bone marrow progenitors, probably by mechanisms that are not strictly related to control of cell cycle progression.

scholarly journals Transendothelial Migration of CD34+ and Mature Hematopoietic Cells: An In Vitro Study Using a Human Bone Marrow Endothelial Cell Line

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 72-80 ◽  
Author(s):  
Robert Möhle ◽  
Malcolm A.S. Moore ◽  
Ralph L. Nachman ◽  
Shahin Rafii
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Transendothelial Migration ◽  
Plating Efficiency ◽  
Cd34 Cells ◽  
Upper Chamber

Abstract To study the role of bone marrow endothelial cells (BMEC) in the regulation of hematopoietic cell trafficking, we have designed an in vitro model of transendothelial migration of hematopoietic progenitor cells and their progeny. For these studies, we have taken advantage of a human BMEC-derived cell line (BMEC-1), which proliferates independent of growth factors, is contact inhibited, and expresses adhesion molecules similar to BMEC in vivo. BMEC-1 monolayers were grown to confluency on 3 μm microporous membrane inserts and placed in 6-well tissue culture plates. Granulocyte-colony stimulating factor (G-CSF )–mobilized peripheral blood CD34+ cells were added to the BMEC-1 monolayer in the upper chamber of the 6-well plate. After 24 hours of coincubation, the majority of CD34+ cells remained nonadherent in the upper chamber, while 1.6 ± 0.3% of the progenitor cells had transmigrated. Transmigrated CD34 cells expressed a higher level of CD38 compared with nonmigrating CD34+ cells and may therefore represent predominantly committed progenitor cells. Accordingly, the total plating efficiency of the transmigrated CD34+ cells for lineage-committed progenitors was higher (14.0 ± 0.1 v 7.8% ± 1.5%). In particular, the plating efficiency of transmigrated cells for erythroid progenitors was 27-fold greater compared with nonmigrating cells (8.0% ± 0.8% v 0.3% ± 0.1%) and 5.5-fold compared with unprocessed CD34+ cells (2.2% ± 0.4%). While no difference in the expression of the β1-integrin very late activation antigen (VLA)-4 and β2-integrin lymphocyte function-associated antigen (LFA)-1 was found, L-selectin expression on transmigrated CD34+ cells was lost, suggesting that shedding had occurred during migration. The number of transmigrated cells was reduced by blocking antibodies to LFA-1, while L-selectin and VLA-4 antibodies had no inhibitory effect. Continuous coculture of the remaining CD34+ cells in the upper chamber of the transwell inserts resulted in proliferation and differentiation into myeloid and megakaryocytic cells. While the majority of cells in the upper chamber comprised proliferating myeloid precursors such as promyelocytes and myelocytes, only mature monocytes and granulocytes were detected in the lower chamber. In conclusion, BMEC-1 cells support transmigration of hematopoietic progenitors and mature hematopoietic cells. Therefore, this model may be used to study mechanisms involved in mobilization and homing of CD34+ cells during peripheral blood progenitor cell transplantation and trafficking of mature hematopoietic cells.

scholarly journals Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1591-1591
Author(s):  
Juliana M. Xavier ◽  
Lauremilia Ricon ◽  
Karla Priscila Vieira ◽  
Longhini Ana Leda ◽  
Carolina Bigarella ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Bone Marrow Niche ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown. In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/-mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays. Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant. The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/-recipient (mean± SD) (2.07 ± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 ± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 ±1.39; Arhgap21+/-: 2.17 ± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 ± 3.85; Arhgap21+/-: 14.07 ±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/-bone marrow and spleen were retained in the blood stream. In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 ±0.03; Arhgap21+/-0.12±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 ±1.1; Arhgap21+/-94.3±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal. The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation. In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. Figure 1 Reduced homing and short term reconstitution in Arhgap21 +/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Figure 1. Reduced homing and short term reconstitution in Arhgap21+/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Disclosures No relevant conflicts of interest to declare.

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