In Vitro and In Vivo Induction of Human Hematopoietic Stem Cell Migration by Extracellular UTP.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1730-1730
Author(s):  
Lara Rossi ◽  
Rossella Manfredini ◽  
Francesco Bertolini ◽  
Davide Ferrari ◽  
Miriam Fogli ◽  
...  
Keyword(s):  
Cell Types ◽  
P2y Receptors ◽  
Extracellular Nucleotides ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stromal Cell Derived Factor

Abstract Regulatory mechanisms governing homing and engraftment of hematopoietic stem cells (HSCs) involve a complex interplay between chemokines, cytokines, growth factors and adhesion molecules in the intricate architecture of bone marrow (BM) microenvironment. HSCs express P2Y and P2X receptors for extracellular nucleotides, which activation by ATP and UTP has been recently demonstrated (Lemoli et al. Blood. 2004) to produce potent stimulatory effects on HSCs. Moreover extracellular nucleotides are emerging as key factors of flogosis phenomena and related chemotactic responses of several cell types, such as dendritic cells, monocytes and endothelial cells. In this study we investigated the biologic activity of extracellular ATP and UTP and their capacity to cooperatively promote SDF-1 (stromal cell-derived factor-1)-stimulated cell chemotaxis. Low concentrations of UTP (10uM) significantly improved, in vitro, HSCs migration. Moreover, UTP inhibits CXCR4 down-regulation of migrating CD34+ cells and increased cell adhesion to fibronectin filaments. Furthermore, in vivo competitive repopulation assays showed that preincubation with UTP significantly improved the homing efficiency of human CD34+ HSCs in nonobese diabetic/severe combined immunodeficient mice. Inhibition assays with Pertussis Toxin from B. Pertussis blocked SDF-1- and UTP-dependent chemotactic responses, suggesting that Gαi proteins may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP-treated CD34+ cells and subsequent in vitro inhibition assays with Toxin B from C. Difficile suggest that RhoGTPase Rac2 and his downstream effectors ROCK1 and ROCK2 are involved in the UTP-promoted, SDF-1-dependent HSCs migration. Taken together, our data suggest that UTP may physiologically modulate HSC migration and homing to the BM, in concert with the chemotactic peptide SDF-1, via the activation of converging signaling transduction pathways between CXCR4 and P2Y receptors, involving Gαi proteins and RhoGTPases.

The extracellular nucleotide UTP is a potent inducer of hematopoietic stem cell migration

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 533-542 ◽  
Author(s):  
Lara Rossi ◽  
Rossella Manfredini ◽  
Francesco Bertolini ◽  
Davide Ferrari ◽  
Miriam Fogli ◽  
...  
Keyword(s):  
Rho Gtpase ◽  
P2y Receptors ◽  
Extracellular Nucleotides ◽  
Potent Inducer ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Homing and engraftment of hematopoietic stem cells (HSCs) to the bone marrow (BM) involve a complex interplay between chemokines, cytokines, and nonpeptide molecules. Extracellular nucleotides and their cognate P2 receptors are emerging as key factors of inflammation and related chemotactic responses. In this study, we investigated the activity of extracellular adenosine triphosphate (ATP) and uridine triphosphate (UTP) on CXCL12-stimulated CD34+ HSC chemotaxis. In vitro, UTP significantly improved HSC migration, inhibited cell membrane CXCR4 down-regulation by migrating CD34+ cells, and increased cell adhesion to fibronectin. In vivo, preincubation with UTP significantly enhanced the BM homing efficiency of human CD34+ cells in immunodeficient mice. Pertussis toxin blocked CXCL12- and UTP-dependent chemotactic responses, suggesting that G-protein alpha-subunits (Gαi) may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP- and CXCL12-treated CD34+ cells and in vitro inhibition assays demonstrated that Rho guanosine 5′-triphosphatase (GTPase) Rac2 and downstream effectors Rho GTPase–activated kinases 1 and 2 (ROCK1/2) are involved in UTP-promoted/CXCL12-dependent HSC migration. Our data suggest that UTP may physiologically modulate the homing of HSCs to the BM, in concert with CXCL12, via the activation of converging signaling pathways between CXCR4 and P2Y receptors, involving Gαi proteins and RhoGTPases.

MicroRNA hsa-mir-155 Blocks Myeloid and Erythroid Differentiation of Human CD34+ Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1337-1337
Author(s):  
Robert W. Georgantas ◽  
Richard Hildreth ◽  
Sebastien Morisot ◽  
Jonathan Alder ◽  
Curt I. Civin
Keyword(s):  
K562 Cells ◽  
Erythroid Differentiation ◽  
Expression Data ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Megakaryocyte Differentiation

Abstract In a large microRNA-array and bioinformatics study, we determined all of the microRNAs (miRs) expressed by human CD34+ hematopoietic stem-progenitor cells (HSPCs) from bone marrow and G-CSF mobilized blood. When we combined miR expression data, mRNA expression data from a previous study (Georgantas et al, Cancer Research 64:4434), and data from various published miR-target prediction algorithms, we were able to bioinformaticly predict the actions of miRs within the hematopoietic system. MircoRNA hsa-mir-155 was highly expressed in CD34+ HSPCs, and was predicted by our bioinformatics database to target several HSPC-expressed mRNAs (CREBBP, CXCR4, Jun, Meis-1, PU.1, AGTRI, AGTRII, Fos, and GATA3) that encode proteins known to be involved in myeloid and/or erythroid differentiation. We used luciferase-3′UTR reporter constructs to confirm that protein expression from these mRNAs were in fact down regulated by microRNA. As an initial test of mir-155′s effect on hematopoietic differentiation, K562 cells were transduced with hsa-mir-155 lentivirus and then exposed to TPA to induce megakaryocyte differentiation, or to hemin to induce erythroid differentiation. Compared to controls, miR-155 reduced K562 megakaryocyte differentiation by ~70%, and erythroid differentiation by >90%. Thus, mir-155 appears to be sufficient to inhibit both megakayrocyte and erythroid differentiation. K562 proliferation was not affected by mir-155, showing that the differentiation block was not due to cell cycle arrest. MicroRNA hsa-mir-155-transduced human mobilized blood CD34+ cells generated >70% fewer myeloid and erythroid colonies than controls in colony forming (CFC) assays, further indicating that mir-155 blocks both myeloid and erythroid differentiation. We are currently further testing the effects of mir-155 on differentiation of CD34+ cells in vitro, and also in vivo on their ability to engraft immunodeficient mice.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2370-2370
Author(s):  
Daniel Ewerth ◽  
Stefanie Kreutmair ◽  
Birgit Kügelgen ◽  
Dagmar Wider ◽  
Julia Felthaus ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Research Funding ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

scholarly journals The Chemokine SDF-1 Is a Chemoattractant for Human CD34+ Hematopoietic Progenitor Cells and Provides a New Mechanism to Explain the Mobilization of CD34+ Progenitors to Peripheral Blood

1997 ◽  
Vol 185 (1) ◽  
pp. 111-120 ◽  
Author(s):  
A. Aiuti ◽  
I.J. Webb ◽  
C. Bleul ◽  
T. Springer ◽  
J.C. Gutierrez-Ramos
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stromal Cell Derived Factor ◽  
Transient Elevation

Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34+ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34+ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34+ cells is mediated by seven transmembrane receptors coupled to Gi proteins. CD34+ cells migrating to SDF-1 include cells with a more primitive (CD34+/CD38− or CD34+/DR−) phenotype as well as CD34+ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34+ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34+ progenitors from peripheral blood than in CD34+ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.

Rapid and efficient homing of human CD34+CD38−/lowCXCR4+stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2mnull mice

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3283-3291 ◽  
Author(s):  
Orit Kollet ◽  
Asaf Spiegel ◽  
Amnon Peled ◽  
Isabelle Petit ◽  
Tamara Byk ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Immunodeficient Mice ◽  
Human Cd34 ◽  
Kinase C ◽  
Cd34 Cells

Abstract Stem cell homing into the bone microenvironment is the first step in the initiation of marrow-derived blood cells. It is reported that human severe combined immunodeficient (SCID) repopulating cells home and accumulate rapidly, within a few hours, in the bone marrow and spleen of immunodeficient mice previously conditioned with total body irradiation. Primitive CD34+CD38−/lowCXCR4+ cells capable of engrafting primary and secondary recipient mice selectively homed to the bone marrow and spleen, whereas CD34−CD38−/lowLin− cells were not detected. Moreover, whereas freshly isolated CD34+CD38+/high cells did not home, in vivo stimulation with granulocyte colony-stimulating factor as part of the mobilization process, or in vitro stem cell factor stimulation for 2 to 4 days, potentiated the homing capabilities of cytokine-stimulated CD34+CD38+ cells. Homing of enriched human CD34+ cells was inhibited by pretreatment with anti-CXCR4 antibodies. Moreover, primitive CD34+CD38−/lowCXCR4+cells also homed in response to a gradient of human stromal cell-derived factor 1 (SDF-1), directly injected into the bone marrow or spleen of nonirradiated NOD/SCID mice. Homing was also inhibited by pretreatment of CD34+ cells with antibodies for the major integrins VLA-4, VLA-5, and LFA-1. Pertussis toxin, an inhibitor of signals mediated by Gαiproteins, inhibited SDF-1–mediated in vitro transwell migration but not adhesion or in vivo homing of CD34+ cells. Homing of human CD34+ cells was also blocked by chelerythrine chloride, a broad-range protein kinase C inhibitor. This study reveals rapid and efficient homing to the murine bone marrow by primitive human CD34+CD38−/lowCXCR4+cells that is integrin mediated and depends on activation of the protein kinase C signal transduction pathway by SDF-1.

The CXCR4 Antagonist, AMD3100, Inhibits AML Transmigratory Activity but Does Not Alter Blast Proliferation or Survival.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2881-2881
Author(s):  
Jane L. Liesveld ◽  
Jeffrey E. Lancet ◽  
Karen E. Rosell ◽  
Jeremy Bechelli ◽  
Camille N. Abboud ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
In Vivo Studies ◽  
Cxcr4 Antagonist ◽  
Healthy Human ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stromal Cell Derived Factor

Abstract Stromal cell derived factor-1 (SDF-1α) and its receptor, CXCR4 play a role in the trafficking of CD34+ cells. AMD3100, a selective CXCR4 antagonist, can mobilize hematopoietic progenitors from marrow to peripheral blood in healthy human volunteers and in patients with multiple myeloma and non-Hodgkin’s lymphoma (Flomenberg et al, Blood 102, 39a, 2003). Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation and NOD/SCID repopulating capacity (Kahn et al. Blood 103:2942, 2004). Since CXCR4 has been found to regulate the migration and development of AML stem cells in NOD/SCID mice, we studied the effect of AMD3100 on AML cells from the standpoint of proliferation and in vitro transendothelial transmigration utilizing a transwell system. AMD3100 (from AnorMED, Inc.), at concentrations from 0.1 to 1.0 ng/ml did not affect the viability or porliferation of purified AML blasts (n=4). AMD3100 did not influence the adherence of AML blasts to endothelial monolayers. In the presence of 0.1 to 1 ng/ml AMD-3100, the transmigration of normal CD34+ cells stimulated by 100 ng/ml SDF-1α through a human umbilical vein endothelial cell (HUVEC) monolayer was completely inhibited. Likewise, the transmigration of AML blasts through HUVECs was not altered by AMD3100 exposure, but the SDF-1α mediated transmigration was inhibited by AMD3100 from 0.1 to 1 ng/ml. The same effect was noted with AML transmigration through marrow stromal layers. The increase in transmigration through endothelial cells stimulated with G-CSF was not inhibited by AMD3100 whereas the transmigration stimulated by interleukin-8 was inhibited. When AMD3100 was placed in the bottom of the migration chamber, no independent effects on AML transmigration were noted. Co-culture of AML blasts with stromal monolayers protected blasts from apoptosis. This protection was not altered by SDF-1α, AMD3100, nor by the combination. These in vitro results demonstrate that AMD3100 can influence the migratory capacity of AML cells but has no direct effects on their proliferation or survival. Further in vitro and in vivo studies will be required to elucidate the role that this unique chemokine antagonist has in the mobilization potential of AML blasts or progenitors or in the interactions of AML cells with their microenvironment. Such studies have implications for AML autografting and AML blast interactions with extramedullary endothelial cells.

Interleukin-27 directly induces differentiation in hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 1903-1912 ◽  
Author(s):  
Jun Seita ◽  
Masayuki Asakawa ◽  
Jun Ooehara ◽  
Shin-ichiro Takayanagi ◽  
Yohei Morita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Extramedullary Hematopoiesis ◽  
Multiple Roles ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Common Signal

Interleukin (IL)-27, one of the most recently discovered IL-6 family cytokines, activates both the signal transducer and activator of transcription (STAT)1 and STAT3, and plays multiple roles in pro- and anti-inflammatory immune responses. IL-27 acts on various types of cells including T, B, and macrophage through the common signal-transducing receptor gp130 and its specific receptor WSX-1, but the effect of IL-27 on hematopoietic stem cells (HSCs) remains unknown. Here, we show that IL-27 together with stem cell factor (SCF) directly acts on HSCs and supports their early differentiation in vitro and in vivo. CD34−/lowc-Kit+Sca-1+lineage marker− (CD34−KSL) cells, a population highly enriched in mouse HSCs, were found to express both IL-27 receptor subunits. In vitro cultures of CD34−KSL cells with IL-27 and SCF resulted in an expansion of progenitors including short-term repopulating cells, while some of their long-term repopulating activity also was maintained. To examine its in vivo effect, transgenic mice expressing IL-27 were generated. These mice exhibited enhanced myelopoiesis and impaired B lymphopoiesis in the bone marrow with extramedullary hematopoiesis in the spleen. Moreover, IL-27 similarly acted on human CD34+ cells. These results suggest that IL-27 is one of the limited cytokines that play a role in HSC regulation.

The sphingosine 1-phosphate receptor agonist FTY720 supports CXCR4-dependent migration and bone marrow homing of human CD34+ progenitor cells

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4478-4486 ◽  
Author(s):  
Takafumi Kimura ◽  
Andreas M. Boehmler ◽  
Gabriele Seitz ◽  
Selim Kuçi ◽  
Tina Wiesner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Actin Polymerization ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Sphingosine 1 Phosphate ◽  
Stem And Progenitor Cells ◽  
Stromal Cell Derived Factor

Abstract The novel immunosuppressant FTY720 activates sphingosine 1-phosphate receptors (S1PRs) that affect responsiveness of lymphocytes to chemokines such as stromal cell-derived factor 1 (SDF-1), resulting in increased lymphocyte homing to secondary lymphoid organs. Since SDF-1 and its receptor CXCR4 are also involved in bone marrow (BM) homing of hematopoietic stem and progenitor cells (HPCs), we analyzed expression of S1PRs and the influence of FTY720 on SDF-1/CXCR4-mediated effects in human HPCs. By reverse transcriptase-polymerase chain reaction (RT-PCR), S1PRs were expressed in mobilized CD34+ HPCs, particularly in primitive CD34+/CD38- cells. Incubation of HPCs with FTY720 resulted in prolonged SDF-1-induced calcium mobilization and actin polymerization, and substantially increased SDF-1-dependent in vitro transendothelial migration, without affecting VLA-4, VLA-5, and CXCR4 expression. In nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, the number of CD34+/CD38- cells that homed to the BM after 18 hours was significantly raised by pretreatment of animals and cells with FTY720, tending to result in improved engraftment. In addition, in vitro growth of HPCs (week-5 cobblestone area-forming cells [CAFCs]) was 2.4-fold increased. We conclude that activation of S1PRs by FTY720 increases CXCR4 function in HPCs both in vitro and in vivo, supporting homing and proliferation of HPCs. In the hematopoietic microenvironment, S1PRs are involved in migration and maintenance of HPCs by modulating the effects of SDF-1. (Blood. 2004;103:4478-4486)

Extracellular nucleotides are potent stimulators of human hematopoietic stem cells in vitro and in vivo

Blood ◽  
2004 ◽  
Vol 104 (6) ◽  
pp. 1662-1670 ◽  
Author(s):  
Roberto M. Lemoli ◽  
Davide Ferrari ◽  
Miriam Fogli ◽  
Lara Rossi ◽  
Cinzia Pizzirani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Severe Combined Immunodeficiency ◽  
Extracellular Nucleotides ◽  
Hematopoietic Stem ◽  
Nonobese Diabetic ◽  
Cd34 Cells ◽  
Wide Range

Abstract Although extracellular nucleotides support a wide range of biologic responses of mature blood cells, little is known about their effect on blood cell progenitor cells. In this study, we assessed whether receptors for extracellular nucleotides (P2 receptors [P2Rs]) are expressed on human hematopoietic stem cells (HSCs), and whether activation by their natural ligands, adenosine triphosphate (ATP) and uridine triphosphate (UTP), induces HSC proliferation in vitro and in vivo. Our results demonstrated that CD34+ HSCs express functional P2XRs and P2YRs of several subtypes. Furthermore, stimulation of CD34+ cells with extracellular nucleotides caused a fast release of Ca2+ from intracellular stores and an increase in ion fluxes across the plasma membrane. Functionally, ATP and, to a higher extent, UTP acted as potent early acting growth factors for HSCs, in vitro, because they strongly enhanced the stimulatory activity of several cytokines on clonogenic CD34+ and lineage-negative CD34- progenitors and expanded more primitive CD34+-derived long-term culture-initiating cells. Furthermore, xenogenic transplantation studies showed that short-term preincubation with UTP significantly expanded the number of marrow-repopulating HSCs in nonobese diabetic/severe combined immunodeficiency mice. Our data suggest that extracellular nucleotides may provide a novel and powerful tool to modulate HSC functions. (Blood. 2004;104:1662-1670)

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