PACAP and its receptor VPAC1 regulate megakaryocyte maturation: therapeutic implications

Megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase–activating peptide (PACAP) and the vasointestinal peptide (VIP) are agonists. We here demonstrate a regulatory role for VPAC1 signaling during megakaryopoiesis. A total of 2 patients with trisomy 18p with PACAP overexpression and transgenic mice overexpressing PACAP in megakaryocytes have thrombopathy, a mild thrombocytopenia, and a reduced number of mature megakaryocytes in their bone marrow. In vitro differentiation of hematopoietic stem cells from the patient and transgenic mice shows a reduced number of megakaryocyte colonies compared with controls. The addition of PACAP, VIP, or the adenylyl cyclase activator forskolin to CD34+ cells inhibits megakaryocyte differentiation. In contrast, neutralizing monoclonal anti-PACAP (PP1A4) or anti-VPAC1 (23A11) antibodies inhibit cAMP formation and stimulate megakaryopoiesis in a thrombopoietin-independent manner. Moreover, wild-type mice obtain an increased platelet count after subcutaneous injection of PP1A4 or 23A11. These antibodies also elevate platelet numbers in animal models of myelosuppressive therapy and in GATA1-deficient mice with congenital thrombocytopenia. Furthermore, 23A11 stimulates the in vitro megakaryocyte differentiation of both normal and GATA1-deficient human CD34+ cells. Together, our data strongly suggest that VPAC1 signaling tempers normal megakaryopoiesis, and that inhibition of this pathway stimulates megakaryocyte differentiation, enhancing platelet recovery after myelosuppressive therapy and in GATA1 deficiency.

Download Full-text

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

Blood ◽

10.1182/blood.v108.11.1337.1337 ◽

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.

Download Full-text

In Vitro and In Vivo Evidence That Ex Vivo Cytokine Priming of Donor Marrow Cells May Ameliorate Posttransplant Thrombocytopenia

Blood ◽

10.1182/blood.v91.1.353 ◽

1998 ◽

Vol 91 (1) ◽

pp. 353-359 ◽

Cited By ~ 34

Author(s):

Mariusz Z. Ratajczak ◽

Janina Ratajczak ◽

Boguslaw Machalinski ◽

Rosemarie Mick ◽

Alan M. Gewirtz

Keyword(s):

Mononuclear Cells ◽

Recovery Period ◽

Hematopoietic Stem ◽

Platelet Recovery ◽

Serum Free ◽

Cd34 Cells ◽

In Vivo Animal Model ◽

Marrow Cells

AbstractThrombocytopenia is typically observed in patients undergoing hematopoietic stem cell transplantation. We hypothesized that delayed platelet count recovery might be ameliorated by increasing the number of megakaryocyte colony- forming units (CFU-Meg) in the hematopoietic cell graft. To test this hypothesis, we evaluated cytokine combinations and culture medium potentially useful for expanding CFU-Meg in vitro. We then examined the ability of expanded cells to accelerate platelet recovery in an animal transplant model. Depending on the cytokine combination used, we found that culturing marrow CD34+cells for 7 to 10 days in serum-free cultures was able to expand CFU-Meg ∼40 to 80 times over input number. Shorter incubation periods were also found to be effective and when CD34+ cells were exposed to thrombopoietin (TPO), kit ligand (KL), interleukin-1α (IL-1α), and IL-3 in serum-free cultures for as few as 48 hours, the number of assayable CFU-Meg was still increased ∼threefold over input number. Of interest, cytokine primed marrow cells were also found to form colonies in vitro more quickly than unprimed cells. The potential clinical utility of this short-term expansion strategy was subsequently tested in an in vivo animal model. Lethally irradiated Balb-C mice were transplanted with previously frozen syngeneic marrow mononuclear cells (106/mouse), one tenth of which (105) had been primed with [TPO, KL, IL-1a, and IL-3] under serum-free conditions for 36 hours before cryopreservation. Mice receiving the primed frozen marrow cells recovered their platelet and neutrophil counts 3 to 5 days earlier than mice transplanted with unprimed cells. Mice which received marrow cells that had been primed after thawing but before transplantation had similar recovery kinetics. We conclude that pretransplant priming of hematopoietic cells leads to faster recovery of all hematopoietic lineages. Equally important, donor cell priming before transplant may represent a highly cost-effective alternative to constant administration of cytokines during the posttransplant recovery period.

Download Full-text

Abstract P019: Exosomes from Human CD34+ Cells: Critical Mediators of Proangiogenic Paracrine Effect of EPCs

Circulation Research ◽

10.1161/res.109.suppl_1.ap019 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Susmita Sahoo ◽

Sol Misener ◽

Tina Thorne ◽

Meredith Millay ◽

Kathryn M Schultz ◽

...

Keyword(s):

Cell Transplantation ◽

Human Peripheral Blood ◽

Limb Ischemia ◽

Therapeutic Effects ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Paracrine Effect ◽

Human Cd34 ◽

Cd34 Cells

Local transplantation of human CD34+ hematopoietic stem cells has been shown to promote neovascularization in pre-clinical studies in models of myocardial and limb ischemia. In early phase clinical trials, transplantation of CD34+ cells has been associated with reduced angina, improved exercise time and reduced amputation rates. Several studies have suggested that paracrine effects by these pro-angiogenic cells mediate the effects induced by cell transplantation. We hypothesized that CD34+ cells secrete exosomes (Exo), which mediate at least a part of the therapeutic function of the cells. Methods and Results: We isolated Exo from the conditioned media of adult human peripheral blood (PB) CD34+ cells. The angiogenic and therapeutic potency of CD34+ Exo was compared with the intact CD34+ cells and also with PB mononuclear cell (MNC) Exo. Exo from both CD34+ cells and MNC are 50–90nm in size, have cup shaped morphology, and carry known Exo-marker proteins such as CD63, TSG101 and Annexin V as shown by electron microscopy, Western blot and flow cytometry. Compared to CD34+ cells or MNC Exo, CD34+ Exo significantly induces in vitro angiogenic activities such as viability, proliferation and tube formation of HUVECs on matrigel- in a dose dependent manner. In vivo, CD34+ Exo stimulated significant neovascularization in mouse corneal angiogenesis assay (14±4 mm v MNC Exo, 4±1 mm, p<0.01) and incorporation of endothelial (CD31+) cells in mouse matrigel-plug assay (6±1.7% v CD34+ cells, 2±0.8%, p<0.01). Finally, in a mouse model of hind limb ischemia (HLI), CD34+ Exo significantly improved perfusion (ratio: 1.01±0.04 v 0.57±0.1, P<0.05), increased capillary density (1.8±0.3/HPF v 0.9±0.1/HPF, p<0.001) and prevented ischemic leg amputation (16% v 100%), as compared with MNC Exo. Conclusions: These data demonstrate that CD34+ Exo induce angiogenic activity and ischemic tissue repair in the absence of CD34+ cells, and suggest that Exo represent important mediators of the therapeutic effects associated with CD34+ cell therapy. We speculate that Exo derived from CD34+ cells may represent a significant component of the paracrine effect of progenitor-cell transplantation for therapeutic angiogenesis.

Download Full-text

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

Blood ◽

10.1182/blood.v95.9.2813.009k20_2813_2820 ◽

2000 ◽

Vol 95 (9) ◽

pp. 2813-2820 ◽

Cited By ~ 215

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.

Download Full-text

Interferon-α2b–induced thrombocytopenia is caused by inhibition of platelet production but not proliferation and endomitosis in human megakaryocytes

Blood ◽

10.1182/blood-2007-12-125906 ◽

2008 ◽

Vol 112 (3) ◽

pp. 542-550 ◽

Cited By ~ 53

Author(s):

Akiko Yamane ◽

Takanori Nakamura ◽

Hidenori Suzuki ◽

Mamoru Ito ◽

Yasuyuki Ohnishi ◽

...

Keyword(s):

Human Platelets ◽

Human Interferon ◽

Inhibitory Effects ◽

Hematopoietic Stem ◽

Platelet Production ◽

Human Cd34 ◽

Cd34 Cells ◽

Nog Mice ◽

Thrombopoietin Mimetic

AbstractHuman interferon (IFN)–α is the standard therapy for chronic hepatitis C to prevent its progression to liver cirrhosis and hepatocellular carcinoma. Thrombocytopenia is one of the major adverse effects of IFN-α and often leads to dose reduction or treatment discontinuation. However, there is little information on how IFN-α inhibits human megakaryopoiesis. In this study, we demonstrated that IFN-α did not inhibit colony formation of megakaryocytes from human CD34+ hematopoietic stem cells. IFN-α did not inhibit endomitosis but did inhibit cytoplasmic maturation of megakaryocytes and platelet production in vitro. IFN-α suppressed the expression of transcription factors regulating late-stage megakaryopoiesis, such as GATA-1, p45NF-E2, MafG. IFN-α also significantly reduced the number of human platelets but not megakaryocytes, and did not inhibit endomitosis of human megakaryocytes in immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice transplanted with human CD34+ cells (hu-NOG). We also demonstrated that a novel thrombopoietin mimetic, NIP-004, was effective for treating IFN-α–induced thrombocytopenia in hu-NOG mice. From ultrastructural study, IFN-α inhibited the maturation of demarcation membranes in megakaryocytes, although NIP-004 prevented the inhibitory effects of IFN-α. These results defined the pathogenesis of IFN-α–induced thrombocytopenia and suggested possible future clinical applications for thrombopoietin mimetics.

Download Full-text

Clinical stem-cell sources contain CD8+CD3+ T-cell receptor–negative cells that facilitate bone marrow repopulation with hematopoietic stem cells

Blood ◽

10.1182/blood-2007-02-076000 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1735-1738 ◽

Cited By ~ 10

Author(s):

Stephanie Bridenbaugh ◽

Linda Kenins ◽

Emilie Bouliong-Pillai ◽

Christian P. Kalberer ◽

Elena Shklovskaya ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Hematopoietic Stem ◽

Cd8 Cells ◽

Human Cd34 ◽

Cd34 Cells

Abstract Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8+ cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8+ cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8α+CD3ϵ+ T-cell receptor– negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34+ cells. In vitro, cFCs cocultured with CD34+ cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8+CD3+TCR− cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.

Download Full-text

In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells

Blood ◽

10.1182/blood.v87.10.4040.bloodjournal87104040 ◽

1996 ◽

Vol 87 (10) ◽

pp. 4040-4048 ◽

Cited By ~ 48

Author(s):

M Rosenzweig ◽

DF Marks ◽

H Zhu ◽

D Hempel ◽

KG Mansfield ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

T Cell ◽

T Lymphocytes ◽

Progenitor Cells ◽

T Cell Differentiation ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG- 2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome.

Download Full-text

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

Blood ◽

10.1182/blood.v106.11.1730.1730 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood.v126.23.2370.2370 ◽

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.

Download Full-text

Immobilized Glycosaminoglycans Reduce Thrombopoietin-Induced Apoptosis during In Vitro Expansion of Megakaryocyte Precursors from Cord Blood CD34+ Cells.

Blood ◽

10.1182/blood.v108.11.1127.1127 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1127-1127

Author(s):

Vipuil Kishore ◽

James F. Eliason ◽

Howard W.T. Matthew

Keyword(s):

Cell Expansion ◽

Dextran Sulfate ◽

Dermatan Sulfate ◽

Annexin V ◽

Hematopoietic Stem ◽

Platelet Recovery ◽

Cd34 Cells ◽

Chitosan Membranes ◽

Carboxy Methyl

Abstract Umbilical cord blood (UCB) provides a rich source of hematopoietic stem cells for transplantation after high dose chemotherapy. However, delayed platelet recovery is a serious limitation of UCB transplantation in adults. A suggested potential solution is the transfusion of expanded megakaryocyte progenitors derived from hematopoietic stem cells (HSCs). Previous work has shown that HSC proliferation and differentiation can be influenced by the use of glycosaminoglycans (GAGs). Specifically, GAGs are known to bind and modulate the activity of many cytokines and growth factors [Gupta et al. 1998, Madihally et al. 1999]. Direct GAG-receptor interactions are also believed to play a role in this modulation. Thrombopoietin (TPO), a relatively specific megakaryocyte/platelet cytokine, plays an important role in early hematopoiesis and megakaryopoiesis. However, it has been reported that TPO induces apoptosis in cells belonging to the megakaryocyte lineage [Ryu et al. 2001]. In this study, we examined the effects of various immobilized GAGs on the expansion and apoptosis of CD41+ megakaryocyte progenitors in vitro. GAG-derivatized, chitosan membranes were prepared in 24 well culture plates by first casting chitosan membranes from acetic acid solution, and then covalently binding the GAG component using carbodiimide chemistry. Saturating GAG surface densities were employed in all studies. The GAGs studied were heparin, hyaluronic acid, chondroitin-4-sulfate, dermatan sulfate, heparan sulfate and the GAG analogue carboxy-methyl dextran sulfate. Freshly isolated CD34+ cells from UCB were cultured in 24 well plates at a density of 25,000 cells/well using serum free media supplemented with bovine serum albumin, rh-insulin, human transferrin, FL (50 ng/ml), TPO (50 ng/ml) and SCF (10 ng/ml). Half medium changes were done twice per week. Wells were demidepopulated at days 7, 11, and 14 and simultaneous assays of viability, CD41 antigen expression, and apoptosis (via Annexin V expression) were conducted by flow cytometry. A rapid CD41+ cell expansion was observed on all GAG surfaces except chondroitin-4-sulfate from day 7 to day 11. Plastic and chondroitin-4-sulfate showed delayed CD41+ cell expansion but by day 14 the number of viable CD41+ cells on all surfaces were comparable. An initial high expansion of CD41+ cells on the carboxy-methyl dextran sulfate surface appeared to plateau between days 11 and 14. The Annexin V analysis revealed that the GAG surfaces had a substantially lower proportion of apoptotic megakaryocytes compared to the plastic and chitosan controls. In particular, heparin, chondroitin-4-sulfate and dermatan sulfate showed two fold lower levels (p<0.05) of apoptotic megakaryocytes. These results suggest that GAGs have a substantial potential to reduce the TPO induced megakaryocyte apoptosis. The use of GAGs along with an optimal cytokine combination may accelerate the application of ex vivo expanded megakaryocyte transfusion, thereby shortening the time of platelet recovery in the thrombocytopenia induced by radiotherapy and chemotherapy. Figure Figure

Download Full-text