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

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.

Abstract 16186: Hypoxic Pre-conditioning on Human CD34+ Stem Cells Enhances Exosome Therapeutics of Ischemic Tissue Repair Through ETS-1-Regulated Pathway

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Yaxuan Liang ◽  
Prabhu Mathiyalagan ◽  
David Kim ◽  
Douglas Losordo ◽  
Roger Hajjar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Limb Ischemia ◽  
Limb Amputation ◽  
Hematopoietic Stem ◽  
Angiogenic Potential ◽  
Ischemic Limb ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Hypoxic Treatment

Previous studies in our lab have revealed a novel mechanism that therapeutically important human CD34+ stem cells secrete exosomes (Exo) to extracellular milieu that induce angiogenic activity independent of the cells. Further studies demonstrated that CD34+ exosomes (CD34Exo) carry and transfer of proangiogenic miRNAs, such as miR-126, which affect the therapeutic function of the exosomes. Herein we hypothesized that hypoxic treatment of CD34+ stem cells can modulate the miRNA content and regenerative efficacy of CD34Exo. Methods and Results: Exosomes from human CD34+ cells cultured under hypoxia (H-Exo) were more proliferative, anti-apoptotic and angiogenic in vitro, as compared to exosomes from cells under normoxia (N-Exo). In a mouse model of hind limb ischemia, H-Exo treatment significantly enhanced perfusion (ratio: 0.93±0.05 v 0.77±0.02), increased capillary density (1.6±0.2 v 1.1±0.1/HPF) and prevented ischemic limb amputation (0% v 37.5%) as compared to N-Exo (p<0.05; n=7-8). Flow cytometry and confocal microscopy indicated that H-Exo was uptaken by endothelial cells in the ischemic limb. Interestingly, hypoxic treatment did not alter the size and quantity of Exo or expression of major proteins of CD34Exo compared with normoxic treatment. However, hypoxic treatment altered the proangiogenic miRNA expression in H-Exo including miR-210 and miR-126. ETS-1, a transcription factor induced by hypoxia-inducible fator-1 (HIF-1) is known to regulate expression of miR-126. We have examined the protein and RNA expression of HIF-1 and ETS-1 in Sca-1 positive mouse hematopoietic stem cells, and propose that HIF-1/ETS-1 regulatory mechanisms affect the expression of exosomal miR-126 under hypoxia. These results are being confirmed in human CD34+ cells using siRNA silencing and HIF hydroxylase inhibitor dimethyloxalylglycine. Conclusion: Hypoxia induced miR-126 expression in CD34 cell-derived exosomes stimulating exosomes-mediated angiogenesis and therapeutic recovery via ETS-transcriptional pathway. Our work has important clinical implications for therapeutic angiogenesis, especially in diabetic and cardiovascular patients who have stem cells with diminished angiogenic potential.

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.

Vitamin K2 Supports Hematopoiesis through Acting on Bone Marrow Mesenchymal Stromal/Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1192-1192 ◽  
Author(s):  
Aya Fujishiro ◽  
Yasuo Miura ◽  
Masaki Iwasa ◽  
Sumie Fujii ◽  
Akihiro Tamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Cells ◽  
Vitamin K2 ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Cd34 Cells

Abstract [Background] Myelodysplastic syndrome is an intractable disorder characterized by ineffective hematopoiesis. Although allogeneic hematopoietic stem cell transplantation is the only curative therapy for eligible patients, hematopoiesis-supportive pharmacotherapy is practically important for transplant-ineligible patients to overcome transfusion dependency and infections. Vitamin K2 (VK2, menatetrenone) is a drug used to aim at improvement of hematopoiesis in MDS patients (Leukemia 14: 1156, 2000). However, the exact mechanism how VK2 improves hematopoiesis remains largely unknown. It was reported that VK2 induces MDS cells to undergo apoptosis (Leukemia 13: 1399, 1999). Here, we investigated our hypothesis that VK2 exerts its hematopoiesis-supportive effects through acting on mesenchymal stem/stromal cells (BM-MSCs) in the bone marrow microenvironment. [Methods] Normal bone marrow (BM) samples from healthy adult volunteers were purchased from AllCells (Emeryville, CA). BM-CD34+ cells were isolated from BM-mononuclear cells using anti-CD34 immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Human BM-MSCs were isolated according to our previously published methods (Stem Cells 32:2245, 2014). In co-culture experiments, BM-MSCs with or without VK2 treatment were seeded on a 24-well culture plate. BM-CD34+ cells were applied on the MSC-grown plate and co-cultured in SFEM (StemCell Technologies, Vancouver, Canada) supplemented with 100 ng/mL SCF, 100 ng/mL Flt-3 ligand, 50 ng/mL TPO and 20 ng/mL IL-3. After 10 days of co-culture, the number and surface marker expression of the expanded hematopoietic cells were examined by flow cytometric analysis. [Results] We first tested the direct effect of VK2 on BM-CD34+ cells. BM-CD34+ cells were treated with VK2 at various concentrations ranged from 0 µM to 10 µM for 24 hours and then cultured in SFEM in combinations with cytokines. Surprisingly, viable hematopoietic cells were hardly detected in the expansion culture of BM-CD34+ cells treated with 10 µM VK2. Even with 1 µM treatment, the number of CD45+ cells was decreased, as compared to that of expansion culture of untreated BM-CD34+ cells. The apoptosis analysis showed that the percentage of AnnexinV+ PI+ cells in the expanded hematopoietic cells is increased by VK2 treatment. We next examined the effect of VK2 on the hematopoiesis-supportive capability of BM-MSCs. BM-MSCs were pretreated with VK2 at various concentrations and then co-cultured with BM-CD34+ cells. The numbers of CD34+ cells and CD45+ cells were increased in a VK2 dose-dependent manner. These results demonstrated that VK2 shows different effects on distinct stem/progenitor cells: the induction of apoptosis in BM-CD34+ cells and the enhancement of hematopoiesis-supportive capability of BM-MSCs. We then investigated whether apoptosis-related cell death of BM-CD34+ cells by VK2 treatment is ameliorated in the presence of BM-MSCs. Both BM-CD34+ cells and BM-MSCs were treated with VK2 for 24 hours, and then co-cultured. The number of CD34+ cells was not decreased significantly in contrast to its severe decrease in single culture of VK2-treated BM-CD34+ cells. We further analyzed the effect of VK2 on BM-MSCs. Subpopulation analysis in co-culture of CD34+ cells with VK2-treated BM-MSCs showed that the expansion efficacy of CD34+CD38+ cells is higher in comparison to that of CD34+CD38- cells. In addition, the percentages of CD34-CD33+ cells and CD34-CD13+ cells were higher than those in co-cultures with untreated BM-MSCs. Therefore, VK2-treated BM-MSCs supported the expanded CD34+ cells to skew their phenotype toward myeloid lineage. The presence of a transwell in the co-culture system was unrelated to the expansion pattern of CD34+ cells, which suggested the involvement of soluble factors with respect to the underlining mechanism. We therefore compared the levels of hematopoiesis-supporting cytokine mRNA expression in VK2-treated and untreated BM-MSCs: VK2-treated BM-MSCs showed lower expression of CXCL12/SDF-1 mRNA and a trend toward higher expression of GM-CSF mRNA. [Summary] VK2 acted on BM-MSCs to support their ability to enhance expansion and myeloid differentiation of BM-CD34+ cells probably via altered GM-CSF and CXCL12/SDF-1 expression in MSCs. These findings may help to identify the mechanisms of therapeutic effects of VK2 in patients with MDS (Figure). Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 542-550 ◽  
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.

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

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1735-1738 ◽  
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.

PACAP and its receptor VPAC1 regulate megakaryocyte maturation: therapeutic implications

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 1885-1893 ◽  
Author(s):  
Kathleen Freson ◽  
Karen Peeters ◽  
Rita De Vos ◽  
Christine Wittevrongel ◽  
Chantal Thys ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Adenylyl Cyclase ◽  
Independent Manner ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Therapeutic Implications ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Megakaryocyte Differentiation

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.

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

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4040-4048 ◽  
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.

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.

Extracellular HMGB1 Enhances Proliferation, Differentiation and Migration of Human CD34+ Hematopoietic Stem Cells in Vitro

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4744-4744
Author(s):  
Xingbing Wang ◽  
Xin Chen ◽  
Weihua Ren ◽  
Xiucai Xu ◽  
Kaidi Song ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Proliferation And Differentiation ◽  
Cord Blood Cd34 ◽  
And Migration

Abstract High-mobility group box 1 protein (HMGB1) is a chromatin protein and acts as a cytokine involved in inflammation, cell proliferation, differentiation, migration and stem cell recruitment. So far, little is known about its effect on hematopoietic stem cells (HSCs). In this study, we investigated whether receptors for HMGB1 are expressed on human CD34+ HSCs, and whether HMGB1 could affect HSCs proliferation, differentiation and migration in vitro. As examined by FACS analysis and RT-PCR, cord blood CD34+ HSCs express the HMGB1 receptors RAGE (receptor for advanced glycation end products), TLR2 (Toll-like receptor 2) and TLR4. To study the effects of HMGB1 on CD34+ HSCs proliferation and differentiation, freshly isolated cord blood CD34+ HSCs were cultured for 7 days in medium alone or in the presence of HMGB1. Flow cytometric analysis showed that HMGB1 (50ng/ml) can induce the differentiation of CD34+ HSCs along the granulo-monocytic (CD14+, CD13+) lineage and erythropoiesis (CD71+). In contrast, HMGB1 did not induce the expression of CD41, a marker for megakaryocyte lineage. The numbers of cells cultured in the presence of HMGB1 were always increased in comparison with controls. Furthermore, higher numbers of granulomonocytic progenitors (CFU-GM), erythroid progenitors (BFU-E), and CFU-MIX were confirmed by CFC assays in a HMGB1 dose dependent manner after 14-day culture. The results suggest that HMGB1 enhances CD34+ HSCs proliferation and differentiation. We next assessed the effects of HMGB1 on HSCs migration by chemotaxis assay using Boyden chambers. HMGB1 dose-dependently increased the chemotactic migration of CD34+ HSCs. A neutralizing anti-RAGE antibody significantly blocked the HMGB1-induced migration of HSCs, whereas neutralizing TLR2 and TLR4 antibodies did not significantly influence HMGB1-stimulated HSCs migration, suggesting that the migratory effect of HMGB1 on human HSCs is predominantly mediated by RAGE. In summary, our results provide the first report of HMGB1 receptors expression profile of human cord blood CD34+ HSCs and demonstrate that HMGB1 can increase the proliferation and migration of HSCs and directly induce of HSCs along myeloid differentiation and erythropoiesis in vitro. Further studies will be needed to clarify the mechanism of HMGB1 activation and the physiological function of HMGB1 in HSCs in vivo.

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.

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