PDGF-BB and Its Role in Megakaryopoiesis and Thrombocythemia

Abstract Background: Essential Thrombocythemia (ET) is characterized by persistently elevated platelet counts in the context of a normal red cell mass. However, the molecular mechanism of ET is still under investigation. Our previous studies demonstrated the presence of functional PDGF receptors (PDGFR) on megakaryocytes and their ability to mediate hematopoiesis and megakaryopoiesis (Ye et al, Haematologica, 2010). The role of PDGF-BB on megakaryocytic progenitors (CD41+, CD34+ cells) and its mechanisms on ET will be further studied in this project. Methods: ELISA, CFU assay, immunofluorescence microscope, flow cytometry and NOD/SCID mice were used in this study. Results: Bone marrow plasma levels of PDGF-BB in ET patients (n=18) and normal control (n=10) were tested and found an increased PDGF-BB levels in ET patients (2071.2±124.8 pg/ml), compared with normal control (1382.5±128.3pg/ml) (P=0.002). In vitro experiment, PDGF-BB promoted the ex vivo expansion of human hematopoietic stem (CD34 +) and progenitor (CD41 + CD61 +) cells. More significantly, PDGF enhanced the engraftment of human CD45 + cells and their myeloid subsets (CD33 +, CD14 + cells) in NOD/SCID mice. PDGF-BB stimulated megakaryopoiesis via PDGFR and its signalling. It also showed a direct stimulatory effect of PDGF-BB on c-Fos, GATA-1 and NF-E2 expressions in megakaryocytes. We speculate that these transcription factors might be involved in the signal transduction of PDGF-BB on the regulation of megakaryopoiesis. PDGF-BB also enhanced platelet recovery in mice model with radiation-induced thrombocytopenia. Studies showed that PDGF, like TPO, significantly promoted platelet recovery and the formation of CFU-MK in this irradiated-mouse. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow histology sections. In the CHRF apoptotic model, PDGF-BB had a similar anti-apoptotic effect as TPO on megakaryocytes. We also demonstrated that PDGF-BB activated the p -Akt , p-Jak2 and p-Stat3 expression, while addition of imatinib mesylate reduced p-Akt, p-Jak2 and p-Stat3 expression in CHRF cells. Conclusion: Our findings suggested that PDGF-BB is likely to be mediated via PDGF receptors with subsequent activation of the Akt and Jak2/ Stat3 pathways in megakaryopoiesis. These studies provide a possible explanation that PDGF-BB and its signaling may be involved in the molecular mechanism of essential thrombocythemia. Disclosures No relevant conflicts of interest to declare.

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PDGF/PDGFR May be Involved in the Physiopathologic Mechanism of Essential Thrombocythemia

Blood ◽

10.1182/blood.v126.23.5208.5208 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5208-5208

Author(s):

Li Xia Zhou ◽

Jieyu Ye ◽

En Yu Liang ◽

Chunfu Li ◽

Beng H Chong ◽

...

Keyword(s):

Bone Marrow ◽

Normal Control ◽

Essential Thrombocythemia ◽

Cell Mass ◽

Potential Effect ◽

Mice Model ◽

Hematopoietic Stem ◽

Platelet Recovery ◽

Bone Marrow Histology

Abstract Our previous studies have demonstrated that PDGF (Platelet-derived growth factor) has a potential effect in the regulation of hematopoiesis and megakaryopoiesis (Yang et al, Thromb Haemastasis, 1997; Ye et al, Haematologica, 2010). Essential Thrombocythemia (ET) is characterized by persistently elevated platelet counts in the context of a normal red cell mass. However, the physiopathologic mechanism of ET is still under investigation. Here, we tested the bone marrow plasma levels of PDGF-BB in essential thrombocythemia patients (n=16) and normal control (n=8), and found an increased PDGF-BB levels in ET patients (2070.92±123.98 pg/ml), compared with normal control (1381.85±128.37pg/ml) (P=0.002). Furthermore, we have demonstrated the presence of functional PDGF receptors (PDGFR) in human megakaryocytes, and their ability to mediate a mitogenic response by bone marrow colony-forming unit-megakaryocyte (CFU-MK) formation assay (n=6). PDGF-BB stimulated in vitro megakaryopoiesis via PDGFR. It also showed a direct stimulatory effect of PDGF-BB on c-Fos expressions in megakaryocytic cells, CHRF. We speculate that these transcription factors might be involved in the signal transduction of PDGF on the regulation of megakaryopoiesis. PDGF also enhanced platelet recovery in mice model with radiation-induced thrombocytopenia. Studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow CFU-MK in this irradiated-mouse. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow histology sections. We also demonstrated that PDGF activated the p- Akt, p-Jak2 and p-Stat3 expression, while addition of imatinib mesylate reduced p-Akt, p-Jak2 and p-Stat3 expression in CHRF cells. Our findings suggested that the PDGF-initiated megakaryopoiesis is likely to be mediated via PDGF receptors with subsequent activation of the Akt and Jak2/ Stat3 pathways. These studies provide a possible explanation that PDGF/PDGFR may be involved in the physiopathologic mechanism of essential thrombocythemia. Disclosures Yang: National Natural Science Foundation of China(81270580): Research Funding.

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Role of PDGF/PDGFR in Regulation of Thrombopoiesis: A Possible Explanation for Imatinib Mesylate-Induced Thrombocytopenia in the Treatment of CML

Blood ◽

10.1182/blood.v120.21.3348.3348 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3348-3348

Author(s):

Mo Yang ◽

Fanyi Meng ◽

Jie yu Ye ◽

Yue Xu ◽

Bin Xiao ◽

...

Keyword(s):

Bone Marrow ◽

Imatinib Mesylate ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Potential Effect ◽

Mice Model ◽

Hematopoietic Stem ◽

Platelet Recovery ◽

Bone Marrow Histology

Abstract Abstract 3348 Platelet-derived growth factor (PDGF), a platelet alpha-granule molecule, imply their potential effect in the regulation of megakaryocytopoiesis and thrombopoiesis, which also intimates the existence of an autocrine and/or paracrine loop constructed by megakaryocytes/platelets and their granular constituents. Our previous studies demonstrated the presence of functional PDGF receptors (PDGFR) on human megakaryocytes and platelets (Yang et al, Thromb Haemastasis, 1997) and CD34+ cells, and their ability to mediate a mitogenic response. PDGF promoted the ex vivo expansion of human hematopoietic stem (CD34+) and progenitor (CD41+ CD61+) cells. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in NOD/SCID mice. PDGF stimulated in vitro megakaryocytopoiesis via PDGFR and/or the indirect effect on bone marrow microenvironment to produce TPO and other cytokines. It also showed a direct stimulatory effect of PDGF on c-Fos, GATA-1 and NF-E2 expressions in megakaryocytes. We speculate that these transcription factors might be involved in the signal transduction of PDGF on the regulation of megakaryocytopoiesis. PDGF also enhanced platelet recovery in mice model with radiation-induced thrombocytopenia. Studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow colony-forming unit-megakaryocyte (CFU-MK) in this irradiated-mouse. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow histology sections. In the M-07e apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on megakaryocytes. We also demonstrated that PDGF activated the PI3k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Our findings suggested that the PDGF-initiated radioprotective effect is likely to be mediated via PDGF receptors with subsequent activation of the PI3k/Akt pathway. The study provides a possible explanation that blockage of PDGFR may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia in the treatment of CML. Disclosures: No relevant conflicts of interest to declare.

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Application of Mesenchymal Stem Cells Derived From the Umbilical Cord Instead of Bone Marrow In Hematopoietic Stem Cells Transplantation.

Blood ◽

10.1182/blood.v116.21.4544.4544 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4544-4544

Author(s):

Ching-Tien Peng

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Umbilical Cord ◽

Acute Gvhd ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

Cell Based Therapy

Abstract Abstract 4544 Bone marrow-derived mesenchymal stem cells (BMMSCs) have been found to enhance engraftment of hematopoietic stem cell transplantation (HSCT), plus show effect against graft-versus host disease (GVHD) because of their immunosuppressive properties. However, harvesting these cells is an invasive and painful procedure. To substitute BMMSCs from alternative sources is necessary. We intravenously infused ex vivo-expanded third-party umbilical cord-derived mesenchymal stem cells (UCMSCs) obtained from a bank 8 times in 3 patients who developed severe, steroid-resistant acute GVHD after allogeneic HSCT. The acute GVHD improved with each infusion of UCMSCs. Besides, after cotransplantation of cord blood and UCMSCs in 5 patients, we found UCMSCs enhanced absolute neutrophil counts and platelet counts recovery. No adverse effects after UCMSCs infusions were noted. We also found that UCMSCs had superior proliferative potential and greater immunosuppressive effects than BMMSCs in vitro. This is the first report of UCMSCs in human clinical application. These findings suggest UCMSCs are effective in treating aGVHD and can enhance hematopoiesis after HSCT. Considering that they are not only easy to obtain but also proliferate rapidly, UCMSCs would be the ideal candidate for cell-based therapy, especially for diseases associated with immune responses because of their immunosuppressive effects. Disclosures: No relevant conflicts of interest to declare.

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Engraftment Effects of Intra Bone Marrow Compared to Intravenous Transplantation of Allogeneic Hematopoietic Stem Cells Following a Reduced Intensity Conditioning in a DLA-Identical Canine Littermate Model

Blood ◽

10.1182/blood.v124.21.1089.1089 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1089-1089

Author(s):

Juliane Werner ◽

Stephanie Schaefer ◽

Sandra Lange ◽

Christoph Machka ◽

Gudrun Knuebel ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Control Group ◽

Reduced Intensity Conditioning ◽

Hematopoietic Stem ◽

Platelet Recovery ◽

Time Points ◽

Donor Chimerism ◽

Cell Counts ◽

Reduced Intensity

Abstract Introduction: Successful engraftment following hematopoietic stem cell transplantation (HSCT) depends on factors like immunosuppression, graft composition and number of infused HSC. Whereas the immunosuppression as well as the type and composition of the graft are influenceable low numbers of available HSCs i.e. “weak grafts” remain a clinical challenge. Weak grafts are accompanied by increased graft failure rates and longer cytopenias associated with increased morbidity. Intra bone marrow (IBM) infusion of HSC might be an approach to overcome these problems. Studies in rodents demonstrated faster engraftment with an IBM HSCT approach compared to intravenous (IV) HSCT following myeloablative conditioning. Studies of IBM HSCT following non-myeloablative or reduced intensity conditioning (RIC) are missing. Aims: Exploring the feasibility and efficiency of IBM allogeneic HSCT in comparison to IV HSCT in dog leukocyte antigen (DLA) identical canine littermates using a RIC regimen. Methods: DLA-identical siblings were used as donor/recipient pairs for HSCT. Recipient dogs were conditioned with 4.5 Gy total body irradiation before HSCT (d0) and received 15 mg/kg Cyclosporin A BID as pre- and postgrafting immunosuppression (d-1 to d+35). BM grafts were harvested at d0. In the control group (CON, n=7) unmodified BM was transplanted IV. In the IBM group (n=7) BM harvests were centrifuged and buffy coat of the BM was then transfused simultaneously into the recipient humeri and femura (50 ml, 10 min). 10 dogs are currently evaluable. Chimerism of the peripheral blood mononuclear cells (PBMC) and granulocytes (G) were tested weekly until week 8 and afterwards in larger intervals. Blood cell counts and clinical toxicities such as weight loss were monitored. Results: Infusion of BM directly into the bone was feasible. All animals engrafted. Median number of infused total nucleated cells was 4.0*108/kg (range 2.3-6.0*108/kg, IBM) and 3.3*108/kg (range 1.9-5.0*108/kg, CON, IBM vs CON: p=0.4). Median CD34+ numbers infused were 3.1*106/kg (range:1.2-10.0*106/kg, IBM) and 3.9*106/kg (range: 1.0-7.2*106/kg, CON; IBM vs CON: p= 0.8). Hematopoietic recovery in the IBM and CON groups were similar. Leukocytes recovery (>1.0*109/l) occurred at median d+11 (range: d+10 - d+16, IBM) and d+10 (range: d+9-d+12, CON; IBM vs CON: p=0.3). Median leukocytes nadirs amounted to 0.23*109/l (IBM) and 0.28*109/l (CON; IBM vs CON: p=0.3) and median duration of leukopenia (<1.0*109/l) were 6 days (range: 5.0–11.0, IBM) and 4 days (range: 3.0–6.0, CON; IBM vs CON: p=0.1). Median platelet nadir after IBMT was 10.0*109/l (range: 0.0 - 25.0*109/l) and 6.0*109/l (range: 3.0-15.0*109/l, CON; IBM vs CON: p=0.8). Period of thrombocytopenia (≤50.0*109/l) lasted for 12 days in both groups (p=0.7). Chimerism analyses showed an early and fast increase in donor chimerism in both groups. The PBMC donor chimerism at d+14, d+28 and d+56 were 46% (range: 30-53%), 57% (range: 40-73%), 64% (range: 60-83%) for IBM. Results in CON were 37% (range: 17-93%), 60% (range: 49-100%), 57% (range: 40-100%) (IBM vs CON, p=n.s. (all time points)). The G chimerism values at that specific points were 95% (range: 53-100%), 100% (range: 53-100%), 96% (range: 88-100%) for IBM and 100% (range: 93-100%), 99% (range: 92-100%), 98% (range: 93-100%) for CON (IBM vs CON, p=n.s. (all time points)). Primary goal of the study was the feasibility of the IBM approach. Ethics regulations did not allow to use weak grafts (≤2.0*106/kg) intentionally. However, 4 animals received weak grafts (CON n=2, 1.0 and 2.0*106/kg; IBM n=2, 1.2 and 1.3 *106/kg). Of interest, comparing data of these dogs showed that durations of leukopenia were similar (median 10 days, both groups), but duration of thrombocytopenia were different (median 8 days, IBM vs 22 days, CON). Additionally, long term donor chimerism was higher in the IBM (median 80% PBMC, 100% G) vs CON (median 61% PBMC, 42% G). Conclusion: First, IBM HSCT is a feasible and effective method to deliver HSC directly into the bone marrow following RIC in a canine HSCT model. Second, our preliminary data suggest that IBM HSCT reveals advantageous engraftment differences in regards to platelet recovery and donor chimerism kinetics compared to the IV HSCT when grafts with low HSC numbers were infused. Follow up data of this study and future studies will have to clarify these observations further. Disclosures No relevant conflicts of interest to declare.

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An Ex Vivo Bioengineered Human Liver Microenvironment to Support Hematopoietic Stem Cell Maintenance and Expansion

Blood ◽

10.1182/blood-2020-141437 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-10

Author(s):

Na Yoon Paik ◽

Grace E. Brown ◽

Lijian Shao ◽

Kilian Sottoriva ◽

James Hyun ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Fetal Liver ◽

Human Umbilical Cord ◽

Hematopoietic Stem ◽

Main Site

Over 17,000 people require bone marrow transplants annually, based on the US department of Health and Human Services (https://bloodcell.transplant.hrsa.gov). Despite its high therapeutic value in treatment of cancer and autoimmune disorders, transplant of hematopoietic stem cells (HSC) is limited by the lack of sufficient source material due primarily inadequate expansion of functional HSCs ex vivo. Hence, establishing a system to readily expand human umbilical cord blood or bone marrow HSCs in vitro would greatly support clinical efforts, and provide a readily available source of functional stem cells for transplantation. While the bone marrow is the main site of adult hematopoiesis, the fetal liver is the primary organ of hematopoiesis during embryonic development. The fetal liver is the main site of HSC expansion during hematopoietic development, furthermore the adult liver can also become a temporary extra-medullary site of hematopoiesis when the bone marrow is damaged. We have created a bioengineered micropatterned coculture (MPCC) system that consists of primary human hepatocytes (PHHs) islands surrounded and supported by 3T3-J2 mouse embryonic fibroblasts. Long-term establishment of stable PHH-MPCC allows us to culture and expand HSC in serum-free medium supplemented with pro-hematopoietic cytokines such as stem cell factor (SCF) and thrombopoietin (TPO). HSCs cultured on this PHH-MPCC microenvironment for two weeks expanded over 200-fold and formed tight clusters around the periphery of the PHH islands. These expanded cells also retained the expression of progenitor markers of Lin-, Sca1+, cKit+, as well as the long-term HSC phenotypic markers of CD48- and CD150+. In addition to the phenotypic analysis, the expanded cells were transplanted into lethally irradiated recipient mice to determine HSC functionality. The expanded cells from the PHH-MPCC microenvironment were able to provide multi-lineage reconstitution potential in primary and secondary transplants. With our bioengineered MPCC system, we further plan to scale up functional expansion of human HSC ex vivo and to better understand the mechanistic, cell-based niche factors that lead to maintenance and expansion HSC. Disclosures No relevant conflicts of interest to declare.

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Osteogenic- and Adipogenic- Differentiated Bone Marrow-Derived Mesenchymal Stem Cells Fail to Support Expansion of Adult Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.4810.4810 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4810-4810

Author(s):

Olga Kulemina ◽

Izida Minullina ◽

Sergey Anisimov ◽

Renata Dmitrieva ◽

Andrey Zaritskey

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Cord Blood ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Feeder Layers

Abstract Abstract 4810 Ex vivo expansion and manipulation of primitive hematopoietic cells has become a major goal in the experimental hematology, because of its potential relevance in the development of therapeutic strategies aimed at treating a diverse group of hematologic disorders. Osteoblasts, mesenchymal stem/progenitor cells (MSC/MPC), adipocytes, reticular cells, endothelial cells and other stromal cells, have been implicated in regulation of HSC maintenance in endosteal and perivascular niches. These niches facilitate the signaling networks that control the balance between self-renewal and differentiation. In the present study, we evaluated and compared the effects of three different stromal feeder layers on expansion of HSPC derived from BM and cord blood (CB): BM mesenchymal stem cells (MSC), osteoblast-differentiated BM mesenchymal stem cells (Ost-MSC) and adipocyte-differentiated BM mesenchymal stem cells (Ad-MSC). BM-MSC cultures were established from plastic adherent BM cell fractions and analyzed for immunophenotype, frequency of colony forming units (CFU-F), frequency of osteo- (CFU-Ost) and adipo- (CFU-Ad) lineage progenitors. Cultures with similar clonogenity (CFU-F: 26,4 ± 4,5%) and progenitors frequency (CFU-Ost: 14,7 ± 4,5%; CFU-Ad: 13,3 ± 4,5%) were selected for co-culture experiments. All MSC were positive for stromal cell-associated markers (CD105, CD90, CD166, CD73) and negative for hematopoietic lineage cells markers (CD34, CD19, CD14, CD45). CD34+ cells were separared from BM and CB samples by magnetic cell sorting (MACS) and analyzed for CD34, CD38 and CD45 expression. Feeder layers (MSC, Ost-MSC, Ad-MSC) were prepared in 24-well plates prior to co-culture experiments: MSCs (4×104 cells/well) were cultured for 24 h and either used for following experiments or stimulated to differentiate into either osteoblasts or adipoctes according to standard protocols. CD34+ cells (3500-10000 cells per well) were co-cultured in Stem Span media with or without a feeder layers and in the presence of cytokines (10 ng/mL Flt3-L, 10 ng/mL SCF, 10ng/mL IL-7) for 7 days. Expanded cells were analyzed for CD34, CD38 and CD45 expression. Results are shown on figures 1 and 2. As expected, CB-derived HSPC expanded much more effectively than BM-derived HSPC. The similar levels of expansion were observed for both, the total number of HSPC, and more primitive CD34+CD38- fraction in the presence of all three feeder layers. Ost-MSC supported CB-derived HSPC slightly better than MSC and Ad-MSC which is in a good agreement with data from literature (Mishima et.al., European Journal of Haematology, 2010), but difference was not statistically significant. In contrast, whereas BM-MSC feeder facilitated CD34+CD38- fraction in BM-derived HSPC, Adipocyte-differentiated MSC and osteoblast-differentiated MSC failed to support BM-derived CD34+CD38- expansion (11,4 ±.4 folds for MSC vs 0,9 ±.0,14 for Ad-MSC, n=5, p<0,01 and 0,92 ±.0,1 for Ost-MSC, n=5, p<0,01).Figure 1.Cord Blood HSPC ex vivo expansionFigure 1. Cord Blood HSPC ex vivo expansionFigure 2.Bone Marrow HSPC ex vivo expansionFigure 2. Bone Marrow HSPC ex vivo expansion Conclusion: BM- and CB-derived CD34+CD38- cells differ in their dependence of bone marrow stroma. Coctail of growth factors facilitate CB HSPC expansion irrespective of lineage differentiation of supporting MSC feeder layer. In contrast, primitive BM CD34+CD38- HSPC were able to expand only on not differentiated MSC. Disclosures: No relevant conflicts of interest to declare.

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RUNX1C-Mediated Reprogramming Of Human Bone Marrow Stromal Cells into Early Blood Progenitors

Blood ◽

10.1182/blood.v122.21.2463.2463 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2463-2463

Author(s):

Weihong Yin ◽

Christopher D Porada ◽

Stephen Walker ◽

Colin Bishop ◽

Graca Almeida-Porada

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Hematopoietic Cells ◽

Dermal Fibroblasts ◽

Hematopoietic Stem ◽

Surface Marker Expression ◽

Somatic Cell Reprogramming ◽

Hematopoietic Lineage

Abstract Somatic cell reprogramming to the hematopoietic lineage, either through a pluripotent state or directly, opens the possibility of production of a ready source of autologous hematopoietic stem cells (HSC) that can be used to treat/cure a wide variety of blood disorders. While it has previously been shown that dermal fibroblasts (HFF) can be directly reprogrammed to the hematopoietic lineage, the efficiency was relatively low and the resultant hematopoietic cells lacked multilineage differentiative potential. Stro1(+) isolated stromal progenitors (SIPs) can easily be isolated from the bone marrow (BM) and expanded ex-vivo to obtain clinically significant numbers of cells. In similarity to HSC, SIPs are derived from the mesoderm, and are intimately linked with HSC specification during ontogeny. As such, they are likely to be epigenetically closer to HSC than HFF, and therefore good candidates for reprogramming into hematopoietic cells. To verify the uniqueness of SIPs for reprogramming, we transduced SIPs and HFF with OCT4 and/or RUNX1C, a master transcription factor (TF) that triggers the developmental onset of definitive hematopoiesis, in the following combinations: 1) OCT4 alone; 2) RUNX1C alone; or 3) OCT4+RUNX1C. We then performed a timeline of gene/cell surface marker expression (using microarray, qRT-PCR, and flow cytometry) from day 3-16 post-transduction. Visual inspection of the cultures showed that, while reprogrammed colonies began to appear in SIPs cultures at day 9, no colonies were seen during this time period in HFF cultures. Flow cytometry and molecular analyses of colonies obtained from OCT4+RUNX1C combination demonstrated that expression of CD41, the earliest marker of commitment to the hematopoietic lineage, commenced within only 3-4 days and peaked at day 5-6, by which time ∼20% of SIPs expressed this marker. This peak in CD41 expression coincided with commencement of expression of CD34 and CD45, and maximal induction of several hematopoiesis-specific TFs and phenotypic markers such as PU.1, HOXB4, GATA2, MIXL, WNT3, KDR, CDX4, which occurred at 1-3 logs higher levels in SIPs than HFF. Further studies demonstrated that the chromatin remodeling function of OCT4 could be replaced with the histone methyltransferase inhibitor Bix-01294, with the combination of RUNX1C and Bix-01294 inducing levels of CD34 and CD41 expression by day 5 that were similar to those achieved with RUNX1C plus OCT4. The present studies thus take several important steps towards making the promise of producing autologous hematopoietic cells for transplantation via direct reprogramming a reality. Disclosures: No relevant conflicts of interest to declare.

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Ontogeny of Hematopoiesis

Blood ◽

10.1182/blood.v122.21.sci-4.sci-4 ◽

2013 ◽

Vol 122 (21) ◽

pp. SCI-4-SCI-4

Author(s):

Elaine Dzierzak

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Transcription Factors ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Developmental Time ◽

Hematopoietic Cell ◽

Conditional Knockout ◽

Hematopoietic Stem ◽

And Function

Abstract The current challenge in hematopoietic transplantation and regeneration therapies is acquiring and/or producing a reliable and plentiful source of hematopoietic stem cells (HSCs). Given that HSCs from bone marrow, peripheral, or umbilical cord blood undergo only limited/no expansion ex vivo, there is a high interest in understanding how the adult cohort of multipotent self-renewing HSCs are generated and expanded during embryonic development. The development of HSCs in vertebrate embryos begins in the major vasculature. HSCs are generated in a short window of developmental time starting at embryonic day E10.5 until E12 in the mouse embryo, and from gestational weeks four to six in the human embryo. The first HSCs, which are as potent as bone marrow HSCs in transplantation procedures, are generated in the aorta-gonad-mesonephros (AGM) region. HSCs are found in the major vasculature – aorta, vitelline artery, and umbilical artery – subsequent to the appearance of hematopoietic cell clusters closely associated with the lumenal walls of these vessels. The relationship of HSCs to these clusters and the identification of the precursors to HSCs have been recently established through genetic, phenotypic, and real-time imaging studies. Remarkably, HSCs and hematopoietic progenitors arise directly from a subset of endothelial cells (hemogenic endothelial cells) in a natural transdifferentiation event. They are made through a process called endothelial to hematopoietic cell transition (EHT). EHT and HSC generation is in part regulated through ventral-derived developmental signals and a group of pivotal (core) transcription factors, including Runx1 and Gata2. Conditional knockout strategies show that these transcription factors are required for the generation of vascular hematopoietic clusters and HSCs, suggesting a role in hematopoietic fate induction and/or cell expansion. Interestingly, whereas both Runx1 and Gata2 are required for HSC generation, only Gata2 remains essential in HSCs after their production. We are profiling hemogenic endothelial and HSCs by RNA sequencing so as to understand the complete genetic program that leads to generation of HSCs. These results will be discussed in the context of developmental signaling pathways (BMP4, Hedgehog, etc.) that appear to impact HSC generation and expansion, and the localized dynamic expression and function of Gata2 and Runx1 in vascular endothelial and hematopoietic cluster cells. Disclosures: No relevant conflicts of interest to declare.

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Down-Regulation of Homeobox Gene Ventx Promotes Expansion of Human Bone Marrow Hematopoietic Stem Cells (HSC)

Blood ◽

10.1182/blood.v118.21.1272.1272 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1272-1272

Author(s):

Hong (Jenny) Gao ◽

Xiaoming Wu ◽

Yan Sun ◽

Jiayun Lu ◽

Leslie E Silberstein ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Cell Fate ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Great Promise ◽

Down Regulation ◽

Hematopoietic Stem ◽

Proliferation And Differentiation

Abstract Abstract 1272 Hematopoietic stem cells (HSC) give rise to mature cells of all lineages of blood and immune systems. HSC transplantation has shown great promise in the treatment of malignancies, reconstitution of hematopoietic systems and HSC-based gene therapy. Cell intrinsic factors/pathways have been the targets of intensive investigation for its potential application in HSC expansion. Over the past decades, several critical cell fate determination pathways, such as the Wnt signaling pathways and senescence pathways have been implicated in the proliferation and differentiation of HSC. Moreover, overexpression of HoxB4 and BMI1 was found to be able to expand human HSC 2∼3 folds. Nevertheless, the regulatory mechanisms of HSC proliferation and differentiation remain incompletely understood and safe and efficacious expansion of human HSC remains as a fundamental challenge that limits the clinical application of HSC-based therapy. VentX is a human homologue of the Xenopus homeobox protein Xom of the BMP4 signaling pathway. Using Xenopus model and methods of reverse genetics, our recent work showed that VentX is a LEF/TCF associated Wnt repressor and an activator of senescence pathways. VentX expression is highly regulated and restricted in hematopoietic cells and serves a major regulator of hematopoietic cell differentiation. To explore the potential role of VentX in proliferation and differentiation of HSC during hematopoiesis, we quantified VentX expression during hematopoiesis, using qRT-PCR methods and examined the effects of altered VentX expression on HSC properties in vitro and in vivo. Our data showed that VentX expression is significantly up-regulated during oncogenesis of hematopioetic cells. We demonstrated that lentiviral knockdown of VentX allowed for more than 5 fold ex vivo expansion of human HSC with balanced lineage development. Importantly, transient knockdown of VentX by siRNA also led to expansion of HSC. The effect of VentX down-regulation on the expansion of human HSC was also demonstrated by enhanced engraftment in the SCID/NODγ2null mouse model. Consistent with its role as a novel regulator of HSC, overexpression of VentX significantly inhibited clonal genesis of HSC. Mechanistically, we demonstrated that VentX controls the expression of cell cycle regulators downstream of the Wnt and senescence pathways, such as the C-myc, CyclinD1 and p21. In summary, using methods of reverse genetic and developmental modeling, we identified VentX as a novel regulator for expansion of human BM HSC. The results of our investigations provide novel insight in regulating HSC proliferation and differentiation. In addition, the findings that transient down-regulation of VentX by SiRNA lead to efficient expansion of bone marrow HSC suggests that VentX may serve as a novel target for safe expansion of HSC for its potential clinical applications. Disclosures: No relevant conflicts of interest to declare.

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Growth Factor Independence 1b (Gfi1b) Regulates The Commitment, Differentiation and Expansion Of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v122.21.2433.2433 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2433-2433

Author(s):

Tarik Moroy ◽

Cyrus Khandanpour ◽

Joseph Krongold

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Mouse Bone Marrow ◽

Paracrine Factors ◽

Self Renewal ◽

Hematopoietic Stem

Abstract The efficacy of bone marrow stem cell transplantation is the therapy of choice for many hematopoietic diseases, in particular leukemia and lymphoma. This therapy is critically dependent on the transfer of sufficient numbers of hematopoietic stem cells (HSCs), which possess the capacity for self-renewal and can fully reconstitute the hematopoietic system. As such, the development of techniques for the expansion of fully functional HSCs is of significant clinical interest. By transiently manipulating the factors that govern HSC homeostasis it has been proposed that HSCs can be expanded without the loss of essential stem cell characteristics. Previously we have observed that ablation of the gene encoding the transcription factor Gfi1b in-vivo results in a dramatic expansion and mobilization of hematopoietic stem cells in the bone marrow and periphery. More recent data suggest that the blood mobilization of Gfi1b deficient HSCs is very likely mediated by a deregulation of the integrin expression. These data led us to hypothesize that Gfi1b could be a potential target for ex-vivo treatment and expansion of HSCs. Indeed, when deletion of Gfi1b was induced in whole bone marrow ex-vivo, HSCs showed a significant expansion in both in absolute number and in terms of proportion of bone marrow. We followed HSCs in ex-vivo expansion cultures from mouse bone marrow by tracking expression of the surface marker CD48, which indicates whether an HSC has transitioned to a differentiation committed multi-potent progenitor. We observed that Gfi1b null HSCs expanded without up-regulating CD48 in contrast to wt HSCs. This suggests that Gf11b deficient HSCs underwent symmetric self-renewal type cell divisions at a significantly increased frequency, when compared to wt HSCs. We had previously shown that HSCs lacking Gfi1b cycle at a faster rate than control HSCs. The combination of increased cell division and preferential self-renewal of Gfi1b-/- HSCs indicates that inhibition of Gfi1b may be the ideal strategy for ex-vivo HSC expansion. As well, in accordance with this preference for self-renewal, Gfi1b null HSCs that were cultured under myeloid differentiation conditions remained primarily in an undifferentiated state as defined by a lack of the myeloid surface markers Gr1 and Mac1. These cultures also demonstrated increased long term colony forming capacity versus controls, further supporting an undifferentiated phenotype in Gfi1b-/- cells. Because the stem cell niche is a highly complex and heterogeneous environment we also investigated whether bone marrow in which Gfi1b has been deleted exerts paracrine effects that contributed to HSC expansion. Co-Culture assays demonstrated that Gfi1b-/- bone marrow was able to induce an expansion of progenitors in wild-type bone marrow of more than 10 fold compared to Gfi1b-/+ bone marrow. Interestingly cells co-cultured with Gfi1b null bone marrow also exhibited an overall proliferation advantage after short-term cultures. This suggests that not only does Gfi1b deletion induce HSC expansion via cell intrinsic mechanisms, but also points to the possibility that this occurs through paracrine factors that alter bone marrow homeostasis. Disclosures: No relevant conflicts of interest to declare.

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