Senescent Mesenchymal Stem Cells Present at the Early Post Allogeneic Hematopoietic Stem Cell Transplantation Period Are Associated with an Increased Incidence of Acute Graft-Versus Host Disease and Increased Plasma Levels of Fas Ligand

Abstract Introduction Mesenchymal stem cells (MSC) are a key component of the hematopoietic niche. In the allogeneic hematopoietic stem cell transplantation (allo-HSCT) setting, the bone marrow stroma, and thus, their MSC remain of host origin. In a preliminary study we observed that, some patients at the early post allo-HSCT period, presented senescent bone marrow (BM) MSC, a finding that has not been previously described nor studied. The aims of our current study were: a) To multiparametrically characterize BM MSC at the early post allo-HSCT period (day +21). b) To confirm senescence of MSC and correlate with clinical and biological parameters (including biomarkers). c) To compare these cells with those from healthy donors. Methods We obtained BM samples on the day +21 post allo-HSCT from 136 patients. MSC were isolated, ex-vivo expanded and characterized, according to the criteria of the International Society for Cellular Therapy. We also obtained samples from peripheral blood at same day +21, for the study of biomarkers, which were analyzed by Luminex technique. The data were correlated with information from complete blood counts (CBC), and the morphological study of the bone marrow the same day. MSC from healthy donors were used as control. Results Patient baseline and transplant related characteristics are detailed in table 1. MSC were expanded ex-vivo showing normal growth, which were cryopreserved in passage 3 in the 33% (n=45/136) of the patients (Group-MSC-N). On the other hand, the remaining 67% (n=91/136), MSC showed premature signs of senescence, thus, not reaching to passage 1 (Group-MSC-S). Full BM chimerism on day+21 was seen in both groups (p=0.03). Concerning acute graft versus-host disease (aGVHD), in the Group-MSC-S, the incidence was 73% compared to 44% in the Group-MSC-N (p=0.001). The median time of the onset of aGVHD was 43 and 37 days respectively (p=0.04). The majority of cases presented with grades I-II in both groups. Plasma levels of biomarkers showed increased levels of Fas Ligand in the Group-MSC-S (p=0.009). There were no statistically significant differences regarding mortality nor relapse rates. Conclusions Bone marrow Mesenchymal stem cells may be severely damaged in some allo-HSCT recipients early after transplantation (day+21). This fact strongly correlates with the risk of development of acute GVHD. Disclosures Díez-Campelo: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Treatment of Intractable Graft-Versus-Host Disease with Bone Marrow Derived Mesenchymal Stem Cells - A Pilot Clinical Trial.

Blood ◽

10.1182/blood.v110.11.4974.4974 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4974-4974 ◽

Cited By ~ 1

Author(s):

Je-Hwan Lee ◽

Seong-Jun Choi ◽

Jung-Hee Lee ◽

Miee Seol ◽

Young-Shin Lee ◽

...

Keyword(s):

Clinical Trial ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Graft Versus Host Disease ◽

Ex Vivo ◽

Performance Status ◽

Hematopoietic Stem ◽

Host Disease ◽

Graft Versus Host

Abstract Recent studies have shown that mesenchymal stem cells (MSCs) have profound immunomodulatory function, both in vitro and in vivo. There are several reports to treat effectively therapy-resistant graft-versus-host disease (GVHD) using ex vivo expanded MSCs. We performed a pilot clinical trial to treat intractable GVHD with bone marrow derived MSCs. All of 5 patients, 3 males and 2 females, with steroid-refractory GVHD were included in this study. Age range was 27 to 48 years old. Diagnosis of underlying disease was CML in 2, AML in 1, ALL in 1, and MDS in 1. Hematopoietic stem cell (HSC) donor was a sibling in 2 and an unrelated volunteer in 3. The donor of MSCs was a sibling in all patients: two donors were the same to HSC donors, but other three were not. About 20 mL of bone marrow was aspirated from donors and MSCs were cultured ex vivo. After about 3 weeks, MSCs were harvested for the first infusion, and 4 more weeks’ culture was done for the planned second infusion. The infused doses of MSCs were 5.3 to 6.9 x 106/kg for the first infusion and 1.8 to 7.0 x 106/kg for the second infusion. The onset of GVHD was post-transplant day 24 to 191 and the times from the onset of GVHD to the infusion of MSCs were 73 to 2469 days. There were no adverse events related to the infusion of MSCs. Three patients did not show any response to the treatment of MSCs for GVHD and two showed minimal response: transient improvement of jaundice and diarrhea without improvement of skin GVHD lesions in one patient, and improvement of performance status without significant increase of pulmonary function test parameters in another patient with lung involvement of GVHD. There were no significant changes in hemoglobin, and peripheral blood counts of platelets, leukocytes, lymphocytes, CD3+ cells, CD4+ cells, CD8+ cells, NK cells and B cells over 6 months’ period after the infusion of MSCs. Results of our pilot study suggest that the treatment effects of MSCs may be limited in patients with chronic established GVHD. Further studies on MSCs for GVHD should be focused on acute or exacerbated GVHD.

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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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Ex Vivo Gene Therapy: Graft-versus-host Disease (GVHD) in NSG™ (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) Mice Transplanted with CD34+ Human Hematopoietic Stem Cells

Toxicologic Pathology ◽

10.1177/0192623319844484 ◽

2019 ◽

Vol 47 (5) ◽

pp. 656-660 ◽

Cited By ~ 3

Author(s):

Sundeep Chandra ◽

Patrizia Cristofori ◽

Carlos Fonck ◽

Charles A. O’Neill

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Graft Versus Host Disease ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Host Disease ◽

Graft Versus Host ◽

Human Cd34

A therapeutic option for monogenic disorders is gene therapy with ex vivo-transduced autologous hematopoietic stem cells (HSCs). Safety or efficacy studies of ex vivo-modified HSCs are conducted in humanized mouse models after ablation of the murine bone marrow and transfer of human CD34+ HSCs. Engrafted human CD34+ cells migrate to bone marrow and differentiate into various human hematopoietic lineages. A 12-week study was conducted in NSG™ mice to evaluate engraftment, differentiation, and safety of human CD34+ cells that were transduced ( ex vivo) with a proprietary lentiviral vector encoding a human gene (BMRN-1) or a mock (green fluorescent protein) vector. Several mice intravenously injected with naive CD34+ cells or transduced CD34+ cells had variable lymphohistiocytic inflammatory cell infiltrates and microgranulomas in the liver and lungs consistent with graft-versus-host disease (GVHD). Spleen, bone marrow, stomach, reproductive tract, but not the skin had similar inflammatory changes. Ex vivo viral transduction of CD34+ cells did not impact engraftment or predispose to xenogeneic GVHD.

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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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Wnt/β-catenin signaling mediates the abnormal osteogenic and adipogenic capabilities of bone marrow mesenchymal stem cells from chronic graft-versus-host disease patients

Cell Death and Disease ◽

10.1038/s41419-021-03570-6 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Han-zhou Qi ◽

Yi-ling Ye ◽

Yuan Suo ◽

Hong Qu ◽

Hai-yan Zhang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Graft Versus Host Disease ◽

Underlying Mechanism ◽

Hematopoietic Stem ◽

Host Disease ◽

Graft Versus Host ◽

Ppar Γ ◽

Marrow Mesenchymal Stem Cells

AbstractChronic graft-versus-host disease (cGVHD) is the main cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mesenchymal stem cells (MSCs) in bone marrow (BM) remain unclear in the pathophysiology of cGVHD. In this study, we analyzed BM-MSCs from 66 patients after allo-HSCT, including 33 with active cGVHD and 33 without cGVHD. BM-MSCs showed similar morphology, frequency, phenotype, and proliferation in patients with or without cGVHD. MSCs from the active cGVHD group showed a decreased apoptosis rate (P < 0.01). Osteogenic capacity was increased while adipogenic capacity was decreased in the active cGVHD MSCs compared with no-cGVHD MSCs. The expressions of osteogenic gene RUNX2 and COL1A1 were higher (P < 0.001) while adipogenic gene PPAR-γ and FABP4 were lower (P < 0.001) in the active cGVHD MSCs than no-cGVHD MSCs. These changes were associated with the severity of cGVHD (P < 0.0001; r = 0.534, r = 0.476, r = −0.796, and r = −0.747, respectively in RUNX2, COL1A1, PPAR-γ, and FABP4). The expression of Wnt/β-catenin pathway ligand Wnt3a was increased in cGVHD-MSCs. The dysfunction of cGVHD-MSCs could be reversed by Dickkopf related protein 1(DKK1) to inhibit the binding of Wnt3a. In summary, the differentiation of BM-MSCs was abnormal in active cGVHD, and its underlying mechanism is the upregulated of Wnt3a through Wnt/β-catenin signaling pathway of MSCs.

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Angiogenin Is a Niche-Secreted RNase Which Regulates Hematopoietic Regeneration

Blood ◽

10.1182/blood.v128.22.171.171 ◽

2016 ◽

Vol 128 (22) ◽

pp. 171-171

Author(s):

Lev Silberstein ◽

Kevin Goncalves ◽

Nicholas Severe ◽

Guo-fu Hu ◽

David T. Scadden

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Board Of Directors ◽

Advisory Committees ◽

Self Renewal ◽

Cellular Source ◽

Equity Ownership ◽

Ng2 Cells

Abstract Background. Identification of novel niche factors is critical for understanding of regulatory mechanisms which control HSPC cell fate decisions and design of novel pro-regenerative therapies. We have developed a proximity-based differential single cell analysis approach to the study of the bone marrow niche, which showed that individual osteolineage cells located in close proximity to transplanted HSPC are enriched for expression of niche factors, and have previously reported identification of IL18 and Embigin as regulators of HSPC quiescence. Here we describe the results of in vivo validation of Angiogenin (ANG) - the third molecule highlighted using the above strategy - as a potent regulator of HSPC quiescence and regeneration. Results. ANG is a secreted RNase which is known to promote angiogenesis, proliferation of cancer cells and also enhance cell survival in response to stress. Analysis of primitive cells subsets in the ANG knock-out mice (AngKO mice) revealed a 1.4-fold increase in the frequency and absolute number of long-term hematopoietic stem cells (LT-HSCs). Subsequent BrdU incorporation and cell cycle studies demonstrated increased proliferative activity in the primitive HSPC compartment indicating that ANG regulates HSPC quiescence. To confirm these findings functionally and to assess the effect of ANG on self-renewal, we exposed AngKO animals to weekly 5-FU injections and performed serial transplantation experiments of WT LT-HSCs into AngKO hosts. We noted significantly increased mortality of AngKO mice in both experimental settings; in a competitive transplant assay, we observed almost complete absence of engraftment by WT cells in the secondary hosts, in keeping with the exhaustion phenotype. Consistently, exposure of AngKO animals to a different type of hematopoietic stress, such as ageing, resulted in development of peripheral blood cytopenias and marked reduction in the number and frequency of HSPC. ANG is expressed in multiple non-hematopoietic cell types in the bone marrow, including osteoprogenitors, mature osteoblasts and nestin-positive mesenchymal stem cells and NG2-positive arteriolar sheath cells. To establish the predominant cellular source of ANG in the niche, we crossed Ang "floxed" mice with the animals in which tamoxifen-inducible Cre-recombinase was driven by the promoters targeting these cell subsets and examined the effect on hematopoiesis. We found that deletion of ANGfrom Osx+, Nes+ and NG2+ cells resulted in an increase of the number of LT-HSC and more active cycling of LT-HSC, short-term HSC (ST-HSC) and multi-potent progenitors (MPP) while ANGdeletion in mature osteoblasts had no effect on these cell populations, but was associated with an increase in number and more active cycling of common lymphoid progenitors (CLP), as was also seen upon ANGdeletion from Nes+ and NG2+ cells. These results indicate that the target cell population which is regulated by ANG depends on the cellular source. Interestingly, transplantation of WT bone marrow into the animals with Osx-specific ANG deletion resulted in development of macrocytic anemia and neutropenia at 6 months, thus indicating that Angiogenin deficiency in the niche is sufficient for the development of the hematopoietic failure. Impaired long-term reconstitution was also observed when ANG was deleted from Nestin+ mesenchymal stem cells but not col1+ mature osteoblasts in the recipient mice. Our findings that the absence of ANG negatively impacts HSPC self-renewal prompted us to investigate whether exposure of HSPC to recombinant ANG protein will have the opposite effect and enhance hematopoietic regeneration. We therefore treated mouse LT-HSCs with recombinant ANG or vehicle control ex-vivo for 2 hours and competitively transplanted them into lethally irradiated WT recipients. We found that ANG-treated LT-HSCs displayed a significantly higher long-term reconstituting ability, which persisted into the secondary transplants. Similar data were obtained following treatment of CD34+ cord blood cells with human ANG. Conclusion. Our work defines ANG as a previously unrecognized regulator of HSPC quiescence and self-renewal and suggests that it can be explored as a potential therapeutic agent to promote hematopoietic regeneration. Disclosures Scadden: Teva: Consultancy; Apotex: Consultancy; Bone Therapeutics: Consultancy; GlaxoSmithKline: Research Funding; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Dr. Reddy's: Consultancy.

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Cotransplantation of HLA-Identical Mesenchymal Stem Cells and Hematopoietic Stem Cells in Chinese Patients with Hematologic Diseases.

Blood ◽

10.1182/blood.v110.11.5061.5061 ◽

2007 ◽

Vol 110 (11) ◽

pp. 5061-5061

Author(s):

Xiaoyan Zhang ◽

Jianyong Li ◽

Kejiang Cao ◽

Hanxin Wu ◽

Hua Lu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Hematopoietic Stem Cells ◽

Mononuclear Cells ◽

Ex Vivo ◽

Chronic Gvhd ◽

Chinese Patients ◽

Hematopoietic Stem ◽

Hematologic Diseases

Abstract Background: Mesenchymal stem cells(MSCs) can be isolated from bone-marrow and expanded ex-vivo, may support hematopoietic reconstruction and mitigate graft-versus-host disease (GVHD) in hematopoietic stem cells transplantation (HSCT). We hope to explore the feasibility and safety of cotransplantation culture-expanded MSCs and hematopoietic stem cells (HSCs) from the same human leucocyte antigen (HLA)-identical sibling donor in patients with hematologic diseases. Methods: Bone marrow mononuclear cells from healthy donors were cultured and expanded ex-vivo. Immunophenotype, karyotye, immunosuppressive property of the harvested MSCs were characterized. Patients were cotransplanted with HSCs and MSCs from the same donor. Hematopoietic reconstruction, complications and clinical outcomes after transplantation in these patients were observed. Results: (1.77±0.40)×106/kg (donor’s weight) MSCs were successfully expanded from 23.6±5.96ml bone marrow samples. They had normal karyotype and were CD73, CD90, CD105 positive and CD34, CD45, HLA-DR negative. They can inhibit mixed lymphocyte reactions (MLRs). Twelve patients were undergoing cotransplantation. No adverse response was observed during and after the infusion of allogenic MSCs. Hematopoietic reconstruction were rapid. Two patients developed grade II∼IV acute GVHD. Two patients developed systermatic chronic GVHD. Four patients suffered from cytomegalovirus (CMV) infection but were cured at last. Till now, seven patients have been alive for 29∼57 months and five patients died. Conclusion: MSCs identified by immunophenotype analysis can be isolated from human bone marrow, expanded effectively by culture. Their quality and quantity are suitable for clinical use. It is safe and feasible to cotransplant patients with allogenic culture-expanded MSCs and HSCs.

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Ex Vivo Expansion and Transplantation of Hematopoietic Stem/Progenitor Cells Supported by Mesenchymal Stem Cells from Human Umbilical Cord Blood

Cell Transplantation ◽

10.3727/000000007783465073 ◽

2007 ◽

Vol 16 (6) ◽

pp. 579-585 ◽

Cited By ~ 48

Author(s):

Guo-Ping Huang ◽

Zhi-Jun Pan ◽

Bing-Bing Jia ◽

Qiang Zheng ◽

Chun-Gang Xie ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Umbilical Cord Blood ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Cd34 Cells

Human mesenchymal stem cells (MSCs) are multipotential and are detected in bone marrow (BM), adipose tissue, placenta, and umbilical cord blood (UCB). In this study, we examined the ability of UCB-derived MSCs (UCB-MSCs) to support ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs) from UCB and the engraftment of expanded HSPCs in NOD/SCID mice. The result showed that UCB-MSCs supported the proliferation and differentiation of CD34+ cells in vitro. The number of expanded total nucleated cells (TNCs) in MSC-based culture was twofold higher than cultures without MSC (control cultures). UCB-MSCs increased the expansion capabilities of CD34+ cells, long-term culture-initiating cells (LTC-ICs), granulocyte-macrophage colony-forming cells (GM-CFCs), and high proliferative potential colony-forming cells (HPP-CFCs) compared to control cultures. The expanded HSPCs were transplanted into lethally irradiated NOD/SCID mice to assess the effects of expanded cells on hematopoietic recovery. The number of white blood cells (WBCs) in the peripheral blood of mice transplanted with expanded cells from both the MSC-based and control cultures returned to pretreatment levels at day 25 posttransplant and then decreased. The WBC levels returned to pretreatment levels again at days 45–55 posttransplant. The level of human CD45+ cell engraftment in primary recipients transplanted with expanded cells from the MSC-based cultures was significantly higher than recipients transplanted with cells from the control cultures. Serial transplantation demonstrated that the expanded cells could establish long-term engraftment of hematopoietic cells. UCB-MSCs similar to those derived from adult bone marrow may provide novel targets for cellular and gene therapy.

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Bone Marrow Derived Mesenchymal Stem Cells and Radiation Response: a Kinetic Study of Gene Response Using Microarray Analysis.

Blood ◽

10.1182/blood.v114.22.4575.4575 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4575-4575

Author(s):

Philippe Garrigou ◽

Jean-Francois Mayol ◽

Catherine Mouret ◽

Christophe Delaunay ◽

Michel Drouet ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Target Genes ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Short Term ◽

Hematopoietic Stem ◽

Pai 1

Abstract Abstract 4575 Mesenchymal Stem cells (MSC) are an important radiosensitive component of the so called hematopoietic stem cell niche. Importantly this supportive microenvironment influences the stem cell repopulation capacity as well as the quiescent/non proliferative state of hematopoietic cells. Senescence is considered as a major process in MSC response following irradiation. However, other studies have reported in mice the reduction of the pool of bone marrow mesenchymal stem/progenitor cells following TBI independently of senescence. An altered osteoblastic differentiation was pointed out in these studies. Furthermore, MSC have been shown to be involved in the repair of ionizing radiation damage of distant epithelial sites which requires adherence genes mitigation. The aim of this study was to clarify some of these points using an in vitro model of irradiation and short term culture. Briefly, confluent human BM-MSC were irradiated at the dose of 2.5 Gy (dose rate: 95 cGy.min-1) and immediately put into culture (Minimum essential medium supplemented with 10% FCS and 10 μg/ml of ciprofloxacin, penicillin and streptomycin). Six, 12, 24, 48 and 72 hours after irradiation, cells were harvested and lysed. Total RNAs were purified using the automatic Qiacube system (Qiagen,Courtaboeuf, France) and processed on DNA microarray scanner (Agilent technologies Inc.) according to supplier's recommendations. Data were analyzed with GeneSpring GX Expresion Analysis software version 10.0 (Agilent) in order to identify the transduction pathways involved. No apoptosis was observed during this short term incubation. Among other genes we identified plasminogen activator inhibitor 1 (PAI-1) as a factor highly upregulated after irradiation, in addition to CD151. This is in accordance with MSC response to nutrient-poor, hypoxic stress environment (Copland et al, Stem cells 2009). As MSC are radiosensitive cells, this may indicate that PAI impacts MSC survival through the mitigation of their adhesiveness to surrounding matrices. As PAI-1 is an important factor involved in the balance of blood coagulation and fibrinolysis as well as in the regulation of angiogenesis, one may speculate the consequences of PAI-1 release from MSC on blood homeostasis. Work is going on to describe the main response target genes. This could allow us to identify therapeutic strategies based on ex-vivo or in vivo manipulation of MSC in a purpose of tissue remodelling. Disclosures: No relevant conflicts of interest to declare.

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Msi2 Maintains Normal Hematopoietic Stem Cell Self-Renewal

Blood ◽

10.1182/blood.v118.21.222.222 ◽

2011 ◽

Vol 118 (21) ◽

pp. 222-222 ◽

Cited By ~ 1

Author(s):

Michael G Kharas ◽

Christopher Lengner ◽

Fatima Al-Shahrour ◽

Benjamin L. Ebert ◽

George Q. Daley

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Board Of Directors ◽

Bone Marrow Cells ◽

Advisory Committees ◽

Self Renewal ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Marrow Cells

Abstract Abstract 222 Genes that regulate normal hematopoietic stem cells are commonly dysregulated in hematopoietic malignancies. Recently we published that the Msi2 RNA binding protein is an important modulator in both normal hematopoietic stem cells and leukemia (Kharas et al, Nat. Medicine 2010). The closely related Msi1 protein has been shown to regulate mRNA translation through binding to the 3'UTR. Based on the high homology in the RNA recognition motifs, Msi2 has been considered to have similar functions. Moreover, increased MSI2 expression in chronic myelogenous leukemia blast crisis and acute myeloid leukemia predicts a worse clinical prognosis. Previous studies have mainly utilized shRNAs to functionally assess the role of Msi2 in the hematopoietic compartment. However, it remains unclear how Msi2 affects hematopoietic stem cells (HSC) and what are its critical mRNA targets. To develop a model focusing on the HSC compartment and to avoid potential compensatory mechanisms during development, we created Msi2 conditional knockout mice and crossed them with Mx1-Cre mice. We induced excision with poly(I):poly(C), (pIpC), and tested the peripheral blood, bone marrow cells and splenocytes by Southern blotting and QPCR analysis to verify Msi2 deletion. Loss of Msi2 mRNA was confirmed in the Lineagelo, Sca1+ and c-Kit+ (LSK) population. Msi2 deleted bone marrow contained reduced myeloid colony forming capacity and replating efficiency. Mice conditionally deleted for Msi2 had normal white blood cell counts but smaller spleens. In addition, we observed normal percentages of the mature hematopoietic populations, including the myeloid and lymphoid compartments. Nevertheless, absolute numbers of long-term HSCs in the bone marrow were reduced by 3-fold. Bone marrow cells non-competitively transplanted into primary and secondary recipient mice showed a dramatic reduction in HSC chimerism. This defect was also observed when bone marrow was transplanted first to allow engraftment followed by Msi2 deletion. Furthermore, we were able to recapitulate this defect in vitro using the cobblestone-forming activity assay. These results indicate that Msi2 is both an important regulator of normal HSC maintenance and required for efficient engraftment. Most interestingly, Msi2 HSCs failed to maintain a normal quiescent HSC population. We performed microarrays to identify the pathways altered in the LSK population. The Msi2 deficient LSKs showed a reduced self-renewal and increased differentiation gene signature. Gene expression analysis indicates a defective self-renewal program in Msi2-deficient HSCs that is identical to the program gained in leukemic stem cells. These data suggest that MSI2 is a critical modulator of HSCs and may help explain its requirement in the most aggressive myeloid leukemias. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.

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