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Bucar S, Branco ADDM, Mata MF, Milhano JC, Caramalho Í, Cabral JMS, Fernandes-Platzgummer A, da Silva CL. Influence of the mesenchymal stromal cell source on the hematopoietic supportive capacity of umbilical cord blood-derived CD34 +-enriched cells. Stem Cell Res Ther 2021; 12:399. [PMID: 34256848 PMCID: PMC8278708 DOI: 10.1186/s13287-021-02474-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Accepted: 06/24/2021] [Indexed: 12/18/2022] Open
Abstract
Background Umbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem/progenitor cells (HSPC). To overcome the low cell number per UCB unit, ex vivo expansion of UCB HSPC in co-culture with mesenchymal stromal cells (MSC) has been established. Bone marrow (BM)-derived MSC have been the standard choice, but the use of MSC from alternative sources, less invasive and discardable, could ease clinical translation of an expanded CD34+ cell product. Here, we compare the capacity of BM-, umbilical cord matrix (UCM)-, and adipose tissue (AT)-derived MSC, expanded with/without xenogeneic components, to expand/maintain UCB CD34+-enriched cells ex vivo. Methods UCB CD34+-enriched cells were isolated from cryopreserved mononuclear cells and cultured for 7 days over an established feeder layer (FL) of BM-, UCM-, or AT-derived MSC, previously expanded using fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (HPL) supplemented medium. UCB cells were cultured in serum-free medium supplemented with SCF/TPO/FLT3-L/bFGF. Fold increase in total nucleated cells (TNC) as well as immunophenotype and clonogenic potential (cobblestone area-forming cells and colony-forming unit assays) of the expanded hematopoietic cells were assessed. Results MSC from all sources effectively supported UCB HSPC expansion/maintenance ex vivo, with expansion factors (in TNC) superior to 50x, 70x, and 80x in UCM-, BM-, and AT-derived MSC co-cultures, respectively. Specifically, AT-derived MSC co-culture resulted in expanded cells with similar phenotypic profile compared to BM-derived MSC, but resulting in higher total cell numbers. Importantly, a subpopulation of more primitive cells (CD34+CD90+) was maintained in all co-cultures. In addition, the presence of a MSC FL was essential to maintain and expand a subpopulation of progenitor T cells (CD34+CD7+). The use of HPL to expand MSC prior to co-culture establishment did not influence the expansion potential of UCB cells. Conclusions AT represents a promising alternative to BM as a source of MSC for co-culture protocols to expand/maintain HSPC ex vivo. On the other hand, UCM-derived MSC demonstrated inferior hematopoietic supportive capacity compared to MSC from adult tissues. Despite HPL being considered an alternative to FBS for clinical-scale manufacturing of MSC, further studies are needed to determine its impact on the hematopoietic supportive capacity of these cells.
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Affiliation(s)
- Sara Bucar
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,Associate Laboratory i4HB - Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - André Dargen de Matos Branco
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,Associate Laboratory i4HB - Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Márcia F Mata
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - João Coutinho Milhano
- Hospital São Francisco Xavier, Centro Hospitalar de Lisboa Ocidental, Lisboa, Portugal
| | | | - Joaquim M S Cabral
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,Associate Laboratory i4HB - Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Ana Fernandes-Platzgummer
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,Associate Laboratory i4HB - Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal. .,Associate Laboratory i4HB - Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
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Harada T, Hirabayashi Y, Hatta Y, Tsuboi I, Glomm WR, Yasuda M, Aizawa S. Kinetics of hematopoietic stem cells and supportive activities of stromal cells in a three-dimensional bone marrow culture system. Growth Factors 2015; 33:347-55. [PMID: 26431462 DOI: 10.3109/08977194.2015.1088534] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
In the bone marrow, hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment. Previously, we established a 3D bone marrow culture system. In this study, we analyzed the kinetics of hematopoietic cells, and more than 50% of hematopoietic progenitor cells, including CFU-Mix, CFU-GM and BFU-E in 3D culture were in a resting (non-S) phase. Furthermore, we examined the hematopoietic supportive ability of stromal cells by measuring the expression of various mRNAs relevant to hematopoietic regulation. Over the 4 weeks of culture, the stromal cells in the 3D culture are not needlessly activated and "quietly" regulate hematopoietic cell proliferation and differentiation during the culture, resulting in the presence of resting hematopoietic stem cells in the 3D culture for a long time. Thus, the 3D culture system may be a new tool for investigating hematopoietic stem cell-stromal cell interactions in vitro.
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Affiliation(s)
| | - Yukio Hirabayashi
- a Department of Functional Morphology and
- b Department of Medicine , Nihon University School of Medicine , Tokyo , Japan
| | - Yoshihiro Hatta
- b Department of Medicine , Nihon University School of Medicine , Tokyo , Japan
| | | | - Wilhelm Robert Glomm
- c Department of Chemical Engineering , Norwegian University of Science and Technology , Trondheim , Norway , and
| | - Masahiro Yasuda
- d Department of Chemical Engineering , Osaka Prefecture University , Osaka , Japan
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Simic P, Zainabadi K, Bell E, Sykes DB, Saez B, Lotinun S, Baron R, Scadden D, Schipani E, Guarente L. SIRT1 regulates differentiation of mesenchymal stem cells by deacetylating β-catenin. EMBO Mol Med 2013; 5:430-40. [PMID: 23364955 PMCID: PMC3598082 DOI: 10.1002/emmm.201201606] [Citation(s) in RCA: 211] [Impact Index Per Article: 17.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2012] [Revised: 11/12/2012] [Accepted: 11/14/2012] [Indexed: 12/13/2022] Open
Abstract
Mesenchymal stem cells (MSCs) are multi-potent cells that can differentiate into osteoblasts, adipocytes, chondrocytes and myocytes. This potential declines with aging. We investigated whether the sirtuin SIRT1 had a function in MSCs by creating MSC specific SIRT1 knock-out (MSCKO) mice. Aged MSCKO mice (2.2 years old) showed defects in tissues derived from MSCs; i.e. a reduction in subcutaneous fat, cortical bone thickness and trabecular volume. Young mice showed related but less pronounced effects. MSCs isolated from MSCKO mice showed reduced differentiation towards osteoblasts and chondrocytes in vitro, but no difference in proliferation or apoptosis. Expression of β-catenin targets important for differentiation was reduced in MSCKO cells. Moreover, while β-catenin itself (T41A mutant resistant to cytosolic turnover) accumulated in the nuclei of wild-type MSCs, it was unable to do so in MSCKO cells. However, mutating K49R or K345R in β-catenin to mimic deacetylation restored nuclear localization and differentiation potential in MSCKO cells. We conclude that SIRT1 deacetylates β-catenin to promote its accumulation in the nucleus leading to transcription of genes for MSC differentiation.
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Affiliation(s)
- Petra Simic
- Glenn Laboratory for the Science of Aging, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
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Becquart P, Cambon-Binder A, Monfoulet LE, Bourguignon M, Vandamme K, Bensidhoum M, Petite H, Logeart-Avramoglou D. Ischemia is the prime but not the only cause of human multipotent stromal cell death in tissue-engineered constructs in vivo. Tissue Eng Part A 2012; 18:2084-94. [PMID: 22578283 DOI: 10.1089/ten.tea.2011.0690] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Local tissue ischemia is a prime cause responsible for the massive cell death in tissue-engineered (TE) constructs observed postimplantation. To assess the impact of ischemia on the death of implanted human multipotent stromal cells (hMSCs), which have great potential for repairing damaged tissues, we hereby investigated the in vivo temporal and spatial fate of human Luc-GFP-labeled MSCs within fibrin gel/coral scaffolds subcutaneously implanted in nude mice. In vivo bioluminescence imaging monitoring and histological analyses of the constructs tested confirmed the irremediable death of hMSCs over 30 days postimplantation. The kinetics of expression of three hypoxic/ischemic markers (HIF-1α, LDH-A, and BNIP3) was also monitored. Our results provided evidence that hMSCs located within the core of implanted constructs died faster and predominantly and strongly expressed the aforementioned ischemic markers. In contrast, cells located in the outer regions of TE constructs were reperfused by neovascularization and were still viable (as evidenced by their ex-vivo proliferative potential) at day 15 postimplantation. These results support the explanation that in the central part of the constructs tested, death of hMSCs was due to ischemia, whereas in the periphery of these constructs, cell death was due to another mechanism that needs to be elucidated.
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Affiliation(s)
- Pierre Becquart
- Laboratory of Bioengineering and Biomechanics for Bone and Articulations, UMR 7052 CNRS, Université Paris Diderot, Sorbonne Paris Cité, Paris, France
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Diaz-Solano D, Wittig O, Ayala-Grosso C, Pieruzzini R, Cardier JE. Human olfactory mucosa multipotent mesenchymal stromal cells promote survival, proliferation, and differentiation of human hematopoietic cells. Stem Cells Dev 2012; 21:3187-96. [PMID: 22471939 DOI: 10.1089/scd.2012.0084] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors and CD34(+) cells were found, at 43 days of co-culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines.
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Affiliation(s)
- Dylana Diaz-Solano
- Unidad de Terapia Celular-Laboratorio de Patología Celular y Molecular, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela
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Hirabayashi Y, Hatta Y, Takeuchi J, Tsuboi I, Harada T, Ono K, Glomm WR, Yasuda M, Aizawa S. Novel three-dimensional long-term bone marrow culture system using polymer particles with grafted epoxy-polymer-chains supports the proliferation and differentiation of hematopoietic stem cells. Exp Biol Med (Maywood) 2011; 236:1342-50. [DOI: 10.1258/ebm.2011.011075] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Hematopoiesis occurs in the bone marrow, where primitive hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment composed of stromal cells. We examined the hematopoietic supportive ability of stromal cells in a 3D culture system using polymer particles with grafted epoxy polymer chains. Umbilical cord blood-derived CD34+ cells were co-cultivated with MS-5 stromal cells. They formed a 3D structure in the culture dish in the presence of particles, and the total numbers of cells and the numbers of hematopoietic progenitor cells, including colony-forming unit (CFU)-Mix, CFU-granulocyte-macrophage, CFU-megakaryocyte and burst-forming unit-erythroid, were measured every seven days. The hematopoietic supportive activity of the 3D culture containing polymer particles and stromal cells was superior to that of 2D culture, and allowed the expansion and maintenance of hematopoietic progenitor cells for more than 12 weeks. Various types of hematopoietic cells, including granulocytes, macrophages and megakaryocytes at different maturation stages, appeared in the 3D culture, suggesting that the CD34+ cells were able to differentiate into a range of blood cell types. Morphological examination showed that MS-5 stromal cells grew on the surface of the particles and bridged the gaps between them to form a 3D structure. Hematopoietic cells slipped into the 3D layer and proliferated within it, relying on the presence of the MS-5 cells. These results suggest that this 3D culture system using polymer particles reproduced the hematopoietic phenomenon in vitro, and might thus provide a new tool for investigating hematopoietic stem cell–stromal cell interactions.
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Affiliation(s)
- Yukio Hirabayashi
- Department of Internal Medicine
- Department of Functional Morphology, Nihon University School of Medicine, 30-1 Oyaguchikamicho, Itabashi-ku, Tokyo 173-8610
| | | | | | - Isao Tsuboi
- Department of Functional Morphology, Nihon University School of Medicine, 30-1 Oyaguchikamicho, Itabashi-ku, Tokyo 173-8610
| | - Tomonori Harada
- Department of Functional Morphology, Nihon University School of Medicine, 30-1 Oyaguchikamicho, Itabashi-ku, Tokyo 173-8610
| | - Kentaro Ono
- Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
| | - Wilhelm Robert Glomm
- Department of Chemical Engineering, Norwegian University of Science and Technology, Sem Sælands vei 4, Trondheim N-7491, Norway
| | - Masahiro Yasuda
- Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
| | - Shin Aizawa
- Department of Functional Morphology, Nihon University School of Medicine, 30-1 Oyaguchikamicho, Itabashi-ku, Tokyo 173-8610
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Bedel R, Thiery-Vuillemin A, Grandclement C, Balland J, Remy-Martin JP, Kantelip B, Pallandre JR, Pivot X, Ferrand C, Tiberghien P, Borg C. Novel role for STAT3 in transcriptional regulation of NK immune cell targeting receptor MICA on cancer cells. Cancer Res 2011; 71:1615-26. [PMID: 21257710 DOI: 10.1158/0008-5472.can-09-4540] [Citation(s) in RCA: 86] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The role of natural killer group 2, member D receptor (NKG2D)-expressing natural killer (NK) cells in tumor immunosurveillance is now well established. Nevertheless, tumor progression occurs despite tumor immunosurveillance, leading to cancer persistence in immunocompetent hosts. STAT3 plays a pivotal role both in oncogenic functions and in immunosuppression. In this study, we investigated the role of STAT3 in suppressing NK cell-mediated immunosurveillance. Using a colorectal cancer cell line (HT29) that can poorly activate NK, we neutralized STAT3 with pharmacologic inhibitors or siRNA and found that this led to an increase in NK degranulation and IFN-γ production in a TGF-β1-independent manner. Exposure to NKG2D-neutralizing antibodies partially restored STAT3 activity, suggesting that it prevented NKG2D-mediated NK cell activation. On this basis, we investigated the expression of NKG2D ligands after STAT3 activation in HT29, mesenchymal stem cells, and activated lymphocytes. The NK cell recognition receptor MHC class I chain-related protein A (MICA) was upregulated following STAT3 neutralization, and a direct interaction between STAT3 and the MICA promoter was identified. Because cross-talk between DNA damage repair and NKG2D ligand expression has been shown, we assessed the influence of STAT3 on MICA expression under conditions of genotoxic stress. We found that STAT3 negatively regulated MICA expression after irradiation or heat shock, including in lymphocytes activated by CD3/CD28 ligation. Together, our findings reveal a novel role for STAT3 in NK cell immunosurveillance by modulating the MICA expression in cancer cells.
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Affiliation(s)
- Romain Bedel
- INSERM (Institut National de la Santé et de la Recherche Médicale) UMR 645, Besançon, France
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da Silva CL, Gonçalves R, dos Santos F, Andrade PZ, Almeida-Porada G, Cabral JMS. Dynamic cell-cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38−and early lymphoid CD7+cells. J Tissue Eng Regen Med 2010; 4:149-58. [DOI: 10.1002/term.226] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
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Higuchi A, Yang ST, Li PT, Chang Y, Tsai EM, Chen YH, Chen YJ, Wang HC, Hsu ST. Polymeric Materials for Ex vivo Expansion of Hematopoietic Progenitor and Stem Cells. POLYM REV 2009. [DOI: 10.1080/15583720903048185] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
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Baek EJ, Kim HS, Kim S, Jin H, Choi TY, Kim HO. In vitro clinical-grade generation of red blood cells from human umbilical cord blood CD34+ cells. Transfusion 2008; 48:2235-45. [PMID: 18673341 DOI: 10.1111/j.1537-2995.2008.01828.x] [Citation(s) in RCA: 71] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
BACKGROUND There is no appropriate alternative source of red blood cells (RBCs) to relieve the worsening shortage of blood available for transfusion. Therefore, in vitro generation of clinically available RBCs from hematopoietic stem cells could be a promising new source to supplement the blood supply. However, there have been few studies about the generation of clinical-grade RBCs by coculture on human mesenchymal stem cells (MSCs) and various cytokine supplements, even though the production of pure RBCs requires coculture on stromal cells and proper cytokine supplements. STUDY DESIGN AND METHODS Umbilical cord blood (CB) CD34+ cells were cultured in serum-free medium supplemented with two cytokine sets of stem cell factor (SCF) plus interleukin-3 (IL-3) plus erythropoietin (EPO) and SCF plus IL-3 plus EPO plus thrombopoietin (TPO) plus Flt-3 for 1 week, followed by coculture upon MSCs derived from bone marrow (BM) or CB for 2 weeks. RESULTS Almost pure clinical-grade RBCs could be generated by coculturing with CB-MSCs but not BM-MSCs. Expansion fold and enucleation rate were significantly higher in coculture with CB-MSCs than BM-MSCs. Despite a 2.5-fold expansion of erythroblasts in the presence of TPO and Flt-3 for 8 days, the final RBC count was higher without TPO and Flt-3. CONCLUSIONS This study is the first report on generating clinical-grade RBCs by in vitro culture with human MSCs and compared effectiveness of several cytokines for RBC production. This provides a useful basis for future production of clinically available RBCs and a model of erythropoiesis that is analogous to the in vivo system.
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Affiliation(s)
- Eun Jung Baek
- The Department of Laboratory Medicine, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
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Dorn I, Lazar-Karsten P, Boie S, Ribbat J, Hartwig D, Driller B, Kirchner H, Schlenke P. In vitro proliferation and differentiation of human CD34+ cells from peripheral blood into mature red blood cells with two different cell culture systems. Transfusion 2008; 48:1122-32. [DOI: 10.1111/j.1537-2995.2008.01653.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Gonçalves R, Lobato da Silva C, Cabral JMS, Zanjani ED, Almeida-Porada G. A Stro-1(+) human universal stromal feeder layer to expand/maintain human bone marrow hematopoietic stem/progenitor cells in a serum-free culture system. Exp Hematol 2006; 34:1353-9. [PMID: 16982328 DOI: 10.1016/j.exphem.2006.05.024] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2006] [Accepted: 05/15/2006] [Indexed: 11/26/2022]
Abstract
OBJECTIVE To compare the ability of allogeneic versus autologous purified human Stro-1(+) mesenchymal stem cell (MSC) populations from different human donors to support the ex vivo expansion and maintenance of human hematopoietic stem/progenitor cells (HSCs). Furthermore, we compared the results obtained with MSC as a feeder layer to traditional allogeneic stromal layers grown in long-term bone marrow culture media (LT-ST). METHODS Adult human bone marrow CD34(+)-enriched cells were cultured in serum-free medium for 2 to 3 weeks over the respective MSC-irradiated feeder layers or over traditional allogeneic LT- ST stromal layers in the presence of stem cell factor, basic fibroblast growth factor, leukemia inhibitory factor, and Flt-3 and analyzed every 2 to 4 days for expansion, phenotype, and clonogenic ability. RESULTS There was a progressive expansion of total numbers of cells in all the experimental groups; however, allogeneic MSCs were more efficient at expanding CD34(+)CD38(-) cells and showed a higher clonogenic potential than both allogeneic LT-ST and autologous MSCs. The differentiative potential of cells cultured on both MSC and LT-ST was primarily shifted toward myeloid lineage; however, only MSCs were able to maintain/expand a CD7(+) population with lymphocytic potential. Importantly, transplantation into preimmune fetal sheep demonstrated that the HSCs cultured over MSCs retained their engraftment capability. CONCLUSION These results indicate that purified Stro-1(+) MSCs may be used as a universal and reproducible stromal feeder layer to efficiently expand and maintain human bone marrow HSCs ex vivo.
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Affiliation(s)
- Raquel Gonçalves
- Department of Animal Biotechnology, University of Nevada, Reno, NV 89557, USA
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Feng Q, Chai C, Jiang XS, Leong KW, Mao HQ. Expansion of engrafting human hematopoietic stem/progenitor cells in three-dimensional scaffolds with surface-immobilized fibronectin. J Biomed Mater Res A 2006; 78:781-91. [PMID: 16739181 PMCID: PMC2396227 DOI: 10.1002/jbm.a.30829] [Citation(s) in RCA: 102] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
An efficient and practical ex vivo expansion methodology for human hematopoietic stem/progenitor cells (HSPCs) is critical in realizing the potential of HSPC transplantation in treating a variety of hematologic disorders and as a supportive therapy for malignant diseases. We report here an expansion strategy using a three-dimensional (3D) scaffold conjugated with an extracellular matrix molecule, fibronectin (FN), to partially mimic the hematopoietic stem cell niche. FN-immobilized 3D polyethylene terephthalate (PET) scaffold was synthesized and evaluated for HSPC expansion efficiency, in comparison with a FN-immobilized 2D PET substrate and a 3D scaffold with FN supplemented in the medium. Covalent conjugation of FN produced substrate and scaffold with higher cell expansion efficiency than that on their unmodified counterparts. After 10 days of culture in serum-free medium, human umbilical cord blood CD34+ cells cultured in FN-conjugated scaffold yielded the highest expansion of CD34+ cells (approximately 100 fold) and long-term culture initiating cells (approximately 47-fold). The expanded human CD34+ cells successfully reconstituted hematopoiesis in NOD/SCID mice. This study demonstrated the synergistic effect between the three-dimensionality of the scaffold and surface-conjugated FN, and the potential of this FN-conjugated 3D scaffold for ex vivo expansion of HSPCs.
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Affiliation(s)
- Qi Feng
- Division of Biomedical Sciences, Johns Hopkins in Singapore, #02-01, The Nanos, 31 Biopolis Way, Singapore 138669
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Xie C, Jia B, Xiang Y, Wang L, Wang G, Huang G, McNiece IK, Wang J. Support of hMSCs transduced with TPO/FL genes to expansion of umbilical cord CD34+ cells in indirect co-culture. Cell Tissue Res 2006; 326:101-10. [PMID: 16685532 DOI: 10.1007/s00441-006-0203-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2005] [Accepted: 03/14/2006] [Indexed: 11/27/2022]
Abstract
A novel indirect co-culture system was established to support ex vivo expansion of hematopoietic progenitors in umbilical cord blood (UCB) by using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human-marrow-derived mesenchymal stem cells (tfhMSCs) as a feeder. UCB CD34+ cells were isolated and cultured by using five culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were taken to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient mouse (SCID) repopulating cell (SRC) assay was performed to confirm the ability of the indirect co-cultured cells from the tfhMSCs system to reconstitute long-term hematopoiesis. Results showed significant differences in the number of total nucleated cells (TNCs) among the culture systems with respect to serum-containing medium or serum-free medium during 14-day culture. In addition, on day 14, the outputs of CD34+ cells, the colony-forming units (CFUs) in culture, and the CFUs in mixed colonies containing erythroid and myeloid cells and megakaryocytes in the tfhMSC indirect co-culture system were significantly enhanced. The LTC-IC assay demonstrated that the tfhMSCs indirect co-culture system had the strongest activity. The SCID-SRC assay confirmed the extensive ability of the expanded cells from the tfhMSCs indirect co-culture systems to reconstitute long-term hematopoiesis. Furthermore, polymerase chain reaction analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of non-obese diabetic/SCID mice. Thus, hMSCs transduced with TPO/FL, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro. The tfhMSC indirect co-culture system may therefore be a suitable system for ex vivo manipulation of primitive progenitor cells under non-contact culture conditions.
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Affiliation(s)
- Chungang Xie
- College of Life Sciences, Zhejiang University, 232 Wen San Road, Hangzhou, Zhejiang Province, 310012, People's Republic of China
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Xie CG, Wang JF, Xiang Y, Qiu LY, Jia BB, Wang LJ, Wang GZ, Huang GP. Cocultivation of umbilical cord blood CD34 + cells with retro-transduced hMSCs leads to effective amplification of long-term culture-initiating cells. World J Gastroenterol 2006; 12:393-402. [PMID: 16489638 PMCID: PMC4066057 DOI: 10.3748/wjg.v12.i3.393] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To establish a novel coculture system for ex vivo expansion of umbilical cord blood(UCB) hematopoietic progenitors using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human marrow-derived mesenchymal stem cells (tfhMSCs) as feeder.
METHODS: UCB CD34+ cells were isolated and cultured using four culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay was performed to confirm ability of the cultured cells to reconstitute long-term hematopoiesis.
RESULTS: There were no significant differences in the number of total nucleated cells among different culture systems in serum-containing medium during 21-d culture. However, on d 14, the outputs of CD34+ cells, CFU-C and CFU-GEMM in tfhMSCs coculture system were significantly enhanced. LTC-IC assay demonstrated that the tfhMSCs coculture system had the most powerful activity. The severe-combined immunodeficient (SCID) mouse repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Furthermore, PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD/SCID mice.
CONCLUSION: The TPO/FL-transduced hMSCs, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.
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Affiliation(s)
- Chun-Gang Xie
- College of Life Sciences, Zhejiang University, 232 Wen San Road, Hangzhou 310012, Zhejiang Province, China
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16
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Kobune M, Kato J, Chiba H, Kawano Y, Tanaka M, Takimoto R, Hamada H, Niitsu Y. Telomerized human bone marrow-derived cell clones maintain the phenotype of hematopoietic-supporting osteoblastic and myofibroblastic stromal cells after long-term culture. Exp Hematol 2005; 33:1544-53. [PMID: 16338498 DOI: 10.1016/j.exphem.2005.09.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2005] [Revised: 08/11/2005] [Accepted: 09/12/2005] [Indexed: 11/20/2022]
Abstract
OBJECTIVE Gene transfer of the telomerase catalytic subunit (TERT) into primary human stromal cells prolonged their lifespan. However, primary human stromal cells are actually composed of adipocytes, myofibroblasts, osteoblasts, etc. Our objective was to investigate the phenotype and hematopoietic-support of the human telomerized stromal cell (HTS) in clonal level. MATERIALS AND METHODS We established HTS clones (HTS-1 to HTS-9) from a parental population of HTSs by limiting dilution. Hematopoietic-supporting activity of the HTS clones was examined by coculturing with CD34(+) cells. RESULTS HTS-1 to HTS-3 contained alkaline phosphatase (ALP)(+) cells, and HTS-4 to HTS-9 were composed of both ALP(+) and alpha-smooth muscle actin-positive cells. Although all HTS clones exhibited normal growth kinetics, one of the HTS clones exhibited a chromosomal abnormality. Moreover, the parental population of the HTS cells acquired an increased growth rate and anchorage independence after 4 years of culturing. Expression of hematopoietic growth factors, such as stem cell factor, angiopoietin-1, and hedgehog mRNA was detected in all HTS clones. The degree of hematopoietic progenitor support differed between the HTS clones, and the expansion level of CD34(+) cells was the highest in HTS-8. CONCLUSION Human telomerized stromal cell clones exhibited the phenotype of hematopoietic supporting osteoblastic and myofibroblastic cells after long-term culture. Clinical application of HTS cells should be limited because of their potential for neoplastic transformation after hTERT gene transfer. HTS cells may be useful for analyzing the molecular mechanism of hematopoietic support of human stromal cells.
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Affiliation(s)
- Masayoshi Kobune
- Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine, Japan.
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Hamada H, Kobune M, Nakamura K, Kawano Y, Kato K, Honmou O, Houkin K, Matsunaga T, Niitsu Y. Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy. Cancer Sci 2005; 96:149-56. [PMID: 15771617 PMCID: PMC11159137 DOI: 10.1111/j.1349-7006.2005.00032.x] [Citation(s) in RCA: 167] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
We developed human mesenchymal stem cell (MSC) lines that could differentiate into various tissue cells including bone, neural cells, bone marrow (BM) stromal cells supporting the growth of hematopoietic stem cell (HSC), and so-called 'tumor stromal cells' mixing with tumor cells. We investigated the applicability of MSC as therapeutic cell transplanting reagents (cytoreagents). Telomerized human BM derived stromal cells exhibited a prolonged lifespan and supported the growth of hematopoietic clonogenic cells. The gene transfer of Indian hedgehog (Ihh) remarkably enhanced the HSC expansion supported by the human BM stromal cells. Gene-modified MSC are useful as therapeutic tools for brain tissue damage (e.g. brain infarction) and malignant brain neoplasms. MSC transplantation protected the brain tissue from acute ischemic damage in the midcerebral artery occlusion (MCAO) animal model. Brain-derived neurotrophic factor (BDNF)-gene transduction further enhanced the protective efficacy against the ischemic damage. MSC possessed excellent migratory ability and exerted inhibitory effects on the proliferation of glioma cells. Gene-modification of MSC with therapeutic cytokines clearly augmented the antitumor effect and prolonged the survival of tumor-bearing animals. Gene therapy employing MSC as a tissue-protecting and targeting cytoreagent would be a promising approach.
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Affiliation(s)
- Hirofumi Hamada
- Department of Molecular Medicine, Sapporo Medical University, Sapporo 060-8556, Japan.
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Gogusev J, Telvi L, Murakami I, Lepelletier Y, Nezelof C, Stojkoski A, Glorion C, Jaubert F. DOR-1, A novel CD10+ stromal cell line derived from progressive Langerhans cell histiocytosis of bone. Pediatr Blood Cancer 2005; 44:128-37. [PMID: 15390308 DOI: 10.1002/pbc.20090] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
BACKGROUND Langerhans cell histiocytosis (LCH) is granulomatous proliferative disorder characterized by the presence of activated Langerhans cells admixed with macrophages, lymphocytes, and eosinophils. In an effort to obtain an LCH ex vivo model, we succeeded in establishing the DOR-1 cell line from an LCH lesion of bone in a 3-year-old girl. PROCEDURE The DOR-1 cell line was established from a CD1a immunoreactive LCH lesion of bone maintained in long-term cell culture. The phenotypic characteristics were assessed by immuno-cytochemistry and fluorescence activated cell sorter (FACS) analysis. Cytogenetic analysis was performed by RHG-banding that was supplemented by fluorescence in situ hybridization (FISH). RESULTS The DOR-1 cells grew in vitro as a poorly differentiated mesenchymal-like cells with a doubling time between 72 and 96 hr. The cells exhibited pleomorphism and consistent immuno-reactivity for CD10 (50%), CD13 (55%), CD68 (65%), and CD117 (70%) while CD1a, Langerin and HLA-DR were not detected. By RHG-banding, several aberrant chromosomes were detected including the t (9; 17) (p23; p13) translocation and a pair of long dicentric marker chromosomes indicating clonal abnormality. Functionally, exposure to 33 nM 12-O-tetradecanoyl phorbol mirystate-13-acetate (TPA) induced DOR-1 cell differentiation with appearance of cytoplasmic extensions. CONCLUSIONS The DOR-1 cell line exhibits distinct immuno-cytochemical features and carries the t (9; 17) (p23; p13) translocation suggesting involvement of stromal-like cell lineage in LCH initiation and progression.
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Giarratana MC, Kobari L, Lapillonne H, Chalmers D, Kiger L, Cynober T, Marden MC, Wajcman H, Douay L. Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nat Biotechnol 2004; 23:69-74. [PMID: 15619619 DOI: 10.1038/nbt1047] [Citation(s) in RCA: 441] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2004] [Accepted: 10/13/2004] [Indexed: 12/22/2022]
Abstract
We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.
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Affiliation(s)
- Marie-Catherine Giarratana
- Laboratoire d'Hématologie EA1638, Université Paris VI, CHU Saint Antoine, 27 rue de Chaligny-75571 Paris Cedex 12, France
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20
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Kobune M, Kawano Y, Ito Y, Chiba H, Nakamura K, Tsuda H, Sasaki K, Dehari H, Uchida H, Honmou O, Takahashi S, Bizen A, Takimoto R, Matsunaga T, Kato J, Kato K, Houkin K, Niitsu Y, Hamada H. Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells. Exp Hematol 2003; 31:715-22. [PMID: 12901977 DOI: 10.1016/s0301-472x(03)00177-2] [Citation(s) in RCA: 80] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
OBJECTIVE To compare the hematopoietic support provided by telomerized human mesenchymal stem cells (MSCs) and telomerized MSC-derived stromal cells. METHODS We transfected the human telomerase catalytic subunit (hTERT) gene into primary MSCs to establish hTERT-transduced MSCs (hTERT-MSCs). Stromal induction of hTERT-MSCs was performed by replacing the culture medium with Dexter-type culture medium. Hematopoietic support was examined by coculture with cord blood CD34(+) cells. RESULTS The hTERT-MSCs were morphologically identical with the primary MSCs and expressed surface antigens including CD105, CD73, and CD166. hTERT-MSCs showed a similar doubling time as primary MSCs and continued to proliferate to over 80 population doublings (PD), although the primary MSCs underwent crisis in vitro at 16 PD. The osteogenic, chondrogenic, adipogenic, neurogenic, and stromal differentiation potential of hTERT-MSCs were maintained up to at least 40 PD. The degree of expansion of CD34(+) cells and total number of colony-forming units in culture (CFU-C) upon 12-day coculture with the hTERT-MSC-derived stromal cells were nearly the same as those upon 12-day coculture with hTERT-MSCs (CD34, 33.0-fold+/-2.8-fold vs 36.1-fold+/-1.7-fold of the initial cell number; CFUs, 344.4-fold+/-62.5-fold vs 239.3-fold+/-87.0-fold; CFU-mix, 368.4-fold+/-113.7-fold vs 341.3-fold+/-234.3-fold). However, on day 18 of coculture, the number of cobblestone areas (CA) observed beneath the stromal cells was 15 times higher than that beneath hTERT-MSCs (CA, 146.9+/-54.6 vs 9.4+/-8.1, p<0.01). CONCLUSION Stromal induction of hTERT-MSCs exclusively enhanced the support of CA formation provided by hTERT-MSCs. Our human hTERT-MSCs will be useful for elucidating the mechanism of the formation of CAs.
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Affiliation(s)
- Masayoshi Kobune
- Dept. of Molecular Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan
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Clark AD, Jørgensen HG, Mountford J, Holyoake TL. Isolation and therapeutic potential of human haemopoietic stem cells. Cytotechnology 2003; 41:111-31. [PMID: 19002948 PMCID: PMC3466700 DOI: 10.1023/a:1024822722285] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
The haemopoietic stem cell (HSC) has long been regarded as an archetypal, tissue specific, stem cell, capable of completely regenerating haemopoiesis after myeloablation. It has proved relatively easy to harvest HSC, from bone marrow or peripheral blood. In turn, isolation of these cells has allowed therapeutic stem cell transplantation protocols to be developed, that capitalise on their prodigious self renewal and proliferative capabilities. Ex vivo approaches have been described to isolate, genetically manipulateand expand pluripotent stem cell subsets. These techniques have been crucial to the development of gene therapy, and may allow adults to enjoy the potential advantages of cord blood transplantation. Recently, huge conceptual changes have occurred in stem cell biology. In particular, the dogma that, in adults, stem cells are exclusively tissue restricted has been questioned and there is great excitement surrounding the potential plasticity of these cells, with the profound implications that this has, for developing novel cellular therapies. Mesenchymal stem cells, multipotent adult progenitor cells and embryonic stem cells are potential sources of cells for transplantation purposes. These cells may be directed toproduce HSC, in vitro and in the future may be used for therapeutic, or drug development, purposes.
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Affiliation(s)
- Andrew D. Clark
- Cancer Research Beatson Laboratories, University of Glasgow, Glasgow, U.K
- Department of Haematology, Royal Infirmary, North Glasgow Hospital University Trust, Glasgow, U.K
| | - Heather G. Jørgensen
- Division of Cancer Sciences and Molecular Pathology, Royal Infirmary, University of Glasgow, Glasgow, U.K
| | - Joanne Mountford
- Division of Cancer Sciences and Molecular Pathology, Royal Infirmary, University of Glasgow, Glasgow, U.K
| | - Tessa L. Holyoake
- Cancer Research Beatson Laboratories, University of Glasgow, Glasgow, U.K
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Kawano Y, Kobune M, Yamaguchi M, Nakamura K, Ito Y, Sasaki K, Takahashi S, Nakamura T, Chiba H, Sato T, Matsunaga T, Azuma H, Ikebuchi K, Ikeda H, Kato J, Niitsu Y, Hamada H. Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT)-transfected human stromal cells. Blood 2003; 101:532-40. [PMID: 12393449 DOI: 10.1182/blood-2002-04-1268] [Citation(s) in RCA: 117] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
We developed a new human stromal cell line that could expand human hematopoietic progenitor/stem cells. Primary human bone marrow stromal cells were infected with retrovirus containing the human telomerase catalytic subunit (hTERT) gene, resulting in increased population doubling and the acquisition of cell immortalization. Characteristics of the hTERT-transduced stromal (hTERT-stromal) cells were identical with those of the primary stromal cells in terms of morphologic appearance and expression of surface antigens. Human cord blood (CB) CD34(+) cells were expanded by coculture with primary stromal or hTERT-stromal cells in the presence of stem cell factor, thrombopoietin, and Flk-2/Flt-3 ligand under serum-free condition. The degree of expansion of CD34(+) cells and total number of colony-forming units in culture (CFU-Cs) after 2 weeks' coculture with the hTERT-stromal cells were nearly the same as those after 2 weeks' coculture with primary stromal cells (CD34(+) cells, 118-fold +/- 8-fold versus 117-fold +/- 13-fold; CFU-Cs, 71-fold +/- 5-fold versus 67-fold +/- 5-fold of initial cell number). CB expansion on hTERT-stromal cells occurred at a similar rate through 7 weeks. In contrast, the rate of CB expansion on primary stromal cells had drastically declined at 7 weeks. In nonobese diabetic/severe combined immunodeficiency (SCID) mice, the degree of engraftment of SCID-repopulating cells that had been cocultured with hTERT-stromal cells for 4 weeks was significantly higher than that of precocultured CB cells. These results indicate that this hTERT-stromal cell line could be useful for ex vivo expansion of hematopoietic progenitor/stem cells and for analyzing the microenvironment of human bone marrow.
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Affiliation(s)
- Yutaka Kawano
- Department of Molecular Medicine, Sapporo Medical University, Japan
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