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      Selective Expansion of CD34+ Cells from Mouse Bone Marrow Cultured on LH/P MP-Coated Plates with Adequate Cytokines

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          Abstract

          Low-molecular-weight heparin/protamine microparticles (LH/P MPs) serve as carriers for controlled release of heparin-binding cytokines. LH/P MPs were stably coated onto plastic surfaces by drying. The purpose of this study is to evaluate a culture method for selective expansion of CD34+ cells using LH/P MPs as cytokine-binding matrix. Ficoll-purified mouse bone marrow cells (mouse FP-BMCs) containing CD34+ cells were cultured on LH/P MP-coated plates in the presence of stem cell factor (SCF), thrombopoietin (Tpo), and Flt-3 ligand (Flt-3) in hematopoietic progenitor growth medium (HPGM) supplemented with 4% heat-inactivated fetal bovine serum (FBS). After 8 days of culture, the total cell count increased 4.6-fold, and flow cytometry analyses revealed that 23.8% of the initial cells and 57.4% of the expanded cells were CD34 positive. Therefore, CD34+ cells were estimated to have increased 11.0-fold. In contrast, cultured CD34+ cells on uncoated tissue culture plates increased 5.8-fold in an identical medium.

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          Most cited references 30

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          Stem cells and their niches.

          A constellation of intrinsic and extrinsic cellular mechanisms regulates the balance of self-renewal and differentiation in all stem cells. Stem cells, their progeny, and elements of their microenvironment make up an anatomical structure that coordinates normal homeostatic production of functional mature cells. Here we discuss the stem cell niche concept, highlight recent progress, and identify important unanswered questions. We focus on three mammalian stem cell systems where large numbers of mature cells must be continuously produced throughout adult life: intestinal epithelium, epidermal structures, and bone marrow.
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            Bone-marrow haematopoietic-stem-cell niches.

            Adult stem cells hold many promises for future clinical applications and regenerative medicine. The haematopoietic stem cell (HSC) is the best-characterized somatic stem cell so far, but in vitro expansion has been unsuccessful, limiting the future therapeutic potential of these cells. Here we review recent progress in characterizing the composition of the HSC bone-marrow microenvironment, known as the HSC niche. During homeostasis, HSCs, and therefore putative bone-marrow HSC niches, are located near bone surfaces or are associated with the sinusoidal endothelium. The molecular crosstalk between HSCs and the cellular constituents of these niches is thought to control the balance between HSC self-renewal and differentiation, indicating that future successful expansion of HSCs for therapeutic use will require three-dimensional reconstruction of a stem-cell-niche unit.
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              Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia.

              The interaction of stem cells with their bone marrow microenvironment is a critical process in maintaining normal hematopoiesis. We applied an approach to resolve the spatial organization that underlies these interactions by evaluating the distribution of hematopoietic cell subsets along an in vivo Hoechst 33342 (Ho) dye perfusion gradient. Cells isolated from different bone marrow regions according to Ho fluorescence intensity contained the highest concentration of hematopoietic stem cell (HSC) activity in the lowest end of the Ho gradient (i.e., in the regions reflecting diminished perfusion). Consistent with the ability of Ho perfusion to simulate the level of oxygenation, bone marrow fractions separately enriched for HSCs were found to be the most positive for the binding of the hypoxic marker pimonidazole. Moreover, the in vivo administration of the hypoxic cytotoxic agent tirapazamine exhibited selective toxicity to the primitive stem cell subset. These data collectively indicate that HSCs and the supporting cells of the stem cell niche are predominantly located at the lowest end of an oxygen gradient in the bone marrow with the implication that regionally defined hypoxia plays a fundamental role in regulating stem cell function.
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                Author and article information

                Journal
                J Tissue Eng
                TEJ
                sptej
                Journal of Tissue Engineering
                SAGE Publications (Sage UK: London, England )
                2041-7314
                2011
                30 October 2011
                : 2
                : 1
                Affiliations
                [1 ]Research Institute, National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan
                [2 ]Research Fellow of the Japan Society for the Promotion of Science, Tokyo 102-8472, Japan
                [3 ]Department of Policy Science, National Institute of Public Health, Wako, Saitama 351-0197, Japan
                [4 ]Department of Plastic and Reconstructive Surgery, National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan
                [5 ]Department of Surgery, National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan
                [6 ]Department of Biosciences and Informatics, School of Fundamental Science and Technology, Keio University, Yokohama, Kanagawa 223-8522, Japan
                Author notes
                Masayuki Ishihara Research Institute National Defense Medical College 3-2 Namiki Tokorozawa Saitama 359-8513 Japan Email: ishihara@ 123456ndmc.ac.jp
                Article
                10.1177_2041731411425419
                10.1177/2041731411425419
                3258842
                22292106
                © The Author(s) 2011

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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