Comparative flow cytometric analysis of mesenchymal stem cells isolated by different methods for regenerative medicine

Comparative flow cytometric analysis of mesenchymal stem cells isolated by different methods for regenerative medicine

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Title: Comparative flow cytometric analysis of mesenchymal stem cells isolated by different methods for regenerative medicine
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Article_Title: Comparative flow cytometric analysis of mesenchymal stem cells isolated by different methods for regenerative medicine
Authors: Andreea Ciotec, Rodica Tatia, Ana-Maria Dobre, Nicolae Efimov, Catalin Iordachel, Daniela Bratosin, Elena Iulia Oprita
Affiliation: National Institute of Research and Development for Biological Science, Bucharest, Romania
CFR 2 Hospital, Bucharest, Romania
”Vasile Goldis” Western University of Arad, Faculty of Biology, Arad, Romania
Abstract: The term stem cell is used to denote an unspecialized progenitor cell residing in niches in various organs and tissues from which it can be recruited to replenish specific tissue as a self-renewal cell source with application in cell-based therapies and tissue engineering. In our study, human mesenchymal stem cells (multipotent mesenchymal stromal stem cells) (hMSC), plastic-adherent and fibroblastic-like cells, have been obtained from multiple biological sources: umbilical cord blood (UCB), bone marrow aspirate (BM) and cancellous bone (CB) and by several existing methods in the literature, in order to compare the cells to be used by coculture with differentiated cells for regeneration of damaged tissues. The magnetic separation method for isolating hMSC led to obtaining a pure culture compared to enzymatic digestion with collagenase and the separation on density gradient (Ficoll) resulted in a pure culture much smaller than the other two methods used. Flow cytometric analysis showed that after 3 weeks of cell cultivation, cells were CD73 positive and CD34 negative, suggesting that their stem phenotype has been preserved. By light microscopy images no significant differences have been observed in colony formation between the different hMSC isolation methods.
Keywords: mesenchymal stem cells, flow cytometry, regenerative medicine
References: Alexanian, A.R., Sieber-Blum, M., Differentiating adult hippocampal stem cells into neural crest derivatives. Neuroscience 118, 1-5, 2003.
Alhadlaq, A., and Mao, J. J., Mesenchymal stem cells: Isolation and therapeutics, Stem Cells Dev. 13, 436-448, 2004.
Alsalameh, S., Amin, R., Gemba, T., Lotz, M., Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage. Arthritis Rheum., 50, 1522-1532, 2004.
Aoki, M., Yasutake, M., Murohara, T., Derivation of functional endothelial progenitor cells from human umbilical cord blood mononuclear cells isolated by a novel cell filtration device. Stem cells, 22, 994-1002, 2004.
Bieback, K., Kern, S., Kluter, H., Eichler, H., Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem cells, 22, 625-634, 2004.
Bottai, D., Fiocco, R., Gelain, F., Defilippis, L., Galli, R., Gritti, A., Vescovi, L.A., Neural stem cells in the adult nervous system. J. Hematother. Stem Cell Res., 12, 655-670, 2003.
Caplan, A.I., Mesenchymal stem cells. J. Orthop. Res., 9, 641-650, 1991.
Cancedda, R., Dozin, B., Giannoni, P., Quarto, R., Tissue engineering and cell therapy of cartilage and bone. Matrix Biol., 22, 81-91, 2003.
Caplan, A.I., Elyaderani, M., Mochizuki, Y., Wakitani, S., Goldberg, V.M., Principles of cartilage repair and regeneration. Clin. Orthop. Relat. Res., 342, 254-269, 1997.
Csaki, C., Matis, U., Mobasheri, A., Ye, H., Shakibaei, M., Chondrogenesis, osteogenesis and adipogenesis of canine mesenchymal stem cells: a biochemical, morphological and ultrastructural study. Histochem. Cell Biol., 128, 507-520, 2007.
Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D., Horwitz, E., Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8, 315-317, 2006.
Friedenstein, A.J., Piatetzky II, S., Petrakova, K.V., Osteogenesis in transplants of bone marrow cells. J. Embryol. Exp. Morphol., 16, 381-390, 1966.
Friedenstein, A.J., Chailakhyan, R.K., Gerasimov, U.V., Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet., 20, 263-272, 1987.
Friedenstein, A. J., Chailakhjan, R. K., and Lalykina, K. S., The development of fibroblast colonies in monolayer cultures of guinea‐pig bone marrow and spleen cells, Cell Tissue Kinet., 3, 393-403, 1970.
Fuchs, E., Tumbar, T., Guasch, G., Socializing with the neighbors: stem cells and their niche. Cell, 116, 769–778, 2004.
Giovannini, S., Brehm, W., Mainil-Varlet, P., Nesic, D., Multilineage differentiation potential of equine blood-derived fibroblast-like cells. Differentiation, 76, 118-129, 2008.
Helder, M.N., Knippenberg, M., Klein-Nulend, J., Wuisman, P.I., Stem cells from adipose tissue allow challenging new concepts for regenerative medicine. Tissue Eng., 13, 1799-1808, 2007.
Huss, R., Lange, C., Weissinger, E.M., Kolb, H.J., Thalmeier, K., Evidence of peripheral bloodderived, plastic-adherent
CD34(-/low) hematopoietic stem cell clones with mesenchymal stem cell characteristics. Stem cells, 18, 252-260, 2000.
Jorgensen C, Gordeladze, Noël D, Tissue engineering through autologous mesenchymal stem cells, Current Opinion in Biotechnology, 15, 406-410, 2004.
Kern, S., Eichler, H., Stoeve, J., Kluter, H., Bieback, K., Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem cells, 24, 1294-1301, 2006.
Kogler, G., Sensken, S., Airey, J.A., Trapp, T., Muschen, M., Feldhahn, N., Liedtke, S., Sorg, R.V., Fischer, J., Rosenbaum, C., Greschat, S., Knipper, A., Bender, J., Degistirici, O., Gao, J., Caplan, A.I., Colletti, E.J., Almeida-Porada, G., Muller, H.W., Zanjani, E., Wernet, P., A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J. Exp. Med., 200, 123-135, 2004.
Koerner, J., Nesic, D., Romero, J.D., Brehm, W., Mainil- Varlet, P., Grogan, S.P., Equine peripheral blood-derived progenitors in comparison to bone marrow-derived mesenchymal stem cells. Stem cells, 24, 1613-1619, 2006.
Lange, C., Schroeder, J., Stute, N., Lioznov, M.V., Zander, A.R., High-potential human mesenchymal stem cells. Stem Cells Dev., 14, 70-80, 2005.
Lee, R.H., Kim, B., Choi, I., Kim, H., Choi, H.S., Suh, K., Bae, Y.C., Jung, J.S., Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol. Biochem., 14, 311-324, 2004.
Martin, D.R., Cox, N.R., Hathcock, T.L., Niemeyer, G.P., Baker, H.J., Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow. Exp. Hematol., 30, 879-886, 2002.
Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R., Multilineage potential of adult human mesenchymal stem cells. Science, 284, 143-147, 1999.
Qu, C.Q., Zhang, G.H., Zhang, L.J., Yang, G.S., Osteogenic and adipogenic potential of porcine adipose mesenchymal stem cells. In Vitro Cell Dev. Biol. Anim., 43, 95-100, 2007.
Till, J.E., McCulloch, E.A., Hemopoietic stem cell differentiation. Biochim. Biophys. Acta., 605, 431-459, 1980.
Tuli R, Tuli S, Nandi S, Wang ML, Alexander PG, Haleem-Smith H, Hozack WJ, Manner PA, Caplan, A.I., Dennis, J.E., Mesenchymal stem cells as trophic mediators. J. Cell Biochem., 98, 1076-1084, 2006.
Ukai, R., Honmou, O., Harada, K., Houkin, K., Hamada, H., Kocsis, J.D., Mesenchymal stem cells derived from peripheral blood protects against ischemia. J. Neurotrauma, 24, 508-520, 2007.
Wagner, W., Wein, F., Seckinger, A., Frankhauser, M., Wirkner, U., Krause, U., Blake, J., Schwager, C., Eckstein, V., Ansorge, W., Ho, A.D., Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp. Hematol., 33, 1402-1416, 2005.
Wickham, M.Q., Erickson, G.R., Gimble, J.M., Vail, T.P., Guilak, F., Multipotent stromal cells derived from the infrapatellar fat pad of the knee. Clin. Orthop. Relat. Res., 412, 196-212, 2003.
Williams, J.T., Southerland, S.S., Souza, J., Calcutt, A.F., Cartledge, R.G., Cells isolated from adult human skeletal muscle capable of differentiating into multiple mesodermal phenotypes. Am. Surg., 65, 22-26, 1999.
Yamamoto, N., Akamatsu, H., Hasegawa, S., Yamada, T., Nakata, S., Ohkuma, M., Miyachi, E.I., Marunouchi, T., Matsunaga, K., Isolation of multipotent stem cells from mouse adipose tissue. J. Dermatol. Sci., 48, 43-52, 2007.
Zuk, P.A., Zhu, M., Mizuno, H., Huang, J., Futrell, J.W., Katz, A.J., Benhaim, P., Lorenz, H.P., Hedrick, M.H., Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng., 7, 211-228, 2001.
Zuk, P.A., Zhu, M., Ashjian, P., De Ugarte, D.A., Huang, J.I., Mizuno, H., Alfonso, Z.C., Fraser, J.K., Benhaim, P., Hedrick, M.H., Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell, 13, 4279-4295, 2002.
Zuk, P.A., Zhu, M., Mizuno, H., Huang, J., Futrell, J.W., Katz, A.J., Benhaim, P., Lorenz, H.P., Hedrick, M.H., Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng., 7, 211-228, 2001.
Zvaifler, N.J., Marinova-Mutafchieva, L., Adams, G., Edwards, C.J., Moss, J., Burger, J.A., Maini, R.N., Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res., 2, 477-488, 2000.
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Correspondence: Iulia Oprita, National Institute for Biological Science Research & Development, Bucharest, Romania; Splaiul Independentei no. 296, district 6, Bucharest, Romania, Tel/Fax +40-(021)-2200881, email: iulia.oprita@yahoo.com

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Article Title: Comparative flow cytometric analysis of mesenchymal stem cells isolated by different methods for regenerative medicine
Authors: Andreea Ciotec, Rodica Tatia, Ana-Maria Dobre, Nicolae Efimov, Catalin Iordachel, Daniela Bratosin, Elena Iulia Oprita
Affiliation: National Institute of Research and Development for Biological Science, Bucharest, Romania
CFR 2 Hospital, Bucharest, Romania
”Vasile Goldis” Western University of Arad, Faculty of Biology, Arad, Romania
Abstract: The term stem cell is used to denote an unspecialized progenitor cell residing in niches in various organs and tissues from which it can be recruited to replenish specific tissue as a self-renewal cell source with application in cell-based therapies and tissue engineering. In our study, human mesenchymal stem cells (multipotent mesenchymal stromal stem cells) (hMSC), plastic-adherent and fibroblastic-like cells, have been obtained from multiple biological sources: umbilical cord blood (UCB), bone marrow aspirate (BM) and cancellous bone (CB) and by several existing methods in the literature, in order to compare the cells to be used by coculture with differentiated cells for regeneration of damaged tissues. The magnetic separation method for isolating hMSC led to obtaining a pure culture compared to enzymatic digestion with collagenase and the separation on density gradient (Ficoll) resulted in a pure culture much smaller than the other two methods used. Flow cytometric analysis showed that after 3 weeks of cell cultivation, cells were CD73 positive and CD34 negative, suggesting that their stem phenotype has been preserved. By light microscopy images no significant differences have been observed in colony formation between the different hMSC isolation methods.
Keywords: mesenchymal stem cells, flow cytometry, regenerative medicine
References: Alexanian, A.R., Sieber-Blum, M., Differentiating adult hippocampal stem cells into neural crest derivatives. Neuroscience 118, 1-5, 2003.
Alhadlaq, A., and Mao, J. J., Mesenchymal stem cells: Isolation and therapeutics, Stem Cells Dev. 13, 436-448, 2004.
Alsalameh, S., Amin, R., Gemba, T., Lotz, M., Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage. Arthritis Rheum., 50, 1522-1532, 2004.
Aoki, M., Yasutake, M., Murohara, T., Derivation of functional endothelial progenitor cells from human umbilical cord blood mononuclear cells isolated by a novel cell filtration device. Stem cells, 22, 994-1002, 2004.
Bieback, K., Kern, S., Kluter, H., Eichler, H., Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem cells, 22, 625-634, 2004.
Bottai, D., Fiocco, R., Gelain, F., Defilippis, L., Galli, R., Gritti, A., Vescovi, L.A., Neural stem cells in the adult nervous system. J. Hematother. Stem Cell Res., 12, 655-670, 2003.
Caplan, A.I., Mesenchymal stem cells. J. Orthop. Res., 9, 641-650, 1991.
Cancedda, R., Dozin, B., Giannoni, P., Quarto, R., Tissue engineering and cell therapy of cartilage and bone. Matrix Biol., 22, 81-91, 2003.
Caplan, A.I., Elyaderani, M., Mochizuki, Y., Wakitani, S., Goldberg, V.M., Principles of cartilage repair and regeneration. Clin. Orthop. Relat. Res., 342, 254-269, 1997.
Csaki, C., Matis, U., Mobasheri, A., Ye, H., Shakibaei, M., Chondrogenesis, osteogenesis and adipogenesis of canine mesenchymal stem cells: a biochemical, morphological and ultrastructural study. Histochem. Cell Biol., 128, 507-520, 2007.
Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D., Horwitz, E., Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8, 315-317, 2006.
Friedenstein, A.J., Piatetzky II, S., Petrakova, K.V., Osteogenesis in transplants of bone marrow cells. J. Embryol. Exp. Morphol., 16, 381-390, 1966.
Friedenstein, A.J., Chailakhyan, R.K., Gerasimov, U.V., Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet., 20, 263-272, 1987.
Friedenstein, A. J., Chailakhjan, R. K., and Lalykina, K. S., The development of fibroblast colonies in monolayer cultures of guinea‐pig bone marrow and spleen cells, Cell Tissue Kinet., 3, 393-403, 1970.
Fuchs, E., Tumbar, T., Guasch, G., Socializing with the neighbors: stem cells and their niche. Cell, 116, 769–778, 2004.
Giovannini, S., Brehm, W., Mainil-Varlet, P., Nesic, D., Multilineage differentiation potential of equine blood-derived fibroblast-like cells. Differentiation, 76, 118-129, 2008.
Helder, M.N., Knippenberg, M., Klein-Nulend, J., Wuisman, P.I., Stem cells from adipose tissue allow challenging new concepts for regenerative medicine. Tissue Eng., 13, 1799-1808, 2007.
Huss, R., Lange, C., Weissinger, E.M., Kolb, H.J., Thalmeier, K., Evidence of peripheral bloodderived, plastic-adherent
CD34(-/low) hematopoietic stem cell clones with mesenchymal stem cell characteristics. Stem cells, 18, 252-260, 2000.
Jorgensen C, Gordeladze, Noël D, Tissue engineering through autologous mesenchymal stem cells, Current Opinion in Biotechnology, 15, 406-410, 2004.
Kern, S., Eichler, H., Stoeve, J., Kluter, H., Bieback, K., Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem cells, 24, 1294-1301, 2006.
Kogler, G., Sensken, S., Airey, J.A., Trapp, T., Muschen, M., Feldhahn, N., Liedtke, S., Sorg, R.V., Fischer, J., Rosenbaum, C., Greschat, S., Knipper, A., Bender, J., Degistirici, O., Gao, J., Caplan, A.I., Colletti, E.J., Almeida-Porada, G., Muller, H.W., Zanjani, E., Wernet, P., A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J. Exp. Med., 200, 123-135, 2004.
Koerner, J., Nesic, D., Romero, J.D., Brehm, W., Mainil- Varlet, P., Grogan, S.P., Equine peripheral blood-derived progenitors in comparison to bone marrow-derived mesenchymal stem cells. Stem cells, 24, 1613-1619, 2006.
Lange, C., Schroeder, J., Stute, N., Lioznov, M.V., Zander, A.R., High-potential human mesenchymal stem cells. Stem Cells Dev., 14, 70-80, 2005.
Lee, R.H., Kim, B., Choi, I., Kim, H., Choi, H.S., Suh, K., Bae, Y.C., Jung, J.S., Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol. Biochem., 14, 311-324, 2004.
Martin, D.R., Cox, N.R., Hathcock, T.L., Niemeyer, G.P., Baker, H.J., Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow. Exp. Hematol., 30, 879-886, 2002.
Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R., Multilineage potential of adult human mesenchymal stem cells. Science, 284, 143-147, 1999.
Qu, C.Q., Zhang, G.H., Zhang, L.J., Yang, G.S., Osteogenic and adipogenic potential of porcine adipose mesenchymal stem cells. In Vitro Cell Dev. Biol. Anim., 43, 95-100, 2007.
Till, J.E., McCulloch, E.A., Hemopoietic stem cell differentiation. Biochim. Biophys. Acta., 605, 431-459, 1980.
Tuli R, Tuli S, Nandi S, Wang ML, Alexander PG, Haleem-Smith H, Hozack WJ, Manner PA, Caplan, A.I., Dennis, J.E., Mesenchymal stem cells as trophic mediators. J. Cell Biochem., 98, 1076-1084, 2006.
Ukai, R., Honmou, O., Harada, K., Houkin, K., Hamada, H., Kocsis, J.D., Mesenchymal stem cells derived from peripheral blood protects against ischemia. J. Neurotrauma, 24, 508-520, 2007.
Wagner, W., Wein, F., Seckinger, A., Frankhauser, M., Wirkner, U., Krause, U., Blake, J., Schwager, C., Eckstein, V., Ansorge, W., Ho, A.D., Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp. Hematol., 33, 1402-1416, 2005.
Wickham, M.Q., Erickson, G.R., Gimble, J.M., Vail, T.P., Guilak, F., Multipotent stromal cells derived from the infrapatellar fat pad of the knee. Clin. Orthop. Relat. Res., 412, 196-212, 2003.
Williams, J.T., Southerland, S.S., Souza, J., Calcutt, A.F., Cartledge, R.G., Cells isolated from adult human skeletal muscle capable of differentiating into multiple mesodermal phenotypes. Am. Surg., 65, 22-26, 1999.
Yamamoto, N., Akamatsu, H., Hasegawa, S., Yamada, T., Nakata, S., Ohkuma, M., Miyachi, E.I., Marunouchi, T., Matsunaga, K., Isolation of multipotent stem cells from mouse adipose tissue. J. Dermatol. Sci., 48, 43-52, 2007.
Zuk, P.A., Zhu, M., Mizuno, H., Huang, J., Futrell, J.W., Katz, A.J., Benhaim, P., Lorenz, H.P., Hedrick, M.H., Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng., 7, 211-228, 2001.
Zuk, P.A., Zhu, M., Ashjian, P., De Ugarte, D.A., Huang, J.I., Mizuno, H., Alfonso, Z.C., Fraser, J.K., Benhaim, P., Hedrick, M.H., Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell, 13, 4279-4295, 2002.
Zuk, P.A., Zhu, M., Mizuno, H., Huang, J., Futrell, J.W., Katz, A.J., Benhaim, P., Lorenz, H.P., Hedrick, M.H., Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng., 7, 211-228, 2001.
Zvaifler, N.J., Marinova-Mutafchieva, L., Adams, G., Edwards, C.J., Moss, J., Burger, J.A., Maini, R.N., Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res., 2, 477-488, 2000.
*Correspondence: Iulia Oprita, National Institute for Biological Science Research & Development, Bucharest, Romania; Splaiul Independentei no. 296, district 6, Bucharest, Romania, Tel/Fax +40-(021)-2200881, email: iulia.oprita@yahoo.com