냉동 보존 전후의 사람 탯줄유래 줄기세포의 특성 분석
Stem Cell Properties of Human Umbilical Cord-derived Stem Eells after Cryopreservation
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For the clinical application, it is needed to keep characteristics of stem cells after storage for a long time. In the present study, we examined stem cell properties of human cord-derived stem cells(HUC) after cryopreservation. Cells were isolated from human umbilical cord and cultured in vitro. At passage 2 or 3, HUC were suspended at a concentration of 1.0×106/mL in cryomedium consisting of DMSO and FBS. After freezing at －80℃ overnight, HUC were cryopreserved at －196℃ nitrogen gas. After 6 months, HUC were thawed and cultured in vitro. Assessment for the stem cell properties was made upon the morphology, population doubling time, and expression profiles of genes and various proteins. Cryopreserved HUC showed more than 70% viability and maintained fibroblast-like morphology similar to HUC before cryopreservation. Throughout the culture, they underwent average 42.8 doublings and produced 6.75×1018 cells. RT-PCR analyses showed that cryopreserved HUC expressed Oct-4, nanog, SCF, NCAM, nestin, GATA-4, BMP4, and HLA-1 genes. They did not express Brachyury and HLA-DR genes. Immunocytochemical studies showed that cryopreserved HUC reacted with antibodies against SSEA-3, -4, Thy-1, vimentin, fibronectin, HCAM, ICAM, HLA-1 proteins. They did not react with antibody against HLA-DR protein. Theses genes and proteins expression patterns of cryopresserved HUC were similar to those of HUC before cryopreservation. These results suggest that cryopreserved HUC could retain proliferative potential and they expressed various genes and proteins similar to HUC before cryopreservation. Thus, cryopreservation might be useful for HUC for future research and clinical application
Abstract
For the clinical application, it is needed to keep characteristics of stem cells after storage for a long time. In the present study, we examined stem cell properties of human cord-derived stem cells(HUC) after cryopreservation. Cells were isolated from human umbilical cord and cultured in vitro. At passage 2 or 3, HUC were suspended at a concentration of 1.0×106/mL in cryomedium consisting of DMSO and FBS. After freezing at －80℃ overnight, HUC were cryopreserved at －196℃ nitrogen gas. After 6 months, HUC were thawed and cultured in vitro. Assessment for the stem cell properties was made upon the morphology, population doubling time, and expression profiles of genes and various proteins. Cryopreserved HUC showed more than 70% viability and maintained fibroblast-like morphology similar to HUC before cryopreservation. Throughout the culture, they underwent average 42.8 doublings and produced 6.75×1018 cells. RT-PCR analyses showed that cryopreserved HUC expressed Oct-4, nanog, SCF, NCAM, nestin, GATA-4, BMP4, and HLA-1 genes. They did not express Brachyury and HLA-DR genes. Immunocytochemical studies showed that cryopreserved HUC reacted with antibodies against SSEA-3, -4, Thy-1, vimentin, fibronectin, HCAM, ICAM, HLA-1 proteins. They did not react with antibody against HLA-DR protein. Theses genes and proteins expression patterns of cryopresserved HUC were similar to those of HUC before cryopreservation. These results suggest that cryopreserved HUC could retain proliferative potential and they expressed various genes and proteins similar to HUC before cryopreservation. Thus, cryopreservation might be useful for HUC for future research and clinical application
참고문헌 (33)
- Assady S, "Insulin production by human embryonic stem cells", Diabetes, 50, pp.1691~1697 (2001)
- Baksh D, "Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow", Stem Cells, 25, pp.1384~1392 (2007)
- Bieback K, "Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood", Stem Cells, 22, pp.625~634 (2004)
- Bilic G, "In vitro lesion repair by human amnion epithelial and mesenchymal cells", Am J Obstet Gynecol, 190, pp.87~92 (2004)
- Bruder SP, "Growth kinetics self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation", J Cell Biochem, 64, pp.278~294 (1997)
- Buchanan SS, "Cryopreservation of stem cells using trehalose: Evaluation of the method using a human hematopoietic cell line", Stem Cells Dev, 13, pp.295~305 (2004)
- Campagnoli C, "Idenification of mesenchymal stem", progenitor cells in human first-trimester fetal blood, liver, and bone marrow, 2001, pp.98, 2396~2402
- Chu Q, "Mechanism of in vitro differentiation of bone marrow stromal cells into neuron-like cells", J Huazhong Univ Sci Technol Med Sci, 24, pp.259~261 (2004)
- Fu YS, "Conversion of human umbilical cord mesenchymal stem cells in Wharton’s jelly to dopaminergic neurons in vitro: potential therapeuric application of Parkinsonism", Stem Cells, 24, pp.115~124 (2006)
- Fukuchi Y, "Human placenta-derived cells have mesenchymal stem", progenitor cell potential, 2004, pp.22, 649~658
- Grepin C, "Enhanced cardiogenesis in embryonic stem cells overexpressing the GATA-4 transcription factor", Develoment, 124, pp.2387~2395 (1997)
- Hu Y, "Isolation and identification of mesnechymal stem cells from human fetal pancreas", J Lab Clin Med, 141, pp.342~349 (2003)
- Izadpanah R, "Long-term in vitro expansion alters the biology of adult mesenchymal stem cells", Cancer Res, 68, pp.4229~4238 (2008)
- Kaufman DS, "Hematopoietic colony-forming cells derived from human embryonic stem cells", Proc Natl Acad Sci USA, 98, pp.10716~10721 (2001)
- Kern S, "Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue", Stem Cells, 24, pp.1294~1301 (2006)
- Kotobuki N, "Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells", Tissue Engineering, 11, pp.663~673 (2005)
- Kuroda R, "Cartilage repair using bone morphogenetic protein 4 and muscle-derived stem cells", Arthritis Rheum, 54, pp.433~442 (2006)
- Lee MW, "Isolation of mesenchymal stem cells from cryopreserved human umbilical cord blood", Int J Hematol, 81, pp.126~130 (2005)
- Lund RD, "Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease", Stem Cells, 25, pp.602~611 (2007)
- Moon JH, "Successful vitrification of human amnion- derived mesenchymal stem cells", Hum. Reprod, 23, pp.1760~1770 (2008)
- Mueller SM, "Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges", J Cell Biochem, 82, pp.583~590 (2001)
- Pittenger MF, "Multilineage potential of adult human mesenchymal stem cells", Science, 284, pp.143~147 (1999)
- Reubinoff BE, "Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro", Nat Biotechnol, 18, pp.399~404 (2000)
- Romanov YA, "Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord", Stem Cells, 21, pp.105~110 (2003)
- Schwartz RE, "Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells", J Clin Invest, 109, pp.1291~1302 (2002)
- Sjoberg G, "Colocalization of nestin and vimentin", desmin in skeletal muscle cells demonstrated by three-dimensional fluorescence digital imaging microscopy, 1994, pp.214, 447~458
- Stenderup K, "Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells", Bone, 33, pp.919~926 (2003)
- Takechi K, "Ultrastructural and immunohistochemical studies of Wharton's jelly umbilical cord cells", Placenta, 14, pp.235~245 (1993)
- Troyer DL, "Wharton’s jelly-derived cells are a primitive stromal cell population", Stem cells, 26, pp.591~599 (2008)
- Wang HS, "Mesenchymal stem cells in the Wahrton’s jelly of the human umbilical cord", Stem Cells, 22, pp.1330~1337 (2004)
- Weiss ML, "Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease", Stem Cells, 24, pp.781~792 (2006)
- Young DA, "Expression of metalloproteinases and inhibitors in the differentiation of P19CL6 cells into cardiac myocytes", Biochem Biophys Res Commun, 24, pp.759~765 (2004)
- Zuk PA, "Human adipose tissue is a source of multipotent stem cells", Mol Biol Cell, 13, pp.4279~4295 (2002)