In vitro isolation, culture and identification of adipose-derived stem cells

doi:10.3969/j.issn.2095-4344.2013.40.006

Abstract

Abstract:

BACKGROUND: Adipose-derived stem cells are easily collected and abundantly cultured, which can proliferate rapidly when being cultured in vitro. With multi-directional differentiation potential, adipose-derived stem cells are expected as seed cells for tissue engineering.
OBJECTIVE: To isolate, culture and identify of adipose-derived stem cells from Sprague-Dawley rats in vitro.
METHODS: The subcutaneous adipose tissue was obtained from the iliac region of rats under the aseptic condition, and then was digested with 0.075% type Ⅰ collagenase and cultured in vitro. The morphology and proliferation characteristics of the cells were observed under an inverted phase contrast microscope. The third passage was put into gauge for growth curve by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and the cells were also identified by CD44, a stem cell marker, with immunofluorescence staining. Adipose-derived stem cells were induced and differentiated into adipocytes in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10% fetal bovine serum, dexamethasone and insulin, and then the cells were identified with oil red “O” staining. Adipose-derived stem cells were induced and differentiated into neural cells, and then the cells were identified with immunohistochemical staining.
RESULTS AND CONCLUSION: The growth curve of adipose-derived stem cells was opposite-like “S” shape, and it strongly expressed CD44 that can designate a stem cell. The passage cells were exposed to a defined medium for adipocyte differentiation, and then could be stained with oil red. After being induced and differentiated into nerve cells, the cells expressed neuron-specific enolase. The adipose-derived stem cells of Sprague-Dawley rats are characterized by easy isolation, culture and proliferation in vitro, expressing related phenotypes of mesenchymal stem cells, as well as induction and differentiation under certain conditions.

Key words: stem cells, adult stem cells, cell differentiation, mesenchymal stem cells, adipogenesis, collagenases

CLC Number:

R318

Du Guo-jia, Chen Xiao-hong, Zhu Guo-hua, Fan Yan-dong, Wang Yun, Dang Mu-ren. In vitro isolation, culture and identification of adipose-derived stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(40): 7054-7060.

Figures/Tables 4

References

[1]Rodbell M. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem. 1964;239:375-380.
[2]Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7(2):211-228.
[3]Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002;13(12): 4279-4295.
[4]Lin XH, Liu N, Xiao YC, et al. Effects of adipose-derived stem cells transplantation on the neuronal apoptosis and the expression of Bcl-2 and caspase-12 in the brain post focal cerebral ischemia in rats. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2011;27(1):40-43.
[5]Graham SH, Chen J, Clark RS. Bcl-2 family gene products in cerebral ischemia and traumatic brain injury. J Neurotrauma. 2000;17(10):831-841.
[6]Plesnila N. Role of mitochondrial proteins for neuronal cell death after focal cerebral ischemia. Acta Neurochir Suppl. 2004;89:15-19.
[7]Ferrer I, Planas AM. Signaling of cell death and cell survival following focal cerebral ischemia: life and death struggle in the penumbra. J Neuropathol Exp Neurol. 2003;62(4): 329-339.
[8]Cai YN, Yuan XD, Ou Y, et al. Apoptosis duringβ-mercaptoethanol-induced differentiation of adult adipose-derived stromal cells into neurons. Neural Regen Res. 2011;6(10):750-755.
[9]Gao X, Dong T, Li Z Q, et al. Survival of human adipose-derived mesenchymal stem cells transplanted via the tail vein in rat brain with cerebral ischemia. Zhongguo Zuzhi Gongchang Yanjiu. 2012;16(27):5067-5071.
[10]The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.
[11]Stanford WL, Caruana G, Vallis KA, et al. Expression trapping: identification of novel genes expressed in hematopoietic and endothelial lineages by gene trapping in ES cells. Blood. 1998;92(12):4622-4631.
[12]Forrai A, Robb L. The gene trap resource: a treasure trove for hemopoiesis research. Exp Hematol. 2005;33(8): 845-856.
[13]Stanford WL, Cohn JB, Cordes SP. Gene-trap mutagenesis: past, present and beyond. Nat Rev Genet. 2001;2(10): 756-768.
[14]Joyner AL, Auerbach A, Skarnes WC. The gene trap approach in embryonic stem cells: the potential for genetic screens in mice. Ciba Found Symp. 1992;165:277-297.
[15]Clay TM, Custer MC, Spiess PJ, et al. Potential use of T cell receptor genes to modify hematopoietic stem cells for the gene therapy of cancer. Pathol Oncol Res. 1999;5(1):3-15.
[16]Filippi MD, Sainteny F. Hematopoietic differentiation of embryonic stem cells in mice: a model to study the biology of hematopoiesis. Transfus Clin Biol. 2001;8(1):6-16.
[17]Kitajima K, Takeuchi T. Mouse gene trap approach: identification of novel genes and characterization of their biological functions. Biochem Cell Biol. 1998;76(6): 1029-1037.
[18]Conquet F. Inactivation in vivo of metabotropic glutamate receptor 1 by specific chromosomal insertion of reporter gene lacZ. Neuropharmacology. 1995;34(8):865-870.
[19]Guan C, Ye C, Yang X, et al. A review of current large-scale mouse knockout efforts. Genesis. 2010;48(2):73-85.
[20]Araki K, Yamamura K. Genetic screening for novel genes by insertional mutagenesis with gene trap method in ES cells. Tanpakushitsu Kakusan Koso. 1995;40(14): 2017- 2024.
[21]Vasileiskii SS. Fetal and embryonic hemoglobins. Sov J Dev Biol. 1972;3(1):27-32.
[22]Schnütgen F. Generation of multipurpose alleles for the functional analysis of the mouse genome. Brief Funct Genomic Proteomic. 2006;5(1):15-18.
[23]Reubinoff BE, Pera MF, Fong CY, et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 2000;18(4):399-404.
[24]Kørbling M, Estrov Z. Adult stem cells for tissue repair - a new therapeutic concept? N Engl J Med. 2003;349(6): 570-582.
[25]Mitchell JB, McIntosh K, Zvonic S, et al. Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers. Stem Cells. 2006;24(2):376-385.
[26]Qin YW, Zhao JM, Su W, et al. Isolation, culture and identification of rat adipose-derived stem cells. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011; 15(23):4242-4246.
[27]Choi YS, Dusting GJ, Stubbs S, et al. Differentiation of human adipose-derived stem cells into beating cardiomyocytes. J Cell Mol Med. 2010;14(4):878-889.
[28]Fang B, Song Y, Lin Q, et al. Human adipose tissue-derived mesenchymal stromal cells as salvage therapy for treatment of severe refractory acute graft-vs.-host disease in two children. Pediatr Transplant. 2007;11(7):814-817.
[29]Campagnoli C, Roberts IA, Kumar S, et al. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood. 2001;98(8): 2396-2402.
[30]Schieker M, Pautke C, Haasters F, et al. Human mesenchymal stem cells at the single-cell level: simultaneous seven-colour immunofluorescence. J Anat. 2007;210(5):592-599.
[31]Zhou MH, Zhang XW, Li L. Research of rat's adipose-derived stem cells induced differentiation into adipogenic cells. Shiyong Fuchan Ke Zazhi. 2011;27(7): 515-518.
[32]Cheng WD, Zhao Y, He JS, et al. Adipogenic and osteogenic potential of rat adipose tissue-derived stromal cells in vitro. Anhui Yike Daxue Xuebao. 2007;42(5): 501-504.
[33]Yang XF, HE X, He J, et al. Human adipose-derived stem cells into adipocytes directed differentiation. Jilin Daxue Xuebao: Yixue Ban. 2009;35(6):987-991.
[34]Kirchner S, Kieu T, Chow C, et al. Prenatal exposure to the environmental obesogen tributyltin predisposes multipotent stem cells to become adipocytes. Mol Endocrinol. 2010; 24(3):526-539.
[35]Li HX, Luo X, Liu RX, et al. Roles of Wnt/beta-catenin signaling in adipogenic differentiation potential of adipose-derived mesenchymal stem cells. Mol Cell Endocrinol. 2008;291(1-2):116-124.
[36]Liu Q, Cen L, Zhou H, et al. The role of the extracellular signal-related kinase signaling pathway in osteogenic differentiation of human adipose-derived stem cells and in adipogenic transition initiated by dexamethasone. Tissue Eng Part A. 2009;15(11):3487-3497.
[37]Ashjian PH, Elbarbary AS, Edmonds B, et al. In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast Reconstr Surg. 2003;111(6): 1922-1931.