Isolation and culture of mouse adipose-derived stem cells and their homing to the intestinal tract

doi:10.3969/j.issn.2095-4344.2015.06.007

Abstract

Abstract:

BACKGROUND: Adipose-derived mesenchymal stem cells have gained more and more attention due to their high safety, less invasiveness, easy purification and rapid proliferation.
OBJECTIVE: To isolate and culture adipose-derived mesenchymal stem cells efficiently and rapidly with high purity and then to explore the cell homing to the intestinal tract after fluorescence labeling.
METHODS: Mouse adipose tissue obtained from the groin and epididymis was aseptically isolated and digested with 0.1% collagenase I. Then the digested cells and undigested adipose tissue were cultured together in the dish to harvest adipose-derived mesenchymal stem cells. The cells were identified by morphology, surface markers, growth kinetics, osteogenic and adipogenic differentiation potential, and then labeled by PKH67 before injected into ulcerative colitis mouse models through the tail vein to observe their homing to the intestinal tract.
RESULTS AND CONCLUSION: In vitro, adipose-derived mesenchymal stem cells isolated by this method exhibited spindle-like appearance, grew intensively and arranged in a swirling shape. Adipose-derived mesenchymal stem cells expressed CD29, CD44 and CD90, but not expressed CD45. After osteogenic induction, alkaline phosphatase staining showed black particles and alizarin red S staining showed red mineralized nodules. After adipogenic induction, oil red O staining showed many lipid droplets were dyed red. Cell growth curve showed cells at 3-5 days were in logarithmic growth phase and they were active. Under fluorescence microscopy, frozen sections of the colon were found green fluorescence points that were increased with time. Results suggest that adipose-derived mesenchymal stem cells isolated and cultured in vitro can proliferate rapidly, purify easily and can be induced to osteoblasts and adipocytes; in vivo, PKH67-labeled cells can home to and proliferate in the colon.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: Adipose Tissue, Mesenchymal Stem Cells, Colitis, Ulcerative

CLC Number:

R394.2

Hou Xiao-lin, Yu Wei-dong, Cui Mei-hua, He Xiang-jun, Liang Jun. Isolation and culture of mouse adipose-derived stem cells and their homing to the intestinal tract[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(6): 854-860.

Figures/Tables 3

References

[1] Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol. 1976;4(5):267-274.
[2] 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.
[3] Igura K, Zhang X, Takahashi K, et al. Isolation and characterization of mesenchymal progenitor cells from chorionic villi of human placenta. Cytotherapy. 2004;6(6): 543-553.
[4] Lee OK, Kuo TK, Chen WM, et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood. 2004;103(5):1669-1675.
[5] Pham PV, Vu NB, Pham VM, et al. Good manufacturing practice-compliant isolation and culture of human umbilical cord blood-derived mesenchymal stem cells. J Transl Med. 2014;12:56.
[6] Zhao Z, Chen Z, Zhao X, et al. Sphingosine-1-phosphate promotes the differentiation of human umbilical cord mesenchymal stem cells into cardiomyocytes under the designated culturing conditions. J Biomed Sci. 2011;18:37.
[7] Bae KS, Park JB, Kim HS, et al. Neuron-like differentiation of bone marrow-derived mesenchymal stem cells. Yonsei Med J. 2011;52(3):401-412.
[8] 杨乃龙,杨芬,徐丽丽.共培养法神经细胞诱导骨髓间充质干细胞向神经元的分化[J]. 中国组织工程研究与临床康复,2008, 12(29): 5611-5614.
[9] Bai K, Huang Y, Jia X, et al. Endothelium oriented differentiation of bone marrow mesenchymal stem cells under chemical and mechanical stimulations. J Biomech. 2010; 43(6): 1176-1181.
[10] Pournasr B, Mohamadnejad M, Bagheri M, et al. In vitro differentiation of human bone marrow mesenchymal stem cells into hepatocyte-like cells. Arch Iran Med. 2011;14(4):244-249.
[11] English K. Mechanisms of mesenchymal stromal cell immunomodulation. Immunol Cell Biol. 2013;91(1):19-26.
[12] Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood. 2002;99(10):3838-3843.
[13] Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol. 2008;8(9):726-736.
[14] Voswinkel J, Francois S, Gorin NC, et al. Gastro-intestinal autoimmunity: preclinical experiences and successful therapy of fistulizing bowel diseases and gut Graft versus host disease by mesenchymal stromal cells. Immunol Res. 2013; 56(2-3):241-248.
[15] Fouillard L, Chapel A, Bories D, et al. Infusion of allogeneic-related HLA mismatched mesenchymal stem cells for the treatment of incomplete engraftment following autologous haematopoietic stem cell transplantation. Leukemia. 2007;21(3):568-570.
[16] Giordano A, Galderisi U, Marino IR. From the laboratory bench to the patient's bedside: an update on clinical trials with mesenchymal stem cells. J Cell Physiol. 2007;211(1):27-35.
[17] Li H, Guo Z, Jiang X, et al. Mesenchymal stem cells alter migratory property of T and dendritic cells to delay the development of murine lethal acute graft-versus-host disease. Stem Cells. 2008;26(10):2531-2541.
[18] Ding DC, Shyu WC, Lin SZ. Mesenchymal stem cells. Cell Transplant. 2011;20(1):5-14.
[19] Phinney DG, Kopen G, Isaacson RL, et al. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation. J Cell Biochem. 1999;72(4):570-585.
[20] Nakanishi C, Nagaya N, Ohnishi S, et al. Gene and protein expression analysis of mesenchymal stem cells derived from rat adipose tissue and bone marrow. Circ J. 2011;75(9): 2260-2268.
[21] Toyoda M, Matsubara Y, Lin K, et al. Characterization and comparison of adipose tissue-derived cells from human subcutaneous and omental adipose tissues. Cell Biochem Funct. 2009;27(7):440-447.
[22] Liu TM, Martina M, Hutmacher DW, et al. Identification of common pathways mediating differentiation of bone marrow- and adipose tissue-derived human mesenchymal stem cells into three mesenchymal lineages. Stem Cells. 2007;25(3):750-760.
[23] Melief SM, Zwaginga JJ, Fibbe WE, et al. Adipose tissue-derived multipotent stromal cells have a higher immunomodulatory capacity than their bone marrow-derived counterparts. Stem Cells Transl Med. 2013;2(6):455-463.
[24] Strioga M, Viswanathan S, Darinskas A, et al. Same or not the same? Comparison of adipose tissue-derived versus bone marrow-derived mesenchymal stem and stromal cells.Stem Cells Dev. 2012;21(14):2724-2752.
[25] Nadri S, Soleimani M, Hosseni RH, et al. An efficient method for isolation of murine bone marrow mesenchymal stem cells. Int J Dev Biol. 2007;51(8):723-729.
[26] 李朝中,肖践明,陈丽星,等.大鼠骨髓间充质干细胞体外分离培养与CM-DiI荧光标记[J].中国组织工程研究,2014,18(1):39-44.
[27] 林楚伟,周胜华,杜优优,等. 全骨髓贴壁并差速传代分离纯化大鼠骨髓间充质干细胞:与密度梯度离心法的比较[J].中国组织工程研究与临床康复, 2010,14(14):2508-2512.
[28] Zohar R, Sodek J, McCulloch CA. Characterization of stromal progenitor cells enriched by flow cytometry. Blood. 1997;90(9): 3471-3481.
[29] 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.
[30] Klingbeil MF, Herson MR, Cristo EB, et al. Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes. Cell Tissue Bank. 2009;10(3):197-204.
[31] 国强华,宋维鹏,王庆胜,等.组织块贴壁法培养人肠系膜动脉平滑肌细胞的研究[J].武警医学,2012,23(12):1033-1035.
[32] Yang XF, He X, He J, et al. High efficient isolation and systematic identification of human adipose-derived mesenchymal stem cells. J Biomed Sci. 2011;18:59.
[33] 刘琴,王丽平,喻晶,等.组织块贴壁法扩增兔脂肪干细胞[J].中国组织工程研究,2014,18(1):88-93.
[34] Park JR, Kim E, Yang J, et al. Isolation of human dermis derived mesenchymal stem cells using explants culture method: expansion and phenotypical characterization. Cell Tissue Bank. 2014 Aug 28. [Epub ahead of print]
[35] Mori Y, Ohshimo J, Shimazu T, et al. Improved Explant Method to Isolate Umbilical Cord-Derived Mesenchymal Stem Cells and Their Immunosuppressive Properties.Tissue Eng Part C Methods. 2014 Sep 13. [Epub ahead of print]
[36] Gittel C, Brehm W, Burk J, et al. Isolation of equine multipotent mesenchymal stromal cells by enzymatic tissue digestion or explant technique: comparison of cellular properties. BMC Vet Res. 2013;9:221.
[37] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-317.
[38] P M, S H, R M, et al. Adult mesenchymal stem cells and cell surface characterization - a systematic review of the literature. Open Orthop J. 2011;5(Suppl 2):253-260.
[39] Baer PC. Adipose-derived mesenchymal stromal/stem cells: An update on their phenotype in vivo and in vitro. World J Stem Cells. 2014;6(3):256-265.
[40] Ciulla MM, Lazzari L, Pacchiana R, et al. Homing of peripherally injected bone marrow cells in rat after experimental myocardial injury. Haematologica. 2003;88(6):614-621.
[41] Kim PJ, Mahmoudi M, Ge X, et al. Direct Evaluation of Myocardial Viability and Stem Cell Engraftment Demonstrates Salvage of the Injured Myocardium. Circ Res. 2015. [Epub ahead of print]
[42] Banas A, Teratani T, Yamamoto Y, et al. IFATS collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury. Stem Cells. 2008;26(10):2705-2712.
[43] Castelo-Branco MT, Soares ID, Lopes DV, et al. Intraperitoneal but not intravenous cryopreserved mesenchymal stromal cells home to the inflamed colon and ameliorate experimental colitis. PLoS One. 2012;7(3):e33360.
[44] Nagyova M, Slovinska L, Blasko J, et al. A comparative study of PKH67, DiI, and BrdU labeling techniques for tracing rat mesenchymal stem cells. In Vitro Cell Dev Biol Anim. 2014; 50(7):656-663