Method for selective quantification of adipose-derived stromal/stem cells in tissue
Main Article Content
Keywords
Adipose-derived stromal/stem cells, adipose tissue, ASC isolation technique, fat graft, selective quantification
Abstract
Fat grafts are valuable for soft-tissue regeneration and augmentation. However, fat graft systems require further improvement for the prediction of graft retention. The concentration of adipose-derived stromal/stem cells (ASCs) is one of the most important factors that affect graft retention; however, current cell quantification techniques have not been applied to adipose tissue. Here we developed a method for the selective quantification of ASCs in tissue (SQAT). We identified a characteristic methylated site in the CD31 promoter after searching for specific markers of ASCs. This DNA methylation was not detected in any cell type other than ASCs in adipose tissue. Therefore, analyzing this methylation may be a suitable approach for quantifying ASCs in tissues because DNA is readily extracted from tissues. SQAT is based on quantifying this methylation by qPCR using methylation-sensitive HapII-treated DNA as the template. SQAT was validated based on the numbers of ASCs determined by CD31−/CD34+-based flow cytometry. The results obtained by both methods were perfectly correlated, thereby demonstrating that SQAT is a useful tool for quantifying ASCs. SQAT analysis using ASCs isolated from suctioned fat according to the standard protocol (i.e., collagenase treatment) showed that the yield of ASCs was 59% ± 21%, which suggests that the ASC isolation technique requires further improvement. Furthermore, SQAT is an excellent method for quantifying ASCs in arbitrary samples (particularly tissue), which could dramatically improve ASC isolation technologies and fat graft systems, thereby facilitating the prediction of graft retention.
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2.Hsu VM, Stransky CA, Bucky LP, Percec I (2012) Fat grafting's past, present, and future: why adipose tissue is emerging as a critical link to the advancement of regenerative medicine. Aesthet Surg J 32: 892-899. doi: 10.1177/1090820X12455658. PMID: 22942117
3.Choi M, Small K, Levovitz C, Lee C, Fadl A, et al. (2013) The volumetric analysis of fat graft survival in breast reconstruction. Plast Reconstr Surg 131: 185-191. doi: 10.1097/PRS.0b013e3182789b13. PMID: 23076412
4.Gause 2nd TM, Kling RE, Sivak WN, Marra KG, Rubin JP, et al. (2014) Particle size in fat graft retention: A review on the impact of harvesting technique in lipofilling surgical outcomes. Adipocyte 3: 273-279. doi: 10.4161/21623945.2014.957987. PMID: 26317051
5.Ersek RA (1991) Transplantation of purified autologous fat: a 3-year follow-up is disappointing. Plast Reconstr Surg 87: 219-227. PMID: 1750860
6.Trojahn Kølle S, Oliveri RS, Glovinski PV, Elberg JJ, Fischer-Nielsen A, et al. (2012) Importance of mesenchymal stem cells in autologous fat grafting: a systematic review of existing studies. J Plast Surg Hand Surg 46: 59-68. doi: 10.3109/2000656X.2012.668326. PMID: 22471250
7.Feisst V, Meidinger S, Locke MB (2015) From bench to bedside: use of human adipose-derived stem cells. Stem Cells Cloning 8: 149-162. doi: 10.2147/SCCAA.S64373. PMID: 26586955
8.Yoshimura K, Suga H, Eto H (2009) Adipose-derived stem/progenitor cells: roles in adipose tissue remodeling and potential use for soft tissue augmentation. Regen Med 4: 265-273. doi: 10.2217/17460751.4.2.265. PMID: 19317645
9.Matsumoto D, Sato K, Gonda K, Takaki Y, Shigeura T, et al. (2006) Cell-assisted lipotransfer: supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection. Tissue Eng 12: 3375-3382. doi: 10.1089/ten.2006.12.3375. PMID: 17518674
10.Yoshimura K, Sato K, Aoi N, Kurita M, Hirohi T, et al. (2007) Cell-assisted lipotransfer for cosmetic breast augmentation: supportive use of adipose-derived stem/stromal cells. Aesthetic Plast Surg 32: 48-55. doi: 10.1007/s00266-007-9019-4. PMID: 17763894
11.Philips BJ, Grahovac TL, Valentin JE, Chung CW, Bliley JM, et al. (2013) Prevalence of endogenous CD34+ adipose stem cells predicts human fat graft retention in a xenograft model. Plast Reconstr Surg 132: 845-858. doi: 10.1097/PRS.0b013e31829fe5b1. PMID: 23783061
12.Li H, Zimmerlin L, Marra KG, Donnenberg VS, Donnenberg AD, et al. (2011) Adipogenic potential of adipose stem cell subpopulations. Plast Reconstr Surg 128: 663-672. doi: 10.1097/PRS.0b013e318221db33. PMID: 21572381
13.Yoshimura K, Shigeura T, Matsumoto D, Sato T, Takaki Y, et al. (2006) Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol 208: 64-76. doi: 10.1002/jcp.20636. PMID: 16557516
14.Woodfin A, Voisin M, Nourshargh S (2007) PECAM-1: a multi-functional molecule in inflammation and vascular biology. Arterioscler Thromb Vasc Biol 27: 2514-2523. doi: 10.1161/ATVBAHA.107.151456. PMID: 17872453
15.Gumina RJ, Kirschbaum NE, Piotrowski K, Newman PJ (1997) Characterization of the human platelet/endothelial cell adhesion molecule-1 promoter: identification of a GATA-2 binding element required for optimal transcriptional activity. Blood 89: 1260-1269. PMID: 9028949
16.Baumann M, Pontiller J, Ernst W (2010) Structure and basal transcription complex of RNA polymerase II core promoters in the mammalian genome: an overview. Mol Biotechnol 45: 241-247. doi: 10.1007/s12033-010-9265-6. PMID: 20300884
17.Tao Q, Robertson KD, Manns A, Hildesheim A, Ambinder RF (1998) The Epstein-Barr virus major latent promoter Qp is constitutively active, hypomethylated, and methylation sensitive. J Virol 72: 7075-7083. PMID: 9696800
18.Boquest AC, Noer A, Sørensen AL, Vekterud K, Collas P (2006) CpG methylation profiles of endothelial cell-specific gene promoter regions in adipose tissue stem cells suggest limited differentiation potential toward the endothelial cell lineage. Stem Cells 25: 852-861. doi: 10.1634/stemcells.2006-0428. PMID: 17170064
19.Noer A, Sørensen AL, Boquest AC, Collas P (2006) Stable CpG hypomethylation of adipogenic promoters in freshly isolated, cultured, and differentiated mesenchymal stem cells from adipose tissue. Mol Biol Cell 17: 3543-3556. doi: 10.1091/mbc.E06-04-0322. PMID: 16760426
20.Brake DK, Smith CW (2008) Flow cytometry on the stromal-vascular fraction of white adipose tissue. Methods Mol Biol 456: 221-229. doi: 10.1007/978-1-59745-245-8_16. PMID: 18516564
21.Oberbauer E, Steffenhagen C, Wurzer C, Gabriel C, Redl H, et al. (2015) Enzymatic and non-enzymatic isolation systems for adipose tissue-derived cells: current state of the art. Cell Regen (Lond) 4: 7. doi: 10.1186/s13619-015-0020-0. PMID: 26435835
22.Varma MJO, Breuls RGM, Schouten TE, Jurgens WJFM, Bontkes HJ, et al. (2007) Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells. Stem Cells Dev 16: 91-104. doi: 10.1089/scd.2006.0026. PMID: 17348807
23.Zuk P (2013) Adipose-derived stem cells in tissue Regeneration: a review. ISRN Stem Cells 2013: 713959. doi: 10.1155/2013/713959.
24.Fittipaldi M, Nocker A, Codony F (2012) Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification. J Microbiol Methods 91: 276-289. doi: 10.1016/j.mimet.2012.08.007. PMID: 22940102
25.Andorrà I, Esteve-Zarzoso B, Guillamón JM, Mas A (2010) Determination of viable wine yeast using DNA binding dyes and quantitative PCR. Int J Food Microbiol 144: 257-262. doi: 10.1016/j.ijfoodmicro.2010.10.003. PMID: 21036413
26.Stoltz J, de Isla N, Li YP, Bensoussan D, Zhang L, et al. (2015) Stem Cells and Regenerative Medicine: Myth or Reality of the 21th Century. Stem Cells Int 2015: 734731. doi: 10.1155/2015/734731. PMID: 26300923
27.Minteer DM, Marra KG, Rubin JP (2015) Adipose stem cells: biology, safety, regulation, and regenerative potential. Clin Plast Surg 42: 169-179. doi: 10.1016/j.cps.2014.12.007. PMID: 25827561
28.Kapur SK, Dos-Anjos Vilaboa S, Llull R, Katz AJ (2015) Adipose tissue and stem/progenitor cells: discovery and development. Clin Plast Surg 42: 155-167. doi: 10.1016/j.cps.2014.12.010. PMID: 25827560
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