The Tumorigenic Risk Of Adipose Tissue Derived Stem Cells In Vitro

Backgound:Stem cells are a kind of primitive undifferentiated cells which have self-renewal and multilineage differentiation capacity, can be classified as Embryonic Stem Cells and Adult Stem Cells according to the source. Mesenchymal stem cells (MSCs) are the main direction of today's stem cell research as a key component of adult stem cells, which have been also identified in several other accessible tissues, including bone marrow, fat, liver, cord blood, etc. and the research in the area of Bone marrow-derived mesenchymal stem cells (BMSCs) is relatively concentrated and mature. In vitro, BMSCs have multilineage differentiation capacity and can be rapidly expanded, which present great prospects for clinical application in tissue engineering, cell therapy and regenerative medicine.Like BMSCs, Adipose tissue-derived stem cells (ASCs) share many of the characteristics of its counterpart in marrow including extensive proliferative potential and the ability to undergo multilineage differentiation in vitro. Significantly, large amounts of ASCs can be obtained from adipose tissue, which, compared with other tissues, contained high numbers of MSCs that can be easily expanded in vitro [2], this advantage makes ASCs become a new hot spot of stem cell research rapidly. However, report had shown that the mesenchymal stem cells derived from lipoma (Lipoma-derived mesenchymal stem cells, LMSCs) had several similar characteristics with those of ASCs, like multilineage differentiation potential, self-renewal and high proliferation. This brings us doubts about the clinical applications of ASCs which involved cell therapy and tissue engineering. Whether ASCs will transform into LMSCs? Whether their proliferation and differentiation could be controlled? Whether the adipose tissue they had formation will appear a pathological growth like lipoma in long-term? These questions had become the key issue associated with the clinical application of ASCs. However, the problem whether ASCs have tumorigenicity have not been studied at homeland and abroad in this area. Therefore, in this study, we cultured and expanded ASCs and LMSCs in vitro, compared eight biological characteristics of the two types of cells, including morphology, differentiation potential, tissue sections, growth kinetics, surface markers, cell cycle distribution, tumor-specific genes and telomerase expression, conducted a preliminary assessment about the tumorigenicity of ASCs. Aims to provide the basis and supports for the clinical applications of ASCs.Objective:To investigate the tumorigenicity of adipose tissue derived stem cells (ASCs) cultured in vitro. By comparing normal ASCs and The lipomas mesenchymal stem cells in morphology, differentiation potential and tissue sections, growth kinetics, surface markers, cell cycle distribution, tumor-specific genes and telomerase expression in terms of identification.Methods:ASCs and Lipoma-derived mesenchymal stem cells (LMSCs) were isolation from the two tissues by means of enzymatic digestion, and appearance of the cultured cells was observed. The adipogenic, osteogenic and chondrogenic ability of ASCs and LMSCs were assessed by oil red O staining, alizarin bordeaux staining and alcian blue staining, respectively. Regular slice and stain of two tissues; the cell viability was evaluated with MTS chromatometry and cell growth curve was generated. Flow cytometry was performed for cell cycle analysis and the expression of the cell surface marker profiles. QRT-PCR was used to detect the expression of tumor-specific genes (the high-mobility group AT-hook 2, HMGA2 and cell death-inducing def45-like effectors, CIDEA), and immunocytochemistry was used to detect the expression of telomerase.Results:ASCs and LMSCs both showed a fusiform shape, like fibroblast cell, which displayed a similar characteristic in morphology, and LMSCs growth faster than ASCs at later time; ASCs and LMSCs both had the ability to differentiate into mature adipocyte, osteoblast and chondrocyte; Apparent differences was observed in histologic sections of adipose tissue and lipoma tissue, lipomas tissue has a complete envelope, bundles of spindle cells and rich fiber interval, but the normal adipose tissue sections shows a large number of fat vacuolese.ASCs shown a significant lower proliferation capacity than LMSCs by MTS chromatometry (p<0.01); The expressions of CD29, CD44, CD105 were observed in ASCs was similar to LMSCs while the level of CD133 was a significant lower in ASCs (5.35%) than in LMSCs (26.87%). ASCs shown a lower proliferation capacity than LMSCs by cell cycle analysis; The expression of HMGA2 shown a lower level in ASCs (RQ=1) than in LMSCs (1.79±0.279) (t=-6.329,P<0.01) and CIDEA shown a higher level in ASCs (RQ=1) than in LMSCs (0.64±0.060) (t=13.324,P<0.01) by qRT-PCR, they both have statistically difference between them(P<0.01); And in ASCs and LMSCs, the integrated optical intensity(IOD) values of hTERT expression are 1379.597±498.617 and 3328.108±902.856 (t=-7.317, P<0.01), size(area) are 132390.27±35568.945 and 238000.53±49264.289 (t=-6.732, P<0.01), density(mean) are 0.009±0.003 and 0.014±0.003 (t=-4.683, P<0.01), revealed the expression of hTERT also shown a significant lower level in ASCs than in LMSCs by immunocytochemistry, all the indexes have significant statistically difference.Conclusions:It presented significant differences between ASCs and LMSCs in the biological characteristics in vitro experiment, and no evidence indicate the malignant transformation of ASCs, however, to confirm the conclusion, it needs further long-term series of experiments.