Adipose Derived Stem Cells Isolated from Premature Aging Mice Show Sustained Stemness: Implications for Regenerative Medicine
Sudheer K. Ravuri, PhD1, Xiaodong Mu, PhD2, Wanqun Chen, PhD2, Johnny Huard, PhD2.
1Steadman Philippon Research Institute, Vail, CO, USA, 2Department of Orthopaedic Surgery, University of Texas Health Science Center, Houston, TX, USA.
PURPOSE: Exhaustion of functional stem cells in tissues may be caused by the effects of aging and stem cell senescence, which may ultimately lead to decreased rejuvenation and increased age progression. Adipose tissues and mature adipocytes may become dysfunctional and acquire a pro-inflammatory, senescent-like phenotype with aging, but the influence of the aging process on adipose stem cells is not well understood. Mice deficient in Zmpste24 (Zmpste24-/- or Z24-/-), a zinc metalloproteinase involved in the formation of mature lamin A for the nuclear membrane, demonstrate premature onset of age-related changes and have been studied as a model for human Hutchinson-Gilford Progeria Syndrome (HGPS). Using this mouse model, our current study is focused on investigating the characteristics and rejuvenative potential of ASCs from Z24-/- premature mice in vitro with implications for regenerative medicine.
METHODS: Subcutaneous adipose tissues were harvested from Z24-/- and age-matched wild-type (WT) control mice and adipose tissue was minced, enzymatically digested for 1 hr at 37°C and ASCs were separated by centrifugation. ASCs were cultured in adipocyte growth medium. We compared the culture characteristics of Z24-/- and WT (wild-type) ASCs, and performed qPCR to determine senescence and quiescent properties of ASCs. Co-cultured ASCs with muscle progenitor cells (MPCs) in a Transwell system for 2 days to determine the potential influence of Z24-/- ASCs on Z24-/- muscle progenitor cells and eventual rescuing and rejuvenation of defective phenotypes.
RESULTS: Z24-/- ASCs grew much slower rate than WT ASCs, but cell senescence-associated β-galactosidase (SA-β-Gal) staining showed <5% in Z24-/- ASCs. RT-qPCR results showed mTORC1 (cell senescence-promoting factor) down-regulation and p21 (negative cell cycle regulator) upregulation with decreased proliferation potential of Z24-/- ASCs. These results suggest that Z24-/- ASCs are more quiescent but not more senescent. Furthermore, TGF-β1expression was upregulated and pro-inflammatory factors (IL-1β and IL-6) were downregulated, suggesting that Z24-/- ASCs may exhibit decreased pro-inflammatory potential. VEGF gene expression was upregulated, suggesting sustained regenerative potential of Z24-/- ASCs. Co-culture of WT or Z24-/- ASCs with Z24-/- MPCs in a Transwell system for 2 days showed that Z24-/- ASCs rescued the defective phenotypes of Z24-/- MPCs in a manner comparable to that of WT ASCs. WT ASCs as well as Z24-/- ASCs were able to repress the expression of pro-inflammatory factors and pro-fibrogenic factors, and increase the expression of anti-inflammatory factors and energy metabolism factors.
CONCLUSION: Our preliminary results showed that ASCs from Z24-/- prematurely aged mice were not as defective as MPCs and exhibited rejuvenative potential comparable to WT ASCs. Z24-/- ASCs were generally not senescent, but may be more quiescent compared to WT ASCs. Both WT and Z24-/- ASCs were able to repress pro-inflammatory and pro-fibrogenic factors, and promoted anti-inflammatory and energy metabolism factors. We believe that ASCs may show sustained stemness and lower senescence, in which ASCs could perform better in anti-aging therapy and tissue rejuvenation, with greater implications for regenerative medicine.
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