3D in Suspension versus 2D in Adhesion: molecular profiles in stemness and mesenchymal differentiation of Spheroids from Adipose-derived Stem Cells

Purpose: Adipose stem cells (ASCs) represent a reliable source of stem cells with a widely demonstrated potential in regenerative medicine and tissue engineering applications. New recent insights suggest that three-dimensional (3D) models may closely mimic the native tissue properties; spheroids from adipose derived stem cells (S-ASCs) exhibit enhanced regenerative abilities compared with those of 2D models. Stem cell therapy success is determined by “cell-quality”; for this reason, microRNA profiles, the involvement of stress signals and cellular aging need to be further investigated. Material and Methods: Adipose tissue was collected from healthy individuals, 44 females and 17 males, after signing informed consent. Mean age was 50, 25 years (range: 18-77). Lipoaspirate samples were harvested from different body areas such as abdomen, breast, flanks, trochanteric region, and knee. Here, we performed a comparative analysis, molecular and functional, of miRNA expression pattern profile “stemness and differentiation associated”, genes connected with stemness, aging, telomeric length and oxidative stress, of adipose stem cells in three-dimensional and adhesion conditions, SASCs-3D and ASCs-2D cultures. Results: We have demonstrated that Spheroids from Adipose-derived Stem Cells (SASCs-3D) present express high level of the typical miRNAs and mRNAs of iPS cells, such as miR-142-3p and SOX2/POU5F1/NANOG, in canonical and in long term in vitro culture condition, express low level of the early and late miRNAs and mRNAs typical of chondrocytic, adipocytic and osteoblastic lineages in canonical and in long term in vitro culture condition. The expression levels of stemness-related markers and anti-aging Sirtuin1 were significantly up-regulated (P < 0.001) in SASC-3D while gene expression of aging-related p16INK4a was increased in ASCs-2D (P < 0.001). We found that 3D and 2D cultures also presented a different gene expression profile for those genes related to telomere maintenance (Shelterin complex, RNA Binding proteins and DNA repair genes) (P < 0.01 and P < 0.001) and oxidative stress (aldehyde dehydrogenase class1 and 3) (P < 0.05, P < 0.01 and P < 0.001) and presented a striking large variation in their cellular redox state. Conclusion: Based on our findings, we propose a “cell quality” model of SASCs, highlighting a precise molecular expression of microRNA pattern profiles, several genes involved with stemness (SOX2, POU5F1 and NANOG), anti-aging (SIRT1), oxidative stress (ALDH3) and telomeres maintenance.Purpose: Adipose stem cells (ASCs) represent a reliable source of stem cells with a widely demonstrated potential in regenerative medicine and tissue engineering applications. New recent insights suggest that three-dimensional (3D) models may closely mimic the native tissue properties; spheroids from adipose derived stem cells (S-ASCs) exhibit enhanced regenerative abilities compared with those of 2D models. Stem cell therapy success is determined by “cell-quality”; for this reason, microRNA profiles, the involvement of stress signals and cellular aging need to be further investigated. Material and Methods: Adipose tissue was collected from healthy individuals, 44 females and 17 males, after signing informed consent. Mean age was 50, 25 years (range: 18-77). Lipoaspirate samples were harvested from different body areas such as abdomen, breast, flanks, trochanteric region, and knee. Here, we performed a comparative analysis, molecular and functional, of miRNA expression pattern profile “stemness and differentiation associated”, genes connected with stemness, aging, telomeric length and oxidative stress, of adipose stem cells in three-dimensional and adhesion conditions, SASCs-3D and ASCs-2D cultures. Results: We have demonstrated that Spheroids from Adipose-derived Stem Cells (SASCs-3D) present express high level of the typical miRNAs and mRNAs of iPS cells, such as miR-142-3p and SOX2/POU5F1/NANOG, in canonical and in long term in vitro culture condition, express low level of the early and late miRNAs and mRNAs typical of chondrocytic, adipocytic and osteoblastic lineages in canonical and in long term in vitro culture condition. The expression levels of stemness-related markers and anti-aging Sirtuin1 were significantly up-regulated (P < 0.001) in SASC-3D while gene expression of aging-related p16INK4a was increased in ASCs-2D (P < 0.001). We found that 3D and 2D cultures also presented a different gene expression profile for those genes related to telomere maintenance (Shelterin complex, RNA Binding proteins and DNA repair genes) (P < 0.01 and P < 0.001) and oxidative stress (aldehyde dehydrogenase class1 and 3) (P < 0.05, P < 0.01 and P < 0.001) and presented a striking large variation in their cellular redox state. Conclusion: Based on our findings, we propose a “cell quality” model of SASCs, highlighting a precise molecular expression of microRNA pattern profiles, several genes involved with stemness (SOX2, POU5F1 and NANOG), anti-aging (SIRT1), oxidative stress (ALDH3) and telomeres maintenance