3D co-culture model of endothelial colony-forming cells (ECFCs) reverses late passage adipose-derived stem cell senescence for wound healing

Abstract

Background: Extensive passage of adipose-derived stem cells (ASCs) in vitro leads to loss of function. Endothelial colony-forming cells (ECFCs) can be isolated from adult peripheral blood. A 3D co-culture system may rescue in vitro ASC senescence. Methods: A 3D co-culture model was successfully established using hyaluronic acid (HA) gel and a 10:1 ratio of late-passage ASCs and ECFCs. Cell density and culture conditions were optimized. Stem cell phenotype was characterized by flow cytometry. ELISA was used to measure the trophic effect of angiogenic growth factors and compare the effects of these factors between the 3-D co-culture and single-cell culture. Therapeutic potential of ASC/ECFC 3-D co-cultures was evaluated in a mouse chronic injury model. Results: Following incubation in a HA substrate 3D co-culture system, ASC morphology, phenotype, secretory profile, and differentiation capacity were restored. The ASC/ECFC co-culture increased the secretion of cytokines, such as hepatocyte growth factor, compared with single-cell 3D culture or monolayer culture. Mice radiation-ulcer wounds treated with ASC/ECFC 3-D co-cultures (spheroids) showed epithelialization and improved healing compared with wounds treated with ASCs or ECFCs alone. Further, transplanted ASC/ECFC spheroids exhibited superior angiogenic potential due to the ability of the ASCs to transdifferentiate into pericytes. Conclusion: 3D co-culture of ECFCs and ASCs in vitro restored native ASC properties by reversing cellular senescence and loss of trophic function. Transplant of ASC/ECFC 3D spheroids in vivo demonstrated proangiogenic capacity with improved therapeutic potential.

Systematic review on spheroids from adipose‐derived stem cells: Spontaneous or artefact state?

“…Several studies tested different hydrogels to form ASC spheroids by employing state of the art materials in the field of tissue engineering such as hyaluronic acid (Feng et al, 2017 ; Hu et al, 2020 ) and chitosan (Chen et al, 2018 ; Cheng, Chang, et al, 2012 ; Cheng, Wang, et al, 2012 ; Hsu et al, 2014 ; Huang et al, 2011 ; Lin et al, 2020 ), demonstrating an improved stemness gene expression compared to the traditional cultures in adhesion plates. The combination of these materials, or with other materials (FGF‐2 and PVA), was able to form 3D spheroids in a short period of time and showed better properties in stemness maintenance and an increased ability in stimulating angiogenic (Hsu et al, 2014 ; Hu et al, 2020 ) and chondrogenic (Huang et al, 2011 ; Lin et al, 2020 ) differentiations. Many materials were thermoresponsive such as GNF (glycosyl‐nucleosyl‐fluorinated) hydrogel (Ziane et al, 2012 ) or poly‐N‐isopropylacrylamide (PNIPAAm) (G. Kim, Jung et al, 2020 ).…”

The Rejuvenation and Functional Restoration of Aged Adipose Stem Cells by DUXAP10 Knockdown via the Regulation of the miR-214-3p/RASSF5 Axis

“… 43 , 44 However, the regenerative capabilities of these cells diminish with donor aging, diabetes, and prolonged in vitro culture, thereby limiting their clinical utility. 45 Previous research has explored several strategies to enhance the wound healing properties of ASCs, including the upregulation of miR-1248 46 or an anti-aging gene β-Klotho, 47 cultivation in a 3D coculture system, 48 and pre-treatment with a NOX1/4 inhibitor GKT137831 49 to mitigate reactive oxygen species production. In the current investigation, we have identified a novel approach wherein the knockdown of DUXAP10 enhances the therapeutic potential of aged ASCs in skin wound healing, as evidenced by improved wound collagen deposition, neovascularization, and accelerated wound closure.…”

Concentrated Growth Factors Promote hBMSCs Osteogenic Differentiation in a Co-Culture System With HUVECs

“…In addition, bone repair and reconstruction are biological processes regulated by many cellular interactions and numerous signaling pathways ( Kim et al, 2021 ). In an indirect co-culture system, the biological behavior of cells is only regulated by paracrine signaling whereas in direct co-culture system, cells are influenced by both paracrine and intercellular signaling between cells, which better reflects the actual situation compared to traditional single cell culture ( Villars et al, 2000 ; Hu et al, 2020 ). It is an ideal experimental model to evaluate interactions between different cells.…”