Gene expression in human adipose-derived stem cells: comparison of 2D films, 3D electrospun meshes or co-cultured scaffolds with two-way paracrine effects

Abstract

Finding the appropriate cues to trigger the desired differentiation is a challenge in tissue engineering when stem cells are involved. In this regard, three-dimensional environments are often compared to cells' twodimensional culture behaviour (plastic culture dish). Here, we compared the gene expression pattern of human adipose-derived stem cells (ASC) seeded in a three-dimensional (3D) electrospun mesh and on a two-dimensional (2D) film -both of exactly the same material. Additionally, we conducted experiments with a scaffold floating above a film to investigate two-way paracrine effects (co-system). Electrospun meshes (3D scaffolds) and films (2D), consisting either of pristine poly-lactic-co-glycolic acid (PLGA) or of PLGA-containing dispersed amorphous calcium phosphate nanoparticles (PLGA/aCaP), were seeded with ASCs and cultured either in Dulbecco Minimum Essential Medium (DMEM) or in osteogenic medium. After two weeks, minimum stem cell criteria markers as well as typical markers for osteogenesis, endothelial cell differentiation, adipogenesis and chondrogenesis were analysed by quantitative real-time PCR. Interestingly, mostly osteogenic genes of cells seeded on 3D meshes were upregulated compared to those on 2D films, while stem cell markers seemed to be only slightly affected. Runx2 and osteocalcin showed an especially strong upregulation under all conditions, while most other factors analysed for 2D/3D changes were highly dependent on the material composition, the culture medium and on paracrine signalling effects. The beneficial 3D environment for stem cells found in many studies has therefore not to be attributed to the third dimension alone and should carefully be compared to 2D films fabricated of the same material. Furthermore, paracrine interactions triggering differentiation are not negligible.

Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration

“…Lineage-specific differentiation of ASCs towards the osteoblast, the endothelial, the adipogenic and the chondrogenic cell lineage had been achieved previously [33] using cell culture media supplementation according to Zuk et al [6]. We used Von Kossa and Alizarin red staining in order to semi-quantitatively assess the osteogenic differentiation extent; CD31 immunohistochemical staining to evaluate the endothelial cell differentiation; Alcian Blue staining to determine the ability of the ASCs to differentiate towards chondrocytes; Oil Red O staining to obtain information about the adipogenic differentiation.…”

“…The fat for these primary cells had been received from a 29-year-old woman by abdominal liposuction. This donor was chosen because it seemed to be a bigger challenge to evoke an osteogenic commitment in ASCs, induced by the nanocomposite, than in ASCs that had previously shown an easy osteogenic differentiation in the osteogenic culture medium, as tested during multilineage differentiation [33].…”

“…We used Von Kossa and Alizarin red staining in order to semi-quantitatively assess the osteogenic differentiation extent; CD31 immunohistochemical staining to evaluate the endothelial cell differentiation; Alcian Blue staining to determine the ability of the ASCs to differentiate towards chondrocytes; Oil Red O staining to obtain information about the adipogenic differentiation. These four differentiations had been carried out before for the primary cells under view, using induction media, and results were presented earlier [33].…”

“…In our previous work, we focused on a nanocomposite based on poly(lactic-co-glycolic acid) (PLGA) and amorphous calcium phosphate nanoparticles (PLGA/aCaP, weight ratio 60:40) seeded with human adipose-derived stem cells (ASCs) [11,[30][31][32]. Moreover, the gene expression of primary human ASCs seeded on a thin casted film made of either PLGA or PLGA/aCaP or on electrospun random fiber meshes (PLGA or PLGA/aCaP) was assessed to investigate the effects of the structure and material composition [33].…”

Tissue Regeneration

“…Many polymers have been synthesized and modified in order to get suitable materials in terms of implants for tendon repair and regeneration; polyglycolic acid (PGA) [75,77], polylactic-co-glycolic acid (PLGA) [78], PLGA/alginate composite [6], polylactic acid (PLA) [79][80][81], poly-l-lactic acid (PLLA) [82][83][84], polycaprolactone (PCL) [85], polycaprolactone/ polyethylene oxide (PCL/PEO) [7], polyurethane (PU) [86,87], polyethylene terephthalate (PET) [88,89], DegraPol ® [90,91] (Figure 2), nanocarbon fiber [92], and polyurea [93], among others. The architecture of the synthetic materials has to be chosen carefully, as gene expression of (stem) cells may be significantly influenced by the microenvironment that the cells encounter [94].…”