Large scale mesenchymal stem cells production from pluripotent stem cells on microcarriers.

OLIVER BLOND; LUCIA MORO; ALESSANDRA NORRIS; ALEJANDRO LA GRECA; LEONARDO ROMORINI; DARÍO FERNÁNDEZ ESPINOSA; MARÍA ELIDA SCASSA; GUSTAVO SEVLEVER; CARLOS LUZZANI; SANTIAGO G. MIRIUKA

Mar del Plata

Congreso; SAIC SAE SAFE 2016; 2016

SAIC

Mesenchymal stem cells (MSC) are strong candidates for regenerative therapies, because of their involvement in stemness for tissue repair and their low immunogenicity. Among their main applications are exosomal preparations or direct cell injections. Since MSC expansion for clinical applications has some issues, due to their limited number of divisions, it is more convenient to derive and amplify mesenchymal stem cells from pluripotent stem cells (PSC).In a previous work done in our laboratory, we have adapted this protocol of differentiation for clinical purposes, using platelet lysate instead of animal serum, making it more affordable and biocompatible. We now demonstrate that it is possible to scale up this protocol by growing cells in 3D, on microcarriers.Once the most suitable microcarrier was found, the most challenging aspects were to adapt the agitation settings and to obtain individualized cells.The best microcarrier and coating combination was cytodex 3 coated with or without geltrex. The most efficient agitation setting (discontinuous shaking at less than 100 rpm) was the one dedicated to mesenchymal stem cells from conventional sources. Furthermore, the dissociation step was optimized by combining strong enzyme digestion with an agitation at 80 rpm and a Rho kinase inhibitor (Y27632) to relax actin-mediated aggregate compaction.After thirty to forty days of differentiation, the MSC phenotype was analyzed by different methods. The combination of cell surface markers, including CD90, CD73 and CD105 and quantitative RT-PCR allowed us to conclude that these cells are indeed MSC.We have now proved that mesenchymal stem cells can be produced from PSC with this differentiation protocol in 3D, on cytodex 3 microcarriers. As the phenotype was confirmed with specific markers, our next objectives will be to confirm that MSC are fully functional by immunomodulation experiments and multilineage differentiation assays.