Generating mesenchymal stem cells in 3D from human embryonic stem cell spheroids

Background: Mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC), named EMSC here, have been found efficacious in animal models of autoimmune, inflammatory, and degenerative diseases. However, all the EMSC derivation methods reported so far are in two-dimensional (2D) culture systems, which are of low efficiency and high cost, difficult for large-scale production for research and therapeutic applications. Methods: We established a 3D system that allowed differentiation of hESC spheroids into MSC spheroids (EMSCSp) following treatment with BMP4 and A8301 for 5 days and subsequent culture in a MSC medium for about 15 days. All the procedures were conducted in one vessel without intermediate passaging. Results: EMSCsp cells were efficiently derived from hESC spheroids within 20 days in the 3D culture system, which could be scaled up from a small culture vessel to a 100-ml plastic bag. EMSCSp could further differentiate into spheroids of chondrocytes or adipocytes. EMSCSp could also reattach and form a 2D-monolayer culture (EMSCSp-ML). Compared to EMSC differentiated in monolayer, EMSCSp-ML had faster proliferation and higher yield, and developed less apoptosis and slower senescence. EMSCSp-ML also retained immune-modulatory effects in vitro and therapeutic effects on two mouse models of colitis. Conclusions: The 3D method provides a simple and economic system for large-scale production of EMSC as an unlimited source of the therapeutically promising cells. In this study, we show that hESC cultured as spheroids (hESCSp) could differentiate into EMSC spheroids (EMSCSp) also via an intermediate trophoblast step. Unlike the 2D differentiation systems, our new method allows all the multi-step differentiation procedures to be completed in one vessel in 3D without any need for cell passaging. EMSCSp could further differentiate into spheroids of chondrocytes and adipocytes or reattached and passaged in monolayer (EMSCSp-ML) without compromising their multipotency. Compared to EMSC differentiated in monolayer (EMSCML) as we reported before [9], EMSCSp-ML had faster proliferation and higher yield, and developed less apoptosis and slower senescence. EMSCSp-ML also retained immune-modulatory effects in vitro and therapeutic effects on a mouse colitis model. Thus, the 3D method is advantageous over the 2D methods for large-scale production of EMSCs for potential clinic application.