Enhanced multi-lineage differentiation of human mesenchymal stem/stromal cells within poly(N-isopropylacrylamide-acrylic acid) microgel-formed three-dimensional constructs.

Human mesenchymal stem/stromal cells (hMSCs) are a potential cell source of stem cell therapy for many serious diseases and hMSC spheroids have emerged to replace single cell suspensions for cell therapy. Three-dimensional (3D) scaffolds or hydrogels which can mimic properties of the extracellular matrix (ECM) have been widely explored for their application in tissue regeneration. However, there are considerably less studies on inducing differentiation of hMSC spheroids using 3D scaffolds or hydrogels. This study is the first to explore multi-lineage differentiation of a stem cell line and primary stem cells within poly(N-isopropylacrylamide) (p(NIPAAm))-based thermosensitive microgel-formed constructs. We first demonstrated that poly(N-isopropylacrylamide-co-acrylic acid) (p(NIPAAm-AA)) was not toxic to hMSCs and the microgel-formed constructs facilitated formation of uniform stem cell spheroids. Due to functional enhancement of cell spheroids, hMSCs within the 3D microgel-formed constructs were induced for multi-lineage differentiation as evidenced by significant up-regulation of messenger RNA (mRNA) expression of chondrogenic and osteogenic genes even in the absence of induction media on day 9. When induction media were in situ supplied on day 9, mRNA expression of chondrogenic, osteogenic and adipogenic genes within the microgel-formed constructs were significantly higher than that in the pellet and 2D cultures, respectively, on day 37. In addition, histological and immunofluorescent images also confirmed successful multi-lineage differentiation of hMSCs within the 3D microgel-formed constructs. Hence, the thermosensitive p(NIPAAm-AA) microgel can be potentially used in an in vitro model for cell differentiation or in vivo transplantation of pre-differentiated human mesenchymal stromal cells into patients for specific lineage differentiation.