Induced Chondrogenic Differentiation of hESCs by hESC-Derived MSCs Conditioned Medium and Sequential ۳D-۲D Culture System

Background and Aims: It has been proven that human mesenchymal stem cells (MSCs) conditioned medium (hMSCs-CM) can influence human embryonic stem cells (hESCs) chondrogenic differentiation. In this study, we hypothesized that conditioned medium (CM) from hESCs-derived MSCs in a sequential ۳D-۲D culture system could facilitate the induction of chondrogenesis in hESCs. Materials and Methods: CM was collected from Yazd۲ (hESCs; ۴۶, XY) derived MSCs confluent cultures and stored at -۲۰ °C. Yazd۴ hESC line (۴۶, XX) was induced for differentiation using EB formation as ۳D culture into SD (spontaneously differentiation) and CM groups (differentiation using conditioned medium) for four days. Cell culture continued in a ۲D (monolayer) culture system for both groups till day ۱۴. Ultimately, chondrogenic differentiation was assessed by Alcian blue and masson′s trichrome staining at ۴ and ۱۴ days of differentiation, and quantitative real-time polymerase chain reaction (PCR) for NANOG, MEOX۱, SOX۵, SOX۶, SOX۹, ACAN, COL۲A۱ and RUNX۲ genes for SD and CM groups on days ۰ and ۴. Results:  The gene expression profile for chondrogenic genes in the CM group was significantly more than the SD group (p< ۰.۰۵). Furthermore, chemical staining assessment illustrated a significant GAG and collagen II difference between the CM and SD groups at days ۴ and ۱۴ (p< ۰.۰۵). Conclusions: Our findings would pave the way for creating an in vitro human chondrogenesis model for further studies in the developmental biology of articular cartilage tissue, which lend itself to cell-based therapy to cure joint diseases such as osteoarthritis.

کلیدواژه ها:

Chondrogenesis ، Conditioned medium ، Human embryonic stem cells ، Mesenchymal stem cells