Chondrogenesis of human embryonic stem cells using a sequential 3D and 2D culture

Background: Osteoarthritis (OA) is one of the most prevalent joint diseases around the world. Cell-based-therapies considered as useful approach for OA treatment. Autologous chondrocyte implantation (ACI) despite non-immunogenicity, can results in disrupting chondrocyte morphology and fibrocartilage formation. Hence, Due to their innate pluripotency, unlimited self-renewal and human source, human embryonic stem cells (hESCs) can be used as an appropriate cell source for OA cell therapy. According to the different reports, culture methods using embryoid body (EB) formation (3D) and monolayer (2D) can be effective for hESCs chondrogenic differentiation. Chondrogenesis from hESCs can provide promising in vitro model for developmental biology studies, drug discoveries and ultimately regenerative medicine applications for human articular cartilage.Objective: Here, chondorgenesis of hESCs was induced using a sequential 3D-2D culture system and assessed by alcian blue and trichrom staining.Materials and Methods: Human ESCs (Yazd4; 46,XX) were induced for differentiation using EB formation in a non-adherent culture condition as 3D culture for 4 days. Then EBs were transferred and cultured further in adherent monolayer culture condition as 2D culture system till day 14 of differentiation. Differentiated EBs and cells were assessed for chondrogenesis induction using alcian blue and trichrom staining following 4 and 14 days of differentiation.Results: Our data has indicated that sequential 3D-2D culture of hESCs induced homogenous population of chondrocytes as identified by alcian blue and trichrom staining following 4 and 14 days in culture.Conclusion: Sequential 3D-2D culture of hESCs can induce spontaneously in vitro chondrogenesis. This method can be improved using defined culture condition and conditioned medium for more efficient outcome and further studies in cell-based therapeutic studies.

کلیدواژه ها:

Chondrogenesis ، Embryoid body ، Human embryonic stem cells ، Monolayer culture