Synergistic Effects of Hypoxia and Morphogenetic Factors on Early Chondrogenic Commitment of Human Embryonic Stem Cells in Embryoid Body Culture

Abstract

Derivation of articular chondrocytes from human stem cells would advance our current understanding of chondrogenesis, and accelerate development of new stem cell therapies for cartilage repair. Chondrogenic differentiation of human embryonic stem cells (hESCs) has been studied using supplemental and cell-secreted morphogenetic factors. The use of bioreactors enabled insights into the effects of physical forces and controlled oxygen tension. In this study, we investigated the interactive effects of controlled variation of oxygen tension and chondrocyte-secreted morphogenetic factors on chondrogenic differentiation of hESCs in the embryoid body format (hESC-EB). Transient hypoxic culture (2 weeks at 5 % O2 followed by 1 week at 21 % O2) of hESC-EBs in medium conditioned with primary chondrocytes up-regulated the expression of SOX9 and suppressed pluripotent markers OCT4 and NANOG. Pellets derived from these cells showed significant up-regulation of chondrogenic genes (SOX9, COL2A1, ACAN) and enhanced production of cartilaginous matrix (collagen type II and proteoglycan) as compared to the pellets from hESC-EBs cultured under normoxic conditions. Gene expression profiles corresponded to those associated with native cartilage development, with early expression of N-cadherin (indicator of cell condensation) and late expression of aggrecan (ACAN, indicator of proteoglycan production). When implanted into highly vascularized subcutaneous area in immunocompromised mice for 4 weeks, pellets remained phenotypically stable and consisted of cartilaginous extracellular matrix (ECM), without evidence of dedifferentiation or teratoma formation. Based on these results, we propose that chondrogenesis in hESC can be synergistically enhanced by a control of oxygen tension and morphogenetic factors secreted by chondrocytes.

Augmentation of Musculoskeletal Regeneration: Role for Pluripotent Stem Cells

“…One method is via the formation of embryoid bodies [70,71]. One study found that hypoxia and morphogenetic factors had synergistic effects on chondrogenesis in EBs [71].…”

“…One method is via the formation of embryoid bodies [70,71]. One study found that hypoxia and morphogenetic factors had synergistic effects on chondrogenesis in EBs [71]. It is established that ESCs are more pluripotent when cultured under hypoxic conditions [72], and in vivo cartilage is subjected to hypoxia, and thus may account for the enhanced chondrogenesis observed under hypoxic conditions.…”

“…This highlights the distinct differences that exist between ASCs and ESCs in terms of chondrogenic differentiation, and gives an insight into the potential complexity of generating chondrocytes from PSCs. Chondrogenic differentiation of EBs has been achieved via various culture methods, such as pellet or micromass culture [71] methods, also used for cartilage generation using primary chondrocytes and MSCs [76].…”

“…However, since the native tissue has a low capacity for self-repair, it is desirable to create in vitro constructs that are functionally and morphologically identical to native cartilage to improve the ease of integration. Scaffold-free techniques for cartilage tissue engineering have been successful in producing in vitro cartilage constructs via chondrogenic differentiation of hPSCs in 3D pellet culture [71,79,81,84,87]. However, limited testing of these cartilage constructs has been performed in a physiologically relevant environment, for example, load-bearing animal model.…”

Challenges in engineering osteochondral tissue grafts with hierarchical structures

“…Synergistic effects of hypoxic conditioning and morphogenetic factors are also investigated in detail in the context of generating chondrocytes/osteoblasts from hESCs and hMSCs. Yodmuang et al showed that chondrogenesis in hESCs can be synergistically enhanced by controlling oxygen tension and morphogenic factors secreted by chondrocytes 44 . In their directed differentiation protocol, Oldershaw et al demonstrated that hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic cell aggregates 43 .…”