Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis.

Osteoarthritis is the most common degenerative joint condition, leading to articular cartilage (AC) degradation, chronic pain and immobility. The lack of appropriate therapies that provide tissue restoration combined with the limited lifespan of joint-replacement implants indicate the need for alternative AC regeneration strategies. Differentiation of human pluripotent stem cells (hPSCs) into AC progenitors may provide a long-term regenerative solution but is still limited due to the continued reliance upon growth factors to recapitulate developmental signalling processes. Recently, TTNPB, a small molecule activator of retinoic acid receptors (RARs), has been shown to be sufficient to guide mesodermal specification and early chondrogenesis of hPSCs. Here, we modified our previous differentiation protocol, by supplementing cells with TTNPB and administering BMP2 at specific times to enhance early development (referred to as the RAPID-E protocol). Transcriptomic analyses indicated that activation of RAR signalling significantly upregulated genes related to limb and embryonic skeletal development in the early stages of the protocol and upregulated genes related to AC development in later stages. Chondroprogenitors obtained from RAPID-E could generate cartilaginous pellets that expressed AC-related matrix proteins such as Lubricin, Aggrecan, and Collagen II, but additionally expressed Collagen X, indicative of hypertrophy. This protocol could lay the foundations for cell therapy strategies for osteoarthritis and improve the understanding of AC development in humans.

Optogenetic manipulation of BMP signaling to drive chondrogenic differentiation of hPSCs.

Optogenetics is a rapidly advancing technology combining photochemical, optical, and synthetic biology to control cellular behavior. Together, sensitive light-responsive optogenetic tools and human pluripotent stem cell differentiation models have the potential to fine-tune differentiation and unpick the processes by which cell specification and tissue patterning are controlled by morphogens. We used an optogenetic bone morphogenetic protein (BMP) signaling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We engineered light-sensitive hESCs through CRISPR-Cas9-mediated integration of the optoBMP system into the AAVS1 locus. The activation of optoBMP with blue light, in lieu of BMP growth factors, resulted in the activation of BMP signaling mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells in the dark. Furthermore, cells differentiated with light could form chondrogenic pellets consisting of a hyaline-like cartilaginous matrix. Our findings indicate the applicability of optogenetics for understanding human development and tissue engineering.