Human pluripotent stem cells (hPSCs) are powerful tools to aid in the interrogation of the mechanisms of pathology of developmental disorders as well as modeling the development of embryonic and adult tissues. In this work, we attempted a patient-specific disease in a dish model of the craniofacial disorder Treacher Collins Syndrome (TCS) and the directed differentiation of brown adipocytes from hPSCs. The TCS model was unable to replicate the expected phenotype of reduced neural crest cell numbers or migratory potential. This shortcoming has been putatively attributed to reducing and high antioxidant conditions of the culture medium. Modulating brown adipocyte activity in humans has become an attractive therapeutic target for obesity and related metabolic disorders such as type 2 diabetes. Murine models suggest the brown adipose organ is capable of significant changes in body mass and circulating levels of insulin and glucose. Methods to interrogate human brown adipocyte functionality have been based on adult primary cells or overexpression systems. Here we report a robust and efficient chemically-defined method to differentiate human pluripotent stem cells into brown adipocytes through a developmentally appropriate paraxial mesoderm intermediate. These adipocytes display characteristics of mature brown adipocytes including marker expression, responsiveness to stimulation, enhanced metabolic activity, thermogenic capacity, lipolysis, and utilization of fatty acids. These cells are capable of being maintained in culture for several weeks, are amenable to passage, and upon transplantation in mice can give rise to adipose depots that maintain both morphological and functional properties. We propose that these cells are appropriate for modeling brown adipocyte development in vivo, elucidating basic biological cell fate decisions and branch points and as a platform for high-throughput drug screening to identify putative therapeutic targets to combat obesity and related disorders.

Skeletal muscle myofibers contain muscle stem cells called satellite cells that express the transcription factor PAX7, and regenerate muscle after acute injury. These skeletal muscle progenitor cells (SMPCs), or satellite cells in the adult muscle, lie outside the muscle fiber and refurbish the endogenous muscle stem cells upon damage. Human pluripotent stem cells (hPSCs) have enormous potential for use in regenerative medicine, especially for muscle wasting diseases like DMD. In DMD, the satellite cells become exhausted leading to failed regenerative ability. Once these muscle stem cells are depleted, damaged muscles are replaced by excessive fat and extracellular matrix deposition leading to muscle deterioration and fibrosis. One potential target to replenish exhausted muscle stem cells in patients with DMD is to generate an equivalent source from hPSCs. The myogenic activity from SMPCs derived from hPSCs is currently not well understood. We evaluated human fetal muscle in order to better understand the timing and specification of human SMPCs during human development and compare to hPSC-derived SMPCs for in vitro and in vivo myogenic activity. We have also shown that PAX7 is present in week 9 and 17 fetal skeletal muscle tissue, which may be indicative of when SMPC differentiation occurs. hPSC-derived SMPCs will be developed by the use of directed differentiation and synthetic mRNA. We have cloned PAX7 coding region into a VEE vector to enable synthetic RNA mediated overexpression (OE) of PAX7 into pre-differentiated hiPSCs in various plating conditions. PAX7 expression exceeded 700 fold in some conditions. Understanding the developmental and molecular underpinnings of human skeletal myogenesis will provide roads into generating SMPCs from hPSCs for use in regenerative medicine.

In Situ Tissue Regeneration: Host Cell Recruitment and Biomaterial Design explores the body's ability to mobilize endogenous stem cells to the site of injury and details the latest strategies developed for inducing and supporting the body's own regenerating capacity. From the perspective of regenerative medicine and tissue engineering, this book describes the mechanism of host cell recruitment, cell sourcing, cellular and molecular roles in cell differentiation, navigational cues and niche signals, and a tissue-specific smart biomaterial system that can be applied to a wide range of therapies. The work is divided into four sections to provide a thorough overview and helpful hints for future discoveries: endogenous cell sources; biochemical and physical cues; smart biomaterial development; and applications. - Explores the body's ability to mobilize endogenous stem cells to the site of injury - Details the latest strategies developed for inducing and supporting the body's own regenerating capacity - Presents smart biomaterials in cell-based tissue engineering applications—from the cell level to applications—in the first unified volume - Features chapter authors and editors who are authorities in this emerging field - Prioritizes a discussion of the future direction of smart biomaterials for in situ tissue regeneration, which will affect an emerging and lucrative industry