Retinoic Acid Receptor Signaling During Paraxial Mesoderm Differentiation of Pluripotent Stem Cells

The treatment of severe musculoskeletal injuries is currently limited to surgical intervention and natural healing during recovery, which are not always sufficient to repair critical defects. Various cell-based approaches for repair augmentation have gained traction, however a reliable cell therapy approach has not been realized. Pluripotent stem cells (PSCs) retain the developmental potential to become any cell in the human body and are a valuable model for studying cell differentiation that hold tremendous promise for treating orthopedic injuries. The goal of our research has been to develop an efficient in vitro differentiation method to generate skeletal progenitor cells, the forerunners of osteoblasts, chondrocytes and tenocytes, responsible for creation and maintenance bone, cartilage and tendon, respectively. We have focused on generating paraxial mesoderm, a specific type of mesoderm whose derivatives give rise to all cell types comprising the axial skeleton. Our approach progressively differentiates PSCs to mimic their natural maturation during embryonic development in order to study the mechanisms directing these events. We have genetically engineered mouse and human embryonic stem cells to express fluorescent protein reporters that enable us to visualize expression of critical genes during differentiation. This allows us to have a rapid diagnostic readout on the effectiveness of experimental conditions and subsequently sort out individual cells of interest for further analysis. The outcomes of my thesis work demonstrate that activation of the Wnt pathway coupled with inverse agonism of retinoic acid receptor (RAR) signaling is capable of inducing paraxial mesoderm, an intermediate cell type arising early in development and a key phase in the progression from PSCs to skeletal progenitor cells. Our studies reveal paraxial mesoderm induction is significantly more efficient from the “primed” or epiblast stem cell state compared to the “naïve” inner cell mass like state. Further, inverse agonism of RARs during the formation of epiblast-like cells favors a paraxial mesoderm fate. Collectively, my thesis work has provided a more complete understanding of the molecular mechanisms prompting stem cell specification into paraxial mesoderm, as well as an earlier than appreciated role for RARs in epiblast cells where they potentially function as transcriptional repressors