Postdoctoral Scientist Cedars-Sinai Board of Governors Regenerative Medicine Institute Los Angeles, California, United States
Abstract: Tendinopathy is the most prevalent among the musculoskeletal injuries. Due to hypocellularity and hypovascularity, adult tendon healing is compromised. This leads to incomplete tissue remodeling and as a result, scar tissue forms and a vicious cycle of chronic inflammation ensues. On the contrary, young tendon heals faster and deposit tendon tissue rapidly. In this context, we studied young and adult rat Achilles tendon healing. Young tendon demonstrated faster improvement in gait and tendon biomechanics, while single cell data revealed a significant increase of tenogenic precursors as early as 1 week compared to adult rat tendon. Informed by tenogenic cell clusters in young rat tendon, we differentiated the iPSCs towards triple positive Scleraxis+ (Scx+), Tenomodulin+ (Tnmd+) and early growth response-1+ (Egr1+) iTenocytes using developmental pathways in a high purity, xeno-free and stepwise manner. First, iPSCs were induced to form primitive streak through activation of TGF, WNT and FGF signaling. Then, WNT/FGF were activated and TGF/BMP were suppressed to complete paraxial mesodermal differentiation. Next, somitogenesis was induced by inhibition of WNT, FGF, TGF, BMP at the same time. Upon formation of somites, we established of PAX1>>PAX3 sclerotome (SCL) cells by activation of Hedgehog while inhibiting WNT and BMP. Subsequently, we achieved >90% Scx+ tenogenic precursor-like syndetome (SYN) cells by context dependent activation of TGF3/BMP14/FGF8. The SYN cells clearly illustrated the tendon trajectory at single cell level. Finally, we obtained readily scalable Tnmd+/Mkx+/Egr1+ iTenocytes within 12 days. We showed that two different iPSC lines can be used for the iTenocyte production with abolished side products. In addition to the remarkable efficiency of iTenocyte induction, the cells can be frozen stored and scaled up without loss of phenotype. Currently, we are investigating the translation potential of iTenocytes in full thickness adult rat tendon defect model. Collectively, the iTenocytes are highly translatable and scalable for future clinical applications in scarless tendon/ligament regeneration and functional restoration.
Funding Source: The study is supported by CIRM-DISC0-14350 and CIRM Scholarship CIRM EDUC4-12751
