968 Enhancing the efficiency of engineered hair follicles with master regulators and extrinsic factors

Abstract

Each year, more than one million patients are hospitalized in the U.S. for significant skin loss due to thermal and pressure injuries, chronic diabetic ulcers or genetic skin diseases. The ability to generate engineered human skin constructs (HSCs) has provided a promising therapy for these patients. However, it remained an unsolved challenge to incorporate hair follicles (HFs) into HSCs in order to improve wound healing and skin function. We initially employed a 3D-printing strategy to induce human HF formation within HSCs through guiding physiological 3D organization of cells in the HF microenvironment. Grafting our HF-bearing HSCs onto immunodeficient mice after their vascularization in vitro resulted in efficient growth of human HFs. However, when HSCs were maintained in vitro instead of grafting, the efficiency of HF induction was as low as 19%, due in part to incomplete restoration of DPC hair inductive gene signature achieved solely through 3D spheroid formation. To further enhance hair inductivity, we leveraged two master regulator (MR) genes, Lef1 and Fli1, which we previously identified as the key regulators of the intact DPC gene signature, as well as extrinsic factors targeting Wnt-signaling. Using RNA-sequencing, we confirmed that Lef1 overexpression and 3D-spheroid culture synergistically restored the intact DPC gene signature. Lef1 overexpression in DPCs markedly increased the efficiency of HF induction from 19% to 70%. Treatment of HSCs with recombinant Wnt10b and Wnt-activator small molecule, CHIR99021, also increased the efficiency to 50%, albeit lower than that was achieved with Lef1 overexpression. Overall, this data suggested that overexpressing Lef1 in DPCs or extrinsically targeting Wnt-signaling in HSCs can be an effective strategy to promote the generation of engineered human HFs from cultured cells.