Engineered skin tissues must reproduce the biological and mechanical functions of their native counterparts if they are to provide health benefits to society. However, engineered skin substitute (ESS) only fulfills basic skin functions and fails to match the structural and biophysical characteristics of the human skin, such as missing hair follicle and sweat gland, limiting its use in vivo. Their absences are due to a lack of functioning cell types that instruct human keratinocytes in ESS to make a hair follicle and a lack of an in-depth understanding of essential epithelial-mesenchymal interactions that drive hair follicle formation. In the case of hair follicle engineering, the epithelial-mesenchymal interactions between keratinocytes and their immediate dermal environment need to be precisely modulated to govern hair follicle lineage commitment. The major cell type that constitutes a unique dermal “niche” is a specialized population of fibroblasts, which are located at the base of the hair follicle, called the dermal papilla (DP), and are different from normal human dermal fibroblasts. However, it is of great difficulty to isolate and expand human DP fibroblasts in vitro while maintaining their inductive capacity for tissue engineering purposes. The long-term goal is to develop novel bioengineering approaches to produce a fully functional human skin equivalent with normal microanatomy. The central hypothesis is that hair follicle induction is an emergent property of skin constructs, which requires the interplay of multiple signals and cell types in an inductive microenvironment. The objectives are to systemically explore how to create an inductive microenvironment in ESS to induce hair follicle formation. To achieve this, we have devised a three-pronged strategy addressing hair follicle bioengineering in ESS: 1) mimic the original niche by fabricating composite keratinocyte-DP cell spheroids in micropatterned ESS; 2) enhance intercellular interactions by adding BMP6 and 3) drive the DP phenotype by re-activating master transcription factors. In the first aim, we will determine if multi- cell type spheroids combined with premade hair canals can mimic a natural niche in ESS. We have developed two 3D composite spheroid models and will determine whether they will allow DP fibroblasts to expand in vitro while maintaining DP inductivity to induce hair follicles in a laser micropatterned skin substitute model. In the second aim, we will determine the inductive functions of Bmp6 in stimulating hair follicle formation in ESS. We will dissect the roles of Bmp6 in hair follicle formation and growth. In the third aim, we will determine whether human DP fibroblasts can be reprogrammed to reestablish hair inductivity. We will assess whether in vitro genetic reprogramming of human DP fibroblasts by CRISPRa-mediated expression of master transcription factors promote the ability of composite keratinocyte-DP cell spheroids to induce hair follicles in ESS. This ...