Generating morphogen gradients to engineer human integumentary organoids

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Project Summary Recent success in generating human organoids from embryonic bodies (EBs) of induced pluripotent stem cells (iPSCs) offers a new tool to understand human organ morphogenesis and genetic diseases. However, the current organoid generating approaches, where cells are symmetrically exposed to differentiation factors/morphogens in culture, only allow for the generation of partial components of a tissue, thereby do not support the spatially-controlled generation of multicomponent tissues. The current challenge in organoid research is to achieve a physiologically-relevant organization of cells, tissue components, and anatomical features. In this project, we postulate that generating defined asymmetrical chemical gradients in EBs will lead to controlled differentiation of iPSCs into multicomponent organoids comprising anatomical features. This approach will overcome the current limitations of symmetrical culture conditions. The skin represents a great model organ to test this hypothesis, because (i) the skin morphogenesis strongly relies on interactions of cells from multiple lineages; and (ii) generation of hair follicles and pigmentation can be used as functional read-outs to assess the robustness of this bioengineering approach, which can later be adapted for other organoid systems. In Specific Aim 1, we will generate precise cross-gradients of differentiation factors at the single EB level using microfluidics. We postulate that this will induce simultaneous generation of epidermal cells and neural crest cell- derived melanocytes and dermal papilla cells. Our success criteria will be the recapitulation of the early events of skin morphogenesis, such as the formation of skin appendages and pigmentation of the epidermis. In Specific Aim 2, we will extend this approach to model a genetic disease. We will focus on Hutchinson-Gilford progeria syndrome (HGPS), which is a rare monogenic premature aging disease with distinct skin abnormalities including sclerotic skin, dyspigmentation, and alopecia. We will first induce skin morphogenesis using patient iPSCs to develop a skin disease phenotype. Subsequently, we will use our microphysiological skin model to identify early developmental abnormalities in progeria skin, which are largely unknown for humans, and further evaluate the efficacy and toxicity of three drugs with different molecular targets on the reversal of HGPS skin phenotype in a human relevant-context. This innovative approach represents a critical step towards engineering fully-developed integumentary organoids and will have an immediate and overwhelming impact on our understanding of human skin morphogenesis and developmental skin diseases.