Acoustic Tweezing Cytometry for Efficient Neural Differentiation

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Project Summary Acoustic Tweezing Cytometry for Efficient Neural Differentiation Human pluripotent stem cells (hPSCs) have been hailed as a promising cell source for treating degenerative, malignant, and genetic diseases, or injuries due to inflammation, infection, and trauma. hPSCs have also been proven as an invaluable discovery tool to study human development and for developing and testing new drugs. However, to fully realize the tremendous potential of hPSCs, the first and perhaps the most critical step is the directed differentiation of hPSCs to specific functional cell types with high efficiency and purity. Motor neurons (MNs) are a specialized class of neurons that reside in the spinal cord and project axons to muscles to control their activity. MNs are damaged in diseases such as spinal cord injury, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). While there are significant interests in differentiating hPSCs into functional MNs for cell therapies and understanding of MN degenerative diseases, poorly defined culture conditions and inefficient protocols of MN differentiation from hPSCs have significantly hindered their broad use. Given that embryonic development is a dynamic process involving constantly changing physical environments, the central hypothesis of this proposed research is that hPSCs, which is equivalent to the epiblast in the peri-implantation human embryo, are intrinsically mechanosensitive, and biophysical cues in the cell microenvironment can provide potent regulatory signals to control their differentiation and functional maturation towards specific neuronal subtypes such as MNs. This proposal is strongly motivated by our exciting preliminary data showing that a novel ultrasound-based technology, acoustic tweezing cytometry (ATC), which can apply controlled dynamic subcellular mechanical forces to hPSCs, can indeed elicit neuroepithelial and even MN differentiation of hPSCs much more rapidly compared to conventional protocols that solely rely on soluble factors. Thus we propose in this research to fully develop the ATC technology to not only elucidate the intrinsic mechanosensitive properties of hPSCs, but also utilize the technology to improve large-scale production of functional MNs. In this research we propose to (Aim 1) develop high-throughput ATC technology with improved capability for mechanical stimulation of hPSCs; (Aim 2) elucidate the role of a regulatory network comprising mechanosensitive pathways (BMP/YAP activity, RhoA/ROCK/cytoskeleton contractility, and Hippo/LATS) in regulating ATC-facilitated neuroepithelial differentiation of hPSCs; (Aim 3) apply ATC for high-efficiency functional MN generation from hPSCs. Successful completion of this research will establish a new, novel approach for hPSC neural differentiation and MN generation, potentially enabling drastic advances in large- scale production of clinical-grade MNs for cell-based therapies and drug screens. Our proposed research will also help establish a novel mechanistic framework for understanding mechanosensitive hPSC properties and will chart a path to unravel their full complexity for their future regenerative medicine applications.