Cell Intrinsic and Extrinsic Factors Driving Maturation in Human PSC-derived Neurons

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Project Summary Human embryonic (hESC) and human induced pluripotent stem cells (hiPSC) offer great promise for basic research and for applications in disease modeling. The initial challenge for exploiting this potential was to direct stem cell differentiation towards specific nerve cell or glial fates relevant to disease. Over the last few years, we have developed many such protocols that now enable researchers to generate > 50 distinct human cell types in a dish. However, a major remaining challenge is the fetal rather than adult-like features exhibited by the resulting cells, which limits their usefulness. The reason for their immaturity is unclear but may be linked to a cell-intrinsic, clock-like mechanism that controls the timing of maturation. While it takes 9 months for a human baby to develop, from conception to birth, the same process takes only 20 days in a mouse. Those dramatic timing differences are recapitulated in a dish, where the maturation of human cells may require many months to reach adult-like properties. Interestingly, we observe such timing differences in both 2D and 3D culture including in neural organoids. Even after transplanting human cells into the mouse brain, cells continue to follow a human-specific maturation trajectory, despite being surrounded by an adult host microenvironment. Here we will address this challenge by building assays to measure and quantify neuronal and glial maturation and by developing strategies to override the intrinsic maturation clock. Towards these goals, we have established a unique stem cell-based assay to produce nerve cells at very high precision and in a temporally synchronized manner. The resulting cells then progressively mature from fetal to adult-like stages over a period of several months allowing us to define markers that predict the neuronal maturation state. In Aim 1, we will build on these preliminary data and establish stage-specific “fingerprints” of nerve cell maturation to determine maturation states at unprecedented precision. In addition, we will characterize the maturation of glial cells (astrocytes and microglia) in a novel tri-culture system to test whether the presence of glia can improve neuronal maturation. In Aim 2, we will apply maturation “fingerprints” as a readout for identifying factors that can accelerate maturation timing. In preliminary studies, we have identified chemicals and genes that are strong candidates for driving neuronal maturation. We will further validate those findings in the tri-culture system to determine the combined effect of intrinsic and extrinsic maturation factors. Finally, we will perform mechanistic studies to understand how those factors induce more adult-like features in human cells. In Aim3, we will test our optimized maturation strategies in more complex 3D culture systems to assess whether induced maturation strategies impact other developmental processes such as cell migration and organization. Finally, we will assess whether “induced maturation” strategies can be adopted to achieve accelerated timelines of neuronal maturation upon transplantation into the developing murine brain, a strategy that could enable new human disease models for the study of neurodevelopmental or neuropsychiatric disorders in vivo.