Poster V3 - Neuronal Differentiation Acceleration through Modulation of the Cell Cycle

Introduction: A significant challenge in creating pluripotent stem cell (PSC)-derived neuron cultures is the extensive amount of time necessary for differentiation and maturation of the tissues. Prior efforts have focused on optimizing the efficiency and functionality of the differentiated neuronal tissues, often treating the long differentiation times as an unavoidable consequence. Depending on the neuron type, the differentiation process can take several months, demanding substantial resources and limiting the repeatability and throughput necessary for studying neuron development and disease modeling. These long differentiation durations also reduce the feasibility of using PSC derived stem cell therapeutics in clinical applications. To overcome these challenges, it is crucial to consider differentiation time scales as a variable that can be engineered, and to develop new methodologies to accelerate the differentiation process.

Materials and

Methods: We demonstrate such a methodology by controlling the pluripotent stem cell cycle. While the specific mechanisms that link cell cycle, pluripotency, and differentiation are unknown, these cellular properties are closely related. To control the cell cycle, we screened a diverse set of cyclin-dependent kinase inhibitors (CDKi). To quantify the pluripotent protein expression, PSCs were analyzed through flow cytometry and proteins of interest were antibody stained. Cell cycle was analyzed through Hoechst33342 DNA staining. Differentiation was carried out through a previously established three-step protocol that includes induction, differentiation, and maturation. To track the differentiation efficiency over time, a Hb9-GFP mESC line was also differentiated to motor neurons.

Results, Conclusions, and Discussions: We found that the cell cycle distribution of PSC populations can be adjusted based on the CDKi concentration. Using PSCs transfected with Hb9-GFP, we observed that treated cells differentiated into Hb9+ cells more rapidly and efficiently than untreated cells. Untreated cells required 13 days to reach a comparable number of Hb9+ cells present in treated conditions at 11 days. On day 11 of differentiation, treated populations had a 2.54 (±0.28) fold increase in Hb9+ cells compared to untreated cells. Additionally, by inducing partial arrest in the G2 cell cycle phase before differentiation, treated cells demonstrated coordinated burst firing events by day 11, compared to untreated controls at day 16, as measured through calcium flux imaging. By day 11, treated PSCs consistently showed a 1.65 (±0.26) fold increase in the number of actively firing neurons and a 1.99 (±0.86) fold increase in connectivity, defined by the number of connections each neuron has with others. These findings illustrate that cell cycle control is a viable method to accelerate neuronal differentiation, supporting the concept that differentiation time scales can indeed be engineered.

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