(553d) Improving Homogeneous Differentiation of Stem Cells By CRISPR-Mediated Knockout

Induced differentiation of stem cells in culture can result in heterogeneous mixtures of cells contaminated with off-target cell types, a reflection of intrinsic heterogeneity within the stem cell population or stochastic events. This hinders efficiency of stem cell differentiation in vitro by requiring extra selection and sorting protocols. Control of cell fate during differentiation is strongly influenced by the activity of transcription factors. We hypothesized that we could eliminate generation of off-target cell types by controlling the availability of transcription factors.

Previously, using forskolin and IBMX to induce dopaminergic differentiation of mesenchymal stem cells, we observed a small population of glutamate sensitive cells indicating heterogeneous stem cell differentiation. We hypothesized that by creating mesenchymal stem cell lines null for transcription factors specifying the glutamatergic phenotype we could produce a more homogeneous mixture of dopaminergic-like cells. We show that MSCs exhibit heterogeneous differentiation during dopaminergic differentiation and aim to create Tbr1^{–/–} MSCs that could potentially yield a more homogeneous dopaminergic population after induction with forskolin and IBMX.