Abstract:
Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional somatic cells for therapies. One approach to achieve this goal is to use transcription factors (TFs) to differentiate hPSCs into desired cell lineages. Using scRNA-seq, we investigated TFs expressed in endothelial progenitors (EPs) derived from hPSCs and identified upregulated expression of SOXF factors SOX7, SOX17, and SOX18 in the EP population. To test whether overexpression of these factors increases differentiation efficiency, we established inducible hPSC lines for each SOXF factor and found only SOX17 overexpression robustly increased the percentage of cells expressing CD34 and vascular endothelial cadherin (VEC). Intriguingly, we discovered SOX17 overexpression alone was sufficient to generate CD34+VEC+CD31− cells, and, when combined with FGF2 treatment, more than 90% of CD34+VEC+CD31+ EP was produced. These cells are capable of further differentiating into functional endothelial cells. To better assist directed differentiation, precise insertion of fluorescent proteins into lineage-specific genes will be needed. However, this approach presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.

Bio:
Dr. Lian is an Associate Professor of Biomedical Engineering & Biology at Penn State. His lab is working on engineering stem cell differentiation with small molecules and gene editing tools (modified mRNA-based CRISPR systems) to develop new cell-based therapies. Dr. Lian received his PhD from University of Wisconsin in 2012, focusing on development of novel stem cell differentiation protocols. Dr. Lian's small-molecule cardiomyocyte differentiation method paper was awarded the Cozzarelli Prize from the National Academy of Science, representing the best biomedical science paper in 2012 in PNAS. After independence in 2015, he received several awards from BME society, including Young Innovator Award from CMBE journal, Junior Investigator Award from BMES advanced biomanufacturing meeting, Rising Star Award from BMES cellular and molecular bioengineering conference. Dr. Lian also received awards from NIH and NSF, including NIH Trailblazer Award, and NSF CAREER award. ​​​​​​​

Zoom:
https://umich.zoom.us/j/94337625486