Call for Pilot and Feasibility (P & F) Grants

Jian Huang use of the Pilot & Feasibility Funds

The signaling pathways regulated by GSK-3 in Hematopoietic Stem Cells

We are approaching our goal by treating mice with a GS3 inhibitor (lithium) and an mTOR inhibitor (Rapamycin) for 2 weeks and then harvesting HSCs to perform microarray and /or whole genome sequencing to identify the genes up-regulated and down-regulated.  Furthermore, I intend to perform an RNAi-based loss of function screen (using a commercial shRNA library) to identify new genes/factors that control the expansion and maintenance of HSC ex-vivo.  This large-scale study will certainly provide new mechanistic insights into how stem cells regulate themselves in culture.  Both studies will use Flow Cytometry and Cell Sorting Resource Laboratory, mouse facility, Penn DNA Sequencing Facility, Penn Molecular profiling Facility and Bioinformatics core facility.

Spencer Sullivan use of the Pilot & Feasibility Funds

Studies of human iPS Cells in Megakaryopoiesis

I intend to use the resources provided by the P30 to study transgene expression in megakaryocyte-derived iPS cells using Glanzmann Thrombasthenia (GT) as a model of targeted expression in this lineage.  I have created three iPS cell lines from patients with GT, all have alpha IIb mutations. I have corrected the defect using a megakaryocytic-specific promoter (GPIb alpha) driving alpha IIb cDNA and targeted to the AAVS1 safe harbor locus in iPS cells.  I have differentiated the iPS cells (control, corrected and uncorrected) into megakaryocytes and examined transgene expression.  The money from the P30 grant is being used for supplies for this project as I set-up iPS cells.  Specifically, the money will be used for tissue culture related supplies and equipment, including media, cytokines, and plasti wre.  Simultaneously, I am also using the same promoter to drive FLI1 expression in an iPS cell line derived from a patient with Jacobsen's syndrome, which involves FLI1 halpoinsufficiency.  In addition to correcting the FLI1 defect, I will also use TALENs to knockout FLI1 in WT iPS cells as a control.  The money from the P30 grant will also be used to purchase appropriate TALENS for this targeted gene knockout.

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