Determination of Cytokine Receptor Profiles for the Enhancement of In Vitro Differentiation of Embryonic Stem Cell-Derived Hematopoietic Progenitors

Cytokines play a critical role in hematopoietic development in vivo and in vitro. Our goal is to establish an in vitro microenvironment that will produce high yields of transplantable self-renewing embryonic stem cell (ESC)-derived hematopoietic progenitors (ES-HP). We will determine the cytokine receptor profiles of ES-HP that are generated via the co-culture of distinct ESC lines with the OP9 bone marrow cell line. In our hands, ES-HP that are generated with this system resemble the definitive hematopoietic progenitors found in vivo in murine fetal liver and adult bone marrow. We hypothesize that determination of the ES-HP cytokine receptor expression profiles (IL-2Rγ, IL-3R𝛼, IL-6R, IL-7R, TGFβR, TPOR, SCFR, LIFR, and Flt3LR) can be utilized to improve production of ES-HP by addition of the appropriate cytokines to the cultures. ES-HPs will be stained for the developmental maturity markers, CD41 and CD45. Fluorescence activated cell sorting (FACS) will be used to isolate ES-HP populations from Day 7 and Day 16 co-cultures. RNA will be extracted from these purified ES-HPs and reverse transcriptase PCR (RT-PCR) will be used to determine the relative expression of cytokine receptors present in ES-HP derived from four different murine ESC lines (D3, 129-GFP, R1, and B6). Positive RT-PCR results will be further confirmed by cell surface level protein expression on ES-HP using FACS. These studies will provide critical data on the cytokine receptor expression patterns and developmental kinetics of their expression in vitro. Data can then be used for the strategic improvement of ES-HP culture techniques.