Year 1

A key limitation in transplanting human embryonic stem cell (hESC)-derived cells remains their potential to elicit a host immune response with subsequent graft rejection due to immune mismatch between host and donor cells. In order to realize the enormous clinical promise of hESCs, novel cell lines capable of evading immune rejection by immunocompetent hosts are desperately needed. Our team has been focused on addressing this critical unmet need, and has had preliminary success in developing and validating an immune override mechanism for human adult stem cells and somatic cells. In the first year of this 3-year project, our main objective was to create a novel non-viral-based gene delivery construct that contains an enhanced red fluorescent protein in combination with an engineered tolerogenic molecule that confers immune protection to cells expressing it on their extracellular membranes. In Q3 of the first budget period, we succeeded in developing the full non-viral-based gene constructs. In total, we have 5 control and 5 tolerogenic gene constructs in our inventory. In the remaining portion of the first budget period, we proceeded to introduce this gene construct into hESCs utilizing a special technique called nucleofection. Through this process, we have developed 11 control cell lines, at least 2 of which appear positive for the fluorescent marker. Expression of this marker ensures that we can track microscopically those hESCs which have integrated the delivered gene constructs into their genome. Moreover, we have succeeded in developing 6 hESC tolerogenic lines, at least 3 of which appear positive for the fluorescent marker. In budget period 2, we will continue to characterize the clones to determine their viability as stably expressing hESC lines as well as whether the engineered override mechanism leads to immune tolerance in a series of in vitro studies. We believe that the progress achieved in the first budget period provides the basis for creating a platform immune tolerant hESC technology that can be employed for the future development of regenerative medicine and curative therapies.