Year 2

Pluripotent stem cells (hPSCs) carry the potential to grow and expand indefinitely while concomitantly harboring the unique ability to generate all mature cell types. These two properties of hPSCs present a double-edged sword: on one hand, hPSCs provide an unlimited supply of cells to replace dead, damaged or diseased cells and tissues, while on the other hand, this property of hPSCs lies at the heart of their dangerous potential to seed and produce tumors. Controlling the behavior of hPSCs and specifically direct their differentiation into cells of interest is of the utmost importance to produce pure and mature cell populations that lack tumor initiating potential and that are suitable for transplantation and cell based therapies.
Stem cell behavior can be regulated and controlled using a number of approaches. One particular approach utilizes gene therapy methods in which expression of specific genes is increased or decreased. While this approach has yielded significant insights into the inner workings of stem cells, such gene therapy methods are inherently problematic as they permanently alter the genetic composition of the targeted cell lines.
An alternative method to affect stem cell behavior is by treating cells with factors that are known to specify cell fates during embryonic development and to maintain adult tissues that are continuously growing and repairing (for example, skin, intestines and blood). Such factors can be supplied from the outside of the cell so that the genome of the targeted cell remains unaltered. Wnt proteins are a class of factors that act on the outside of the cell, regulate many biological processes and potently affect stem cell behavior. We hypothesize that Wnt proteins can be utilized to control and regulate stem cell behavior, thereby overcoming the risks associated with methods involving gene transduction. As potent stem cell factors, Wnt proteins can be employed to instruct hPSCs to adapt a differentiated state and thereby diminish their tumor initiating potential.
In this grant application we propose to investigate the effect of Wnt proteins on hPSC behavior. Specifically, we are examining the effect of Wnt proteins on the proliferation and differentiation state of hPSC. To this end, we have developed and isolated several recombinant proteins that either activate or block Wnt signaling. In preliminary studies we found that treatment of hPSC with Wnts instructs cells to exit the undifferentiated state and adopt a more restricted function. In addition, we are exploring the role of the various cell surface proteins that receive and process the signaling input from Wnt proteins. These studies have led us to identify a set of cell surface molecules with expression patterns that correlate closely with the differentiation status of hPSCs. In an additional line of investigation, we are exploring the role of Wnt signaling in the process of reprogramming and in the induction of the pluripotent stem cell state. The goal is to increase reprogramming efficiencies and to generate induce pluripotent stem cells independently of gene transduction, thereby yielding “safer” stem cell populations. Finally, we are using a cellular microarray platform previously developed in our laboratory to interrogate the interactions between Wnt proteins and the extracellular environment. These experiments are aimed at optimizing the biological and biochemical activities of Wnt proteins.
A long term goal of these studies is to gain a better understanding of the mechanism of action of Wnt on stem cells, which will enable studies to specifically direct cells into a particular fate.