WNT signaling and the control of cell fate decisions in human pluripotent stem cells.

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. 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. 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 fate 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 genes are a class of factors that 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 undifferentiated hPSC populations. 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. Preliminary results indicate 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 in stem cell assays.

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.

Human pluripotent stem cells (hPSCs) and their derivatives represent the only research tool to study human development. As such, these cells allow us to study the progression of diseases at the cellular level in a dish, probe how specific genetic defects contribute to the myriad of developmental and birth defects, and generate the “raw material” for the development of cell-based therapies of presently incurable diseases, such as cancer, cardiovascular disease, and neurodegenerative disorders. However, our understanding of the basic mechanisms underlying stem cell biology is incomplete, and the processes by which individual cells organize each other to give rise to the complexity of multi-cellular life remain mysterious. At the heart of embryonic development, and hence stem cell biology, lies an intricate process of cell communication. Individual cells produce and release signals, known as growth factors, that instruct neighboring cells to assume specific behaviors and properties. Wnt proteins represent a major class of growth factors with potent effects on stem cells and developmental processes. We have examined the role of these Wnt proteins in hPSCs and their derivatives, including neural stem cells, a cell type that can generate many of the cells found in the central nervous system. The goal of this research project was to gain a better understanding of these WNT growth factors so that we can apply them to affect stem cell behavior. Identifying the mechanisms by which Wnt proteins act has allowed us to contribute valuable tools and protocols for the manipulation of hPSC and neural stem cells into mature cell types suitable for cell replacement therapies and disease modeling. With a rise in life expectancy to over 80 years an increased number of people are suffering from age-related diseases, such as cancer and neurodegenerative disorders. Current medical treatments do not cure such diseases. Recent advances in the study of hPSCs have provided the opportunity to develop novel cell replacement therapies for the treatment of many such diseases. Development of novel cell-based therapies will also overcome the inadequacy of conventional drug-based treatments. A major challenge in the study and use hPSCs is to develop robust methods for the directed differentiation of hPSC into functionally mature cell types suitable for therapeutic applications. Our research has been aimed at gaining a better understanding of how to manipulate hPSCs and neural stem cells. The research has led to fundamental insights into hPSC biology and produced protocols and reagents of critical importance in regenerative medicine. In addition, this research has broad benefits to sceintists with a wide spectrum of interests. The research has provided not only the foundation for the scientific advances in regenerative medicine to improve health, but also technology advancement and financial profit for the people of California.

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 hypothesized 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 proposed to investigate the effect of Wnt proteins on hPSC behavior and on reprogramming. Specifically, we examined the effect of Wnt proteins on the proliferation and differentiation state of hPSC. To this end, we developed and isolated several recombinant proteins that either activate or block Wnt signaling. We found that treatment of hPSC with Wnts instructs cells to exit the undifferentiated state and adopt a more restricted function. In addition, we explored 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 explored the role of Wnt signaling in the process of reprogramming and in the induction of the pluripotent stem cell state. The goal of this research was to increase reprogramming efficiencies and to generate induced pluripotent stem cells (iPSCs) independently of gene transduction, thereby yielding “safer” stem cell populations. In a surprising series of experiments we found that Wnt signaling is absolutely required for reprogramming. Using fibroblasts from patients with a rare genetic defect called Focal Dermal Hypoplasia (FDH), we were able to demonstrate that Wnt signaling is required during the process of reprogramming. These experiments have also allowed us to initiate a new direction in our research where we will employ these FDH-iPSCs to model the disease in a dish and perform drug screens that will correct the molecular defects associated with this disease.