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

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

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01406
Approved funds: 
$1,329,298
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
iPS Cell
Public Abstract: 
With their ability to develop into virtually all mature cell types, human pluripotent stem cells (hPSC) represent a unique and powerful research tool to study the fundamental mechanisms regulating human development. In addition, hPSC provide 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 multicellular life remain mysterious. At the heart of embryonic development lies an intricate process of cell communication. Individual cells within the developing organism produce and release signals, known as growth factors, that instruct neighboring cells to assume specific behaviors and properties. Unique combinations of such growth factors regulate a multitude of developmental processes, including the growth and differentiation of hPSC. Wnt proteins represent a major class of growth factors with potent effects on stem cells and developmental processes. However, despite nearly 30 years of research on these proteins with over 1,500 publications in 2008 alone, the mechanisms by which Wnt proteins elicit specific cellular responses and regulate stem cell biology remain poorly understood. Two major shortcomings have impeded progress in the study of Wnt proteins. First, Wnt proteins long resisted biochemical characterization so that their manipulation in biological systems was difficult and even impossible. Second, Wnt proteins do not act alone but rather exert their activities in combination with thousands of biological molecules. Present day technologies are insufficient in to re-creating these complex cellular microenvironments in which Wnts act. We have developed powerful technologies that allow us to systematically dissect the role of Wnt signaling in regulating the behavior of hPSC. By developing the means to isolate Wnt proteins we are now able to examine their effects on stem cell growth and differentiation. In addition, we established a novel technology platform with which we can interrogate the effect of thousands of combinations of Wnt proteins and other biological molecules on hPSC. This combination of technological advances and expertise puts us in an ideal position to define the role of Wnts in developmental processes. By identifying the mechanisms by which Wnt proteins act we will contribute valuable tools and protocols for the manipulation and specific differentiation of hPSC into mature cell types that can be utilized in cell replacement therapies.
Statement of Benefit to California: 
The rise in life expectancy to over 80 years will likely lead to an increase in the number of people suffering from age-related diseases, such as cancer, heart disease and neurodegenerative disorders. Current medical treatments can control, but not cure, such diseases. Recent advances in the study of human pluripotent stem cells (hPSC) 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. Several scientific obstacles need to be overcome before the full potential of hPSC-based therapies can be realized. First, sufficiently large numbers of clinical grade hPSC that can be thoroughly tested and characterized need to be derived. Second, robust protocols for the directed differentiation of hPSC into functionally mature cell types suitable for transplantation need to be developed. To address these challenges we propose a set of experiments that will significantly expand our understanding of basic biology of hPSC. To this end we will examine the role of a major class of stem cell factors, called Wnt proteins, in regulating hPSC growth and differentiation. In addition, we will expand on a technology that allows us to interrogate the effect of thousands of combinations, including Wnt proteins, on hPSC behavior. These experiments will enable the development of cost-effective protocols for the large-scale production of undifferentiated hPSC and functionally mature cell types. Our technology, which we will make freely available, will additionally benefit many other lines of scientific inquiry, such as defining growth conditions of rare adult stem cell populations and modeling the cellular basis of diseases. Thus, our proposed research is fundamental to applications of hPSC in regenerative medicine and has broad benefits to researchers with a wide spectrum of scientific interests. This research will not only benefit the health of Californians, but also the California economy by developing new reagents, protocols and technologies that will be adopted by existing companies as well as seed and complement novel business ideas. The outcome of this project will contribute to the development of a biotechnology platform that can provide great benefits to the advancement of California biotechnology. The patents, royalties and licensing fees that result from the advances in the proposed research will provide California tax revenues. Thus, the current proposed research provides not only the essential foundation for the scientific advances in regenerative medicine to improve health and quality of life, but also potential technology advancement and financial profit for the people in California.
Progress Report: 

Year 1

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.

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.

Year 3

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.

Year 4

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.

© 2013 California Institute for Regenerative Medicine