Molecular determinants of accurate differentiation from human pluripotent stem cells

Funding Type: 
Basic Biology III
Grant Number: 
Award Value: 
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Public Abstract: 
The use of human pluripotent stem cells for cell-based therapeutics is predicated on the ability to convert these cells into functional equivalents of those lost in disease or injury. However, there is only scant evidence that either human embryonic stem cells or human induced pluripotent stem cells make differentiated progeny that are functionally equivalent to those found in tissues. Our preliminary results, gathered over several years suggest that in fact human pluripotent stem cells may make authentic tissue derived cells, but they appear to be most similar to cells found only during very early fetal development. As a result, it is unclear if these cells will suitably replace tissue derived cells in postnatal therapies. We have also uncovered several genes whose expression appears to distinguish mature tissue derived cells and those generated from human pluripotent stem cells. We have designed this project to determine whether manipulating expression of those genes in pluripotent derivatives can bring them closer to postnatal tissue derived cells. We also propose to discover small molecule compounds that can have the same effect. Upon successful completion of these aims, we will bring to the community compounds that allow for appropriate maturation of all types of pluripotent derivatives. Therefore, these reagents will facilitate cell based therapeutics enormously and perhaps allow for successful transplantation of pluripotent derivatives for the treatment of a wide variety of diseases and injuries.
Statement of Benefit to California: 
For human pluripotent stem cells to reach their full potential in cell based therapeutics it is absolutely essential that their differentiation to particular cell types produces a product with the functional capacity to replace lost tissue. As California is a leader in the adoption of pluripotent stem cell therapies, it is vital that it also be a leader in the generation of derivatives from pluripotent stem cells that accurately mimic cells found in tissue. Our data suggests that instead of generating cells that would normally be found in adult tissue, human pluripotent stem cells instead produce cells found during very early fetal development. Not only does this suggest that cell based therapies with such cells might be hampered, but in fact the proliferative nature of these cells could even make them dangerous. We propose a series of experiments that will test the idea that manipulating the gene expression of pluripotent derivatives during differentiation will make cells that more accurately reflect those found in tissues that would typically require stem cell based treatments. We will develop tools that will be easily applicable for a wide range of cell types and yield mature, functional cells that not only mimic tissue derived cells, but can also functionally replace cells that are lost in disease or injury. This work will be particularly beneficial to ongoing efforts in California to apply pluripotent stem cells in therapeutic settings, and will therefore greatly benefit Californians requiring such treatment.
Progress Report: 
Our goal for this award was to determine how various genes contribute to inherent differences between cells made in vivo (tissue) versus in vitro (from pluripotent stem cells). Our previous work identified a number of genes that always appeared to be expressed differently between cells made in different contexts. For pluripotent stem cells and their progeny to be useful clinically, it is imperative that the cells generated be as close as possible to their tissue derived counterparts. Therefore, in the last year we have begun to experimentally turn some of these genes on or off depending on their variance with cells found in tissue. We have have found that some of these manipulations do indeed bring these cells closer together, while others seem to have no effect. In the coming year, we are going to combine those manipulations that are effective to see if they can have an additive or perhaps even a synergistic effect. We aim to eventually create cells in vitro that perfectly mimic those found in tissue and develop methods to make this process universally applicable across cell types to facilitate both disease modeling and regenerative medicine.
In the last year, we have made significant progress in finding new ways to improve the developmental maturity of the progeny of human pluripotent stem cells. We now have several methods to drive maturity of human neural progenitors forward to a more gliogenic state. We are now working to combine these different methods to see if they can be additive or even synergistic. We hope that this work will yield more clinically relevant pluripotent progeny.
We have made significant progress on our work designed to improve differentiation from human pluripotent stem cells. Starting with an extensive characterization of the differences between tissue and pluripotent derived neural cells, we identified a list of candidates of important molecular differences between cells born in tissue versus those born in a cell culture dish. In the last three years we have systematically been testing those candidates with the highest potential to alter differentiation potential. This has led to the discovery of a handful of genes that affect transcription of other genes. Among these is a gene that is important to regulate how a cell responds to the level of oxygen in culture. We found that the level of oxygen in culture can in fact affect the differentiation potential of human pluripotent stem cells. This important observation is significant because it allows researchers a simply tool to alter differentiation and make different types of cells more quickly. Building on this, we found two small molecules with the ability to mimic fluctuations in oxygen levels, and therefore provide a very simple tool to affect differentiation. We are now in the process of combining various tools together to see if human pluripotent derived cells can be made even more similar to their tissue derived equivalents.