Genetically modified human ES cells as novel tools for developing human cell replacement therapy

Funding Type: 
Tools and Technologies I
Grant Number: 
ICOC Funds Committed: 
Public Abstract: 

Human ES cells (hESCs) are capable of unlimited duplication (self-renewal) and retain the ability to differentiation into all cell types in the body. Therefore, hESCs hold great promise for the human cell replacement therapy. While significant progress has been made to differentiate hESCs into various cell types, major obstacles remain that hinder clinic trial in human patients. First, during the cell type-specific differentiation of hESCs, a small fraction of undifferentiated hESCs frequently remain mixed with the differentiated cells. These undifferentiated hESCs pose serious cancer risk by forming tumors after transplanted into patients. Second, the cell type-specific differentiation of hESCs is usually consisted of multiple steps, resulting in a mixture of cells of various lineages and developmental stages. Approach to purify the cells of interest from this mixture of differentiated cells, a prerequisite for human therapy, is generally not established. Our overall goal is to develop novel tools and strategies to overcome these major obstacles by genetic manipulation of hESCs through efficient homologous recombination, a technology already established in my lab.

Since the federally approved hESCs cannot be used for efficient homologous recombination due to their growth requirement as cluster of cells, our proposed research must use non-federally approved hESCs and thus cannot be supported by any federal agencies.

To eliminate the undifferentiated hESCs during cell type-specific differentiation, I propose to employ homologous recombination to genetically modify hESCs so that the undifferentiated hESCs but not their differentiated derivatives can be effectively killed by existing FDA-approved drugs.

One of a few clinically proven human cell replacement therapies is the transplantation of β cells harvested from human donors into diabetic patients. Due to the limited source of human donors, hESCs have become a very promising source for β cells. Recent studies have provided definite evidence that hESCs can be differentiated into β cell precursors, which differentiate into functional β cells in mice. However, the lack of purification steps leads to tumor formation and highly mixed cell populations in the transplants. To solve these problems, I propose a strategy to easily purify the hESC-derived β cell precursors by genetically modifying hESCs through homologous recombination so that these β cell precursors are marked with fluorescence.

These genetic modifications will introduce genes into the specific sites of the hESC genome that should have no impact on the expression of the endogenous genes. Products of these genes should have little toxicity on hESCs and their differentiated derivatives. Therefore, once validated, these tools and strategies could be directly applied to the therapeutic grade hESCs and improve their safety and efficacy for human therapy.

Statement of Benefit to California: 

Human ES cell (hESC)-based therapy holds great promise for curing many life-long devastating diseases such as the Type 1 diabetes. Type 1 diabetes is one of the most common chronic diseases in children and young adults in California. This life-long disease, caused by the autoimmune destruction of the insulin-secreting pancreatic β cells, is the major cause of blindness and kidney failure despite existing insulin replacement therapy. Recent success in restoring long-term insulin independence by transplanting β cells harvested from human donors into diabetic patients provides the proof-of-concept evidence that human cell replacement therapy could cure the disease. Due to the very limited supply of β cells from human donors, hESCs have become a very promising source of unlimited supply of β cells. Recent studies have provide definite evidence that hESCs can be differentiated into functional β cells. However, the cancer risk associated with the undifferentiated hESCs mixed with the differentiated cells and the lack of an approach to purify the β cell precursors differentiated from hESCs pose serious obstacles for the clinic trials in human patients. Our proposed research is aimed to overcome these major obstacles so that clinic trial of this highly promising human ES cell based therapy becomes feasible in the very near future. If successful, our proposed tools and strategies should be applicable to eliminate cancer risk associated with all other hESC-based cell replacement therapies of major human diseases.