New Technology for the Derivation of Human Pluripotent Stem Cell Lines for Clinical Use

New Technology for the Derivation of Human Pluripotent Stem Cell Lines for Clinical Use

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00667
Award Value: 
$1,266,134
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Other
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
Other
Status: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

Since their discovery eleven years ago, there has been steady progress towards the application of human embryonic stem (ES) cells in medicine. Now, the field is on the threshold of a new era. Recent results from several laboratories show that human skin cells can be converted to cells resembling ES cells through simple genetic manipulations in the laboratory. There is currently much excitement about these induced pluripotent stem (iPS) cells, which might have advantages over ES cells in studying and treating disease. However, we do not yet sufficiently understand their nature and potential to be certain that they can replace (ES) cells in research and therapy. Because of this, it is important to continue to develop new ES cell lines, and to compare their properties with those of iPS cells. Technological advances in ES cell research now enable us to grow stem cells under conditions that are much more suitable for future patient use than those used to develop the first ES cell lines. However, these new methodologies have for the most part been developed with, and tested on, a handful of the long-established ES cell lines on the NIH Registry. In the first year of this grant, we have focused on new technologies for deriving and propagating ES and iPS cell lines. One of our goals was to derive these lines under defined conditions (where all the chemical and protein additives used in the cell cultures are known) and without the use of animal products. The use of defined culture systems means that cells can be produced in a standardized consistent fashion and that no unknown and potentially hazardous components are present. Elimination of animal products reduces the possibility that additives like animal serum or animal helper cells could transmit disease causing agents like viruses to the stem cells. We first adapted laser technology to isolate the part of the embryo that gives rise to ES cell cultures, an important step in stem cell derivation. This technology replaces the use of antibodies made in animals. Then we developed and successfully tested a defined medium for stem cell growth. The new medium supported robust long term propagation of stem cells without causing deleterious genetic changes to the cells, and it is a very simple formulation free of any animal products and containing only a few defined protein components. Current techniques for deriving iPS cells require genetic modification of the adult cells, a procedure that carries a risk of inducing changes in the stem cells that cause them to form tumors when injected into a host. We have successfully used these techniques to make iPS cell lines in our laboratory. However, because of the risks associated with genetic modification, we have explored alternative methods for making iPS cells. One promising approach is to introduce the contents of an ES cell into the adult cell by a technique called cytoplast fusion. This method is similar to that used in cloning animals, when the contents of an egg are introduced into an adult cell to reprogram it to behave like an early embryo cell. Components in the egg or the ES cell can reset the adult cell back to an embryonic state. We overcame some technical challenges and successfully developed techniques for mixing ES cell contents into adult cells. Unfortunately, although the ES cell material did cause some reprogramming of the adult cell back to the embryonic state, the process was incomplete, and no bona fide iPS cells were obtained. We are now pursuing alternative technology developed by several groups during the past year, which uses a type of genetic modification in adult cells that can be erased once the iPS cells are developed. In the next year of our grant, we will apply our new cell derivation and propagation technology to produce ES and iPS cells under optimal conditions, and begin to compare the properties of the two cell types to see if indeed they are identical in their behavior.

Year 2

Since their discovery eleven years ago, there has been steady progress towards the application of human embryonic stem (ES) cells in medicine. Now, the field is on the threshold of a new era. Recent results from several laboratories show that human skin cells can be converted to cells resembling ES cells through simple genetic manipulations in the laboratory. There is currently much excitement about these induced pluripotent stem (iPS) cells, which might have advantages over ES cells in studying and treating disease. However, we do not yet sufficiently understand their nature and potential to be certain that they can replace (ES) cells in research and therapy. Because of this, it is important to continue to develop new ES cell lines, and to compare their properties with those of iPS cells. Technological advances in ES cell research now enable us to grow stem cells under conditions that are much more suitable for future patient use than those used to develop the first ES cell lines. In the second year of this grant, we have refined and validated new technologies for deriving and propagating ES and iPS cell lines. One of our goals was to derive these lines under defined conditions (where all the chemical and protein additives used in the cell cultures are known) and without the use of animal products. The use of defined culture systems means that cells can be produced in a standardized consistent fashion and that no unknown and potentially hazardous components are present. Elimination of animal products reduces the possibility that additives like animal serum or animal helper cells could transmit disease causing agents like viruses to the stem cells. Our culture system is fully defined, and is based on a totally new molecular pathway for maintaining stem cell propagation that we have discovered. We have now fully validated the system for long-term support of human pluripotent stem cells. Deriving new ES cell lines from embryos can be more demanding than maintaining existing lines. We are in the process of deriving new ES cell lines using another system that still employs helper cells (feeder cells) but has no animal products (xeno-free conditions). We have successfully used this system to derive iPSC (below). Current techniques for deriving iPS cells require genetic modification of the adult cells, a procedure that carries a risk of inducing changes in the stem cells that cause them to form tumors when injected into a host. We have successfully used these techniques to make iPS cell lines in our laboratory and have developed four new iPSC lines that are being distributed through our CIRM Shared Laboratory Facility. However, because of the risks associated with genetic modification, we have explored alternative methods for making iPS cells. We have now made 5 iPSC lines using technology that enables us to remove the genetic modification after the reprogramming event. These cell lines were derived under xeno-free conditions. In the final year of this grant, we will complete the derivation and characterization of ES and iPS cell lines under identical conditions, and we will carefully compare the properties of these two types of pluripotent stem cell.

Year 3

The overall goal of this study was to develop new technologies to produce human pluripotent stem cell lines under conditions that are optimal for future clinical use. In the final year of this study, we used a new culture system validated in our lab in Years 1 and 2 of this grant to derive a human embryonic stem cell line and approximately 25 human induced pluripotent stem cell lines under conditions that are completely free of animal products, an important consideration for therapy. The cell lines derived under these conditions showed all the features expected of human pluripotent stem cell lines, demonstrating clear proof of concept for this approach. In a second set of experiments, we addressed another roadblock to the use of induced pluripotent stem cells in research and therapy. Efficient, high throughput production of human induced pluripotent stem cells for the establishment of banks for tissue matching will require very efficient selection of fully reprogrammed cell lines. However, after reprogramming, only a small proportion of stem cell lines are truly pluripotent. We identified new cell surface markers that enable us to prospectively isolate colonies of stem cells that are fully reprogrammed to the pluripotent state.

© 2013 California Institute for Regenerative Medicine