New Technology ForThe Derivation of Human Pluripotent Stem Cell Lines For Clinical Use

New Technology ForThe Derivation of Human Pluripotent Stem Cell Lines For Clinical Use

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00667
Approved funds: 
$1,266,134
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Other
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
Other
Public Abstract: 
Since their discovery almost ten 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 this proposal, we will rigorously test improved techniques for growing stem cells, developed in our lab and elsewhere, to see how well they work in deriving new ES cell lines. We will ensure that these new methods allow for production and maintenance of ES cells free of genetic abnormalities. Current technology for producing iPS cell lines also has significant limitations. Published procedures run the risk of producing genetic damage in the stem cells, and rely on the use of cancer causing genes. Also, using current techniques it is more difficult to produce of iPS cells from adult tissue (the most appropriate source for therapy) than from fetal tissue. This proposal examines alternate approaches to generating iPS cells aimed to circumvent these limitations. Using these improved technologies, we will carefully compare the properties of ES cells and iPS cells derived and propagated under identical conditions. This is important, because to find out if iPS cells really are equivalent to ES cells, we have to compare examples of both cell types made and grown under the same conditions. Otherwise, differences that we see may be related to how the cells were produced and propagated, or how long they have been grown in the laboratory, rather than to inherent differences between them. The significance of this research is threefold. First, new ES cell lines suitable for therapeutic use will be derived from embryos and made available to any researcher who requires them. Second, we will answer the important scientific question over the equivalency of ES and iPS cells under experimental conditions that remove variables related to cell culture techniques and cell age. Finally, we will improve and validate the technology for derivation and maintenance of ES and iPS cells for future clinical use.
Statement of Benefit to California: 
Through their support of Proposition 71 the citizens of California recognized the fundamental importance of stem cell research to the future of biotechnology and regenerative medicine. Funding from this initiative enables California scientists to work outside of limits imposed on federally funded research, to develop and study new pluripotent stem cell lines from spare embryos. These embryonic stem (ES) cell lines have the property of pluripotency, or the ability to give rise to any type of body cell. Now, rapid technological progress in methodology for growing embryonic stem cells, and for developing cells with the properties of embryonic stem cells from adult tissue, provide new opportunities for producing stem cell lines that are safe for patient use, or for producing cell lines that will avoid the problems of tissue rejection during transplantation. However, in many ways these new technologies are as yet untested. This proposal is aimed at applying and assessing new methods for making pluripotent stem cells from embryos or from adult tissue. We will capitalize on our extensive experience in deriving and characterizing stem cell lines, to achieve three goals: first, the development of new cell lines under conditions that ensure they will be as safe as possible for use in patients; second, development of improved techniques for making pluripotent cell lines from embryos or adult tissues; third, a clear answer to the key question of whether stem cells from adult tissues are equivalent in potential to those from embryos. These advances will ensure that the best possible stem cell lines are available to Californian scientists, physicians and their future patients. Through this program, stem cell research in the State will remain at the forefront of the field and will lead in technological innovation in cell culture biotechnology, as well as in basic science and translational and clinical research. Finally the ability to derive new cell lines from embryos and carefully compare them to cell lines from adult tissue will provide data that is critical to the future of the field and to directing the research efforts of the California Institute for Regenerative Medicine.
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