The goals of this proposal are to investigate endodermal differentiation and proliferation in human ES cell cultures. Endodermal cells give rise to the epithelial lining of the respiratory and digestive tract as well as to the liver and pancreas. The future treatment of diseases such as type I diabetes using stem cell therapy relies on our ability to differentiate stem cells into endoderm, a prerequisite step to forming pancreatic beta cells. In 2005, D’Amour et al. reported the efficient differentiation of human ES cells into endoderm. This report provides a potentially effective protocol that needs to be further evaluated (specific aim 1). In addition, given that the success of stem-cell therapy depends on our ability to generate large numbers of differentiated cells (e.g. 200-700 million beta cells per patient are currently being used in the Edmonton protocol), we will investigate the ability of the endodermal generated in specific aim 1 cells to proliferate in culture (specific aim 2).
Stem cell therapy relies on the development of efficient and reproducible protocols to differentiate stem cells into various cell types such as pancreatic beta cells. The first step to making pancreatic beta cells is the differentiation of stem cells into so-called endodermal cells, one of the 3 basic cell types of the body. In addition, in order to make stem cell therapy a viable option, one needs to be able to generate large numbers of differentiated cells from stem cells. Our proposal aims to establish such protocols. The health of California and its citizens will ultimately benefit from this research as it will help develop stem cell therapies.
SYNOPSIS: The proposed research by an accomplished Principal Investigator (PI) addresses issues relevant to the production of insulin-secreting cells for transplant therapy of diabetes. The first goal is to determine whether the protocol of D’Amour et al. for generating endodermal cells from hESCs can be replicated and is applicable to a wider variety of hESC lines. Aim 1 will examine whether 4 NIH-approved and 6 unapproved human ES cell lines (one of which overlaps with the 8 tested by D’Amour) activate definitive but not visceral endoderm markers efficiently when stimulated by a combination of Activin A and low serum. The second goal is to discover culture conditions for expanding this endodermal cell population, a critical step to produce the number of differentiated cells required for transplant therapies. Aim 2 will test whether growth factors for common developmental signaling pathways can stimulate proliferation of endodermal cells derived in Aim 1 and enriched by FACS for CXCR4 expression.
INNOVATION AND SIGNIFICANCE: This application proposes to examine the potential of several established hESC lines for differentiating to definitive endoderm. Despite recently reported advances, the technology for producing functional beta-like cells from hEScs is woefully incomplete. This is such an important issue that a dedicated, independent and critical effort to reproduce the results of D’Amour, Baetge and their colleagues is crucial. It is important to determine whether the endodermal differentiation protocol is generally applicable and has the potential to be successful for a wide range of human ES cell lines and therefore possibly individual ES cell donors. The PI will use this published protocol, optimize it, then attempt to guide the cells more efficiently to endodermal fates. These studies have the potential of closing the gap in our ability to differentiate hESCs to pancreatic beta cells. Such a source of insulin-secreting cells could then serve as a source for cell replacement therapy. The approach is only moderately innovative in that most of the first Specific Aim is a screen of hESC lines grown using a published protocol. The potential for innovation lies in discovering new experimental conditions to expand the endodermal progenitor cell population by inducing cell replication. However, there is no insightful strategy derived from an understanding of the growth and development of the vertebrate endoderm development, other than to apply several common growth factors and other unspecified one to enriched endoderm cultures and examine the effects on cell propagation. Overall, the results are potentially very significant in furthering our ability to treat diabetes.
STRENGTHS: This project is basically a screen of several hESC lines to determine which can better differentiate to endoderm. The PI has much experience with endoderm formation. He has worked toward liver, gut and pancreas differentiation. Most of his experience, however, is in the zebrafish system. The major strength of this proposal is the opportunity to recruit an aggressive experimenter to apply novel perspectives on endoderm and pancreatic development from the zebrafish to an important therapeutic application of ES cell technology. Dr. Stainier has recruited excellent accomplished collaborators at UCSF with appropriate expertise and inherent scientific interest in endoderm development from human ES cells that should help ensure success. Thus, Drs. Fisher and Reijo-Pera, co-directors of the UCSF Human Embryonic Stem Cell Research Center, will provide collaborative help, technical support and training individually, and through the ES Cell core facility. Both collaborators are highly experienced hESC researchers.
The methodologies proposed are straight forward and do not pose a problem in performing the work. The PI has written a thoughtful proposal and has considered potential difficulties, as well as possible outcomes and interpretations. Since the submission of this application, D’Amour and colleagues have reported the next major step for the production of insulin-secreting beta-like cells from the enriched endoderm cell culture. Despite this recent advance, the goals of this proposal are still valid: general applicability and the need to expand the progenitor population, such as endoderm, to provide sufficient insulin-secreting cells for transplantation therapy.
WEAKNESSES: The underlying hypothesis is not clear. More rationale and more experimental details are needed. There are many references to the D'Amour (2005) protocol but no details given. If the protocol does not work on these hESC lines, the PI doesn't provide details on what he will try next. He states that the protocol will be optimized and that he will try alternate strategies, but no details are given. There is a lack of detail in general in the experimental design. For example, in Specific Aim 2 it states that "...test the ability of various signaling pathways.... Cell proliferation will be assessed by BrdU labeling as well as immunostaining for phospo-histone." Details as to how this will be accomplished would be nice. Additionally, no mention is made of over how many passages and how much time the compounds will be tested, th concentrations of the growth factors used, how the results will be obtained and quantified, etc.
The D’Amour study briefly compared a total of eight human ESC lines and showed that the expression kinetics for four key markers for gastrulation, definitive endoderm and visceral endoderm had similar trends. This proposal will examine nine additional lines plus HUES7, which was in the D’Amour study and should provide a base-line for comparison. It is not clear how much further insight that testing an equal number of additional lines will give, and precisely which developmental parameters among the many examined by D’Amour et al. will be examined and which are thought to be most critical to replicate.
One reviewer felt that the absence of an explicit experimental plan for Aim 2, the discovery of growth factors that could be used to expand the derived endoderm cell population is a major weakness. Although the PI has assembled a talented staff and collaborators for training and advice, similar work is not ongoing in his laboratory and there will be a significant learning curve for human ES culture and manipulation that will delay initial progress.
The PI has allocated only 5% of his time to this project. It is stated that he will supervise all aspects of the research. There is concern that 5% is not enough of a commitment.
DISCUSSION: This is a very simple, potentially very important proposal that is grounded in developmental biology concepts. The applicant seeks to reproduce and test with multiple hESC lines, the Novocell observation that immature beta-like cells can be derived from hESC differentiated along endodermal lineage. This step-wise, simplistic approach with straight-forward methodology will yield a starting point for making beta cells, and Novocell has shown the insulin production capability of immature beta cells. In this work, the PI could also verify the results from Novocell. Novocell tested 8-9 lines and found significant differences in efficiency of beta cell generation; these investigators will be testing yet more lines to further understand variation in differentiation potential. Here, there are no new methods to challenge the published protocol and data, and reviewers disagreed over the benefits of characterizing a battery of hESCs. One reviewer argued that studying nine more lines beyond the D'Amour work would not yield more information since in the earlier work there were no significant differences in the efficiency of hESCs differentiating into endoderm, and added that while there were a few efficiency differences, the expression and phenotype data were the same. Another reviewer stated that the work needs to be done with multiple lines under controlled conditions by the same group so that the results can be compared. A comment was made that there is still much to be learned regarding endodermal differentiation into beta cells. This is an opportunity to recruit an external scientist (external to Novocell) who is a very aggressive experimentalist to verify a key finding. Another strength is that the applicant has expertise in studying endodermal differentiation in zebrafish. A final question was brought up regarding whether the people of California would benefit from reproducing this protocol, and the generation of new lines was mentioned as a benefit, but it was unclear how this would help. The absence of an explicit plan for Aim 2, the use of growth factors to maintain endoderm, is a major weakness. The PI's strength is from zebrafish, and this may be the reason that there was not insightful thought put into Aim 2. Details were often "sketchy" with no alternative method described should the D'Amour protocol fail.
PROGRAMMATIC REVIEW: During programmatic review, this proposal was recommended for special consideration if additional funding should become available on the basis that the proposed study is directly relevant to the search for beta cell replacement therapy for diabetes. It also has implications for diverse diseases given the second aim of developing conditions for endodermal proliferation in culture.