Regulation of apoptosis during endocrine development

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Widespread clinical application of cell-based therapies for type 1 diabetes is limited by a serious shortage of islet tissue for engraftment. Therefore, alternative approaches to beta (β) cell generation are required. Presently, much of the promise of regenerative and restorative medicine lies in stem cells. Understanding the molecular events that drive the genesis of functional insulin-producing cells from undifferentiated stem cells is critical to devise strategies that will help restore and/or maintain β cell mass. One avenue of research directed towards this goal is the in vitro expansion of human embryonic stem cells (hESC). Realizing this potential requires a better understanding of the mechanisms that occur as hESCs differentiate into endoderm and subsequently into pancreatic lineage. Embryonic stem cells face several key developmental decisions in the pathway to forming a mature organ. In pancreatic development, the earliest critical steps are gastrulation and subsequent formation of definitive endoderm. Recently, a method to generate definitive endoderm was described (1); however the process is associated with massive cell loss that occurs by the induction of apoptosis, or programmed cell death. The net result of the differentiation protocol is a small population of endodermal cells that limits the experimental and applicable use of these cells. In the proposed studies, we have assembled a multi-disciplined team to explore the mechanisms of apoptosis during differentiation. We will specifically examine the role of the chemokine/chemokine receptor SDF-1/CXCR4 in this process. The appearance of the CXCR4 receptor is a marker for endodermal development. The receptor plays a plays a critical role initiating cell survival or cell death pathways. We hypothesize that inadequate levels of the CXCR4 ligand, SDF-1α, during endodermal development initiates apoptosis and we will test whether the addition of recombinant SDF-1 protects the cells from death. Additionally, we will study the mechanism by which apoptosis is induced during differentiation. We have identified a novel protein kinase A interacting protein, AKIP, that we hypothesize plays a critical role in initiating apoptotic program. Taken together, these studies will help elucidate the molecular mechanisms associated with hESC differentiation and survival.

1. K. A. D’Amour et al., Nat Biotechnol 23, 1534 (Dec, 2005).

Statement of Benefit to California (provided by applicant)

Although Type 1 diabetes can be managed with daily injections of insulin, no cure for this disease exists. Islet transplants offer hope for many with type 1 diabetes, however, a lack of sufficient tissue for transplantation has restricted the number of patients who can receive therapy. Furthermore, a recent report found that more than 75% of islet transplant patients required insulin treatment within 2 years following the transplant. This underscores the need for a renewable and sustainable source for insulin-producing beta (β) cells. To address this problem, we have initiated studies on the mechanisms of islet growth and function in the hopes of generating a renewable cell-based system for transplant by directing differentiation of human embryonic stem cells (hESC) or pancreatic progenitor cells into insulin producing β cells. Before hESC can be routinely used as therapeutic agents, we must unravel the complicated molecular processes underlying differentiation. A critcal first step in differentiation is the transition of hESC from a pluripotent state to a cell of endodermal lineage. An effective protocol for endodermal differentiation was recently developed, however during differentiation many of the cells initiated a cell suicide program, termed apoptosis. The work proposed here will elucidate the molecular mechanisms that drive endodermal apoptosis. Identification of the key enzymes and proteins in this process will ultimately result in our ability to control differentiation, which will in turn generate larger pools of islet precursor cells.

The phenomenon of apoptosis is not unique to endodermal formation. Massive apoptosis has been documented in many differentiation protocols. Therefore, this study has broad implications for all aspects of hESC research. By answering a fundamental question about how hESCs survive during the differentiation process, the state and citizens of California will benefit and its citizens because more laboratories will have greater access to a larger population of cells to study various diseases.

Review

SYNOPSIS: The goal of this project is to optimize the survival of committed definitive endoderm from hES cells, to ultimately enhance development of insulin-producing pancreatic beta cells. Current protocols to generate endoderm have confirmed a chemokine receptor is a relevant marker, although these protocols can be associated with a high level of cell death. The hypothesis put forth is that cell death occurs due to activation by CXCR4 of apoptotic pathways due to limiting ligand. The project seeks to characterize receptor/ligand expression during endoderm induction, evaluate the use of ligand, and characterize downstream apoptotic signaling pathways. Initially, serum-free conditions will be used to commit hESC to endoderm fate and the expression of various markers will be followed and correlated with cell death. It is expected that apoptosis will correlate with a relative lack of ligand. The Prinicipal Investigator (PI) then will test if the ligand provides a cell survival signal for definitive endoderm. Finally, he/she will determine if chemokine receptor-induced apoptosis functions through specific downstream signalling pathways.

STRENGTHS: The ability to efficiently generate pancreatic cells from hESC is an important and significant goal. Progress in enhancing the numbers of committed cells will be useful. It may be a good idea to incorporate known regulatory mechanisms from other systems to bear on this problem.

The proposal is well-designed and architectured. An adequate collection of markers were utilized to assess the differentiation of stem cells and their progression toward endodermal lineages. A combination of RT-PCR, western blotting, enzyme assays and immunocytochemistry will be utilized.

The preliminary data as well as the background information provides an innovative and provocative yet tangible model for the maintenance of the endoderm.

The primary strength of the PI is in the structure/function of chemokine receptors (protein biophysics) and not in stem cells nor apoptosis per se. However, the PI has assembled a strong cast of contributors and collaborators to study the role of relevant factor in apoptosis of endodermal lines derived from embryonic stem cells, that will cumulatively be critical to the success of this proposal. The PI is also strongly funded and not in danger of lacking needed resources.

The data accumulated within the funding period of this grant should provide adequate preliminary data for larger scale, federally-funded studies.

WEAKNESSES: A major weakness of the application is a lack of definition for the relative role of the PI and others. Protocols are referred to as “we developed” but it is not apparent that the PI or his/her laboratory was involved in these experiments. Rather it would appear that a collaborator and his/her colleagues are carrying out the embryonic stem cell work. The only facilities that are described are for another colleague. The collaboration section is filled out as Not/Apllicable, and there are no letters to attest to the collaboration. It was not clear why the PI is designated as such, considering that the majority of the work will be performed in the laboratory and presumably in the hands of his/her collaborator.

It is also not clear what is anticipated by the inhibition of cell death. Perhaps the generation of definitive endoderm is facilitated by selection through death of other cell types. If the embryonic stem cells are blocked from cell death, will this generate more definitive endoderm, or instead dilute the lineage? The logic is also not entirely clear. If the endoderm progenitors express CXCR4 and survive, non-expressing cells may be the ones that die. It is equally conceivable that induction of cell death is an essential component of this particular protocol to enrich for endoderm.

If the ligand is not limiting in the system, the project has little left to test. This would be a very easy experiment to carry out, and it is surprising that it hasn’t been tested.

The later experiments described have relevance for understanding basic mechanisms of signaling but are less clearly relevant to the focus of endoderm development.

The investigator alludes to the supposition that the primary anti-apoptotic role of the ligand, mediated through CXCR4, is to modulate signaling. As noted by the PI, the ligand has other proliferative roles and pathways. Since both pathways may be regulated it is clear that the investigators propose to investigate this cross regulation. Perhaps an antagonist approach may be an appropriate model to study the capacity of the ligand to serve an anti-apoptotic role. Likewise no attempts to monitor other signaling pathways were proposed for the studies described for late stages of the project.

There is a pervasive lack of attention to detail and proofreading in the proposal in its current form. In some cases the lack of attention to detail detracted from the overall strength of the proposal, since there was some confusion about the preliminary data listed in the feasibility section.

The structure of the collaboration needs to be clearly laid out. Recent progress addressing this problem has been published by several groups, including the generation of beta cells, and this needs to be taken into account.

DISCUSSION: The innovation in this proposal lies in testing the effects of a ligand on downstream cell death pathways. One reviewer claims that there appears to be a flaw in logic in that if CXCR4 cells survive, then adding ligand won’t help. To affect CXCR4 signaling, downstream effectors would need to be modulated. In similar experiments done using mouse cells, the ligand doesn’t do anything. This reviewer also noted that there were no letters of collaboration included, and commented that this was a poorly written proposal. The second reviewer commented on the fact that not much work has been published on effects of the ligand in this area, and agrees that the proposal’s success depends upon the treatment having some effect. There was concern that since much of the work would be done in the lab of the collaborator, it wasn’t clear who the prinicipal investigator really would be. It was also mentioned that the numerous grammatical errors and poor labeling of figures detracted from the proposal.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Lansing, Sherry