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RC1-00146-1: Endoderm-specific integration for human embryonic stem cells: towards tissue regenerative solutions for lung and pancreas

Recommendation: Not recommended for funding 

Public Abstract (provided by applicant)

Human embryonic stem cells (hESC) show great promise for tissue regeneration because of their propensity to make many of the cell types in the body. However, exactly how hESC can be directed to make specific parts of the body remains unclear. This is a major roadblock to harnessing the striking potential of hESC as a regenerative engineering solution for organ failure. Regeneration of complex tissues derived from the inner layer of the body called the endoderm such as the lung and pancreas is particularly challenging.

We already have in hand several novel hESC lines that have a strong propensity to enter endodermal tissues in small scale culture systems. We also have already enjoyed preliminary success with other kinds of stem cells in getting them to enter and make lung in special kinds of mice. Now we plan to see if we can make lung and pancreas with our new hESC lines.

• Aim 1 We will try to force hESC to enter endoderm cell lineages by driving them with organ specific signals.
• Aim 2. We will see how hESC do when they are introduced into developing lung or pancreas because we think there are special signals that come from naturally growing tissue.
• Aim 3. We will see whether hESC can actually replace damaged lung or pancreatic tissue to simulate the repair of diseased tissue.

This project brings together an experienced team of investigators with complementary sets of expertise in hESC, tissue development, tissue repair and tissue regeneration. We expect to discover a toolbox of critical factors that will drive hESC to integrate into and function as part of endoderm derived tissues and hence are likely to directly bring forward hESC based regeneration solutions for failure of the lung and pancreas.

This work cannot be funded by the Federal Government because we will utilize novel hESC lines.

Statement of Benefit to California (provided by applicant)

Diabetes and pulmonary failure are two serious and increasingly common problems among the children and adults of California. These serious public health problems lead to much loss of schooling and productivity every year. This proposal seeks to discover a toolbox of critical factors that will enable hESC to enter, integrate in and function as part of tissue regenerative solutions for lung and pancreatic failure. If we are successful, these critical factors may become medicines that will be critical to harnessing the potential of hESC as tissue regeneration solutions. The know-how for these critical processes will reside in California, giving the State a clear competitive advantage.

Review

SYNOPSIS: The specific aims of this proposal are first to create endoderm lineage-specific stem/progenitor cells from human Embryonic Stem Cells (hESCs); then to develop novel assay systems to determine how to optimize the functional endoderm-specific engraftment of hESCs into endoderm-derived organs including lung and pancreas; and finally, to optimize the contribution of precursor cells to lung and pancreas regeneration in vivo. Methodologies to be used include: lentiviral infection or electroporation to establish expression of transcription factors, pharmacologic enhancers of differentiation; immunohistology; flow cytometry; Affymetrix and microarrays; murine genetic mutants, hESC microinjection into embryonic lung and pancreatic tissue; functional studies of key regulatory factors; and in vivo treatment with hESCs of animals whose lungs and pancreatic islets have been injured by various modalities.

SIGNIFICANCE AND INNOVATION: The goal is to investigate the possibility that hESCs can be driven to express key endodermal phenotypes that then can integrate into adult tissue and repair damage, all without forming teratomas. The specific tissues focused on are the lung epithelia and the pancreatic islets. If the hypothesis were supported, it would be very exciting with important implications for the field.

Reviewers agree that creating new approaches to using hESCs to enhance endoderm lineage-specific stem/progenitor cells, developing new assay systems to determine approaches to optimize functional endoderm-specific engraftment of hESCs into lung and pancreas, and optimizing in vivo rescue of damaged lung and islet tissue in vivo would be innovative and important contributions.

QUALITY OF RESEARCH PLAN: The proposal is considered an unrealistic, ambitious plan to drive hESCs to endoderm by lineage-specific ectopic transcription factor expression and to develop means of having them incorporate in vivo to damaged lung or pancreas. As steps to do this, the hESC will be genetically manipulated to express stably lung or pancreas lineage genes, endoderm will be differentiated, a novel assay for high throughput determinations of optimizing the niche in explanted mouse tissues developed, and in vivo non-invasive imaging for endoderm differentiation developed. However, the under-developed status of the preliminary data in the proposal and the sketchily described experimental plans themselves raise questions about feasibility (see below).

According to reviewers, the proposal is not formulated as a proper research proposal and the quality of the research plan is seriously compromised by the lack of sufficient methodological detail, coherence, and preliminary data. Lack of this information makes the plan seem overly simplistic.

The initial conception of the grant, i.e. that lung and pancreas arise relatively simply from foregut under the influence of one or a small number of known factors, is considered simply not correct. The aims are sweeping and unobtainable, that through a candidate gene approach (and a small number at that) lineage-specific lines will be obtained for lung and pancreas. Further, the applicant plans to apply these results to multiple tissue regeneration models. Throughout the proposal, there is essentially no strategic rationale or design. One reviewer noted that the discussion of statistics was particularly weak.

While the concept of combining lung and pancreas as two endodermal tissues in the same project has some rationale, it dilutes the focus. Preliminary data is weak. Not enough information is provided for how to obtain specificity of endodermal differentiation using the specific strategy proposed by the PI. Many investigators have been trying to get endoderm populations under manipulated conditions and the protocols are improving, but no details about how this group plans to approach this are given.

STRENGTHS: The expertise of the PI is cited as a strength. The PI is an expert on lung development and has a team with expertise in ESC, viral vectors, and bioinformatics. (The pancreatic development aspect is less well represented.)

The lung explant model and the in vivo injury models are excellent for this project (however, those of the pancreas are less well developed; usually for pancreas explants by E12, the stomach and duodenum are dissected away, leaving just the two pancreatic buds for culture.)

There are 12 novel hESC lines to be used (but no rationale is provided for using so many or nor how they will be tested is described.)

The environment and facilites are excellent and all that is needed for this work.

WEAKNESSES: Reviewers agree that overall the proposal is too broad and lacks details, appreciation of likely difficulties and a proper scientific design. The proposal presents unconvincing preliminary data. The lack of preliminary data using murine ESCs and hESCs that are available to the PI makes this proposal premature.

Specific points raised by reviewers include the following. For Aim 1, the successful use of specific vectors (which introduce new cDNAs into cells transiently) to provide sufficient new protein to cells is marginal at best. Preliminary data supports the PI’s proposal but no images are shown, nor are survival data. Results from studies on glucose-stimulated insulin secretion were not described which would demonstrate physiologic beta cell function. Also, no work was done with murine ESCs or hESCs. The latter omission is difficult to understand since the PI states s/he has 12 lines of hESCs to use. Why hasn’t this already been attempted as a preliminary experiment? Another example in this Aim is a description of experiments for lung tissue and the use of differentiation enhancers; no information about drug concentrations or culture conditions are provided. It is also indicated that development of novel vectors will be carried out in collaboration with another investigator via her/his current CIRM program proposal, which at this point is not been reviewed or funded.

For Aim 2, plans are presented to study the effects of key growth factor signaling using mutants and inhibitors, but no experimental details are provided.

For Aim 3, it is proposed to inject hESCs via a specific route. Cells injected via this route might reach injured lung tissue, but almost certainly will not reach injured pancreatic islets because of lung capillary clearance. A superior mesenteric artery or even aortic injection approach would be much more feasible. How was the number of hESCs to be injected established? No functional studies of pancreatic islets are proposed to ensure that the hESC treatment will improve glucose-stimulated insulin secretion, which could be done in vivo and in vitro. No preliminary data using ESCs, mouse or human is provided, and easily could have been. No consideration of immune rejection by mouse recipients of hESCs is even considered.

One reviewer points out that preliminary data are given but are weak, and sufficient technical details are not provided (including controls used and cycles) that would lead the reviewer to accept the startling results. Why are two different labeling procedures proposed for non-invasive imaging without trying to do any correlation between the two? None of the necessary controls are mentioned. How many cells can be detected? How reproducibly? How well is the pancreas localized in either modality? Can there be distinction between pancreas (or lung) and the surrounding tissues?

Teratoma risk is high for injecting hESCs in vivo. There is some mention of this risk but monitoring of this over both time and tissues is unclear.

Intraperitoneal placement of islets does not work well; many more islets are needed. Additionally one might expect more risk of teratomas in the less specific niche.

A reviewer suggests the following for improving the proposal: limit the studies to lung or pancreas; be more thorough in writing the experimental plan; collect preliminary data using ESCs that will allow an informed assessment as to the feasibility of the proposal.

DISCUSSION: There was no further discussion beyond the reviewers’ comments.

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:

• None