SYNOPSIS: This is an interesting project to develop strategies for cardiac regeneration following myocardial infaction (MI), with a goal of maximizing functional implantation and overcoming the scarring effect which can lead to heart failure. The idea is to integrate the generation of functional cardiac progenitors from embryonic stem cells (ESC) with the development of effective cell delivery mechanisms into the host tissue. The goal of the proposed work is to make cardiac patches starting from stem cells of mouse, swine and human. There are two aims:
1) Use a 3D microarray approach to evaluate a limited number of biochemical and electrical stimulations to control and maximize the directed differentiation of hESC to functional cardiomyocytes (CM). Embroid bodies (EBs) will be settled into a 3D microsystem with multiplexed electrical, biological, and chemical stimuli (azaC, bmp2, NO, tgfalpha, etc.). Measurements can be taken on the grid (patch-clamp, Ca+ imaging) or off the array for IHC or RT-PCR. Two cell sources will be tested, using either Flk1+ generation from Flk1-/Bra+ populations, or ES cells that express the cardiac Ik1 channel and will mature into an adult phenotype.
2) To develop a delivery vehicle in an infarct model to compare direct injection of cells with a 3D material construct derived from porcine urinary bladder matrix (UBM). A swine infarct model will be used, with or without endothelial cells.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The significance of deriving bona fide cardiomyocytes from hESCs and demonstrating their utility in an animal model of cardiac repair would be high. In particular, a cardiac patch could in principle provide major improvements in cell retention, increased viability, and enhanced integration with the host. The utilization of combined molecular and physical signaling for cardiac differentiation of hESCs is also novel. It is also a good idea to test the benefit of including functional endothelium. However, the experimental design, as explained below, does not give sufficient confidence that the stated research goals will be met, and that the potential significance and innovation will in fact be achieved.
The stated goals of the project are quite exciting: combination of molecular and electrical signals to direct differentiation of hESCs into cardiac lineages, and the implantation of hESC-derived cells using a native tissue matrix to achieve cardiac repair. The aims are logical for achieving these goals. Aim 1 is to develop methodologies for derivation of functional cardiomyocytes by controlled and high-yield differentiation of hESCs. Aim 2 is to develop delivery vehicles for implantation of cardiomyocytes, either alone or in combination with endothelial cells.
However, the enthusiasm for this proposal is diminished once one starts to read the Research Design and Methods and Preliminary Studies. It is unclear what will be done, why and how. In summary, the proposal has a very high significance but is very poorly written and suffers from many scientific shortcomings, some of which are listed below.
All three reviewers had major concerns that embryonic cells from three species are used: murine, human and swine, and that it is totally unclear why these choices were made and how will methods be translated from one cell source to another. All three reviewers were skeptical that the human cell protocols for derivation of cardiomyocytes can be optimized as stated by extension of established protocols for murine vascular cells. Two reviewers pointed out that it seems that this Aim is funded already by a CIRM SEED grant, for which Dr. McCloskey is a co-PI.
The microarray platform for in vitro studies is neither described nor referenced, and the reviewer could not understand what are the design features that enable medium perfusion and electrical stimulation. There are statements that the PI has the capability to perfuse and stimulate cell cultures long term, but these statements are not documented in any way (published work, preliminary studies, detailed design).
A list of molecular agents has six different factors for testing for impact on differentiation to CM, but their selection, roles, concentrations and combinations are not specified. Electrical signals are not specified either, and the whole body of literature on electrical stimulation of individual cells and three-dimensional tissues is ignored.
The second Aim describes a swine infarct model that will be carried out by the co-PI at Davis. The PI states that swine ESC will be used for this purpose, yet there is no indication these cells are available or are being tested at all in the first aim. One reveiwer commented that they would like to see a discussion of how swine stem cells differ from mouse and human. Swine cardiomyocytes derived from embryonic stem cells will be tested, but it is totally unclear how will these cells be derived, and what are the compatibility issues (e.g. inbred population).
At another point the PI indicates that mES (GFP+) or hESC (Ik1+) will be used, but its not clear how this relates to the swine model. Overall, the ES side of the transplant model were not clearly indicated, and priorities for translating the results from Aim1 with the transplant model were not discussed.
Overall, the research design is poorly developed, and reflects insufficient understanding of cardiac differentiation. Also, preliminary studies are limited to mouse vascular cells and the swine model of cardiac repair, and do not establish the feasibility of the proposed work.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. McCloskey is an Asst. Professor in the School of Engineering at Merced (2005). She trained as a postdoctoral fellow with Dr. Robert Nerem at Georgia Institute of Technology and developed expertise with derivation of vascular cells from ES. She published 3 first author papers in solid specialty journals on post-doctoral work. Since 2005, the PI has modest publication record, with two articles in the last two years (2005 and 2006) on derivation of endothelial cells from murine embryonic stem
The candidate is clearly able to carry out the ES work and has recruited collaborators to assist with the microarray platforms (Khine, who is PI on the SEED grant) and animal models (Li at Davis).
Dr. McCloskey is fully independent and has developed collaborators and mentors to assist in her development as a stem cell biologist. She is committed to develop tissue engineering approaches using ES cells integrated into animal disease models. The career development plan is not particularly strong. Most of the description relates to the past experience during her post doc at Georgia Tech and very little to her future plans. Also, she specifically says that mentorship at her University is limited.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The institutional commitment is strong, but the institution is new (only two years) so assessing a track record in supporting young faculty is not applicable. UC Merced is able to provide most of necessary resources and centers to successfully execute the proposed specific aims. Dr. McCloskey was provided with startup and 600 sqft laboratory space with TC facilities and small equipment, microscopes, etc. The animal work would be done at Davis with the co-PI Dr. Li. The Dean, Dr. Wright commits to provide additional funds (10K per year) and teaching relief (1 course per year) in support of this grant. There is a clear commitment to stem cell biology and so far 5 faculty are on campus. Additional mentorship can be recruited from outside.
DISCUSSION: Reviewers concurred that the research design is poorly developed. A major complication in this study was the lack of clarity and rationale for choosing specific cells for each study. It was not clear to reviewers which cells would be used in each experiment, and why.