SYNOPSIS: The hypothesis of this proposal is that CXCR4/SDF1 signaling could be used to improve targeted transplantation of human embryonic stem cells (hESCs) to injured and pathological tissues. There are three aims: 1) To establish by FACS designer lines of hESCs that express high, low or undetectable levels of the chemokine receptor CXCR4 while maintaining self-renewal, “stemness” and myogenic capacity; 2) To develop non-invasive imaging methods with MRI to monitor and optimize the visualization of these cells in skeletal muscle after their ex vivo labeling with super-paramagnetic Fe contrast agent; 3) To develop non-invasive MRI methods to monitor and optimize the CXCR4/SDF-1 based delivery of hESCs to muscle regenerating after injury or during chronic dystrophic pathology, in vivo. The parameters to be tested are local or systemic vascular introduction of cells, different models of muscle injury and the balance for concentration of labeled cells, resolution and total imaging time as well as image registration for longitudinal studies.
IMPACT AND SIGNIFICANCE: The major goal of this proposal is to develop MRI imaging methods for monitoring systemic delivery and manipulation of stem cell responses in living skeletal muscle. Such visualization techniques will be extremely important for investigators. With disease and aging, muscles lose their ability to regenerate due partially to a loss of satellite cells, a muscle stem cell. The introduction of hESCs in muscle injury models is proposed to foster targeting to the injured tissue and myogenic differentiation in situ. If such replacement therapy worked, there would be benefit to many with muscular dystrophies as well as an aging population. Potentially of more general benefit would be the development of non-invasive monitoring of such introduced cells.
QUALITY OF RESEARCH PLAN: This proposal is the collaboration of two imaging scientists (Majumdar and Conolly) and one stem cell researcher with expertise in muscle regeneration (Conboy). All the investigators have considerable expertise in their respective areas. Their hypothesis is that CXCR4/SDF1 signaling could be used to improve targeted transplantation of hESCs to injured and pathological tissues independently of the hESC origin, method of derivation and line –to-line variation. Indeed, the premise of injecting undifferentiated hESCs into an animal and expect targeting just to the injured tissue needs further supporting data.
The procedures to improve imaging are very detailed, with preliminary data on imaging injected mesenchymal stem cells (to also be run as controls throughout). Attention is given to the balance of MRI for concentration of labeled cells, resolution and total imaging time as well as image registration for longitudinal studies. However, the experimental design of hESCs into the muscle is more sketchy. While hESCs can respond to soluble factors from supernatants of myofiber explant cultures with some resultant myogenic differentiation, it is unclear that the injection of undifferentiated hESCs, whether locally into a muscle or systemically into the vasculature would result in similar differentiation or limited engraftment just in the injured muscle. Yet little attention is given to other engraftment sites even though SDF1 is expressed in multiple places, not just the injured muscle. In addition, there is no discussion of whether the use of immune competent rodents will have an immune response when injected with hESCs. It was suggested by Li et al. (Stem Cells 2004) that hESCs might have immune-privileged properties; however, these experiments were very short-term (48 h). The applicants plan to do MRI experiments out to 5 days post-transplantation. This potential caveat was not discussed. Finally, three NIH approved hESC lines are to be used in this project. There is a suggestion that several other lines, which were more recently generated by the Melton group at Harvard, will also be used but no justification given for these.
Another concern is in regard to the MRI studies themselves. The method used for cell labeling will depend on the detection of the Feridex IV (Fe) in combination with protamine sulfate (Pro) to visualize the cells. As cells divide, this agent will not be carried forth with the dividing cells. In addition, when cells die, the agent may remain behind, and it will not be clear whether live or dead cells are being imaged. This may significantly confound quantification. The applicants outline methods of quantification of the hESCs in the animal models. However, they do not explain whether quantification of potentially small differences between the designer hESCs with varying CXCR4 levels will be problematic. They will be correlating their results to ex vivo markers, such as viability and cell morphology, and they will measure the expression of myogenic markers, such as myf-5, eMHC and cell proliferation.
Overall, this is a sketchy and unsophisticated research plan. The first specific aim, to establish "designer"lines of hESCs with varying levels of CXCR4 and to then test them for "stemness" vs myogenic potential, is without firm scientific foundation, assay precision, or experimental accuracy. The imaging aims are valuable, but ignore issues of xenotransplantion and rejection, and do not present detailed plans, analyses, or alterative approaches.
STRENGTHS: Reviewers cited several strengths. First, the team of investigators is synergistic, each an expert in a relevant field. Second, the procedures to improve imaging are very detailed, with preliminary data on imaging injected mesenchymal stem cells (to also be run as controls throughout). Third, the work has clear separation of responsibilities but planned communications on a regular basis are described with dedicated website, conference calls and monthly meetings. Fourth, the idea of correlating CXCR4/SDF-1 expression with hESC delivery to injured muscle tissue is an innovative idea. Finally, many control and validation experiments to compare MR imaging data with ex vivo histology and various markers are proposed.
WEAKNESSES: The work plan is weak and the experimental design for introducing hESCs into the muscle is sketchy with conflicting comments on the models to be used suggesting only the cardiotoxin injury model (rat and mouse) and/or mdx mice from Jackson. Initial ex vivo studies will be used for improving the imaging parameters, but parallel experiments to see if labeled cells still have the same efficiency of in vitro myogenic differentiation are needed. While hESCs can respond to soluble factors from supernatants of myofiber explant cultures with some resultant myogenic differentiation, it is unclear that the injection of undifferentiated hESCs, whether locally into a muscle or systemically into the vasculature would result in similar differentiation. Also, there is no demonstration that the PI or the group can make 'designer lines,' or that they will plan to carefullly assay and characterize those lines once made.
CXCR4 is expressed on many cells within the body, particularly endothelial cells of the glomeruli and intestines, CD8+ T lymphocytes and muscle satellite cells. A higher expression of CXCR4 is also seen in cancerous cells of many types and is thought to influence their metastatic ability. The premise that within a hESC line there is marked heterogeneity of expression of the cell surface chemokine receptor CXCR4 that is unchanging needs to be clearly shown. Furthermore, there is no reason to think that establishing fixed levels of CXCR4 expression in hESC lines will be specifically useful to direct muscle differentiation. The expectation is that there will be steady levels of CXCR4 over 48 hours in culture but the in vivo experiments are over 5 days.
The question of whether the immune response to these foreign cells influence the results is not addressed, particular given the potential difficulties of using hESCs in immune competent mice out to 5 days post-transplantation.
The specific approaches that will be taken to improve imaging, and how will they be assayed are not discussed nor are caveats of imaging hESCs using the proposed Fe-Pro labeling methods.
DISCUSSION: This proposal has two goals: a scientific goal to improve the targeting of transplanted cells to injured tissue using the chemokine signaling pathway CDCR4/SDF1; and a technology goal to develop improved methods for non-invasive monitoring of cells. The hypothesis is strange in that CDCR4 (receptor)/SDF1 is found in a lot of tissues; it is a widespread signaling system. Given this, it is unclear why the investigators expect injury will result in enhanced targeting of hESC to the injured site. There was a huge amount of experimental detail on imaging; but very little on biology despite an expert biology collaborator, suggesting little evidence of intellectual collaboration, at least for the development of this proposal. One reviewer noted that despite the experimental detail on imaging, the problem of dilution of the label in dividing cells was not addressed. The potential for immune response to the transplanted cells in the model also was not addressed.