Early Translational I
Devastating consequences of limbal stem cell deficiency, caused by wide range of diseases such as burn injury or Stevens-Johnson syndrome, include corneal conjunctivalization, vascularization, and visual blindness. Extensive literature review suggests that stem cell transplant results in better outcomes based on objective measures. Temporizing options, such as medical management, or transplantation of amniotic membrane or non-corneal epithelial cells, rarely restore useful vision. Limbal stem cell transplantation restores partial vision by repopulating the corneal epithelium. Yet, there are many limitations, e.g. limited autologous donor tissue, lack of a specific marker and optimal culture protocol for ex vivo expansion, or immunosuppression for allografts. Here, we present an innovative two-pronged approach to this bottleneck. We identified a unique cell surface marker Notch-1 for activated corneal stem cells. This marker increases our efficiency in isolating corneal stem cells from limbus tissue, as well as other mixed cell populations. It also facilitates tracking of corneal stem cells in in vivo and animal models. We propose to demonstrate the stemness of isolated Notch-1+ ABGC2+ cells from limbal tissue, characterize colony forming capacity and life span, and optimize culture conditions for ex vivo expansion. To date, the lack of specific marker for isolation of corneal stem cells from the dissociated limbal tissue severely limit ex vivo expansion of corneal stem cell for autologous transplantation. Contaminant cells likely decrease quality of the transplant and, thus, visual restoration. Our Notch-1 marker will vastly increase sorting efficiency of high-purity corneal stem cells, resulting in cost effective industrial purification methods. The marker will allow us to track the fate of the in vivo transplanted cells, allowing monitor of rejection, in vivo migration and life span, and early detection of proliferation of undesirable stem cells. We have also developed methodologies to induce a cluster of miRNA into skin epithelial cells to revert these cells back to embryonic-like state, i.e. miRNA-induced pluripotent stem cells. We propose to differentiate these cells into corneal epithelial-like cells, isolate them using the Notch-1 marker, followed by ex vivo expansion. The complex religious, political, and ethical climate of using human embryonic stem cells makes the reprogrammed miRNA-induced pluripotent stem cells the superior alternative for autologous transplantation, obviating the need for limbal tissue in case of severe limbal stem cell deficiency or the need for immunosuppression. Vision loss is a debilitating condition, significantly decreases quality of life and exacting an enormous burden on the society. This proposal aspires to address these issues, as well as contributing to the stem cell biology knowledge base.
Statement of Benefit to California:
California is the most populated state in the USA. Currently, more than 36 million people reside in the State of California, with a projected population of more than 46 million in 2030. Accordingly, the number of citizens with ocular surface diseases and limbal stem cell deficiency is disproportionately elevated, compared to other states in the Union. This is compounded by environmental risk factors, such as fire in [REDACTED]. Ocular surface diseases and limbal stem cell deficiency may lead to complete loss of useful vision, a debilitating condition. Such conditions exact an enormous psychological stress on the patients and their friends and families. The financial burden to California economy in terms of loss of productivity and health care costs is prohibitive over the lifetime of a patient. The State of California also consistently ranked in the top ten largest economies worldwide if it was an independent country. Agriculture, information technology, high technology, and biotechnology are some of the engines powering the state's economy. Education, innovation, and research and development (R&D) comprise the backbone of our competitive edge. In an economy downturn, such as the current recession, it is of the utmost important to continue investment in R&D to develop new ideas, processes, and technologies rather than cutting costs. Such investments will pay dividends in the future when the world economy recovers and people turn to the USA and California as the leaders of innovations and ideas. There are many ways our proposed research will benefit the State of California. Our research explores the cure for corneal blindness, helping sufferers restore their eyesight, improve quality of life, and be productive members of the State. Contributions of our and others' research will help California retain its competitive edge in economy, technology, and medicine.
This is a proposal focused on development of stem cell therapies for corneal injuries and diseases. The first aim describes experiments to isolate and characterize a population of stem cells from the limbus of the eye, based on the expression of a cell surface marker, Notch-1, which is expressed in a subpopulation of limbal cells in vivo. In the second aim, the applicant will examine the role of the putative limbal stem cells in repairing a damaged cornea, using a preclinical animal model. As part of this aim, inhibitors of Notch-1 will be tested for therapeutic potential. Finally, in the third aim, skin fibroblasts reprogrammed using microRNAs (mirPS cells) will be differentiated into limbal cells, and microarrays will be conducted to identify genes involved in the differentiation of the mirPS cells into corneal epithelial cells. Reviewers agreed that stem cell therapies might be a good option for treatment of injuries and diseases of the cornea, and therefore, that this project might yield a stem cell development candidate. However, because the field is not very mature, the applicant did not adequately convince reviewers that the experiments described in this proposal would impact the near-term translation of stem cell therapies to the clinic. The putative stem cells observed in the limbus have not yet been isolated or extensively characterized (outside of one lab), and only weak inferential evidence supports their therapeutic potential. The experiments described in this proposal aim to purify limbal cells and to determine whether they are stem cells (in other words, whether they are pluripotent and self-renewing) and whether they are capable of differentiating into mature corneal epithelial cells in vitro and in vivo. These goals were considered very mechanistic, and reviewers commented that the work would be more adequately supported under a different (non-translational) funding initiative. In addition to a potential lack of responsiveness, reviewers raised some technical issues that impact the project’s feasibility. First, although the applicant does provide data showing that Notch-positive cells can be found in the limbus and corneal region, it is not clear that that the marker is expressed exclusively on a true stem cell population. If Notch is not specific to the pluripotent stem cell population, the first aim would be difficult to achieve. Second, there were insufficient data presented in the application supporting the claim that mirPS cells were pluripotent (iPS-like) cells, or that they can be induced to express markers of limbal stem cells or the corneal lineage, or that they can survive in the preclinical eye-wounding model. Although the applicant proposes to do many of these experiments if funded, reviewers commented that these data should be in hand before embarking on a translational project. One reviewer highlighted experiments to assess the therapeutic potential of Notch-1 inhibitors as potentially interesting, but this aim seemed somewhat of an aside from the cell therapy focus of the application and, again, it was not clear that the studies would lead to a significant advance, as no supporting preliminary data were provided. Finally, one reviewer stated that no consideration was given to how mirPS-derived limbal cells would be characterized for the residual presence of potentially teratoma-forming pluripotent cells, a crucial translational issue. In addition, reviewers commented that the application was disjointed and lacked cohesion. Reviewers commented that the applicant is an expert in corneal and developmental biology but lacks a strong track record in stem cell research. Similarly, collaborators were seen as strong but lacking experience in stem cell science and therapeutic development. Given the complexity of Notch biology in stem cell behavior, this lack of expertise was troubling. Finally, the applicant did not highlight the necessary environment, facilities and expertise available at the applicant institution that are important for the translational aspects of the program - scale up of cell production, GMP capability, safety assessment, etc. In summary, this application focuses on development of stem cell therapies for corneal pathologies. Although the therapeutic target is important, reviewers commented that the project is in too early a stage of development to justify funding in the Early Translational initiative. The lack of preliminary data and the weak stem cell experience of the research team led the reviewers to question the feasibility of the project. In addition, reviewers were not convinced that the applicant team had the expertise or access to all the facilities required to translate this project to the clinic.