Early Translational I
ICOC Funds Committed:
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.