Human embryonic stem (hES) cells are the first type of human cell cultured in the laboratory that have the potential to become any of the several hundred cell types in the developing gestational human. Wound healing of human skin has been likened to "a recapitulation of gestation". Therefore, if wound healing is like the gestation of a developing infant, it is likely that hES cells could be used to heal chronic skin wounds and provide the many types of cells in human skin with names such as keratinocytes, fibroblasts, endothelial cells, melanocytes and others. Essentially, the wound healing process must fill in a hole in the skin and regenerate skin tissue. Elderly people (especially those with obesity, spinal cord injuries, diabetes, vascular diseases and leg vein diseases) frequently have non-healing skin wounds. The annual cost to care for chronic wounds exceeds $11 billion. Although we think of chronic wounds mainly in elderly adults, there exists a rare genetic disease in children called recessive dystrophic epidermolyis bullosa (RDEB) which causes skin fragility and chronic blistering wounds that heal with scarring. RDEB children die around age 20 – 30 from an aggressive cancer forming in the chronic wounds. The cause of RDEB is a gene defect in a skin collagen called type VII collagen (C7) that makes structures that hold together to two main layers of the skin – the epidermis and dermis. Without this collagen, the epidermis and dermis do not stick together and a chronic blistering wound ensues. We have grafted RDEB skin onto mice and injected the grafts with RDEB gene-corrected fibroblasts and reversed the RDEB in the grafts. Likewise, we have injected into the circulation of the grafted animals RDEB-gene corrected fibroblasts and found that they "home" to the RDEB skin and reverse the RDEB disease. Nevertheless, there are limitations to these cells such as their long-term survival. hES cells are immortal and will continue in the RDEB skin and keep it disease free. Since hES cells are primitive, they will be less likely to be rejected by the host. We will determine their longevity in the host and determine where in the skin these hES cells will reside as they deliver C7 to the skin and wounds. The RDEB hES strategies in this proposal essentially address many of the issues with healing of chronic wounds. We will deliver hES cells to wounded human skin and evaluate where they reside in the healing wound, what connective tissue molecules they deliver to the wound, if there is an acceleration of wound healing and if the wounds heal with less scaring since the hES cells express genes thought to be involved in "scarless" wound healing. Therefore, this proposal will provide insights and possible therapy for an orphan genetic disease in children and chronic, non-healing skin wounds which are an enormous health care problem in the general populaous.
Statement of Benefit to California:
Our proposal has the potential to cure a rare, incurable, genetic, skin blistering disease in children called Recessive Dystrophic Epidermolysis Bullosa (RDEB). Stanford University in Palo Alto has had an EB Registry with many RDEB patients. The care of these children is arduous and a huge economic burden on both the families and the State. To date, the care of these children is only palliative and primitive. Because RDEB is rare, pharmaceutical companies have not invested in RDEB since the market is so small. We hypothesize that RDEB children could be treated with novel strategies based on human embryonic stem cells (hES cells) or hES cells genetically engineered by our laboratory. The main advantage of hES cells is their unlimited reproductive capacity and their primitive nature making them less immunogenic. RDEB is due to a gene defect in type VII collagen (C7) which resides between the two main layers of the skin, the epidermis and the dermis where it is organized into anchoring fibril structures that literally "anchor" the epidermis and dermis together. RDEB children lack a normal complement of functioning anchoring fibrils. Therefore they have poor epidermal-dermal adherence, skin fragility and chronic scarring blistering wounds. We have delivered gene-corrected RDEB fibroblasts into RDEB skin grafted onto mice. The gene-corrected RDEB cells "home" to the RDEB skin grafts and deposit C7 which organizes into anchoring fibrils which corrects the genetic RDEB skin phenotype. There is a RDEB-like C7 knock out mouse that because of wounds in the skin, mouth and esophagus only live 2 – 5 days. We intravenously injected gene- corrected RDEB fibroblasts into these mice and corrected their genetic blistering phenotype. These mice now live more than 2 months. If we had primitive skin cells like those derived from hES cells that artificially express C7 and have a long replicative capacity, these mice might have a normal life span which is one goal in this proposal. The hES technology generated for RDEB can be applied to chronic skin wounds which are an enormous health care problem costing over $11 billion per annum. Our data shows that intravenous administration of cells over-expressing C-7 will "home" to human skin wounds and markedly enhance wound healing. The proposed technology, with the unique regenerative properties of the embryonic dermis present in hES-derived dermal progenitor cells, may help RDEB children and adults with chronic non-healing wounds thus saving considerable health care costs for California.