A skeletal stem cell orchestrating limb regeneration

Project Summary Approximately .5% of children are born missing a limb, and 2.1 million in the US alone are living with limb loss. Current management for these patients largely relies on the use of prostheses, however most prosthetic use is marked by significant patient dissatisfaction due to functional limitations. Only regeneration of an intact limb can offer optimal function and quality of life for these patients. While it is well known that select non- mammalian organisms have remarkable capabilities for complete limb regeneration, it is sometimes overlooked that similar capacity for regeneration, at least in latent or incomplete form, exists in mammals including humans and mice. Digit tip amputation in both humans and mice can lead to blastema formation and composite regeneration of the digit tip. Understanding the mechanism of these mammalian limb regenerative responses is a necessary step to inducing or extending these responses to promote limb regeneration. However, methods to achieve this goal have been elusive, in part because the specific stem cells mediating regenerative blastema formation and organizing the regenerative response were unknown. Recently, we have identified a new stem cell forming the skeleton that is specialized for skeletal healing responses, and we find that these stem cells are the cell of origin of the blastema forming after digit amputation, undergoing injury induced plasticity that then confers upon them the capability to not only mediate bone regeneration, but to more over orchestrate a composite tissue regeneration program. We further find that when this specific stem cell is harvested from blastema tissue and transplanted to a site of tibial amputation, they the capacity to mediate true limb regeneration at this new site, thereby allowing initiation of a true limb regeneration response to be initiated at any amputation site. Here, we will develop these observations into a new approach for limb regeneration, first (Aim 1) we will determine how this stem cell acts in the blastema forming after digit tip amputation orchestrate multi-tissue regeneration and how these cells are transcriptionally reprogrammed in the blastema to acquire the capacity to orchestrate regenerative responses. Next (Aim 2), we will further develop the method of blastema stem cell implantation into amputation sites to initiate regeneration, evaluating whether the true multi-tissue regeneration occurs at the transplantation site, and whether new joint tissue can be patterned in the transplanted cells, thereby allowing for true limb reconstruction, including joints. Ultimately, this proposal will develop key initial elements of a new approach for the regeneration of amputated limbs, by transducing a specific population of autologous skeletal stem cells to induce a blastema-like state, and then implanting them into the amputation site to initiate regeneration of the missing limb.