Direct reprogramming towards vascular progenitors for the treatment of ischemia
Angiogenesis or the generation of new blood vessels is a critical part of the normal healing process. Newly created vessels ensure the delivery of oxygen, nutrients, and specific repair signals to injured tissues. Indeed, even though additional repair mechanisms are required, such as replenishment of tissue-specific cell types, angiogenesis contributes to the healing of a number of different processes such as nerve regeneration, skin, muscle and bone repair among others. Insufficient angiogenesis is a hallmark of chronic wounds and often present in the elderly, people with high cholesterol, diabetes, and heavy drinkers and smokers. More than $4 billion has been invested in research and development and over 600 clinical trials targeting a variety of disorders including diabetes-related complications, peripheral arterial disease, stroke and wound healing as a few examples, are currently under way. Most of the studies have focused on the development of molecules facilitating self-repair by increasing endogenous angiogenesis. Stem cell technologies open the possibility for the generation and engineering of blood vessels in vitro suitable for transplantation in specific local areas. Thus, even though stem cells technologies can allow for the large-scale generation of vessels suitable for the healing of a wide variety of disorders, safety concerns hamper their translation into the clinic.
Rarely does a new technology come along that has the potential to make such a major impact on human health like stem cell reprogramming does. The successful development of stem cell therapies for disorders in which the generation of new vessels is necessary for healing will have an obvious and direct effect on the patients and families affected. Furthermore, regenerative medicine techniques are not limited to the generation of new vasculature and bear the potential to treat a vast array of diseases including Parkinson’s, Alzheimer’s, diabetes and blood disorders. All of these disorders place a tremendous burden on the State in terms of health care cost. The idea of connecting basic discoveries in stem cell research to clinical applications is new and unique to the California initiative. As such, California is the primary beneficiary of this technological investment. We envision 2 major positive effects for Californians: (1) California patients will be privileged once stem cell therapies are developed and ready to be implemented. This will create a positive wave in the general perception and awareness as to the position of California and its medical institutions worldwide. (2) California will witness the growth of its technological/industrial infrastructure to develop new forms of treatments on the wave of new basic discoveries. This combination is powerful and dividends will be generated in due time in the form of revenue from health care delivery and intellectual property.
The development of lineage conversion strategies for the generation of vascular progenitor cells (VPCs) and their differentiated derivatives, endothelial and smooth muscle cells, will facilitate the clinical implementation of reprogramming strategies and future personalized medicine. We have previously developed methodologies facilitating the high efficient and rapid generation of human VPCs with potential to give rise to functional human vasculature. Our long-term goals involve the refinement and further development of strategies that not only eliminates the risk of tumor formation due to residual pluripotent stem cell transplantation (a risk inherent to the use of embryonic and induced pluripotent stem cells), but also reduces the time necessary for generation of vessels for the efficient translation and application of these methodologies in acute ischemic situations. Ultimately, we are testing different cell sources as well as refining our reprogramming methodologies to generate: 1) a single and safe methodology for the generation of human vessels; 2) in vivo preclinical data on the functionality and tumor formation potential of the different methodologies used; 3) provide in vivo pre-clinical data on immunocompromised animal models as well as in syngeneic models of cell transplantation to evaluate potential immune rejection of autologous material and the role of inflammation in potential tumor formation. Our in vivo experiments will additionally take place in two different injury models of ischemia: hind limb ischemia and models of cardiac infarction. During the first year of the funded period, we have successfully completed the evaluation of different cell types as a source for the generation of vascular cells and determined that fibroblasts, cells from the skin that can be easily obtained in a patient-specific manner, to be the most reliable source of somatic cells for indirect lineage conversion into vascular progenitor cells. Additionally, we have generated different inducible constructs for the different genes used during reprogramming to a dedifferentiated state, namely Oct4, Sox2, KLF4 and c-Myc. Each gene has been evaluated independently for their potential to generate CD34+ cells, and we have found Sox2 to be indispensable for the process. We have also started the experiments addressing functionality as well as initiated experiments on tumor formation capacity. So far most of the different methodologies tested gave rise to functional cells (albeit with varying efficiencies and timing) and, most importantly, did not result in tumor formation so far. Lastly, we have started to analyze the genetic mutations present in converted endothelial cells and found no significant copy number variations even when integrative approaches were employed.
Reprogramming technologies allow for defining cell identity a la carte. Whereas reprogramming to induced Pluripotent Stem Cells (iPSCs) have attracted most of the attention due to their potential for regenerative medicine and disease modeling applications, while avoiding the need for embryonic material required for generating Embryonic Stem Cell (ESC) lines, iPSC generation and their further differentiation to specific cell lineages and tissues do not represent the sole reprogramming strategy available. Indeed, the use of iPSCs, per se, possesses a priori the inherent risk of tumor formation due to residual pluripotent cell transplantation. In addition, the long time required for iPSC reprogramming and further differentiation make iPSCs prone to accumulation of random somatic mutations that, whereas mostly silenced and irrelevant, might in some cases provoke cellular anomalies leading to malfunction or carcinogenesis. Among all different lineages that can be derived from iPSCs and ESCs, vascular lineages present perhaps the broadest catalogue of potential clinical use. Vascularization, or the generation of new vessels, does not only provide an alternative for the treatment of, for example diabetic patients in where the vasculature is being destroyed, but also extends to a number of vascular diseases, and most importantly, represents the cornerstone for any traumatic injury treatment as well as for cardiovascular disease and infarction. Vascularization will, in this case, allow for replenishing oxygen and nutrient supplies to the injured area. This in turn allows for healing and avoids necrosis, which so often leads to amputation procedures.
Our proposal therefore focuses on generating functional vascular cells for human use and their testing in different ischemia and infarction models. We have focused first on the deciphering of a methodology for deriving vascular cells from both iPSCs and ESCs with high efficiencies. So far, our results indicate that vascular cells derived from pluripotent cells are functional and do not lead to tumors upon transplantation. In addition, our differentiation methodologies do not seemingly compromise the human genome. Therefore, this represents a safe approach for clinical translation. To avoid any potential problem that might arise in the long term, we have additionally identified a set of different conditions that allow us to directly convert human skin cells into functional vascular cells. In this case, the generated vascular cells do not transition through an iPSC state and are therefore safer by definition. To further provide a safe cellular product for clinical applications we have furthermore translated all our methodologies to non-integrative gene delivery systems that further secure and contribute to the absence of genetic mutations in the converted vascular cells. To date, we have been able to produce high quality vascular cells in a large-scale manner and are now ready for initiating our safety studies in injury models of myocardial infarction and limb ischemia.
Strategies for generating human tissues for regenerative medicine have recently been boosted by the development of novel stem cell technologies. However, current technologies are not exempt of risk and require further optimization for their safe application in humans. During the course of this project we have been able to identify novel genetic elements and laboratory conditions facilitating the conversion of human skin cells to vessels comprising the vascular system. The generation of vessels in the laboratory may allow for the treatment of multiple human maladies including ischemic situations, vascular defects and other diseases affecting the cardiovascular system, which collectively, remain the major cause of mortality in the developed world. Our methodologies increased the overall efficiency (by shortening the time required to generate vessels and increasing the amount of cells yielded), and thus future availability for patients, as well as demonstrated a safer profile when compared to previous technologies.
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