Human stem cell reprogramming for tissue engineering and regenerative medicine
Bajpai, Vivek K.
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Stem cells, due to their ability to differentiate into mature cell types, represent attractive solution to restore tissue or organ functions that are lost due to diseases. However, use of both adult and pluripotent stem cells in regenerative medicine is limited by following factors: 1) lack of easily accessible autologous multipotent stem cells sources; 2) lack of rigorous characterization of stemness potential of usable adult stem cells; 3) lack of efficient and robust methods of pluripotent stem cell differentiation into desired cell fate. My thesis attempts to address all the aforementioned problems primarily in the context of vascular tissue engineering, regeneration and directed neural crest development. First chapter of thesis focuses on our current understanding of the stem cell field, reprograming, differentiation, transdifferentiation including various stem cell sources with their differentiation potential towards vascular lineages. The first clinical studies using tissue engineered vascular grafts are already under way supporting the potential of this technology in the treatment of cardiovascular and other diseases. Despite progress in engineering biomaterials with the appropriate mechanical properties and biological cues as well as bioreactors for generating the correct tissue microenvironment, the source of cells that make up the vascular tissues remains a major challenge for tissue engineers and physicians. Mature cells from the tissue of origin may be difficult to obtain and suffer from limited proliferative capacity, which may further decline as a function of donor age. On the other hand, multipotent and pluripotent stem cells have great potential to provide large numbers of autologous cells with great differentiation capacity. Here, I discuss the adult multipotent as well as embryonic and induced pluripotent stem cells, their differentiation potential towards vascular lineages and their use in engineering functional and implantable vascular tissues. I also discuss the associated challenges that need to be addressed in order to facilitate the transition of this technology from the bench to the bedside. Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) including hair follicle derived MSC (hHF-MSC) serve as a source of contractile SMC, they suffer from limited proliferation and culture senescence, especially when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional autologous SMC for downstream applications. In this work, I developed step-wise, controlled differentiation method of hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC by exploiting epithelial to mesenchymal transition (EMT) transcriptional machinery. hiPSC derived MSC restored ageing induced loss of function which was observed in their parental hHF-MSC population phenotypic rejuvenation by induced pluripotency. hiPSC derived MSC gave rise to robust SMC phenotype as observed by molecular and functional characterization including vasoreactivity analysis. Step-wise differentiation strategy of hiPSC provides a controlled approach of phenotypic regulation and thus derived MSC or SMC can be potentially useful for varied biomedical application including vascular disease modelling, tissue engineering and regenerative medicine. Our quest to identify multipoetnt stem cell sources with broader differentiation potential (beyond MSC fate) motivated the next section of my work. During embryonic development, neural crest stem cells (NC) migrate laterally along the length of developing notochord and give rise to diverse cell types (e.g. peripheral neurons, Schwann cells, melanocytes and skeletal and connective tissue) (1) (1) (1). In this work, I have demonstrated that human epidermal stem cells (Keratinocytes (KC)) derived from neonatal and adult skin can be coaxed to acquire functional NC fate under defined culture conditions without any transgene overexpression. We further established that bona fide KC give rise to NC (KC-NC) by demonstrating that clones arise from single KC can give rise to NC. KC-NC differentiated into whole repertoire of mature NC derivatives including peripheral neuron, Schwann cell, melanocyte, endothelial and mesenchymal cells (osteocytes, myocyte, chondrocytes, adipocytes) both in-vitro and in-vivo in a chicken embryo model system confirming bona fide character of KC-NC. Next, we engineered blood vessels using KC-NC derived vascular cells and implanted them successfully into ovine arterial circulation. Furthermore, KC-NC derived Schwann cells were transplanted into a shiverer (Rag2(-/-)) mice model of dysmyelination to test their ability to remyelinate neurons. Taken together, these results establishes KC-NC as novel multipoetnt stem cell source with applications in vascular tissue engineering and neural/glial regenerative therapies.