Cellular cardiomyoplasty is a cell therapy using stem cells or progenitor cells for myocardial regeneration to improve cardiac function and mitigate heart failure. Since we first published cellular cardiomyoplasty in 1989, this procedure became the innovative method to treat damaged myocardium other than heart transplantation. A significant improvement in cardiac function, metabolism, and perfusion is generally observed in experimental and clinical studies, but the improvement is mild and incomplete. Although safety, feasibility, and efficacy have been well documented for the procedure, the beneficial mechanisms remain unclear and optimization of the procedure requires further study. This chapter briefly reviews the stem cells used for cellular cardiomyoplasty and their clinical outcomes with possible improvements in future studies.

Cellular cardiomyoplasty is a cell therapy using stem cells or progenitor cells for myocardial regeneration to improve cardiac function and mitigate heart failure. Since we first published cellular cardiomyoplasty in 1989, this procedure became the innovative method to treat damaged myocardium other than heart transplantation. A significant improvement in cardiac function, metabolism, and perfusion is generally observed in experimental and clinical studies, but the improvement is mild and incomplete. Although safety, feasibility, and efficacy have been well documented for the procedure, the beneficial mechanisms remain unclear and optimization of the procedure requires further study. This chapter briefly reviews the stem cells used for cellular cardiomyoplasty and their clinical outcomes with possible improvements in future studies.

Cardiovascular disease is the leading cause of death in the United States, and coronary artery disease (CAD) kills over 370,000 people annually. There are available therapies that offer a temporary solution; however, only a heart transplant can fully resolve heart failure, and donor organ shortages severely limit this therapy. C-kit+ human cardiac stem cells (hCSCs) offers a viable alternative therapy to treat cardiovascular disease by replacing damaged cardiac tissue; however, low cell viability, low retention/engraftment, and uncontrollable in vivo differentiation after transplantation has limited the efficacy of stem cell therapy. Tissue engineering solutions offer potential tools to overcome current limitations of stem cell therapy. Materials derived from natural sources such as keratin from human hair offers innate cellular compatibility, bioactivity, and low immunogenicity. Keratin proteins extracted using oxidative chemistry known as keratose (KOS) have shown therapeutic potential in a wide range of applications including cardiac regeneration. My studies utilize KOS hydrogels to modulate c-kit+ hCSC differentiation, and explore the capability of differentiated cells to regenerate vascular tissue. In the first Chapter, we reviewed literature relevant to keratin-based biomaterials and their biomedical applications, the use of stem cells in cardiovascular research, and the differentiation of vascular smooth muscle cells (VSMCs). The section on biomedical applications of keratin biomaterials focuses on the oxidized form of keratin known as keratose (KOS), because this was the material used for our research. Since we planned to use this material in conjunction with c-kit+ hCSCs, we also briefly explored the use of stem cells in cardiovascular research. Additionally, we examined some key signaling pathways, developmental origins, and the cell phenotype of VSMCs for reasons that will become clear after observing results from chapters 2 and 3. Based upon our review of the literature, KOS biomaterials and c-kit+ hCSCs were determined to be promising as a combined therapeutic for the regeneration of cardiac tissue. Research in Chapter 2 focused on characterizing the effects of KOS hydrogel on c-kit+ hCSC cell viability, proliferation, morphology, and differentiation. Results demonstrated that KOS hydrogels could maintain hCSC viability without any observable toxic effects, but it modulated cell size, proliferation, and differentiation compared to standard tissue culture polystyrene cell culture (TCPS). KOS hydrogel produced gene and protein expression consistent with a VSMC phenotype. Further, we also observed novel "endothelial cell tube-like" microstructures formed by differentiated VSMCs only on KOS hydrogel, suggesting a potential capability of the hCSC-derived VSMCs for in vitro angiogenesis. Results from this study lead us to question what signaling pathways might be responsible for the apparent VSMC differentiation pattern we observed on KOS hydrogels. Research in Chapter 3 explored the time course of VSMC differentiation, cell contractility, inhibition of VSMC differentiation, and measured protein expression of transforming growth factor beta 1 (TGF-β1) and its associated peptides for hCSCs cultured on KOS hydrogels, tissue culture polystyrene, and collagen hydrogels. A review of VSMC differentiation signaling pathways informed our decision to investigate the role of TGF-β1 in VSMC differentiation. Results demonstrated that KOS hydrogel differentiated hCSCs significantly increased expression for all three vascular smooth muscle (VSM) markers compared to TCPS differentiated cells. Additionally, KOS differentiated hCSCs were significantly more contractile than cells differentiated on TCPS. Recombinant human (rh) TGF-β1 was able to induce VSM differentiation on TCPS. VSM differentiation was successfully inhibited using TGF-β NABs and A83-01. Enzyme-Linked Immunosorbent Assay (ELISA) analysis revealed that both TCPS and KOS hydrogel differentiated cells produced TGF-β1, with higher levels being measured at early time points on TCPS and later time points on KOS hydrogels. Results from supplementing rhTGF-β1 to TCPS and KOS hydrogels revealed that KOS seems to interact with TGF-β to a greater extent than TCPS. Western blot results revealed that latency TGFβ binding protein (LTBP-1) and latency associated peptide (LAP) had elevated levels early during differentiation. Further, the levels of LTBP-1 and LAP were higher on KOS differentiated hCSCs than TCPS hCSCs. This study reaffirms previous results of a VSM phenotype observed on KOS hydrogels, and provides convincing evidence for TGF-β1 inducing VSM differentiation on KOS hydrogels. Additionally, results from ELISA and western blot provide evidence that KOS plays a direct role in this pathway via interactions with TGF-β]1 and its associated proteins LTBP-1 and LAP. Results from chapter 2 and 3 offered significant evidence that our cells exhibited a VSMC phenotype, and that TGF-β1 signaling was a key contributor for the observed phenotype, but we still needed an animal model to explore the therapeutic potential of our putative VSMCs. Research in Chapter 4 investigated a disease model to test the ability of KOS hydrogel differentiated cells to regenerate vascular tissue. To measure vascular regenerative capability, we selected a murine model of critical limb ischemia (CLI). CLI was induced in 3 groups (n=15/group) of adult mixed gender NSG mice by excising the femoral artery and vein, and then treated the mice with either PBS (termed as PBS-treated), Cells differentiated on TCPS (termed as Cells from TCPS), or KOS hydrogel-derived VSMCs (termed as Cells from KOS). Blood perfusion of the hind limbs was measured immediately before and after surgery, then 14, and 28 days after surgery using Laser Doppler analysis. Tissue vascularization, cell engraftment, and skeletal muscle regeneration were measured using immunohistochemistry, 1,1'-Dioctadecyl3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiL) vessel painting, and hematoxylin and eosin (HandE) pathohistological staining. During the 4-week period, both cell treatment groups showed significant increases in blood perfusion compared to the PBS-treated control, and at day 28 the Cells from KOS group had significantly better blood flow than the Cells from TCPS group. Additionally, the Cells from KOS group demonstrated a significant increase in the ratio of DiL positive vessels, capillary density, and a greater density of small diameter arterioles compared to the PBS-treated group. Further, both cell-treated groups had similar levels of engraftment into the host tissue. We conclude that Cells from KOS therapy increases blood perfusion in an NSG model of CLI, but does not lead to increased cell engraftment compared to other cell based therapies. Overall, the results from this dissertation demonstrated that KOS hydrogels produce VSMC differentiation from c-kit+ hCSCs mediated by TGF-β1 signaling, and that the differentiated cells are able to increase blood perfusion in a CLI model by increasing capillary density, suggesting enhanced angiogenesis. Future studies should explore potential protein-protein interactions between KOS, TGF-β1 and its associated proteins. Additionally, we should plan animal studies that examine the efficacy of our cells to regenerate cardiac tissue following an acute myocardial infarction (AMI). / PHD

L'objectif de cette thèse était de développer et de caractériser un modèle de cellules souches cardiaques humaines dans un contexte de thérapie cellulaire. Après avoir sélectionné et caractérisé une population de cellules souches d'origine mésenchymateuse, isolée à partir d'auricules humaines, exprimant le marqueur W8B2 (CSCs W8B2+), nous nous sommes focalisés (par les techniques de RT-qPCR à haut rendement, d'immuno-marquage, de western-blot et de fluorescence calcique) sur ; 1. la caractérisation génique des canaux ioniques et des acteurs de la signalisation calcique et 2. l'étude de leur différenciation in vitro en parallèle à l'activité calcique intracellulaire. Les résultats montrent que CSCs W8B2+ tendent à se différencier en cellules pacemaker. Certains gènes spécifiques nodaux, comme Tbx3, HCN, ICaT,L, Kv, NCX, s'expriment durant la différenciation. L'enregistrement de l'activité calcique (via une sonde optogénétique) montre la présence d'oscillations calciques qui évoluent en fréquence et en intensité pendant la différenciation. Les stocks-IP3 sensibles et l'échangeur NCX joueraient un rôle fondamental.Nous avons ensuite étudié l'importance du canal BKCa et des récepteurs sphingosine 1-phosphate (S1P) dans la régulation des propriétés fondamentales des CSCs W8B2+. L'inhibition du BKCa diminue la prolifération cellulaire en accumulant les cellules à la phase G0/G1, réprime l'auto-renouvellement mais n'affecte pas la migration. Quant à la S1P elle freine la prolifération et l'auto-renouvellement via une voie différente de celles des récepteurs S1P1,2,3.Ce travail fait ressortir des cibles moléculaires fondamentales dans un contexte de thérapie cellulaire cardiaque. / The aim of this thesis was to develop and characterize a model of human heart stem cells in a context of cell therapy.A population of mesenchymal stem cells, expressing the W8B2 marker (CSCs W8B2+), was first isolated from human auricles and characterized using high-throughput RT-qPCR techniques, immuno-labeling, western-blot and calcium fluorescence imaging. These experiments were focused on 1. the gene expression of ion channels and calcium signaling proteins; and 2. the study of CSCs W8B2+ in vitro differentiation and associated intracellular calcium activity changes.The results show that CSCs W8B2+ tend to differentiate into pacemaker cells. Some nodal specific genes such as Tbx3, HCN, ICaT, L, Kv, NCX, are expressed during differentiation. The recording of calcium activity (via an optogenetic probe) shows the presence of calcium oscillations that change in frequency and intensity during differentiation. IP3 sensitive calcium stocks and the NCX exchanger would play a fundamental role in these variations.Then we studied the importance of the BKCa channel and the sphingosine 1-phosphate (S1P) receptors in the regulation of the fundamental properties of the W8B2+ CSCs. Inhibition of BKCa reduces cell proliferation by accumulating cells in the G0 / G1 phase, suppresses cell self-renewal but does not affect migration properties. Concerning S1P, it decreases proliferation and self-renewal without stimulate S1P1,2,3 receptors.This work highlights fundamental potential molecular targets in a context of cardiac cell therapy.