Stem or progenitor cell populations are often established in unique niche microenvironments that regulate cell fate decisions. Although niches have been shown to be critical for the normal development of several tissues, their role in the cardiovascular system is poorly understood. In this study, we characterized the cardiovascular progenitor cell (CPC) niche in developing human and mouse hearts, identifying signaling pathways and extracellular matrix (ECM) proteins that are crucial for CPC maintenance and expansion. We demonstrate that collagen IV (ColIV) and β-catenin-dependent signaling are essential for maintaining and expanding undifferentiated CPCs. Since niches are three-dimensional (3D) structures, we investigated the impact of a 3D microenvironment that mimics the in vivo niche ECM. Employing electrospinning technologies, 3D in vitro niche substrates were bioengineered to serve as culture inserts. The threedimensionality of these structures increased mouse embryonic stem cell differentiation into CPCs when compared to 2D control cultures, which was further enhanced by incorporation of ColIV into the substrates. Inhibiting p300-dependent β-catenin signals with the small molecule IQ1 facilitated further expansion of CPCs. Our study represents an innovative approach to bioengineer

While stem cell niches in vivo are complex three-dimensional (3D) microenvironments, the relationship between the dimensionality of the niche to its function is unknown. We have created a 3D microenvironment through electrospinning to study the impact of geometry and different extracellular proteins on the development of cardiac progenitor cells (Flk-1(+)) from resident stem cells and their differentiation into functional cardiovascular cells. We have investigated the effect of collagen IV, fibronectin, laminin and vitronectin on the adhesion and proliferation of murine ES cells as well as the effects of these proteins on the number of Flk-1(+) cells cultured in 2D conditions compared to 3D system in a feeder free condition. We found that the number of Flk-1(+) cells was significantly higher in 3D scaffolds coated with laminin or vitronectin compared to colIV-coated scaffolds. Our results show the importance of defined culture systems in vitro for studying the guided differentiation of pluripotent embryonic stem cells in the field of cardiovascular tissue engineering and regenerative medicine.

Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers—hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1—at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes.

Synthetic polymers or naturally-derived extracellular matrix (ECM) proteins have been used to create tissue engineering scaffolds; however, the need for surface modification in order to achieve polymer biocompatibility and the lack of biomechanical strength of constructs built using proteins alone remain major limitations. To overcome these obstacles, we developed novel hybrid constructs composed of both strong biosynthetic materials and natural human ECM proteins. Taking advantage of the ability of cells to produce their own ECM, human foreskin fibroblasts were grown on silicon-based nanostructures exhibiting various surface topographies that significantly enhanced ECM protein production. After 4 weeks, cell-derived sheets were harvested and histology, immunochemistry, biochemistry and multiphoton imaging revealed the presence of collagens, tropoelastin, fibronectin and glycosaminoglycans. Following decellularization, purified sheet-derived ECM proteins were mixed with poly(ε-caprolactone) to create fibrous scaffolds using electrospinning. These hybrid scaffolds exhibited excellent biomechanical properties with fiber and pore sizes that allowed attachment and migration of adipose tissue-derived stem cells. Our study represents an innovative approach to generate strong, non-cytotoxic scaffolds that could have broad applications in tissue regeneration strategies.

T cells are the predominant immune cell population in the pancreatic tumor microenvironment. High CD8+ and Th1-polarized CD4+ T cell infiltration is associated with prolonged survival in human pancreatic ductal adenocarcinoma (PDAC). However, the expression pattern of co-stimulatory and inhibitory receptors by PDAC-infiltrating T cells and their prognostic significance are not well defined. In this study, we employed multiplex immunofluorescence to investigate the intratumoral expression of the co-stimulatory receptor inducible T-cell co-stimulator (ICOS), the inhibitory receptors lymphocyte-activation gene 3 (LAG-3), programmed death 1 (PD-1), and V-domain immunoglobulin suppressor of T cell activation (VISTA) by tumor-infiltrating T cells (CD3) in a cohort of 69 patients with resected PDAC. T cells were enriched particularly within the stromal area and were highly heterogeneous across tumors. Further, T cells were associated with prolonged disease-free survival (DFS). However, LAG-3 expression by PDAC-infiltrating T cells was correlated with reduced DFS. Our study highlights the biological importance of LAG-3 expression by tumor-infiltrating T cells. LAG-3+ T cells may represent a novel prognostic marker and a particularly attractive target for immunotherapeutic strategies in PDAC.