Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease.

Modeling and experimental methods to predict oxygen distribution in bone defects following cell transplantation.

We have developed a mathematical model that allows simulation of oxygen distribution in a bone defect as a tool to explore the likely effects of local changes in cell concentration, defect size or geometry, local oxygen delivery with oxygen-generating biomaterials (OGBs), and changes in the rate of oxygen consumption by cells within a defect. Experimental data for the oxygen release rate from an OGB and the oxygen consumption rate of a transplanted cell population are incorporated into the model. With these data, model simulations allow prediction of spatiotemporal oxygen concentration within a given defect and the sensitivity of oxygen tension to changes in critical variables. This information may help to minimize the number of experiments in animal models that determine the optimal combinations of cells, scaffolds, and OGBs in the design of current and future bone regeneration strategies. Bone marrow-derived nucleated cell data suggest that oxygen consumption is dependent on oxygen concentration. OGB oxygen release is shown to be a time-dependent function that must be measured for accurate simulation. Simulations quantify the dependency of oxygen gradients in an avascular defect on cell concentration, cell oxygen consumption rate, OGB oxygen generation rate, and OGB geometry.

Slowing the Onset of Hypoxia Increases Colony Forming Efficiency of Connective Tissue Progenitor Cells In Vitro.

BACKGROUND: Survival and colony formation by transplanted tissue derived connective tissue progenitor cells (CTPs) are thought to be important factors in the success of clinical tissue engineering strategies for bone regeneration. Transplantation of cells into defects larger than a few millimeters expose cells to a profoundly hypoxic environment. This study tested the hypothesis that delaying the onset of hypoxia will improve the survival and performance of CTPs in vitro. METHODS: To mimic declines seen in an avascular in vivo bone defect, colony forming efficiency by marrow derived nucleated cells was assessed under osteogenic conditions. Variation in the rate of oxygen decline from an oxygen tension of 21% to 0.1% oxygen was explored using an incubator with programmable active control of gas concentrations. The effect of doping cultures with defined concentrations of RBCs was also used to evaluate the potential for RBCs to serve as a natural buffer in the setting of declining oxygen levels. RESULTS: A delay in onset of hypoxia over 96 hours resulted in a 3-fold increase in the relative colony forming efficiency (rCFE) of CTPs as compared to an immediate onset of hypoxia. The presence of RBCs in vitro inhibited the rCFE of CTPs. Given the negative effects of RBCs, methods of RBC removal were evaluated and compared for their effectiveness of RBC removal and retention of colony forming efficiency. CONCLUSIONS: These data suggest that conditions of hypoxia compromise colony forming efficiency in marrow derived CTPs. However, slowing the rate of decline of oxygen preserved colony forming efficiency at levels achieved in a stable normoxic (3% O2) environment. These data also suggest that RBCs are detrimental to the rCFE of CTPs and that buffy coat is an effective and preferred method for removing RBCs from marrow aspirates while preserving CTPs. These findings may inform clinical strategies for CTP transplantation.