Multiplexed Optical Sensors in Arrayed Islands of Cells for multimodal recordings of cellular physiology.

Cells typically respond to chemical or physical perturbations via complex signaling cascades which can simultaneously affect multiple physiological parameters, such as membrane voltage, calcium, pH, and redox potential. Protein-based fluorescent sensors can report many of these parameters, but spectral overlap prevents more than ~4 modalities from being recorded in parallel. Here we introduce the technique, MOSAIC, Multiplexed Optical Sensors in Arrayed Islands of Cells, where patterning of fluorescent sensor-encoding lentiviral vectors with a microarray printer enables parallel recording of multiple modalities. We demonstrate simultaneous recordings from 20 sensors in parallel in human embryonic kidney (HEK293) cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and we describe responses to metabolic and pharmacological perturbations. Together, these results show that MOSAIC can provide rich multi-modal data on complex physiological responses in multiple cell types.

Scaffold Composition Determines the Angiogenic Outcome of Cell-Based Vascular Endothelial Growth Factor Expression by Modulating Its Microenvironmental Distribution.

Delivery of genetically modified cells overexpressing Vascular Endothelial Growth Factor (VEGF) is a promising approach to induce therapeutic angiogenesis in ischemic tissues. The effect of the protein is strictly modulated by its interaction with the components of the extracellular matrix. Its therapeutic potential depends on a sustained but controlled release at the microenvironmental level in order to avoid the formation of abnormal blood vessels. In this study, it is hypothesized that the composition of the scaffold plays a key role in modulating the binding, hence the therapeutic effect, of the VEGF released by 3D-cell constructs. It is found that collagen sponges, which poorly bind VEGF, prevent the formation of localized hot spots of excessive concentration, therefore, precluding the development of aberrant angiogenesis despite uncontrolled expression by a genetically engineered population of adipose tissue-derived stromal cells. On the contrary, after seeding on VEGF-binding egg-white scaffolds, the same cell population caused aberrantly enlarged vascular structures after 14 d. Collagen-based engineered tissues also induced a safe and efficient angiogenesis in both the patch itself and the underlying myocardium in rat models. These findings open new perspectives on the control and the delivery of proangiogenic stimuli, and are fundamental for the vascularization of engineered tissues/organs.

Engineered mesenchymal cell-based patches as controlled VEGF delivery systems to induce extrinsic angiogenesis.

UNLABELLED: Therapeutic over-expression of Vascular Endothelial Growth Factor (VEGF) by transduced progenitors is a promising strategy to efficiently induce angiogenesis in ischemic tissues (e.g. limb muscle and myocardium), but tight control over the micro-environmental distribution of the dose is required to avoid induction of angioma-like tumors. Therapeutic VEGF release was achieved by purified transduced adipose mesenchymal stromal cells (ASC) that homogeneously produce specific VEGF levels, inducing only normal angiogenesis after injection in non-ischemic tissues. However, the therapeutic potential of this approach mostly in the cardiac field is limited by the poor cell survival and the restricted area of effect confined to the cell-injection site. The implantation of cells previously organized in vitro in 3D engineered tissues could overcome these issues. Here we hypothesized that collagen sponge-based construct (patch), generated by ASC expressing controlled VEGF levels, can function as delivery device to induce angiogenesis in surrounding areas (extrinsic vascularization). A 7-mm-thick acellular collagen scaffold (empty), sutured beneath the patch, provided a controlled and reproducible model to clearly investigate the ongoing angiogenesis in subcutaneous mice pockets. VEGF-expressing ASC significantly increased the capillary in-growth inside both the patch itself and the empty scaffold compared to naïve cells, leading to significantly improved survival of implanted cells. These data suggest that this strategy confers control (i) on angiogenesis efficacy and safety by means of ASC expressing therapeutic VEGF levels and (ii) over the treated area through the specific localization in an engineered collagen sponge-based patch. STATEMENT OF SIGNIFICANCE: Development of efficient pro-angiogenic therapies to restore the micro-vascularization in ischemic tissues is still an open issue. Although extensively investigated, the promising approach based on injections of progenitors transduced to over-express Vascular Endothelial Growth Factor (VEGF) has still several limitations: (i) need of a tight control over the microenvironmental VEGF dose to avoid angioma-like tumor growth; (ii) poor implanted cell survival; (iii) effect area restricted mainly to the injection sites. Here, we aimed to overcome these drawbacks by generating a novel cell-based controlled VEGF delivery device. In particular, transduced mesenchymal cells, purified to release a sustained, safe and efficient VEGF dose, were organized in three-dimensional engineered tissues to improve cell survival and provide a uniform vascularization throughout both the mm-thick implanted constructs themselves and the surrounding area.

A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.

Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

Disease modeling and phenotypic drug screening for diabetic cardiomyopathy using human induced pluripotent stem cells.

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

Controlled angiogenesis in the heart by cell-based expression of specific vascular endothelial growth factor levels.

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

Cell and gene therapy approaches for cardiac vascularization.

Despite encouraging preclinical results for therapeutic angiogenesis in ischemia, a suitable approach providing sustained, safe and efficacious vascular growth in the heart is still lacking. Vascular Endothelial Growth Factor (VEGF) is the master regulator of angiogenesis, but it also can easily induce aberrant and dysfunctional vascular growth if its expression is not tightly controlled. Control of the released level in the microenvironment around each cell in vivo and its distribution in tissue are critical to induce stable and functional vessels for therapeutic angiogenesis. The present review discusses the limitations and perspectives of VEGF gene therapy and of different cell-based approaches for the implementation of therapeutic angiogenesis in the treatment of cardiac ischemia.