A standardized method to purify cardiomyocytes from individual mouse hearts of any age

Rationale: Primary cardiomyocytes are invaluable for understanding postnatal heart development and elucidating disease mechanisms in genetic and pharmacological models, however, a method to obtain freshly purified cardiomyocytes at any postnatal age, without using different age-dependent isolation procedures and cell culture, is lacking. Objective: To develop a standardized method that allows rapid isolation and purification of cardiomyocytes in high yield and viability from individual neonatal, infant, and adult mice. Methods and Results: Hearts of C57BL/6J mice were cannulated using a novel in situ aortic cannulation procedure optimized to allow cannulation of even the very small vessel of neonates (postnatal day 0-2, P0-2). Hearts were then subjected to Langendorff retrograde perfusion and enzymatic digestion. Cardiomyocytes were isolated after subsequent tissue disaggregation and filtration, in high yield (1.56-2.2x106 cardiomyocytes/heart) and viability (~70-100%). The larger size of infant (P10 and P13) and adult (P70), but not neonatal, cardiomyocytes relative to non-myocytes, allowed enrichment by differential centrifugation. Cardiomyocytes from all ages were further purified by immunomagnetic bead-based depletion of non-myocytes. Together, these procedures resulted in the isolation of highly purified cardiomyocytes (~94%) within 1 hour, enabling experiments using individual replicates. For example, RNA-sequencing of cardiomyocytes purified from one P2 male and female heart per litter (n=4 litters) showed distinct clustering by litters and sex differences for nine differentially expressed genes (FDR<0.005). In situ fixation via coronary perfusion, performed immediately after tissue digestion, preserved the cytoarchitecture of isolated cardiomyocytes (yielding ~94% rod-shaped cardiomyocytes at all ages), allowing capture of spindle-shaped neonatal cells undergoing mitosis, as well as enabling accurate quantitation of cardiomyocyte area and nucleation state. Conclusion: The procedures developed here provide a universal protocol for the rapid isolation and purification of high-quality cardiomyocytes from hearts of any postnatal age, even those of neonates, thereby enabling direct comparisons between individual hearts.

CD31 Positive-Extracellular Vesicles from Patients with Type 2 Diabetes Shuttle a miRNA Signature Associated with Cardiovascular Complications

Innovative biomarkers are needed to improve the management of patients with type 2 diabetes mellitus (T2DM). Blood circulating miRNAs have been proposed as a potential tool to detect T2DM complications but the lack of tissue specificity, among other reasons, has hampered their translation to clinical settings. Extracellular vesicle (EV)-shuttled miRNAs have been proposed as an alternative approach. Here, we adapted an immunomagnetic bead-based method to isolate plasma CD31 positive (⁺) EVs to harvest vesicles deriving from tissues relevant for T2DM complications. Surface marker characterization showed that CD31⁺ EVs were also positive for a range of markers typical of both platelets and activated endothelial cells. After characterization, we quantified 11 candidate miRNAs associated with vascular performance and shuttled by CD31⁺EVs in a large (n=218), cross-sectional cohort of patients categorized as T2DM without complications, T2DM with complications, and controls. We found that 10 of the tested miRNAs are affected by T2DM, while the signature composed by miR-146a, -320a, -422a, -451a efficiently identified T2DM patients with complications. Furthermore, another CD31⁺EV-shuttled miRNA signature, i.e. miR-155, -320a, -342-3p, -376, and -422a, detected T2DM patients with a previous major adverse cardiovascular event. Many of these miRNAs significantly correlate with clinical variables held to play a key role in the development of complications. In addition, we show that CD31⁺ EVs from patients with T2DM are able to promote the expression of selected inflammatory mRNAs, <i>i.e.</i> <i>CCL2</i>, <i>IL-1α</i>, and <i>TNFα</i>, when administered to endothelial cells <i>in vitro</i>. Overall, these data suggest that the miRNA cargo of plasma CD31⁺ EVs is largely affected by T2DM and related complications, <a>encouraging further research to explore the diagnostic potential and the functional role of these alterations. </a>

CD31 Positive-Extracellular Vesicles from Patients with Type 2 Diabetes Shuttle a miRNA Signature Associated with Cardiovascular Complications

Innovative biomarkers are needed to improve the management of patients with type 2 diabetes mellitus (T2DM). Blood circulating miRNAs have been proposed as a potential tool to detect T2DM complications but the lack of tissue specificity, among other reasons, has hampered their translation to clinical settings. Extracellular vesicle (EV)-shuttled miRNAs have been proposed as an alternative approach. Here, we adapted an immunomagnetic bead-based method to isolate plasma CD31 positive (⁺) EVs to harvest vesicles deriving from tissues relevant for T2DM complications. Surface marker characterization showed that CD31⁺ EVs were also positive for a range of markers typical of both platelets and activated endothelial cells. After characterization, we quantified 11 candidate miRNAs associated with vascular performance and shuttled by CD31⁺EVs in a large (n=218), cross-sectional cohort of patients categorized as T2DM without complications, T2DM with complications, and controls. We found that 10 of the tested miRNAs are affected by T2DM, while the signature composed by miR-146a, -320a, -422a, -451a efficiently identified T2DM patients with complications. Furthermore, another CD31⁺EV-shuttled miRNA signature, i.e. miR-155, -320a, -342-3p, -376, and -422a, detected T2DM patients with a previous major adverse cardiovascular event. Many of these miRNAs significantly correlate with clinical variables held to play a key role in the development of complications. In addition, we show that CD31⁺ EVs from patients with T2DM are able to promote the expression of selected inflammatory mRNAs, <i>i.e.</i> <i>CCL2</i>, <i>IL-1α</i>, and <i>TNFα</i>, when administered to endothelial cells <i>in vitro</i>. Overall, these data suggest that the miRNA cargo of plasma CD31⁺ EVs is largely affected by T2DM and related complications, <a>encouraging further research to explore the diagnostic potential and the functional role of these alterations. </a>

Abstract 455: The Roles of VEGF Receptors in Human Cardiac Progenitor Cell Contribution to New Vascular Formation

Background: Human cardiac progenitor cells (CPCs) have been shown to play a valuable role in myocardial tissue maintenance, including their ability to develop into endothelial and vascular smooth muscle cells. The vascular endothelial growth factor (VEGF) ligand family has been identified as essential for angiogenesis. VEGF receptors (VEGFRs) comprise three main subtypes, VEGFRs1,2,3; our prior data identified CPC expression of both VEGFRs and of pro-angiogenic secreted growth factors. Hypothesis: Human CPCs utilise VEGFR signalling to potentiate CPC-driven angiogenesis, directly via CPC differentiation and indirectly via secrotome. Methods: Human adult myocardial tissue samples were collected during cardiac surgery and c-Kit-positive (c-Kit + ), CD45-negative (CD45 - ) CPCs isolated by immunomagnetic bead sorting, with five CPC lines generated from individual-donor samples. The c-Kit + / CD45 - CPC population was then characterised by clonogenicity assay, immunocytochemistry (ICC), and real-time RT-qPCR. Human CPC lines were FACS sorted into 3 lineage groups: endothelial (CD31 + ), smooth muscle (CD91 + /CD140b + /CD31 - ) and uncommitted (CD91 - /CD140b - /CD31 - ). Expression of VEGFRs and markers (SDF1; TGF-β) in CPC sub-populations was assessed by: qPCR; Western blot; ICC. Impact on signal transduction by VEGF-A stimulation was identified by Western blot and ICC. Results: Human CPCs were sorted into populations of: endothelial linage CD31 + (1.26% of total cells), smooth muscle lineage CD91 + /CD140b + /CD31 - (13.77%) and CD91 - /CD140b - /CD31 - (31.28%) cells. Analysis of gene expression identified VEGFRs 1, 2 and 3 in all three sub-populations, but only VEGFR1 expression was confirmed at protein level, seen in all three sub-populations. High expression levels of growth factors secreted by CPCs (SDF, TGF-β, VEGFs, FGF-2) were identified in human CPCs, also in all three sub-populations. Conclusion: Human CPC lines were isolated and analysed in bulk and sub-populations, identifying VEGFR1 expression at both gene and protein levels, but not VEGFR2 and VEGFR3. Our further work will identify signalling pathways in human CPCs linked to VEGF-A stimulation, along with the impact of VEFG-A stimulation on CPC secretome and linked angiogenic potential.

Integrated Immunomagnetic Bead-Based Microfluidic Chip for Exosomes Isolation

Exosomes are essential early biomarkers for health monitoring and cancer diagnosis. A prerequisite for further investigation of exosomes is the isolation, which is technically challenging due to the complexity of body fluids. This paper presents the development of an integrated microfluidic chip for exosomes isolation, which combines the traditional immunomagnetic bead-based protocol and the recently emerging microfluidic approach, resulting in benefits from both the high-purity of the former and the automated continuous superiority of the latter. The chip was designed based on an S-shaped micromixer with embedded baffle. The excellent mixing efficiency of this micromixer compared with Y-shaped and S-shaped micromixers was verified by simulation and experiments. The photolithography technique was employed to fabricate the integrated microfluidic chip, and the manufacturing process was elucidated. We finally established an experimental platform for exosomes isolation with the fabricated microfluidic chip built in. Exosomes isolation experiments were conducted using this platform. The distribution and morphology of the isolated exosomes were observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Quantitative size analyses based on transmission electron micrographs indicated that most of the obtained particles were between 30 and 150 nm. Western blot analyses of the isolated exosomes and the serum were conducted to verify the platform’s capability of isolating a certain subpopulation of exosomes corresponding to specified protein markers (CD63). The complete time for isolation of 150 μL serum samples was approximately 50 min, which was highly competitive with the reported existing protocols. Experimental results proved the capacity of the established integrated microfluidic chip for exosomes isolation with high purity, high integrity, and excellent efficiency. The platform can be further developed to make it possible for practical use in clinical applications as a universal exosomes isolation and characterization tool.