Yamada, Satsuki

Abstract Cardiopoiesis-primed human stem cells exert sustained benefit in treating heart failure despite limited retention following myocardial delivery. To assess potential paracrine contribution, the secretome of cardiopoiesis conditioned versus naïve human mesenchymal stromal cells was decoded by directed proteomics augmented with machine learning and systems interrogation. Cardiopoiesis doubled cellular protein output generating a distinct secretome that segregated the conditioned state. Altering the expression of 1035 secreted proteins, cardiopoiesis reshaped the secretome across functional classes. The resolved differential cardiopoietic secretome was enriched in mesoderm development and cardiac progenitor signaling processes, yielding a cardiovasculogenic profile bolstered by upregulated cardiogenic proteins. In tandem, cardiopoiesis enhanced the secretion of immunomodulatory proteins associated with cytokine signaling, leukocyte migration, and chemotaxis. Network analysis integrated the differential secretome within an interactome of 1745 molecules featuring prioritized regenerative processes. Secretome contribution to the repair signature of cardiopoietic cell-treated infarcted hearts was assessed in a murine coronary ligation model. Intramyocardial delivery of cardiopoietic cells improved the performance of failing hearts, with undirected proteomics revealing 50 myocardial proteins responsive to cell therapy. Pathway analysis linked the secretome to cardiac proteome remodeling, pinpointing 17 cardiopoiesis-upregulated secretome proteins directly upstream of 44% of the cell therapy-responsive cardiac proteome. Knockout, in silico, of this 22-protein secretome-dependent myocardial ensemble eliminated indices of the repair signature. Accordingly, in vivo, cell therapy rendered the secretome-dependent myocardial proteome of an infarcted heart indiscernible from healthy counterparts. Thus, the secretagogue effect of cardiopoiesis transforms the human stem cell secretome, endows regenerative competency, and upregulates candidate paracrine effectors of cell therapy-mediated molecular restitution.

Background The purpose of this study was to investigate a therapeutic approach targeting the inflammatory response and consequent remodeling from ischemic myocardial injury. Methods and Results Coronary thrombus aspirates were collected from patients at the time of ST‐segment–elevation myocardial infarction and subjected to array‐based proteome analysis. Clinically indistinguishable at myocardial infarction (MI), patients were stratified into vulnerable and resilient on the basis of 1‐year left ventricular ejection fraction and death. Network analysis from coronary aspirates revealed prioritization of tumor necrosis factor‐α signaling in patients with worse clinical outcomes. Infliximab, a tumor necrosis factor‐α inhibitor, was infused intravenously at reperfusion in a porcine MI model to assess whether infliximab‐mediated immune modulation impacts post‐MI injury. At 3 days after MI (n=7), infliximab infusion increased proregenerative M2 macrophages in the myocardial border zone as quantified by immunofluorescence (24.1%±23.3% in infliximab versus 9.29%±8.7% in sham; P <0.01). Concomitantly, immunoassays of coronary sinus samples quantified lower troponin I levels (41.72±7.34 pg/mL versus 58.11±10.75 pg/mL; P <0.05) and secreted protein analysis revealed upregulation of injury‐modifying interleukin‐2, ‐4, ‐10, ‐12, and ‐18 cytokines in the infliximab‐treated cohort. At 4 weeks (n=12), infliximab treatment resulted in significant protective influence, improving left ventricular ejection fraction (53.9%±5.4% versus 36.2%±5.3%; P <0.001) and reducing scar size (8.31%±10.9% versus 17.41%±12.5%; P <0.05). Conclusions Profiling of coronary thrombus aspirates in patients with ST‐segment–elevation MI revealed highest association for tumor necrosis factor‐α in injury risk. Infliximab‐mediated immune modulation offers an actionable pathway to alter MI‐induced inflammatory response, preserving contractility and limiting adverse structural remodeling.

Abstract Patients with heart failure experience limitations in daily activity and poor quality-of-life. Prospective surveillance of health-related quality-of-life supplemented traditional death and hospitalization outcomes in the multinational, randomized, double-blinded CHART-1 clinical trial that assessed cardiopoiesis-guided cell therapy in ischemic heart failure patients with reduced left ventricular ejection fraction. The Minnesota Living with Heart Failure Questionnaire (MLHFQ), a Food and Drug Administration qualified instrument for evaluating therapeutic effectiveness, was applied through the 1-year follow-up. Cell treated (n = 109) and sham procedure (n = 140) cohorts reported improved MLHFQ scores comparable between the 2 study arms (mean treatment difference with baseline adjustment −3.2 points, P = .107). Superiority of cell treatment over sham in betterment of the MLHFQ score was demonstrated in patients with pre-existing advanced left ventricular enlargement (baseline-adjusted mean treatment difference −6.4 points, P = .009). In this highly responsive subpopulation, benefit on the MLHFQ score paralleled reduction in death and hospitalization post-cell therapy (adjusted Mann-Whitney odds 1.43, 95% CI, 1.01-2.01; P = .039). The potential of cell therapy in addressing the quality-of-life dimension of heart failure requires further evaluation for disease relief.

Heart failure pathobiology is permissive to reparative intent. Regenerative therapies exemplify an emerging disruptive innovation aimed at achieving structural and functional organ restitution. However, mixed outcomes, complexity in use, and unsustainable cost have curtailed broader adoption, mandating the development of novel cardio-regenerative approaches. Lineage guidance offers a standardized path to customize stem cell fitness for therapy. A case in point is the molecular induction of the cardiopoiesis program in adult stem cells to yield cardiopoietic cell derivatives designed for heart failure treatment. Tested in early and advanced clinical trials in patients with ischemic heart failure, clinical grade cardiopoietic cells were safe and revealed therapeutic improvement within a window of treatment intensity and pre-treatment disease severity. With the prospect of mass customization, cardiopoietic guidance has been streamlined from the demanding, recombinant protein cocktail-based to a protein-free, messenger RNA-based single gene protocol to engineer affordable cardiac repair competent cells. Clinical trial biobanked stem cells enabled a systems biology deconvolution of the cardiopoietic cell secretome linked to therapeutic benefit, exposing a paracrine mode of action. Collectively, this new knowledge informs next generation regenerative therapeutics manufactured as engineered cellular or secretome mimicking cell-free platforms. Launching biotherapeutics tailored for optimal outcome and offered at mass production cost would contribute to advancing equitable regenerative care that addresses population health needs.

Abstract Stem cell paracrine activity is implicated in cardiac repair. Linkage between secretome functionality and therapeutic outcome was here interrogated by systems analytics of biobanked human cardiopoietic cells, a regenerative biologic in advanced clinical trials. Protein chip array identified 155 proteins differentially secreted by cardiopoietic cells with clinical benefit, expanded into a 520 node network, collectively revealing inherent vasculogenic properties along with cardiac and smooth muscle differentiation and development. Next generation RNA sequencing, refined by pathway analysis, pinpointed miR-146 dependent regulation upstream of the decoded secretome. Intracellular and extracellular integration unmasked commonality across cardio-vasculogenic processes. Mirroring the secretome pattern, infarcted hearts benefiting from cardiopoietic cell therapy restored the disease proteome engaging cardiovascular system functions. The cardiopoietic cell secretome thus confers a therapeutic molecular imprint on recipient hearts, with response informed by predictive systems profiling.

Abstract To optimize the regenerative proficiency of stem cells, a cardiopoietic protein-based cocktail consisting of multiple growth factors has been developed and advanced into clinical trials for treatment of ischemic heart failure. Streamlining the inductors of cardiopoiesis would address the resource intensive nature of the current stem cell enhancement protocol. To this end, the microencapsulated-modified-mRNA (M3RNA) technique was here applied to introduce early cardiogenic genes into human adipose-derived mesenchymal stem cells (AMSCs). A single mesodermal transcription factor, Brachyury, was sufficient to trigger high expression of cardiopoietic markers, Nkx2.5 and Mef2c. Engineered cardiopoietic stem cells (eCP) featured a transcriptome profile distinct from pre-engineered AMSCs. In vitro, eCP demonstrated protective antioxidant capacity with enhanced superoxide dismutase expression and activity; a vasculogenic secretome driving angiogenic tube formation; and macrophage polarizing immunomodulatory properties. In vivo, in a murine model of myocardial infarction, intramyocardial delivery of eCP (600 000 cells per heart) improved cardiac performance and protected against decompensated heart failure. Thus, heart repair competent stem cells, armed with antioxidant, vasculogenic, and immunomodulatory traits, are here engineered through a protein-independent single gene manipulation, expanding the available regenerative toolkit.

Abstract Heritable mitochondrial DNA (mtDNA) mutations are common, yet only a few recurring pathogenic mtDNA variants account for the majority of known familial cases in humans. Purifying selection in the female germline is thought to be responsible for the elimination of most harmful mtDNA mutations during oogenesis. Here we show that deleterious mtDNA mutations are abundant in ovulated mature mouse oocytes and preimplantation embryos recovered from PolG mutator females but not in their live offspring. This implies that purifying selection acts not in the maternal germline per se, but during post-implantation development. We further show that oocyte mtDNA mutations can be captured and stably maintained in embryonic stem cells and then reintroduced into chimeras, thereby allowing examination of the effects of specific mutations on fetal and postnatal development.