Cellular therapy to treat heart failure is an ongoing focus of intense research, but progress toward structural and functional recovery remains modest. Engineered augmentation of established cellular effectors overcomes impediments to enhance reparative activity. Such 'next generation' implementation includes delivery of combinatorial cell populations exerting synergistic effects. Concurrent isolation and expansion of three distinct cardiac-derived interstitial cell types from human heart tissue, previously reported by our group, prompted design of a 3D structure that maximizes cellular interaction, allows for defined cell ratios, controls size, enables injectability, and minimizes cell loss. Herein, mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs) and c-Kit + cardiac interstitial cells (cCICs) when cultured together spontaneously form scaffold-free 3D microenvironments termed Cardi-oClusters. scRNA-Seq profiling reveals CardioCluster expression of stem cell-relevant factors, adhesion/extracellular-matrix molecules, and cytokines, while maintaining a more native transcriptome similar to endogenous cardiac cells. CardioCluster intramyocardial delivery improves cell retention and capillary density with preservation of cardiomyocyte size and long-term cardiac function in a murine infarction model followed 20 weeks. CardioCluster utilization in this preclinical setting establish fundamental insights, laying the framework for optimization in cell-based therapeutics intended to mitigate cardiomyopathic damage.

1Background: Cellular therapy to treat heart failure is an ongoing focus of intense 2 research and development, but progress has been frustratingly slow due to limitations of 3 current approaches. Engineered augmentation of established cellular effectors 4 overcomes impediments, enhancing reparative activity with improved outcomes relative 5 to conventional techniques. Such 'next generation' implementation includes delivery of 6 combinatorial cell populations exerting synergistic effects. Concurrent isolation and 7 expansion of three distinct cardiac-derived interstitial cell types from human heart tissue, 8 as previously reported by our group, prompted design of a three-dimensional (3D) 9 structure that maximizes cellular interaction, allows for defined cell ratios, controls size, 10 enables injectability, and minimizes cell losses upon delivery. 11 Methods: Three distinct populations of human cardiac interstitial cells including 12 mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and c-Kit + cardiac 13 interstitial cells (cCICs) when cultured together spontaneously form scaffold-free 3D 14 microenvironments termed CardioClusters. Biological consequences of CardioCluster 15 formation were assessed by multiple assays including single cells RNA-Seq 16 transcriptional profiling. Protective effects of CardioClusters in vitro were measured 17 using cell culture models for oxidative stress and myocardial ischemia in combination 18 with freshly isolated neonatal rat ventricular myocytes. Long-term impact of adoptively 19 transferred CardioClusters upon myocardial structure and function in a xenogenic model 20 of acute infarction using NOD scid mice was assessed over a longitudinal time course of 21 20-weeks. 22 Results: CardioCluster design enables control over composite cell types, cell ratios, 1 size, and preservation of structural integrity during delivery. Profound changes for 2 biological properties of CardioClusters relative to constituent parental cell populations 3 include enhanced expression of stem cell-relevant factors, adhesion/extracellular-matrix 4 molecules, and cytokines. The CardioCluster 3D microenvironment maximizes cellular 5 interaction while maintaining a more native transcriptome similar to endogenous cardiac 6 cells. CardioCluster delivery improves cell retention following intramyocardial injection 7 with preservation of long-term cardiac function relative to monolayer-cultured cells when 8 tested in an experimental murine infarction model followed for up to 20 weeks post-9 challenge. CardioCluster-treated hearts show increases in capillary density, 10 preservation of cardiomyocyte size, and reduced scar size indicative of blunting 11 pathologic infarction injury. 12 Conclusions: CardioClusters are a novel 'next generation' development and delivery 13 approach for cellular therapeutics that potentiate beneficial activity and enhance 14 protective effects of human cardiac interstitial cell mixed populations. CardioClusters 15 utilization in this preclinical setting establishes ...

No abstract