Rationale The relative actions and synergism between distinct myocardial-derived stem cell populations remains obscure. Ongoing debates regarding optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Objective Establish a robust cardiac stem cell isolation and culture protocol to consistently generate three distinct stem cell populations from a single human heart biopsy. Methods and Results Isolation of three endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device (LVAD). Tissue explants were mechanically minced into 1 mm3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes (CMs) and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting (MACS) technology. Initial sorting is based upon c-Kit expression that enriches for two c-kit+ cell populations yielding a mixture of cardiac progenitor cells (CPCs) and endothelial progenitor cells (EPCs). Flow through c-Kit− mesenchymal stem cells (MSCs) are positively selected by surface expression of markers CD90 and CD105. After one week of culture the c-Kit+ population is further enriched by selection for a CD133+ EPC population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Conclusions Three distinct cardiac cell populations with individualized phenotypic properties consistent with CPCs, EPCs and MSCs can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients.

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 ...

Existing approaches to modify stem cells for myocardial regeneration desperately need innovative solutions that enhance cell engraftment and persistence. Although this deficiency has been attacked through combinatorial stem cell delivery, there is no evidence that these stem cell injections provide for direct cellular crosstalk to promote stem cell survival and proliferation. Therefore, we created a CardioCluster, a 3D microenvironment consisting of three defined cell populations from the human heart: c-kit + cardiac progenitor cells (CPCs), CD90 + /CD105 + mesenchymal stem cells (MSCs) and CD133 + endothelial progenitor cells (EPCs). The size of the CardioCluster can be controlled by the quantity of cells used to create the cluster, allowing them to be infused into the heart without being reduced to single cell suspensions as is the case for cardiosphere-derived cells where the structural and cell-cell contact information is lost when delivered. Unlike cardiospheres, these cardiac cells are combined into a rationally designed cluster with MSCs and CPCs in the central core and EPCs forming the outer layer. EPCs play a vital role in forming neovasculature that will connect the CardioClusters to living heart tissue not damaged by ischemia and allow for revascularization of the damaged myocardium. In vitro we have shown that EPCs are better able to form tubular networks on matrigel-coated plates compared to either CPCs or MSCs. MSCs reinforce the 3D structure by releasing growth factors that attract and maintain cells within the cluster, as well as release immunomodulatory signaling factors. Upon induction of an oxidative stress by hydrogen peroxide CardioClusters show improved cell survival with a lower percentage of apoptotic and/or necrotic cell populations compared to the three populations individually. Upon myocardial injection CardioClusters have been shown to maintain their 3D structural integrity. Future directions will be to assess long-term engraftment and regenerative potential in a mouse model of myocardial infarction.