Differentiation, Metabolism, and Cardioprotective Secretory Functions of Human Cardiac Stromal Cells from Ischemic and Endocarditis Patients.

This study investigates the characteristics of cardiac mesenchymal stem cell-like cells (CMSCLCs) isolated from the right atrial appendage of human donors with ischemia and a young patient with endocarditis (NE-CMSCLCs). Typical CMSCLCs from ischemic heart patients were derived from coronary artery bypass grafting procedures and compared against bone marrow mesenchymal stromal cells (BM-MSCs). NE-CMSCLCs had a normal immunophenotype, but exhibited enhanced osteogenic differentiation potential, rapid proliferation, reduced senescence, reduced glycolysis, and lower reactive oxygen species generation after oxidative stress compared with typical ischemic CMSCLCs. These differences suggest a unique functional status of NE-CMSCLCs, influenced by the donor health condition. Despite large variances in their paracrine secretome, NE-CMSCLCs retained therapeutic potential, as indicated by their ability to protect hypoxia/reoxygenation-injured human cardiomyocytes, albeit less effectively than typical CMSCLCs. This research describes a unique cell phenotype and underscores the importance of donor health status in the therapeutic efficacy of autologous cardiac cell therapy.

Engineering stable and non-immunogenic immunoenzymes for cancer therapy via in situ generated prodrugs.

Engineering human enzymes for therapeutic applications is attractive but introducing new amino acids may adversely affect enzyme stability and immunogenicity. Here we used a mammalian membrane-tethered screening system (ECSTASY) to evolve human lysosomal beta-glucuronidase (hBG) to hydrolyze a glucuronide metabolite (SN-38G) of the anticancer drug irinotecan (CPT-11). Three human beta-glucuronidase variants (hBG3, hBG10 and hBG19) with 3, 10 and 19 amino acid substitutions were identified that display up to 40-fold enhanced enzymatic activity, higher stability than E. coli beta-glucuronidase in human serum, and similar pharmacokinetics in mice as wild-type hBG. The hBG variants were two to three orders of magnitude less immunogenic than E. coli beta-glucuronidase in hBG transgenic mice. Intravenous administration of an immunoenzyme (hcc49-hBG10) targeting a sialyl-Tn tumor-associated antigen to mice bearing human colon xenografts significantly enhanced the anticancer activity of CPT-11 as measured by tumor suppression and mouse survival. Our results suggest that genetically-modified human enzymes represent a good alternative to microbially-derived enzymes for therapeutic applications.

Extracellular vesicles purified from serum-converted human platelet lysates offer strong protection after cardiac ischaemia/reperfusion injury.

Extracellular vesicles (EVs) from cultured cells or bodily fluids have been demonstrated to show therapeutic value following myocardial infarction. However, challenges in donor variation, EV generation and isolation methods, and material availability have hindered their therapeutic use. Here, we show that human clinical-grade platelet concentrates from a blood establishment can be used to rapidly generate high concentrations of high purity EVs from sero-converted platelet lysate (SCPL-EVs) with minimal processing, using size-exclusion chromatography. Processing removed serum carrier proteins, coagulation factors and complement proteins from the original platelet lysate and the resultant SCPL-EVs carried a range of trophic factors and multiple recognised cardioprotective miRNAs. As such, SCPL-EVs protected rodent and human cardiomyocytes from hypoxia/re-oxygenation injury and stimulated angiogenesis of human cardiac microvessel endothelial cells. In a mouse model of myocardial infarction with reperfusion, SCPL-EV delivery using echo-guided intracavitary percutaneous injection produced large improvements in cardiac function, reduced scar formation and promoted angiogenesis. Since platelet-based biomaterials are already widely used clinically, we believe that this therapy could be rapidly suitable for a human clinical trial.

Degradable Biocompatible Porous Microtube Scaffold for Extended Donor Cell Survival and Activity.

Cell therapy has significant therapeutic potential but is often limited by poor donor cell retention and viability at the host implantation site. Biomaterials can improve cell retention by providing cells with increased cell-cell and cell-matrix contacts and materials that allow three-dimensional cell culture to better recapitulate native cell morphology and function. In this study, we engineered a scaffold that allows for cell encapsulation and sustained three-dimensional cell culture. Since cell therapy is largely driven by paracrine secretions, the material was fabricated by electrospinning to have a large internal surface area, micrometer-thin walls, and nanoscale surface pores to allow for nutrient exchange without early cell permeation. The material is degradable, which allows for less invasive removal of the implant. Here, a biodegradable poly(lactic-co-glycolic acid) (PLGA) microtube array membrane was fabricated. In vitro testing showed that the material supported the culture of human dermal fibroblasts for at least 21 days, with paracrine secretion of pro-angiogenic FGF2. In vivo xenotransplantation of human cells in an immunocompetent mouse showed that donor cells could be maintained for more than one month and the material showed no obvious toxicity. Analysis of gene expression and tissue histology surrounding the implant showed that the material produced muted inflammatory and immune responses compared to a permanent implant and increased markers of angiogenesis.

Assessment of circulating extracellular vesicles from calorie-restricted mice and humans in ischaemic injury models.

Calorie restriction (CR) and fasting affect lifespan, disease susceptibility and response to acute injury across multiple animal models, including ischaemic injuries such as myocardial infarction or kidney hypoxia. The cargo and function of circulating extracellular vesicles (EV) respond to changes in host physiology, including exercise, injury, and other interventions. Thus, we hypothesised that EVs induced following CR may reflect some of the beneficial properties of CR itself. In a pilot study, EVs were isolated from mice following 21 days of 30 % CR, and from eight human donors after 72 h water-only fasting. EV size, concentration and morphology were profiled by NTA, western blot and cryoEM, and their function was assessed using multiple assays related to ischaemic diseases. We found that EVs from post-fasting samples better protected cardiac cells from hypoxia/reperfusion (H/R) injury compared to pre-fasting EVs. However, there was no difference when used to treat H/R-injured kidney epithelial cells. Post-fasting derived EVs slowed the rate of fibroblast migration and slightly reduced macrophage inflammatory gene expression compared to pre-fasting derived EVs. Lastly, we compared miRNA cargos of pre- and post-fasting human serum EVs and found significant changes in a small number of miRNAs. We conclude that fasting appears to influence EV cargo and function, with varied effects worthy of further exploration.

Porous scaffold for mesenchymal cell encapsulation and exosome-based therapy of ischemic diseases.

Ischemic diseases including myocardial infarction (MI) and limb ischemia are some of the greatest causes of morbidity and mortality worldwide. Cell therapy is a potential treatment but is usually limited by poor survival and retention of donor cells injected at the target site. Since much of the therapeutic effects occur via cell-secreted paracrine factors, including extracellular vesicles (EVs), we developed a porous material for cell encapsulation which would improve donor cell retention and survival, while allowing EV secretion. Human donor cardiac mesenchymal cells were used as a model therapeutic cell and the encapsulation system could sustain three-dimensional cell growth and secretion of therapeutic factors. Secretion of EVs and protective growth factors were increased by encapsulation, and secreted EVs had hypoxia-protective, pro-angiogenic activities in in vitro assays. In a mouse model of limb ischemia the implant improved angiogenesis and blood flow, and in an MI model the system preserved ejection fraction %. In both instances, the encapsulation system greatly extended donor cell retention and survival compared to directly injected cells. This system represents a promising therapy for ischemic diseases and could be adapted for treatment of other diseases in the future.

Liposome-encapsulated anthraquinone improves efficacy and safety in triple negative breast cancer.

Breast cancer is the most common cancer among women and a leading cause of death worldwide. Triple negative breast cancer (TNBC) is a highly aggressive subtype which is the most challenging to treat. Due to heterogeneity and a lack of specific molecular targets, small molecule-based chemotherapy is the preferred course of treatment. However, these drugs have high toxicity due to off-target effects on healthy tissues, and tumors may develop resistance. Here, we present a polyethylene glycol-modified nanoscale liposomal formulation (LipoRV) of a new anthraquinone derivative which has potent effects on multiple TNBC cell lines. LipoRV readily inhibited the cell cycle, induced cell apoptosis, and reduced long-term proliferative potential of TNBC cells. In a xenograft animal model, LipoRV successfully cleared tumors and demonstrated a good safety profile, without detrimental effects on biochemical markers. Finally, RNA sequencing of LipoRV-treated TNBC cells was carried out, indicating that LipoRV may have immunomodulatory properties. These findings demonstrate that a liposomal anthraquinone-based molecule has excellent promise for TNBC therapy in the future.