Newt A1 cell-derived extracellular vesicles promote mammalian nerve growth.

Newts have the extraordinary ability to fully regenerate lost or damaged cardiac, neural and retinal tissues, and even amputated limbs. In contrast, mammals lack these broad regenerative capabilities. While the molecular basis of newts' regenerative ability is the subject of active study, the underlying paracrine signaling factors involved remain largely uncharacterized. Extracellular vesicles (EVs) play an important role in cell-to-cell communication via EV cargo-mediated regulation of gene expression patterns within the recipient cells. Here, we report that newt myogenic precursor (A1) cells secrete EVs (A1EVs) that contain messenger RNAs associated with early embryonic development, neuronal differentiation, and cell survival. Exposure of rat primary superior cervical ganglion (SCG) neurons to A1EVs increased neurite outgrowth, facilitated by increases in mitochondrial respiration. Canonical pathway analysis pinpointed activation of NGF/ERK5 signaling in SCG neurons exposed to A1EV, which was validated experimentally. Thus, newt EVs drive neurite growth and complexity in mammalian primary neurons.

Targeting extracellular vesicles to injured tissue using membrane cloaking and surface display.

BACKGROUND: Extracellular vesicles (EVs) and exosomes are nano-sized, membrane-bound vesicles shed by most eukaryotic cells studied to date. EVs play key signaling roles in cellular development, cancer metastasis, immune modulation and tissue regeneration. Attempts to modify exosomes to increase their targeting efficiency to specific tissue types are still in their infancy. Here we describe an EV membrane anchoring platform termed "cloaking" to directly embed tissue-specific antibodies or homing peptides on EV membrane surfaces ex vivo for enhanced vesicle uptake in cells of interest. The cloaking system consists of three components: DMPE phospholipid membrane anchor, polyethylene glycol spacer and a conjugated streptavidin platform molecule, to which any biotinylated molecule can be coupled for EV decoration. RESULTS: We demonstrate the utility of membrane surface engineering and biodistribution tracking with this technology along with targeting EVs for enhanced uptake in cardiac fibroblasts, myoblasts and ischemic myocardium using combinations of fluorescent tags, tissue-targeting antibodies and homing peptide surface cloaks. We compare cloaking to a complementary approach, surface display, in which parental cells are engineered to secrete EVs with fusion surface targeting proteins. CONCLUSIONS: EV targeting can be enhanced both by cloaking and by surface display; the former entails chemical modification of preformed EVs, while the latter requires genetic modification of the parent cells. Reduction to practice of the cloaking approach, using several different EV surface modifications to target distinct cells and tissues, supports the notion of cloaking as a platform technology.

Newt cells secrete extracellular vesicles with therapeutic bioactivity in mammalian cardiomyocytes.

Newts can regenerate amputated limbs and cardiac tissue, unlike mammals which lack broad regenerative capacity. Several signaling pathways involved in cell proliferation, differentiation and survival during newt tissue regeneration have been elucidated, however the factors that coordinate signaling between cells, as well as the conservation of these factors in other animals, are not well defined. Here we report that media conditioned by newt limb explant cells (A1 cells) protect mammalian cardiomyocytes from oxidative stress-induced apoptosis. The cytoprotective effect of A1-conditioned media was negated by exposing A1 cells to GW4869, which suppresses the generation of extracellular vesicles (EVs). A1-EVs are similar in diameter (~100-150 nm), structure, and share several membrane surface and cargo proteins with mammalian exosomes. However, isolated A1-EVs contain significantly higher levels of both RNA and protein per particle than mammalian EVs. Additionally, numerous cargo RNAs and proteins are unique to A1-EVs. Of particular note, A1-EVs contain numerous mRNAs encoding nuclear receptors, membrane ligands, as well as transcription factors. Mammalian cardiomyocytes treated with A1-EVs showed increased expression of genes in the PI3K/AKT pathway, a pivotal player in survival signaling. We conclude that newt cells secrete EVs with diverse, distinctive RNA and protein contents. Despite ~300 million years of evolutionary divergence between newts and mammals, newt EVs confer cytoprotective effects on mammalian cardiomyocytes.

Cardiac and systemic rejuvenation after cardiosphere-derived cell therapy in senescent rats.

AIM: The aim is to assess the effects of CDCs on heart structure, function, gene expression, and systemic parameters in aged rats. Diastolic dysfunction is characteristic of aged hearts. Cardiosphere-derived cell (CDC) therapy has exhibited several favourable effects on heart structure and function in humans and in preclinical models; however, the effects of CDCs on aging have not been evaluated. METHODS AND RESULTS: We compared intra-cardiac injections of neonatal rat CDCs to vehicle (phosphate-buffered saline, PBS) in 21.8 ± 1.6 month-old rats (mean ± standard deviation; n = 23 total). Ten rats 4.1 ± 1.5 months of age comprised a young reference group. Blood, echocardiographic, haemodynamic and treadmill stress tests were performed at baseline in all animals, and 1 month after treatment in old animals. Histology and the transcriptome were assessed after terminal phenotyping. For in vitro studies, human heart progenitors from older donors, or cardiomyocytes from aged rats were exposed to human CDCs or exosomes secreted by CDCs (CDC-XO) from paediatric donors. Transcriptomic analysis revealed that CDCs, but not PBS, recapitulated a youthful pattern of gene expression in the hearts of old animals (85.5% of genes differentially expressed, P < 0.05). Telomeres in heart cells were longer in CDC-transplanted animals (P < 0.0001 vs. PBS). Cardiosphere-derived cells attenuated hypertrophy by echo (P < 0.01); histology confirmed decreases in cardiomyocyte area (P < 0.0001) and myocardial fibrosis (P < 0.05) vs. PBS. Cardiosphere-derived cell injection improved diastolic dysfunction [lower E/A (P < 0.01), E/E' (P = 0.05), end-diastolic pressure-volume relationship (P < 0.05) compared with baseline), and lowered serum brain natriuretic peptide (both P < 0.05 vs. PBS). In CDC-transplanted old rats, exercise capacity increased ∼20% (P < 0.05 vs. baseline), body weight decreased ∼30% less (P = 0.05 vs. PBS) and hair regrowth after shaving was more robust (P < 0.05 vs. PBS). Serum biomarkers of inflammation (IL-10, IL-1b, and IL-6) improved in the CDC group (P < 0.05 for each, all vs. PBS). Young CDCs secrete exosomes which increase telomerase activity, elongate telomere length, and reduce the number of senescent human heart cells in culture. CONCLUSION: Young CDCs rejuvenate old animals as gauged by cardiac gene expression, heart function, exercise capacity, and systemic biomarkers.

Therapeutic benefits of intravenous cardiosphere-derived cell therapy in rats with pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive condition characterized by occlusive pulmonary arteriopathy, in which survival remains poor despite pharmacologic advances. The aim of this study was to evaluate the ability of cardiosphere-derived cells (CDCs), cardiac progenitor cells with potent anti-inflammatory and immunomodulatory properties, to attenuate hemodynamic and morphometric remodeling of the right ventricle (RV) and pulmonary arterioles in rats with established monocrotaline (MCT)-induced PAH. Animals were divided into 3 groups: 1) Control (CTL), 2) PAH in which CDCs were centrally infused (CDC) and 3) PAH in which saline was given (Sham). Significant increments in RV systolic pressure (RVSP) and RV hypertrophy were noted in Sham animals compared to CTL. In CDC rats at day 35, RSVP fell (- 38%; p< 0.001) and RV hypertrophy decreased (-26%; p< 0.01). TAPSE and cardiac output were preserved in all 3 groups at day 35. Pulmonary arteriolar wall thickness was greater in Sham rats compared to CTL, and reduced in CDC animals for vessels 20-50 µm (P<0.01; back to CTL levels) and 50-80µm (P<0.01) in diameter. The macrophage population was increased in Sham animals compared to CTL (P< 0.001), but markedly reduced in CDC rats. In conclusion, infusion of CDCs markedly attenuated several key pathophysiologic features of PAH. As adjunctive therapy to PAH-specific agents, CDCs have the potential to impact on the pathobiology of adverse pulmonary arteriolar remodeling, by acting on multiple mechanisms simultaneously.

Durable Benefits of Cellular Postconditioning: Long-Term Effects of Allogeneic Cardiosphere-Derived Cells Infused After Reperfusion in Pigs with Acute Myocardial Infarction.

BACKGROUND: Infusion of allogeneic cardiosphere-derived cells (allo-CDCs) postreperfusion elicits cardioprotective cellular postconditioning in pigs with acute myocardial infarction. However, the long-term effects of allo-CDCs have not been assessed. We performed a placebo-controlled pivotal study for long-term evaluation, as well as shorter-term mechanistic studies. METHODS AND RESULTS: Minipigs underwent 1.5-hour mid-left anterior descending balloon occlusion followed by reperfusion and were randomized to receive intracoronary allo-CDCs or vehicle 30 minutes postreperfusion. Left ventriculography (LVG) demonstrated preserved ejection fraction (EF) and attenuation of LV remodeling in CDC-treated pigs. Pigs underwent cardiac magnetic resonance imaging (MRI) and LVG 1 hour and 8 weeks after therapy to evaluate efficacy. MRI showed improvement of EF and attenuation of LV remodeling immediately after allo-CDC infusion. In addition, allo-CDCs improved regional function and decreased hypertrophy 2 months post-treatment. Histological analysis revealed increased myocardial salvage index, enhanced vascularity, sustained reductions in infarct size/area at risk and scar transmurality, and attenuation of collagen deposition in the infarct zone of allo-CDC-treated pigs at 2 months. Allo-CDCs did not evoke lymphohistiocytic infiltration or systemic humoral memory response. Short-term experiments designed to probe mechanism revealed antiapoptotic effects of allo-CDCs on cardiomyocytes and increases in cytoprotective macrophages, but no increase in overall inflammatory cell infiltration 2 hours after cell therapy. CONCLUSIONS: Allo-CDC infusion postreperfusion is safe, improves cardiac function, and attenuates scar size and remodeling. The favorable effects persist for at least 2 months after therapy. Thus, cellular postconditioning confers not only acute cardioprotection, but also lasting structural and functional benefits.

Cellular postconditioning: allogeneic cardiosphere-derived cells reduce infarct size and attenuate microvascular obstruction when administered after reperfusion in pigs with acute myocardial infarction.

BACKGROUND: Intracoronary delivery of cardiosphere-derived cells (CDCs) has been demonstrated to be safe and effective in porcine and human chronic myocardial infarction. However, intracoronary delivery of CDCs after reperfusion in acute myocardial infarction has never been assessed in a clinically-relevant large animal model. We tested CDCs as adjunctive therapy to reperfusion in a porcine model of myocardial infarction. METHODS AND RESULTS: First, escalating doses (5, 7.5, and 10 million cells) of allogeneic CDCs were administered intracoronary 30 minutes after reperfusion. Forty-eight hours later, left ventriculography was performed and animals euthanized to measure area at risk, infarct size (IS), and microvascular obstruction. Second, identical end points were measured in a pivotal study of minipigs (n=14) that received 8.5 to 9 million allogeneic CDCs, placebo solution, or sham. Multiple indicators of cardioprotection were observed with 7.5 and 10 million allogeneic CDCs, but not 5 million CDCs, relative to control. In the pivotal study, IS, microvascular obstruction, cardiomyocyte apoptosis, and adverse left ventricular remodeling were all smaller in the CDC group than in sham or placebo groups. In addition, serum troponin I level at 24 hours was lower after CDC infusion than that in the placebo or sham groups, consistent with the histologically-demonstrated reduction in IS. CONCLUSIONS: Intracoronary delivery of allogeneic CDCs is safe, feasible, and effective in cardioprotection, reducing IS, preventing microvascular obstruction, and attenuating adverse acute remodeling. This novel cardioprotective effect, which we call cellular postconditioning, differs from previous strategies to reduce IS in that it works even when initiated with significant delay after reflow.

Allogeneic cardiospheres delivered via percutaneous transendocardial injection increase viable myocardium, decrease scar size, and attenuate cardiac dilatation in porcine ischemic cardiomyopathy.

BACKGROUND: Epicardial injection of heart-derived cell products is safe and effective post-myocardial infarction (MI), but clinically-translatable transendocardial injection has never been evaluated. We sought to assess the feasibility, safety and efficacy of percutaneous transendocardial injection of heart-derived cells in porcine chronic ischemic cardiomyopathy. METHODS AND RESULTS: We studied a total of 89 minipigs; 63 completed the specified protocols. After NOGA-guided transendocardial injection, we quantified engraftment of escalating doses of allogeneic cardiospheres or cardiosphere-derived cells in minipigs (n = 22) post-MI. Next, a dose-ranging, blinded, randomized, placebo-controlled ("dose optimization") study of transendocardial injection of the better-engrafting product was performed in infarcted minipigs (n = 16). Finally, the superior product and dose (150 million cardiospheres) were tested in a blinded, randomized, placebo-controlled ("pivotal") study (n = 22). Contrast-enhanced cardiac MRI revealed that all cardiosphere doses preserved systolic function and attenuated remodeling. The maximum feasible dose (150 million cells) was most effective in reducing scar size, increasing viable myocardium and improving ejection fraction. In the pivotal study, eight weeks post-injection, histopathology demonstrated no excess inflammation, and no myocyte hypertrophy, in treated minipigs versus controls. No alloreactive donor-specific antibodies developed over time. MRI showed reduced scar size, increased viable mass, and attenuation of cardiac dilatation with no effect on ejection fraction in the treated group compared to placebo. CONCLUSIONS: Dose-optimized injection of allogeneic cardiospheres is safe, decreases scar size, increases viable myocardium, and attenuates cardiac dilatation in porcine chronic ischemic cardiomyopathy. The decreases in scar size, mirrored by increases in viable myocardium, are consistent with therapeutic regeneration.

c-kit+ cells minimally contribute cardiomyocytes to the heart.

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine whether endogenous c-kit(+) cells contribute differentiated cardiomyocytes to the heart during development, with ageing or after injury in adulthood. A complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008. By contrast, c-kit(+) cells amply generated cardiac endothelial cells. Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

Stimulation of endogenous cardioblasts by exogenous cell therapy after myocardial infarction.

Controversy surrounds the identity, origin, and physiologic role of endogenous cardiomyocyte progenitors in adult mammals. Using an inducible genetic labeling approach to identify small non-myocyte cells expressing cardiac markers, we find that activated endogenous cardioblasts are rarely evident in the normal adult mouse heart. However, myocardial infarction results in significant cardioblast activation at the site of injury. Genetically labeled isolated cardioblasts express cardiac transcription factors and sarcomeric proteins, exhibit spontaneous contractions, and form mature cardiomyocytes in vivo after injection into unlabeled recipient hearts. The activated cardioblasts do not arise from hematogenous seeding, cardiomyocyte dedifferentiation, or mere expansion of a preformed progenitor pool. Cell therapy with cardiosphere-derived cells amplifies innate cardioblast-mediated tissue regeneration, in part through the secretion of stromal cell-derived factor 1 by transplanted cells. Thus, stimulation of endogenous cardioblasts by exogenous cells mediates therapeutic regeneration of injured myocardium.

Small heat shock protein HSPB1 regulates growth of embryonic zebrafish craniofacial muscles.

The small heat shock protein HspB1 (Hsp27) is abundantly expressed in embryonic muscle tissues of a wide variety of vertebrate species. However, the functional significance of this expression pattern is not well established. In the present study, we observed specific, high level expression of HspB1 protein and an HspB1 gene reporter in developing craniofacial muscles of the zebrafish, Danio rerio, and examined the consequences of reducing HspB1 expression to the development and growth of these muscles. Quantitative morphometric analyses revealed a reduction in the cross-sectional area of myofibers in embryos expressing reduced HspB1 levels by as much as 47% compared to controls. In contrast, we detected no differences in the number of myofibrils or associated nuclei, nor the number, size or development of chondrocytes in surrounding tissues. We also did not detect changes to the overall organization of sarcomeres or myofibrils in embryos expressing reduced levels of HspB1. Together our results reveal a critical role for HspB1 in the growth of myofibrils and provide new insight into the mechanism underlying its developmental function.

HSF1 is essential for the resistance of zebrafish eye and brain tissues to hypoxia/reperfusion injury.

Ischemia and subsequent reperfusion (IR) produces injury to brain, eye and other tissues, contributing to the progression of important clinical pathologies. The response of cells to IR involves activation of several signaling pathways including those activating hypoxia and heat shock responsive transcription factors. However, specific roles of these responses in limiting cell damage and preventing cell death after IR have not been fully elucidated. Here, we have examined the role of heat shock factor 1 (HSF1) in the response of zebrafish embryos to hypoxia and subsequent return to normoxic conditions (HR) as a model for IR. Heat shock preconditioning elevated heat shock protein expression and protected zebrafish embryo eye and brain tissues against HR-induced apoptosis. These effects were inhibited by translational suppression of HSF1 expression. Reduced expression of HSF1 also increased cell death in brain and eye tissues of embryos subjected to hypoxia and reperfusion without prior heat shock. Surprisingly, reduced expression of HSF1 had only a modest effect on hypoxia-induced expression of Hsp70 and no effect on hypoxia-induced expression of Hsp27. These results establish the zebrafish embryo as a model for the study of ischemic injury in the brain and eye and reveal a critical role for HSF1 in the response of these tissues to HR. Our results also uncouple the role of HSF1 expression from that of Hsp27, a well characterized heat shock protein considered essential for cell survival after hypoxia. Alternative roles for HSF1 are considered.