Crespo‐Diaz, Ruben

Objectives/HypothesisRadiation plays a prominent role in advanced stage head and neck tumors. Often, the radiated area includes adjacent nonmalignant mesenchymal tissue, which contains a mixture of cells that has been shown to accelerate wound healing. The purpose of this study is to determine the long‐term effect of radiation on the expansion potential of adipose‐derived stromal/stem cell (ADSC) tissue and on the ability of resident stem cells in this fraction to undergo phenotypic differentiation.Study Design/Methods: After institutional review board approval, 12 patients with a history of head and neck radiation and pending surgery were enrolled. Adipose tissue was collected from irradiated tissue (XRT) and nonirradiated tissue (NRT) sites. Mesenchymal stem cells were isolated from these populations, with subsequent assessment of cellular kinetics and differentiation potential between harvest sites.ResultsAdipose‐derived stromal/stem cells could not be isolated from XRT in six patients due to lack of in vitro cell proliferation. For the remaining six patients, overall cumulative population‐doubling time was longer for XRT relative to NRT (29.3 vs. 11.5 days; P = 0.02). However, no significant differences were observed in cell generation time or viability. When XRT and NRT ADSC fractions were grown to standardized concentrations and incubated under conditions that induce phenotypic differentiation of resident stem cells, no significant changes in chondrogenic, adipogenic, or osteogenic differentiation were observed.ConclusionThese preliminary observations suggest that irradiated ADSCs close to the surgical site undergo long‐term changes in proliferative capacity. The potential for phenotypic differentiation is retained, however, in ADSCs that survive the irradiation process.Level of Evidence2b.

Background Cardiac resynchronization therapy using bi‐ventricular pacing is proven effective in the management of heart failure (HF) with a wide QRS ‐complex. In the absence of QRS prolongation, however, device‐based resynchronization is reported unsuitable. As an alternative, the present study tests a regenerative cell‐based approach in the setting of narrow QRS ‐complex HF. Methods and Results Progressive cardiac dyssynchrony was provoked in a chronic transgenic model of stress‐triggered dilated cardiomyopathy. In contrast to rampant end‐stage disease afflicting untreated cohorts, stem cell intervention early in disease, characterized by mechanical dyssynchrony and a narrow QRS ‐complex, aborted progressive dyssynchronous HF and prevented QRS widening. Stem cell‐treated hearts acquired coordinated ventricular contraction and relaxation supporting systolic and diastolic performance. Rescue of contractile dynamics was underpinned by a halted left ventricular dilatation, limited hypertrophy, and reduced fibrosis. Reverse remodeling reflected a restored cardiomyopathic proteome, enforced at systems level through correction of the pathological molecular landscape and nullified adverse cardiac outcomes. Cell therapy of a dyssynchrony‐prone cardiomyopathic cohort translated prospectively into improved exercise capacity and prolonged survivorship. Conclusions In narrow QRS HF, a regenerative approach demonstrated functional and structural benefit, introducing the prospect of device‐autonomous resynchronization therapy for refractory disease.

Background— Regenerative cell-based therapies are associated with limited myocardial retention of delivered stem cells. The objective of this study is to develop an endocardial delivery system for enhanced cell retention. Methods and Results— Stem cell retention was simulated in silico using 1- and 3-dimensional models of tissue distortion and compliance associated with delivery. Needle designs, predicted to be optimal, were accordingly engineered using nitinol, a nickel and titanium alloy displaying shape memory and superelasticity. Biocompatibility was tested with human mesenchymal stem cells. Experimental validation was performed with species-matched cells directly delivered into Langendorff-perfused porcine hearts or administered percutaneously into the endocardium of infarcted pigs. Cell retention was quantified by flow cytometry and real-time quantitative polymerase chain reaction methodology. Models, computing optimal distribution of distortion calibrated to favor tissue compliance, predicted that a 75°-curved needle featuring small-to-large graded side holes would ensure the highest cell retention profile. In isolated hearts, the nitinol curved needle catheter (C-Cath) design ensured 3-fold superior stem cell retention compared with a standard needle. In the setting of chronic infarction, percutaneous delivery of stem cells with C-Cath yielded a 37.7±7.1% versus 10.0±2.8% retention achieved with a traditional needle without effect on biocompatibility or safety. Conclusions— Modeling-guided development of a nitinol-based curved needle delivery system with incremental side holes achieved enhanced myocardial stem cell retention.

With favorable regenerative and immunotolerant profiles, patient-derived human mesenchymal stem cells (hMSCs) are increasingly considered in cell therapy. Derived from bone marrow (BM) and standardized with culture in fetal bovine serum (FBS), translation of hMSC-based approaches is impeded by protracted expansion times, risk of xenogenic response, and exposure to zoonoses. Here, human platelet lysate adherent to good manufacturing practices (GMP-hPL) provided a nonzoonotic adjuvant that enhanced the capacity of BM-hMSC to proliferate. The nurturing benefit of GMP-hPL was generalized to hMSC from adipose tissue evaluated as an alternative to bone marrow. Long-term culture in GMP-hPL maintained the multipotency of hMSC, while protecting against clonal chromosomal instability detected in the FBS milieu. Proteomic dissection identified TGF-β, VEGF, PDGF, FGF, and EGF as highly ranked effectors of hPL activity, revealing a paradigm of healing that underlies platelet lysate adjuvancy. Thus, GMP-adherent human platelet lysate accelerates hMSC proliferation with no chromosomal aberrancy, through an innate repair paradigm.

Abstract Stem cells are an emerging strategy for treatment of myocardial infarction, limited however to postinjury intervention. Preventive stem cell-based therapy to augment stress tolerance has yet to be considered for lifelong protection. Here, pluripotent stem cells were microsurgically introduced at the blastocyst stage of murine embryo development to ensure stochastic integration and sustained organ contribution. Engineered chimera displayed excess in body weight due to increased fat deposits, but were otherwise devoid of obesity-related morbidity. Remarkably, and in sharp contrast to susceptible nonchimeric offspring, chimera was resistant to myocardial infarction induced by permanent coronary occlusion. Infarcted nonchimeric adult hearts demonstrated progressive deterioration in ejection fraction, while age-matched 12–14-months-old chimera recovered from equivalent ischemic insult to regain within one-month preocclusion contractile performance. Electrical remodeling and ventricular enlargement with fibrosis, prominent in failing nonchimera, were averted in the chimeric cohort characterized by an increased stem cell load in adipose tissue and upregulated markers of biogenesis Ki67, c-Kit, and stem cell antigen-1 in the myocardium. Favorable outcome in infarcted chimera translated into an overall benefit in workload capacity and survival. Thus, prenatal stem cell transplant yields a cardioprotective phenotype in adulthood, expanding cell-based indications beyond traditional postinjury applications to include pre-emptive therapy. Disclosure of potential conflicts of interest is found at the end of this article.

Abstract Pathogenic causes underlying nonischemic cardiomyopathies are increasingly being resolved, yet repair therapies for these commonly heritable forms of heart failure are lacking. A case in point is human dilated cardiomyopathy 10 (CMD10; Online Mendelian Inheritance in Man #608569), a progressive organ dysfunction syndrome refractory to conventional therapies and linked to mutations in cardiac ATP-sensitive K+ (KATP) channel subunits. Embryonic stem cell therapy demonstrates benefit in ischemic heart disease, but the reparative capacity of this allogeneic regenerative cell source has not been tested in inherited cardiomyopathy. Here, in a Kir6.2-knockout model lacking functional KATP channels, we recapitulated under the imposed stress of pressure overload the gene-environment substrate of CMD10. Salient features of the human malignant heart failure phenotype were reproduced, including compromised contractility, ventricular dilatation, and poor survival. Embryonic stem cells were delivered through the epicardial route into the left ventricular wall of cardiomyopathic stressed Kir6.2-null mutants. At 1 month of therapy, transplantation of 200,000 cells per heart achieved teratoma-free reversal of systolic dysfunction and electrical synchronization and halted maladaptive remodeling, thereby preventing end-stage organ failure. Tracked using the lacZ reporter transgene, stem cells engrafted into host heart. Beyond formation of cardiac tissue positive for Kir6.2, transplantation induced cell cycle activation and halved fibrotic zones, normalizing sarcomeric and gap junction organization within remuscularized hearts. Improved systemic function induced by stem cell therapy translated into increased stamina, absence of anasarca, and benefit to overall survivorship. Embryonic stem cells thus achieve functional repair in nonischemic genetic cardiomyopathy, expanding indications to the therapy of heritable heart failure. Disclosure of potential conflicts of interest is found at the end of this article.

Abstract Pluripotent stem cells demonstrate an inherent propensity for unrestricted multi-lineage differentiation. Translation into regenerative applications requires identification and isolation of tissue-specified progenitor cells. From a comprehensive pool of 11,272 quality-filtered genes, profiling embryonic stem cells at discrete stages of cardiopoiesis revealed 736 transcripts encoding membrane-associated proteins, where 306 were specifically upregulated with cardiogenic differentiation. Bioinformatic dissection of exposed surface biomarkers prioritized the chemokine receptor cluster as the most significantly over-represented gene receptor family during pre cardiac induction, with CXCR4 uniquely associated with mesendoderm formation. CXCR4+ progenitors were sorted from the embryonic stem cell pool into mesoderm-restricted progeny according to co-expression with the early mesoderm marker Flk-1. In contrast to CXCR4−/Flk-1− cells, the CXCR4+/Flk-1+ subpopulation demonstrated overexpressed cardiac lineage transcription factors (Mef2C, Myocardin, Nkx2.5), whereas pluripotent genes (Oct4, Fgf4, Sox2) as well as neuroectoderm (Sox1) and endoderm alpha-fetoprotein markers were all depleted. In fact, the CXCR4+/Flk-1+ biomarker combination identified embryonic stem cell progeny significantly enriched with Mesp-1, GATA-4, and Tbx5, indicative of pre cardiac mesoderm and the primary heart field. Although the CXCR4+/Flk-1+ transcriptome shared 97% identity with the CXCR4−/Flk-1− counterpart, the 818 divergent gene set represented predominantly cardiovascular developmental functions and formed a primitive cardiac network. Differentiation of CXCR4+/Flk-1+ progenitors yielded nuclear translocation of myocardial transcription factors and robust sarcomerogenesis with nascent cardiac tissue demonstrating beating activity and calcium transients. Thus, the CXCR4/Flk-1 biomarker pair predicts the emergence of cardiogenic specification within a pluripotent stem cell pool, enabling targeted selection of cardiopoietic lineage. Disclosure of potential conflicts of interest is found at the end of this article.