Ruben Crespo-Diaz

Guided Cardiopoiesis Enhances Therapeutic Benefit of Bone Marrow Human Mesenchymal Stem Cells in Chronic Myocardial Infarction

OBJECTIVES The goal of this study was to guide bone marrow-derived human mesenchymal stem cells (hMSCs) into a cardiac progenitor phenotype and assess therapeutic benefit in chronic myocardial infarction. BACKGROUND Adult stem cells, delivered in their naïve state, demonstrate a limited benefit in patients with ischemic heart disease. Pre-emptive lineage pre-specification may optimize therapeutic outcome. METHODS hMSC were harvested from a coronary artery disease patient cohort. A recombinant cocktail consisting of transforming growth factor-beta(1), bone morphogenetic protein-4, activin A, retinoic acid, insulin-like growth factor-1, fibroblast growth factor-2, alpha-thrombin, and interleukin-6 was formulated to engage hMSC into cardiopoiesis. Derived hMSC were injected into the myocardium of a nude infarcted murine model and followed over 1 year for functional and structural end points. RESULTS Although the majority of patient-derived hMSC in their native state demonstrated limited effect on ejection fraction, stem cells from rare individuals harbored a spontaneous capacity to improve contractile performance. This reparative cytotype was characterized by high expression of homeobox transcription factor Nkx-2.5, T-box transcription factor TBX5, helix-loop-helix transcription factor MESP1, and myocyte enhancer factor MEF2C, markers of cardiopoiesis. Recombinant cardiogenic cocktail guidance secured the cardiopoietic phenotype across the patient cohort. Compared with unguided counterparts, cardiopoietic hMSC delivered into infarcted myocardium achieved superior functional and structural benefit without adverse side effects. Engraftment into murine hearts was associated with increased human-specific nuclear, sarcomeric, and gap junction content along with induction of myocardial cell cycle activity. CONCLUSIONS Guided cardiopoiesis thus enhances the therapeutic benefit of bone marrow-derived hMSC in chronic ischemic cardiomyopathy.

Increased expression of BubR1 protects against aneuploidy and cancer and extends healthy lifespan

The BubR1 gene encodes for a mitotic regulator that ensures accurate segregation of chromosomes through its role in the mitotic checkpoint and the establishment of proper microtubule–kinetochore attachments. Germline mutations that reduce BubR1 abundance cause aneuploidy, shorten lifespan and induce premature ageing phenotypes and cancer in both humans and mice. A reduced BubR1 expression level is also a feature of chronological ageing, but whether this age-related decline has biological consequences is unknown. Using a transgenic approach in mice, we show that sustained high-level expression of BubR1 preserves genomic integrity and reduces tumorigenesis, even in the presence of genetic alterations that strongly promote aneuplodization and cancer, such as oncogenic Ras. We find that BubR1 overabundance exerts its protective effect by correcting mitotic checkpoint impairment and microtubule–kinetochore attachment defects. Furthermore, sustained high-level expression of BubR1 extends lifespan and delays age-related deterioration and aneuploidy in several tissues. Collectively, these data uncover a generalized function for BubR1 in counteracting defects that cause whole-chromosome instability and suggest that modulating BubR1 provides a unique opportunity to extend healthy lifespan.

Platelet lysate consisting of a natural repair proteome supports human mesenchymal stem cell proliferation and chromosomal stability

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.

CXCR4+/FLK-1+ Biomarkers Select a Cardiopoietic Lineage from Embryonic Stem Cells

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.

Embryonic Stem Cell Therapy of Heart Failure in Genetic Cardiomyopathy

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.

Optimized Delivery System Achieves Enhanced Endomyocardial Stem Cell Retention

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.

Induced pluripotent stem cell intervention rescues ventricular wall motion disparity, achieving biological cardiac resynchronization post-infarction

•  The pumping function of the heart depends on ordered initiation and propagation of myocardial excitation. Cardiac output is compromised by inconsistent timing and direction of wall motion, leading to dyssynchrony and organ failure. •  Myocardial infarction induces irreversible heart damage. Extensive damage hampers effective pacemaker‐based cardiac resynchronization therapy, the current standard‐of‐care. Establishment of alternative approaches is thus warranted. •  High‐resolution imaging was here utilized to non‐invasively map suitable therapeutic targets within a dyssynchronous heart. Speckle‐tracking echocardiography unmasked the source of progressive cardiac dyssynchrony within the primary infarcted region. •  Bioengineered stem cells with a capacity to induce a regenerative response were implanted into infarcted areas. Speckle‐tracking echocardiography and histology assessment revealed that cell therapy achieved cardiac resynchronization and long‐term repair. •  This proof‐of‐concept study thus introduces a stem cell‐based regenerative solution to address cardiac dyssynchrony post‐infarction.

Stem cell transplant into preimplantation embryo yields myocardial infarction-resistant adult phenotype.

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. STEM CELLS 2009;27:1697–1705

Reply: The C-CURE Randomized Clinical Trial (Cardiopoietic stem Cell therapy in heart failURE).

We appreciate the interest of Dr. Mielewczik and colleagues in the C-CURE (Cardiopoietic stem Cell therapy in heart failURE) trial. As outlined in our paper [(1)][1], feasibility and safety were the primary endpoints in this first-in-man study that assessed cardiogenically-oriented, autologous bone

Lineage specification of Flk-1+ progenitors is associated with divergent Sox7 expression in cardiopoiesis

Embryonic stem cell differentiation recapitulates the diverse phenotypes of a developing embryo, traceable according to markers of lineage specification. At gastrulation, the vascular endothelial growth factor (VEGF) receptor, Flk-1 (KDR), identifies a mesoderm-restricted potential of embryonic stem cells. The multi-lineage propensity of Flk-1(+) progenitors mandates the mapping of fate-modifying co-factors in order to stratify differentiating cytotypes and predict lineage competency. Here, Flk-1-based selection of early embryonic stem cell progeny separated a population depleted of pluripotent (Oct4, Sox2) and endoderm (Sox17) markers. The gene expression profile of the Flk-1(+) population was notable for a significant upregulation in the vasculogenic Sox7 transcription factor, which overlapped with the emergence of primordial cardiac transcription factors GATA-4, Myocardin and Nkx2.5. Sorting the parental Flk-1(+) pool with the chemokine receptor CXCR4 to enrich the cardiopoietic subpopulation uncovered divergent Sox7 expression, with a 7-fold induction in non-cardiac compared to cardiac progenitors. Bioinformatic resolution sequestered a framework of gene expression relationship between Sox transcription factor family members and the Flk-1/CXCR4 axes with significant integration of beta-catenin signaling. Thus, differential Sox7 gene expression presents a novel biomarker profile, and possible regulatory switch, to distinguish cardiovascular pedigrees within Flk-1(+) multi-lineage progenitors.