Satsuki Yamada

Cardiopoietic programming of embryonic stem cells for tumor-free heart repair

Embryonic stem cells have the distinct potential for tissue regeneration, including cardiac repair. Their propensity for multilineage differentiation carries, however, the liability of neoplastic growth, impeding therapeutic application. Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-α, enhancing the cardiogenic competence of recipient heart. The in vivo aptitude of TNF-α to promote cardiac differentiation was recapitulated in embryoid bodies in vitro. The procardiogenic action required an intact endoderm and was mediated by secreted cardio-inductive signals. Resolved TNF-α–induced endoderm-derived factors, combined in a cocktail, secured guided differentiation of embryonic stem cells in monolayers produce cardiac progenitors termed cardiopoietic cells. Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny. Recruited cardiopoietic cells delivered in infarcted hearts generated cardiomyocytes that proliferated into scar tissue, integrating with host myocardium for tumor-free repair. Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration.

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

OBJECTIVESnThe 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.nnnBACKGROUNDnAdult 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.nnnMETHODSnhMSC 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.nnnRESULTSnAlthough 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.nnnCONCLUSIONSnGuided cardiopoiesis thus enhances the therapeutic benefit of bone marrow-derived hMSC in chronic ischemic cardiomyopathy.

Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency.

Expression of p16Ink4a and p19Arf increases with age in both rodent and human tissues. However, whether these tumour suppressors are effectors of ageing remains unclear, mainly because knockout mice lacking p16Ink4a or p19Arf die early of tumours. Here, we show that skeletal muscle and fat, two tissues that develop early ageing-associated phenotypes in response to BubR1 insufficiency, have high levels of p16Ink4a and p19Arf. Inactivation of p16Ink4a in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19Arf inactivation exacerbates senescence and ageing in BubR1 mutant mice. Thus, we identify BubR1 insufficiency as a trigger for activation of the Cdkn2a locus in certain mouse tissues, and demonstrate that p16Ink4a is an effector and p19Arf an attenuator of senescence and ageing in these tissues.

iPS Programmed Without c-MYC Yield Proficient Cardiogenesis for Functional Heart Chimerism

Rationale: Induced pluripotent stem cells (iPS) allow derivation of pluripotent progenitors from somatic sources. Originally, iPS were induced by a stemness-related gene set that included the c-MYC oncogene. Objective: Here, we determined from embryo to adult the cardiogenic proficiency of iPS programmed without c-MYC, a cardiogenicity-associated transcription factor. Methods and Results: Transgenic expression of 3 human stemness factors SOX2, OCT4, and KLF4 here reset murine fibroblasts to the pluripotent ground state. Transduction without c-MYC reversed cellular ultrastructure into a primitive archetype and induced stem cell markers generating 3-germ layers, all qualifiers of acquired pluripotency. Three-factor induced iPS (3F-iPS) clones reproducibly demonstrated cardiac differentiation properties characterized by vigorous beating activity of embryoid bodies and robust expression of cardiac Mef2c, α-actinin, connexin43, MLC2a, and troponin I. In vitro isolated iPS-derived cardiomyocytes demonstrated functional excitation-contraction coupling. Chimerism with 3F-iPS derived by morula-stage diploid aggregation was sustained during prenatal heart organogenesis and contributed in vivo to normal cardiac structure and overall performance in adult tumor-free offspring. Conclusions: Thus, 3F-iPS bioengineered without c-MYC achieve highest stringency criteria for bona fide cardiogenesis enabling reprogrammed fibroblasts to yield de novo heart tissue compatible with native counterpart throughout embryological development and into adulthood.

Protection conferred by myocardial ATP-sensitive K+ channels in pressure overload-induced congestive heart failure revealed in KCNJ11 Kir6.2-null mutant

Ventricular load can precipitate development of the heart failure syndrome, yet the molecular components that control the cardiac adaptive response to imposed demand remain partly understood. Compromised ATP‐sensitive K+ (KATP) channel function renders the heart vulnerable to stress, implicating this metabolic sensor in the homeostatic response that would normally prevent progression of cardiac disease. Here, pressure overload was imposed on the left ventricle by transverse aortic constriction in the wild‐type and in mice lacking sarcolemmal KATP channels through Kir6.2 pore knockout (Kir6.2‐KO). Despite equivalent haemodynamic loads, within 30 min of aortic constriction, Kir6.2‐KO showed an aberrant prolongation of action potentials with intracellular calcium overload and ATP depletion, whereas wild‐type maintained ionic and energetic handling. On catheterization, constricted Kir6.2‐KO displayed compromised myocardial performance with elevated left ventricular end‐diastolic pressure, not seen in the wild‐type. Glyburide, a KATP channel inhibitor, reproduced the knockout phenotype in the wild‐type, whereas the calcium channel antagonist, verapamil, prevented abnormal outcome in Kir6.2‐KO. Within 48 h following aortic constriction, fulminant biventricular congestive heart failure, characterized by exercise intolerance, cardiac contractile dysfunction, hepatopulmonary congestion and ascites, halved the Kir6.2‐KO cohort, while no signs of organ failure or mortality were seen in wild‐type. Surviving Kir6.2‐KO developed premature and exaggerated fibrotic myocardial hypertrophy associated with nuclear up‐regulation of calcium‐dependent pro‐remodelling MEF2 and NF‐AT pathways, precipitating chamber dilatation within 3 weeks. Thus, KATP channels appear mandatory in acute and chronic cardiac adaptation to imposed haemodynamic load, protecting against congestive heart failure and death.

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.

Stem Cell Platforms for Regenerative Medicine

The pandemic of chronic degenerative diseases associated with aging demographics mandates development of effective approaches for tissue repair. As diverse stem cells directly contribute to innate healing, the capacity for de novo tissue reconstruction harbors a promising role for regenerative medicine. Indeed, a spectrum of natural stem cell sources ranging from embryonic to adult progenitors has been recently identified with unique characteristics for regeneration. The accessibility and applicability of the regenerative armamentarium has been further expanded with stem cells engineered by nuclear reprogramming. Through strategies of replacement to implant functional tissues, regeneration to transplant progenitor cells or rejuvenation to activate endogenous self‐repair mechanisms, the overarching goal of regenerative medicine is to translate stem cell platforms into practice and achieve cures for diseases limited to palliative interventions. Harnessing the full potential of each platform will optimize matching stem cell‐based biologics with the disease‐specific niche environment of individual patients to maximize the quality of long‐term management, while minimizing the needs for adjunctive therapy. Emerging discovery science with feedback from clinical translation is therefore poised to transform medicine offering safe and effective stem cell biotherapeutics to enable personalized solutions for incurable diseases.

Sarcolemmal ATP-Sensitive K+ Channels Control Energy Expenditure Determining Body Weight

Metabolic processes that regulate muscle energy use are major determinants of bodily energy balance. Here, we find that sarcolemmal ATP-sensitive K(+) (K(ATP)) channels, which couple membrane excitability with cellular metabolic pathways, set muscle energy expenditure under physiological stimuli. Disruption of K(ATP) channel function provoked, under conditions of unaltered locomotor activity and blood substrate availability, an extra energy cost of cardiac and skeletal muscle performance. Inefficient fuel metabolism in K(ATP) channel-deficient striated muscles reduced glycogen and fat body depots, promoting a lean phenotype. The propensity to lesser body weight imposed by K(ATP) channel deficit persisted under a high-fat diet, yet obesity restriction was achieved at the cost of compromised physical endurance. Thus, sarcolemmal K(ATP) channels govern muscle energy economy, and their downregulation in a tissue-specific manner could present an antiobesity strategy by rendering muscle increasingly thermogenic at rest and less fuel efficient during exercise.

Administration of Allogenic Stem Cells Dosed to Secure Cardiogenesis and Sustained Infarct Repair

Abstract: The mitotic capacity of heart muscle is too limited to fully substitute for cells lost following myocardial infarction. Emerging stem cell‐based strategies have been proposed to overcome the self‐renewal shortfall of native cardiomyocytes, yet there is limited evidence for their capability to achieve safe de novo cardiogenesis and repair. We present our recent experience in treating long‐term, infarcted hearts with embryonic stem cells, a prototype source for allogenic cell therapy. The cardiogenic potential of the engrafted murine embryonic stem cell colony was pre‐tested by in vitro differentiation, with derived cells positive for nuclear cardiac transcription factors, sarcomeric proteins and functional excitation‐contraction coupling. Eight weeks after infarct, rats were randomized into sham‐ or embryonic stem cell‐treated groups. Acellular sham controls or embryonic stem cells, engineered to express enhanced cyan fluorescent protein (ECFP) under control of the cardiac actin promoter, were injected through a 28‐gauge needle at three sites into the peri‐infarct zone for serial assessment of functional and structural impact. In contrast to results with sham‐treated animals, stem cell therapy yielded, over the 5‐month follow‐up period, new ECFP‐positive cardiomyocytes that integrated with the infarcted myocardium. The stem cell‐treated group showed a stable contractile performance benefit with normalization of myocardial architecture post infarction. Transition of embryonic stem cells into cardiomyocytes required host signaling to support cardiac‐specific differentiation and could result in tumorigenesis if the stem cell dose exceeded the hearts cardioinductive capacity. Supported by the host environment, proper dosing and administration of embryonic stem cells is thus here shown useful in the chronic management of cardiac injury promoting sustained repair.

Nuclear Reprogramming with c-Myc Potentiates Glycolytic Capacity of Derived Induced Pluripotent Stem Cells

Reprogramming strategies influence the differentiation capacity of derived induced pluripotent stem (iPS) cells. Removal of the reprogramming factor c-Myc reduces tumorigenic incidence and increases cardiogenic potential of iPS cells. c-Myc is a regulator of energy metabolism, yet the impact on metabolic reprogramming underlying pluripotent induction is unknown. Here, mitochondrial and metabolic interrogation of iPS cells derived with (4F) and without (3F) c-Myc demonstrated that nuclear reprogramming consistently reverted mitochondria to embryonic-like immature structures. Metabolomic profiling segregated derived iPS cells from the parental somatic source based on the attained pluripotency-associated glycolytic phenotype and discriminated between 3F versus 4F clones based upon glycolytic intermediates. Real-time flux analysis demonstrated a greater glycolytic capacity in 4F iPS cells, in the setting of equivalent oxidative capacity to 3F iPS cells. Thus, inclusion of c-Myc potentiates the pluripotent glycolytic behavior of derived iPS cells, supporting c-Myc-free reprogramming as a strategy to facilitate oxidative metabolism-dependent lineage engagement.