Approach For Cardiac Regeneration Research Articles

Membrane remodelling triggers maturation of excitation–contraction coupling in 3D-shaped human-induced pluripotent stem cell-derived cardiomyocytes

The prospective use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac regenerative medicine strongly depends on the electro-mechanical properties of these cells, especially regarding the Ca2+-dependent excitation–contraction (EC) coupling mechanism. Currently, the immature structural and functional features of hiPSC-CM limit the progression towards clinical applications. Here, we show that a specific microarchitecture is essential for functional maturation of hiPSC-CM. Structural remodelling towards a cuboid cell shape and induction of BIN1, a facilitator of membrane invaginations, lead to transverse (t)-tubule-like structures. This transformation brings two Ca2+ channels critical for EC coupling in close proximity, the L-type Ca2+ channel at the sarcolemma and the ryanodine receptor at the sarcoplasmic reticulum. Consequently, the Ca2+-dependent functional interaction of these channels becomes more efficient, leading to improved spatio-temporal synchronisation of Ca2+ transients and higher EC coupling gain. Thus, functional maturation of hiPSC-cardiomyocytes by optimised cell microarchitecture needs to be considered for future cardiac regenerative approaches.

Synthetic Fibroblasts: Terra Incognita in Cardiac Regeneration.

Ischemic heart disease, a major risk factor for myocardial infarction (MI), occurs when the blood vessels supplying oxygen-rich blood to the heart become partially or fully occluded by lipid-rich plaques, resulting in myocardial cell death, remodeling, and scarring. In addition, MI occurs as result of lipid-rich plaque rupture, resulting in thrombosis and vessel occlusion. Cardiac fibroblasts (CFs) and CF-derived growth factors are crucial post-MI in myocardial remodeling. Information regarding the regenerative phenotypes of CFs is scarce; however, regenerative CFs are translationally relevant in myocardial regeneration following MI. The emerging technologies in regenerative cardiology offer cutting-edge translational opportunities, including synthetic cells. In this review, we critically reviewed the current knowledge and the ongoing research efforts on application of synthetic cells for improving cardiac regeneration post-MI. Impact statement Synthetic cells offer tremendous regenerative potential in otherwise deleterious cardiac remodeling postmyocardial infarction. Understanding the role of fibroblasts in cardiac healing and the therapeutic applications of synthetic cells would open a multitude of novel cardiac regenerative approaches. The novel concept of synthetic fibroblasts that emulate native cardiac fibroblasts can provide an effective solution in cardiac healing.

A Brief History in Cardiac Regeneration, and How the Extra Cellular Matrix May Turn the Tide.

Tissue homeostasis is perturbed by stressful events, which can lead to organ dysfunction and failure. This is particularly true for the heart, where injury resulting from myocardial infarction or ischemic heart disease can result in a cascading event ultimately ending with the loss of functional myocardial tissue and heart failure. To help reverse this loss of healthy contractile tissue, researchers have spent decades in the hopes of characterizing a cell source capable of regenerating the injured heart. Unfortunately, these strategies have proven to be ineffective. With the goal of truly understanding cardiac regeneration, researchers have focused on the innate regenerative abilities of zebrafish and neonatal mammals. This has led to the realization that although cells play an important role in the repair of the diseased myocardium, inducing cardiac regeneration may instead lie in the composition of the extra cellular milieu, specifically the extra cellular matrix. In this review we will briefly summarize the current knowledge regarding cell sources used for cardiac regenerative approaches, since these have been extensively reviewed elsewhere. More importantly, by revisiting innate cardiac regeneration observed in zebrafish and neonatal mammals, we will stress the importance the extra cellular matrix has on reactivating this potential in the adult myocardium. Finally, we will address how we can harness the ability of the extra cellular matrix to guide cardiac repair thereby setting the stage of next generation regenerative strategies.

Abstract 532: Isolation of Human Placenta-derived Cdx2 Cells for Cardiac Regeneration

Coronary artery disease and heart failure are among the leading causes of mortality in United States and worldwide. The limited proliferation of the adult mammalian heart has prompted the need to recognize novel strategies that can restore contractile function in heart disease. We recently reported that cells expressing trophoblast marker Cdx2 isolated from murine end-gestation placentas are multipotent and immune-privileged, with homing ability to travel to sites of cardiac injury to regenerate injured myocardium. Since murine Cdx2 cells are immunologically naïve and trophoblast-mediated placentation is a conserved phenomenon, our findings are ripe for translational application. Here we demonstrate the expression of CDX2 and isolation of CDX2 cells from human term placentas highlighting a potentially critical cell source for allogeneic therapy for cardiac regeneration. We studied de-identified human term placenta tissues from three different anatomical sites from three different patients. Formalin-fixed paraffin embedded samples, second trimester chorionic villus samples and de-identified fresh chorion samples were utilized. Using a multiparametric approach including transcript analysis and Sanger sequencing, immunoblot and immunofluorescence analysis, with the subsequent screening of different anatomical sites, we observed that CDX2 is present in the chorionic (fetal cytotrophoblast) portion of human term placentas including the chorionic villous samples (CVS). Subsequent validation on positive control cells DLD1 (human colon carcinoma cell line), versus HeLa cells (low endogenous CDX2 expression) was performed. Visualization of CDX2 expression showed the nuclear localization in the villi region. We have further demonstrated using a CDX2 promoter-driven fluorescence-based lentiviral expression system, we can isolate viable CDX2 cells from fresh term placenta. We demonstrate for the first time that CDX2 cells are present and can be isolated from term human placentas for prospective cardiomyocgenic differentiation. A step closer to the translational approach, our results herein may represent a paradigmatic shift in the way we approach early embryonic lineages and cell fate choices towards the identification of an unexplored human cell-based approach for cardiac regeneration.

Toward Cardiac Regeneration: Combination of Pluripotent Stem Cell-Based Therapies and Bioengineering Strategies.

Cardiovascular diseases represent the major cause of morbidity and mortality worldwide. Multiple studies have been conducted so far in order to develop treatments able to prevent the progression of these pathologies. Despite progress made in the last decade, current therapies are still hampered by poor translation into actual clinical applications. The major drawback of such strategies is represented by the limited regenerative capacity of the cardiac tissue. Indeed, after an ischaemic insult, the formation of fibrotic scar takes place, interfering with mechanical and electrical functions of the heart. Hence, the ability of the heart to recover after ischaemic injury depends on several molecular and cellular pathways, and the imbalance between them results into adverse remodeling, culminating in heart failure. In this complex scenario, a new chapter of regenerative medicine has been opened over the past 20 years with the discovery of induced pluripotent stem cells (iPSCs). These cells share the same characteristic of embryonic stem cells (ESCs), but are generated from patient-specific somatic cells, overcoming the ethical limitations related to ESC use and providing an autologous source of human cells. Similarly to ESCs, iPSCs are able to efficiently differentiate into cardiomyocytes (CMs), and thus hold a real regenerative potential for future clinical applications. However, cell-based therapies are subjected to poor grafting and may cause adverse effects in the failing heart. Thus, over the last years, bioengineering technologies focused their attention on the improvement of both survival and functionality of iPSC-derived CMs. The combination of these two fields of study has burst the development of cell-based three-dimensional (3D) structures and organoids which mimic, more realistically, the in vivo cell behavior. Toward the same path, the possibility to directly induce conversion of fibroblasts into CMs has recently emerged as a promising area for in situ cardiac regeneration. In this review we provide an up-to-date overview of the latest advancements in the application of pluripotent stem cells and tissue-engineering for therapeutically relevant cardiac regenerative approaches, aiming to highlight outcomes, limitations and future perspectives for their clinical translation.

Design and characterization of an electroconductive scaffold for cardiomyocytes based biomedical assays.

Cardiovascular diseases (CVD) are a major cause of mortality worldwide. Accessibility to heart tissue is limited due to sampling issues and lack of appropriate culture conditions. In addition, animal models are not an ideal choice for physiological, pharmacological, and fundamental evaluations in the cardiovascular field due to interspecies differences. Hence, there is an inevitable need for functional in vitro cardiac models. In this study, we have synthesized a novel electroconductive scaffold comprised of cardiac extracellular matrix (ECM) derived pre-cardiogel (pCG) blended with polypyrrole (Ppy). Our data revealed that 2.5% (w/v) pyrrole (Py) had the highest possible Py ratio that provided pCG-Ppy gel formation. The prepared mixture was fabricated into a scaffold by using the freeze-dried method. The scaffolds had open interconnected pores that ranged from 55±24μm for the cardiogel (CG)-Ppy to 74±26μm for the CG scaffolds, with no alterations in vital ECM components of collagen, polysaccharides, and glycosaminoglycans (GAGs). Incorporation of Ppy increased the CG stiffness with a final complex modulus from 80 pa to 140 pa. The CG-Ppy group had significantly greater electrical conductivity than the CG group. Scaffolds supported neonatal mouse cardiomyocyte (NMCM) adhesion, viability, cardiac-specific gene expression, and spontaneous beating up to 14days after seeding. Among the fabricated hydrogels, the CG-Ppy group resulted in the synchronous beating of cardiomyocyte clusters and upregulation of cardiac genes involved in cardiac muscle contraction (cardiac troponin T [cTNT]) and cardiomyocyte electrical coupling (connexin 43 [Cx43]). Thus, this ECM-based electro-conductive scaffold might provide a promising substrate for constructing in vitro cardiac models for drug testing, disease modeling, developmental studies, and cardiac regenerative approaches.

Abstract 478: Small Molecule ICG-001, Sodium Butyrate, and Retinoic Acid Enhanced Direct Cardiac Reprogramming of Induced Cardiomyocytes (iCMs)

Background: Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) in situ represents a promising strategy for cardiac regeneration. A combination of three cardiac transcription factors Gata4, Mef2c and Tbx5 (GMT), and microRNAs can convert fibroblasts into iCMs, albeit with low efficiency in vitro . We aimed to increase the efficiency, maturation and speed of direct reprogramming using a unique combination of small molecules, in combination with previously identified reprogramming transcription factors, that we selected and combined in a cocktail specifically formulated to effect optimal impact on the aspects of reprogramming pathways thought to represent “roadblocks” or points of opportunity in these pathways. Methods: We screened a variety of such candidate compounds in primary rat/human cardiac fibroblast. Cardiac reprogramming was induced by lentivirus encoding Gata4, Mef2c and Tbx5 plus small molecules, and the presence of iCM was confirmed 10 days later by QRT-PCR, IFs and FACS analysis for the activation of endogenous cardiac troponin T (cTnT). Results: After validating and optimizing the dose and conditions for adding small molecules to the reprogramming cocktail, we treated GMT-overexpressing fibroblast with a combination of sodium butyrate (SB, 1mM; HDAC inhibitor), ICG-001(1μM; WNT inhibitor), and retinoic acid (RA, 1μM). We found that that a combination of SB, ICG-001, and RA significantly increased GMT-induced reprogramming efficiency to 22 % cTnT + iCMs from primary rat cardiac fibroblasts compared to 8% in the presence of single compounds and 3.5% without any added small molecule compound alone. QRT-PCR and IFs, analysis shows that a combination of the SB, ICG-001, and RA increased cTnT gene expression 4-6 fold when added to GMT-overexpressing rat cardiac fibroblasts. We also demonstrated that combined addition of SB, ICG-001, and RA increased cardiac fibroblast reprogramming efficiency 4 fold when added to GMTHM plus miR-590-overexpressing human cardiac fibroblasts. Conclusions: Thus, HDAC and WNT inhibitors along with RA, jointly accelerate GMT-induced cardiac reprogramming from rat/human cardiac fibroblasts and pave the way for new translational approaches for cardiac regeneration.

Cardiomyocyte Proliferation for Therapeutic Regeneration.

Current pharmacologic treatments for cardiovascular disease do not correct the underlying cellular defect, the loss of cardiomyocytes. With recent advancements in cardiac regenerative approaches, the induction of endogenous mature cardiomyocyte proliferation has emerged as a new possibility. Here, we review progress made toward the regeneration of cardiac tissue in the mammalian heart through the stimulation of mature cardiomyocyte renewal. The targeting of several developmental and signaling pathways has been shown to stimulate cell cycle re-entry in mature cardiomyocytes. In animal models of cardiac regeneration, various strategies have been used to target these pathways to stimulate cardiomyocyte renewal and have relied on the delivery of signaling factors via systemic delivery, epicardial patches, or direct intramyocardial injection. Gene therapy techniques involving the viral delivery of transgenes by using adenoviral or adeno-associated viral vectors have been used to successfully target cardiac gene expression. The delivery of nucleic acids in the form of anti-microRNAs and microRNA mimetics has also been shown to be effective in stimulating cardiomyocyte renewal. As the field of cardiac regeneration continues to progress, an important ongoing challenge in developing clinically translatable therapies is limiting the stimulation of growth pathways in non-cardiomyocytes.

Abstract 35: Enhanced Reprogramming of Human Fibroblasts into Cardiomyocytes Using Minimal Transcription Factors

The adult human heart has limited regenerative capacity and is thus an important target for novel regenerative approaches to replenish lost cardiomyocytes after cardiac injury. Cardiac reprogramming that converts fibroblasts to contractile induced cardiomyocytes (iCMs) by overexpressing cardiac lineage specific transcription factors holds great promise as an alternative approach for cardiac regeneration and disease modeling. Significant advance has been made to generate mouse iCMs; however, human iCM (hiCM) generation remains challenging and the yield is low for clinical applications. Here, we leveraged the knowledge that we learned from studying mouse iCM reprogramming to define the optimal condition for hiCM induction. We titrated the dosage of the human reprogramming factors systematically and surprisingly found the minimal core components GATA4, MEF2C and TBX5 were sufficient to induce cardiac fate in human primary fibroblasts. This is in sharp contrast to what has been reported in the literature. Subsequently, we cloned these three factors into a polycistronic vector separated by 2A peptides for defined ratio of protein expression. By optimizing the growth condition, we further improved the efficiency of hiCM reprogramming. Mechanistically, we found the balanced expression of this minimal combination of transcription factors with tailored microenvironment enhanced the establishment of cardiac program in non-myocytes. In sum, our study demonstrates that the use of a single vector with only three transcription factors simplifies generation and improves the yield of hiCMs for potential future clinical applications.

Towards regenerating the mammalian heart: challenges in evaluating experimentally induced adult mammalian cardiomyocyte proliferation

In recent years, there has been a dramatic increase in research aimed at regenerating the mammalian heart by promoting endogenous cardiomyocyte proliferation. Despite many encouraging successes, it remains unclear if we are any closer to achieving levels of mammalian cardiomyocyte proliferation for regeneration as seen during zebrafish regeneration. Furthermore, current cardiac regenerative approaches do not clarify whether the induced cardiomyocyte proliferation is an epiphenomena or responsible for the observed improvement in cardiac function. Moreover, due to the lack of standardized protocols to determine cardiomyocyte proliferation in vivo, it remains unclear if one mammalian regenerative factor is more effective than another. Here, we discuss current methods to identify and evaluate factors for the induction of cardiomyocyte proliferation and challenges therein. Addressing challenges in evaluating adult cardiomyocyte proliferation will assist in determining 1) which regenerative factors should be pursued in large animal studies; 2) if a particular level of cell cycle regulation presents a better therapeutic target than another (e.g., mitogenic receptors vs. cyclins); and 3) which combinatorial approaches offer the greatest likelihood of success. As more and more regenerative studies come to pass, progress will require a system that not only can evaluate efficacy in an objective manner but can also consolidate observations in a meaningful way.

Abstract 17262: MicroRNA-23b Mediated Inhibition of Gsk-3β and Nischarin Promotes Proliferation of Cardiomyocytes

Background: Promoting proliferation of cardiomyocytes (CMs) following myocardial infarction is a promising approach for cardiac regeneration. Recent studies from our lab identified a panel of microRNAs inducing proliferation of adult CMs; of these miR-23b was one of the most robust CM proliferation-inducing miR and is conserved across species. In silico analysis identified binding sites for miR-23b on the 3’UTRs of both GSK-3β (GSK) and nischarin (NISCH). Here, we hypothesize that miR-23b promotes both in vitro and in vivo proliferation of CMs by regulating the expression of cell cycle inhibitors GSK and NISCH. Methods and Results: Adult CMs isolated from 8-12 week old female rats were transfected with hsa-miR-23b, cel-miR-67 (control), siGSK, siNISCH, siGSK + siNISCH (si=siRNA). EdU was added (5 μM) each day post-transfection and cells were fixed on day 6 for IF staining (Troponin-I, EdU, pH3, and DAPI), protein, and RNA analysis. A significant increase in EdU+ CMs was found in cells transfected with miR-23b (8.51%±0.78), siGSK (8.30%±1.31) and siNISCH (5.73%±1.52) when compared to control miR cel-67 (3.13%±0.84). Interestingly, simultaneous inhibition of both GSK and NISCH exhibited robust proliferation (13.88%±1.15) similar to miR-23b transfection. Luciferase assay confirmed binding sites for miR-23b on the 3’UTR of GSK with a 0.85±0.04 fold reduction of chemiluminescent activity. Compared to control, western blotting following miR-23b transfection revealed an increase in pERK (2.46±0.69 fold), a decrease in GSK (0.18±0.12 fold), and an increase in β-catenin (4.17±2.03 fold), indicating miR-23b mediated inhibition of GSK and subsequent increase in β-catenin. For in vivo analysis, 6 day old Fischer rats received intraperitoneal adenoviral miR-23b (10^11 viral particles) followed by EdU injections on alternate days. On day 6, rats were sacrificed, and the hearts were fixed/sectioned for IF staining. Rats injected with miR-23b expressed higher levels of EdU (2.42±0.46 fold), and decreased fold of GSK (0.32±0.10) and NISCH (0.29±0.17) compared to control as confirmed via IHC staining. Conclusion: MiR-23b promotes proliferation of CMs by simultaneous inhibition of both GSK and NISCH and upregulation of downstream targets pERK and β-catenin.

Reduced matrix rigidity promotes neonatal cardiomyocyte dedifferentiation, proliferation and clonal expansion.

Cardiomyocyte (CM) maturation in mammals is accompanied by a sharp decline in their proliferative and regenerative potential shortly after birth. In this study, we explored the role of the mechanical properties of the underlying matrix in the regulation of CM maturation. We show that rat and mouse neonatal CMs cultured on rigid surfaces exhibited increased myofibrillar organization, spread morphology, and reduced cell cycle activity. In contrast, compliant elastic matrices induced features of CM dedifferentiation, including a disorganized sarcomere network, rounding, and conspicuous cell-cycle re-entry. The rigid matrix facilitated nuclear division (karyokinesis) leading to binucleation, while compliant matrices promoted CM mitotic rounding and cell division (cytokinesis), associated with loss of differentiation markers. Moreover, the compliant matrix potentiated clonal expansion of CMs that involves multiple cell divisions. Thus, the compliant microenvironment facilitates CM dedifferentiation and proliferation via its effect on the organization of the myoskeleton. Our findings may be exploited to design new cardiac regenerative approaches.

A New Paradigm in Cardiac Regeneration: The Mesenchymal Stem Cell Secretome.

The potentialities to apply mesenchymal stem cells (MSCs) in regenerative medicine have been extensively studied over the last decades. In the cardiovascular disease (CVD) field, MSCs-based therapy is the subject of great expectations. Its therapeutic potential has been already shown in several preclinical models and both the safety and efficacy of MSCs-based therapy are being evaluated in humans. It is now clear that the predominant mechanism by which MSCs participate in heart tissue repair is through a paracrine activity. Via the production of a multitude of trophic factors endowed with different properties, MSCs can reduce tissue injury, protect tissue from further adverse effects, and enhance tissue repair. The present review discusses the current understanding of the MSCs secretome as a therapy for treatment of CVD. We provide insights into the possible employment of the MSCs secretome and their released extracellular vesicles as novel approaches for cardiac regeneration that would have certain advantages over injection of living cells.

Concise Review: Engineering Myocardial Tissue: The Convergence of Stem Cells Biology and Tissue Engineering Technology

Abstract Advanced heart failure represents a leading public health problem in the developed world. The clinical syndrome results from the loss of viable and/or fully functional myocardial tissue. Designing new approaches to augment the number of functioning human cardiac muscle cells in the failing heart serve as the foundation of modern regenerative cardiovascular medicine. A number of clinical trials have been performed in an attempt to increase the number of functional myocardial cells by the transplantation of a diverse group of stem or progenitor cells. Although there are some encouraging suggestions of a small early therapeutic benefit, to date, no evidence for robust cell or tissue engraftment has been shown, emphasizing the need for new approaches. Clinically meaningful cardiac regeneration requires the identification of the optimum cardiogenic cell types and their assembly into mature myocardial tissue that is functionally and electrically coupled to the native myocardium. We here review recent advances in stem cell biology and tissue engineering and describe how the convergence of these two fields may yield novel approaches for cardiac regeneration. Stem Cells 2013;31:2587–2598

Poly(glycerol sebacate)\Poly(butylene succinate‐dilinoleate) Blends as Candidate Materials for Cardiac Tissue Engineering

SummaryPoly (glycerol sebacate) (PGS) and poly (butylene succinate‐dilinoleate) (PBS‐DLA) are biodegradable polymers with potential application in cardiac tissue engineering. In the present study novel blends comprising PGS prepolymer and PBS‐DLA were prepared with varying compositions (70/30, 60/40, 50/50, 40/60, 30/70 and 0/100 in weight percentage). The physical, chemical, and mechanical properties of the PGS/PBS‐DLA blends were measured and compared. By adding PBS‐DLA to PGS the need for curing PGS prepolymer was eliminated, as the blended films are chemically stable. With increasing amount of PBS‐DLA the hydrophobicity of the blend system increased reaching values close to that of neat PBS‐DLA films. Furthermore, addition of PBS‐DLA significantly affected the mechanical properties of the blends, i.e. the elastic modulus of the blends was enhanced with increasing PBS‐DLA addition from 1.2 MPa to 54 MPa. At the same time, PBS‐DLA addition led to decreased degradation rate of the films. Furthermore the PBS‐DLA counteracted the acidity of the free carboxylic groups on the free end chains of the PGS prepolymer. In vitro cytocompatibility studies indicated high biocompatibility. Taken together the results confirm that the novel PGS/PBS‐DLA matrices exhibit promising characteristics as a biomaterial for application in cardiac regeneration approaches.

The Non-coding Road Towards Cardiac Regeneration

Our understanding of cardiovascular disease has evolved rapidly, leading to a number of treatments that have improved patient quality of life and mortality rates. However, there is still no cure for heart failure. This has led to the pursuit of cardiac regeneration to prevent, and ultimately cure, this debilitating condition. To this end, several approaches have been proposed, including activation of cardiomyocyte proliferation, activation of endogenous or exogenous stem/progenitor cells, delivery of de novo cardiomyocytes, and in situ direct reprogramming of cardiac fibroblasts. While these different methodologies are currently being intensely investigated, there are still a number of caveats limiting their application in the clinic. Given the emerging regulatory potential of non-coding RNAs for controlling diverse cellular processes, these molecules may offer potential solutions in this pursuit of cardiac regeneration. In this concise review, we discuss the potential role of non-coding RNAs in a variety of different cardiac regenerative approaches.

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Cardiospheres (CSs) are self-assembling multicellular clusters from the cellular outgrowth from cardiac explants cultured in nonadhesive substrates. They contain a core of primitive, proliferating cells, and an outer layer of mesenchymal/stromal cells and differentiating cells that express cardiomyocyte proteins and connexin 43. Because CSs contain both primitive cells and committed progenitors for the three major cell types present in the heart, that is, cardiomyocytes, endothelial cells, and smooth muscle cells, and because they are derived from percutaneous endomyocardial biopsies, they represent an attractive cell source for cardiac regeneration. In preclinical studies, CS-derived cells (CDCs) delivered to infarcted hearts resulted in improved cardiac function. CDCs have been tested safely in an initial phase-1 clinical trial in patients after myocardial infarction. Whether or not CDCs are superior to purified populations, for example, c-kit+ cardiac stem cells, or to gene therapy approaches for cardiac regeneration remains to be evaluated.

Abstract P015: Hypoxia Regulates Embryonic Cardiac Myocyte Proliferation and Differentiation

Mammalian cardiac myocytes (CM) proliferate readily during development but exit the cell cycle perinatally. The molecular mechanism for this is poorly understood could lead to new approaches for cardiac regeneration. We employed a novel bioinformatics approach to identify transcription factors that may control perinatal cell-cycle arrest and identified hypoxia inducible factor 1α (Hif1α) as a potential regulator of CM proliferation and differentiation. To test our hypothesis, e12.5d embryonic heart cells were cultured in atmospheric (17%) or hypoxic (3%) [O2] and then assessed for proliferation over 4 days. Total cell numbers were significantly increased (∼2 fold, P<0.005) in cells cultured in 3% vs. 17% [O2] after 4 days. Hypoxia increased numbers of cTn+ cells labeled with p-Histone-H3 indicating an increase in CM proliferation. Carboxyfluorescein succinimidyl ester staining indicated that the majority (70%) of cells cultured in 3% [O2] divided 2–3 times while the majority (73%) of cells in 17% [O2] divided 0–1 times after 3 days of culture. As expected, hypoxia stabilized the Hif1α protein and induced the expression of Vegf. Messenger RNA for the G1-S phase cyclin dependent kinase Ccnd2 was increased (>2 fold) in hypoxic vs. atmospheric cultured cells indicating that hypoxia my increase proliferation by regulating the expression of cell cycle machinery. Furthermore, early mesodermal transcription factors Brachyury and Mesp1 and the CM progenitor marker Nkx2.5 were induced (>5 fold) in hypoxic vs. atmospheric [O2] cultured cells suggesting that hypoxia positively regulates early mesodermal and CM progenitor cell differentiation. To validate this hypothesis, we differentiated mouse embryonic stem cells in atmospheric and hypoxic [O2] and observed similar increases in the expression of Brachyury, Mesp1 and Nkx2.5 in hypoxic differentiated cells. Transient culture (2 d) of embryoid bodies (EBs) in hypoxia increased the frequency of beating EBs and the % of cTn+ cells compared to cells differentiated in atmospheric O2. Taken together these data suggest that the hypoxic environment in which CMs develop may serve as a key signal that regulates mesodermal progenitor marker expression and CM progenitor cell differentiation and proliferation.

Direct Reprogramming of Fibroblasts into Cardiomyocytes-A New Approach for Cardiac Regeneration

Generating iPS cells by a combination of defined factors demonstrated that somatic cells can be induced to change their cell fate. Many fibroblasts exist in the post-natal heart, but no single master regulator of cardiac reprogramming has been identified. We postulated that not a single, but a combination of cardiogenic factors may reprogram fibroblasts into cardiomyocytes. We selected 14 candidate factors which are specifically expressed in embryonic cardiomyocytes and exhibited severe developmental cardiac defects and embryonic lethality when mutated. Among them, a combination of three developmental transcription factors rapidly and efficiently reprogrammed post-natal cardiac fibroblasts directly into cardiomyocytes. Induced cardiomyocytes expressed cardiac-specific proteins, exhibited a global gene expression profile and an epigenetic state similar to cardiomyocytes, and contracted spontaneously. Induced cardiomyocytes were not converted into the cardiac progenitor state for reprogramming, but rather they were directly reprogrammed into differentiated cardiomyocytes by defined factors. Moreover, fibroblast cells transplanted into mouse hearts one day after transduction of the three factors differentiated into cardiomyocytes in vivo. These findings demonstrate that functional cardiomyocytes can be directly reprogrammed from differentiated somatic cells by defined factors. Reprogramming the vast pool of endogenous cardiac fibroblasts might provide a source of cardiomyocytes for regenerative therapies.

The Next Generation of Review for Cardiac Cell Therapies

Heart failure is the leading cause of morbidity and mortality in the Western World, affecting approximately 5 million people in the United States [1] and at least 10 million people in Western Europe [2]. Heart transplantation remains the best available treatment option for end-stage heart disease; however, donor availability is limited. Therapeutic options currently available to clinicians have a limited role in improving outcomes in patients with heart failure. Cell-based therapies for heart disease have shown some promise to restore cardiac function in both preclinical and early clinical trials though many scientists and clinicians are still unable to determine which the most appropriate cell to utilize is and the clear mechanism of action within the cardiac tissue. With the excitement of cell therapy for heart disease came numerous papers touting the effectiveness of many different cell types in various small and large animal models. Along with the plethora of experimental data regarding cellular therapy for heart disease, many reviews were published to help both researchers and clinicians understand the types of cells under investigation for heart disease. Most of these reviews were organized by types of cells (i.e., endothelial progenitor cell, mesenchymal stem cells, embryonic stem cells, etc.) and detailed the various positive data, the limitations of the cells, and the future clinical relevance of the cells for different myocardial applications. The review by Stamm et al. [3] not only gives researchers and clinicians the most current up-to-date information regarding cell therapies for cardiac disease, but is organized by the type of regenerative therapy and not the type of cell. Stamm et al. group the different cell therapies into three areas of cardiac regeneration: direct, indirect, and intrinsic. Stamm et al. go on to comment on the potential clinical applicability of each of the three approaches for cardiac regeneration with direct regeneration holding the most promise. The reader is not bombarded with lineage markers but is given just enough technical description of each cell type so as to allow for clear understanding of which cell type is being reviewed. Stamm et al. also sprinkle into the text some illustrative data and insightful comments regarding their own experience in the cardiac cell therapy arena. In addition, Stamm et al. include recent updates on the legal status of the therapeutic use of cells in both North America and Europe along with keen perspective on the progress and pitfalls industrial and academic forays into cell therapy. Overall, Stamm et al. have taken years of experimental and clinical data in the field of regenerative cell therapy for cardiac disease and condensed it into a smartly organized review of the current and next generation cell therapies for heart disease.