Jamie L. Ifkovits

Injectable hydrogel properties influence infarct expansion and extent of postinfarction left ventricular remodeling in an ovine model

A recent trend has emerged that involves myocardial injection of biomaterials, containing cells or acellular, following myocardial infarction (MI) to influence the remodeling response through both biological and mechanical effects. Despite the number of different materials injected in these approaches, there has been little investigation into the importance of material properties on therapeutic outcomes. This work focuses on the investigation of injectable hyaluronic acid (MeHA) hydrogels that have tunable mechanics and gelation behavior. Specifically, two MeHA formulations that exhibit similar degradation and tissue distribution upon injection but have differential moduli (~8 versus ~43 kPa) were injected into a clinically relevant ovine MI model to evaluate the associated salutary effect of intramyocardial hydrogel injection on the remodeling response based on hydrogel mechanics. Treatment with both hydrogels significantly increased the wall thickness in the apex and basilar infarct regions compared with the control infarct. However, only the higher-modulus (MeHA High) treatment group had a statistically smaller infarct area compared with the control infarct group. Moreover, reductions in normalized end-diastolic and end-systolic volumes were observed for the MeHA High group. This group also tended to have better functional outcomes (cardiac output and ejection fraction) than the low-modulus (MeHA Low) and control infarct groups. This study provides fundamental information that can be used in the rational design of therapeutic materials for treatment of MI.

Optimization of Direct Fibroblast Reprogramming to Cardiomyocytes Using Calcium Activity as a Functional Measure of Success

Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persist for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods.

Injectable acellular hydrogels for cardiac repair.

Injectable hydrogels are being developed as potential translatable materials to influence the cascade of events that occur after myocardial infarction. These hydrogels, consisting of both synthetic and natural materials, form through numerous chemical crosslinking and assembly mechanisms and can be used as bulking agents or for the delivery of biological molecules. Specifically, a range of materials are being applied that alter the resulting mechanical and biological signals after infarction and have shown success in reducing stresses in the myocardium and limiting the resulting adverse left ventricular (LV) remodeling. Additionally, the delivery of molecules from injectable hydrogels can influence cellular processes such as apoptosis and angiogenesis in cardiac tissue or can be used to recruit stem cells for repair. There is still considerable work to be performed to elucidate the mechanisms of these injectable hydrogels and to optimize their various properties (e.g., mechanics and degradation profiles). Furthermore, although the experimental findings completed to date in small animals are promising, future work needs to focus on the use of large animal models in clinically relevant scenarios. Interest in this therapeutic approach is high due to the potential for developing percutaneous therapies to limit LV remodeling and to prevent the onset of congestive heart failure that occurs with loss of global LV function. This review focuses on recent efforts to develop these injectable and acellular hydrogels to aid in cardiac repair.

Inhibition of TGFβ Signaling Increases Direct Conversion of Fibroblasts to Induced Cardiomyocytes

Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ∼5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic.

Influence of Injectable Hyaluronic Acid Hydrogel Degradation Behavior on Infarction Induced Ventricular Remodeling

Increased myocardial wall stress after myocardial infarction (MI) initiates the process of adverse left ventricular (LV) remodeling that is manifest as progressive LV dilatation, loss of global contractile function, and symptomatic heart failure, and recent work has shown that reduction in wall stress through injectable bulking agents attenuates these outcomes. In this study, hyaluronic acid (HA) was functionalized to exhibit controlled and tunable mechanics and degradation once cross-linked, in an attempt to assess the temporal dependency of mechanical stabilization in LV remodeling. Specifically, two hydrolytically degrading (low and high HeMA-HA, degrading in ~3 and 10 weeks, respectively) and two stable (low and high MeHA, little mass loss even after 8 weeks) hydrogels with similar initial mechanics (low: ~7 kPa; high: ~35-40 kPa) were evaluated in an ovine model of MI. Generally, the more stable hydrogels maintained myocardial wall thickness in the apical and basilar regions more efficiently (low MeHA: apical: 6.5 mm, basilar: 7 mm, high MeHA: apical: 7.0 mm basilar: 7.2 mm) than the hydrolytically degrading hydrogels (low HeMA-HA: apical: 3.5 mm, basilar: 6.0 mm, high HeMA-HA: apical: 4.1 mm, basilar: 6.1 mm); however, all hydrogel groups were improved compared to infarct controls (IC) (apical: 2.2 mm, basilar: 4.6 mm). Histological analysis at 8 weeks demonstrated that although both degradable hydrogels resulted in increased inflammation, all treatments resulted in increased vessel formation compared to IC. Further evaluation revealed that while high HeMA-HA and high MeHA maintained reduced LV volumes at 2 weeks, high MeHA was more effective at 8 weeks, implying that longer wall stabilization is needed for volume maintenance. All hydrogel groups resulted in better cardiac output (CO) values than IC.

Light‐Induced Temperature Transitions in Biodegradable Polymer and Nanorod Composites

Shape-memory materials (including polymers, metals, and ceramics) are those that are processed into a temporary shape and respond to some external stimuli (e.g., temperature) to undergo a transition back to a permanent shape.[1, 2] Shape memory polymers are finding use in a range of applications from aerospace to fabrics, to biomedical devices and microsystem components.[3–5] For many applications, it would be beneficial to initiate heating with an external trigger (e.g., transdermal light exposure). In this work, we formulated composites of gold nanorods (<1% by volume) and biodegradable networks, where exposure to infrared light induced heating and consequently, shape transitions. The heating is repeatable and tunable based on nanorod concentration and light intensity and the nanorods did not alter the cytotoxicity or in vivo tissue response to the networks.

Biodegradable fibrous scaffolds with tunable properties formed from photo-cross-linkable poly(glycerol sebacate).

It is becoming increasingly apparent that the architecture and mechanical properties of scaffolds, particularly with respect to mimicking features of natural tissues, are important for tissue engineering applications. Acrylated poly(glycerol sebacate) (Acr-PGS) is a material that can be cross-linked upon exposure to ultraviolet light, leading to networks with tunable mechanical and degradation properties through simple changes during Acr-PGS synthesis. For example, the number of acrylate functional groups on the macromer dictates the concentration of cross-links formed in the resulting network. Three macromers were synthesized that form networks that vary dramatically with respect to their tensile modulus ( approximately 30 kPa to 6.6 MPa) and degradation behavior ( approximately 20-100% mass loss at 12 weeks) based on the extent of acrylation ( approximately 1-24%). These macromers were processed into biodegradable fibrous scaffolds using electrospinning, with gelatin as a carrier polymer to facilitate fiber formation and cell adhesion. The resulting scaffolds were also diverse with respect to their mechanics (tensile modulus ranging from approximately 60 kPa to 1 MPa) and degradation ( approximately 45-70% mass loss by 12 weeks). Mesenchymal stem cell adhesion and proliferation on all fibrous scaffolds was indistinguishable from those of controls. The scaffolds showed similar diversity when implanted on the surface of hearts in a rat model of acute myocardial infarction and demonstrated a dependence on the scaffold thickness and chemistry in the host response. In summary, these diverse scaffolds with tailorable chemical, structural, mechanical, and degradation properties are potentially useful for the engineering of a wide range of soft tissues.

Integration of Bmp and Wnt signaling by Hopx specifies commitment of cardiomyoblasts

Making cardiomyocytes In the heart, multiple cell types work together. Cardiac progenitor cells give rise to cardiomyocyte, endothelial, or smooth muscle lineages. However, the identity of a marker specific to cardiomyocyte formation has been elusive. Jain et al. now identify a specialized progenitor population that is committed exclusively to forming cardiomyocytes. They also identify the niche signals that promote lineage commitment and the mechanisms involved in making cardiomyocytes. The findings may help in the development of future cell-based regenerative therapeutics for heart disease. Science, this issue 10.1126/science.aaa6071 Identification of the committed cardiomyoblast that retains proliferative potential may inform cardiac regenerative therapeutics. INTRODUCTION Cardiac progenitor cells are multipotent, and lineage analyses of murine and chick cardiac development have demonstrated that these cells give rise to the cardiac endothelium, smooth muscle, and cardiomyocytes. However, the mechanisms governing commitment to the myocyte lineage in vivo remain largely unknown. Further understanding of these mechanisms, and of the identity of progenitors committed to the myocyte lineage, may advance cardiac regenerative therapies. RATIONALE Hopx is an atypical homeodomain expressed in cardiac mesoderm shortly after cardiac progenitor cells are first evident. Previous studies have demonstrated that Hopx functions as a nuclear transcription co-repressor and is expressed in adult, +4 intestinal stem cells and hair follicle bulge stem cells. We compare lineage tracing of multipotent cardiac progenitor cells marked by Islet1 and Nkx2-5 expression with lineage tracing of Hopx+ cells. We also perform functional studies of Hopx from endogenous tissue and differentiated embryoid bodies to identify mechanisms promoting commitment and myogenesis. RESULTS We define and characterize a Hopx-expressing cardiomyoblast intermediate that is committed to the cardiomyocyte fate. Hopx+ is initially expressed in a subset of cardiac progenitor cells residing in the precardiac mesoderm prior to the expression of troponin T, a component of the contractile sarcomere apparatus of myocytes. Lineage-tracing experiments demonstrate that Hopx+ cells give rise to cardiac myocytes exclusively. Early Hopx+ cardiomyoblasts expand during cardiogenesis. Overexpression of Hopx in cardiac progenitor cells leads to an increase in myocytes, whereas Hopx deficiency compromises myogenesis. Whole-genome analysis reveals that Hopx occupies regulatory regions of multiple Wnt-related genes, and Hopx–/– cardiac tissues are characterized by an expansion of Wnt signaling. Restoration of Wnt levels during differentiation of Hopx–/– embryoid bodies partially rescues myogenesis. Wnt signaling is a potent regulator of stemness of cardiac progenitor cells, and our data suggest that Hopx promotes myogenesis by repressing Wnt signaling. Cardiac progenitor cells down-regulate Wnt signaling as they enter the cardiac outflow tract, coincident with the expression of Hopx. The outflow tract is also enriched for bone morphogenetic protein (Bmp) signaling, known to influence differentiation of myocytes. Hopx physically interacts with activated Smad complexes in vitro and in vivo. Exogenous Bmp4 represses Wnt signaling in cardiac explants, and Bmp4-mediated Wnt repression requires Hopx. Thus, Hopx functions to couple Bmp signaling to repression of Wnt. CONCLUSION Our work defines an intermediate cardiac progenitor that expresses Hopx and is committed exclusively to the myocyte fate. Therefore, akin to an erythroblast in hematopoietic differentiation, we have termed these committed cardiac progenitor cells “cardiomyoblasts.” The ability to identify committed, but undifferentiated, cardiomyocyte precursors may facilitate development of cardiac regenerative therapies, including those using embryonic stem cells and induced pluripotent stem cells. Hopx functions to promote myogenesis by physically interacting with Smad proteins to repress Wnt signaling. Our findings raise the possibility that Hopx-mediated integration of Bmp signaling to repress Wnt may be active in other progenitor populations and may potentially underlie the tumor suppressor function of Hopx. Lineage tracing of Hopx+ cells. Images depicting lineage tracing of early Hopx+ cardiomyoblasts that give rise to myocytes in the left ventricle and atria. Some images are duplicated and pseudocolored. Cardiac progenitor cells are multipotent and give rise to cardiac endothelium, smooth muscle, and cardiomyocytes. Here, we define and characterize the cardiomyoblast intermediate that is committed to the cardiomyocyte fate, and we characterize the niche signals that regulate commitment. Cardiomyoblasts express Hopx, which functions to coordinate local Bmp signals to inhibit the Wnt pathway, thus promoting cardiomyogenesis. Hopx integrates Bmp and Wnt signaling by physically interacting with activated Smads and repressing Wnt genes. The identification of the committed cardiomyoblast that retains proliferative potential will inform cardiac regenerative therapeutics. In addition, Bmp signals characterize adult stem cell niches in other tissues where Hopx-mediated inhibition of Wnt is likely to contribute to stem cell quiescence and to explain the role of Hopx as a tumor suppressor.

Electrospinning of photocrosslinked and degradable fibrous scaffolds

Electrospun fibrous scaffolds are being developed for the engineering of numerous tissues. Advantages of electrospun scaffolds include the similarity in fiber diameter to elements of the native extracellular matrix and the ability to align fibers within the scaffold to control and direct cellular interactions and matrix deposition. To further expand the range of properties available in fibrous scaffolds, we developed a process to electrospin photocrosslinkable macromers from a library of multifunctional poly(beta-amino ester)s. In this study, we utilized one macromer (A6) from this library for initial examination of fibrous scaffold formation. A carrier polymer [poly(ethylene oxide) (PEO)] was used for fiber formation because of limitations in electrospinning A6 alone. Various ratios of A6 and PEO were successfully electrospun and influenced the scaffold fiber diameter and appearance. When electrospun with a photoinitiator and exposed to light, the macromers crosslinked rapidly to high double bond conversions and fibrous scaffolds displayed higher elastic moduli compared to uncrosslinked scaffolds. When these fibers were deposited onto a rotating mandrel and crosslinked, organized fibrous scaffolds were obtained, which possessed higher moduli (approximately 4-fold) in the fiber direction than perpendicular to the fiber direction, as well as higher moduli (approximately 12-fold) than that of nonaligned crosslinked scaffolds. With exposure to water, a significant mass loss and a decrease in mechanical properties were observed, correlating to a rapid initial loss of PEO which reached an equilibrium after 7 days. Overall, these results present a process that allows for formation of fibrous scaffolds from a wide variety of possible photocrosslinkable macromers, increasing the diversity and range of properties achievable in fibrous scaffolds for tissue regeneration.

Biodegradable and radically polymerized elastomers with enhanced processing capabilities

The development of biodegradable materials with elastomeric properties is beneficial for a variety of applications, including for use in the engineering of soft tissues. Although others have developed biodegradable elastomers, they are restricted by their processing at high temperatures and under vacuum, which limits their fabrication into complex scaffolds. To overcome this, we have modified precursors to a tough biodegradable elastomer, poly(glycerol sebacate) (PGS) with acrylates to impart control over the crosslinking process and allow for more processing options. The acrylated-PGS (Acr-PGS) macromers are capable of crosslinking through free radical initiation mechanisms (e.g., redox and photo-initiated polymerizations). Alterations in the molecular weight and % acrylation of the Acr-PGS led to changes in formed network mechanical properties. In general, Youngs modulus increased with % acrylation and the % strain at break increased with molecular weight when the % acrylation was held constant. Based on the mechanical properties, one macromer was further investigated for in vitro and in vivo degradation and biocompatibility. A mild to moderate inflammatory response typical of implantable biodegradable polymers was observed, even when formed as an injectable system with redox initiation. Moreover, fibrous scaffolds of Acr-PGS and a carrier polymer, poly(ethylene oxide), were prepared via an electrospinning and photopolymerization technique and the fiber morphology was dependent on the ratio of these components. This system provides biodegradable polymers with tunable properties and enhanced processing capabilities towards the advancement of approaches in engineering soft tissues.