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Background: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. Methods: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. Results: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN , the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient’s condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. Conclusions: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

Introduction: Hypertrophic cardiomyopathy (HCM) is the most commonly inherited cardiac disease affecting 1:500 to 1:200 individuals worldwide. HCM has a heterogeneous genetic profile and phenotypic expression. More than 1400 known pathogenic variants have been identified in 11 sarcomere genes. In about 40% of HCM patients, the genetic cause may not be identified. The same mutation may lead to different phenotypes and severity in different individuals. Identification of novel HCM genes and modifiers will expand our understanding of the signaling pathways that are responsible for phenotypic expression of HCM. Methods: The UK Biobank comprises clinical and genetic data for greater than 500,000 individuals. We used OASIS, an information system for analyzing, searching, and visualizing associations between phenotype and genotype data to analyze this data. We compared control individuals to HCM individuals identified by ICD-10 code (I42.1 and I42.2) in a 20-to-1 fashion. Related individuals and those with confounding diagnoses were excluded. Results: The analysis was performed with Plink’s GLM option, and we identified 84 variants with a minor allele frequency of 0.5% or greater in 65 genes associated with HCM with a p < 1x10 -6 , including 4 with p < 5x10 -8 . The identified genes encode lncRNAs, miRNAs, and membrane proteins. Variants with high significance were identified in the genes encoding putative ciliary components DNAL4 (dynein axonemal light chain 4; p = 2.9x10 -8 ), MYO1D (unconventional myosin 1D; p = 3.1x10 -8 ), ITFAP (intraflagellar transport associated protein; p = 9.5x10 -8 ), CABCOCO1 (ciliary associated calcium biding coiled-coil 1; p = 3.7x 10 -7 ), EVL (Enah-Vasp-like; p = 4.4x 10 -7 ) and IFT122 (intraflagellar transport 122; p = 8.0 x10 -7 ). Conclusion: While none of these have previously associated with HCM, our findings suggest ciliary structure and function may play a role in disease manifestation. Our method is unique by pooling individuals in a large population set to identify potential causative or contributing mutations. Bioinformatic tools, such as OASIS, allow for the identification of previously unrecognized variants that may play a role in the development of HCM. This approach has identified numerous novel genes as possible risk loci.

Hypertrophic cardiomyopathy (HCM) is characterized by heterogeneous phenotypic expression, natural history, and genetic profile with numerous causative mutations. The relationship between genotype and phenotype in HCM is incompletely understood. Identifying genetic modifiers may expand understanding of the signaling pathways that are responsible for phenotypic expression. UK Biobank comprises clinical and genetic data for greater than 500,000 individuals. We compared control individuals to those with a diagnosis of HCM (identified via ICD-10 code I42.1) in a 20-to-1 fashion to identify genetic differences. Data were compared using Mixed Model Analysis for Pedigrees/Populations (MMAP) mixed-model regression. Variants were assessed using bioinformatics tools including linkage disequilibrium and Eigen scoring. Results were then compared to previously published genome-wide association datasets. Mixed-model regression identified 5 variants of cyclin-dependent kinase inhibitor 1A ( CDKN1A ) as statistically significant with one intronic variant (rs3176326) likely affecting a gene promoter region with an Eigen-PC score of 7.779. This variant was found to have a p-value of 5.08x10 -8 when contrasted to controls with a minor allele frequency of 0.199 in the affected individuals. The 4 other variants were in linkage disequilibrium (r 2 0.90-0.99) with rs3176326. The odds ratio of HCM with rs3176326 is 1.81.To our knowledge, CDKN1A has not previously been associated with HCM. By utilizing bioinformatic tools we were able to identify 5 variants that may be associated with the risk for HCM. One locus affects a promotor region and resulted in a notably high Eigan-PC score suggestive that it has an impact on HCM. When comparing these findings with previous genome-wide association studies utilizing the UK Biobank, we find that CDKN1A has a possible association with HCM, though those results were not statistically significant. CDKN1A has been previously associated with the p53 DNA repair and cell senescence pathway. Animal and human studies have implicated it in the development of fibrosis. A recent genome-wide association metanalysis identified it as a risk locus for heart failure. These findings support our conclusion that CDKN1A is a risk locus for HCM.

Nonischemic dilated cardiomyopathy (DCM) often has a genetic etiology, however, its prevalence and etiologies are not completely understood. The UK Biobank comprises clinical and genetic data for greater than 500,000 individuals with enrollees 40-69 years of age. Our group created a custom phenotype of heart failure using ICD-10 codes for several subtypes of heart failure diagnoses including DCM. We then compared the individuals included in the custom heart failure phenotype to control individuals in a 20-to-1 fashion to identify genetic differences. Data were compared using Mixed Model Analysis for Pedigrees/Populations (MMAP) mixed-model regression. We identified 8 unlinked intronic variants in the dystrophin gene ( DMD ) that, when separated by self-identified race, occurred with a combined minor allele frequency of 0.15 in individuals with heart failure who identified as being of African descent. The combined minor allele frequency of these variants was 0.05 in individuals who self-identified as being of European descent. One variant of DMD in particular (rs139029250), was identified with a minor allele frequency of 0.05 in African British with DCM. The unadjusted odds ratio of a diagnosis of heart failure in individuals with rs129029250 was 4.65. When separated by gender, the unadjusted odds ratios are 2.02 for females and 6.44 for males. DMD is most notably known for its role in Duchenne and Becker muscular dystrophy, both of which are known to cause dilated cardiomyopathy in affected individuals. However, none of the individuals (36 female and 43 male) identified in our analysis with rs129029250 have been diagnosed with Duchenne muscular dystrophy, Becker muscular dystrophy, or a primary disorder of muscle (ICD code G70). Additionally, these individuals have an intronic variant of DMD , while Duchene and Becker muscular dystrophy are both due to exonic mutations. These findings suggest a possible common variant in the DMD gene that may contribute to DCM in individuals of African descent.

Retinoic acid (RA) is a well-known morphogen in human development. However, how an RA gradient distribution influences cardiac development remains obscure due to the lack of appropriate experimental apparatus. To address this issue, a polymeric micelle system with covalently attached RA was engineered to deliver gradient quantities of RA upon photo-irradiation. A photo-degradable polymeric nanoparticle (NP) composed of an amphiphilic methoxy(polyethylene glycol)-b-poly(e-caprolactone)-co-poly(azido-e-caprolactone-g-ortho nitrobenzyl retinoic ester) copolymer was synthesized, and hanging RA was covalently attached through a photo-sensitive o-nitrobenzyl (ONB) linker. The ONB linker was efficiently cleaved when exposed to a light (365 nm)-gradient, and the consequent gradient release of RA from the micelles was demonstrated. The efficacy of the photo-gradient-mediated RA release was validated across different concentrations of polymer micelles over varied irradiation periods. It was confirmed that polymer micelles demonstrated minimal cytotoxicity when exposed to mouse embryoid bodies (EBs). Finally, when the photo-gradient release of polymer micelles was applied, GFP-cardiac troponin T reporter mouse EBs demonstrated a concurrent gradient-like pattern of cardiac differentiation, verifying the utility of our novel photo-gradient approach to study morphogen gradients not only for cardiac development but also for other potential biological microenvironments subject to morphogen presentation with highly defined spatial and temporal geometries.

The study of disease pathophysiology has long relied on model systems, including animal models and cultured cells. In 2006, Shinya Yamanaka achieved a breakthrough by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). This revolutionary discovery provided new opportunities for disease modeling and therapeutic intervention. With established protocols, investigators can generate iPSC lines from patient blood, urine, and tissue samples. These iPSCs retain ability to differentiate into every human cell type. Advances in differentiation and organogenesis move cellular in vitro modeling to a multicellular model capable of recapitulating physiology and disease. Here, we discuss limitations of traditional animal and tissue culture models, as well as the application of iPSC models. We highlight various techniques, including reprogramming strategies, directed differentiation, tissue engineering, organoid developments, and genome editing. We extensively summarize current established iPSC disease models that utilize these techniques. Confluence of these technologies will advance our understanding of pediatric diseases and help usher in new personalized therapies for patients.

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

Congenital dilated cardiomyopathy (cDCM) is a rare but typically fatal disease. In most cases, its etiology is unknown, but a genetic root cause is often suspected. The objective of current study is to determine whether a novel non-structural gene causes cDCM. We hypothesized that rare genetic mutations caused cDCM, which could be modeled using patient-derived induced pluripotent stem cells (iPSCs). We generated the cardiomyocytes from iPSCs of a cDCM proband and found significant impairment in contractility and mitochondrial function, compared to those of healthy controls. To identify the causal mutations, we performed a whole exome sequencing of the cDCM patient and his parents (“trio”). Based on the assumption of recessive mode of inheritance or a de novo mutation, 9 candidate causal genes were identified. On the ground of the expression profiles of trio and pathogenicity predictor algorithms, we identified an indel and a nonsynonymous point mutation in the rotatin (encoded by RTTN ) gene as the putative genetic defect responsible for cDCM. CRISPR/Cas9-mediated knockout of RTTN in healthy control iPSCs recapitulated cardiomyocyte defects, and the correction of the missense mutation in the disease iPSCs restored cardiomyocyte structure and function, confirming causality. Thus, rotatin is a new causal molecule for cDCM and plays an important role in cardiac regulation.

IMPACT: Triple negative breast cancer (TNBC) affects 10-20% of women with breast cancer and is biologically more aggressive than other subtypes. The novel compound we have developed, DL7076, would give clinicians a vital strategy to improve the commonly used cyclophosphamide (CPA) and doxorubicin (DOX) regimen in the treatment of TNBC. OBJECTIVES/GOALS: The objective of this research project is to develop a novel compound which can activate both 1) the constitutive androstane receptor (CAR) and subsequently enhance the CYP2B6-mediated activation of CPA, and 2) the nuclear factor erythroid- related factor-2 (Nrf2) leading to the cardiomyocyte protection from DOX-associated cardiotoxicity. METHODS/STUDY POPULATION: Following the identification of the compound candidate, DL7076 was evaluated for tissue specific induction of CAR and Nrf2 using qPCR, western blot analysis, and luciferase reporter assays.Further, we have developed a multicellular coculture model incorporating human primary hepatocytes for metabolism, TNBC spheroids as the target, and cardiomyocytes as a side target of DOX. We have investigated the anticancer effects of CPA/DOX on TNBC cells and the toxic effects on cardiomyocytes with/without a CAR-Nrf2 activator, in a multicellular environment where hepatic metabolism is well-retained. RESULTS/ANTICIPATED RESULTS: We found that our dual activator of CAR and Nrf2, DL7076, exhibits tissue specific induction of CAR and Nrf2. Inclusion of DL7076 in combination with the CPA/DOX regimen improves anticancer efficacy, through the subsequent increase in the formation of the active CPA metabolite. With the addition of DL7076, DOX-mediated off-target cardiotoxicity was markedly reduced.Lastly, utilizing the novel coculture system with human primary hepatocytes, TNBC spheroids, and cardiomyocytes, the inclusion of DL7076 to the CPA/DOX regimen shows decreased spheroid viability and improved cardiomyocyte viability and function. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our findings suggest that DL7076 can facilitate DOX/CPA containing regimens by increasing CAR-mediated metabolism and subsequent CPA bioactivation while selectively protecting cardiomyocytes from DOX-induced toxicity. This research is expected to translate our basic scientific findings into therapeutic interventions for women with TNBC.

The organization and composition of the extracellular matrix (ECM) have been shown to impact the propagation of electrical signals in multiple tissue types. To date, many studies with electroactive biomaterial substrates have relied upon passive electrical stimulation of the ionic media to affect cell behavior. However, development of cell culture systems in which stimulation can be directly applied to the material – thereby isolating the signal to the cell-material interface and cell–cell contracts – would provide a more physiologically-relevant paradigm for investigating how electrical cues modulate lineage-specific stem cell differentiation. In the present study, we have employed unmodified, directly-stimulated, (un)patterned graphene as a cell culture substrate to investigate how extrinsic electrical cycling influences the differentiation of naïve human mesenchymal stem cells (hMSCs) without the bias of exogenous biochemicals. We first demonstrated that cyclic stimulation does not deteriorate the cell culture media or result in cytotoxic pH, which are critical experiments for correct interpretation of changes in cell behavior. We then measured how the expression of osteogenic and neurogenic lineage-specific markers were altered simply by exposure to electrical stimulation and/or physical patterns. Expression of the early osteogenic transcription factor RUNX2 was increased by electrical stimulation on all graphene substrates, but the mature marker osteopontin was only modulated when stimulation was combined with physical patterns. In contrast, the expression of the neurogenic markers MAP2 and β3-tubulin were enhanced in all electrical stimulation conditions, and were less responsive to the presence of patterns. These data indicate that specific combinations of non- biological inputs – material type, electrical stimulation, physical patterns – can regulate hMSC lineage specification. This study represents a substantial step in understanding how the interplay of electrophysical stimuli regulate stem cell behavior and helps to clarify the potential for graphene substrates in tissue engineering applications.

PURPOSE OF REVIEW: The goal of this review is to highlight the potential of induced pluripotent stem cell (iPSC)-based modeling as a tool for studying human cardiovascular diseases. We present some of the current cardiovascular disease models utilizing genome editing and patient-derived iPSCs. RECENT FINDINGS: The incorporation of genome-editing and iPSC technologies provides an innovative research platform, providing novel insight into human cardiovascular disease at molecular, cellular, and functional level. In addition, genome editing in diseased iPSC lines holds potential for personalized regenerative therapies. SUMMARY: The study of human cardiovascular disease has been revolutionized by cellular reprogramming and genome editing discoveries. These exceptional technologies provide an opportunity to generate human cell cardiovascular disease models and enable therapeutic strategy development in a dish. We anticipate these technologies to improve our understanding of cardiovascular disease pathophysiology leading to optimal treatment for heart diseases in the future.

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo , we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro . In this study, we employed a library of combinatorial polymers comprising of three functional subunits – poly-e-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) – as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-96%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro . Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions.

Background— New drugs are routinely screened for I Kr blocking properties thought to predict QT prolonging and arrhythmogenic liability. However, recent data suggest that chronic (hours) drug exposure to phosphoinositide 3-kinase inhibitors used in cancer can prolong QT by inhibiting potassium currents and increasing late sodium current ( I Na-L ) in cardiomyocytes. We tested the extent to which I Kr blockers with known QT liability generate arrhythmias through this pathway. Methods and Results— Acute exposure to dofetilide, an I Kr blocker without other recognized electropharmacologic actions, produced no change in ion currents or action potentials in adult mouse cardiomyocytes, which lack I Kr . By contrast, 2 to 48 hours of exposure to the drug generated arrhythmogenic afterdepolarizations and ≥15-fold increases in I Na-L . Including phosphatidylinositol 3,4,5-trisphosphate, a downstream effector for the phosphoinositide 3-kinase pathway, in the pipette inhibited these effects. I Na-L was also increased, and inhibitable by phosphatidylinositol 3,4,5-trisphosphate, with hours of dofetilide exposure in human-induced pluripotent stem cell–derived cardiomyocytes and in Chinese hamster ovary cells transfected with SCN5A , encoding sodium current. Cardiomyocytes from dofetilide-treated mice similarly demonstrated increased I Na-L and afterdepolarizations. Other agents with variable I Kr -blocking potencies and arrhythmia liability produced a range of effects on I Na-L , from marked increases (E-4031, d -sotalol, thioridazine, and erythromycin) to little or no effect (haloperidol, moxifloxacin, and verapamil). Conclusions— Some but not all drugs designated as arrhythmogenic I Kr blockers can generate arrhythmias by augmenting I Na-L through the phosphoinositide 3-kinase pathway. These data identify a potential mechanism for individual susceptibility to proarrhythmia and highlight the need for a new paradigm to screen drugs for QT prolonging and arrhythmogenic liability.

The interaction between cells or tissues and natural or synthetic materials which mimic the natural biological environment has been a matter of great interest in tissue engineering. In particular, surface properties of biomaterials (regardless of whether they are natural or synthetic) have been optimized using nanotechnology to improve interactions with cells for regenerative medicine applications. Specifically, in vivo and in vitro studies have demonstrated greater bladder tissue growth on polymeric surfaces with nanoscale to submicron surface features. Improved bladder cell responses on nanostructured polymers have been correlated to unique nanomaterial surface features leading to greater surface energy which influences initial protein interactions. Moreover, coupled with the observed greater in vitro and in vivo bladder cell adhesion as well as proliferation on nanostructured compared to conventional synthetic polymers, decreased calcium stone formation has also been measured. In this article, the importance of nanostructured biomaterial surface features for bladder tissue replacements are reviewed with thoughts on future directions for this emerging field. WIREs Nanomed Nanobiotechnol 2011 3 134–145 DOI: 10.1002/wnan.89 For further resources related to this article, please visit the WIREs website

Future biomaterials must simultaneously enhance tissue regeneration while minimizing immune responses and inhibiting infection. While the field of tissue engineering has promised to develop materials that can promote tissue regeneration for the entire body, such promises have not become reality. However, tissue engineering has experienced great progress due to the recent emergence of nanotechnology. Specifically, it has now been well established that increased tissue regeneration can be achieved on almost any surface by employing novel nano-textured surface features. Numerous studies have reported that nanotechnology accelerates various regenerative therapies, such as those for the bone, vascular, heart, cartilage, bladder and brain tissue. Various nano-structured polymers and metals (alloys) have been investigated for their bio (and cyto) compatibility properties. This review paper discusses several of the latest nanotechnology findings in regenerative medicine (also now called nanomedicine) as well as their relative levels of success.

Rationale: The lack of measurable single-cell contractility of human-induced pluripotent stem cell–derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. Objective: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Methods and Results: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (I Na ). Conclusions: The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.

Primary dilated cardiomyopathy (DCM) is a non-ischemic heart disease with impaired pumping function of the heart. In this study, we used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a healthy volunteer and a primary DCM patient to investigate the impact of DCM on iPSC-CMs׳ responses to different types of anisotropic strain. A bioreactor system was established that generates cardiac-mimetic forces of 150 kPa at 5% anisotropic cyclic strain and 1 Hz frequency. After confirming cardiac induction of the iPSCs, it was determined that fibronectin was favorable to other extracellular matrix protein coatings (gelatin, laminin, vitronectin) in terms of viable cell area and density, and was therefore selected as the coating for further study. When iPSC-CMs were exposed to three strain conditions (no strain, 5% static strain, and 5% cyclic strain), the static strain elicited significant induction of sarcomere components in comparison to other strain conditions. However, this induction occurred only in iPSC-CMs from a healthy volunteer ("control iPSC-CMs"), not in iPSC-CMs from the DCM patient ("DCM iPSC-CMs"). The donor type also significantly influenced gene expressions of cell-cell and cell-matrix interaction markers in response to the strain conditions. Gene expression of connexin-43 (cell-cell interaction) had a higher fold change in healthy versus diseased iPSC-CMs under static and cyclic strain, as opposed to integrins α-5 and α-10 (cell-matrix interaction). In summary, our iPSC-CM-based study to model the effects of different strain conditions suggests that intrinsic, genetic-based differences in the cardiomyocyte responses to strain may influence disease manifestation in vivo.

Induced pluripotent stem cells (iPSCs) from patients’ somatic tissues provide a viable source to create autologous cardiomyocytes (CMs) for potential cardiac-related cell therapies. However, a gap between the generation of iPSC-derived cardiomyocytes (iPSC-CMs) and the successful intra-cardiac engraftment of the cells to restore heart function remains to be bridged. Clinical data reporting engraftment of cells within human heart tissue has not been without its challenges, with significant cell loss from the site of delivery due to the physical stress of the cardiac cycle and the hostile inflammatory response within the infarct zone.[1–3] Hydrogels have been proven to support the survival of multiple cell types and have served as a platform for cell transplantation.[4, 5] Yet the use of tissue-adhesive, temperature-sensitive hydrogels to deliver iPSC-derived cardiomyocytes to infarcted heart remains to be explored. Therefore, we developed a polymer hydrogel to encapsulate, deliver, and integrate iPSC-CMs into infarcted myocardium to restore heart function. A temperature-sensitive biodegradable copolymer (polyethylene glycol-co-poly-e-caprolactone (PEG-PCL)) was synthesized [6] and conjugated with a collagen-binding peptide (SYIRIADTNIT). [7] The polymer was soluble in aqueous solutions at room temperature and underwent solution-to-gel transition at 37°C (Fig. 1 a,b).[6] As the peptide-modified polymer had a strong affinity to collagen I in vitro (Fig. 1c), it was expected that it would significantly increase the binding of the hydrogel to an infarcted heart, thus immobilizing iPSC-CMs within the damaged myocardium. Functional, beating cardiomyocytes were derived from patient fibroblast-derived iPSCs using a “Matrigel sandwich” method.[8] Cardiac differentiation was confirmed by fluorescence-activated cell sorting (FACS) and by immunostaining with known cardiac markers cardiac troponin T (cTNT), ryanodine receptor 2 (RYR) and α-actinin (Fig. 1f). The iPSC-CMs encapsulated in the polymer hydrogel at 37°C for two weeks maintained their viability (Fig. 1g) and cardiac phenotype, as evidenced by strong expression of troponin T and Nkx2.5 (Fig. 1h); thus, PEG-PCL does not appear to have obvious toxic effects on iPSC-CMs. Fig. 1 PEG-PCL copolymer was (a) dissolved in PBS at RT and (b) gelled at 37°C within 5 min (Sol-gel transition). Collagen-binding study was performed by adding peptide-modified polymer to collagen-coated surface at 37°C, and unbound polymer ... Engraftment of iPSC-CMs into infarcted myocardium using the peptide-modified hydrogel and its impact on infarcted heart function and structure were assessed with a rat myocardial infarction (MI) model. All animal surgery and animal care were approved by the Institutional Animal Care and Use Committee (IACUC) at Vanderbilt University (protocol: M/12/074). The left anterior descending (LAD) coronary artery of nude rats was ligated to induce MI. At 30 minutes post-MI, iPSC-CMs alone, or modified copolymer solution with or without iPSC-CMs (2–4 million/rat) were injected around the infarct border zone. A negative control group received the LAD ligation and phosphate-buffered saline (PBS) injection without cells or copolymer. At two weeks post-injection, heart dimensions and functional output were assessed by echocardiography. All groups had ventricular dilation and reduced fractional shortening (FS) (Fig. 2). However, rats treated with iPSC-CM-encapsulated copolymer demonstrated significantly less decline in FS (Δ= −6.37±0.49%) compared to other groups (Δ= −12.77±2.04%, −11.44±2.04% and −12.65±1.53% for PBS control, iPSC-CM only, and polymer only groups, respectively (Fig. 2a). p=0.016 vs. PBS, p=0.021 vs. iPSC-CM only, and p=0.005 vs. polymer only). Overall, the iPSC-CM plus polymer group demonstrated 50.1%, 28.2% and 49.6% improvement in LV systolic function over PBS, iPSC-CM only and polymer only groups (Fig. 2b). Moreover, rats treated with iPSC-CM plus polymer demonstrated a trend toward less LV enlargement (Δ= 15.07±3.24%) compared to other groups (Δ=24.44±3.99%, 25.02±2.03% and 23.17±4.51% for PBS control, iPSC-CM only, and polymer only groups, respectively; Fig. 2c) although this reached statistical significance only in comparison to the iPSC-CM group (p=0.032). Overall, iPSC-CM plus polymer group demonstrated 38.3%, 39.8% and 35.0% less LV enlargement over PBS, iPSC-CM only and polymer only groups, suggesting that iPSC-CM encapsulated in polymer curtailed adverse ventricular remodeling better than other treatment modalities. Fig. 2 The effects of hydrogel-encapsulated iPSC-CMs on left ventricle function and remodeling. (a–c) Echocardiography was performed before the ligation of rat LAD coronary arteries (baseline) and again 2 weeks post-delivery (n=5 rats per group). (a, ... Histological examination of the hearts was performed, and the LV anterior free wall thickness was measured using ImageJ and averaged from 3 randomly selected regions in each rat heart. Result demonstrated that, in addition to LV chamber enlargement, the LAD ligation resulted in dramatic thinning and significant fibrosis of the LV anterior free wall at two weeks in control groups (Fig. 2e). In contrast, heart injected with polymer-encapsulated iPSC-CMs had smaller LV chamber, thicker LV free wall and less fibrosis (Fig. 2e). Overall, hearts in the iPSC-CM plus polymer group were significantly thicker than all other groups; the average LV anterior wall thickness of cell plus polymer group was 2.46±0.06mm, compared with 0.48±0.07, 0.35±0.05 and 0.39±0.02mm in PBS, cell only and polymer only groups, respectively (Fig. 2d, p

Clinical trials utilizing mesenchymal stem cells (MSCs) for severe vascular diseases have highlighted the need to effectively engraft cells and promote pro-angiogenic activity. A functional material accomplishing these two goals is an ideal solution as spatiotemporal and batch-to-batch variability in classical therapeutic delivery can be minimized, and tissue regeneration would begin rapidly at the implantation site. Gelatin may serve as a promising biomaterial due to its excellent biocompatibility, biodegradability, and non-immuno/antigenicity. However, the dissolution of gelatin at body temperature and quick enzymatic degradation in vivo have limited its use thus far. To overcome these challenges, an injectable, in situ crosslinkable gelatin was developed by conjugating enzymatically-crosslinkable hydroxyphenyl propionic acid (GHPA). When MSCs are cultured in 3D in vitro or injected in vivo in GHPA, spontaneous endothelial differentiation occurs, as evidenced by marked increases in endothlelial cell marker expressions (Flk1, Tie2, ANGPT1, vWF) in addition to forming an extensive perfusable vascular network after 2-week subcutaneous implantation. Additionally, favorable host macrophage response is achieved with GHPA as shown by decreased iNOS and increased MRC1 expression. These results indicate GHPA as a promising soluble factor-free cell delivery template which induces endothelial differentiation of MSCs with robust neovasculature formation and favorable host response.

There is a tremendous interest in human cardiomyocytes generated from patient-derived induced pluripotent stem cells (iPSC-CMs) for the study and possible treatment of human heart diseases. Despite their vast potential, a significant impediment to a broader application of iPSC-CMs to study human myocyte biology is the structural and functional immaturity of iPSC-CMs. Growing evidence indicates that synthetic polymers utilized as extracellular substrates can exert significant effects on in vitro tissue generation, although the underlying mechanisms remain largely unknown. Based on the profound impact of the extracellular matrix of developing embryos on in vivo organogenesis, we hypothesize that engineered polymer substrates will likewise influence in vitro maturation of iPSC-CMs. A subset of combinatorial polymers was synthesized by polymerizing poly(ε-caprolacton) (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL), abbreviated as x%PEG-y%PCL-z%cPCL (x, y, and z: molar %). We investigated effects of the polymer composition on maturation of iPSC-CMs with respect to the beating behavior, mitochondrial function and molecular profiles after 30 days in culture on polymer scaffolds. Results showed the 4%PEG-96%PCL scaffold promoted the most active beating in iPSC-CMs at 30 days and further, that the mitochondrial function, as assessed by tetramethyl rhodamine methylester (TMRM) was significantly increased in the iPSC-CMs cultured on 4%PEG-96%PCL over other polymers. Molecular profiling analysis indicates 4%PEG-96%PCL scaffolds enhanced the expression of MYL2 (a commonly accepted marker of mature ventricular myocytes) as well as of components of the intermediate filaments linking the plasma membrane to the myofilament. In summary, although the polymers we used here exhibit similar physicochemical properties, they have divergent effects on iPSC-CM differentiation. Thus, specific chemical compositions of synthetic substrates can exert profound influence on in vitro maturation of hiPSC-CMs. Our work exploring the effects of synthetic biomaterials on human stem cell differentiation could pave the way for a successful translation of ongoing advances in tissue engineering to new treatments for human heart diseases.

Introduction: Regeneration of heart tissue post-infarction is hampered by the limited proliferation of cardiomyocytes. Due to their expandability and pluripotency, human induced pluripotent stem cells (iPSCs) are considered an ideal cell source to produce cardiomyocytes and regenerate heart tissue. However, previous clinical trials revealed the quick loss of injected cells (> 90%) after delivery in aqueous solutions (e.g. phosphate buffered saline, PBS), which significantly limited the clinical outcome. To enhance the retention of cells and promote tissue regeneration, we synthesized a temperature-responsive polymer which is water-soluble to encapsulate cardiomyocytes at room temperature, following injection into epicardium, it quickly forms a gel and holds the cells in situ at body temperature. Functional peptides can be conjugated to this material and facilitate its adhesion to cardiac tissue for optimal cell integration and cardiac regeneration. Materials and Methods: The polymers monomethoxypoly(ethylene glycol) (mPEG) and poly(ε-caprolactone) (PCL) were copolymerized to produce mPEG-PCL. The polymer was further modified with decorin-derived peptide, which can firmly bind to collagenous tissue. The solution-to-gel transition temperature (Ts) was determined by dissolving the polymer in PBS, slowly increasing the temperature from 4 to 40°C and checking the fluidity of the solution. The viability of cardiomyocytes encapsulated in polymer gel at 37°C for 2 weeks was determined by calcein AM. Peptide-modified polymer solution was injected to the epithelium of adult rat heart. Echocardiography was performed to evaluate the heart functions before and two weeks post injection. The tissue response to this material was studied by histological staining. Results and Conclusion: The material successfully underwent solution-to-gel transition at 37°C. Human iPSCs-derived cardiomyocytes encapsulated in the gel were still viable after 2 weeks. There was no heart dysfunction 2 weeks post-injection. No significant inflammatory response was induced in vivo. These demonstrate the safety and feasibility of this material to delivery cardiomyocytes to infarct heart.

An estimated 2.5 percent of the American population has heart valve (HV) disease and more than 100,000 US patients require a prosthetic valve replacement each year [1]. However, prosthetic valves can cause accelerated calcification leading to recurrence of HV disease in patients [2]. Thus, the development of a suitable tissue-engineered heart valve (TEHV) would greatly benefit patients with HV disease. Aortic valve interstitial cells (AVICs) play a crucial role in the progression of aortic valve disease as well as the maintenance of normal valve. Therefore, in order to design a suitable TEHV, these specialized cells need to be better understood. AVICs are known to synthesize ECM and express matrix degrading enzymes and their inhibitors that mediate and regulate remodeling of ECM components [3]. Interestingly, it was recently established that AVICs sense the stiffness of their surrounding ECM in vivo and are phenotypically responsive to mechanical cues with AVICs differentiating into myofibroblasts or osteoblasts, which are pathologic markers. Specifically, soft collagen gels (∼34kPa) caused less differentiation of AVICs than stiffer collagen gel (∼100kPa) [4]. However, for these experiments the AVICs were cultured on tissue culture polystyrene (TCPS) for at least one passage, and it is likely that AVICs cultured on TCPS might retain modified characteristics of AVICs in tissue prior to seed them on soft gels because of the memory to rigid substrate stiffness. Therefore, in this study, we examined the phenotype and function of AVICs on substrates that mimic ECM stiffness of adult leaflet as well as of developing embryo. In addition, we examine the effects of transforming growth factor-β1 (TGF-β1) which has been the most extensively studied cytokine initiator of fibrotic response of AVICs.Copyright © 2012 by ASME

Although artificial prostheses for diseased heart valves have been around for several decades, viable heart valve replacements have yet to be developed due to their complicated nature. The majority of research in heart valve replacement technology seeks to improve decellularization techniques for porcine valves or bovine pericardium as an effort to improve current clinically used valves. The drawback of clinically used valves is that they are nonviable and thus do not grow or remodel once implanted inside patients. This is particularly detrimental for pediatric patients, who will likely need several reoperations over the course of their lifetimes to implant larger valves as the patient grows. Due to this limitation, additional biomaterials, both synthetic and natural in origin, are also being investigated as novel scaffolds for tissue-engineered heart valves, specifically for the pediatric population. Here, we provide a brief overview of valves in clinical use as well as of the materials being investigated as novel tissue-engineered heart valve scaffolds. Additionally, we focus on natural-based biomaterials for promoting cell behavior that is indicative of the developmental biology process that occurs in the formation of heart valves in utero, such as epithelial-to-mesenchymal transition or transformation. By engineering materials that promote native developmental biology cues and signaling, while also providing mechanical integrity once implanted, a viable tissue-engineered heart valve may one day be realized. A viable tissue-engineered heart valve, capable of growing and remodeling actively inside a patient, could reduce risks and complications associated with current valve replacement options and improve overall quality of life in the thousands of patients who received such valves each year, particularly for children.

Polyurethane (PU) is a versatile elastomer that is commonly used in biomedical applications. In turn, materials derived from nanotechnology, specifically carbon nanofibers (CNFs), have received increasing attention for their potential use in biomedical applications. Recent studies have shown that the dispersion of CNFs in PU significantly enhances composite nanoscale surface roughness, tensile properties, and thermal stability. Although there have been studies concerning normal primary cell functions on such nanocomposites, there have been few studies detailing cancer cell responses. Since many patients who require bladder transplants have suffered from bladder cancer, the ideal bladder prosthetic material should not only promote normal primary human urothelial cell (HUC) function, but also inhibit the return of bladder cancerous cell activity. This study examined the correlation between transitional (UMUC) and squamous (or SCaBER) urothelial carcinoma cells and HUC on PU:CNF nanocomposites of varying PU and CNF weight ratios (from pure PU to 4:1 [PU:CNF volume ratios], 2:1, 1:1, 1:2, and 1:4 composites to pure CNF). Composites were characterized for mechanical properties, wettability, surface roughness, and chemical composition by atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, and goniometry. The adhesion and proliferation of UMUC and SCaBER cancer cells were assessed by MTS assays. Cellular responses were further quantified by measuring the amounts of nuclear mitotic protein 22 (NMP-22), vascular endothelial growth factor (VEGF), and tumor necrosis factor alpha. Results demonstrated that both UMUC and SCaBER cell proliferation rates decreased over time on substrates with increased CNF in PU. In addition, with the exception of VEGF from UMUC (which was the same across all materials), composites containing the most CNF activated cancer cells (UMUC and SCaBER) the least, as shown by their decreased expression of NMP-22, tumor necrosis factor alpha, and VEGF. Moreover, the adhesion of HUC increased on composites containing more CNF than PU. Overall levels of NMP-22 were significantly lower in HUC than in cancerous UMUC and SCaBER cells on PU:CNF composites. Thus, this study provided a novel nanocomposite consisting of CNF and PU that should be further studied for inhibiting the return of cancerous bladder tissue and for promoting normal non-cancerous bladder tissue formation.

The interaction between cells or tissues and natural or synthetic materials which mimic the natural biological environment has been a matter of great interest in tissue engineering. In particular, surface properties of biomaterials (regardless of whether they are natural or synthetic) have been optimized using nanotechnology to improve interactions with cells for regenerative medicine applications. Specifically, in vivo and in vitro studies have demonstrated greater bladder tissue growth on polymeric surfaces with nanoscale to submicron surface features. Improved bladder cell responses on nanostructured polymers have been correlated to unique nanomaterial surface features leading to greater surface energy which influences initial protein interactions. Moreover, coupled with the observed greater in vitro and in vivo bladder cell adhesion as well as proliferation on nanostructured compared to conventional synthetic polymers, decreased calcium stone formation has also been measured. In this article, the importance of nanostructured biomaterial surface features for bladder tissue replacements are reviewed with thoughts on future directions for this emerging field.

Lijuan Zhang1, Young Wook Chun2, Thomas J Webster21Department of Chemistry and 2Division of Engineering, Brown University, Providence, RI USAAbstract: Poly(lactic-co-glycolic) acid (PLGA) has been widely used as a biomaterial in regenerative medicine because of its biocompatibility and biodegradability properties. Previous studies have shown that cells (such as bladder smooth muscle cells, chondrocytes, and osteoblasts) respond differently to nanostructured PLGA surfaces compared with nanosmooth surfaces. The purpose of the present in vitro research was to prepare PLGA films with various nanometer surface features and determine whether lung cancer epithelial cells respond differently to such topographies. To create nanosurface features on PLGA, different sized (190 nm, 300 nm, 400 nm, and 530 nm diameter) polystyrene beads were used to cast polydimethylsiloxane (PDMS) molds which were used as templates to create nanofeatured PLGA films. Atomic force microscopy (AFM) images and root mean square roughness (RMS) values indicated that the intended spherical surface nanotopographies on PLGA with RMS values of 2.23, 5.03, 5.42, and 36.90 nm were formed by employing 190, 300, 400, and 530 nm beads. A solution evaporation method was also utilized to modify PLGA surface features by using 8 wt% (to obtain an AFM RMS value of 0.62 nm) and 4 wt% (to obtain an AFM RMS value of 2.23 nm) PLGA in chloroform solutions. Most importantly, lung cancer epithelial cells adhered less on the PLGA surfaces with RMS values of 0.62, 2.23, and 5.42 nm after four hours of culture compared with any other PLGA surface created here. After three days, PLGA surfaces with an RMS value of 0.62 nm had much lower cell density than any other sample. In this manner, PLGA with specific nanometer surface features may inhibit lung cancer cell density which may provide an important biomaterial for the treatment of lung cancer (from drug delivery to regenerative medicine).Keywords: nanotechnology, cancer, nanotopography, lung carcinoma

Purified carbon nanotubes (with removed toxic catalytic particles) have been considered as novel materials for drug delivery and for generating artificial organs more efficiently due to their unique surface features. Traditionally, the surface chemistry of carbon nanotubes has been modified through various functionalization strategies to increase biocompatibility. Importantly, modulating the intrinsic material surface energy of carbon nanotubes (without functionalization, thus, establishing permanent, non degradable chemical, and physical surface properties) can potentially reduce an immune response mediated by macrophages. Herein, we report macrophage responses on different surface energy carbon nanotubes while keeping their nanoscale surface roughness. Specifically, interactions of ultra hydrophobic (bare or unmodified) and hydrophilic carbon nanotubes (due to the formation of oxide layers) with macrophages were investigated. All results clearly support the evidence that tailoring the surface energy of carbon nanotubes mediates a macrophage immune response.

Purified carbon nanotubes (with removed toxic catalytic particles) have been considered as novel materials for drug delivery and for generating artificial organs more efficiently than traditional tissue engineering materials due to their unique surface features. The surface chemistry of carbon nanotubes has been modified through various functionalization strategies to increase biocompatibility. Importantly, modulating the intrinsic material surface energy of carbon nanotubes (without functionalization, thus, establishing permanent, non degradable chemical, and physical surface properties) can potentially reduce an immune response mediated by macrophages. Herein, we report macrophage responses on different surface energy carbon nanotubes while keeping their nanoscale surface roughness. Specifically, interactions of ultra hydrophobic (bare or unmodified) and hydrophilic carbon nanotubes (due to the formation of oxide layers) with macrophages were investigated. It was observed that macrophage density on both carbon nanotube scaffolds were lower than on traditional materials. In particular, the amount of released cytokine (TNF-α) from macrophages cultured on hydrophilic carbon nanotube scaffolds was much smaller than on hydrophobic carbon nanotube scaffolds. All results clearly supported that tailoring the surface energy of carbon nanotubes mediates a macrophage immune response.

Nanomedicine (a division of nanotechnology) is an interdisciplinary research field incorporating biology, chemistry, engineering and medicine with the intention to improve disease prevention, diagnosis, and treatment. Specifically, there have been great strides made in using nanomedicine to enhance the functions of cells necessary to regenerate a diverse number of tissues (such as bone, blood vessels, the bladder, teeth, the nervous system, and the heart to name a few). Traditional (micron-structured or nano-smooth) implants suffer from: (i) infection, (ii) inflammation, and (iii) insufficient prolonged bonding between the implanted material and surrounding tissue. To date, such conventional implants have been improved by implementing nanotopographical features on their surfaces. In this review paper, the application of nanomaterials to regenerate numerous organs (including, as specific examples, bone, neural, and bladder tissues) will be presented with necessary future directions highlighted for the field of nanomedicine to progress.

The present study describes enzymatically cross-linked gelatin-based hydrogels as in situ forming tissue adhesives. A series of gelatin derivatives with different phenolic contents were synthesized by conjugating hydroxyphenyl propionic acid and tyramine to gelatin backbones. Two gelatin derivatives, gelatin–hydroxyphenyl propionic acid (GH) and gelatin–hydroxyphenyl propionic acid–tyramine (GHT) with maximum obtainable phenolic contents (146.6 μmol g−1 GH and 395.7 μmol g−1 GHT), were used to prepare gelatin-based hydrogels via horseradish peroxidase (HRP)-mediated reactions in the presence of hydrogen peroxide (H2O2). By changing the HRP and H2O2 concentrations, the gelation time, mechanical strength, and degradation rate of the hydrogels were fairly well controlled, indicating a tunable rate and degree of cross-linking. In addition, we found that an increase in phenolic content led to increased mechanical strength of the hydrogels. Lap-shear test results clearly showed that the GH and GHT hydrogels exhibited 2–3 times greater tissue adhesiveness compared to fibrin glues. On the basis of these results, we conclude that in situ forming gelatin-based hydrogels, which are both injectable and sprayable, can be used as an alternative to conventional tissue adhesives.

Synthetic polymers have been proposed for replacing resected cancerous bladder tissue. However, conventional (or nanosmooth) polymers used in such applications (such as poly(ether) urethane (PU) and poly-lactic-co-glycolic acid (PLGA)) often fail clinically due to poor bladder tissue regeneration, low cytocompatibility properties, and excessive calcium stone formation. For the successful reconstruction of bladder tissue, polymer surfaces should be modified to combat these common problems. Along these lines, implementing nanoscale surface features that mimic the natural roughness of bladder tissue on polymer surfaces can promote appropriate cell growth, accelerate bladder tissue regeneration and inhibit bladder calcium stone formation. To test this hypothesis, in this study, the cytocompatibility properties of both a non-biodegradable polymer (PU) and a biodegradable polymer (PLGA) were investigated after etching in chemicals (HNO(3) and NaOH, respectively) to create nanoscale surface features. After chemical etching, PU possessed submicron sized pores and numerous nanometer surface features while PLGA possessed few pores and large amounts of nanometer surface roughness. Results from this study strongly supported the assertion that nanometer scale surface roughness produced on PU and PLGA promoted the density of urothelial cells (cells that line the interior of the bladder), with the greatest urothelial cell densities observed on nanorough PLGA. In addition, compared to respective conventional polymers, the results provided evidence that nanorough PU and PLGA inhibited calcium oxalate stone formation; submicron pored nanorough PU inhibited calcium oxalate stone formation the most. Thus, results from the present study suggest the importance of nanometer topographical cues for designing better materials for bladder tissue engineering applications.