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Aligned nanofiber scaffolds improve functionality of cardiomyocytes differentiated from human induced pluripotent stem cell-derived cardiac progenitor cells.

ABSTRACT: Cardiac progenitor cells (CPCs), capable of differentiating into multiple cardiac cell types including cardiomyocytes (CMs), endothelial cells, and smooth muscle cells, are promising candidates for cardiac repair/regeneration. In vitro model systems where cells are grown in a more in vivo-like environment, such as 3D cultures, have been shown to be more predictive than 2D culture for studying cell biology and disease pathophysiology. In this report, we focused on using Wnt inhibitors to study the differentiation of human iPSC-CPCs under 2D or 3D culture conditions by measuring marker protein and gene expression as well as intracellular Ca2+ oscillation. Our results show that the 3D culture with aligned nanofiber scaffolds, mimicing the architecture of the extracellular matrix of the heart, improve the differentiation of iPSC-CPCs to functional cardiomyocytes induced by Wnt inhibition, as shown with increased number of cardiac Troponin T (cTnT)-positive cells and synchronized intracellular Ca2+ oscillation. In addition, we studied if 3D nanofiber culture can be used as an in vitro model for compound screening by testing a number of other differentiation factors including a ALK5 inhibitor and inhibitors of BMP signaling. This work highlights the importance of using a more relevant in vitro model and measuring not only the expression of marker proteins but also the functional readout in a screen in order to identify the best compounds and to investigate the resulting biology.

SUBMITTER: Ding M

PROVIDER: S-EPMC7419298 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Aligned nanofiber scaffolds improve functionality of cardiomyocytes differentiated from human induced pluripotent stem cell-derived cardiac progenitor cells.

Ding Mei M Andersson Henrik H Martinsson Sofia S Sabirsh Alan A Jonebring Anna A Wang Qing-Dong QD Plowright Alleyn T AT Drowley Lauren L

Scientific reports 20200811 1

Cardiac progenitor cells (CPCs), capable of differentiating into multiple cardiac cell types including cardiomyocytes (CMs), endothelial cells, and smooth muscle cells, are promising candidates for cardiac repair/regeneration. In vitro model systems where cells are grown in a more in vivo-like environment, such as 3D cultures, have been shown to be more predictive than 2D culture for studying cell biology and disease pathophysiology. In this report, we focused on using Wnt inhibitors to study th ...[more]

PMID: 32782331

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Project description:IntroductionDilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates.MethodshiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes.ResultsSEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro.ConclusionsOverall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch.

Project description:Recent advances in cardiac tissue engineering have shown that human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in a three-dimensional (3D) micro-environment exhibit superior physiological characteristics compared with their two-dimensional (2D) counterparts. These 3D cultured hiPSC-CMs have been used for drug testing as well as cardiac repair applications. However, the fabrication of a cardiac scaffold with optimal biomechanical properties and high biocompatibility remains a challenge. In our study, we fabricated an aligned polycaprolactone (PCL)-Gelatin coaxial nanofiber patch using electrospinning. The structural, chemical, and mechanical properties of the patch were assessed by scanning electron microscopy (SEM), immunocytochemistry (ICC), Fourier-transform infrared spectroscopy (FTIR)-spectroscopy, and tensile testing. hiPSC-CMs were cultured on the aligned coaxial patch for 2 weeks and their viability [lactate dehydrogenase (LDH assay)], morphology (SEM, ICC), and functionality [calcium cycling, multielectrode array (MEA)] were assessed. Furthermore, particle image velocimetry (PIV) and MEA were used to evaluate the cardiotoxicity and physiological functionality of the cells in response to cardiac drugs. Nanofibers patches were comprised of highly aligned core-shell fibers with an average diameter of 578 ± 184 nm. Acellular coaxial patches were significantly stiffer than gelatin alone with an ultimate tensile strength of 0.780 ± 0.098 MPa, but exhibited gelatin-like biocompatibility. Furthermore, hiPSC-CMs cultured on the surface of these aligned coaxial patches (3D cultures) were elongated and rod-shaped with well-organized sarcomeres, as observed by the expression of cardiac troponin-T and α-sarcomeric actinin. Additionally, hiPSC-CMs cultured on these coaxial patches formed a functional syncytium evidenced by the expression of connexin-43 (Cx-43) and synchronous calcium transients. Moreover, MEA analysis showed that the hiPSC-CMs cultured on aligned patches showed an improved response to cardiac drugs like Isoproterenol (ISO), Verapamil (VER), and E4031, compared to the corresponding 2D cultures. Overall, our results demonstrated that an aligned, coaxial 3D cardiac patch can be used for culturing of hiPSC-CMs. These biomimetic cardiac patches could further be used as a potential 3D in vitro model for "clinical trials in a dish" and for in vivo cardiac repair applications for treating myocardial infarction.

Project description:Retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells, called induced retinal pigment epithelium (iRPE), is being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration. The success of RPE implantation is linked to the use of biomimetic scaffolds that simulate Bruch's membrane and promote RPE maturation and integration as a functional tissue. Due to difficulties associated with animal protein-derived scaffolds, including sterility and pro-inflammatory responses, current practices favor the use of synthetic polymers, such as polycaprolactone (PCL), for generating nanofibrous scaffolds. In this study, we tested the hypothesis that plant protein-derived fibrous scaffolds can provide favorable conditions permissive for the maturation of RPE tissue sheets in vitro. Our natural, soy protein-derived nanofibrous scaffolds exhibited a J-shaped stress-strain curve that more closely resembled the mechanical properties of native tissues than PCL with significantly higher hydrophilicity of the natural scaffolds, favoring in vivo implantation. We then demonstrate that iRPE sheets growing on these soy protein scaffolds are equivalent to iRPE monolayers cultured on synthetic PCL nanofibrous scaffolds. Immunohistochemistry demonstrated RPE-like morphology and functionality with appropriate localization of RPE markers RPE65, PMEL17, Ezrin, and ZO1 and with anticipated histotypic polarization of vascular endothelial growth factor and pigment epithelium-derived growth factor as indicated by enzyme-linked immunosorbent assay. Scanning electron microscopy revealed dense microvilli on the cell surface and homogeneous tight junctional contacts between the cells. Finally, comparative transcriptome analysis in conjunction with principal component analysis demonstrated that iRPE on nanofibrous scaffolds, either natural or synthetic, matured more consistently than on nonfibrous substrates. Taken together, our studies suggest that the maturation of cultured iRPE sheets for subsequent clinical applications might benefit from the use of nanofibrous scaffolds generated from natural proteins. Impact statement Induced retinal pigment epithelium (iRPE) from patient-derived induced pluripotent stem cells (iPSCs) may yield powerful treatments of retinal diseases, including age-related macular degeneration. Recent studies, including early human clinical trials, demonstrate the importance of selecting appropriate biomaterial scaffolds to support tissue-engineered iRPE sheets during implantation. Electrospun scaffolds show particular promise due to their similarity to the structure of the native Bruch's membrane. In this study, we describe the use of electroprocessed nanofibrous soy protein scaffolds to generate polarized sheets of human iPSC-derived iRPE sheets. Our evaluation, including RNA-seq transcriptomics, indicates that these scaffolds are viable alternatives to scaffolds electrospun from synthetic polymers.

Project description:Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alteration has been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes (CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells (iPSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since iPSC preserve the entire patients' genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with iPSC.

Dataset Information

Aligned nanofiber scaffolds improve functionality of cardiomyocytes differentiated from human induced pluripotent stem cell-derived cardiac progenitor cells.

Publications

Aligned nanofiber scaffolds improve functionality of cardiomyocytes differentiated from human induced pluripotent stem cell-derived cardiac progenitor cells.

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