Directed Differentiation of Human Induced Pluripotent Stem Cells to Heart Valve Cells.

BACKGROUND: A main obstacle in current valvular heart disease research is the lack of high-quality homogeneous functional heart valve cells. Human induced pluripotent stem cells (hiPSCs)-derived heart valve cells may help with this dilemma. However, there are no well-established protocols to induce hiPSCs to differentiate into functional heart valve cells, and the networks that mediate the differentiation have not been fully elucidated. METHODS: To generate heart valve cells from hiPSCs, we sequentially activated the Wnt, BMP4, VEGF (vascular endothelial growth factor), and NFATc1 signaling pathways using CHIR-99021, BMP4, VEGF-165, and forskolin, respectively. The transcriptional and functional similarity of hiPSC-derived heart valve cells compared with primary heart valve cells were characterized. Longitudinal single-cell RNA sequencing was used to uncover the trajectory, switch genes, pathways, and transcription factors of the differentiation. RESULTS: An efficient protocol was developed to induce hiPSCs to differentiate into functional hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells. After 6-day differentiation and CD144 magnetic bead sorting, ≈70% CD144+ cells and 30% CD144- cells were obtained. On the basis of single-cell RNA sequencing data, the CD144+ cells and CD144- cells were found to be highly similar to primary heart valve endothelial cells and primary heart valve interstitial cells in gene expression profile. Furthermore, CD144+ cells had the typical function of primary heart valve endothelial cells, including tube formation, uptake of low-density lipoprotein, generation of endothelial nitric oxide synthase, and response to shear stress. Meanwhile, CD144- cells could secret collagen and matrix metalloproteinases, and differentiate into osteogenic or adipogenic lineages like primary heart valve interstitial cells. Therefore, we identified CD144+ cells and CD144- cells as hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells, respectively. Using single-cell RNA sequencing analysis, we demonstrated that the trajectory of heart valve cell differentiation was consistent with embryonic valve development. We identified the main switch genes (NOTCH1, HEY1, and MEF2C), signaling pathways (TGF-ß, Wnt, and NOTCH), and transcription factors (MSX1, SP5, and MECOM) that mediated the differentiation. Finally, we found that hiPSC-derived valve interstitial-like cells might derive from hiPSC-derived valve endothelial-like cells undergoing endocardial-mesenchymal transition. CONCLUSIONS: In summary, this is the first study to report an efficient strategy to generate functional hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells from hiPSCs, as well as to elucidate the differentiation trajectory and transcriptional dynamics of hiPSCs differentiated into heart valve cells.

Bioelectric signaling and the control of cardiac cell identity in response to mechanical forces.

Developing cardiovascular systems use mechanical forces to take shape, but how ubiquitous blood flow forces instruct local cardiac cell identity is still unclear. By manipulating mechanical forces in vivo, we show here that shear stress is necessary and sufficient to promote valvulogenesis. We found that valve formation is associated with the activation of an extracellular adenosine triphosphate (ATP)­dependent purinergic receptor pathway, specifically triggering calcium ion (Ca2+) pulses and nuclear factor of activated T cells 1 (Nfatc1) activation. Thus, mechanical forces are converted into discrete bioelectric signals by an ATP-Ca2+-Nfatc1­mechanosensitive pathway to generate positional information and control valve formation.

Generation and characterization of cardiac valve endothelial-like cells from human pluripotent stem cells.

The cardiac valvular endothelial cells (VECs) are an ideal cell source that could be used for making the valve organoids. However, few studies have been focused on the derivation of this important cell type. Here we describe a two-step chemically defined xeno-free method for generating VEC-like cells from human pluripotent stem cells (hPSCs). HPSCs were specified to KDR+/ISL1+ multipotent cardiac progenitors (CPCs), followed by differentiation into valve endothelial-like cells (VELs) via an intermediate endocardial cushion cell (ECC) type. Mechanistically, administration of TGFb1 and BMP4 may specify VEC fate by activating the NOTCH/WNT signaling pathways and previously unidentified targets such as ATF3 and KLF family of transcription factors. When seeded onto the surface of the de-cellularized porcine aortic valve (DCV) matrix scaffolds, hPSC-derived VELs exhibit superior proliferative and clonogenic potential than the primary VECs and human aortic endothelial cells (HAEC). Our results show that hPSC-derived valvular cells could be efficiently generated from hPSCs, which might be used as seed cells for construction of valve organoids or next generation tissue engineered heart valves.

Vitrification of Heart Valve Tissues.

Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples (1-3 mL total volume) where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, permits preservation of large samples (80-100 mL total volume) with several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at -80 °C; better matrix preservation than freezing with retention of material properties; very low cell viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is not used; and reduced manufacturing costs. More recently, we have developed Protocol 4 in which VS55 is supplemented with sugars resulting in reduced concerns regarding nucleation during cooling and warming. This method can be used for large samples resulting in retention of cell viability and permits short-term exposure to -80 °C with long-term storage preferred at or below -135 °C.

Freeze-Drying of Decellularized Heart Valves for Off-the-Shelf Availability.

Malfunctioning heart valves can cause severe health problems, which if left untreated can lead to death. One of the treatment options is to replace a diseased heart valve with a decellularized valve construct prepared from human or animal material. Decellularized tissue scaffolds closely resemble properties of native tissue, while lacking immunogenic factors of cellular components. After transplantation, circulating stem and progenitor cells of the patient adhere to the scaffold resulting in in vivo tissue regeneration of the valve. Decellularized heart valve scaffold implants need to be stored to be readily available whenever needed, which can be done by freeze-drying. The advantage of freeze-drying is that it does not require bulky and energy-consuming freezing equipment for storage and allows easy transport. This chapter outlines the entire process from decellularization to freeze-drying to obtain dry decellularized heart valves, which after a simple rehydration step, can be used as implants. The protocol is described for porcine heart valves, but procedures can easily be adapted for material obtained from other species.

Nfatc1 Promotes Interstitial Cell Formation During Cardiac Valve Development in Zebrafish.

RATIONALE: The transcription factor NFATC1 (nuclear factor of activated T-cell 1) has been implicated in cardiac valve formation in humans and mice, but we know little about the underlying mechanisms. To gain mechanistic understanding of cardiac valve formation at single-cell resolution and insights into the role of NFATC1 in this process, we used the zebrafish model as it offers unique attributes for live imaging and facile genetics. OBJECTIVE: To understand the role of Nfatc1 in cardiac valve formation. METHODS AND RESULTS: Using the zebrafish atrioventricular valve, we focus on the valve interstitial cells (VICs), which confer biomechanical strength to the cardiac valve leaflets. We find that initially atrioventricular endocardial cells migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the ECM (extracellular matrix) between the 2 endocardial cell monolayers, undergo endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion and a switch from a promigratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. With high-speed microscopy and echocardiography, we show that reduced VIC formation correlates with valvular dysfunction and severe retrograde blood flow that persist into adulthood. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b-a well-known regulator of epithelial-to-mesenchymal transition. CONCLUSIONS: Our study sheds light on the function of Nfatc1 in zebrafish cardiac valve development and reveals its role in VIC formation. It also further establishes the zebrafish as a powerful model to carry out longitudinal studies of valve formation and function.

In vivo tissue engineering of a trilayered leaflet-shaped tissue construct.

Aim: We aimed to develop a leaflet-shaped trilayered tissue construct mimicking the morphology of native heart valve leaflets. Materials & methods: Electrospinning and in vivo tissue engineering methods were employed. Results: We developed leaflet-shaped microfibrous scaffolds, each with circumferentially, randomly and radially oriented three layers mimicking the trilayered, oriented structure of native leaflets. After 3 months in vivo tissue engineering with the scaffolds, the generated leaflet-shaped tissue constructs had a trilayered structure mimicking the orientations of native heart valve leaflets. Presence of collagen, glycosaminoglycans and elastin seen in native leaflets was observed in the engineered tissue constructs. Conclusion: Trilayered, oriented fibrous scaffolds brought the orientations of the infiltrated cells and their produced extracellular matrix proteins into the constructs.

TGF-ß Signaling Promotes Tissue Formation during Cardiac Valve Regeneration in Adult Zebrafish.

Cardiac valve disease can lead to severe cardiac dysfunction and is thus a frequent cause of morbidity and mortality. Its main treatment is valve replacement, which is currently greatly limited by the poor recellularization and tissue formation potential of the implanted valves. As we still lack suitable animal models to identify modulators of these processes, here we used adult zebrafish and found that, upon valve decellularization, they initiate a rapid regenerative program that leads to the formation of new functional valves. After injury, endothelial and kidney marrow-derived cells undergo cell cycle re-entry and differentiate into new extracellular matrix-secreting valve cells. The TGF-ß signaling pathway promotes the regenerative process by enhancing progenitor cell proliferation as well as valve cell differentiation. These findings reveal a key role for TGF-ß signaling in cardiac valve regeneration and establish the zebrafish as a model to identify and test factors promoting cardiac valve recellularization and growth.

Quantifying heart valve interstitial cell contractile state using highly tunable poly(ethylene glycol) hydrogels.

Valve interstitial cells (VIC) are the primary cell type residing within heart valve tissues. In many valve pathologies, VICs become activated and will subsequently profoundly remodel the valve tissue extracellular matrix (ECM). A primary indicator of VIC activation is the upregulation of α-smooth muscle actin (αSMA) stress fibers, which in turn increase VIC contractility. Thus, contractile state reflects VIC activation and ECM biosynthesis levels. In general, cell contraction studies have largely utilized two-dimensional substrates, which are a vastly different micro mechanical environment than 3D native leaflet tissue. To address this limitation, hydrogels have been a popular choice for studying cells in a three-dimensional environment due to their tunable properties and optical transparency, which allows for direct cell visualization. In the present study, we extended the use of hydrogels to study the active contractile behavior of VICs. Aortic VICs (AVIC) were encapsulated within poly(ethylene glycol) (PEG) hydrogels and were subjected to flexural-deformation tests to assess the state of AVIC contraction. Using a finite element model of the experimental setup, we determined the effective shear modulus µ of the constructs. An increase in µ resulting from AVIC active contraction was observed. Results further indicated that AVIC contraction had a more pronounced effect on µ in softer gels (72 ±â€¯21% increase in µ within 2.5 kPa gels) and was dependent upon the availability of adhesion sites (0.5-1 mM CRGDS). The transparency of the gel allowed us to image AVICs directly within the hydrogel, where we observed a time-dependent decrease in volume (time constant τ=3.04 min) when the AVICs were induced into a hypertensive state. Our results indicated that AVIC contraction was regulated by both the intrinsic (unseeded) gel stiffness and the CRGDS peptide concentrations. This finding suggests that AVIC contractile state can be profoundly modulated through their local micro environment using modifiable PEG gels in a 3D micromechanical-emulating environment. Moving forward, this approach has the potential to be used towards delineating normal and diseased VIC biomechanical properties using highly tunable PEG biomaterials. STATEMENT OF SIGNIFICANCE.

Dynamic Bioreactors with Integrated Microfabricated Devices for Mechanobiological Screening.

Biomechanical stimulation is a common strategy to improve the growth, maturation, and function of a variety of engineered tissues. However, identifying optimized biomechanical conditioning protocols is challenging, as cell responses to mechanical stimuli are modulated by other multifactorial microenvironmental cues, including soluble factors and biomaterial properties. Traditional bioreactors lack the throughput necessary for combinatorial testing of cell activity in mechanically stimulated engineered tissues. Microfabricated systems can improve experimental throughput, but often do not provide uniform mechanical loading, are challenging to use, lack robustness, and offer limited amounts of cells and tissue for analysis. To address the need for higher-throughput, combinatorial testing of cell activity in a tissue engineering context, we developed a hybrid approach, in which flexible polydimethylsiloxane microfabricated inserts were designed to simultaneously generate multiple tensile strains when stretched cyclically in a standard dynamic bioreactor. In the embodiment presented in this study, each insert contained an array of 35 dog bone-shaped wells in which cell-seeded microscale hydrogels can be polymerized, with up to eight inserts stretched simultaneously in the bioreactor. Uniformity of the applied strains, both along the length of a microtissue and across multiple microtissues at the same strain level, was confirmed experimentally. In proof-of-principle experiments, the combinatorial effects of dynamic strain, biomaterial stiffness, and transforming growth factor (TGF)-ß1 stimulation on myofibroblast differentiation were tested, revealing both known and novel interaction effects and suggesting tissue engineering strategies to regulate myofibroblast activation. This platform is expected to have wide applicability in systematically probing combinations of mechanobiological tissue engineering parameters for desired effects on cell fate and tissue function. Impact Statement In this study, we introduce a dynamic bioreactor system incorporating microfabricated inserts to enable systematic probing of the effects of combinations of mechanobiological parameters on engineered tissues. This novel platform offers the ease of use, robustness, and well-defined mechanical strain stimuli inherent in traditional dynamic bioreactors, but significantly improves throughput (up to 280 microtissues can be tested simultaneously in the embodiment presented in this study). This platform has wide applicability to systematically probe combinations of dynamic mechanical strain, biomaterial properties, biochemical stimulation, and other parameters for desired effects on cell fate and engineered tissue development.

[The number and subgroups of lymphocytes in valve tissue of rheumatic heart disease combined with diabetes mellitus].

Objective To investigate the effect of diabetes mellitus on lymphocytes in rheumatic heart valve tissue and its mechanism. Methods Valve tissues of 40 patients undergoing heart valve replacement were collected, including 20 patients in rheumatic heart disease group (without diabetes) and 20 patients in diabetic group (rheumatic heart disease combined with diabetes). In addition, 20 cases of valve tissue from control group were collected. HE staining was used to observe the damage of valve tissue and the area of collagen degeneration. CD4+ T cells, CD8+ T cells, B cells and plasma cells were detected by immunohistochemical staining. Flow cytometry was used to detect the proportion of regulatory T cells (Tregs) in peripheral blood. Results Compared with the rheumatic heart disease group, the damage of valve tissue in the diabetic group was further aggravated, the number of infiltrating inflammatory cells increased, and the area of collagen degeneration was enlarged. Compared with the control group, the number of T cells, CD4+ T cells, CD8+ T cells, B cells and plasma cells in valve tissue of patients with rheumatic heart disease increased significantly. Diabetes mellitus further increased the number of T cells, CD4+ T cells, B cells and plasma cells in valve tissue, but had no significant effect on CD8+ T cells. The proportion of Tregs in the peripheral blood of patients with rheumatic heart disease was significantly reduced. Diabetes mellitus could further reduce the proportion of Tregs. Conclusion The number of T cells, CD4+T cells, B cells and plasma cells in heart valves of rheumatic heart disease patients with diabetes mellitus go up significantly, and Treg ratio goes down.

Biologically Inspired Scaffolds for Heart Valve Tissue Engineering via Melt Electrowriting.

Heart valves are characterized to be highly flexible yet tough, and exhibit complex deformation characteristics such as nonlinearity, anisotropy, and viscoelasticity, which are, at best, only partially recapitulated in scaffolds for heart valve tissue engineering (HVTE). These biomechanical features are dictated by the structural properties and microarchitecture of the major tissue constituents, in particular collagen fibers. In this study, the unique capabilities of melt electrowriting (MEW) are exploited to create functional scaffolds with highly controlled fibrous microarchitectures mimicking the wavy nature of the collagen fibers and their load-dependent recruitment. Scaffolds with precisely-defined serpentine architectures reproduce the J-shaped strain stiffening, anisotropic and viscoelastic behavior of native heart valve leaflets, as demonstrated by quasistatic and dynamic mechanical characterization. They also support the growth of human vascular smooth muscle cells seeded both directly or encapsulated in fibrin, and promote the deposition of valvular extracellular matrix components. Finally, proof-of-principle MEW trileaflet valves display excellent acute hydrodynamic performance under aortic physiological conditions in a custom-made flow loop. The convergence of MEW and a biomimetic design approach enables a new paradigm for the manufacturing of scaffolds with highly controlled microarchitectures, biocompatibility, and stringent nonlinear and anisotropic mechanical properties required for HVTE.

Optimizing detergent concentration and processing time to balance the decellularization efficiency and properties of bioprosthetic heart valves.

Decellularization treatment has been widely used to decrease the potential immunogenicity and improve the anticalcification properties of bio-derived materials, which may be utilized as an alternative method for the preparation of bioprosthetic heart valves. However, the excessive decellularization treatments will deteriarate the properties of heart valves. Among the decellularizaton parameters, detergent concentration and processing time are considered as those of the most key factors. Therefore, it should be meaningful to balance the decellularization efficiency and properties of bioprosthetic heart valves by optimizing the detergent concentration and processing time. In this study, three groups of the decellularized heart valves treated by sodium deoxycholate (SD) with different concentration and processing time were investigated through histological, biochemical, and mechanical analysis. Similar decellularization efficiency can be concluded through histological staining, DNA and α-Gal quantification results. Extracellular matrix contents quantification and tensile test results revealed that there is no obvious difference among the three decellularized heart valves. in vitro cytotoxicity assay showed that the remnant detergent is not enough to cause cell death, which indicated that the decellularized porcine aortic heart valves may be suitable for further in vivo research. In conclusion, Triton X-100/SD may be a suitable protocol used for heart valves decellularization. And it is feasible to vary the detergent processing time by changing the detergent concentration without compromising the decellularization efficiency.

VGLL4 plays a critical role in heart valve development and homeostasis.

Heart valve disease is a major clinical problem worldwide. Cardiac valve development and homeostasis need to be precisely controlled. Hippo signaling is essential for organ development and tissue homeostasis, while its role in valve formation and morphology maintenance remains unknown. VGLL4 is a transcription cofactor in vertebrates and we found it was mainly expressed in valve interstitial cells at the post-EMT stage and was maintained till the adult stage. Tissue specific knockout of VGLL4 in different cell lineages revealed that only loss of VGLL4 in endothelial cell lineage led to valve malformation with expanded expression of YAP targets. We further semi-knockout YAP in VGLL4 ablated hearts, and found hyper proliferation of arterial valve interstitial cells was significantly constrained. These findings suggest that VGLL4 is important for valve development and manipulation of Hippo components would be a potential therapy for preventing the progression of congenital valve disease.

Impact of modified gelatin on valvular microtissues.

A significant challenge in the field of tissue engineering is the biofabrication of three-dimensional (3D) functional tissues with direct applications in organ-on-a-chip systems and future organ engineering. Multicellular valvular microtissues can be used as building blocks for the formation of larger scale valvular macrotissues. Yet, for the controlled biofabrication of 3D macrotissues with predefined complex shapes, directed assembly of microtissues through bioprinting is needed. This study aimed to investigate if modified gelatin is an instructive material for valvular microtissues. Valvular microtissues were encapsulated in modified gelatin hydrogels and cross-linked in the presence of a photoinitiator (Irgacure 2959 or VA-086). Hydrogel properties were determined, and valvular interstitial cell functions like phenotype, proliferation, migration, mRNA expression of extracellular matrix (ECM) molecules, ECM deposition, and tissue fusion were characterized by histochemical stainings and RT-qPCR. Encapsulated microtissues remained viable, produced heart valve-related ECM components, and remained in a quiescent state. However, encapsulation induced some changes in ECM formation and gene expression. Encapsulated microtissues showed lower remodeling capacity and increased expression levels of Col I/V, elastin, hyaluronan, biglycan, decorin, and Sox9 compared with nonencapsulated microtissues. Furthermore, this study demonstrated that proliferation, migration, and tissue fusion was more pronounced in softer gels. In general, we evidenced that modified gelatin is an instructive material for physiologically relevant valvular microtissues and provided a proof of concept for the formation of larger valvular tissue by assembling microtissues at random in soft gels.

Endocardially Derived Macrophages Are Essential for Valvular Remodeling.

During mammalian embryogenesis, de novo hematopoiesis occurs transiently in multiple anatomical sites including the yolk sac, dorsal aorta, and heart tube. A long-unanswered question is whether these local transient hematopoietic mechanisms are essential for embryonic growth. Here, we show that endocardial hematopoiesis is critical for cardiac valve remodeling as a source of tissue macrophages. Colony formation assay from explanted heart tubes and genetic lineage tracing with the endocardial specific Nfatc1-Cre mouse revealed that hemogenic endocardium is a de novo source of tissue macrophages in the endocardial cushion, the primordium of the cardiac valves. Surface marker characterization, gene expression profiling, and ex vivo phagocytosis assay revealed that the endocardially derived cardiac tissue macrophages play a phagocytic and antigen presenting role. Indeed, genetic ablation of endocardially derived macrophages caused severe valve malformation. Together, these data suggest that transient hemogenic activity in the endocardium is indispensable for the valvular tissue remodeling in the heart.

Non-pathological Chondrogenic Features of Valve Interstitial Cells in Normal Adult Zebrafish.

In the heart, unidirectional blood flow depends on proper heart valve function. As, in mammals, regulatory mechanisms of early heart valve and bone development are shown to contribute to adult heart valve pathologies, we used the animal model zebrafish (ZF, Danio rerio) to investigate the microarchitecture and differentiation of cardiac valve interstitial cells in the transition from juvenile (35 days) to end of adult breeding (2.5 years) stages. Of note, light microscopy and immunohistochemistry revealed major differences in ZF heart valve microarchitecture when compared with adult mice. We demonstrate evidence for rather chondrogenic features of valvular interstitial cells by histological staining and immunodetection of SOX-9, aggrecan, and type 2a1 collagen. Collagen depositions are enriched in a thin layer at the atrial aspect of atrioventricular valves and the ventricular aspect of bulboventricular valves, respectively. At the ultrastructural level, the collagen fibrils are lacking obvious periodicity and orientation throughout the entire valve.

Dynamic Expression Profiles of Sox9 in Embryonic, Post Natal, and Adult Heart Valve Cell Populations.

Heart valves are dynamic structures and abnormalities during embryonic development can lead to premature lethality or congenital malformations present at birth. The transcription factor Sox9 has been shown to be critical for early and late stages of valve formation, but its defined expression pattern throughout embryonic, post natal, and adult growth and maturation is incomplete. Here we use an antibody to detect 1-100 amino acids of Sox9 and show that in the developing embryo, Sox9 is not detected in valve endothelial cells (VECs) lining the primitive valve structures, but is highly expressed in the endothelial-derived valve interstitial cell population following endothelial-to-mesenchymal transformation. Expression is maintained in this cell population after birth, but is additionally detected in VECs from post natal day 1. Using a specific antibody to detect a phosphorylated form of Sox9 at Serine 181 (pSox9), we note enrichment of pSox9 in VECs at post natal days 1 and 10 and this pattern correlates with the known upstream kinase RockI, and downstream target, Aggrecan. The contribution of Sox9 to post natal growth and maturation of the valve is not known, but this study provides insights for future work examining the differential functions of Sox9 protein in valve cell populations. Anat Rec, 302:108-116, 2019. © 2018 Wiley Periodicals, Inc.

Behavior of valvular interstitial cells on trilayered nanofibrous substrate mimicking morphologies of heart valve leaflet.

Heart valve tissue engineering could be an alternative to the current bioprosthetic heart valve that faces limitations especially in pediatric patients. However, heart valve tissue engineering has remained challenging because leaflets - the primary component of a heart valve - have three layers with three diverse orientations - circumferential, random and radial, respectively. In order to mimic the orientations, we first designed three novel collectors to fabricate three nanofibrous layers with those orientations from a polymeric biomaterial in an electrospinning system. Then, we devised a novel direct electrospinning technique to develop a unified trilayered nanofibrous (TN) substrate comprising those oriented layers. The TN substrate supported the growth and orientations of seeded porcine valvular interstitial cells (PVICs) and their deposited collagen fibrils. After one month culture, the obtained trilayered tissue construct (TC) exhibited increased tensile properties over its TN substrate. Most importantly, the developed TC did not show any sign of shrinkage. Gene expression pattern of the PVICs indicated the developing stage of the TC. Their protein expression pattern was quite similar to that of leaflets. STATEMENT OF SIGNIFICANCE: This manuscript talks about development of a novel trilayered nanofibrous substrate mimicking the morphologies of a heart valve leaflet. It also describes culturing of valvular interstitial cells that reside in a leaflet, in the substrate and compares the behavior of the cultured cells with that in native leaflets in terms cell morphology, protein deposition and its orientation, and molecular signature. This study builds the groundwork for our future trilayered, tissue-engineered leaflet development. This research article would be of great interest to investigators and researchers in the field of cardiovascular tissue engineering especially in cardiac valve tissue engineering through biomaterial-based tissue engineering.