Establishment of A Three-Dimensional Culture Condition for The U266 Cell Line Based on Peripheral Blood Plasma-Derived Fibrin Gels.

OBJECTIVE: The study of pathophysiology as well as cellular and molecular aspects of diseases, especially cancer, requires appropriate disease models. In vitro three-dimensional (3D) structures attracted more attention to recapitulate diseases rather than in vitro two-dimensional (2D) cell culture conditions because they generated more similar physiological and structural properties. Accordingly, in the case of multiple myeloma (MM), the generation of 3D structures has attracted a lot of attention. However, the availability and cost of most of these structures can restrict their use. Therefore, in this study, we aimed to generate an affordable and suitable 3D culture condition for the U266 MM cell line. MATERIALS AND METHODS: In this experimental study, peripheral blood-derived plasma was used to generate fibrin gels for the culture of U266 cells. Moreover, different factors affecting the formation and stability of gels were evaluated. Furthermore, the proliferation rate and cell distribution of cultured U266 cells in fibrin gels were assessed. RESULTS: The optimal calcium chloride and tranexamic acid concentrations were 1 mg/ml and 5 mg/ml for gel formation and stability, respectively. Moreover, the usage of frozen plasma samples did not significantly affect gel formation and stability, which makes it possible to generate reproducible and available culture conditions. Furthermore, U266 cells could distribute and proliferate inside the gel. CONCLUSION: This available and simple fibrin gel-based 3D structure can be used for the culture of U266 MM cells in a condition similar to the disease microenvironment.

Multifunctional Conductive Biomaterials as Promising Platforms for Cardiac Tissue Engineering.

Adult cardiomyocytes are terminally differentiated cells that result in minimal intrinsic potential for the heart to self-regenerate. The introduction of novel approaches in cardiac tissue engineering aims to repair damages from cardiovascular diseases. Recently, conductive biomaterials such as carbon- and gold-based nanomaterials, conductive polymers, and ceramics that have outstanding electrical conductivity, acceptable mechanical properties, and promoted cell-cell signaling transduction have attracted attention for use in cardiac tissue engineering. Nevertheless, comprehensive classification of conductive biomaterials from the perspective of cardiac cell function is a subject for discussion. In the present review, we classify and summarize the unique properties of conductive biomaterials considered beneficial for cardiac tissue engineering. We attempt to cover recent advances in conductive biomaterials with a particular focus on their effects on cardiac cell functions and proposed mechanisms of action. Finally, current problems, limitations, challenges, and suggested solutions for applications of these biomaterials are presented.

Vascular endothelial growth factor sustained delivery augmented cell therapy outcomes of cardiac progenitor cells for myocardial infarction.

Cell therapy has become a novel promising approach for improvement of cardiac functional capacity in the instances of ventricular remodeling and fibrosis caused by episodes of coronary artery occlusion and hypoxia. The challenge toward enhancing cell engraftment as well as formation of functional tissue, however, necessitated combinatorial approaches. Here, we complemented human embryonic stem cell-derived cardiac progenitor cell (hESC-CPC) therapy by heparin-conjugated, vascular endothelial growth factor (VEGF)-loaded fibrin hydrogel as VEGF delivery system. Transplantation of these cardiac committed cells along with sustained VEGF release could surpass the cardiac repair effects of each constituent alone in a rat model of acute myocardial infarction. The histological sections of rat hearts revealed improved vascularization as well as inclusion of hESC-CPC-derived cardiomyocytes, endothelial, and smooth muscle cells in host myocardium. Thus, co-transplantation of hESC-CPC and proangiogenic factor by a suitable delivery rate may resolve the shortcomings of conventional cell therapy.

Expansion of Human Pluripotent Stem Cell-derived Early Cardiovascular Progenitor Cells by a Cocktail of Signaling Factors.

Cardiovascular progenitor cells (CPCs) derived from human pluripotent stem cells (hPSCs) are proposed to be invaluable cell sources for experimental and clinical studies. This wide range of applications necessitates large-scale production of CPCs in an in vitro culture system, which enables both expansion and maintenance of these cells. In this study, we aimed to develop a defined and efficient culture medium that uses signaling factors for large-scale expansion of early CPCs, called cardiogenic mesodermal cells (CMCs), which were derived from hPSCs. Chemical screening resulted in a medium that contained a reproducible combination of three factors (A83-01, bFGF, and CHIR99021) that generated 1014 CMCs after 10 passages without the propensity for tumorigenicity. Expanded CMCs retained their gene expression pattern, chromosomal stability, and differentiation tendency through several passages and showed both the safety and possible cardio-protective potentials when transplanted into the infarcted rat myocardium. These CMCs were efficiently cryopreserved for an extended period of time. This culture medium could be used for both adherent and suspension culture conditions, for which the latter is required for large-scale CMC production. Taken together, hPSC-derived CMCs exhibited self-renewal capacity in our simple, reproducible, and defined medium. These cells might ultimately be potential, promising cell sources for cardiovascular studies.

Chitosan-PVA-CNT nanofibers as electrically conductive scaffolds for cardiovascular tissue engineering.

Conductive scaffolds are suitable candidates for cardiovascular tissue engineering (CTE) due to their similarity to the extracellular matrix of native tissue. Here, nanofiber scaffolds based on polyvinyl alcohol (PVA), chitosan (CS), and different concentrations of carbon nanotube (CNT) were produced using electrospinning. Scanning electron microscopy (SEM) image, mechanical test (elastic modulus: 130 ±â€¯3.605 MPa), electrical conductivity (3.4 × 10-6 S/Cm), water uptake, cell adhesion, and cell viability (>80%) results of the PVA-CS-CNT1 scaffold revealed that the nanofiber containing 1% of CNT has optimal properties for cardiac differentiation. Afterwards, the differentiation of rat mesenchymal stem cells (MSCs) to cardiomyocytes was performed on the optimal scaffold by electrical stimulation in the presence of 5-azacytidine, TGF-ß and ascorbic acid. The real-time qPCR results indicated that the expression of Nkx2.5, Troponin I, and ß-MHC cardiac marker was increased significantly (>3 folds) in comparison to control group. Based on the findings of this study, the incorporation of MSCs, conductive scaffolds, and electrical stimulation seem to be a promising approach in CTE.

Cardioprotective effects of omega-3 fatty acids and ascorbic acid improve regenerative capacity of embryonic stem cell-derived cardiac lineage cells.

One of the major issues in cell therapy of myocardial infarction (MI) is early death of engrafted cells in a harsh oxidative stress environment, which limits the potential therapeutic utility of this strategy in the clinical setting. Increasing evidence implicates beneficial effects of omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and ascorbic acid (AA) in cardiovascular diseases, in particular their role in ameliorating fibrosis. In the current study, we aim to assess the cytoprotective role of EPA + DHA and AA in protecting embryonic stem cell (ESC)-derived cardiac lineage cells and amelioration of fibrosis. Herein, we have shown that preincubation of the cells with EPA + DHA + AA prior to H2 O2 treatment attenuated generation of reactive oxygen species (ROS) and enhanced cell viability. Gene expression analysis revealed that preincubation with EPA + DHA + AA followed by H2 O2 treatment, upregulated heme oxygenase-1 (HO-1) along with cardiac markers (GATA4, myosin heavy chain, α isoform [MYH6]), connexin 43 [CX43]) and attenuated oxidative stress-induced upregulation of fibroblast markers (vimentin and collagen type 1 [Col1]). Alterations in gene expression patterns were followed by marked elevation of cardiac troponin (TNNT2) positive cells and reduced numbers of vimentin positive cells. An injection of EPA + DHA + AA-pretreated ESC-derived cardiac lineage cells into the ischemic myocardium of a rat model of MI significantly reduced fibrosis compared to the vehicle group. This study provided evidence that EPA + DHA + AA may be an appropriate preincubation regimen for regenerative purposes. © 2019 BioFactors, 45(3):427-438, 2019.

Prediction of putative small molecules for manipulation of enriched signalling pathways in hESC-derived early cardiovascular progenitors by bioinformatics analysis.

Human pluripotent stem cell-derived cardiovascular progenitor cells (CPCs) are considered as powerful tools for cardiac regenerative medicine and developmental study. Mesoderm posterior1+ (MESP1+ ) cells are identified as the earliest CPCs from which almost all cardiac cell types are generated. Molecular insights to the transcriptional regulatory factors of early CPCs are required to control cell fate decisions. Herein, the microarray data set of human embryonic stem cells (hESCs)-derived MESP1+ cells was analysed and differentially expressed genes (DEGs) were identified in comparison to undifferentiated hESCs and MESP1-negative cells. Then, gene ontology and pathway enrichment analysis of DEGs were carried out with the subsequent prediction of putative regulatory small molecules for modulation of CPC fate. Some key signalling cascades of cardiogenesis including Hippo, Wnt, transforming growth factor-ß, and PI3K/Akt were highlighted in MESP1+ cells. The transcriptional regulatory network of MESP1+ cells were visualised through interaction networks of DEGs. Additionally, 35 promising chemicals were predicted based on correlations with gene expression signature of MESP1+ cells for effective in vitro CPC manipulation. Studying the transcriptional profile of MESP1+ cells resulted into the identification of important signalling pathways and chemicals which could be introduced as powerful tools to manage proliferation and differentiation of hESC-derived CPCs more efficiently.

Establishment of A Protocol for In Vitro Culture of Cardiogenic Mesodermal Cells Derived from Human Embryonic Stem Cells.

OBJECTIVE: Cardiovascular progenitor cells (CPCs) are introduced as one of the promising cell sources for preclinical studies and regenerative medicine. One of the earliest type of CPCs is cardiogenic mesoderm cells (CMCs), which have the capability to generate all types of cardiac lineage derivatives. In order to benefit from CMCs, development of an efficient culture strategy is required. We aim to explore an optimized culture condition that uses human embryonic stem cell (hESC)-derived CMCs. MATERIALS AND METHODS: In this experimental study, hESCs were expanded and induced toward cardiac lineage in a suspension culture. Mesoderm posterior 1-positive (MESP1+) CMCs were subjected to four different culture conditions: i. Suspension culture of CMC spheroids, ii. Adherent culture of CMC spheroids, iii. Adherent culture of single CMCs using gelatin, and iv. Adherent culture of single CMCs using Matrigel. RESULTS: Although, we observed no substantial changes in the percentage of MESP1+ cells in different culture conditions, there were significantly higher viability and total cell numbers in CMCs cultured on Matrigel (condition iv) compared to the other groups. CMCs cultivated on Matrigel maintained their progenitor cell signature, which included the tendency for cardiogenic differentiation. CONCLUSION: These results showed the efficacy of an adherent culture on Matrigel for hESC-derived CMCs, which would facilitate their use for future applications.

Isolation and characterization of cardiogenic, stem-like cardiac precursors from heart samples of patients with congenital heart disease.

AIMS: Regenerative therapies based on resident human cardiac progenitor cells (hCPCs) are a promising alternative to medical treatments for patients with myocardial infarction. However, hCPCs are rare in human heart and finding efficient source and proper surface marker for isolation of these cells would make them a good candidate for therapy. MAIN METHODS: We have isolated 5.34∗10(6)±2.04∗10(5)/g viable cells from 35 heart tissue samples of 23 patients with congenital heart disease obtained during their heart surgery along with 6 samples from 3 normal subjects during cardiac biopsy. KEY FINDINGS: According to FACS analysis, younger ages, atrial specimen and disease with increased pulmonary vascular resistance were associated with higher percentage of c-kit(+) (CD117) hCPCs. Analysis for other stemness markers revealed increased CD133(+) cells in the hearts of patients with congenital heart disease. By using both immune-labeling and PCR, we demonstrated that these cells express key cardiac lineage and endothelial transcription factors and structural proteins during in vitro differentiation and do express stemness transcription factors in undifferentiated state. Another novel datum of potentially relevant interest is their ability in promoting greater myocardial regeneration and better survival in rat model of myocardial infarction following transplantation. SIGNIFICANCE: Our results could provide evidence for conditions associated with enriched hCPCs in patients with congenital heart disease. Moreover, we showed presence of a significant number of CD133 expressing cardiogenic stem-like cardiac precursors in the heart of patients with congenital heart disease, which could be isolated and stored for future regenerative therapies in these patients.

Cellular and molecular characterization of human cardiac stem cells reveals key features essential for their function and safety.

Cell therapy of heart diseases is emerging as one of the most promising known treatments in recent years. Transplantation of cardiac stem cells (CSCs) may be one of the best strategies to cure adult or pediatric heart diseases. As these patient-derived stem cells need to be isolated from small heart biopsies, it is important to select the best isolation method and CSC subpopulation with the best cardiogenic functionality. We employed three different protocols including c-KIT(+) cell sorting, clonogenic expansion, and explants culture to isolate c-KIT(+) cells, clonogenic expansion-derived cells (CEDCs), and cardiosphere-derived cells (CDCs), respectively. Evaluation of isolated CSC characteristics in vitro and after rat myocardial infarction (MI) model transplantation revealed that although c-KIT(+) and CDCs had higher MI regenerative potential, CEDCs had more commitment into cardiomyocytes and needed lower passages that were essential to reach a definite cell count. Furthermore, genome-wide expression analysis showed that subsequent passages caused changes in characteristics of cells, downregulation of cell cycle-related genes, and upregulation of differentiation and carcinogenic genes, which might lead to senescence, commitment, and possible tumorigenicity of the cells. Because of different properties of CSC subpopulations, we suggest that appropriate CSCs subpopulation should be chosen based on their experimental or clinical use.

Preparation of a porous conductive scaffold from aniline pentamer-modified polyurethane/PCL blend for cardiac tissue engineering.

A novel biodegradable electroactive polyurethane containing aniline pentamer (AP) was blended with polycaprolactone (PCL). The prepared blend (PB) and PCL were further fabricated in to scaffolds using a mixture of poly(ethylene glycol) and salt particles in a double porogen particulate leaching and compression molding methodology. Scaffolds held open and interconnected pores having pore size ranging from several µm to 150 µm. PB scaffolds had compression modulus and strength of 4.1 and 1.3 MPa, respectively. The conductivity of the scaffold was measured as 10(-5) ± 0.09 S .cm(-1) and preserved for at least 100 h post fabrication. Scaffolds supported neonatal cardiomyocytes adhesion and growth with PB showing more extensive effect on the expression of the cardiac genes involved in muscle contraction and relaxation (troponin-T) and cytoskeleton alignment (actinin-4). Our results highlight the potential of incorporation of AP as an electroactive moiety for induction of cardiomyocyte proliferation and repair of damaged heart tissue.

The behavior of cardiac progenitor cells on macroporous pericardium-derived scaffolds.

Cardiovascular diseases hold the highest mortality rate among other illnesses which reveals the significance of current limitations in common therapies. Three-dimensional (3D) scaffolds have been utilized as potential therapies for treating heart failure following myocardial infarction (MI). In particular, native tissues have numerous properties that make them potentially useful scaffolding materials for recreating the native cardiac extracellular matrix (ECM). Here, we have developed a pericardium-derived scaffold that mimics the natural myocardial extracellular environment and investigated its properties for cardiac tissue engineering. Human pericardium membranes (PMs) were decellularized to yield 3D macroporous pericardium scaffolds (PSs) with well-defined architecture and interconnected pores. PSs enabled human Sca-1(+) cardiac progenitor cells (CPCs) to migrate, survive, proliferate and differentiate at higher rates compared with decellularized pericardium membranes (DPMs) and collagen scaffolds (COLs). Interestingly, histological examination of subcutaneous transplanted scaffolds after one month revealed low immunological response, enhanced angiogenesis and cardiomyocyte differentiation in PSs compared to DPMs and COLs. This research demonstrates the feasibility of fabricating 3D porous scaffolds from native ECMs and suggests the therapeutic potential of CPC-seeded PSs in the treatment of ischemic heart diseases.