P3503Transcriptome changes in atrial myocytes during the transition from a proliferative into a contractile phenotype and vice versa

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
J Liu ◽  
R Tsonaka ◽  
H Mei ◽  
B Akerboom ◽  
M Schalij ◽  
...  
Keyword(s):  
Stem Cells ◽  
Rna Sequencing ◽  
Cardiac Contraction ◽  
Neonatal Rat ◽  
Cardiomyocyte Differentiation ◽  
Atrial Myocytes ◽  
Contractile Phenotype ◽  
Cardiomyogenic Differentiation ◽  
Differentiation And Proliferation ◽  
Differentiation And Dedifferentiation

Abstract Background iAM-1 cells are conditionally immortalized neonatal rat atrial myocytes allowing toggling between proliferative and contractile phenotypes by a single-component change in culture medium composition. In the absence of proliferation stimuli, the cells synchronously differentiate into functional cardiomyocytes. Following re-expression of the immortalization factor, the fully differentiated iAM-1 cells dedifferentiate and start to proliferate again. Purpose The aim of our study was to investigate the changes in gene expression profile in iAM-1 cells during one round of cardiac differentiation and dedifferentiation in order to identify potential (new) regulators of atrial myocyte differentiation and proliferation. Methods RNA sequencing was performed on iAM-1 cells at 9 time points during one cycle of cardiomyogenic differentiation and dedifferentiation (20 million 150-bp paired-end reads per sample, 4 samples per time point). The resulting sequence data were analysed by EdgeR. Hierarchical clustering and principle component analysis were performed in R. GO category enrichment was determined using DAVID. Results Approximately 13,000 genes were extracted from the RNA sequencing analysis. In general, dynamic changes in mRNA levels during the transition from a proliferative into a contractile phenotype opposed those that occurred when differentiated iAM-1 were re-exposed to proliferation stimuli. These inverse trends were most evident for genes involved in cell cycle progression, DNA replication, sarcomere formation and cardiac contraction. Moreover, the RNA-SEQ data allowed us to make a distinction between genes contributing to the early and late phases of cardiomyogenic differentiation and dedifferentiation and to identify similarities and differences in the transcriptional programs underlying the cardiomyogenic differentiation of iAM-1 cells versus those of embryonic stem cells and induced pluripotent stem cells. The transcriptome analysis also unveiled several genes with potentially important and previously unrecognized roles in cardiomyocyte differentiation and proliferation. iAM-1 differentiation and dedifferention Conclusions Due to their ability to homogenously and synchronously differentiate and dedifferentiate, iAM-1 cells offer unique new insights into the transcriptional regulation of cardiomyocyte differentiation and proliferation.

scholarly journals Transcriptome analysis of conditionally immortalized atrial myocytes: identification of a novel atrium-enriched protein involved in sarcomere assembly and maintenance

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
P R R Van Gorp ◽  
J Zhang ◽  
J Liu ◽  
R Tsonaka ◽  
H Mei ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Heart Development ◽  
Troponin I ◽  
Specific Role ◽  
Neonatal Rat ◽  
Cardiomyocyte Differentiation ◽  
Atrial Myocytes ◽  
Uncharacterized Protein ◽  
Cardiomyogenic Differentiation ◽  
Cardiac Sarcomeres

Abstract Background Heart development relies on the tight spatiotemporal control of cardiac gene expression. Genes involved in these processes have been identified using mainly (transgenic) animals models and pluripotent stem cell-derived cardiomyocytes (CMs). Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal cell line of conditionally immortalized neonatal rat atrial myocytes (NRAMs) which allows toggling between proliferative and differentiated (i.e. excitable and contractile) phenotypes in a synchronized and homogenous manner. Purpose To identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation, dedifferentiation and proliferation by exploiting the unique properties of conditionally immortalized NRAMs (iAMs). Methods and results RNA sequencing was performed during a full cycle of iAM-1 differentiation and subsequent dedifferentiation, identifying ±13,000 transcripts, of which the dynamic expressional changes during cardiomyogenic differentiation in most cases opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many genes with a known (lineage-specific) role in cardiac muscle formation, thereby confirming the relevance of iAMs as cardiomyogenic differentiation model. Filtering for cardiomyocyte-enriched low abundancy transcripts, resulted in the identification of an uncharacterized protein, which is highly conserved among Nephrozoa and up- and downregulated during cardiomyocyte differentiation and dedifferentiation, respectively. In neonatal and adult rats, this protein is muscle-specific, highly atrium-enriched and localized around the C-zone of cardiac sarcomeres. Lentiviral shRNA-mediated knockdown resulted in loss of sarcomeric organization in both NRAMs and iAMs. Neither knockdown nor overexpression of this protein affected the electrophysiological properties of differentiated iAM monolayers. Conclusions iAM-1 cells offer a relevant model to identify and characterize novel (low abundancy) genes involved in cardiomyocyte (de)differentiation as exemplified by the identification a novel uncharacterized protein that is muscle-specific, highly atrium-enriched, localized around the C-zone of cardiac sarcomeres and plays a specific role in atrial sarcomerigenesis. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Netherlands Organisation for Health Research and Development (ZonMw) Leiden Regenerative Medicine Platform Holding project with number (LRMPH) Figure 1. (A) Experimental setup. At the indicated timepoints iAM-1 cells were fixed for immunostaining and RNA extraction for transcriptome analysis. (B) Immunochemical staining of iAM-1 cells for the proliferation marker Ki-67 and the Z-line marker sarcomeric α-actinin. (C & D) Immunohistological double stainings of longitudinal sections of neonatal rat hearts for the uncharacterized protein (GOI 1) and the sarcomeric protein cardiac troponin I (TNNI3). LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. Scale bar, 250 μm.

P5725Identification of novel cardiomyogenic factors by transcriptome analysis of conditionally immortalized atrial myocytes

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
P R R Van Gorp ◽  
J Liu ◽  
S O Dekker ◽  
J Zhang ◽  
M J Schalij ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Mrna Levels ◽  
Atrial Myocytes ◽  
Rat Cardiomyocytes ◽  
Cardiomyogenic Differentiation ◽  
Whole Transcriptome Analysis ◽  
Whole Transcriptome ◽  
Six Genes

Abstract Background Cardiac development involves the properly timed expression of cardiomyogenic differentiation factors (CDFs). CDFs have mainly been discovered using animal models and, more recently, pluripotent stem cell-derived cardiomyocytes (PSC-CMCs). These models are, however, laborious, time-consuming and costly. Also, cardiomyogenic differentiation of CMCs is heterogeneous and yields phenotypically immature CMCs. Recently, our research group generated a monoclonal line of conditionally immortalized atrial myocytes, called iAM-1. After removal of the proliferation stimulus these cells spontaneously and synchronously differentiate into mature atrial myocytes, making them ideally suited for transcriptome analysis and the discovery of novels factors involved in cardiomyogenic differentiation. Methods Whole transcriptome analysis of iAM-1 cells was performed at 9 different time points during cardiomyogenic differentiation and subsequent dedifferentiation by RNA sequencing. Six genes upregulated during cardiomyogenic differentiation were selected for knockdown studies in differentiating iAM-1 cells. Each of these genes was targeted by a bicistronic lentiviral vector (LV) driving expression of a specific short hairpin RNA (shRNA) and of enhanced green fluorescent protein (eGFP). Knockdown effects during cardiomyogenic differentiation were studied by immunocytology. The LVs were also used in primary neonatal atrial and ventricular rat cardiomyocytes to study the role of the selected genes in cardiomyocyte homeostasis. Results Whole transcriptome analysis of differentiating iAM-1 cells identified the dynamic expression levels of ± 13.000 genes, including the expected profile for genes known to play a role in atrial myocyte differentiation, like Nkx2–5, Tbx3, Tbx5 and Nppa. Six genes with an unknown role in cardiomyocyte differentiation and homeostasis were selected based on significant upregulation during iAM-1 differentiation, substantial mRNA levels and selective expression in cardiac tissue. Inhibiting gene expression by lentiviral RNA interference resulted for Nkx2–5 as well as for 3 out of 6 target genes in disturbed iAM-1 differentiation, as evinced by loss of sarcomeric cross-striations. Similar effects were observed in shRNA-expressing (i.e. eGFP-positive) primary atrial and ventricular neonatal rat myocytes. Taken together, these results highlight the importance of these novel genes during cardiomyogenic differentiation and homeostasis in atrial as well as ventricular myocytes. Conclusions Transcriptome analysis of cardiomyogenic differentiation in conditionally immortalized atrial myocytes combined with genetic knockdown experiments led to the identification of several novel factors involved in the differentiation and homeostasis of atrial and ventricular myocytes. These results highlight the suitability of iAM-1 as model for fundamental research of cardiomyogenic differentiation. Acknowledgement/Funding ZonMW

scholarly journals The Combination of bFGF and Hydrocortisone is a Better Alternative Compared to 5-Azacytidine for Cardiomyogenic Differentiation of Bone Marrow and Adipose Stem Cells

2021 ◽  
Vol 50 (7) ◽  
pp. 1987-1996
Author(s):  
Nadiah Sulaiman ◽  
Nur Qisya Afifah Veronica Sainik ◽  
Shamsul Bin Sulaiman ◽  
Pezhman Hafez ◽  
Min Hwei Ng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cardiac Tissue ◽  
Treated Group ◽  
Cell Number ◽  
Heart Cells ◽  
Cardiomyocyte Differentiation ◽  
Cardiomyogenic Differentiation ◽  
Marrow Stem Cell

Stem cells can be differentiated into cardiomyocytes by induction with 5-azacytidine (5-aza) but its carcinogenicity is of concern for future translational application. Alternatively, growth factors and hormones such as basic fibroblast growth factor (bFGF) and hydrocortisone have been reported to act as a therapeutic inducer for cardiomyocytes differentiation. In this study, we aim to investigate the ability of bFGF and hydrocortisone in combination to stimulate the differentiation of mesenchymal stem cells (MSC) into cardiomyocytes lineage. Sheep adipose tissue stem cell (ATSC) and bone marrow stem cell (BMSC) were isolated, cultured and induced with the three groups of induction factors; 5-aza alone, the combination of hydrocortisone and bFGF and all three factors in combination for cardiomyogenic differentiation. Morphological, protein and functional ability of both ATSC and BMSC were observed and analysed to confirm cardiomyocyte differentiation. Viability of BMSC and ATSC in each treated group was significantly higher (P < 0.05) on both cells after treated with 10 nM of bFGF and 50 μM of hydrocortisone. Cardiomyocyte proteins; α-Sarcomeric actin (αSA) and Phospolamban (Plb) was detected in both ATSC and BMSC exposed to induction factors but not in the control negative group. Both ATSC and BMSC without induction factors showed only minute cell number possesses αSA and Plb. Calcium ion (Ca2+) spark was observed in primary heart cells. Similarly, Ca2+ spark was also detected in induced ATSC and BMSC, proving some functionality of induced cells. In conclusion, bFGF and hydrocortisone are safer induction factor compared to the currently used 5-aza as both showed higher viability after induction, therefore more cells are available for future use in cardiac tissue engineering.

Icariin Promotes In Vitro Cardiomyocyte Proliferation and Differentiation in Human Bone Marrow-Derived Mesenchymal Stem Cells

2022 ◽  
Vol 12 (3) ◽  
pp. 480-488
Author(s):  
Shaoying Liu ◽  
Chengying Zhang ◽  
Jing Hao ◽  
Yuna Liu ◽  
Sidao Zheng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Immunofluorescent Staining ◽  
Cardiomyocyte Differentiation ◽  
Cardiomyogenic Differentiation ◽  
Proliferation And Differentiation

Mesenchymal stem cells (MSCs) are the excellent candidates in myocardial regeneration given their easy accessibility, low immunogenicity and high potential for cardiomyocyte differentiation. This work focused on investigating the role of icariin, a main active component of the Traditional Chinese herb epimedium, in human bone marrow-derived MSCs (BMSCs) proliferation and differentiation into cardiomyocytes In Vitro. Human BMSCs were cultivated In Vitro, and MTT assay was conducted to measure their proliferation. On this basis, we selected the optimal icariin dose for promoting the proliferation to induce cardiomyocyte differentiation of MSCs, which were pretreated with or without 5-azacytidine (5-Aza). Cardiac-specific cardiac troponin I (cTnI) and connexin 43 (Cx43)-positive cells were detected by immunofluorescent staining. The differentiation ratio of MSCs was examined by flow cytometry. This study measured early cardiac transcription factors (TFs) Nkx2.5 and GATA4 levels through RT-PCR and Western blotting (WB). As a result, icariin increased MSC proliferation dependent on its dose, and the optimal dose was determined to be 80 μg/l. Furthermore, MSCs showed minimal cardiomyogenic differentiation when induced by icariin alone as confirmed by the expression of cardiac-related markers. Moreover, a synergic interaction was observed when icariin and 5-Aza cooperated to induce cardiomyocyte differentiation of MSCs. In conclusion, Icariin stimulates proliferation and facilitates cardiomyocyte differentiation of MSCs In Vitro and may be potentially used as a new method for enhancing the MSCs efficacy in cardiovascular disease.

scholarly journals Identification of New Transcription Factors that Can Promote Pluripotent Reprogramming

2021 ◽  
Author(s):  
Ping Huang ◽  
Jieying Zhu ◽  
Yu Liu ◽  
Guihuan Liu ◽  
Ran Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Rna Sequencing ◽  
Human Urine ◽  
Embryonic Stem ◽  
Rna Seq ◽  
Reprogramming Efficiency ◽  
Yamanaka Factors ◽  
Urine Cells

Abstract Background Four transcription factors, Oct4, Sox2, Klf4, and c-Myc (the Yamanka factors), can reprogram somatic cells to induced pluripotent stem cells (iPSCs). Many studies have provided a number of alternative combinations to the non-Yamanaka factors. However, it is clear that many additional transcription factors that can generate iPSCs remain to be discovered. Methods The chromatin accessibility and transcriptional level of human embryonic stem cells and human urine cells were compared by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to identify potential reprogramming factors. Selected transcription factors were employed to reprogram urine cells, and the reprogramming efficiency was measured. Urine-derived iPSCs were detected for pluripotency by Immunofluorescence, quantitative polymerase chain reaction, RNA sequencing and teratoma formation test. Finally, we assessed the differentiation potential of the new iPSCs to cardiomyocytes in vitro. Results ATAC-seq and RNA-seq datasets predicted TEAD2, TEAD4 and ZIC3 as potential factors involved in urine cell reprogramming. Transfection of TEAD2, TEAD4 and ZIC3 (in the presence of Yamanaka factors) significantly improved the reprogramming efficiency of urine cells. We confirmed that the newly generated iPSCs possessed pluripotency characteristics similar to normal H1 embryonic stem cells. We also confirmed that the new iPSCs could differentiate to functional cardiomyocytes. Conclusions In conclusion, TEAD2, TEAD4 and ZIC3 can increase the efficiency of reprogramming human urine cells into iPSCs, and provides a new stem cell sources for the clinical application and modeling of cardiovascular disease. Graphical abstract

Application potential of three-dimensional silk fibroin scaffold using mesenchymal stem cells for cardiac regeneration

2021 ◽  
pp. 088532822110185
Author(s):  
Yuksel Cetin ◽  
Merve G Sahin ◽  
Fatma N Kok
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Silk Fibroin ◽  
Cardiac Tissue ◽  
Troponin I ◽  
Cardiac Tissue Engineering ◽  
Swelling Ratio ◽  
Differentiation Potential ◽  
Cardiomyogenic Differentiation

Cardiac tissue engineering focusing on biomaterial scaffolds incorporating cells from different sources has been explored to regenerate or repair damaged area as a lifesaving approach.The aim of this study was to evaluate the cardiomyocyte differentiation potential of human adipose mesenchymal stem cells (hAD-MSCs) as an alternative cell source on silk fibroin (SF) scaffolds for cardiac tissue engineering. The change in surface morphology of SF scaffolds depending on SF concentration (1–6%, w/v) and increase in their porosity upon application of unidirectional freezing were visualized by scanning electron microscopy (SEM). Swelling ratio was found to increase 2.4 fold when SF amount was decreased from 4% to 2%. To avoid excessive swelling, 4% SF scaffold with swelling ratio of 10% (w/w) was chosen for further studies.Biodegradation rate of SF scaffolds depended on enzymatic activity was found to be 75% weight loss of SF scaffolds at the day 14. The phenotype of hAD-MSCs and their multi-linage potential into chondrocytes, osteocytes, and adipocytes were shown by flow cytometry and immunohistochemical staining, respectively.The viability of hAD-MSCs on 3D SF scaffolds was determined as 90%, 118%, and 138% after 1, 7, and 14 days, respectively. The use of 3D SF scaffolds was associated with increased production of cardiomyogenic biomarkers: α-actinin, troponin I, connexin 43, and myosin heavy chain. The fabricated 3D SF scaffolds were proved to sustain hAD-MSCs proliferation and cardiomyogenic differentiation therefore, hAD-MSCs on 3D SF scaffolds may useful tool to regenerate or repair damaged area using cardiac tissue engineering techniques.

Human adipose‐derived stromal/stem cells are distinct from dermal fibroblasts as evaluated by biological characterization and RNA sequencing

2021 ◽  
Author(s):  
Mariane Izabella Melo ◽  
Pricila Cunha ◽  
Marcelo de Miranda ◽  
Camila Cristina Fraga Faraco ◽  
Joana Lobato Barbosa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Rna Sequencing ◽  
Dermal Fibroblasts ◽  
Biological Characterization ◽  
Stromal Stem Cells
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