Int J Stem Cells.  2019 Jul;12(2):251-264. 10.15283/ijsc18126.

Angiotensin II and TGF-β1 Induce Alterations in Human Amniotic Fluid-Derived Mesenchymal Stem Cells Leading to Cardiomyogenic Differentiation Initiation

• GasiÅ«nienÄ— M
• Petkus G
• Matuzevičius D
• Navakauskas D
• NavakauskienÄ— R

• KMID: 2465896
• DOI: http://doi.org/10.15283/ijsc18126

Abstract

BACKGROUND AND OBJECTIVES
Human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may be a valuable source for cardiovascular tissue engineering and cell therapy. The aim of this study is to verify angiotensin II and transforming growth factor-beta 1 (TGF-β1) as potential cardiomyogenic differentiation inducers of AF-MSCs.
METHODS AND RESULTS
AF-MSCs were obtained from amniocentesis samples from second-trimester pregnant women, isolated and characterized by the expression of cell surface markers (CD44, CD90, CD105 positive; CD34 negative) and pluripotency genes (OCT4, SOX2, NANOG, REX1). Cardiomyogenic differentiation was induced using different concentrations of angiotensin II and TGF-β1. Successful initiation of differentiation was confirmed by alterations in cell morphology, upregulation of cardiac genes-markers NKX2-5, TBX5, GATA4, MYH6, TNNT2, DES and main cardiac ion channels genes (sodium, calcium, potassium) as determined by RT-qPCR. Western blot and immunofluorescence analysis revealed the increased expression of Connexin43, the main component of gap junctions, and Nkx2.5, the early cardiac transcription factor. Induced AF-MSCs switched their phenotype towards more energetic and started utilizing oxidative phosphorylation more than glycolysis for energy production as assessed using Agilent Seahorse XF analyzer. The immune analysis of chromatin-modifying enzymes DNMT1, HDAC1/2 and Polycomb repressive complex 1 and 2 (PRC1/2) proteins BMI1, EZH2 and SUZ12 as well as of modified histones H3 and H4 indicated global chromatin remodeling during the induced differentiation.
CONCLUSIONS
Angiotensin II and TGF-β1 are efficient cardiomyogenic inducers of human AF-MSCs; they initiate alterations at the gene and protein expression, metabolic and epigenetic levels in stem cells leading towards cardiomyocyte-like phenotype formation.

Keyword

Myocytes; Cardiac; Amniotic fluid; Cell differentiation; Chromatin; Histones

MeSH Terms

Amniocentesis
Amniotic Fluid
Angiotensin II*
Angiotensins*
Blotting, Western
Calcium
Cell Differentiation
Cell- and Tissue-Based Therapy
Chromatin
Chromatin Assembly and Disassembly
Connexin 43
Epigenomics
Female
Fluorescent Antibody Technique
Gap Junctions
Glycolysis
Histones
Humans*
Ion Channels
Mesenchymal Stromal Cells*
Muscle Cells
Oxidative Phosphorylation
Phenotype
Polycomb Repressive Complex 1
Pregnant Women
Smegmamorpha
Stem Cells
Tissue Engineering
Transcription Factors
Up-Regulation
Angiotensin II
Angiotensins
Calcium
Chromatin
Connexin 43
Histones
Ion Channels
Polycomb Repressive Complex 1
Transcription Factors

Figure

• 

  Fig. 1 Characterization of human AF-MSCs. (A) The typical spindle-shaped morphology of human amniotic fluid-derived mesenchymal stem cells, cultivated in cell culture. Scale bar=400 μm. (B) The expression of cell surface markers CD44, CD90, CD105 and CD34 as detected by flow cytometry. Unlabeled ctrl–non-labeled, undifferentiated control cells. Results are presented as mean±SD (n=3). (C) The relative expression of pluripotency genes-markers, such as OCT4, SOX2, NANOG and REX1 as determined by RT-qPCR. Data, relative to GAPDH, are presented as mean±SD (n=3).

• 

  Fig. 2 Alterations in morphology and gene expression patterns during decitabine, angiotensin II and TGF-β1 induced cardiac differentiation. (A) Assessment of the cell morphology 12 days after the cardiac differentiation initiation. Dec: decitabine, AngII: angiotensin II, TGF-β1: transforming growth factor-beta 1. Scale bar=400 μm. (B) The connections between proteins that are considered as cardiomyogenic differentiation markers. NKX2-5, TBX5 and GATA4 are the early transcription markers, initiating the expression of structural proteins of cardiomyocytes, such as MYH6 (α-myosin heavy chain), TNNT2 (cardiac troponin T) and DES (Desmin). The data was obtained from the STRING database. (C) The relative expression of the main cardiac : genes-markers, such as NKX2-5, TBX5, GATA4, MYH6, TNNT2 and DES at the days 5 (5 d.) and 12 (12 d.) of induced differentiation. Ctrl–non-differentiated control cells. (D) The relative expression of cardiac ion channels genes at the day 12 of differentiation: SCN5A (sodium voltage-gated channel α-subunit 5), CACNA1D (L-type calcium channel), KCNJ12 and KCND3 (voltage-gated potassium) and HCN2 (hyperpolarization-activated cyclic nucleotide-gated channel). The relative gene expression was determined by RT-qPCR, normalized to GAPDH and presented as a fold change over undifferentiated control. The data are presented as mean±SD (n=3), p≤0.05 (*), p≤0.01 (**), p≤0.001 (***), ns: non-significant.

• 

  Fig. 3 Evaluation of Connexin43 (Cx43) and Nkx2.5 levels in induced AF-MSCs. (A) The levels of Cx43, the main gap junction protein, and Nkx2.5, an early cardiac transcription factor, as determined by Western blot in non-differentiated cells (Ctrl) and differentiated cells at the days 5 and 12. As a positive control mouse heart lysate was used. The relative density of each band was measured using ImageJ software (NIH, USA), normalized to the GAPDH loading control and presented as a fold difference over control. The data are presented as mean±SD (n=3), p≤0.05 (*), p≤0.01 (**), p≤0.001 (***), ns: non-significant. The blots represent one of three independent experiments showing similar results. (B) The distribution of Cx43 in control and induced cells at the 12 day of differentiation as obtained using the immunofluorescence assay. The data from each differentiation sample is presented in two images: the left panel–two channels view (blue nuclei and green Cx43) and the right panel–the same image in three channels view (blue nuclei, green Cx43 and red F-actin). Samples were observed using Zeiss Axio Observer fluorescence microscope, 63X magnification with immersion oil, scale bar=10 μm. (C) The immunofluorescence data of Nkx2.5 protein in control and induced cells at the 12 day of differentiation. Nuclei stained blue, Nkx2.5 – red and F-actin – green. Samples were observed using Zeiss Axio Observer fluorescence microscope, 63X magnification with immersion oil, scale bar=10 μm. (D) Quantitative evaluation of Nkx2.5 nuclear localization at the day 12 of differentiation as calculated from the Nkx2.5 immunofluorescence images. The nuclear quantity of Nkx2.5 was normalized to the median value of undifferentiated Control (Ctrl) and presented as normalized nuclear quantity (NNQ). All NNQs were graphed as Tukey-style box plots with sample size n=66, 31, 57, 58, 64 and 60. Relative change of medians and the statistical significance of changes are presented above the box plots. Wilcoxon rank sum test was used for statistical analysis, p≤0.001 (***).

• 

  Fig. 4 Cell energy phenotype and metabolic alterations during cardiac differentiation of AF-MSCs. (A) Cell energy diagram showing normalized oxygen consumption rate (OCR) and normalized extracellular acidification rate (ECAR) of control and differentiated cells at the basal (the hollow points) and stressed conditions (colored points). (B) The ratio of normalized OCR to normalized ECAR in control and induced AF-MSCs. (C) Metabolic potential (in percent) of undifferentiated and induced AF-MSCs was calculated from stressed OCR/ECAR over Baseline OCR/ECAR. (D) Mitochondrial respiration expressed as normalized OCR of control and differentiated cells after the addition of ETC inhibitors oligomycin, FCCP, rotenone and antimycin A. The time in the x-axis represents time points when each measurement was done. All calculations of mitochondrial function were done referring to these measurements. (E) Maximal respiration, calculated as normalized OCR fold induction over baseline. (F) Spare respiratory capacity, a derivative from Maximal respiration, was expressed in percent for the control and induced cells. (G) ATP production was calculated as the OCR difference between the baseline and after the addition of oligomycin. (H) The relative expression of genes, related to the cell metabolism and respiration: PPARGC1A (Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1 Alpha), NRF1 (nuclear respiratory factor 1) and HIF1A (hypoxia-inducible factor 1-alpha), in control and differentiated cells as determined using RT-qPCR. The data were normalized to GAPDH and presented as a fold change over undifferentiated control. The data in A~G was obtained using Seahorse XFp Extracellular Flux Analyzer (Agilent, USA), “Cell energy phenotype test” (parts A~C) or “Cell Mito Stress Test” kits (parts D~G) and cells differentiated for 12 days. Results are presented as mean±SD. p≤0.05 (*), p≤0.01 (**), p≤0.001 (***), ns: non-significant.

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  Fig. 5 Epigenetic alterations during induced cardiomyogenic differentiation. (A) Total proteins were extracted from control cells (Ctrl) and cells, induced using angiotensin II and TGF-β1 at the days 5 and 12 of differentiation. Alterations in the expression levels of Polycomb repressive complex 2 (PRC2) proteins EZH2 and SUZ12, Polycomb repressive complex 1 (PRC1) component BMI1, DNA and histones modifying proteins DNMT1, HDAC1 and HDAC2 as well as in histone modifications, considered as the markers of active chromatin state (H3K4me3, H3K9Ac and H4hyperAc) are presented as determined by Western blot. Mouse heart lysate was used as a positive control. The blots represent one of three independent experiments showing similar results. (B) Relative band density of chromatin remodeling proteins and (C) histone modifications, as measured using ImageJ software (NIH, USA) and normalized to the GAPDH loading control. The data are presented as mean±SD (n=3), p≤0.05 (*), p≤0.01 (**), p≤0.001 (***), ns: non-significant.

Reference

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