Simple monolayer differentiation of murine cardiomyocytes via nutrient deprivation-mediated activation of β-catenin
Pablo Hofbauer1,6,7, Jangwook P. Jung1,2,7, Tanner J. McArdle1, and Brenda M. Ogle1,2,3,4,5*
Affiliations:
1 Department of Biomedical Engineering, 2 Stem Cell Institute, 3 Masonic Cancer Center, 4Lillehei Heart Institute, 5 Institute for Engineering in Medicine,University of Minnesota – Twin Cities, 312 Church St. SE, Minneapolis, MN 55455, U.S.A.
6Department of Biochemical Engineering, University of Applied Sciences Technikum Wien,Höchstädtplatz 5, 1200, Vienna, Austria
7 Pablo Hofbauer and Jangwook P. Jung contributed equally to this work.
E-mail: Pablo Hofbauer (), JangwookP. Jung (), and Tanner J. McArdle ()
Supplementary Figures
Figure S1.OCT4 staining confirms the pluripotent state of the used iPSC line (iPSC #1).(A) Quantification of 3 independent fields of viewresults in >90% OCT4-positive cells. n=3, meanSD. Representative field of view is shown in (B).
Figure S2.Cell viability in different serum conditions.Culture of miPSCs in 2% FBS led to significant cell death and no proliferation, while increasing concentration to 5% FBS increased survival but still led to significant cell death. No contracting regions were found in either condition when the NDCD-protocol was performed.
Figure S3.Comparison of two different types of FBS shows no significant difference in CTNTfraction.Cells underwent the NDCD-protocol in two different sera (Defined, Cat# SH30070.0, Hyclone vs. Normal, Cat# 26140, Gibco) and showed no difference in covered area of CTNT-positive phenotypes. A total of 303 images for Normal FBS and 488 images for Defined FBS. MeanSD.
Figure S4.Normal morphology and growth of miPSCs subjected to the NDCD-protocol. No significant cell death was observed and became confluent at about days 6-7.
Figure S5.Comparison of NMR and biochemical detection of lactate and glucose.NMR and biochemical assays showed very similar trends of differential concentrations of lactate and glucose. Lactate and glucose concentrations were measured by MAK064-KIT (Sigma-Aldrich) and TR15421 (Thermo-Fisher), respectively. n=3, meanSD.
Figure S6. The concentration differentials of amino acids during the course of NDCD-protocol.Glutamine,an energy source in cultures of PSCs [1], matches the profile for glucose with higher consumption during the nutrient-limiting phase of the culture period (A). Glutamate was barely detected (see HC, Figure 4A), its cyclized form pyroglutamate was released to culture medium at early time points in the culture period. Profiling exometabolomes of human midbrain-derived neural progenitor cells showed a similar pyroglutamate rate of release in in vitro cultures over 48 h[2] (B). The differentials of the amino acids (C) Serine and (D) Glycine display an increase until day 6 followed by a sharp decrease toward a plateau at 0.
Supplementary Tables
Table S1. Formulation of GMEMand NEAA in miPSC culture medium.
GMEM / mg/LInorganic Salts
Calcium chloride anhydrous / 200.00
Magnesium sulfate anhydrous / 98.00
Potassium chloride / 400.00
Sodium bicarbonate / 2750.00
Sodium chloride / 6400.00
Sodium phosphate monobasic monohydrate / 123.80
Amino Acids
L-Arginine·HCl / 42.12
L-cystine free base / 24.02
Glycine / 800.00
L-Histidine·HCl·H2O / 21.00
L-isoleucine / 52.46
L-leucine / 52.46
L-lysine HCl / 73.06
L-methionine / 14.92
L-phenylalanine / 33.02
L-threonine / 47.64
L-tryptophan / 8.16
L-tyrosine free base / 36.22
L-valine / 46.86
Vitamins and others
Choline chloride / 2.00
Folic acid / 2.00
i-inositol / 4.00
Niacinamide / 2.00
D-calcium pantothenate / 2.00
Pyridoxal HCl / 2.00
Riboflavin / 0.20
Thiamine·HCl / 2.00
Dextrose anhydrous / 4500.00
Phenol red sodium salt / 17.00
Sodium pyruvate / 110.00
Non-essential Amino Acids
Glycine / 750.00
L-Alanine / 890.00
L-Asparagine / 1320.00
L-Aspartic acid / 1330.00
L-Glutamic Acid / 1470.00
L-Proline / 1150.00
L-Serine / 1050.00
Table S2. qRT-PCR primer sequences for WNT-responsive genes.
Target gene / Primer sequence (5 3) / Amplicon size (bp)Ccnd1 / FOR: TTCGCAGACCTGACATCCAGTACC
REV: TCCTCGCTTTCCATGTGTGAGG / 279
Axin2 / FOR: AGCCTCATGAAGAGCCTTCCAAC
REV: TCTCCGGAAGAAACCCTTGCATC / 104
Ctnnb1 / FOR: GCTTCCGCTGTCCTGAGA
REV: ATGCCAGCAGATTCCATACC / 101
Gapdh / FOR: TTCCGCGACGTGGACAT
REV: TCAAACTCGTTGACATCGAAGGT / 150
Table S3.Summary of the main features of current and past cardiomyocyte differentiation protocols from murine pluripotent stem cells.
Differentiation Protocol / EB with beating foci / Fraction of Total Cell Number / ReferenceEB formation + Wnt Modulation / >95% EB with beating foci / - / [3]
EB formation + Matrigel + StemPro / >50% EB with beating foci / - / [4]
EB formation + Wnt Modulation / ~ 50% EB with beating foci / ~ 15% NKX2.5+ cells / [5]
EB formation + Activin A + BMP4 + VEGF + bFGF / - / ~ 60% cTnT+ cells / [6]
EB formation + Wnt Modulation / >98% EB with beating foci / ~ 58% α-actinin+ cells / [7]
EB formation + VEGF, Activin A, BMP4 / - / 50-60% cTnT+ cells / [8]
EB formation + IGF / <70% EB with beating foci / - / [9]
Monolayer + N2, B27, VEGF, Activin, BMP4, StemPro, bFGF / - / 60-78% cTnT+ cells / [10]
Supplementary Videos
Supplementary Video 1. Cardiomyocytes derived from miPSCsvia forming EBs and adding WNT3A.
Supplementary Video 2. Cardiomyocytes derived from miPSCsthat underwent the NDCD-protocol.
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