Developmental Changes in Electrophysiological Characteristics of Human Induced Pluripotent

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Developmental changes in electrophysiological characteristics of human induced Pluripotent StemCell-derived cardiomyocytes

*Meital Ben-Ari, M.Sc, a, b, c, *Shulamit Naor, M.Sc, a, b, c, Naama Zeevi-Levin, Ph.D, c, Revital Schick, M.Sc, a, b, c, Ronen Ben Jehuda, M.Sc, a, b, c, d, Irina Reiter, M.Sc, a, b, c, Amit Raveh, B.Sc, e, Inna Grijnevitch, B.Sc, e, Omri Barak, Ph.D, c, Michael R. Rosen, M.D, f, Amir Weissman, M.D, c,g, Ofer Binah, Ph.D, a, b, c

aDepartment of Physiology, BiophysicsandSystems Biology, bThe Rappaport Institute, cRappaport Faculty of Medicine, dDepartment of Biotechnology and eFaculty of Electrical Engineering, Technion, Haifa, Israel. fDepartments of Pharmacology and Pediatrics, Columbia University Medical Center, New York, NY, USA, gDepartment of Obstetrics and Gynecology, Rambam Medical Center, Haifa, Israel.

*These authors contributed equally to this manuscript.

Corresponding author

Ofer Binah, PhD

Department of Physiology

Ruth & Bruce Rappaport Faculty of Medicine
Efron Street, POB 9649, Haifa, 31096 Israel Email: ; Tel: +972-4-8295262; Fax: +972-4-8513919

Methods

Generation of induced Pluripotent Stem Cells (iPSCs) from human hair follicle keratinocytes (HFKTs)

Derivation of keratinocytes from human plucked hair follicles

iPSCs were generated from keratinocytes derived from 2 healthy females (39 and 54 year old) as previously described.1 The study was approved by the local Ethics Committee(permit # 3116), and both subjects gave informed consent. Ten hairs with visible outer root sheathes were plucked from the scalps of each volunteer. The bulk of the hair follicles was cut off and the follicles were immersed in a 10 cm Petri dish with Dulbecco’s modified Eagle’s medium (DMEM) containing 25 mmol/l HEPES, 1 mmol/l L-glutamine and 400 U/ml penicillin, 400 μg/ml streptomycin (PS) for 15 min - 1hr at 37oC. The follicles were washed with PBS (Biological Industries, Beit Haemek, Israel), covered with 0.1% trypsin and 0.02% EDTA (diluted with PBS) and incubated for 30 min at 37oC. A single cell suspension culture was obtained by vigorously pipetting the follicles with DMEM supplemented with 10% fetal bovine serum (FBS). The dissociated keratinocytes were centrifuged for 10 min at 200 g and seeded in 3 wells of a 6-well plate on an inactivated 3T3 feeder layer (2x104 3T3 cells/cm2) with Green medium (60% DMEM, 30% DMEM F-12, 10% FBS, 1 mmol/l sodium pyruvate, 2 mmol/l L-glutamine, 5 μg insulin, 0.5 μg/ml hydrocortisone, 0.2 nmol/l adenine, 2 nmol/l triiodothyronine (T3), 10 ng/ml Epidermal Growth Factor and 100 U/ml penicillin, 100 μg/ml streptomycin). For splitting, 3T3 cells were removed after incubation with 0.02% EDTA for 5 min in 37oC. The culture was washed with PBS, and then the keratinocytes detached and were dissociated into single cells by incubation with 0.1% Trypsin and 0.02% EDTA in PBS at 37°C for 10-15 min.1

Generation of iPSCs from HFKTs

On the 1stday 30,000 HFKTs were seeded on an inactivated 3T3 feeder layer (20,000 cells/cm2) supplemented with Green medium, in one well of a 6-well plate.2 On the 2nd day, viruses were produced as follows: the humanized version of a single lentiviral vector STEMCCA Cassette was generated following the transfection of 293T cells with five plasmids: STEM-CCA: Gag-Pol: REV: TAT: VSVG, at ratios of 20:1:1:1:2, respectively.3 The total plasmid amount was 15 µg DNA. Transfection was done by a jetPEI™ Reagent (Polyplus transfectionTM, France). The medium was replaced with fresh medium 24 hrs post-transfection (day 3). At 48 hrs post-transfection (day 4), the accumulated viral particles were passed through a 0.45 µm filter, supplemented with 2 µg/ml polybrene and used for infection of HFKTs. Immediately before infection 3T3 feeder cells were removed using 0.02% EDTA. The infection was performed during centrifugation for 50 min at 500 g, at 32°C. Thereafter, the medium was replaced with fresh Green medium, and fresh inactivated 3T3 feeder cells were added. The infection was repeated on the following day (day 5). On day 5 post-infection (day 8), infected keratinocytes were detached by 0.1% Trypsin and 0.02% EDTA in PBS at 37°C for 10 min, centrifuged for 10 min at 200 g, and seeded on an inactivated mouse embryonic fibroblast (MEF) feeder with Green medium on a 6-well plate. On the following day the medium was replaced with human embryonic stem cells (hESCs) medium containing 8 ng/ml basic fibroblast growth factor (bFGF) (Invitrogen, NY, USA).4 The medium was replaced every second day. Finally, 21-25 days after seeding the infected keratinocytes, hESC-like colonies emerged and could be further expanded and analyzed.

iPSCs differentiation into cardiomyocytes

For the electrophysiological experiments iPSCs were grown on MEF feeders in 80% DMEM/F-12 (HAM) 1:1 (Biological Industries, Beit Haemek, Israel), 20% knockout serum replacement, 4 ng/ml bFGF, 1 mM glutamine, 0.1 mM β-mercaptoethanol, and 1% essential amino acid stock (all from Gibco-BRL, Gaithersburg, MD, USA, To induce embryoid bodies (EBs) formation, iPSCs were detached using 1 mg/ml type IV collagenase (Gibco-BRL) and transferred to Petri dishes to allow their aggregation. Resultant EBs were grown in 80% DMEM, 20% FBS (HyClone, UT, USA, 1 mmol/l glutamine, and 1% nonessential amino acid stock. The EBs were then cultured in suspension for 7 days and plated on gelatin-coated (0.1%; Sigma-Aldrich, MO, USA) 6-well plates. Daily microscopic observations were conducted to detect the first spontaneous contractions. The contracting areas were then carefully dissected out by micro-scalpel for action potential recordings (detailed below). Our previous publications reported a normal karyotype and electrogram properties recorded from EBs generated from two volunteers (#201201 and #201202, data not shown).1,9,10 Because the FACS analysis required a large number of cardiomyocytes, we used the “Matrix sandwich” protocol for differentiation11. Briefly, iPSCs were cultured on Matrigel (GFR, BD Biosciences, NJ, USA) coated 6-well plates in mTeSR1 (Stemcell Technologies, Canada) medium for 5-6 days. To initiate differentiation, cells were washed with PBS and incubated with 1 ml/well Versene solution (Invitrogen, Life Technologies, MA, USA) at 37oC for 5 minutes and seeded on Matrigel coated plate at 6x106/6 well plate density in mTeSR1 medium supplemented with 10 μmol/l ROCK inhibitor (Cayman Chemical, MI, USA). The medium was changed daily, and after 2 days when the monolayer of cells reached 80-90% confluence, a thin layer of Matrigel was overlaid by freshly mixing 1 mg Matrigel in 15 ml ice cold mTeSR1 medium and replacing the medium in each well of a 6 well plate with 2.5 ml of Matrigel containing mTeSR1. Cells were cultured in mTeSR1 medium for another 1-2 days until the cells were 100% confluent, which is referred to as day 0, when the medium was replaced with 2.5 ml of RPMI 1640 (Invitrogen, Life Technologies) basal medium plus B-27 without insulin supplement (Invitrogen, Life Technologies) containing Activin A (100 ng/ml, R&D Systems) and Matrigel (0.5 mg Matrigel/6-well plate). After 24 hours, the medium was changed to the same medium as day 0 (3 ml/well) without Matrigel but supplemented with BMP4 (5-10 ng/ml, R&D Systems) and bFGF (5-10 ng/ml, R&D Systems) for another 4 days without medium change. At day 5, the medium was changed to RPMI plus B27 complete supplement (Invitrogen, Life Technologies), and the medium was changed every 2-3 days.

Action potential and If recording and analysis

For action potential recordings, spontaneously contracting areas of EBs were mechanically dissociated and enzymatically dispersed (collagenase II 1 mg/ml; Worthington, NJ, USA, This dispersion resulted in single cells-to-small clusters which were plated on gelatin-coated glass coverslips (13 mm diameter) in 24-well plates. The coverslips were incubated at 37°C, and a recovery period of 2 days was allowed before performing electrophysiological experiments.9,12 In all experiments, the coverslips were superfused at 37°C with an external solution containing (in mmol/l): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose and 10 HEPES titrated to pH 7.4 with NaOH (310 mOsm). The patch pipette solution contained (mmol/l): 120 KCl, 1 MgCl2, 3 Mg-ATP, 10 HEPES, and 10 EGTA titrated to pH 7.2 with KOH and adjusted at 290 mOsm with saccharose (all materials were purchased from Sigma-Aldrich). Ifwas recorded from single cardiomyocytes in the presence of 500 μM BaCl2 to block IK1. To record If the membrane was clamped at 15 sec intervals, from a holding potential of -40 mV to -120 mV in 10 mV steps for 2 sec pulse duration. All experiments were performed at 36°C.Axopatch 200B, Digidata 1322 and pClamp10 (Molecular Devices, Sunnyvale, CA) were used for data amplification, acquisition and analysis. Signals were digitized at 10 kHz and filtered at 2 kHz. Patch electrodes with resistances of 4-7 MΩ were pulled from borosilicate glass capillaries (Harvard Apparatus, MA, USA).

Action potential analysis

Action potentials were analyzed for amplitude, overshoot, maximal upstroke velocity (dV/dtmax) and action potential duration (APD) at 90% repolarization (APD90). The electrophysiological experiments were conducted for up to 30 minutes, during which action potential configuration and characteristics were stable. In the course of these experiments we have never witnessed a time-dependent action potential phenotype change in any cell studied. APD90 and dV/dtmax data were fit using a Gaussian model13 (Fig. 5). For each age group, we applied an Expectation Maximization algorithm (MATLAB built-in function gmdistribution.fit, Mathworks) to fit between 1 and 5 Gaussians to the data. Model selection was done using the Bayesian Information Criterion which penalized the log likelihood by the number of parameters. Using the Akaike Information Criterion (a slightly different penalty) yielded similar results except for APD90value distribution in the 4th age group where the data were inconclusive regarding 2 or 3 populations.

Beat rate variability

Using dedicated MATLAB software, action potentials were analyzed for peak detection, from which interbeat-intervals (IBIs) and IBIs coefficient of variance (IBI COV) were calculated.9To analyze beat rate variability (BRV), we used IBI, IBI COV and SD1 and SD2 (i.e., Poincaréplot measures).

Poincaré plot

In the Poincaré plot, each IBI (IBIn+1) is plotted against its predecessor (IBIn), creating a scattered mass of points in a two-dimensional array which reflect the dynamics of the system14. Quantitative analysis of the plot is performed by fitting an ellipse to the group of points, with its center coinciding with the centroid of the ellipse (the point of the average IBI), and adjusting two perpendicular lines traversing the centroid. The longitudinal line designated SD2, represents long-term variability of the data (reflecting the standard deviation of the IBI intervals (SDIBI)), while the perpendicular line designated SD1 represents short-term beat-to-beat variability14(analogous to the standard deviation of successive differences (SDSD) of IBI, or its root-mean-square (RMSSD)).

Fluorescence Activated Cell Sorting (FACS)

Cells were detached from the culture plates by incubation with 0.25% trypsin-EDTA (Invitrogen, Life Technologies) for 10 minutes at 37°C. Next, cell aggregates were further dissociated with plastic tip and neutralized by adding an equal volume of EB medium. About one million cells were used for each sample analysis. For extracellular staining, cells were incubated with primary antibody diluted in 100 μl /sample FACS buffer (PBS without Ca/Mg2+, 2% BSA) for 1 hour at room temperature. Cells were washed twice in 2 ml FACS buffer, centrifuged, and the supernatant was discarded. Cells then were incubated with secondary antibody specific to the primary IgG isotype diluted in 100 μl /sample FACS buffer on ice in dark for 1 hour. For intracellular staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Bioscience, NJ, USA) on ice in dark for 20 minutes. Cells were washed twice in Perm/Wash buffer (BD Bioscience), centrifuged, supernatant discarded and cells were incubated with primary antibody diluted in 100μl/sample Cytofix/Cytoperm buffer solution for 45 minutes at room temperature in dark. Cells were washed twice in 2 ml Cytofix/Cytoperm buffer solution, centrifuged, supernatant discarded and cells were incubated with secondary antibody specific to the primary IgG isotype diluted in 100 μl /sample Cytofix/Cytoperm buffer in dark for 1 hour at room temperature. Cells were washed twice in Perm/Wash buffer, centrifuged, supernatant discarded and resuspended in 400 μl FACS buffer for analysis. Data were collected on a FACS CyAn ADP (Beckman Coulter) flow cytometer and analyzed using FlowJo v10. Primary antibodies used for staining: Troponin T (Thermo Scientific, Waltham, MA, USA), HCN4 (Novus Biological, CO, USA), myosin regulatory light chain 2 (Novus Biological, Colorado, USA) and myosin regulatory light chain 7 (Novus Biological). Secondary antibodies used for staining: anti-mouse conjugated with FITC (Jackson Laboratory, ME, USA), anti-rat conjugated with APC (Jackson Laboratory, USA) and anti-rabbit conjugated with brilliant violet 421 (BioLegend, CA, USA).

Separation into 3 groups according to APD90 and dV/dtmax using Gaussian fit

The analysis was performed by fitting a Gaussian to the entire APD90 and dV/dtmax populations, and the number of populations provided by the lowest Bayesian Information Criteria (BIC, also known as Schwarz criteria) values was registered.

Statistical analysis

Offline data analysis and statistical evaluations were performed with SigmaPlot (Systat Software Inc., CA, USA) and Prism (GraphPad Software, CA, USA). In Figs. 1D and 2B two-way ANOVA was performed. In Figs. 3H,4F-4Jone-way ANOVA was performed on ranks followed by Dunn’s test. In Fig. 3I and Fig.6t-test for unpaired observations was used. In Fig. 1 in the On-line Supplement we preformed:Two-Way ANOVA in panel B and t-test followed by Mann-Whitney Rank Sum Test in panel C. P<0.05 was considered significant. Results were expressed as Mean±SEM.

Results

Developmental changes in APs characteristics

Phenotypic diversity of APs

The experiments were performed on iPSC-CMs generated from two healthy volunteers (cell lines KTN and KTI).Hence, we confirmed that there are no significant differences in AP characteristics between KTN and KTI, thereby permitting us to combine data from the two cell lines. To this end we compared dV/dtmax, APD90, overshoot and amplitude in the first age group of both KTN and KTI iPSC-CMs (On-line Supplement Fig. 1). Action potential characteristics from the two cell lines show very similar scatter. Furthermore, one-way ANOVA revealed P>0.05 for all 4 characteristics (see legend, On-line supplement Fig. 1).

Development changes in electrophysiological characteristics

In addition to APD90, APD20 and APD50 were also characterized and were found to be scattered throughout the age groups (On-line Supplement, Fig. 2A-B). Yet APD20 (P<0.05, Fig. 2C) and APD50 (P<0.05, Fig. 2D) were larger in the 4th age group than in the others.

Can we use APD90 and dV/dtmax as objective measures to separate AP into the 3 groups?

Similarly to APD90, dV/dtmax could not be used to generate 3 distinct groups (On-line Supplement Figs. 3A-D). Specifically, two populations were found in each age group, albeit the 2nd population in the On-line Supplement Fig. 3B is small. In the 1st, 2nd and 4th age groups (On-line Supplement Figs. 3A-D) which include only nodal-to-ventricular phenotypes, the populations represent the nodal phenotype with lower dV/dtmax and the ventricular with higher dV/dtmax. The Mean±SEM dV/dtmax values (V/sec) were: 1st group: 8.1±0.3 and 16.9±1.2; 2nd group: 10.8±0.4 and 40.5±5.6; 4th group: 19.0±0.8 and 37.5±3.0. As expected, the 3rd group (On-line Supplement Fig. 3C) includes 2 clear populations: nodal-to-ventricular (dV/dtmax= 31.8±2.6 V/sec), and the atrial having much faster dV/dtmax of 87.5±2.7 V/sec. In summary, this statistical analysis shows that while neither APD90 nor dV/dtmax per se can discriminate among the 3 AP phenotypes, at certain ages the Gaussian fit of APD90 and dV/dtmax can generate 2 distinct groups.

Supplemental Figures and legends

Figure 1 On-line Supplement

Figure 1: Electrophysiological characteristics of action potentials in 7-to-24 day iPSC-CMs to validate unionof parameters from the 2 cell-lines (KTI, KTN). [A-D] The changes with days in culture of dV/dtmax, APD90, overshoot and amplitude, respectively (n=37 iPSC-CMs). To determine if there is a significant difference between the 2 cell-linesfor each of the 4 measured parameters, one-way ANOVA was performed. In [A] P=0.860, [B] P=0.113, [C] P=0.115, [D] P=0.242.

Figure 2 On-line Supplement

Figure 2: Electrophysiological characteristics of action potentials in 7-to-95 day iPSC-CMs.[A] and [B]: The changes with days in culture of APD20and APD50, respectively (n=203 iPSC-CMs).[C] and [D]: The Mean±SEM APD20 and APD50, respectively, in the 4 age groups (7-24, 25-56, 57-70, 71-95 days in culture; n=37, 44, 70 and 52 iPSC-CMs, respectively). To determine if there is a significant difference between each of the 2 parameters measured among the 4 age groups, one-way ANOVA was performed on ranks followed by Dunn’s test. In [C] *P<0.05, 71-95 vs. 7-24, 25-56 and 57-70 days.***P<0.05, 57-70 vs. 7-24 and 25-56. In [D] *P<0.05.

Figure 3 On-line Supplement

Figure 3: The relationships between dV/dtmax and day in culture. [A-D]Histogram distribution anddV/dtmax and best Gaussian fit in the 4 age groups (7-24, 25-56, 57-70, 71-95 days in culture; n=37, 44, 70 and 52 respectively). The Gaussian fit was calculated to automatically identify and quantify populations, using Schwarz criteria. Mean±SEM dV/dtmax of the populations (V/sec): 8.1±0.3 and 16.9±1.2 (7-24 day iPSC-CMs) [A], 10.8±0.4 and 40.5±5.6 (25-56 iPSC-CMs) [B], 31.8±2.6 and 87.5±2.7 (57-70 iPSC-CMs) [C], 19.0±0.8 and 37.5±3.0 (71-95 iPSC-CMs) [H].

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