Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling
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Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.
KeywordsHuman iPS cells Cardiac differentiation WNT signaling BMP signaling Lactate enrichment Disease modeling
In spite of recent advances in medicine cardiovascular disorders remain a major cause of mortality in the world . Supply with human cardiomyocytes is generally limited due to lack of donors as well as the restricted proliferation rate of adult cardiomyocytes. Thus, with respect to use human cardiomyocytes for regenerative therapies, drug toxicity studies as well as disease modeling alternative sources are highly desired. There have been many attempts in this direction using adult stem cells such as bone marrow derived stem cells (BMSCs) , mesenchymal stem cell (MSCs) , c-kit and isl-1 positive cardiac stem cells (CSCs) [4, 5]. However there is little evidence that BMSCs and MSCs differentiate into cardioymocytes after transplantation since positive effects observed using those cells are mainly due to angiogenesis and paracrine effects . Although it has been shown that CSCs can be differentiated into all cardiovascular lineages in an animal model , in humans there have been rare studies due to lack of donors, limited in vitro amplification as well as complicated isolation procedures of the CSCs [6, 8]. Embryonic stem (ES) cells hold great promise for providing an unlimited source of cardiac cells since ES cells self-renew indefinitely in cell culture and are able to differentiate into any somatic cell type . However ethical considerations associated with the use of human embryos might represent a roadblock for clinical application . Major breakthrough in this field came when Yamanaka and colleagues showed that overexpression of four transcription factors namely Oct-4, Sox2, Klf-4 and c-Myc were able to transform somatic cells into induced pluripotent stem cells (iPS) . iPS technology allows generation of pluripotent stem cells from any somatic cells. Not only it overcomes ethical concerns associated with ES cells but also offers the potential of autologous transplantation since patient-specific cells can be used for cellular reprogramming .
Numerous protocols have been published reporting the derivation of cardiomyocyte-like cells from human ES and iPS cells. Induction of differentiation by co-culture with stroma cells has been demonstrated  as well as the use of embryoid body (EB) based differentiation paradigms [13, 14]. It is similar to embryonic development in some respect and cells from all three germ layers are formed during the course of differentiation. However EBs have complex microenvironments and for this reason signaling pathways are difficult to modulate explaining poor efficiency of cardiac differentiation . Moreover, there is a significant line-to-line variability with respect to the method of reprogramming used and iPS quality resulting in up to 100-fold differences in lineage specific gene expression amongst the lines treated with same protocols . Such variability within a broad range of pluripotent cell lines greatly limits its application [17, 18]. Several approaches have been published utilizing monolayer culture of cells in a serum free condition having growth factors such as BMP4, Activin A, FGF2, VEGF in order to increase the efficiency while reducing the heterogeneity arising during EB based differentiation [19, 20, 21]. However, it has been shown that optimal concentrations of growth factors greatly vary among different iPS lines. A study by Kattman et al. as well as follow up report by Sa et al. systematically showed different requirements of Activin A and BMP4 concentration for efficient cardiomyocytes yield amongst different pluripotent cell lines [22, 23]. Thus, robust and efficient cardiac differentiation requires the optimization of the protocol for each individual line, which makes it laborious .
Recent advances in cell signaling studies have shed light on detailed signaling pathways involved during cardiac differentiation. It has been shown that WNT signaling plays a critical role during cardiogenesis . It has been suggested that during early embryonic differentiation WNT is required for mesodermal specification, however, later on cardiac specification is hampered by WNT signaling and thus inhibition of WNT signaling might be necessary for the formation of cardiomyocytes . Recent studies have shown the use of WNT inhibiting small molecules for increasing the cardiomyocyte yield in the case of EB  as well as monolayer-based protocols [28, 29]. Moreover, two recent studies by Cao et al. have shown importance of ascorbic acid in increasing the cardiac differentiation efficiency by affecting MEK/ERK pathway. By combining the application of BMP4, CHIR99021 and ascorbic acid they were successful in isolation of cardiovascular cells from human pluripotent stem cells [30, 31]. However, varied differentiation propensities of multiple pluripotent cell lines to a particular protocol require more alternative approaches. Hence, still it is highly desired to devise simple and efficient protocols to achieve high robustness and efficacy. Here we show improvement in cardiac differentiation efficiency using precise modulation of WNT and BMP signaling. We identify an optimal combination of concentrations of BMP4 and CHIR99021 and finally perform cardiomyocyte enrichment by lactate supplementation . By that within 15 days we achieve generation of more than 90 % cardiomyocytes as judged by cardiac Tropononin T staining with unmatched low line-to-line variability.
Materials and Methods
Human iPSC Cultivation and Subsequent Cardiac Differentiation
Human iPSC were maintained on hESC-qualified Matrigel(TM) (BD Biosciences) coated plates in mTESR1 (STEMCELL Technologies) medium until they reached 80 to 90 % confluency. Cardiac differentiation was induced by BMP4 (Life technologies) (25 ng/ml) and CHIR99021 (5 μM) (Sigma-Aldrich) in RPMI1640 (Life technologies) medium containing B27 (Life technologies) and 2 mM glutamine (Life technologies) and 50 μg/ml L-Ascorbic acid (Cell culture tested powder; Sigma-Aldrich) as a basal medium. After 24 h cells were kept in the same basal medium with CHIR (5 μM) only for 18–36 h. Afterwards cells were kept in RPMI basal medium with B27 without insulin for 24 h then medium was replaced with similar basal medium having WNT inhibitor either 10 μM of XAV939 (Sigma-Aldrich) or IWR1 (Sigma-Aldrich) for 5 days. Afterwards cells were kept 4 to 5 days in basal medium (B27 + insulin) followed by replacement with cardiac enrichment medium (RPMI 1640 without glucose (Life technologies) + 4 mM sodium L-lactate  (Sigma-Aldrich). Cells were kept in enrichment medium for 4 to 5 days. After enrichment phase medium was switched back to basal medium (RPMI + B27 + glutamine).
del-AR1034ZIMA 001 and fl-AR1034ZIMA 001 are lentiviral reprogramming derived iPS sister clones from human dermal fibroblasts (del: transgene excised, fl: transgene floxed . Human iPS cell line (k-hiPS)  is a lentivirally derived iPS cell line from human keratinocytes kindly gifted by Dr. Stefan Liebau from University of Tübingen, Germany. Human iPS cell line iLB-C-50-s9 is a Sendai virus derived iPS cell line from human cord blood cells. Human iPS cell line iLB-C1-30 m-r12 is a retroviral reprogramming derived iPS cell line from human dermal fibroblasts cells . As WNT reporter cell line we used a neural stem cell line (I3 lt-NES) carrying the 7TGP WNT reporter construct developed by Fuerer & Nusse (2010)  and obtained via Addgene (#24305).
For cardiomyocyte characterization, immunostaining was performed using anti-cTNT (Abcam; 1:100) and anti-alpha-actinin (Sigma-Aldrich; 1:200) as primary antibodies and Alexafluor-488-cojugated anti-mouse IgG as a secondary antibody. Briefly, cells were washed with PBS (Life technologies), fixed with 4 % PFA (Sigma-Aldrich) for 15 min and permeabilized in PBS containing 0.1 % Triton X-100 (Sigma-Aldrich) and 5 % FCS (Life techologies) for 30 min. Cells were then incubated overnight with the primary antibodies. Next day, secondary antibody Alexafluor-488-cojugated anti-mouse IgG (1:1,000; Life technologies) were used to detect and visualize the primary antibodies. All antibodies were diluted in blocking solution. Similar protocol was also used for the staining using ISL1 (Biorbyt; 1:200) antibody. Micrographs were taken with an Axiovert 200 M microscope (Carl Zeiss).
Band size (bp)
Oct4-F: aac ctg gag ttt gtg cca ggg ttt
Oct4-R: tga act tca cct tcc ctc caa cca
Tbrachyury-F: tgt ccc agg tgg ctt aca gat gaa
Tbrachyury-R: ggt gtg cca aag ttg cca ata cac
ISL1-F: cac aag cgt ctc ggg att gtg ttt
ISL1-R: agt ggc aag tct tcc gac aa
Nkx2.5-F: gcg att atg cag cgt gca atg agt
Nkx2.5-R: aac ata aat acg ggt ggg tgc gtg
cTNT-F: ttc acc aaa gat ctg ctc ctc gct
cTNT-R: tta tta ctg gtg tgg agt ggg tgt gg
β-actin (BA)-F: ttt gaa tga tga gcc ttc gtc ccc
β-actin (BA)-R: ggt ctc aag tca gtg tac agg taa gc
1 × 106 cells were trypsinized and fixed with 4 % PFA for 10 min. Cells were then washed with PBS, permeabilized in PBS containing 0.1 % Triton X-100 and 5 % FCS for 30 min and incubated for 2 h with cTNT antibody (Abcam; 1:100). No antibody was taken as a negative control. Cells were then washed once with PBS containing 0.1 % Tween-20 and resuspended in PBS containing 0.1 % Triton-X 100 and 5 % FCS and secondary antibody Alexafluor-488-cojugated anti-mouse IgG (1:1,000; Life technologies) for 1 h in dark. Finally, cells were washed again with PBS containing 0.1 % Tween-20 and measured for FACS analysis. Analysis was performed by Flow Jo program.
Whole-Cell Calcium Current Measurements
Conventional whole-cell patch clamp recordings were performed at room temperature in bath solution containing (mM): NaCl 137, CsCl 5.4, CaCl2 2, MgCl2 1, glucose 10, HEPES 10 (pH 7.4 with NaOH). Borosilicate pipettes (2–3 MΩ) were filled with a solution containing (mM): CsCl 120, MgCl2 1, Mg-ATP 4, EGTA 10, HEPES 5 (pH 7.2 with CsOH). Giga-Ohm seals were formed by gentle suction. Membrane capacitance was automatically displayed in the pClamp 10.2 software (Axon instruments). Cells were depolarized from a holding potential of −80 mV to −40 mV for 45 ms in order to inactivate sodium channels. This prepulse was followed by test voltages ranging from −40 to +50 mV in 10 mV steps (pulse duration 150 ms) with a 3 s interval between single pulses.
Calcium imaging was performed in cell cultures loaded with 5µM of calcium indicator dye fluo-4AM (Life technologies). Briefly, the cells were cultivated in 6 well plates and incubated then with 1 ml of loading dye solution containing fluo-4 AM at room temperature for 20 min in the dark. Movies were captured through the Keyence Microscope BZ-9000 (Keyence, Japan)
Action Potential and Ramp Recordings
Cardiomyocytes were enzymatically dissociated at day 30 and plated at low density on glass cover slips coated with fibronectin. Action potentials and voltage ramps from −100 mV to 60 mV, 250 ms long were recorded on spontaneously beating cardiomyocytes with a EPC 10 amplifier (Heka Electronics) as previously described . Electrode resistance was between 2.5 and 3.5 MΩ, the pipette solution contained (in mM): 50 KCl; 80 KAsparatate; 10 EGTA; 10 Hepes; 3 MgATP; 1 MgCl2 (pH 7.2) and the extracellular solution contained (in mM): 140 NaCl; 5.4 KCl; 1.8 CaCl2; 1 MgCl2; 10 Hepes; 10 Glucose (pH 7.4).
Transmission Electron Microscopy
Undifferentiated iPS cells or differentiated iPS cells at day 21 were fixed in 4.5 % glutaraldehyde in 0.1 M phosphate buffer pH 7.2 (PB). After washing with 0.1 M PB, specimens were subsequently fixed for 1 h with 1 % osmiumtetroxide in PB and washed with water. Specimens were then dehydrated in ascending concentrations of ethanol including en-bloc contrasting using 2 % uranylacetate in 70 % ethanol for 1 h. Subsequently, they were embedded in Epon812 and used for preparation of ultrathin sections which were poststained with 2 % uranylacetate and 0, 2 % lead citrate. The sections were observed using a LEO AB 912 transmission electron microscope (Zeiss NTS, Oberkochen, Germany)
Defining Optimal Window of WNT Modulation to Achieve Efficient Myocardial Induction in Human iPSCs
Lactate Based Cardiac Enrichment Strongly Reduces Line-to-Line Variability of Cardiomyocyte Differentiation
Characterization and Validation of Cardiomyocytes Derived from Human iPSCs
We performed a series of standard immunohistochemical and electrophysiological methods to assess the functionality of cardiomyocyte derived. Obtained cardiomyocytes showed strong cardiac specific alpha-actinin staining with typical striation pattern as well as cTNT staining (Fig. 3c). Moreover, patch-clamp recordings on single beating cardiomyocytes showed typical spontaneous action potentials (Fig. 3d), with atrial (n = 2) or ventricular (n = 4)-like properties, as well as characteristic voltage-dependent inward and outward currents using voltage ramps (Fig. 3e). We recorded Ca2+ currents typical for L-type Ca2+ channels with a half-maximum activation at −13.69 ± 0.97 mV, a maximum current density of −11.55 ± 1.6 pA/pF at 0 mV and nearly complete inactivation during 150 ms of depolarization (n = 4) (Fig. 3f and g). In addition calcium imaging was performed using the fluorescent Ca2+ indicator Fluo-4 AM. Fluo-4 AM fluorescence intensity sparks indicates binding of calcium to fluo-4 upon release of calcium from the sarcoplasmic reticulum in hiPS derived cardiomyocytes. Fluo-4 fluorescence intensity sparks were recorded using fluorescence microscopy (Supplementary movie S2).
Ultra-Structural Analysis of iPS Cell-Derived Cardiomyocytes
Our study reports a novel robust strategy to obtain cardiomyocytes from diverse human iPS lines that originate from a wide spectrum of state-of-the-art reprogramming technology using an optimal combination of well-orchestrated extrinsic stimuli such as BMP4 and CHIR followed by WNT inhibition using XAV939 or IWR1 and enrichment of cardiomyocytes by supplying lactate as an energy source. The protocol described herein divides the whole differentiation process into three phases, namely cardiovascular induction, cardiac specification and cardiomyocyte enrichment. Our analysis revealed that efficient cardiac induction requires appropriate concentrations as well as precise timing of the right window of application of each chemical. We found out that sequential application of each chemical, BMP4 and CHIR, respectively, for up to two days is sufficient to drive the cells into cardiovascular fate which is consistent with earlier studies [28, 29, 31, 42]. Notably, we have used basal media with insulin during first two days of induction phase. Previous studies have reported use of basal medium without insulin during cardiac differentiation due to its negative influence on cardiac specification of early mesoderm [28, 39]. However, our study shows that using basal medium without insulin from the beginning appears very stressful to the cells. Hence we decided to keep insulin for the initial two days and remove it during the specification phase in order to minimize cell death and its negative influence on cardiac specification. In order to achieve cardiac specification of early mesoderm, we used inhibitors of WNT signaling for up to 4 days as described in earlier studies [28, 29]. We performed a comparative validation of molecules exhibiting WNT inhibitory activity using a WNT reporter cell line. It turned out that amongst four different compounds tested, XAV939 showed the strongest WNT inhibitory effect and was therefore used for all differentiation experiments. Moreover, we also achieved the same cardiac differentiation efficiency with previously described WNT inhibitor IWR-1 . We observed different cardiac differentiation efficiencies in multiple iPS lines presumably due to the complexity of the signals, which is in accordance with recent studies [17, 18]. We therefore elaborated further purification steps to improve the yield of cardiomyocytes with reduced line-to-line variability. We decided to assess the potential of lactate enrichment of cardiomyocytes and show a substantial cardiac enrichment of an iPS line (iLB-C1-30 m-r12) that exhibited relatively poor cardiomyocyte yield using our optimized chemical cocktail only. This indicates that the combination of extrinsically induced differentiation stimuli together with metabolic enrichment is an efficient means to overcome line-to-line variability of cardiomyocyte differentiation. There are various published studies showing successful cardiac differentiation of human pluripotent stem cells. However, as depicted in supplementary table 4 three key features separates our protocol from others i.e. i) sequential treatment of cardiac inducing factor with a clearly defined time window together with ii) an insulin switch, which in our experience is very critical to get reproducible results; iii) in addition our protocol includes a rapid and efficient way to enrich the cardiac population with lactate supplement. In order to evaluate the functional properties of cardiomyocytes, comprehensive validation was not only carried out at protein level by alpha-actinin and cTNT stainings but also by ultra-structural analysis. TEM results showed well organized sarcomeric structures in 21 day old cardiomyocytes with distinct I- and A-bands indicating relatively mature phenotype as described in earlier studies [15, 29]. Spontaneous action potentials of differentiated cells showed typical cardiomyocyte behaviour and we identified both ventricular and atrial-like shapes. Voltage ramps identified fast sodium, calcium as well as potassium currents. Characteristics of Ca2+ currents obtained from iPS-derived cardiomyocytes were similar to those recently obtained from murine ventricular myocytes . I-V relationship furthermore perfectly agreed with data from HEK293 cells expressing recombinant human L-type Ca2+-channels suggesting that indeed iPS-derived cells express cardiac-like channel complexes consisting of pore-forming and auxiliary subunits . Moreover calcium imaging further indicated usual calcium current activity seen in the case of human cardiomyocytes. In conclusion, cardiomyocytes obtained by our protocol have the potential for stem cell based therapies, drug toxicity as well as disease modeling studies. Hence we expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes.
We would like to thank all members of the Stem Cell and Regenerative Medicine Group of the University of Wuerzburg for helpful suggestions and outstanding support. We also thank Johannes Jungverdorben from the University of Bonn, Leonhard Linta and Stefan Liebau from the University of Ulm, both Germany, for kindly providing us with human iPS lines. Frank Edenhofer was supported by grants from the Deutsche Forschungsgemeinschaft DFG (ED79/1-2), and the German Ministry of Education and Research, BMBF (01 GN 0813). Daniela Malan and Philipp Sasse were supported by the “StemCellFactory” project which is co-funded by the European Union (European Regional Development Fund - Investing in your future) and the German federal state North Rhine-Westphalia (NRW). Oliver Brüstle and Michael Peitz were supported by the ‘StemCellFactory’ projects (North Rhine Westphalian Programme for Regional Competitiveness and Employment (Bio.NRW); PtJ-Az.: z0911bt027i and North Rhine Westphalian Ministry of Innovation, Science and Research Programme “Translational Stem Cell Research” PtJ-Az.: z1403ts007e).
Disclosure of Potential Conflicts of Interest
O.B. is co-founder and has stock in LIFE & BRAIN GmbH, Bonn. The other authors declare no potential conflicts of interest.
Spontaneously beating cells at day 12 of cardiac differentiation of human iPS line (del-AR1034ZIMA 001) before lactate enrichment. (AVI 4263 kb)
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