Generation of PDGFRα+ Cardioblasts from Pluripotent Stem Cells

Isolating actively proliferating cardioblasts is the first crucial step for cardiac regeneration through cell implantation. However, the origin and identity of putative cardioblasts are still unclear. Here, we uncover a novel class of cardiac lineage cells, PDGFRα+Flk1− cardioblasts (PCBs), from mouse and human pluripotent stem cells induced using CsAYTE, a combination of the small molecules Cyclosporin A, the rho-associated coiled-coil kinase inhibitor Y27632, the antioxidant Trolox, and the ALK5 inhibitor EW7197. This novel population of actively proliferating cells is cardiac lineage–committed but in a morphologically and functionally immature state compared to mature cardiomyocytes. Most important, most of CsAYTE-induced PCBs spontaneously differentiated into functional αMHC+ cardiomyocytes (M+CMs) and could be a potential cellular resource for cardiac regeneration.

C D E Figure S6. PCBs can be just efficiently induced and expanded by CsAYTE within short period rather than expanded keeping their cardiogenic potential for long-term.
(D and E) Representative FACS analysis and the percentages of cTnT + cells to confirm the cardiac differentiation capacity. Expanded PCBs with BIO+LIF were differentiated without BIO+LIF for 5 days, and cTnT + cells were analyzed. Each group, n = 4. **p < 0.01 versus Sorted PCBs.    (A) Delayed rectifying K + current (I K ) evoked by depolarizing test pulses between -100 and +80 mV in 10 mV increment from a holding potential of -80 mV at 5 s interval. I K is effectively inhibited by TEA at test potentials between +50 and +80 mV. Each group, n = 3. *p < 0.05 versus baseline. (B) Na + current (I Na ) evoked by depolarizing test pulses between -70 mV and +50 mV in 10 mV increment from a holding potential of -80 mV at 5 s interval. I Na is effectively inhibited by TTX at test potentials between -50 and -40 mV. Each group, n = 3. *p < 0.05 versus baseline.
(C) T-type Ca 2+ current (I CaT ) activated by depolarizing test pulses between -60 and +50 mV in 10 mV increment from a holding potential of -40 mV at 5 s interval. I CaT is effectively inhibited by mibefradil at test potentials between -30 and +10 mV. Each group, n = 3. *p < 0.05 versus baseline.
(D) Western blot analysis for expressions of ion channel proteins in the indicated cells.    Mouse brachyury Mouse gata4 ESCs, and OP9 cells were generated as described previously 1-3 and transferred to KAIST.
Mouse iPSCs derived from FVB strain, which were generated as described previously 4 , were a generous gift from Drs. Hyun-Jai Cho and Hyo-Soo Kim (Seoul National University Hospital).

Induction of mouse PSC-derived MPCs, cardioblasts and cardiomyocytes
For the induction of Flk1 + MPCs, ESCs and iPSCs were cultured without leukemia inhibitory factor (LIF, Millipore) and plated on a 0.1% gelatin-coated dish at a cell density between Cells were cultured in the differentiation medium which was changed every other day. To validate whether PCBs can be expanded, purified PCBs at day 6.0, were seeded onto a 0.1% gelatin-coated culture plate at a low-density (6.5 × 10 4 cells/cm 2 ), and incubated them with either control vehicle, CsAYTE, BIO (2.5 M) + LIF (10 3 units/ml), or BACS (5 ng/ml BMP4, 10 ng/ml Activin A, 3 M CHIR99021, and 2 M SU5402).

Induction of human iPSC-derived cardioblasts and cardiomyocytes
Human iPSC-derived cardiomyocyte differentiation was induced as previously reported 6  days. At day 4, the culture medium was replaced with RPMI+B27 without growth factors.
The medium was changed every 1-2 days. Beating cardiomyocytes were observed at day 8-9.

Flow cytometry analysis and cell sorting
The cells were harvested with 0.25% trypsin-EDTA or dissociation buffer (Invitrogen). To

Immunofluorescence staining and visualization of cells
The cells were fixed with 4% paraformaldehyde (PFA) and blocked with 5% goat ( Immunofluorescence staining of mitochondria was performed using MitoTracker Orange CMTMRos probe (Invitrogen), with which the cells were incubated for 30 to 60 min at 37°C in serum-free medium before fixation. Immunocytochemistry stained images were obtained using an LSM780 confocal fluorescence microscope (Carl Zeiss). Live images of cardiomyocyte differentiation process and MHC GFP + cardiomyocytes were obtained using Axiovert 200M microscope (Carl Zeiss) equipped with AxioCam MRm (Carl Zeiss). Images were analyzed using ImageJ software (http://imagej.nih.gov/ij/, 1.47V, NIH, USA). Phase-contrast images including beating cardiomyocytes were obtained using an Infinity X digital camera and DpxView LE software (DeltaPix).

Assays for cell cycle
To determine the cell cycle, BrdU/7-AAD cell cycle analysis was performed according to the manufacturer's instructions (BD Biosciences). Briefly, cells were incubated with BrdU (1 mM) for 1 h, dissociated with 0.25% trypsin-EDTA, fixed, permeabilized, and fixed once more, followed by 1 h incubation with DNase I (200 U) at 37C. After incubation with APCconjugated anti-BrdU antibody for 20 min at RT, the cells were stained with 7-AAD. The cells were analyzed by FACS Aria II and the data were analyzed using FlowJo software.

Quantitative real time PCR
Total RNA was extracted using Trizol RNA extraction kit (Invitrogen) according to the manufacturer's instructions. Total RNA was reverse transcribed into cDNA using GoScript TM cDNA synthesis system (Promega). cDNA was applied for quantitative real-time PCR using FastStart SYBR Green Master mix (Roche) and Bio-rad S1000 Thermocycler with the indicated primers (Supplementary Table 2). Beta-actin was used as a reference gene and the results were presented as relative values to control using the ΔΔCt method.

Short Hairpin RNA (shRNA)-Mediated Silencing of PDGFR
PDGFR was silenced in Flk1 + MPCs and PCBs derived from mouse ESC using PDGFR-shRNA lentiviral transduction particles (SHCLNV-NM_011058, Sigma-Aldrich). Control cells were transduced with non-target shRNA lentiviral particles (SHC216V, Sigma-Aldrich) according to the manufacturer's instruction. Transduction of Flk1 + MPCs or PCBs was carried out by spinoculation (1,000 g) for 90 min at 4°C in the presence of 8 g/ml polybrene reagent.

Transmission electron microscopic analysis
The cells were fixed in 2.5% glutaraldehyde in PBS at 4°C overnight, and then with 1% osmium tetroxide in PBS for 2 h. The tissues were washed, dehydrated, embedded, and then semi-thin sections were cut (0.5-1 m). Further ultra-sectioning (60-90 nm) was performed and then the slices were double stained with uranyl acetate and lead citrate, and imaged using a JEM 1200 EX2 electron microscope (Jeol). Developed images were scanned on a flatbed scanner (Umax PowerLook 1100, Fremont) and analyzed using ImageJ software.

Electrophysiology
Action potentials (APs) and ion currents were recorded from cells placed onto the recording at 10 kHz, 4-pole Bessel type low-pass filter and sampled at a rate of 4 kHz for voltage and acquisition, were controlled using our own software (PatchPro). The liquid junction potentials between bathing and pipette filling solution, which were calculated based on ionic mobility, were < 5 mV. TEA, TTX, and mibefradil were used to block delayed rectifying K + channels, voltage-gated Na + channels, and T-type Ca 2+ channels, respectively.

Microarray analysis
For control and test RNAs, synthesis of target cRNA probes and hybridization were performed using Agilent's Low RNA Input Linear Amplification kit (Agilent Technology) according to the manufacturer's instructions. Briefly, each 1 g of total RNA and T7 promoter primer mix were incubated at 65°C for 10 min. cDNA master mix (5X First strand buffer,