Culture of hPSC lines
HES2 (karyotype: 46, XX) was purchased from WiCell (USA). The HES3-NKX-2.5GFP reporter cell line (karyotype: 46, XX) was provided by E. Stanley and A. Elefanty (Monash University, Australia). BYS-0113 (karyotype: 46, XY) was purchased from the American Type Culture Collection (USA). hPSCs were maintained on Matrigel (Corning, USA)– or vitronectin (Thermo Fisher Scientific)–coated well plates in feeder-free hPSC culture medium consisting of DMEM/F12 (Cellgro, Corning) supplemented with 1% penicillin/streptomycin (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific), 1× nonessential amino acids (Thermo Fisher Scientific), 55 μM β-mercaptoethanol (Thermo Fisher Scientific), 20% KnockOut serum (Thermo Fisher Scientific), and rhbFGF (50 ng/ml ) (Thermo Fisher Scientific).
CM differentiation of hPSC lines
Both HES2 and HES3-NKX-2.5GFP cell lines were differentiated into CMs using a modified version of previously published cardiac differentiation protocols (21, 22). Briefly, hPSCs were grown to 80 to 90% confluence and dissociated into single cells and reaggregated to form EBs in StemPro-34 medium (Thermo Fisher Scientific) containing 1% penicillin/streptomycin (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific), transferrin (150 mg/ml; Roche, Switzerland), ascorbic acid (50 mg/ml; Sigma-Aldrich), and monothioglycerol (50 mg/ml; Sigma-Aldrich), 10 mM Y-27632 (ROCK inhibitor, Tocris, UK), and rhBMP4 (1 ng/ml; R&D Systems, USA) for 18 hours on an orbital shaker. At day 1, the EBs were transferred to mesoderm induction media consisting of StemPro-34 medium with above supplements (-Y-27632) and rhBMP4, rhActivinA (R&D Systems), and rhbFGF (R&D Systems) at the optimal cardiac differentiations for each line given in fig. S6. At day 3, the EBs were harvested, washed with Iscove's modified Dulbecco's medium, and transferred to cardiac mesoderm specification medium consisting of StemPro-34 medium, 2 mM IWP2 (Wnt inhibitor, Tocris), and rhVEGF (10 ng/ml; R&D Systems). At day 6, the EBs were transferred to StemPro-34 with rhVEGF (5 ng/ml) for an additional 7 days under hypoxic conditions (5% O2). The cultures were further matured for another 8 to 10 days in StemPro-34 medium without additional cytokines under ambient oxygen conditions. At day 20, the hPSC-derived CMs were analyzed on the basis of the expression of cTNT via FCM. The EBs were cultured in ultralow attachment six-well dishes (Corning) throughout the differentiation, which routinely generated cultures with greater than 85% CMs, as determined by cTNT expression.
Definitive endoderm differentiation of hPSC lines
HES3-NKX-2.5GFP cell lines were differentiated into definitive endodermal cells using a commercially available kit (PSC Definitive Endoderm Induction Kit, A306260, Thermo Fisher Scientific). Briefly, hPSCs were seeded and grown to 10 to 20% confluence. At day 0, the medium was changed to PSC definitive endoderm induction medium A for 24 hours; after which, the medium was changed to PSC definitive endoderm induction medium B for 24 hours. The cells were then recovered for analysis and SCQC capture experiments. The differentiation routinely generates greater than 95% definitive endodermal cells based on the FCM analysis of SOX17 expression.
Generation of samples containing diluted or spiked hPSCs
Confluent hPSCs (50 to 70%) were dissociated by TrypLE (Thermo Fisher Scientific) for 3 min at RT. Dissociated cells were centrifuged, and the cell number was quantified by an automated cell counter (Countess II, Thermo Fisher Scientific) by taking the average of three to five individual counts. Low concentration solutions were achieved by serial dilution (maximal 9:1 ratio per dilution). Day 20 hPSC-derived EBs were dissociated to single cells by collagenase type 2 (300 U/mg; Worthington Biochemical Corp., USA) in Hanks’ buffer (Thermo Fisher Scientific) at 37°C for 90 min, followed by 3 min TrypLE treatment. Confluent hPSCs (50 to 70%) were dissociated by TrypLE for 3 min at RT, quantified by the cell counter, and serially diluted to achieve low concentrations of hPSCs. Populations of hPSCs and hPSC-derived CMs were combined together in the end to generate spiked samples containing 0.0005 to 5% HES2 cells in CMs. Total number of cells for each experiment is indicated in the figure captions.
For surface marker analyses, diluted or spiked samples were fixed by 4% methanol-free paraformaldehyde (PFA; Thermo Fisher Scientific) at RT for 10 min, blocked by 1% BSA (Sigma-Aldrich) in PBS (Wisent Bioproducts, Canada) on ice for 30 min, and stained by antibodies of SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, CD324 (E-cadherin), CD326 (EpCAM), CD9, or CD90 (all from Miltenyi Biotec, Germany) for 10 min at 4°C in a flow buffer containing 1% BSA in PBS. For intracellular marker analyses, samples were fixed by 4% PFA at RT for 10 min, permeabilized by 0.5% Triton X-100 (Sigma-Aldrich) in PBS at RT for 10 min, blocked by 1% BSA in PBS on ice for 30 min, and stained by antibodies of SOX2, Oct3/4, Nanog (all from Miltenyi Biotec), or cTNT (BD Biosciences) for 30 min at RT in flow buffer. Detailed information regarding conjugations and dilutions is given in table S3. Stained samples were analyzed using the FACSCanto flow cytometer (BD Biosciences, USA) or the fluorescence-activated cell sorting (FACS) LSR Fortessa flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (FlowJo LLC., USA). To characterize the LOD of FCM, three individual tubes were prepared for each concentration. The LOD was defined as means + 3 SD.
Droplet digital PCR
Total RNA was isolated from the spiked samples by using a single-cell RNA purification kit (51800, Norgen Biotek Corp., Canada) and used for ddPCR. The isolated RNA was used for cDNA synthesis using the First-Strand DNA Synthesis Kit (Invitrogen, USA), which contained random hexamer primers and Superscript III Reverse Transcriptase, according to the manufacturer’s protocol. The cDNA was submitted to the Centre for Applied Genomics (The Hospital for Sick Children, Toronto, Canada) for a standard ddPCR performed by a QX200 ddPCR system (Bio-Rad, USA). The TaqMan primers for target genes were purchased from Thermo Fisher Scientific: POU5F1 (OCT3/4, Hs00999634_gH), SOX2 (Hs04234836_s1), and CD326 (EpCAM, Hs00901885_m1). The TBP or B2M gene was used as the housekeeping control. The results were analyzed by the Centre for Applied Genomics using QuantaSoft Analysis Pro Software (Bio-Rad).
Characterization of magnetic labeling
Diluted samples were fixed by 4% PFA at RT for 10 min and labeled by anti–TRA-1-60 (dilution: 1:50; Miltenyi Biotec) in 1 ml of 1% BSA for 30 min at RT. Labeled samples were washed with 1% BSA in PBS twice and centrifuged at 2000 rpm for 4 min to form pellets. Pellets were then dehydrated with increasing concentrations of ethanol at 10-min intervals and embedded with Quetol-Spurr resin (Sigma-Aldrich) overnight. Samples were sliced to 70- to 80-nm-thick layers by an ultramicrotome (Ultracut RMC MT6000, Leica Microsystems, Germany) and deposited on electron microscopy grids (Ted Pella Inc.). Samples were observed under a transmission electron microscope (FEI Tecnai 20, Thermo Fisher Scientific) using 100-kV accelerating voltage.
Stem cell quantitative cytometry
Diluted or spiked samples were fixed by 4% PFA at RT for 10 min and labeled by anti–TRA-1-60 or anti-CD326 microbeads (dilution: 1:50; Miltenyi Biotec) in 1 ml of flow buffer for 30 min at RT. Labeled samples were loaded into the chips and profiled at flow rates ranging from 2 to 10 ml/hour. For the quantification of capture and depletion efficiency, captured cells were stained by DAPI and NucDead 488 (Thermo Fisher Scientific) for 10 min at the flow rate of 1 ml/hour. For the quantification of spiked hPSCs in hPSC-derived CMs, captured cells were permeabilized by 0.5% Triton X-100 in PBS at RT for 10 min at the flow rate of 1 ml/hour and stained by cocktails of antibodies [DAPI, NucDead 488, Oct3/4–PE (phycoerythrin), and Nanog–APC (allophycocyamin)] for 30 min at RT in a buffer containing 1% BSA and 0.1% Tween 20 (Bio-Rad, USA) at the flow rate of 400 μl/hour. Detailed information regarding antibody dilutions is given in table S3. After staining, the cells were washed with flow buffer for 10 min at the flow rate of 1 ml/hour. Washed chips were stored at 4°C and scanned within a week of profiling. To quantify the number of captured hPSCs, the chips were tile scanned using a Nikon Ti-E microscope with automated stages. The exposure time is 20 ms for DAPI, 10 ms for NucDead 488, 200 ms for Oct3/4-PE, and 400 ms for Nanog-APC. Scanned images were combined into a large image using Nikon NIS-Elements software (high content analysis version) and quantified using IMARIS software (Bitplane, Oxford Instrument, UK) via colocalization analysis. Cells (hPSCs) were defined as DAPI+, NucDead+, Oct3/4+, and Nanog+. To characterize the LOD of SCQC, three to five individual runs were performed for each concentration. The captured cell numbers were divided by 0.85 to normalize the effect of capture efficiency. The LOD was defined as means + 3 std.
Magnetic-activated cell sorting
For live cell separation, 1 million of HES2 hPSCs or derived CMs were labeled by anti–TRA-1-60 microbeads (dilution: 1:50) in 1 ml of flow buffer for 10 min at RT, as instructed by the manufacturer. Labeled samples were applied to MS columns (Miltenyi Biotec) and washed twice using the flow buffer. TRA-1-60–positive cells were recovered from the column by firmly pushing the plunger into the column twice. Recovered cells were centrifuged and immediately processed for cell counting using an automated cell counter. For stained cell separations, 1 million of HES2 PSCs or derived CMs were fixed, permeabilized, and stained by DAPI, Oct3/4-PE, and Nanog-APC. These cells were subsequently labeled with anti–TRA-1-60 microbeads (dilution: 1:50) in 1 ml of flow buffer for 10 min at RT. Labeled samples were then sorted using MS columns. Recovered cells were centrifuged and immediately processed for cell counting.
Teratoma formation and analysis
All animal experiments were carried out in accordance with the protocol approved by the University of Toronto Animal Care Committee. Male NOD/SCID/interleukin 2 receptor Gamma chain null (NSG) strains of mice at 6 to 8 weeks of age were purchased from the Jackson laboratory (USA) and maintained at the University of Toronto animal facility. Spiked sample with 1 × 106 cells in 15 μl of Matrigel (Corning) was injected into the pericardium of testis. Ten weeks after injection, mice were euthanized, and the formation of teratomas was examined. Extracted teratomas were weighed and fixed in 10% formalin (Sigma-Aldrich). Formalin-fixed, paraffin-embedded teratomas were sectioned (5-μm thickness) and stained with hematoxylin and eosin. Histological examination was performed by a licensed veterinary pathologist blinded to the difference of samples to identify the germ layers in the teratomas.
In vitro colony-forming assay for spiked samples
Spiked samples were labeled by anti–TRA-1-60 microbeads in 1 ml of 1% BSA in PBS for 30 min at RT. Labeled samples were loaded into the chips and profiled at flow rates of 4 ml/hour. After profiling, magnets were removed from the chip. The negative groups were obtained from the syringe, and the positive groups were obtained by withdrawing cells in the chips with a new syringe. The profiled groups were centrifuged and resuspended in the hPSC culture medium for reculturing on vitronectin-coated well plates. At days 3, 6, 10, and 15 after profiling, the recultured cells were fixed by 4% PFA at RT for 10 min, permeabilized by 0.5% Triton X-100 in PBS at RT for 10 min, and stained by antibodies of DAPI, TRA-1-60-Vio488 (Miltenyi Biotec), Oct3/4-PE, and Nanog-APC for 60 min at RT in flow buffer. Detailed information regarding conjugations and dilutions is given in table S3. The plates were tile scanned using the Nikon Ti-E microscope. The exposure time is 20 ms for DAPI, 100 ms for TRA-1-60-Vio488, 200 ms for Oct3/4-PE, and 400 ms for Nanog-APC. hPSCs were defined as DAPI+, NucDead+, Oct3/4+, and Nanog+. Number of colonies (>4 hPSCs per colony) per well was quantified manually.
Isolation and characterization of rare hPSCs isolated from manufactured batches
In addition to the abovementioned EB-based protocol, a commercially available cardiac differentiation kit (A2921201, Thermo Fisher Scientific) was also used to generate batches of CMs from a monolayer. Briefly, HES2 and HES3-NKX-2.5GFP cells were maintained in vitronectin-coated well plates in Essential 8 medium (Thermo Fisher Scientific) for 2 days (days −2 to 0). The confluency of cells at day 0 is between 50 and 70%, as suggested by the manufacturer. Then, the medium was replaced with CM differentiation medium A and B at days 0 and 2, respectively. At day 4, the medium was changed to Cardiomyocyte Maintenance Medium (A2920801, Thermo Fisher Scientific) and changed every 2 days until day 16. Contracting CMs appeared at day 8, and the beating conditions of the CMs were monitored at days 8, 12, and 16 using the Nikon Ti-E microscope. The percentages of cTNT-positive cells at days 8, 12, and 16 were quantified by FCM using the protocol described in the “Flow cytometry” section. At days 4, 8, 12, and 16, monolayers of hPSC-derived CMs were dissociated by TrypLE for 4 min. At days 10 and 20, hPSC-derived CMs grown as EBs were dissociated to single cells by collagenase type 2 (300 U/ml) in Hanks’ buffer at 37°C (30 min for day 10 and 90 min for day 20), followed by 3-min TrypLE treatment. Dissociated cells were labeled by anti–TRA-1-60 microbeads and profiled using the protocol same to the spiked samples. The positive groups were recultured in StemFlexTM medium (A3349401, Thermo Fisher Scientific). At days 2, 6, and 10 after profiling, the positive cells were fixed, stained, and quantified using the same protocol used for the spiked samples. If rare hPSCs (DAPI+, TRA-1-60+, Oct3/4+, and Nanog+) were found at day 2, the same groups were passaged when it reached 80 to 90% confluency up to 14 days to allow rare hPSCs to proliferate. To examine the pluripotency of isolated rare hPSCs, proliferated hPSCs were differentiated into three germ lineages using a trilineage differentiation kit (130-115-660, Miltenyi Biotec), which typically takes 7 days. At day 7, the cells were fixed, permeabilized, and stained with DAPI, FOXA2, and SOX17 (for endoderm); DAPI, smooth muscle actin (SMA), and CD144 (for mesoderm); and DAPI, PAX6, and Nestin (for ectoderm). Detailed information regarding conjugations and dilutions is given in table S3. The stained plates were observed using the Nikon Ti-E microscope. The exposure time is 20 ms for DAPI, 100 ms for FOXA2-PE, 400 ms for SOX17-AF647, 20 ms for SMA-PE, 400 ms for CD144-AF647, 200 ms for PAX6-PE, and 600 ms for Nestin-AF647. To examine the naïveness of isolated rare hPSCs, total RNA was isolated from the proliferated hPSCs following the same protocol used for ddPCR. A comparative CT experiment was performed on an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific) using hPSC naïve-state qPCR array (07521, Stemcell Technologies, Canada). The assay was carried out using 5 μl of TaqMan Universal Mix, 4 μl of nuclease-free water, 1 μl of cDNA (10 ng/μl) for each sample in a 96-well plate. Cycling conditions for the qPCR were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The post-PCR analysis was performed by an online tool provided by the manufacturer (https://stemcell.shinyapps.io/qpcr_tool/).