Research ArticleHEALTH AND MEDICINE

Ultrasensitive and rapid quantification of rare tumorigenic stem cells in hPSC-derived cardiomyocyte populations

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Vol. 6, no. 12, eaay7629

Abstract

The ability to detect rare human pluripotent stem cells (hPSCs) in differentiated populations is critical for safeguarding the clinical translation of cell therapy, as these undifferentiated cells have the capacity to form teratomas in vivo. The detection of hPSCs must be performed using an approach compatible with traceable manufacturing of therapeutic cell products. Here, we report a novel microfluidic approach, stem cell quantitative cytometry (SCQC), for the quantification of rare hPSCs in hPSC-derived cardiomyocyte (CM) populations. This approach enables the ultrasensitive capture, profiling, and enumeration of trace levels of hPSCs labeled with magnetic nanoparticles in a low-cost, manufacturable microfluidic chip. We deploy SCQC to assess the tumorigenic risk of hPSC-derived CM populations in vivo. In addition, we isolate rare hPSCs from the differentiated populations using SCQC and characterize their pluripotency.

INTRODUCTION

Human pluripotent stem cells (hPSCs) hold great promise for cell therapy given their ability to differentiate into many different cell types (1). Numerous studies have demonstrated the considerable potential of hPSC derivatives in treating chronic diseases, including neuron degeneration (2) and chronic heart failure (3). Typically, a dose of cell therapy for treating heart failure requires 0.1 to 1 billion of de novo hPSC-derived functional cells (4); therefore, the translation of cell therapy from bench to bedside heavily depends on reliable manufacturing of high-quality cell products (46).

The self-renewal and pluripotent properties of hPSCs are also associated with a high level of tumorigenicity in vivo (7). Undifferentiated cells can persist in differentiated populations following long periods of time in culture (8, 9), and rare contaminating hPSCs, even at concentrations less than 0.025%, can lead to teratoma formation in animal models (1012). As a result, quantitation of the percentage of rare hPSCs is a key quality control parameter that needs to be monitored in manufactured populations to be used for cell therapy applications (4, 13).

Flow cytometry (FCM) and the polymerase chain reaction (PCR) are powerful methods for the analysis of rare cells. Unfortunately, neither of these methods is sensitive enough to rapidly and accurately identify rare hPSCs in relevant samples. FCM has intrinsic limitations including sampling losses and dead volumes (14) that reduce accuracy at exceptionally low levels of hPSC contamination that may still represent a potential risk of tumor formation (15). PCR-based methods are often problematic due to the high background from differentiated cells relative to the rare undifferentiated controls (16) and reverse transcription–induced artifacts, including primer-independent complementary DNA (cDNA) synthesis and template switching (17).

Here, we report a new approach to the quantitation of rare undifferentiated cells: stem cell quantitative cytometry (SCQC). This method takes advantage of a rare cell profiling approach based on a strategy for monitoring cancer cells (18, 19). SCQC uses a microfluidic chip that is scalable, cost-effective, and compatible with the requirements of manufacturing and quality control. SCQC has excellent sensitivity and is able to profile rare hPSCs robustly even when present at concentrations as low as 0.0005% in populations of hPSC-derived cardiomyocytes (CMs). Through the analysis of CM samples containing defined numbers of spiked hPSCs, we demonstrate that SCQC detects rare contaminants with unprecedented performance. Comparative studies show that SCQC can accurately quantify hPSCs at levels that are not reliably detected by either FCM or droplet digital PCR (ddPCR). Last, we use SCQC to isolate live rare hPSCs from differentiated CM populations and characterize their pluripotency.

RESULTS

Development of microfluidic SCQC

The device used for SCQC relies on immunomagnetic labeling to profile cells based on their surface marker expression (Fig. 1A) (20). Cells are labeled with magnetic nanoparticles (MNPs) that bind to a surface marker expressed by hPSCs, but not CMs. Labeled cells are subsequently introduced into a microfluidic device with a flow velocity gradient and a constant magnetic field (Fig. 1, A to C, and fig. S1, A and B). Cells with more MNPs experience a higher magnetic force to compensate the downstream drag force generated by the flow velocity. As a result, the hPSCs that bind more MNPs than the differentiated CMs can withstand high flow velocities and remain in the high-velocity regions of the device. The CMs with few or no MNPs are captured in the low-velocity regions or flushed away. At the completion of the run, the cells trapped in the microfluidic device are quantified by immunofluorescence and microscopy to generate a cytometric profile that includes number, phenotype, and distribution of trapped cells. The information from the cytometric profile provides an assessment of the total number of hPSCs.

Sensitivity and specificity of SCQC

We used the HES2 hPSC line and derivative CMs to characterize and optimize the performance of SCQC. The CMs were differentiated for at least 20 days following an established protocol (fig. S2) (21, 22). The hPSC-derived CMs contain more than 85% cardiac troponin T (cTNT)–positive cells. In the first suite of experiments, we used FCM to benchmark eight surface markers (SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, EpCAM, CD90, CD9, and E-cadherin) and three intracellular markers (Oct4, SOX2, and Nanog) for distinguishing hPSCs and hPSC-derived CMs (fig. S3). FCM results revealed that TRA-1-60 and EpCAM offer the highest separation (equivalently the best contrast) among all markers. Hence, we chose TRA-1-60 and EpCAM MNPs for on-chip optimization.

HES2 hPSCs or derivative CMs were labeled with antibody-conjugated MNPs, profiled using SCQC and stained by a (4′,6-diamidino-2-phenylindole) DAPI/NucDead 488 cocktail for visualization (fig. S4A). TRA-1-60 MNPs produced higher levels of hPSC capture and hPSC-derived CM depletion (fig. S4B) than EpCAM MNPs. We also investigated the specificity of the TRA-1-60 MNPs for labeling hPSCs by transmission electron microscopy (fig. S4C). More than 10 major clusters of MNPs were observed on the surface of hPSCs, while 0 or 1 major cluster of MNPs was detected on the CMs. On the basis of these findings, we concluded that the TRA-1-60 has enhanced performance for capturing rare hPSCs in hPSC-derived CMs compared with other surface markers. Next, we tested the direct isolation of hPSCs using TRA-1-60 MNPs via magnetic-activated cell sorting (MACS). However, MACS had poor capture and recovery efficiency for both live and fixed cells (fig. S5). Notably, around 20% of cells, regardless of cell type, remained trapped in the columns and could not be recovered. This is consistent with previous studies (23, 24). Therefore, it is necessary to use the microfluidic chip to enable more efficient recovery and in situ immunostaining of rare cells for quantification.

We next assessed the specificity of SCQC to evaluate its utility for the quantitation of undifferentiated hPSCs. In the absence of an external magnetic field or the absence of MNPs, less than 0.05% hPSCs were captured at the flow rate of 2 ml/hour. Subsequently, the flow rate, which dominates the flow velocity on-chip, was optimized to balance the capture of hPSCs and the depletion of hPSC-derived CMs (fig. S4D). At the optimized flow rate (10 ml/hour), we captured 85.7% of hPSCs while depleting 99.7% of CMs (Fig. 2, A and B). Over 90% of captured hPSCs were detected in zones 1 to 5, which generates a reproducible cytometric profile. In addition, we characterized the dynamic range of the SCQC using the optimized flow rate. The chip had a consistent capture efficiency and cytometric profile in the range of 0 to 5000 hPSCs (Fig. 2C and fig. S4E). The chip offered sufficient depletion (>99%) up to 500,000 CMs (fig. S4E). Also, the chip maintained a consistent capture performance when using other hPSC cell lines, including an induced pluripotent stem cell line (fig. S4F) and other hPSC-derived populations (i.e., definitive endodermal cells) (fig. S4G). On the basis of these results, we conclude that SCQC is a highly sensitive and selective method with an excellent dynamic range for the capture and analysis of hPSCs.

We determined the limit of detection (LOD) of SCQC using samples of hPSC-derived CMs spiked with defined numbers of hPSCs. As the SCQC device nonspecifically captures a small fraction of hPSC-derived CMs, immunostaining was used to quantify the number of hPSCs on the fluidic chip. The hPSCs were defined using a cocktail of DAPI, Oct4, and Nanog (Fig. 2D). We found that SCQC can clearly identify the difference between the negative control (zero hPSC in 1,000,000 hPSC-derived CMs) and the 0.0005% sample (five hPSCs in 1,000,000 hPSC-derived CMs as shown in Fig. 2E). Hence SCQC achieves a LOD of 0.0005% for quantifying rare hPSCs.

Quantitative comparison between SCQC, FCM, and ddPCR

We conducted a comparative study to systematically evaluate the performance of SCQC, FCM, and ddPCR for rare hPSC detection. We generated populations of hPSC-derived CMs containing 0.01 to 5% of spiked HES2 hPSCs. For FCM, we used TRA-1-60 and EpCAM as the hPSC markers with a two-laser six-color flow cytometer. For ddPCR, we monitored the expression of three hPSC genes: POU5F1, SOX2, and CD326. TBP or B2M was included as a housekeeping control. For SCQC, we applied the TRA-1-60 MNPs and the flow rate of 10 ml/hour, as detailed above.

The representative profiles obtained by FCM are shown in Fig. 3A and fig. S6A. The hPSC signal (TRA-1-60+ EpCAM+) decreased rapidly with the decreasing number of hPSCs. FCM was unable to detect hPSCs in the 0.1% sample, as the signal for these samples was the same as the signal for the samples that did not contain hPSCs. The combination of the two most sensitive markers (TRA-1-60/EpCAM) yielded the optimal LOD around 0.2% to 0.3% (see fig. S6, A and B). Previous literature suggests that the number of total events that must be collected to detect a population present at a frequency of 0.1% is at least 10 million (25). Routine collection of 10 million events by FCM is neither cost- or time-effective.

The representative ddPCR results are shown in Fig. 3, (B and C) and fig. S6 (C and D). From the three primer sets tested (POU5F1, SOX2, and EpCAM), we found that the combination of EpCAM as the target and TBP as a housekeeping gene yielded the lowest LOD, calculated at 0.2 to 0.4% (Fig. 3B). The LOD obtained using POU5F1 and SOX2, which are the definitive genes for pluripotency, was higher than 1% (fig. S6D). This is not an unexpected result as previous literature has indicated that pluripotent genes (i.e., POU5F1, SOX2, Nanog, Klf-4, and Lin28) are weakly expressed at the RNA level in hPSC-derived cells, typically in the range of 0.01 to 1% relative to undifferentiated hPSCs (16, 26). As a result, the signal from rare hPSCs is concealed by the large background generated by a substantial excess of hPSC-derived cells. Therefore, given the lack of a specific hPSC marker, ddPCR is not a practical method for quantifying rare hPSCs in differentiated population.

Device fabrication

Positive molds were fabricated by a stereolithographic 3D printer (μMicrofluidics Edition 3D Printer, Creative CADworks, Canada) using the “CCW master mold for PDMS” resin (Resinworks 3D, Canada). The layer thickness is set to 50 μm. Negative molds were fabricated by casting PDMS (Dow Chemical, USA) on positive molds and baked at 70°C for 2 hours. Negative molds were then treated by saturated detergent solution (Sparkleen, Thermo Fisher Scientific, USA) in 70% ethanol at room temperature (RT) for at least an hour. PDMS-positive replicas were generated by casting PDMS on negative molds and baked at 70°C for 2 hours. The cured replicas were then peeled off, punched, and plasma bonded to thickness no. 1 glass coverslips (Ted Pella, USA). The bonded chips were left in a 100°C oven for 30 min to secure a robust bonding. Afterward, the silicon tubing was attached to the inlet and outlet of the device. Before use, the devices were conditioned with 1% Pluronic F68 (Sigma-Aldrich, USA) in phosphate-buffered saline (PBS) for at least 1 hour to reduce the nonspecific adsorption. Each device was sandwiched between two arrays of N52 NdFeB magnets (K&J Magnetics, USA; 1.5 mm by 8 mm) with alternating polarity. A syringe pump (Chemyx, USA) was used for the duration of the cell capture process.

Device characterization

For the characterization of microstructures, printed positive molds, PDMS negative molds, and PDMS positive replicas were sputter coated with 20-nm Au (Denton Desk II, Leica, Germany) and observed under field emission scanning electron microscopes (Hitachi SU-5000 or FEI Quanta FEG 250) using 5-kV accelerating voltage. PDMS-positive replicas were also measured by a thickness gage (Mitutoyo, Japan) to determine the thickness of each zone.

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.

Flow cytometry

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/).

SUPPLEMENTARY MATERIALS

Fig. S1. Scalable and cost-effective fabrication of SCQC device.

Fig. S2. Schematic representation of the differentiation strategy used to generate hPSC-derived CMs.

Fig. S3. Benchmarking of surface and intracellular marker candidates for hPSC quantification.

Fig. S4. Characterization and optimization of SCQC for hPSC quantification.

Fig. S5. Quantification of cell capture/depletion via MACS.

Fig. S6. Optimization of FCM and ddPCR for rare hPSC quantification.

Fig. S7. Isolation and characterization of live rare hPSCs.

Table S1. Quantification of the detection limit and linearity (R2) of FCM, SCQC, and ddPCR.

Table S2. General comparison of FCM, SCQC, and ddPCR for manufacturing applications.

Table S3. Details of antibodies and nanobeads used in this study.

Movies S1 to S3. Representative video clips showing the contraction of hPSC-derived CMs at days 8, 12, and 16 using monolayer-based protocol, respectively.

Movies S4 and S5. Representative video clips showing the contraction of hPSC-derived CMs at day 10 and day 20 using EB-based protocol, respectively.

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REFERENCES AND NOTES

Acknowledgments: We would like to thank members of the Kelley and Keller laboratory, especially B. Green, X. Fan, A. Garcia, S. Ogawa, and S. Protze, for experimental advice and critical comments on the manuscript, A. Elefanty and E. Stanley (Monash University) for providing the HES3-NKX-2.5GFP reporter cell line, M. Ly, K. Patel, and H. Patel (Creative CADworks) for establishing the protocol for 3D printing, T. Paton (The Hospital for Sick Children) for assistance in ddPCR, M. Ganguly (University Health Network) for assistance in histology, and M. Larsen (Mbed Pathology) for assistance in pathology. Funding: Research reported in this publication was supported in part by the Canadian Institutes of Health Research (grant FDN-148415 to S.O.K. and grant FDN-159937 to G.M.K.). This research is part of the University of Toronto’s Medicine by Design initiative, which receives funding from the Canada First Research Excellence Fund. Z.W. was supported by a Connaught International Scholarship. Author contributions: Z.W., M.G., E.H.S., G.M.K., and S.O.K. conceived and designed the experiments. Z.W., M.G., R.M.M., S.U.A., M.L., L.Z., S.P., and Y.Z. performed the experiments and analyzed the data. All authors discussed the results and contributed to the preparation and editing of the manuscript. Competing interests: G.M.K is a founding investigator, equity holder, and a paid consultant for BlueRock Therapeutics LP and a paid consultant for VistaGen Therapeutics. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from S.O.K.
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