AI-guided laser purification of human iPSC-derived cardiomyocytes for next-generation cardiac cell manufacturing

Abstract

Current methods for producing cardiomyocytes from human induced pluripotent stem cells (hiPSCs) using 2D monolayer differentiation are often hampered by batch-to-batch variability and inefficient purification processes. Here, we introduce CM-AI, a novel artificial intelligence-guided laser cell processing platform designed for rapid, label-free purification of hiPSC-derived cardiomyocytes (hiPSC-CMs). This approach significantly reduces processing time without the need for chronic metabolic selection or antibody-based sorting. By integrating real-time cellular morphology analysis and targeted laser ablation, CM-AI selectively removes non-cardiomyocyte populations with high precision. This streamlined process preserves cardiomyocyte viability and function, offering a scalable and efficient solution for cardiac regenerative medicine, disease modeling, and drug discovery.

Fig. 1. CM-AI automated detection of CMs and laser ablation of non-myocytes in 2D monolayers.

a Step 1 in this hiPSC-CM enrichment process is to acquire a phase contrast image of a well of cardiac differentiation with AI detection of CM colonies (red). b Step 2 in this easy-to-use process is replating enriched hiPSC-CMs. c Phase contrast and fluorescent imaging of GFP+ CMs enrichment in 2D monolayers. Successful AI recognition of CMs in phase contrast images was validated using time-lapse imaging of monolayers pre-AI laser processing and post-AI laser processing. The overall GFP confluence did decrease significantly in six independent wells processed using CM-AI-laser ablation of the non-myocyte (GFP-) cell population (Pre GFP confluence = 11.23 ± 3.2%; Post GFP confluence = 8.88 ± 2.4%; n = 6 paired t-test, **P = 0.009). The green Mask was used for quantification of the GFP confluence area before and after CM-AI laser processing. d Validation of CM enrichment in replated monolayers. Following CM-AI laser processing and CM enrichment, CMs were replated on CELLvo™ MatrixPlus human ECM-coated plates for analysis. CM-AI laser-processed wells reached purity (>85%) levels significantly greater than unprocessed wells. (n = 4 monolayers per group; unpaired t-test; ****P < 0.0001).

Fig. 2. Validation of hiPSC-CM purity after CM-AI processing of two separate cell lines by flow cytometry.

a Flow cytometry plots of GFP + TTN-GFP hiPSC-CMs following CM-AI purification stained for cardiac troponin T (cTnT-APC). b Flow cytometry of untagged 19.9.11 hiPSC-CMs stained with cTnT-APC following CM-AI purification. c Summary of cTnT+ cell percentages from three independent differentiations using TTN-GFP and 19.9.11 hiPSC lines (n = 3 per line). Average purity was 92.0 ± 1.0% and 94.3 ± 7.2%, respectively. No residual expression of pluripotency markers (NANOG, POU5F1) detected post-CM-AI processing (qRT-PCR; Supplementary Table 1). Data are mean ± s.d.

Fig. 3. Acute structural and electrical functional analysis of CM-AI purified hiPSC-CM monolayers.

a Western blot analysis of atrial-specific CM proteins in purified versus unpurified monolayers. CM-AI + Laser purified monolayers (n = 6, Lanes 1–6) showed significant enrichment of Cx40, cTnI, and mlc2a protein expression compared to time and batch-matched non-purified monolayers. b Cx40 protein expression was significantly enriched in laser-purified hiPSC-CM monolayers compared to non-purified monolayers (0.73 ± 0.13au; n = 6 vs. 0.08 ± 0.01au; n = 3; ****P < 0.0001, unpaired t-test). c cTnI protein expression was significantly greater in laser-purified monolayers compared to non-purified hiPSC-CM atrial monolayers (0.31 ± 0.09au; n = 6 vs. 0.02 ± 0.01au; n = 3; ***P = 0.0009, unpaired t-test). d mlc2a myofilament protein expression was significantly enriched in laser-purified hiPSC-CM monolayers compared to non-purified monolayers (0.16 ± 0.08au; n = 6 vs. 0.03 ± 0.01; n = 3; *P = 0.04, unpaired t-test). e Atrial hiPSC-CM monolayer function was quantified using calcium-sensitive dye (Calbryte 520AM, 5 μM) and high-resolution optical mapping. Representative spontaneous calcium flux traces from re-plated CM-AI + laser processed monolayers (blue) and replated non-purified monolayers (black). f CM-AI  + laser processed monolayers had faster spontaneous beat rate compared to non-purified monolayers (1.84 ± 0.14 Hz; n = 21 monolayers vs. 0.58 ± 0.14 Hz; n = 16 monolayers; ****P < 0.0001, unpaired t-test). g Calcium wave propagation velocity was faster in CM-AI laser processed monolayers compared to non-purified monolayers (22.0 ± 7.6 cm/s; n = 21 monolayers vs. 12.5 ± 3.5 cm/s; n = 15 monolayers; ****P < 0.0001). h Calcium transient duration 80 (CaTD80) was significantly shorter in CM-AI laser processed monolayers compared to non-purified monolayers (0.164 ± 0.02 s; n = 21 vs. 0.379 ± 0.07 s; n = 15; ****P < 0.0001, unpaired t-test).

Fig. 4. Precision and safety of CM-AI laser ablation.

a–d hiPSCs (19-9-11 hiPSC line) were plated as a monolayer on Kataoka Plates and differentiated to CMs. On day 7 hiPSC-CM monolayers were laser cut to make grids (0.4 mm²). a, c phase contrast images show where laser grids were made. b, d Staining with AnnexinV (red apoptosis reagent) shows dead cells are limited to the laser ablated areas. e–h AnnexinV (red) staining following CM-AI and laser processing to purify hiPSC-CMs using two different GFP-tagged cell lines. For each cell line tested, GFP+ CMs are outlined with white dotted line to enhance visualization in (f, h).

Fig. 5. Functional validation of CM-AI processed cardiomyocytes.

a–c Phase contrast images of custom programmed laser ablation test using a purified hiPSC-CM monolayer. The laser was programmed to kill cells in the shape of the block “M” × 2. d Spontaneous calcium activations were recorded using a calcium-sensitive probe (Calbryte520AM) and high-speed imaging system. Spontaneous activation waves navigated around the laser-ablated hiPSC-CMs and through the live hiPSC-CMs. Representative data shown from ≥3 replicates. Supplementary Movie 3 illustrates continuous contraction of live CM networks.

Fig. 6. Post-thaw function and viability of cryopreserved CM-AI processed hiPSC-CMs.

a Post-thaw viability was similar between two hiPSC lines tested (TTN-GFP hiPSC-CM = 74.3 ± 7.4; 19.9.11 hiPSC-CM = 61.7 ± 2.9%, n = 3 separate batches per group). b Time-lapse imaging was used to quantify hiPSC-CM 2D monolayer formation kinetics following thaw, data is mean ± standard deviation at each time point. c Phase contrast and GFP images for each cell line tested in panel b on day 3.5 post thaw. d, e Representative calcium transient recordings from each hiPSC line at baseline and with isoproterenol (ISO, 200 nM) treatment. f Each cell line hiPSC-CMs responded to ISO with increased beat rate (TTN-GFP hiPSC-CM baseline = 68.9 ± 8.1; +ISO = 93.3 ± 10.3 bpm, n = 6) (19.9.11 hiPSC-CM baseline = 100.3 ± 15.6; +ISO = 130.0 ± 20.6bpm, n = 6). Paired t-test, ***P = 0.0001. g Each cell line hiPSC-CMs responded to 200 nM ISO with increased calcium wave propagation velocity (TTN-GFP hiPSC-CM baseline = 33.7 ± 5.9; +ISO = 46.9 ± 4.6 cm/s, n = 6) (19.9.11 hiPSC-CM baseline = 30.1 ± 3.4; +ISO = 37.2 ± 6.3 cm/s, n = 6). Paired t-test, ***P = 0.0003; **P = 0.04.

Fig. 7. Pharmacologic response of CM-AI cryopreserved hiPSC-CMs to PDE4 inhibition in 96-well high-throughput electrophysiology screen.

a Cryopreserved TTN-GFP atrial hiPSC-CMs phase contrast and GFP images, 20×. b Zoomed image of sarcomere structures in hiPSC-CMs. c Representative time-space plots indicate functional monolayer electrophysiological activations across the width of each well of the 96-well plate (9 mm diameter). Rolipram increased hiPSC-CM monolayer beat rate. d Representative calcium transient traces indicating the effect of rolipram to increase calcium transient amplitude. e Rolipram increased 2D monolayer beat rate (VEH = 80.0 ± 11.8; 1.0 µM Rolipram = 113.3 ± 33.0; 10.0 µM Rolipram = 129.2 ± 9.4 bpm, n = 8 per group). One way ANOVA, multiple comparisons, *P = 0.01; ***P = 0.0004. f Rolipram increased calcium transient amplitude (VEH = 0.53 ± 0.17; 1.0 µM Rolipram = 0.72 ± 0.40; 10.0 µM Rolipram=1.22 ± 0.50 ∆F/F₀, n = 8 per group). One way ANOVA, multiple comparisons, **P = 0.004. g Rolipram increased calcium impulse conduction velocity (VEH = 23.5 ± 4.1; 1.0 µM Rolipram = 30.3 ± 17.6; 10.0 µM Rolipram = 47.9 ± 20.4 cm/s, n = 8 per group). One way ANOVA, Brown-Forsythe and Welch tests, multiple comparisons, *P = 0.02.

Fig. 8. Functional assessment of 3D engineered heart tissues (3D EHTs) derived from CM-AI-processed, cryopreserved hiPSC-derived atrial CMs.

aTop: Schematic illustration of the 3D EHT platform used to evaluate contractile function of CM-AI laser-processed hiPSC-derived atrial CMs. Right: Representative 10× brightfield image of a 3D EHT. The embedded magnet for force sensing is outlined in blue. Created in https://BioRender.comb Representative contractile force traces recorded from the same 3D EHT over time, demonstrating maturation of contractile performance. c Quantitative analysis of 3D EHT contractile function across multiple time points (Day 7, 22, and 36). Contraction frequency increased over time (Day 7 = 1.97 ± 0.13; Day 22 = 2.63 ± 0.12; Day 36 = 2.27 ± 0.12 Hz, n = 10). Peak contractile force increased over time (Day 7 = 25.9 ± 2.5; Day 22 = 38.0 ± 4.9; Day 36 = 53.3 ± 5.0 µN, n = 10). Contraction velocity increased over time (Day 7 = 327.3 ± 33.0; Day 22 = 516.4 ± 60.9; Day 36 = 650.5 ± 63.5 µN/s, n = 10). Relaxation velocity increased over time (Day 7 = 102.1 ± 10.6; Day 22 = 164.9 ± 19.7; Day 36 = 201.8 ± 18.9 µN/s, n = 10). Time to 50% peak of contraction (TTP 50%) increased from day 22 to 36 (Day 7 = 0.039 ± 0.0009; Day 22 = 0.034 ± 0.001; Day 36 = 0.040 ± 0.0008 s, n = 10). Time to 50% relaxation increased between days 22 and 36 (Day 7 = 0.048 ± 0.002; Day 22 = 0.040 ± 0.001; Day 36 = 0.051 ± 0.003 s, n = 10). All data represent repeated measurements from the same 10 3D EHTs at each time point. One way ANOVA; Friedman test; multiple comparisons; ****P < 0.0001; ***P = 0.0001. d Ivabradine treatment (0.3 nM) significantly reduced spontaneous beat rate (**P = 0.009, unpaired t-test and Kolmogorov–Smirnov test), while other contractile parameters remained unaffected. e Representative 3D EHT force traces at baseline and after vehicle control (VEH, dashed lines) treatment. f Representative force traces showing ivabradine treatment slowed the spontaneous beat rate of 3D EHTs as quantified in (d).