Are these cardiomyocytes? Protocol development reveals impact of sample preparation on the accuracy of identifying cardiomyocytes by flow cytometry

Abstract

Running Title: Standard flow cytometry protocol for assessing stem cell-derived cardiomyocytes SummaryModern differentiation protocols enable efficient, yet imperfect, differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM). As the number of laboratories and studies implementing this technology expands, the accurate assessment of cell identity in differentiation cultures is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity in hPSC-CM cultures has not yet been established. To address this gap, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in hPSC-CM and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges of interpreting data generated by published methods and informed the development of a robust protocol for routine assessment of hPSC-CM. Overall, the new data, workflow for evaluating fitfor-purpose use of antibodies, and standardized protocol described here should benefit investigators new to this field as well as those with expertise in hPSC-CM differentiation.

A Multi-omics Method Enabled by Sequential Metabolomics and Proteomics for Human Pluripotent Stem Cell-derived Cardiomyocytes

“…Cardiac-specific pathways found in KEGG pathways included Cardiac Muscle Contraction (hsa02460) and Adrenergic Signaling in Cardiomyocytes (hsa02461) comprising 36 and 57 proteins, respectively ( Figure 2G ). Cardiomyocyte-specific marker cardiac troponin T (cTnT, gene: TNNT2 ) [8] was consistently detected in each CM replicate with 0.2% RSD in LFQ intensity ( Figure S11A ). Other cardiac proteins such as slow skeletal troponin I (ssTnI, gene: TNNI1 ), α-cardiac muscle actin (α-CAA, gene: ACTC1 ), cardiac troponin C (cTnC, gene: TNNC1 ), cardiac myosin binding protein C (gene: MYBPC3 ), cardiac phospholamban (gene: PLN ), and cardiomyocyte transcription factor GATA-4 (gene: GATA4 ) [44] were consistently detected as well ( Figure S11 B-F ).…”

“…Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have shown immense promise for patient-specific disease modeling, cardiotoxicity screening, and regenerative therapy development (1)(2)(3)(4)(5). Enabled by recent advances, hPSC-CMs can now be generated consistently with high yield and purity at a clinically relevant scale (6)(7)(8)(9). However, one of the roadblocks impeding the realization of the full potential of hPSC-CMs is their structural and phenotypic immaturity compared to adult CMs (9, 10).…”

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

“…These quality-controlled cultures are cryopreserved for experimental studies. The current differentiation approaches yield a heterogeneous mix of nodal-, atrial-and ventricle-like cells 3,16,17,41 . Therefore, strategies employed for CM subtype population enrichment can further improve the specificity of the cultures.…”

Reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes

“…DF6-9-9T hiPSCs were maintained in monolayer culture and differentiation was performed as described [24], generating predominantly ventricular-like cells. Quality control (QC) evaluation of hiPSC-CM differentiation was performed by following the Standard Operating Procedure for flow cytometry-based assessment of troponin positivity within hiPSC-CM cultures as previously described [25] and experimental details are provided in the Supplementary Methods (Table S3).…”