Dual Function of iPSC-Derived Pericyte-Like Cells in Vascularization and Fibrosis-Related Cardiac Tissue Remodeling In Vitro.

Myocardial interstitial fibrosis (MIF) is characterized by excessive extracellular matrix (ECM) deposition, increased myocardial stiffness, functional weakening, and compensatory cardiomyocyte (CM) hypertrophy. Fibroblasts (Fbs) are considered the principal source of ECM, but the contribution of perivascular cells, including pericytes (PCs), has gained attention, since MIF develops primarily around small vessels. The pathogenesis of MIF is difficult to study in humans because of the pleiotropy of mutually influencing pathomechanisms, unpredictable side effects, and the lack of available patient samples. Human pluripotent stem cells (hPSCs) offer the unique opportunity for the de novo formation of bioartificial cardiac tissue (BCT) using a variety of different cardiovascular cell types to model aspects of MIF pathogenesis in vitro. Here, we have optimized a protocol for the derivation of hPSC-derived PC-like cells (iPSC-PCs) and present a BCT in vitro model of MIF that shows their central influence on interstitial collagen deposition and myocardial tissue stiffening. This model was used to study the interplay of different cell types-i.e., hPSC-derived CMs, endothelial cells (ECs), and iPSC-PCs or primary Fbs, respectively. While iPSC-PCs improved the sarcomere structure and supported vascularization in a PC-like fashion, the functional and histological parameters of BCTs revealed EC- and PC-mediated effects on fibrosis-related cardiac tissue remodeling.

Anti-androgenic therapy with finasteride improves cardiac function, attenuates remodeling and reverts pathologic gene-expression after myocardial infarction in mice.

Maladaptive cardiac remodeling after myocardial infarction (MI) is increasingly contributing to the prevalence of chronic heart failure. Women show less severe remodeling, a reduced mortality and a better systolic function after MI compared to men. Although sex hormones are being made responsible for these differences, it remains currently unknown how this could be translated into therapeutic strategies. Because we had recently demonstrated that inhibition of the conversion of testosterone to its highly active metabolite dihydrotestosterone (DHT) by finasteride effectively reduces cardiac hypertrophy and improves heart function during pressure overload, we asked here whether this strategy could be applied to post-MI remodeling. We found increased abundance of DHT and increased expression of androgen responsive genes in the mouse myocardium after experimental MI. Treatment of mice with finasteride for 21 days (starting 7 days after surgery), reduced myocardial DHT levels and markedly attenuated cardiac dysfunction as well as hypertrophic remodeling after MI. Histological and molecular analyses showed reduced MI triggered interstitial fibrosis, reduced cardiomyocyte hypertrophy and increased capillary density in the myocardium of finasteride treated mice. Mechanistically, this was associated with decreased activation of myocardial growth-signaling pathways, a comprehensive normalization of pathological myocardial gene-expression as revealed by RNA deep-sequencing and with direct effects of finasteride on cardiac fibroblasts and endothelial cells. In conclusion, we demonstrated a beneficial role of anti-androgenic treatment with finasteride in post-MI remodeling of mice. As finasteride is already approved for the treatment of benign prostate disease, it could potentially be evaluated as therapeutic strategy for heart failure after MI.

EMC10 (Endoplasmic Reticulum Membrane Protein Complex Subunit 10) Is a Bone Marrow-Derived Angiogenic Growth Factor Promoting Tissue Repair After Myocardial Infarction.

BACKGROUND: Clinical trials of bone marrow cell-based therapies after acute myocardial infarction (MI) have produced mostly neutral results. Treatment with specific bone marrow cell-derived secreted proteins may provide an alternative biological approach to improving tissue repair and heart function after MI. We recently performed a bioinformatic secretome analysis in bone marrow cells from patients with acute MI and discovered a poorly characterized secreted protein, EMC10 (endoplasmic reticulum membrane protein complex subunit 10), showing activity in an angiogenic screen. METHODS: We investigated the angiogenic potential of EMC10 and its mouse homolog (Emc10) in cultured endothelial cells and infarcted heart explants. We defined the cellular sources and function of Emc10 after MI using wild-type, Emc10-deficient, and Emc10 bone marrow-chimeric mice subjected to transient coronary artery ligation. Furthermore, we explored the therapeutic potential of recombinant Emc10 delivered by osmotic minipumps after MI in heart failure-prone FVB/N mice. RESULTS: Emc10 signaled through small GTPases, p21-activated kinase, and the p38 mitogen-activated protein kinase (MAPK)-MAPK-activated protein kinase 2 (MK2) pathway to promote actin polymerization and endothelial cell migration. Confirming the importance of these signaling events in the context of acute MI, Emc10 stimulated endothelial cell outgrowth from infarcted mouse heart explants via p38 MAPK-MK2. Emc10 protein abundance was increased in the infarcted region of the left ventricle and in the circulation of wild-type mice after MI. Emc10 expression was also increased in left ventricular tissue samples from patients with acute MI. Bone marrow-derived monocytes and macrophages were the predominant sources of Emc10 in the infarcted murine heart. Emc10 KO mice showed no cardiovascular phenotype at baseline. After MI, however, capillarization of the infarct border zone was impaired in KO mice, and the animals developed larger infarct scars and more pronounced left ventricular remodeling compared with wild-type mice. Transplanting KO mice with wild-type bone marrow cells rescued the angiogenic defect and ameliorated left ventricular remodeling. Treating FVB/N mice with recombinant Emc10 enhanced infarct border-zone capillarization and exerted a sustained beneficial effect on left ventricular remodeling. CONCLUSIONS: We have identified Emc10 as a previously unknown angiogenic growth factor that is produced by bone marrow-derived monocytes and macrophages as part of an endogenous adaptive response that can be enhanced therapeutically to repair the heart after MI.

A Synthetic Toolbox for the In Situ Formation of Functionalized Homo- and Heteropolysaccharide-Based Hydrogel Libraries.

A synthetic toolbox for the introduction of aldehydo and hydrazido groups into the polysaccharides hyaluronic acid, alginate, dextran, pullulan, glycogen, and carboxymethyl cellulose and their use for hydrogel formation is reported. Upon mixing differently functionalized polysaccharides derived from the same natural precursor, hydrazone cross-linking takes place, which results in formation of a hydrogel composed of one type of polysaccharide backbone. Likewise, hydrogels based on two different polysaccharide strands can be formed after mixing the corresponding aldehydo- and hydrazido-modified polysaccharides. A second line of these studies paves the way to introduce a biomedically relevant ligand, namely, the adhesion factor cyclic RGD pentapeptide, by using an orthogonal click reaction. This set of modified polysaccharides served to create a library of hydrogels that differ in the combination of polysaccharide strands and the degree of cross-linking. The different hydrogels were evaluated with respect to their rheological properties, their ability to absorb water, and their cytotoxicity towards human fibroblast cell cultures. None of the hydrogels studied were cytotoxic, and, hence, they are in principal biocompatible for applications in tissue engineering.

Higher frequencies of BCRP+ cardiac resident cells in ischaemic human myocardium.

AIMS: Several cardiac resident progenitor cell types have been reported for the adult mammalian heart. Here we characterize their frequencies and distribution pattern in non-ischaemic human myocardial tissue and after ischaemic events. METHODS AND RESULTS: We obtained 55 biopsy samples from human atria and ventricles and used immunohistological analysis to investigate two cardiac cell types, characterized by the expression of breast cancer resistance protein (BCRP)/ABCG2 [for side population (SP) cells] or c-kit. Highest frequencies of BCRP+ cells were detected in the ischaemic right atria with a median of 5.40% (range: 2.48-11.1%) vs. 4.40% (1.79-7.75%) in the non-ischaemic right atria (P = 0.47). Significantly higher amounts were identified in ischaemic compared with non-ischaemic ventricles, viz. 5.44% (3.24-9.30%) vs. 0.74% (0-5.23%) (P = 0.016). Few numbers of BCRP+ cells co-expressed the cardiac markers titin, sarcomeric α-actinin, or Nkx2.5; no co-expression of BCRP and progenitor cell marker Sca-1 or pluripotency markers Oct-3/4, SSEA-3, and SSEA-4 was detected. C-kit+ cells displayed higher frequencies in ischaemic (ratio: 1:25 000 ± 2500 of cell counts) vs. non-ischaemic myocardium (1:105 000 ± 43 000). Breast cancer resistance protein+/c-kit+ cells were not identified. Following in vitro differentiation, BCRP+ cells isolated from human heart biopsy samples (n = 6) showed expression of cardiac troponin T and α-myosin heavy-chain, but no full differentiation into functional beating cardiomyocytes was observed. CONCLUSION: We were able to demonstrate that BCRP+/CD31- cells are more abundant in the heart than their c-kit+ counterparts. In the non-ischaemic hearts, they are preferentially located in the atria. Following ischaemia, their numbers are elevated significantly. Our data might provide a valuable snapshot at potential progenitor cells after acute ischaemia in vivo, and mapping of these easily accessible cells may influence future cell therapeutic strategies.

Murine and human pluripotent stem cell-derived cardiac bodies form contractile myocardial tissue in vitro.

AIMS: We explored the use of highly purified murine and human pluripotent stem cell (PSC)-derived cardiomyocytes (CMs) to generate functional bioartificial cardiac tissue (BCT) and investigated the role of fibroblasts, ascorbic acid (AA), and mechanical stimuli on tissue formation, maturation, and functionality. METHODS AND RESULTS: Murine and human embryonic/induced PSC-derived CMs were genetically enriched to generate three-dimensional CM aggregates, termed cardiac bodies (CBs). Addressing the critical limitation of major CM loss after single-cell dissociation, non-dissociated CBs were used for BCT generation, which resulted in a structurally and functionally homogenous syncytium. Continuous in situ characterization of BCTs, for 21 days, revealed that three critical factors cooperatively improve BCT formation and function: both (i) addition of fibroblasts and (ii) ascorbic acid supplementation support extracellular matrix remodelling and CB fusion, and (iii) increasing static stretch supports sarcomere alignment and CM coupling. All factors together considerably enhanced the contractility of murine and human BCTs, leading to a so far unparalleled active tension of 4.4 mN/mm(2) in human BCTs using optimized conditions. Finally, advanced protocols were implemented for the generation of human PSC-derived cardiac tissue using a defined animal-free matrix composition. CONCLUSION: BCT with contractile forces comparable with native myocardium can be generated from enriched, PSC-derived CMs, based on a novel concept of tissue formation from non-dissociated cardiac cell aggregates. In combination with the successful generation of tissue using a defined animal-free matrix, this represents a major step towards clinical applicability of stem cell-based heart tissue for myocardial repair.

Fabrication of heart tubes from iPSC derived cardiomyocytes and human fibrinogen by rotating mold technology.

Due to its structural and functional complexity the heart imposes immense physical, physiological and electromechanical challenges on the engineering of a biological replacement. Therefore, to come closer to clinical translation, the development of a simpler biological assist device is requested. Here, we demonstrate the fabrication of tubular cardiac constructs with substantial dimensions of 6 cm in length and 11 mm in diameter by combining human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and human foreskin fibroblast (hFFs) in human fibrin employing a rotating mold technology. By centrifugal forces employed in the process a cell-dense layer was generated enabling a timely functional coupling of iPSC-CMs demonstrated by a transgenic calcium sensor, rhythmic tissue contractions, and responsiveness to electrical pacing. Adjusting the degree of remodeling as a function of hFF-content and inhibition of fibrinolysis resulted in stable tissue integrity for up to 5 weeks. The rotating mold device developed in frame of this work enabled the production of tubes with clinically relevant dimensions of up to 10 cm in length and 22 mm in diameter which-in combination with advanced bioreactor technology for controlled production of functional iPSC-derivatives-paves the way towards the clinical translation of a biological cardiac assist device.

Protein-free media for cardiac differentiation of hPSCs in 2000 mL suspension culture.

BACKGROUND: Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins, in particular albumin, which is prone to quality variations and presents a substantial cost factor, hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations, we have developed chemically defined, entirely protein-free media based on RPMI, supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA). METHODS AND RESULTS: Both media compositions enable the efficient, directed differentiation of embryonic and induced hPSCs, matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture, producing > 1.3 × 109 cardiomyocytes in a single process run. Transcriptome analysis, flow cytometry, electrophysiology, and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells. CONCLUSIONS: This study promotes the robustness and upscaling of the cardiomyogenic differentiation process, substantially reduces media costs, and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration.

Generation of human induced pluripotent stem cell line encoding for a genetically encoded voltage indicator Arclight A242.

Genetically encoded voltage indicators (GEVIs) allow for monitoring membrane potential changes in neurons and cardiomyocytes (CMs) as an alternative to patch-clamp techniques. GEVIs facilitate non-invasive, high throughput screening of electrophysiological properties of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). A dual transgenic hiPSC line with Arclight A242 (GEVI) and an antibiotic resistance cardiac selection cassette was successfully generated from an earlier established hiPSC line MHHi001-A. After cardiac differentiation and selection, purified populations of CMs with constitutive GEVI expression can be utilized for studying cardiac development, disease modeling, and drug testing.

Performance and feasibility of three different approaches for computer based semi-automated analysis of ventricular arrhythmias in telemetric long-term ECG in cynomolgus monkeys.

Computer-based analysis of long-term electrocardiogram (ECG) monitoring in animal models represents a cost and time-consuming process as manual supervision is often performed to ensure accuracy in arrhythmia detection. Here, we investigate the performance and feasibility of three ECG interval analysis approaches A) attribute-based, B) attribute- and pattern recognition-based and C) combined approach with additional manual beat-to-beat analysis (gold standard) with regard to subsequent detection of ventricular arrhythmias (VA) and time consumption. ECG analysis was performed on ECG raw data of 5 male cynomolgus monkeys (1000 h total, 2 × 100 h per animal). Both approaches A and B overestimated the total number of arrhythmias compared to gold standard (+8.92% vs. +6.47%). With regard to correct classification of detected VA event numbers (accelerated idioventricular rhythms [AIVR], ventricular tachycardia [VT]) approach B revealed higher accuracy compared to approach A. Importantly, VA burden (% of time) was precisely depicted when using approach B (-1.13%), whereas approach A resulted in relevant undersensing of ventricular arrhythmias (-11.76%). Of note, approach A and B could be performed with significant less working time (-95% and - 91% working time) compared to gold standard. In sum, we show that a combination of attribute-based and pattern recognition analysis (approach B) can reproduce VA burden with acceptable accuracy without using manual supervision. Since this approach allowed analyses to be performed with distinct time saving it represents a valuable approach for cost and time efficient analysis of large preclinical ECG datasets.

A micro-LED array based platform for spatio-temporal optogenetic control of various cardiac models.

Optogenetics relies on dynamic spatial and temporal control of light to address emerging fundamental and therapeutic questions in cardiac research. In this work, a compact micro-LED array, consisting of 16 × 16 pixels, is incorporated in a widefield fluorescence microscope for controlled light stimulation. We describe the optical design of the system that allows the micro-LED array to fully cover the field of view regardless of the imaging objective used. Various multicellular cardiac models are used in the experiments such as channelrhodopsin-2 expressing aggregates of cardiomyocytes, termed cardiac bodies, and bioartificial cardiac tissues derived from human induced pluripotent stem cells. The pacing efficiencies of the cardiac bodies and bioartificial cardiac tissues were characterized as a function of illumination time, number of switched-on pixels and frequency of stimulation. To demonstrate dynamic stimulation, steering of calcium waves in HL-1 cell monolayer expressing channelrhodopsin-2 was performed by applying different configurations of patterned light. This work shows that micro-LED arrays are powerful light sources for optogenetic control of contraction and calcium waves in cardiac monolayers, multicellular bodies as well as three-dimensional artificial cardiac tissues.

Generation of human induced pluripotent stem cell lines encoding for genetically encoded calcium indicators RCaMP1h and GCaMP6f.

Calcium plays a key role in cardiomyocytes (CMs) for the translation of the electrical impulse of an action potential into contraction forces. A rapid, not-invasive fluorescence imaging technology allows for the monitoring of calcium transients in human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) to investigate the cardiac electrophysiology in vitro and after cell transplantation in vivo. The genetically encoded calcium indicators (GECIs) GCaMP6f or RCaMP1h were successfully transfected in the previously established hiPSC line MHHi001-A, together with a cardiac specific antibiotic selection cassette facilitating the monitoring of the calcium handling in highly pure populations of hiPSC-CMs.

Two-photon induced collagen cross-linking in bioartificial cardiac tissue.

Cardiac tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs for transplantation. Besides contractile function, the stiffness of tissue engineered constructs is crucial to generate transplantable tissue surrogates with sufficient mechanical stability to withstand the high pressure present in the heart. Although several collagen cross-linking techniques have proven to be efficient in stabilizing biomaterials, they cannot be applied to cardiac tissue engineering, as cell death occurs in the treated area. Here, we present a novel method using femtosecond (fs) laser pulses to increase the stiffness of collagen-based tissue constructs without impairing cell viability. Raster scanning of the fs laser beam over riboflavin-treated tissue induced collagen cross-linking by two-photon photosensitized singlet oxygen production. One day post-irradiation, stress-strain measurements revealed increased tissue stiffness by around 40% being dependent on the fibroblast content in the tissue. At the same time, cells remained viable and fully functional as demonstrated by fluorescence imaging of cardiomyocyte mitochondrial activity and preservation of active contraction force. Our results indicate that two-photon induced collagen cross-linking has great potential for studying and improving artificially engineered tissue for regenerative therapies.

Rhesus monkey cardiosphere-derived cells for myocardial restoration.

BACKGROUND AIMS: Cardiosphere-derived cells (CDC) have been proposed as a promising myocardial stem cell source for cardiac repair. They have been isolated from human, porcine and rodent cardiac biopsies. However, their usefulness for myocardial restoration remains controversial. We aimed to determine the survival, differentiation and functional effects of Rhesus monkey CDC (RhCDC) in a mouse model of myocardial infarction. METHODS: RhCDC were isolated and characterized by flow cytometry and reverse transcriptase (RT)-polymerase chain reaction (PCR) and compared with human CDC. They were injected intramyocardially into severe combined immune deficiency (SCID) beige mice after ligature of the left anterior descending artery (LAD). Phosphate-buffered saline (PBS) served as placebo. Medium treatment alone was used to distinguish between cellular and non-cellular effects. Animals were divided into a non-infarcted control group (n = 7), infarct control groups (n = 24), medium-treated infarct groups (n = 35) and RhCDC-treated infarct groups (n = 33). Follow-up was either 1 or 4 weeks. LV function was assessed by pressure-volume loop analysis. Differentiation was analyzed by immunhistochemical profiling and RT-PCR. RESULTS: Proliferating RhCDC grafts were detected after transplantation in an acute infarct model. RhCDC as well as medium treatment protected myocardium within the infarct area and improved LV function. RhCDC had a superior regenerative effect than medium alone. CONCLUSIONS: For the first time, RhCDC have been used for the restoration of infarcted myocardium. RhCDC proliferated in vivo and positively influenced myocardial remodeling. This effect could be mimicked by treatment with unconditioned medium alone, emphasizing a non-cellular paracrine therapeutic mechanism. However, as a robust cardiac stem cell source, CDC might be useful to evoke prolonged paracrine actions in cardiac stem cell therapy.

Dextran-based scaffolds for in-situ hydrogelation: Use for next generation of bioartificial cardiac tissues.

In pursuit of a chemically-defined matrix for in vitro cardiac tissue generation, we present dextran (Dex)-derived hydrogels as matrices suitable for bioartificial cardiac tissues (BCT). The dextran hydrogels were generated in situ by using hydrazone formation as the crosslinking reaction. Material properties were flexibly adjusted, by varying the degrees of derivatization and the molecular weight of dextran used. Furthermore, to modulate dextran's bioactivity, cyclic pentapeptide RGD was coupled to its backbone. BCTs were generated by using a blend of modified dextran and human collagen (hColI) in combination with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and fibroblasts. These hColI + Dex blends with or without RGD supported tissue formation and functional maturation of CMs. Contraction forces (hColI + Dex-RGD: 0.27 ± 0.02 mN; hColI + Dex: 0.26 ± 0.01 mN) and frequencies were comparable to published constructs. Thus, we could demonstrate that, independent of the presence of RGD, our covalently linked dextran hydrogels are a promising matrix for building cardiac grafts.

Establishment of MHHi001-A-5, a GCaMP6f and RedStar dual reporter human iPSC line for in vitro and in vivo characterization and in situ tracing of iPSC derivatives.

Transgenic hiPSC lines carrying reporter genes represent valuable tools for functional characterization of iPSC derivatives, disease modelling and clinical evaluation of cell therapies. Here, the hiPSC line 'Phoenix' (Haase et al., 2017) was genetically engineered using TALEN-based integration of the calcium sensor GCaMP6f and RedStarnuc reporter into the AAVS1 site. Characterization of undifferentiated cells and functional investigation of hiPSC-derived cardiomyocytes-containing BCTs showed a strong intracellular calcium transient-dependent GCaMP6f and eminent RedStarnuc signal. Therefore, our dual reporter line provides an excellent tool to facilitate monitoring of engraftment, calcium fluctuations and coupling of iPSC derivatives such as cardiomyocytes in vitro and in vivo in animal models.

iPSC culture expansion selects against putatively actionable mutations in the mitochondrial genome.

Therapeutic application of induced pluripotent stem cell (iPSC) derivatives requires comprehensive assessment of the integrity of their nuclear and mitochondrial DNA (mtDNA) to exclude oncogenic potential and functional deficits. It is unknown, to which extent mtDNA variants originate from their parental cells or from de novo mutagenesis, and whether dynamics in heteroplasmy levels are caused by inter- and intracellular selection or genetic drift. Sequencing of mtDNA of 26 iPSC clones did not reveal evidence for de novo mutagenesis, or for any selection processes during reprogramming or differentiation. Culture expansion, however, selected against putatively actionable mtDNA mutations. Altogether, our findings point toward a scenario in which intracellular selection of mtDNA variants during culture expansion shapes the mutational landscape of the mitochondrial genome. Our results suggest that intercellular selection and genetic drift exert minor impact and that the bottleneck effect in context of the mtDNA genetic pool might have been overestimated.

The Long Non-coding RNA Cyrano Is Dispensable for Pluripotency of Murine and Human Pluripotent Stem Cells.

Pluripotency is tightly regulated and is crucial for stem cells and their implementation for regenerative medicine. Non-coding RNAs, especially long non-coding RNAs (lncRNAs) emerged as orchestrators of versatile (patho)-physiological processes on the transcriptional and post-transcriptional level. Cyrano, a well-conserved lncRNA, is highly expressed in stem cells suggesting an important role in pluripotency, which we aimed to investigate in loss-off-function (LOF) experiments. Cyrano was described previously to be essential for the maintenance of mouse embryonic stem cell (ESC) pluripotency. In contrast, using different genetic models, we here found Cyrano to be dispensable in murine and human iPSCs and in human ESCs. RNA sequencing revealed only a moderate influence of Cyrano on the global transcriptome. In line, Cyrano-depleted iPSCs retained the potential to differentiate into the three germ layers. In conclusion, different methods were applied for LOF studies to rule out potential off-target effects. These approaches revealed that Cyrano does not impact pluripotency.

Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture.

Aiming at clinical translation, robust directed differentiation of human pluripotent stem cells (hPSCs), preferentially in chemically defined conditions, is a key requirement. Here, feasibility of suspension culture based hPSC-cardiomyocyte (hPSC-CM) production in low-cost, xeno-free media compatible with good manufacturing practice standards is shown. Applying stirred tank bioreactor systems at increasing dimensions, our advanced protocol enables routine production of about 1 million hPSC-CMs/mL, yielding ∼1.3 × 108 CM in 150 mL and ∼4.0 × 108 CMs in 350-500 mL process scale at >90% lineage purity. Process robustness and efficiency is ensured by uninterrupted chemical WNT pathway control at early stages of differentiation and results in the formation of almost exclusively ventricular-like CMs. Modulated WNT pathway regulation also revealed the previously unappreciated role of ROR1/CD13 as superior surrogate markers for predicting cardiac differentiation efficiency as soon as 72 h of differentiation. This monitoring strategy facilitates process upscaling and controlled mass production of hPSC derivatives.