Rationale: There are several methods to measure cardiomyocyte (CM) and muscle contraction but these require customized hardware, expensive apparatus and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming and only specialist researchers can quantify data. Objective: Here we describe and validate an automated, open source software tool (MUSCLEMOTION) adaptable for use with standard laboratory- and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes and pharmacological responses. Methods and Results: MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in: (i) 1-dimensional in vitro models such as isolated adult and human pluripotent stem cell-derived CMs (hPSC-CMs); (ii) 2-dimensional in vitro models, such as beating CM monolayers or small clusters of hPSC-CMs; (iii) 3-dimensional multicellular in vitro or in vivo contractile tissues such as cardiac "organoids", engineered heart tissues (EHT), zebrafish- and human hearts. MUSCLEMOTION was effective under different recording conditions (bright field microscopy with simultaneous patch clamp recording, phase contrast microscopy and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement such as optical flow, pole deflection, edge-detection systems or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. Conclusions: Using a single open source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell-, animal- and human models.

To realize the therapeutic potential of human embryonic stem cells (hESCs), it is necessary to regulate their differentiation in a uniform and reproducible manner. We have developed a method in which known numbers of hESCs in serum-free medium were aggregated by centrifugation to foster the formation of embryoid bodies (EBs) of uniform size (spin EBs). These spin EBs differentiated efficiently and synchronously, as evidenced by the sequential expression of molecular markers representing stem cells, primitive streak, and mesoderm. In the presence of hematopoietic growth factors, reproducible differentiation was achieved with blood cells formed in more than 90% of EBs. Using chimeric EBs generated from mixtures of green fluorescence protein–positive (GFP+) and GFP– hESCs in a clonogenic assay, hematopoietic precursor frequency was estimated to be approximately 1:500 input cells. This method of EB formation provides a generally applicable means for modulating and objectively monitoring the directed differentiation of hESCs.

Background—: Pluripotent stem cells (PSCs) offer a new paradigm for modeling genetic cardiac diseases, but it is unclear whether mouse and human PSCs can truly model both gain- and loss-of-function genetic disorders affecting the Na ⁺ current ( I _Na ) because of the immaturity of the PSC-derived cardiomyocytes. To address this issue, we generated multiple PSC lines containing a Na ⁺ channel mutation causing a cardiac Na ⁺ channel overlap syndrome. Method and Results—: Induced PSC (iPSC) lines were generated from mice carrying the Scn5a ^{1798insD/ +} (Scn5a-het) mutation. These mouse iPSCs, along with wild-type mouse iPSCs, were compared with the targeted mouse embryonic stem cell line used to generate the mutant mice and with the wild-type mouse embryonic stem cell line. Patch-clamp experiments showed that the Scn5a-het cardiomyocytes had a significant decrease in I _Na density and a larger persistent I _Na compared with Scn5a-wt cardiomyocytes. Action potential measurements showed a reduced upstroke velocity and longer action potential duration in Scn5a-het myocytes. These characteristics recapitulated findings from primary cardiomyocytes isolated directly from adult Scn5a-het mice. Finally, iPSCs were generated from a patient with the equivalent SCN5A ^{1795insD/ +} mutation. Patch-clamp measurements on the derivative cardiomyocytes revealed changes similar to those in the mouse PSC-derived cardiomyocytes. Conclusion—: Here, we demonstrate that both embryonic stem cell- and iPSC-derived cardiomyocytes can recapitulate the characteristics of a combined gain- and loss-of-function Na ⁺ channel mutation and that the electrophysiological immaturity of PSC-derived cardiomyocytes does not preclude their use as an accurate model for cardiac Na ⁺ channel disease.

Differentiating human embryonic stem cells (HESCs) represent an experimental platform for establishing the relationships between the earliest lineages that emerge during human development. Here we report the targeted insertion in HESCs of sequences encoding green fluorescent protein (GFP) into the locus of MIXL1, a gene transiently expressed in the primitive streak during embryogenesis.^1,2 GFP fluorescence in MIXL1^GFP/w HESCs differentiated in the presence of BMP4 reported the expression of MIXL1, permitting the identification of viable human primitive streak-like cells. The use of GFP as a reporter for MIXL1 combined with cell surface staining for platelet-derived growth factor receptor alpha (PDGFRα) enabled the isolation of a cell population that was highly enriched in primitive hematopoietic precursors, the earliest derivatives of the primitive streak. These experiments demonstrate the utility of MIXL1^GFP/w HESCs for analyzing the previously inaccessible events surrounding the development of human primitive streak-like cells and their subsequent commitment to hematopoiesis.