Comparing the effects of chemical Ca2+ dyes and R-GECO on contractility and Ca2+ transients in adult and human iPSC cardiomyocytes.

We compared commonly used BAPTA-derived chemical Ca2+ dyes (fura2, Fluo-4, and Rhod-2) with a newer genetically encoded indicator (R-GECO) in single cell models of the heart. We assessed their performance and effects on cardiomyocyte contractility, determining fluorescent signal-to-noise ratios and sarcomere shortening in primary ventricular myocytes from adult mouse and guinea pig, and in human iPSC-derived cardiomyocytes. Chemical Ca2+ dyes displayed dose-dependent contractile impairment in all cell types, and we observed a negative correlation between contraction and fluorescence signal-to-noise ratio, particularly for fura2 and Fluo-4. R-GECO had no effect on sarcomere shortening. BAPTA-based dyes, but not R-GECO, inhibited in vitro acto-myosin ATPase activity. The presence of fura2 accentuated or diminished changes in contractility and Ca2+ handling caused by small molecule modulators of contractility and intracellular ionic homeostasis (mavacamten, levosimendan, and flecainide), but this was not observed when using R-GECO in adult guinea pig left ventricular cardiomyocytes. Ca2+ handling studies are necessary for cardiotoxicity assessments of small molecules intended for clinical use. Caution should be exercised when interpreting small molecule studies assessing contractile effects and Ca2+ transients derived from BAPTA-like chemical Ca2+ dyes in cellular assays, a common platform for cardiac toxicology testing and mechanistic investigation of cardiac disease physiology and treatment.

Measurement of Myofilament-Localized Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations.

RATIONALE: Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here, we develop and characterize genetically encoded Ca2+ indicators restricted to the myofilament to directly visualize Ca2+ changes in the sarcomere. OBJECTIVE: To produce and validate myofilament-restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil, and levosimendan) or following cotransduction of 2 established hypertrophic cardiomyopathy disease-causing mutants (cTnT [Troponin T] R92Q and cTnI [Troponin I] R145G) that alter myofilament Ca2+ handling. METHODS AND RESULTS: When expressed in adult ventricular cardiomyocytes RGECO-TnT (Troponin T)/TnI (Troponin I) sensors localize correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localized Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Coadenoviral transduction of RGECO-TnT/TnI with hypertrophic cardiomyopathy causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the 2 mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+. CONCLUSIONS: RGECO-TnT/TnI are functionally equivalent. They visualize Ca2+ within the myofilament and reveal unrecognized aspects of small molecule and disease-associated mutations in living cells.

New cell lines from mouse epiblast share defining features with human embryonic stem cells.

The application of human embryonic stem (ES) cells in medicine and biology has an inherent reliance on understanding the starting cell population. Human ES cells differ from mouse ES cells and the specific embryonic origin of both cell types is unclear. Previous work suggested that mouse ES cells could only be obtained from the embryo before implantation in the uterus. Here we show that cell lines can be derived from the epiblast, a tissue of the post-implantation embryo that generates the embryo proper. These cells, which we refer to as EpiSCs (post-implantation epiblast-derived stem cells), express transcription factors known to regulate pluripotency, maintain their genomic integrity, and robustly differentiate into the major somatic cell types as well as primordial germ cells. The EpiSC lines are distinct from mouse ES cells in their epigenetic state and the signals controlling their differentiation. Furthermore, EpiSC and human ES cells share patterns of gene expression and signalling responses that normally function in the epiblast. These results show that epiblast cells can be maintained as stable cell lines and interrogated to understand how pluripotent cells generate distinct fates during early development.

Embryonic stem cell-derived tissues are immunogenic but their inherent immune privilege promotes the induction of tolerance.

Although human embryonic stem (ES) cells may one day provide a renewable source of tissues for cell replacement therapy (CRT), histoincompatibility remains a significant barrier to their clinical application. Current estimates suggest that surprisingly few cell lines may be required to facilitate rudimentary tissue matching. Nevertheless, the degree of disparity between donor and recipient that may prove acceptable, and the extent of matching that is therefore required, remain unknown. To address this issue using a mouse model of CRT, we have derived a panel of ES cell lines that differ from CBA/Ca recipients at defined genetic loci. Here, we show that even expression of minor histocompatibility (mH) antigens is sufficient to provoke acute rejection of tissues differentiated from ES cells. Nevertheless, despite their immunogenicity in vivo, transplantation tolerance may be readily established by using minimal host conditioning with nondepleting monoclonal antibodies specific for the T cell coreceptors, CD4 and CD8. This propensity for tolerance could be attributed to the paucity of professional antigen-presenting cells and the expression of transforming growth factor (TGF)-beta(2). Together, these factors contribute to a state of acquired immune privilege that favors the polarization of infiltrating T cells toward a regulatory phenotype. Although the natural privileged status of ES cell-derived tissues is, therefore, insufficient to overcome even mH barriers, our findings suggest it may be harnessed effectively for the induction of dominant tolerance with minimal therapeutic intervention.

Non-invasive phenotyping and drug testing in single cardiomyocytes or beta-cells by calcium imaging and optogenetics.

Identification of drug induced electrical instability of the heart curtails development, and introduction, of potentially proarrhythmic drugs. This problem usually requires complimentary contact based approaches such as patch-clamp electrophysiology combined with field stimulation electrodes to observe and control the cell. This produces data with high signal to noise but requires direct physical contact generally preventing high-throughput, or prolonged, phenotyping of single cells or tissues. Combining genetically encoded optogenetic control and spectrally compatible calcium indicator tools into a single adenoviral vector allows the analogous capability for cell control with simultaneous cellular phenotyping without the need for contact. This combination can be applied to single rodent primary adult cardiomyocytes, and human stem cell derived cardiomyocytes, enabling contactless small molecule evaluation for inhibitors of sodium, potassium and calcium channels suggesting it may be useful for early toxicity work. In pancreatic beta-cells it reveals the effects of glucose and the KATP inhibitor gliclazide.

The derivation of highly germline-competent embryonic stem cells containing NOD-derived genome.

It would be extremely advantageous to the analysis of disease mechanisms in the spontaneous mouse model of type 1 diabetes, the nonobese diabetic (NOD) strain, if genes in this strain could be modified in vivo using embryonic stem (ES) cells and homologous recombination. However, a NOD ES cell line with adequate germline transmission has not yet been reported. We report the development of highly germline-competent ES cell lines from the F1 hybrid of NOD and 129 for use in NOD gene targeting. Consequently, we developed ES cell lines derived from (NOD x 129)F1 x 129 backcross 1 mice, which were intercrossed to select for homozygosity of particular regions of NOD genome known to contain disease loci.

Single-cell enzymatic dissociation of hESC lines OxF1-OxF4 and culture in feeder-free conditions.

Experimental manipulation of hESCs has been hampered by their fragility and susceptibility to apoptosis when dissociated into single cells. The OxF lines are particularly robust and may be successfully passaged as single cells, with the inclusion of ROCK inhibitor in the medium. The protocols here describe the enzymatic dissociation of hESCs into a single-cell suspension and the plating of these cells onto either feeder cells or a protein-coated surface.

The Maestro (Mro) gene is dispensable for normal sexual development and fertility in mice.

The mammalian gonad arises as a bipotential primordium from which a testis or ovary develops depending on the chromosomal sex of the individual. We have previously used DNA microarrays to screen for novel genes controlling the developmental fate of the indifferent embryonic mouse gonad. Maestro (Mro), which encodes a HEAT-repeat protein, was originally identified as a gene exhibiting sexually dimorphic expression during mouse gonad development. Wholemount in situ hybridisation analysis revealed Mro to be expressed in the embryonic male gonad from approximately 11.5 days post coitum, prior to overt sexual differentiation. No significant expression was detected in female gonads at the same developmental stage. In order to address its physiological function, we have generated mice lacking Maestro using gene targeting. Male and female mice homozygous for a Mro null allele are viable and fertile. We examined gonad development in homozygous male embryos in detail and observed no differences when compared to wild-type controls. Immunohistochemical analysis of homozygous mutant testes of adult mice revealed no overt abnormalities. Expression profiling using DNA microarrays also indicated no significant differences between homozygote embryonic male gonads and controls. We conclude that Maestro is dispensable for normal male sexual development and fertility in laboratory mice; however, the Mro locus itself does have utility as a site for insertion of transgenes for future studies in the fields of sexual development and Sertoli cell function.