Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Formation of mouse primary cardiomyocyte clusters in agarose-coated wells. ( a ) Schematic drawing of the conventional dish cultivation of cardiomyocytes. The dispersed cells were cultured on the bottom of a 35-mm non-agarose-coated dish. After spread of the 2.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5.0\times 10^{4}\, {\rm cells/mL}$$\end{document} 5.0 × 10 4 cells / mL isolated single cardiomyocytes, the cells attached on the bottom of the 35-mm cultivation dish dispersedly. The cells started to beat 2–3 days after cultivation started. ( b ) A micrograph of dispersed cardiomyocytes in a 35-mm non-agarose-coated dish. ( c ) Schematic drawing of the cultivation of dispersed cells in a 35-mm agarose-coated dish. After spread of the 2.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5.0\times 10^{4}\, {\rm cells/mL}$$\end{document} 5.0 × 10 4 cells / mL isolated single cardiomyocytes, the cells dispersed on the bottom of the agarose layer in the agarose-coated 35-mm cultivation dish. Even after 2–3 days of cultivation, the cells remained isolated with a round shape, and no clusters formed on the bottom. ( d ) A micrograph of cardiomyocytes in an agarose-coated 35-mm cultivation dish. ( e ) Schematic drawing of the cultivation of dispersed cells in a 15.5-mm agarose-coated cultivation well (in a 24-well cultivation plate). After spread of the 1.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5\times 10^{4}\hbox { cells/mL}$$\end{document} 5 × 10 4 cells/mL isolated single cardiomyocytes, dispersed cells gathered and formed small clusters; finally, they gathered into a single large cluster in the 15.5-mm agarose-coated cultivation well. ( f ) A micrograph of a cardiomyocyte cluster in a 15.5-mm agarose-coated cultivation well.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Cell Culture, Isolation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Micrographs of single cells and clusters of mouse primary and hES-derived cardiomyocytes. ( a ) Mouse primary cardiomyocytes (primary) in a 35-mm non-agarose-coated dish (single cell), ( b ) primary cells in a 24-well agarose-coated plate (cluster), ( c ) hES cardiomyocytes in a 35-mm non-agarose-coated dish (single cell), and ( d ) hES in a 24-well agarose-coated plate (cluster).

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Derivative Assay

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Analysis of interbeat interval (IBI) distribution of single and clustered mouse primary and hES cardiomyocytes. ( a )–( d ): Method of measuring interbeat interval (IBI) of single cardiomyocytes and clusters. Temporal change of luminance in the red square area for single cell ( a ) and cluster ( c ) caused by their beating was recorded, as shown in the time-course intensity profiles ( b ) and ( d ), respectively. IBIs of their beating were acquired from the time intervals between two neighboring peaks in the time-course intensity profiles. ( e ), ( f ): Distribution of IBIs of mouse primary cardiomyocytes. ( e ) The relationship between mean IBIs and fluctuations of beating [coefficient of variability (CV) of IBIs] of single isolated primary cardiomyocytes (blue open circles, n = 73) and primary clusters (red filled triangles, n = 6). ( f ) A histogram of all plots in ( e ). The blue filled bars indicate the frequency of IBIs of single cardiomyocytes; the blue arrow and the error bar indicate the corresponding mean value and standard deviation (SD) of single-cardiomyocyte IBIs, respectively. The red filled bars indicate the frequency of IBIs of clusters; the red arrow and the error bar indicate the corresponding mean value and SD of clusters. ( g ), ( h ): Distribution of IBIs in hES cardiomyocytes. ( g ) The relationship between mean IBIs and CV of IBIs in single isolated hES cardiomyocytes (blue open circles, n = 125) and hES clusters (red filled triangles, n = 27). ( h ) A histogram of all plots in ( g ). The blue filled bars indicate the frequency of IBIs of single cardiomyocytes; the blue arrow and the error bar indicate the corresponding mean values and SD of single cardiomyocytes, respectively. The red filled bars indicate the frequency of IBIs of clusters; the red arrow and the error bar indicate the corresponding mean value and SD of clusters.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Isolation, Standard Deviation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Distribution of IBIs and fluctuation of IBI distribution of the hES cardiomyocyte clusters and their constituent cells. ( a )–( c ): Micrographs of hES cardiomyocyte clusters. ( d )–( f ): Distribution of IBIs and the CV of IBIs in the clusters ( a )–( c ) and isolated constituent cells from each cluster (n=50 from among re-cultivated \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1.0\times 10^{3}\hbox { cells}$$\end{document} 1.0 × 10 3 cells ). These plots ( d )–( f ) correspond to each cluster ( a )–( c ). The red filled triangles indicate the cardiomyocyte clusters, and the blue open circles indicate constituent cardiomyocytes of each cluster. Each cluster was measured 2 days after the beating started. Single cardiomyocytes were isolated from each cluster by trypsinization. IBIs of single constituent cardiomyocytes were measured 3 days after their isolation. Median and 95% confidence interval of single cardiomyocytes were 0.971 s and 0.825–1.25 s ( d ), 1.19 s and 1.00–1.28 s ( e ), and 1.12 s and 0.844–1.22 s ( f ), respectively. ( g )–( i ): Histograms of IBIs of each cluster and its isolated constituent cells. The blue filled bars indicate the ratio of frequency for single constituent cardiomyocytes; the blue arrows and error bars indicate the mean IBIs and SDs, and the red arrows also indicate the mean IBIs of clusters.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Isolation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Influence of trypsinization on interbeat intervals in dispersed individual hES cardiomyocytes. ( a ) Distribution of the IBIs and the CV of IBIs in single hES cardiomyocytes before and after trypsinization. The blue open circles indicate the single hES cardiomyocytes (n = 50) before trypsinization. The orange open circles indicate the single cardiomyocytes (n = 50) after trypsinization. ( b ) Histograms of IBIs of hES single cardiomyocytes before and after trypsinization. The blue filled bars indicate the mean IBIs of single cardiomyocytes before trypsinization; the blue arrow and error bar indicate their mean value and SD. The orange filled bars indicate the frequency of mean IBIs of trypsinized single cardiomyocytes; the orange arrow and error bar indicate their mean value and SD.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques:

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Distributions of interbeat intervals (IBIs) and fluctuations of the two hES cardiomyocyte clusters before and after their connection and after re-separation. ( a )–( e ): Micrographs of cardiomyocyte clusters. Micrographs of the large cluster ( a ) and small cluster ( b ) before contact. These clusters were measured when they had been cultivated for 7 days. The two hES cardiomyocyte clusters were connected ( c ). The measurement was performed 3 days after the two clusters contacted each other. Micrographs of the large cluster ( d ) and small cluster ( e ) after separation. The measurements were taken within 5 min of separation. ( f ): Distribution of IBIs and fluctuations of two clusters before contact, during contact, and after separation. Blue filled bar and error bar indicate the mean IBIs and SD of the large cluster. Green filled bar and error bar indicate the mean IBIs and SD of the small cluster.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques:

Journal: Stem Cells and Development

Article Title: Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support

doi: 10.1089/scd.2019.0132

Figure Lengend Snippet: Schematic illustration of the cell culture system. (1) Hematopoietic induction: colonies of undifferentiated hiPSCs were transferred into low-binding plates to induce EB formation. After 5 days, spherical EBs were further cultured on adherent plates in APEL ™ medium containing SCF, EPO, and IL-3. The medium was changed weekly. Within 2 weeks, an HCFC was established, from which hematopoietic cells were continuously released into the supernatant and harvested for further characterization. (2) Erythroid differentiation: cells released into the supernatant were harvested and differentiated into RBCs in a three-phase erythropoiesis system over 18 days. Hematopoietic and erythroid differentiation were monitored by colony formation assay, flow cytometry, microscopy, and hemoglobin analysis. EB, embryoid body; EPO, erythropoietin; HCFC, hematopoietic cell forming complex; hiPSCs, human induced pluripotent stem cells; IL-3, interleukin 3; RBCs, red blood cells; SCF, stem cell factor.

Article Snippet: Red blood cell (RBC) differentiation from human induced pluripotent stem cells (hiPSCs) offers great potential for developmental studies and innovative therapies.

Techniques: Cell Culture, Binding Assay, Colony Assay, Flow Cytometry, Microscopy

Journal: Stem Cells and Development

Article Title: Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support

doi: 10.1089/scd.2019.0132

Figure Lengend Snippet: Schematic overview illustrating the simplified xeno- and feeder free culture model for the ex vivo generation of RBCs from hiPSCs. High cellular output and enhanced enucleation are achieved using only three cytokines (EPO, IL-3, and SCF) and preserving the structure of the initially formed HCFC. The system may further be switched to granulocyte and macrophage generation by slight modification of added cytokines.

Article Snippet: Red blood cell (RBC) differentiation from human induced pluripotent stem cells (hiPSCs) offers great potential for developmental studies and innovative therapies.

Techniques: Ex Vivo, Preserving, Modification