Journal: Genes

Article Title: A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors

doi: 10.3390/genes15050575

Figure Lengend Snippet: SMAR iPSCs can be differentiated into phenotypic natural killer (NK) cells. ( A ) Scheme depicting the differentiation process from iPSC to NK cells. CD34+ haematopoetic progenitor cells (HPCs) were generated from SMAR iPSCs by embryoid body (EB) formation and cultured for 12 days, after which CD34+ cells were sorted and further differentiated into lymphoid progenitor cells and NK cells in a two-step process over 28 days. ( B ) The expression of major NK cell marker CD56 and leukocyte marker CD45 in primary NK cells and SMAR iPSC-derived NK cells as measured by flow cytometry. ( C ) The expression of a panel of NK cell markers in primary NK cells and SMAR iPSC-derived NK cells as measured by flow cytometry, including maturation markers (CD57, CD94), inhibitory receptors (Killer Inhibitory Receptors, KIRs), and activating receptors (CD16, CD69, NKG2D). Data shown are from two independent experiments.

Article Snippet: iPSCs were differentiated into NK cells using the STEMdiff NK Cell Kit (Stem Cell Technologies) according to the manufacturer’s instructions.

Techniques: Generated, Cell Culture, Expressing, Marker, Derivative Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: Hereditary E200K mutation within the prion protein gene alters human iPSC derived cardiomyocyte function

doi: 10.1038/s41598-022-19631-5

Figure Lengend Snippet: E200K and DS cardiomyocytes have altered electrophysiology. ( a ) Tropomyosin staining of the differentiated cardiomyocytes, scale bar = 100 µm. ( b ) Schematic showing measured parameters. ( c ) Representative traces for the control, E200K and DS cardiomyocytes (y axes are not equivalent but scaled to show detail). Graphs showing ( d ) peak to peak amplitude, ( e ) peak to peak duration, ( f ) field potential duration (corrected to RR interval; FPDc), ( g ) conduction velocity, ( h ) RR interval and ( i ) RR coefficient of variance (variability of the RR interval). Graphs show data points of each independent biological repeat ‘ n ’ with mean and SEM, *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Cardiomyocytes were differentiated using STEMdiff Cardiomyocyte Differentiation kit (Stem Cell Technologies) as per the manufacturer’s instructions.

Techniques: Staining, Control

Journal: Scientific Reports

Article Title: Hereditary E200K mutation within the prion protein gene alters human iPSC derived cardiomyocyte function

doi: 10.1038/s41598-022-19631-5

Figure Lengend Snippet: E200K cardiomyocytes show minimal changes in health. Graphs of ( a ) caspase activation and ( b ) prestoblue metabolism assay. ( c ) Example images of β-galactosidase staining (blue; red arrows indicate deposits), an indicator of cellular senescence. Scale bar = 100 µm. Graphs show data points of each independent biological repeat ‘ n ’ with mean and SEM, *p < 0.05 and ***p < 0.001.

Article Snippet: Cardiomyocytes were differentiated using STEMdiff Cardiomyocyte Differentiation kit (Stem Cell Technologies) as per the manufacturer’s instructions.

Techniques: Activation Assay, Staining

Journal: Scientific Reports

Article Title: Hereditary E200K mutation within the prion protein gene alters human iPSC derived cardiomyocyte function

doi: 10.1038/s41598-022-19631-5

Figure Lengend Snippet: E200K cardiomyocytes have increased mitochondrial superoxide. ( a ) Rate of production of DCF fluorescence as an indicator of general ROS production. ( b ) Example images of mitoSOX fluorescence within the cardiomyocyte cultures. Scale bar = 100 µm. ( c ) Quantification of mitoSOX intensity. Graphs show data points of each independent biological repeat ‘ n ’ with mean and SEM, **p < 0.01 and ***p < 0.001.

Article Snippet: Cardiomyocytes were differentiated using STEMdiff Cardiomyocyte Differentiation kit (Stem Cell Technologies) as per the manufacturer’s instructions.

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: Hereditary E200K mutation within the prion protein gene alters human iPSC derived cardiomyocyte function

doi: 10.1038/s41598-022-19631-5

Figure Lengend Snippet: E200K and DS cardiomyocytes demonstrate altered mitochondrial function. ( a ) Example traces showing changes in oxygen consumption rates (OCR) on stimulation or inhibition of the ETC. Traces show the mean and SEM of control (n = 9), E200K (n = 5) and DS (n = 4) cardiomyocytes. Graphs showing ( b ) basal respiration, ( c ) maximum respiration, ( d ) spare capacity, ( e ) ATP production, ( f ) proton leak and ( g ) coupling efficiency calculated from the experimental setup shown in ( a ). ( h ) FCCP titrations of control and E200K cardiomyocytes. Shown are the mean and SEM of six repeats for the control and three repeats for the E200K cardiomyocytes. ( i ) Mitochondrial polarization of control and E200K cardiomyocytes shown as mean fluorescence per cell ( j ). Except for ( a ) and ( h ) as described, graphs show data points of each independent biological repeat ‘ n ’ with mean and SEM, *p < 0.05, **p < 0.01 and ***p < 0.001 change from control, #p < 0.05 change from basal respiration.

Article Snippet: Cardiomyocytes were differentiated using STEMdiff Cardiomyocyte Differentiation kit (Stem Cell Technologies) as per the manufacturer’s instructions.

Techniques: Inhibition, Control, Fluorescence

Journal: Scientific Reports

Article Title: Hereditary E200K mutation within the prion protein gene alters human iPSC derived cardiomyocyte function

doi: 10.1038/s41598-022-19631-5

Figure Lengend Snippet: A 200 E/E , 200 E/K and 200 K/K genetically matched cardiomyocytes demonstrate changes in electrophysiology. Schematic representation of the cloning strategies for cells from the donor carrying the PRNP E200K mutation ( a ) and for cells from a donor with no known hereditary disease ( b ) to produce the three genotypes (200 E/E , 200 E/K and 200 K/K ) on the same genetic background (see S13 for detailed information). Graphs showing ( c ) peak to peak amplitude, ( d ) peak to peak duration, ( e ) field potential duration (corrected to RR interval), ( f ) RR interval and ( g ) RR coefficient of variance (variability of the RR interval). Graphs show data points of each independent biological repeat ‘ n ’ with mean and SEM, *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Cardiomyocytes were differentiated using STEMdiff Cardiomyocyte Differentiation kit (Stem Cell Technologies) as per the manufacturer’s instructions.

Techniques: Cloning, Mutagenesis

Journal: Scientific Reports

Article Title: Hereditary E200K mutation within the prion protein gene alters human iPSC derived cardiomyocyte function

doi: 10.1038/s41598-022-19631-5

Figure Lengend Snippet: A 200 E/E , 200 E/K and 200 K/K genetically matched cardiomyocytes display altered mitochondrial function. Example traces showing changes in oxygen consumption rates on stimulation or inhibition of the ETC in the 200 E/E , 200 E/K and 200 K/K genetically matched cardiomyocytes from the PRNP E200K donor ( a ) or no known mutation donor ( b ) cells. Traces show the mean and SEM (n ≥ 6). Graphs showing ( c ) basal respiration, ( d ) maximum respiration, ( e ) spare capacity, ( f ) ATP production, ( g ) proton leak and ( h ) coupling efficiency calculated from the experimental setup shown in ( a ). ( i ) Mitochondrial polarization of 200 E/E , 200 E/K and 200 K/K cardiomyocytes shown as mean fluorescence intensity per cell. ( j ) MitoSOX intensity measurements. Except for A & B as described, graphs show data points of each independent biological repeat ‘ n ’ with mean and SEM, *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Cardiomyocytes were differentiated using STEMdiff Cardiomyocyte Differentiation kit (Stem Cell Technologies) as per the manufacturer’s instructions.

Techniques: Inhibition, Mutagenesis, Fluorescence

Journal: Scientific Reports

Article Title: Hereditary E200K mutation within the prion protein gene alters human iPSC derived cardiomyocyte function

doi: 10.1038/s41598-022-19631-5

Figure Lengend Snippet: Schematic summary of findings. Diagrammatic representation of the differences between the E200K donor-derived cardiomyocytes and the cardiomyocytes from the control no-known-disease donors (left) and the genetically matched single or double mutation (right). For the isotopically matched lines, only the control mutation inserted lines are shown but the conditions that were consistently changed across both CRISPR-Cas9 engineered lines are indicated. Arrows indicate increased or decreased accordingly.

Article Snippet: Cardiomyocytes were differentiated using STEMdiff Cardiomyocyte Differentiation kit (Stem Cell Technologies) as per the manufacturer’s instructions.

Techniques: Derivative Assay, Control, Mutagenesis, CRISPR