Journal: Circulation

Article Title: Purification of Cardiomyocytes From Differentiating Pluripotent Stem Cells Using Molecular Beacons That Target Cardiomyocyte-Specific mRNA

doi: 10.1161/circulationaha.113.004228

Figure Lengend Snippet: Figure 4. Purification of cardiomyocytes from differentiating mouse embryonic stem cells (mESCs) with cardiomyocyte-specific molecular beacons (MBs). A, Schematic of the protocol used for differentiating mESCs to the cardiac lineage. EB indicates embryoid body; and ES, embryonic stem cell. B, Percent expression of Tnnt2 at days 4 and 9 during mESC differentiation into cardiomyocytes; n=3. C, Immunocytochemistry results of mESC-derived cardiomyocytes at day 9 for Tnnt2, Tnnt3, and Actn2. Scale bars, 20 µm. D, Flow cytometry analyses of MHC1-MB (an MB that targeted myosin heavy chain 6/7 mRNA) signals in mESC differentiation culture at day 9; n=6. FSC indicates forward scatter. E, Flow cytometry analysis of Tnnt2 expression in mESCs sorted with MHC1-MB and fluorescence- activated cell sorting (FACS) at differentiation day 9; n=3. Numbers represent percentages of MB-positive cells (D, E). F, FACS-sorted MHC1-MB–positive cells exhibited Tnnt2 and Actn2 in immunocytochemistry. Scale bars, 20 µm. G, Quantitative reverse-transcriptase polymerase chain reaction analyses showing difference in gene expression levels between mouse tail tip fibroblasts (MTTF) and presorted (PRE) and postsorted (POST) mESCs. The cardiomyocyte genes Tnnt2, Myh6, Myh7, and Myl2 were significantly enriched in postsorted cells with MHC1-MB, and noncardiac lineage genes (Acta2, Ddr2, Gata1, and Sox17) were substantially reduced compared with presorted cells. The y axis represents relative mRNA expression of target genes to GAPDH. A.U. indicates arbitrary units. *P<0.05 compared with presorted group; n=3.

Article Snippet: mESCs (J1) were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Atlanta Biologicals), 1% non-essential amino acids solution, 1% L-glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin and 2,000 U ml−1 mouse LIF (Millipore) on feeder layers of mitotically inactivated STO cells, a mouse embryonic fibroblast line (ATCC).

Techniques: Purification, Expressing, Immunocytochemistry, Derivative Assay, Flow Cytometry, Fluorescence, FACS, Reverse Transcription, Polymerase Chain Reaction, Gene Expression

Journal: Circulation

Article Title: Purification of Cardiomyocytes From Differentiating Pluripotent Stem Cells Using Molecular Beacons That Target Cardiomyocyte-Specific mRNA

doi: 10.1161/circulationaha.113.004228

Figure Lengend Snippet: Figure 5. Purification of cardiomyocytes from differentiating human embryonic stem cells (hESCs) by cell sorting via molecular beacon (MB) and fluorescence-activated cell sorting (FACS). A, Schematic of the protocol to differentiate human pluripotent stem cells (hPSCs) to the cardiac lineage. BMP4 indicates bone morphogenetic protein 4; END-2, mouse endoderm-like cells; ES, embryonic stem cell; and FGF2, basic fibroblast growth factor. B, Percent expression of TNNI3 at days 9 and 13 determined by flow cytometry during hESC differentiation into cardiomyocytes; n=3. C, Flow cytometry results of MHC1-MB–positive cells in hESC differentiation culture at day 13; n=6. FSC indicates forward scatter; and MHC1-MB, an MB that targeted myosin heavy chain 6/7 mRNA. D, Flow cytometry results showing TNNI3 expression of FACS-sorted hESCs at day 13 of differentiation after applying MHC1-MB. Numbers represent percentages of MB-positive cells (C, D). n=3.

Article Snippet: mESCs (J1) were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Atlanta Biologicals), 1% non-essential amino acids solution, 1% L-glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin and 2,000 U ml−1 mouse LIF (Millipore) on feeder layers of mitotically inactivated STO cells, a mouse embryonic fibroblast line (ATCC).

Techniques: Purification, FACS, Fluorescence, Expressing, Flow Cytometry

Journal: Circulation

Article Title: Purification of Cardiomyocytes From Differentiating Pluripotent Stem Cells Using Molecular Beacons That Target Cardiomyocyte-Specific mRNA

doi: 10.1161/circulationaha.113.004228

Figure Lengend Snippet: Figure 5. (continued) E, Immunocytochemistry for TNNI3 and TNNT2 on MHC1-MB–positive cells sorted from hESC cultures. Scale bars, 20 µm. F, mRNA expression levels of cardiac and noncardiac genes measured by quantitative reverse-transcriptase polymerase chain reaction. Comparisons were made for human dermal fibroblast (HDF), presorted hESCs at day 13 (PRE), and post-FACS–sorted cells at day 13 (POST) using MHC1-MB. Cardiomyocyte genes (TNNT2, MYH6, MYH7, and MYL2) were significantly higher in sorted hESCs than in presorted hESCs and HDF. Expression of the noncardiac lineage genes (PECAM1, CALPONIN, THY1, MYOD, and NEUROD) was significantly lower in sorted cells than in others. The y axis represents relative mRNA expression of target genes to GAPDH. A.U. indicates arbitrary units. *P<0.05 compared with presorted group; n=3. G, mRNA expression levels of cardiac genes from both MHC1-MB–positive and –negative cells measured by quantitative reverse-transcriptase polymerase chain reaction. H, Action potentials of MHC1-MB–positive cardiomyocytes. Shown are representative configurations of nodal (top), atrial (middle), and ventricular (bottom) action potentials.

Article Snippet: mESCs (J1) were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Atlanta Biologicals), 1% non-essential amino acids solution, 1% L-glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin and 2,000 U ml−1 mouse LIF (Millipore) on feeder layers of mitotically inactivated STO cells, a mouse embryonic fibroblast line (ATCC).

Techniques: Immunocytochemistry, Expressing, Reverse Transcription, Polymerase Chain Reaction

Journal: Circulation

Article Title: Purification of Cardiomyocytes From Differentiating Pluripotent Stem Cells Using Molecular Beacons That Target Cardiomyocyte-Specific mRNA

doi: 10.1161/circulationaha.113.004228

Figure Lengend Snippet: Figure 6. Purification of cardiomyocytes from differentiating human induced pluripotent stem cells (hiPSCs) by cell sorting via molecular beacon (MB) and fluorescence-activated cell sorting (FACS). A, Schematic of the protocol to differentiate hiPSCs to the cardiac lineage. BMP4 indicates bone morphogenetic protein 4; END-2, mouse endoderm-like cells; ES, embryonic stem cells; and FGF2, basic fibroblast growth factor. B, Percent expression of TNNI3 at days 9 and 13 during hiPSC differentiation into cardiomyocytes determined by flow cytometry; n=3. C, Flow cytometric analysis of MHC1-MB signals in hiPSC differentiation culture at day 13; n=6. MHC1-MB indicates an MB that targeted myosin heavy chain 6/7 mRNA. D, Flow cytometric results showing TNNI3 expression of FACS-sorted hiPSCs at day 13 of differentiation after applying MHC1-MB. Numbers represent percentages of MB-positive cells (C, D). n=3. E, Immunocytochemistry for TNNI3 and TNNT2 on MHC1-MB–positive cells sorted from hiPSC cultures. Scale bars, 20 µm. F, mRNA expression levels of cardiac and noncardiac genes measured by quantitative reverse-transcriptase polymerase chain reaction. Comparisons were made among human dermal fibroblast (HDF), presorted hiPSCs at day 13 (PRE), and MB-based FACS-sorted hiPSCs at day 13 (POST). The y axis represents relative mRNA expression of target genes to GAPDH. A.U. indicates arbitrary units. *P<0.05 compared to presorted group; n=3. G, Calcium imaging of MHC1-MB–positive cardiomyocytes sorted from hiPSCs. Left, Confocal scan of representative cells loaded with Fluo-4 AM, with magnification of line-scanned region indicated with red dashed line (white scale bars, 20 µm; legend shows increasing calcium levels, with blue being low calcium). Right, Time course of [Ca]2+I, measured at line-scan region in cell pictured and paced at 0.5 Hz by field stimulation. [Ca]2+ is plotted as fluorescence intensity normalized to baseline (F/F0).

Article Snippet: mESCs (J1) were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Atlanta Biologicals), 1% non-essential amino acids solution, 1% L-glutamine, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin and 2,000 U ml−1 mouse LIF (Millipore) on feeder layers of mitotically inactivated STO cells, a mouse embryonic fibroblast line (ATCC).

Techniques: Purification, FACS, Fluorescence, Expressing, Flow Cytometry, Immunocytochemistry, Reverse Transcription, Polymerase Chain Reaction, Imaging

Journal: Journal of nanobiotechnology

Article Title: Recombinant high-density lipoprotein targeted delivery of celastrol to promote foam cells lipophagy against early atherosclerosis.

doi: 10.1186/s12951-025-03327-9

Figure Lengend Snippet: Fig. 11 Lipophagy induced by CEL-rHDL is related to the Ca2+/CaMKKβ/AMPK/mTOR signaling pathway. (A) The changes of relevant proteins in foam cells treated with CEL or CEL-rHDL in the presence or absence of 25 µM STO-609. (B) The changes of relevant proteins in foam cells treated with CEL or CEL-rHDL in the presence or absence of 5 µM CC. (C) Schematic presentation of the signaling cascade to lipophagy. *p < 0.05, **p < 0.01 and ***p < 0.001

Article Snippet: Thapsigargin (TG), Compound C (CC), BAPTA/AM, STO‐609, Bafilomycin A1 (Baf ) and 3-Methyladenine (3-MA) were obtained from Topscience Co., Ltd (Shanghai, China).

Techniques: