Journal: European cells & materials

Article Title: DESIGNER FAT CELLS: ADIPOGENIC DIFFERENTIATION OF CRISPR-CAS9 GENOME-ENGINEERED INDUCED PLURIPOTENT STEM CELLS

doi: 10.22203/eCM.v046a09

Figure Lengend Snippet: Criteria for chosen guide RNAs for Leptin KO. Score was computed as 100 % minus a weighted sum of off-target hit-scores in the target genome (full ranked list not shown). Single nucleotide polymorphism (SNP) NA indicates no common SNP found in the gRNA. Leptin KO iPSCs were then differentiated into PDiPSCs using the micromass procedure as described above.

Article Snippet: Leptin knockout PDiPSCs in culture were supplemented with 1,000 ng/mL of murine leptin (498-OB-05M; R&D Systems, Minneapolis, MN, USA), a concentration that was determined after performing a leptin dose analysis.

Techniques:

Journal: European cells & materials

Article Title: DESIGNER FAT CELLS: ADIPOGENIC DIFFERENTIATION OF CRISPR-CAS9 GENOME-ENGINEERED INDUCED PLURIPOTENT STEM CELLS

doi: 10.22203/eCM.v046a09

Figure Lengend Snippet: Leptin KO PDiPSCs were cultured in expansion or adipogenic media for 14 days. ( a ) Timeline of culture conditions. ( b ) All groups were stained with Oil Red O and Days 5, 9, and 14 are shown here. Red stain indicates lipid content in cells. The yellow arrows point to lipid-containing cells. Scale bar is 200 μm. ( c ) All Groups were stained with BODIPY/DAPI BODIPY, green, stains for lipids and DAPI, blue, stains nuclei. Scale bar is 100 μm. ( d ) WT and leptin KO PDiPSCs were collected at various timepoints for gene expression characterization. Pparg, Adip , and Lep , were target genes evaluated. Values represent fold change ± SEM ( n = 3 ). Letters represent significance (A p < 0.05, WT adipogenic media; B p < 0.05 leptin KO expansion media; C p < 0.05 leptin KO adipogenic media) from WT PDiPSCs expansion media control. In an effort to restore adipogenic differentiation, murine leptin was added to the culture media as a supplement. ( e ) Leptin KO PDiPSCs with leptin added were cultured in expansion or adipogenic media and for 14 days and were stained with Oil Red O and days 5, 9, and 14 as shown here. Scale bar is 200 μm. ( f ) The same groups as ( e ) were stained with BODIPY/DAPI. Scale bar is 100 μm. ( g ) Lipid content of the PDiPSCs (leptin KO and leptin KO with leptin added) was measured at day 14 using the BODIPY images. Bars represent lipid content (%) ± SEM ( n = 3 ). Asterisks represent significance (* p < 0.01) compared with the leptin KO PDiPSCs in adipogenic media. ( h ) Cells from leptin KO with and without leptin added were collected at various timepoints for gene expression characterization, day 9 is shown here. Pparg, Apn , and Lep , were target genes evaluated. Values represent fold change ± SEM ( n = 3 ). Asterisks represent significance (** p < 0.0001 , *** p < 0.00001) compared with the leptin KO PDiPSCs expansion media control. ( i ) For 9 days, Leptin KO PDiPSCs were treated with adipogenic media and supplemented with varying doses of leptin. Samples were fixed and stained with Oil Red O. 1,000 ng/mL of leptin added to culture media displays the highest concentration of lipid-containing cells. Scale bar is 200 μm. All statistics were run using a 2-way ANOVA with Sidak’s post-hoc test. SEM, standard error of the mean.

Article Snippet: Leptin knockout PDiPSCs in culture were supplemented with 1,000 ng/mL of murine leptin (498-OB-05M; R&D Systems, Minneapolis, MN, USA), a concentration that was determined after performing a leptin dose analysis.

Techniques: Cell Culture, Staining, Gene Expression, Control, Concentration Assay