Figure 4
Figure 4. Overexpression of the Lycat transgene increased the formation of BL-CFCs and hematopoietic and endothelial cells by in vitro function assay. (A) D4 EBs from FC5 and FC22 were dissociated and determined to have 3-fold more potency than D4 EBs from VC to generate BL-CFCs in methylcellulose cultures. Top left, morphology of a representative BL-CFC (×100) by 10× objective lens. (B) D6 EBs from FC5 and FC22 were dissociated and determined to have 3-fold more potency than D6 EBs from VC to generate primitive erythroids in methylcellulose cultures in the presence of hEPO. Top left, morphology of 2 representative BFU-E colonies (×100) by 10× objective lens. (C) D9 EBs from VC, FC5, and FC22 were dissociated and assayed for multilineage hematopoietic colony formation. The Lycat transgene in FC5 and FC22 increased the formation of CFU-Es (4-fold), BFU-Es (5-fold), CFU-Gs (2-fold), CFU-Ms (2-fold), CFU-GMs (2-fold) and CFU-GEMMs (2-fold). (D) D11 EBs from FC5 and FC22 had better potential than those from VC to form long endothelial sprouting. (i) The EBs producing much long endothelial sprouting as shown in panel Fi (×100) by 10× objective lens. (ii) The EBs producing only some endothelial sprouting as shown in panel Fii (×40) by 4× objective lens. (iii) The EBs producing no endothelial sprouting as shown in panel Fiii (×100) by 10× objective lens. (E) Representative colonies of BFU-Es (×200), CFU-Es (×200), CFU-GMs (×200) by 20× objective lens, CFU-Gs (×40) by 4× objective lens, CFU-Ms (×100) by 10× objective lens, and CFU-GEMMs (×40) by 4× objective lens. Live images were taken under a Nikon ECLIPSE TE2000-U fluorescence microscope (Nikon, Yokohama, Japan). Images were acquired with a SPOT charge-coupled device camera (Diagnostic Instruments, Sterling Heights, MI), imported with SPOT software version 4.6.4.4, and prepared with Adobe Photoshop 7.0 (Adobe, San Jose, CA). Results are presented as the mean plus or minus the standard error of the mean (SEM). Statistical significance in Figure 4A,B was determined using an unpaired Student t test. P values were calculated from quantitative effects in 3 independent experiments, compared with those of FC5 and FC22 with those of VC, respectively. (A-D) Error bars represent standard deviations.

Overexpression of the Lycat transgene increased the formation of BL-CFCs and hematopoietic and endothelial cells by in vitro function assay. (A) D4 EBs from FC5 and FC22 were dissociated and determined to have 3-fold more potency than D4 EBs from VC to generate BL-CFCs in methylcellulose cultures. Top left, morphology of a representative BL-CFC (×100) by 10× objective lens. (B) D6 EBs from FC5 and FC22 were dissociated and determined to have 3-fold more potency than D6 EBs from VC to generate primitive erythroids in methylcellulose cultures in the presence of hEPO. Top left, morphology of 2 representative BFU-E colonies (×100) by 10× objective lens. (C) D9 EBs from VC, FC5, and FC22 were dissociated and assayed for multilineage hematopoietic colony formation. The Lycat transgene in FC5 and FC22 increased the formation of CFU-Es (4-fold), BFU-Es (5-fold), CFU-Gs (2-fold), CFU-Ms (2-fold), CFU-GMs (2-fold) and CFU-GEMMs (2-fold). (D) D11 EBs from FC5 and FC22 had better potential than those from VC to form long endothelial sprouting. (i) The EBs producing much long endothelial sprouting as shown in panel Fi (×100) by 10× objective lens. (ii) The EBs producing only some endothelial sprouting as shown in panel Fii (×40) by 4× objective lens. (iii) The EBs producing no endothelial sprouting as shown in panel Fiii (×100) by 10× objective lens. (E) Representative colonies of BFU-Es (×200), CFU-Es (×200), CFU-GMs (×200) by 20× objective lens, CFU-Gs (×40) by 4× objective lens, CFU-Ms (×100) by 10× objective lens, and CFU-GEMMs (×40) by 4× objective lens. Live images were taken under a Nikon ECLIPSE TE2000-U fluorescence microscope (Nikon, Yokohama, Japan). Images were acquired with a SPOT charge-coupled device camera (Diagnostic Instruments, Sterling Heights, MI), imported with SPOT software version 4.6.4.4, and prepared with Adobe Photoshop 7.0 (Adobe, San Jose, CA). Results are presented as the mean plus or minus the standard error of the mean (SEM). Statistical significance in Figure 4A,B was determined using an unpaired Student t test. P values were calculated from quantitative effects in 3 independent experiments, compared with those of FC5 and FC22 with those of VC, respectively. (A-D) Error bars represent standard deviations.

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