Journal: Scientific Reports
Article Title: Direct conversion of fibroblasts into urothelial cells that may be recruited to regenerating mucosa of injured urinary bladder
doi: 10.1038/s41598-019-50388-6
Figure Lengend Snippet: Exploration of a procedure to induce urothelial cell-like phenotypes in aHDFs. ( a ) Cell conversion procedures. ( b ) aHDFs were seeded in laminin-coated 12-well plates. On the next day (day 0), retroviral vectors containing FOXA1 (F), TP63 (T), MYCL (L) and KLF4 (K) genes were transduced into the cells as indicated by “+”. Cells were cultured in Standard Medium (days 1 to 3) and CnT-Prime (days 4 to 21). RNA was extracted from the cells and subjected to real-time RT-PCR to evaluate mRNA levels for the UPK1b and UPK2 genes. * P < 0.05 and *** P < 0.001 vs. non-transduced aHDFs. # P < 0.05 and ### P < 0.001 vs. HUCs. ( c , d ) aHDFs were seeded in non-coated (N) culture plates, or culture plates coated with collagen (C), poly-L-lysine (P) or laminin (L). After transduction with FTLK retroviral vectors (day 0), cells were cultured as in ( a ), and confocal microscopic imaging ( c ) and real-time RT-PCR analysis ( d ) were performed on day 21. * P < 0.05 and *** P < 0.001 vs. N. ## P < 0.01 and ### P < 0.001 vs. HUCs. ( e, f ) aHDFs were seeded in laminin-coated plates, infected with or without FTLK retroviral vectors, and cultured for days 1 to 3 in Standard Medium. Cells were then cultured in Standard Medium (S) or CnT-Prime (C) until day 21, when confocal microscopic imaging ( e ) and real-time RT-PCR analysis ( f ) were performed. *** P < 0.001 vs. non-transduced aHDFs cultured in Standard Medium. ## P < 0.01 and ### P < 0.001 vs. HUCs. Values are mean +/− SD (n = 3).
Article Snippet: Full length cDNA clones for human FOXA1, IRF1, TP63 and SHH genes were purchased from DNAFORM library, and coding sequences were amplified by RT-PCR with KOD-Plus-Neo DNA polymerase (Toyobo) and primers specific for FOXA1 (5′-CAGCAGTGTGGTGGTACGGGATGTTAGGAACTGTGAAGATG-3′ and 5′-ACCGGCGCTCAGCTGGCTAGGAAGTGTTTAGGACGGG-3′), IRF1 (5′-CAGTGTGGTGGTACGGGATGCCCATCACTCGGATGCGC-3′ and 5′-ACCGGCGCTCAGCTGGACCGGCGCTCAGCTGG-3′), TP63 (5′-CAGTGTGGTGGTACGGGATGAATTTTGAAACTTCACGG-3′ and 5′-ACCGGCGCTCAGCTGGCTATCACTCCCCCTCCTCTTT-3′) and SHH (5′-CAGTGTGGTGGTACGGGATGCTGCTGCTGGCGAGATGT-3′ and 5′-ACCGGC GCTCAGCTGGCTAGCTGGACTTGACCGCCAT-3′).
Techniques: Cell Culture, Quantitative RT-PCR, Transduction, Imaging, Infection
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