Pegip

MSCs were transfected with lentiviral particles containing the Wnt5a gene and empty vectors. The lentiviral system consisted of three vectors: the packaging plasmids psPAX2 (Addgene, USA), the envelope plasmids pMD2.G (Addgene, USA), and the expression vector pEGIP (Addgene, USA). To generate the Wnt5a expression vector, the cDNA encoding mouse Wnt5a (NM_009524.4) was amplified by RT-PCR using specific primers, digested with restriction enzymes, and subcloned into the expression vector plasmid pEGIP. The recombinant lentivirus was produced by co-transfecting psPAX2, pMD2.G, and the expression vector containing Wnt5a or empty vector into HEK 293 T cells using calcium phosphate transfection. The supernatant containing the virus was collected 48 h after transfection, centrifuged at 1,500 rpm for 5 min to remove debris, and then filtered through a 0.45 μm pore-size filter. For lentiviral transduction, MSCs were incubated with lentiviral solution and 10 μg/mL polybrene (Sigma-Aldrich, USA, H9268) in complete medium for 9 h. Subsequently, the culture medium was changed, and MSCs were selected in complete medium containing 1.6 μg/mL of puromycin (Genechem, China) for 72 h. Finally, the MSC^Wnt5a and MSC^Vector cell lines were obtained and expanded for subsequent experiments.

A 2nd-generation lentiviral system was used, and non-replicative lentiviral particles were produced by transient transfection of HEK293FT cells with psPAX2 (Addgene #12260), pMD2.G (Addgene #12259), and expression vector, at a 2:1:3 ratio, as previously described [9 (link)]. Three expression vectors were constructed: pFOXA2IP, pFHIG, and pCWFOXA2. Foxa2 was amplified from pGCDNsam_Foxa2 (Addgene #33004) with the primers mmFoxa2_BamHI_F and mmFoxa2_BsrGI_R and subcloned between BamHI and BsrGI sites in pEGIP (Addgene #26777). For dox-inducible expression studies, Foxa2 was amplified with mmFoxa2_NheI_F and mmFoxa2_BamHI_R primers and subcloned into the pCW-cas9 (Addgene # 50661) Tet-on expression vector in the NheI/BamHI flanked region. Hnf4a was amplified from pGCDNsam_HNF4α (Addgene #33002) with primers mmHnf4a_XbaI_F and mmHnf4a_BamHI_R and subcloned into pFUWOSKM (Addgene #20328) vector in the XbaI/BamHI flanked region, while IRES-GFP sequence was subcloned in frame, within BamHI/AscI restriction sites, after amplification from MSCVPIG (Addgene #18751) using IRES-GFP_BamHI_F and IRES-GFP_AscI_R primers. Primer sequences are listed in Table S1.