Fig. 1: Generation of an isogenic iPSC panel for disease modelling of DS.
Schematic overview depicting the strategy used to generate isogenic iPSC lines by combining XIST-induced chromosome silencing and genome/chromosome-editing technologies. a Cord blood mononuclear cells were derived from a male patient with DS. Then, a DS-specific iPSC line (Tri21) was directly established using Sendai virus. A corrected-disomy cell line (cDi21) was generated by eliminating a single copy of chromosome 21 from a Tri21 iPSC line. b A 4-Mb region corresponding to a ‘Down syndrome critical region’ was selectively deleted only from the paternal chromosome 21 in Tri21 iPSCs to generate Partial-Tri21 iPSC. NPCs differentiated from Partial-Tri21 iPSCs were transfected with piggyBac (PB) transposon vectors containing human PIGP and/or DSCR3 cDNA under the regulation of the Tet-inducible system. NPCs were differentiated into APCs for further analysis. c Dox-inducible human XIST cDNA was inserted into one copy of chromosome 21 in Tri21 iPSCs (XIST-Tri21 iPSC) for chromosome silencing. rtTA was additionally introduced into NPCs differentiated from XIST-Tri21 iPSCs, using a PB vector. Transcriptional and phenotypic analysis was conducted in Dox-untreated (D−) and Dox-treated (D+) cells, and in NPCs or APCs at 3 weeks after Dox removal (Dremov). The location of the XIST transgene is indicated by the red rectangle. The black and striped chromosomes indicate a silenced and a reverted chromosome 21, respectively. d The DYRK1A gene was targeted in one or two chromosomes in the XIST-Tri21 iPSC line to generate DY+/+/m- and DY+/m/m-XIST-Tri21 iPSCs, respectively. Three types of genome-edited cell lines were generated in each single- or double-targeted clone, based on the parental origin of the three chromosomes. Black stars indicate the DYRK1A target sites. Two maternal copies and one paternal copy of chromosome 21 are indicated as M1/M2 and P, respectively. e DYRK1A-targeted iPSC lines were differentiated to APCs and subjected to phenotypic analysis. Light yellow panels and light green panels indicate iPSC lines and NPC/APC lines, respectively.
