Fig 2: Differentiation of hiPSCs into chondrogenic cells. (A) Schematic representation of the refined 14-day differentiation protocol. Different factors are added to the media during the three stages. Cells are passaged at the end of stage 1 and 2 at day 4 and 8, respectively. (B) Expression analysis of pluripotent and chondrogenic genes show a loss of pluripotency and an increase in expression of chondrogenic genes after the chondrogenic differentiation. Expression of POU5F1 was very high in hiPSC Line #3 EV relative to all other hiPSC lines, resulting in a shift of the mean expression value for this gene used to generated the heat map. However, all hiPSC lines showed downregulation of POU5F1 upon differentiation. (C) ARSB gene expression remained high after chondrogenic differentiation (n = 3). (D) Biochemical analysis showed a rescue of ARSB enzyme activity in chondrogenic cells (n = 3). GE: Gene edited with ARSB cDNA, EV: Gene edited with empty vector control. Data are expressed as means ± SE.

Fig 3: The 2logFC of genes involved in (A) Bone and Cartilage development, (B) WNT signaling, (C) Cell growth and apoptosis, (D) Metabolic processes, (E) Ion transport and regulation. GE: Gene edited with ARSB cDNA, EV: Gene edited with empty vector control.

Fig 4: Normalization of inclusions after gene correction. (A) periodic acid–Schiff (PAS) staining of day 14 differentiated chondrogenic cells showed the accumulation of inclusions in EV cells, indicated by the increase of purple signal. The insert shows a higher magnification of selected cells to visualize the inclusions. (B) Quantification of PAS positive cells showed the normalization of inclusions after gene correction. Data represent means ± SD of 4 cytospins of independent cultures of chondrogenic cells. GE: Gene edited with ARSB cDNA, EV: Gene edited with empty vector control. Data are expressed as means ± SE. Statistical tests were performed with two-way ANOVA and Šídák multiple comparisons correction, *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig 5: Gene editing in hiPSCs. (A) The gene editing strategy was designed to express ARSB from the AAVS1 locus. PAM sequence indicated in purple, gRNA target indicated in blue. (B) The donor construct generated for insertion of the ARSB cDNA in the AAVS1 safe harbor by CRISPR/Cas9-mediated gene editing. The neomycin cassette enables G418 selection of targeted colonies. (C) The empty vector construct used to generate isogenic controls. (D) Strategy for the PCR based genotyping of targeted colonies, primer set 1 spans the insertion site and only gives a product in the absence of targeting, primer set 2 amplifies a product only in the presence of the construct at the target site. (E) Typical genotyping result of picked colonies. With primer set 2, 12/15 colonies show successful mono-allelic or bi-allelic targeting. With primer set 1, 6/15 lack a PCR product, indicating a loss of the endogenous sequence. In combination colonies 3, 12, 15, 16, 21 and 22 show bi-allelic insertion of the construct. DNA Ladder: 1 Kb Plus DNA Ladder (Invitrogen) (F) Quantification of mono-allelic, bi-allelic and unsuccessfully targeted colonies. (G) ARSB mRNA expression in the selected bi-allelic targeted clones, determined using RNAseq. GE: Gene edited with ARSB cDNA, EV: Gene edited with empty vector control.