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. 2022 Dec 6:10:949063.
doi: 10.3389/fbioe.2022.949063. eCollection 2022.

Modeling cartilage pathology in mucopolysaccharidosis VI using iPSCs reveals early dysregulation of chondrogenic and metabolic gene expression

M Broeders  1   2   3 , Jgj van Rooij  4 , E Oussoren  1   3 , Tjm van Gestel  1   2   3 , Ca Smith  5 , Sj Kimber  5 , Rm Verdijk  6 , Maem Wagenmakers  3   4 , Jmp van den Hout  1   3 , At van der Ploeg  1   3 , R Narcisi  7 , Wwmp Pijnappel  1   2   3
Affiliations

Modeling cartilage pathology in mucopolysaccharidosis VI using iPSCs reveals early dysregulation of chondrogenic and metabolic gene expression

M Broeders et al. Front Bioeng Biotechnol. .

Abstract

Mucopolysaccharidosis type VI (MPS VI) is a metabolic disorder caused by disease-associated variants in the Arylsulfatase B (ARSB) gene, resulting in ARSB enzyme deficiency, lysosomal glycosaminoglycan accumulation, and cartilage and bone pathology. The molecular response to MPS VI that results in cartilage pathology in human patients is largely unknown. Here, we generated a disease model to study the early stages of cartilage pathology in MPS VI. We generated iPSCs from four patients and isogenic controls by inserting the ARSB cDNA in the AAVS1 safe harbor locus using CRISPR/Cas9. Using an optimized chondrogenic differentiation protocol, we found Periodic acid-Schiff positive inclusions in hiPSC-derived chondrogenic cells with MPS VI. Genome-wide mRNA expression analysis showed that hiPSC-derived chondrogenic cells with MPS VI downregulated expression of genes involved in TGF-β/BMP signalling, and upregulated expression of inhibitors of the Wnt/β-catenin signalling pathway. Expression of genes involved in apoptosis and growth was upregulated, while expression of genes involved in glycosaminoglycan metabolism was dysregulated in hiPSC-derived chondrogenic cells with MPS VI. These results suggest that human ARSB deficiency in MPS VI causes changes in the transcriptional program underlying the early stages of chondrogenic differentiation and metabolism.

Keywords: cartilage; disease modeling; induced pluripotent stem cells; lysosomal storage disease; mucopolysaccharidosis type VI.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Cartilage pathology in a 25 year old diseased MPS VI patient. (A) X-ray and (B) CT scan of the right hip. Macroscopic pictures from autopsy of the (C) right acetabulum and (D) femoral head. (E) X-ray of the lumbar vertebrae. (F) A macroscopic picture from autopsy of one vertebra. (G) HE stain of cartilage from this vertebra (Magnification ×100), (H) details from the inset in G (Magnification ×400). (I,J) Electron microscopy analysis showing examples of chondroyctes with different types of vacuoles and in addition lipid storage (indicated with *). (K) Characteristics of included patients.
FIGURE 2
FIGURE 2
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.
FIGURE 3
FIGURE 3
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.
FIGURE 4
FIGURE 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.
FIGURE 5
FIGURE 5
Enrichment of gene ontology (GO) terms. (A) 25 top dysregulated GO biological processes in EV and GE cells. (B) Significantly dysregulated GO molecular functions. (C) Significantly dysregulated GO cellular components. Upregulated processes in GE cells are indicated in green. Upregulated processes in EV cells are indicated in red. GE: Gene edited with ARSB cDNA, EV: Gene edited with empty vector control.
FIGURE 6
FIGURE 6
Expression analysis of differentially expressed genes between GE and EV. Expression analysis of differentially expressed 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.
FIGURE 7
FIGURE 7
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.
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