Using human induced pluripotent stem cells to reveal the molecular pathology involved in osteogenesis imperfecta resulting from two type I collagen C-propeptide mutations

Abstract

While the genes underlying the genetic brittle bone disease, osteogenesis imperfecta (OI), are well established, the precise pathological mechanisms are unclear. Recent studies have suggested that ER-stress resulting from mutations in COL1A1 or COL1A2 that cause protein misfolding may be a significant contributor to the pathology. If this proves correct, then the ER-stress pathways may offer new drug targets to modify disease severity. However, to understand these pathological pathways in the proper cellular context, we need to study patient bone cells (osteoblasts). Since these are unavailable, we have developed two mutant human iPSC lines with severe OI collagen I (COL1A1) C-propeptide misfolding mutations and corresponding isogenic controls. To introduce the OI26 mutation (c.3969_3970insT) in exon 49 of the COL1A1 gene into an isogenic control iPS cell line (OI26-IC), we used a CRISPR-Cas9 system where Cas9 is fused to a human geminin peptide (SpCas9-Gem) to facilitate transient Cas9 expression and reduce NHEJ-mediated indels. Simultaneous conventional CRISPR-Cas9 and reprogramming were applied to generate OI64 mutant (c.3936 G>T) and isogenic control iPS lines from OI64 patient fibroblasts. Sequencing confirmed targeting of one allele without mutations in the other allele, and SNP arrays demonstrated chromosomal integrity. The colonies of targeted iPSCs have normal stem cell morphology. Immunocytochemistry and flow cytometry confirmed the expression of pluripotency markers. The ability of these iPSC lines to differentiate into three main germ layers was confirmed. Osteogenic differentiation was achieved via initial differentiation via the paraxial mesoderm-sclerotome pathway followed by air-liquid interface-based culture in osteogenic medium for three weeks. These cultures express sentinel osteoblast markers, including RUNX2, SP7, BGLAP, ALPL, COL1A1, and produce a mineralized matrix toward the end of differentiation. Moreover, the osteoblast-like cells in this system demonstrated high similarities with cultured human osteoblast in the expression of several osteoblast-specific markers. Finally, we dissected the intra-and extracellular effects of OI26 and OI64 mutations using mutant and isogenic iPS cell-derived osteoblast-like cells. Compared to OI26 and OI64 isogenic control cells, mutant osteoblast-like cells had reduced extracellular matrix mineralization, reduced extracellular type I collagen protein, and lower expression of ALPL, all hallmarks of osteogenesis imperfecta. A marked increase in the ratio of intracellular to extracellular type I collagen was observed in OI osteoblast-like cells reflecting the increased intracellular retention of the mutant collagen and/or the increased extracellular collagen degradation. Despite this intracellular accumulation of the misfolded OI collagens, there was no evidence of the activation of a canonical cellular unfolded protein. While this raises important questions about the inevitability of a UPR due to collagen misfolding and the role of the UPR in OI pathology, it is important to acknowledge that the lack of the UPR could also be due to limitations in our experimental system. As type I collagen expression is lower in our OI models compared to cultured primary human osteoblasts, the level of mutant chains may be inadequate to trigger ER stress pathways in osteoblasts, whose translational and folding machinery is adapted to high levels of collagen synthesis. Thus, the osteogenic differentiation protocol will require to be optimized to generate more robust iPSC-derived OI bone models. Illumination of the more detailed molecular pathology of OI resulting from two C-propeptide type I collagen mutations will allow us to screen potential disease-modifying drugs.