Induced pluripotent stem cells (IPSCS) for modelling mucopolysaccharidosis type I (Hurler syndrome).

Mucopolysaccharidosis type I (MPS-IH or Hurler syndrome) is a rare lysosomal storage disease caused by mutations in the IDUA gene, resulting in the deficiency of alpha-L-iduronidase (IDUA) enzyme activity with a consequent intracellular accumulation of glycosaminoglycans (GAGs). Among a broad spectrum of clinical manifestations, MPS-IH is characterized by a range of skeletal abnormalities known as dysostosis multiplex. To date, the skeletal pathogenesis of the MPSs has been assumed to be directly related to the progressive storage of GAGs. It is now clear that more complex cellular and molecular mechanisms underlie the patient clinical symptoms. Therefore, an appropriate humanized in vitro model is highly recommended to highlight these mechanisms. Compared to mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs) represent a useful tool to achieve this purpose, due to their high proliferation capability in culture and, mostly, to their ability to mimic development. Thus, they demonstrate great potential for investigating the osteogenic differentiation process. In this study, we generated MPS-IH patient-specific iPS cells (MPS-IH iPSCs) which maintained the genetic mutation in the IDUA gene and, as a consequence, reduced IDUA enzyme activity and GAGs intracellular accumulation. In order to assess if the osteogenic differentiation phenotype is already compromised in MPS-IH iPSCs cell, we focused on their bone differentiation capability. Thus, we developed an osteogenic differentiation protocol through the generation of mesenchymal stromal cells from iPSC (hereafter named MSCs-like cells). We designed a robust, multistep differentiation method to isolate MSCs-like cells, both from wild-type iPSCs (WT-iPSCs) and MPS-IH iPSCs. The process included: embryoid body (EB) formation, cell outgrowth from EBs, monolayer culture of sprouted cells from EBs, and a serial of passages in culture until they reached a fibroblast-like morphology and the full expression of mesenchymal surface markers. Firstly, we characterized WT and patient derived-MSCs-like cells in terms of morphology, phenotype, proliferation kinetics and differentiation capacity in mesodermal tissues. WT and patient derived-MSCs-like cells showed the capacity to differentiate in adipocytes, as confirmed by Oil Red O staining. Moreover, MSCs-like cells derived-chondrogenic pellets exhibited a spherical, compact morphology. Histological analysis revealed an initial chondrogenic differentiation, as confirmed by q-RT-PCR for key early chondrogenic markers, such as SOX9 and COLII. Subsequently, we developed an osteogenic differentiation protocol for the obtained MSC-like cells. In order to verify if the differentiation process was accomplished, we performed Alizarin Red staining and quantified the hydroxyapatite production by colorimetric detection at 405 nm both on WT and MPS-IH iPSCs-derived osteoblasts. At the same time, we examined the expression for key osteogenic markers, such as OPN, RUNX2 OTC, OTN, ALP and COL 1A2, through q-RT-PCR. Recently, our group isolated MSCs from bone marrow (BM-MSCs) of both healthy donors and MPS-IH patients, studying a possible involvement of MSCs in the skeletal abnormalities affecting Hurler patients. We previously observed the ability of WT, MPS-IH BM-MSCs and MSCs-derived osteoblasts to stimulate osteoclastogenesis in vitro by measuring the molecular levels of receptor activator of nuclear factor-Kb ligand (RANKL) and osteoprotegerin (OPG), two key partners of the system directly regulating osteoclast differentiation. MPS-IH MSCs and osteoblasts derived from MPS-IH MSCs, expressed a higher level of RANKL compared to HD-MSCs and osteoblasts. OPG level, instead, was similar. In the present study, the osteogenic differentiation protocol developed allowed us to assess if this altered phenotype is already evident in both MSCs-like cells MSCs-like derived osteoblasts, by evaluating the OPG and RANKL expression levels.