Impaired neural differentiation of MPS IIIA patient induced pluripotent stem cell-derived neural progenitor cells

Abstract

Mucopolysaccharidosis type IIIA (MPS IIIA) is characterised by a progressive neurological decline leading to early death. It is caused by bi-allelic loss-of-function mutations in SGSH encoding sulphamidase, a lysosomal enzyme required for heparan sulphate glycosaminoglycan (HS GAG) degradation, that results in the progressive build-up of HS GAGs in multiple tissues most notably the central nervous system (CNS). Skin fibroblasts from two MPS IIIA patients who presented with an intermediate and a severe clinical phenotype, respectively, were reprogrammed into induced pluripotent stem cells (iPSCs). The intermediate MPS IIIA iPSCs were then differentiated into neural progenitor cells (NPCs) and subsequently neurons. The patient derived fibroblasts, iPSCs, NPCs and neurons all displayed hallmark biochemical characteristics of MPS IIIA including reduced sulphamidase activity and increased accumulation of an MPS IIIA HS GAG biomarker. Proliferation of MPS IIIA iPSC-derived NPCs was reduced compared to control, but could be partially rescued by reintroducing functional sulphamidase enzyme, or by doubling the concentration of the mitogen fibroblast growth factor 2 (FGF2). Whilst both control heparin, and MPS IIIA HS GAGs had a similar binding affinity for FGF2, only the latter inhibited FGF signalling, suggesting accumulated MPS IIIA HS GAGs disrupt the FGF2:FGF2 receptor:HS signalling complex. Neuronal differentiation of MPS IIIA iPSC-derived NPCs was associated with a reduction in the expression of neuronal cell marker genes βIII-TUBULIN, NF-H and NSE, revealing reduced neurogenesis compared to control. A similar result was achieved by adding MPS IIIA HS GAGs to the culture medium during neuronal differentiation of control iPSC-derived NPCs. This study demonstrates the generation of MPS IIIA iPSCs, and NPCs, the latter of which display reduced proliferation and neurogenic capacity. Reduced NPC proliferation can be explained by a model in which soluble MPS IIIA HS GAGs compete with cell surface HS for FGF2 binding. The mechanism driving reduced neurogenesis remains to be determined but appears downstream of MPS IIIA HS GAG accumulation.

Neurological Disease Modeling Using Pluripotent and Multipotent Stem Cells: A Key Step Towards Understanding and Treating Mucopolysaccharidoses

“…So, the same parameters, which were initially assessed in fibroblasts, were also analysed after the neurodifferentiation protocol. Not surprisingly, the MPS IIIA cells exhibited higher levels of HS, a consequence of lower enzyme activity compared to the controls, further validating the disease modeling value of this kind of cells [123].…”

“…Still on Sanfilippo syndrome, for the most frequent type, MPS IIIA, a comprehensive study was carried out by R. J. Lehmann et al in 2021 [123], to investigate the ability of fibroblasts-derived iPSCs to differentiate into a neuronal cell line and discover intrinsic mechanisms of the disease. After properly characterizing the pluripotency phase, the authors performed a neurodifferentiation protocol.…”

Neurological Disease Modeling Using Pluripotent and Multipotent Stem Cells: A Key Step towards Understanding and Treating Mucopolysaccharidoses

“…So, the same parameters, which were initially assessed in fibroblasts, were also analyzed after the neurodifferentiation protocol. Not surprisingly, the MPS IIIA cells exhibited higher levels of HS, a consequence of lower enzyme activity compared to the controls, further validating the disease modeling value of this kind of cells [ 63 ].…”

“…Still in Sanfilippo syndrome, for its most frequent type MPS IIIA, a comprehensive study was carried out by R. J. Lehmann et al in 2021 [ 63 ], to investigate the ability of fibroblasts-derived iPSCs to differentiate into a neuronal cell line and discover intrinsic mechanisms of the disease. After properly characterizing the pluripotent phase, the authors performed a neurodifferentiation protocol.…”

A novel CRISPR/Cas9-based iduronate-2-sulfatase (IDS) knockout human neuronal cell line reveals earliest pathological changes

“…One of the major findings of this investigation was that full IDS loss of function negatively impact on the neuronal differentiation, particularly into dopaminergic neurons. Analyses of neuronal differentiation have been previously made in MPS II and MPS IIIA mice 26 , 27 . Notably, De Risi and colleagues, using primary cultures from mesencephalic neurons of MPS IIIA mice showed progressively decreased TH + -cell density, which Authors pointed as a result of increased cell death 25 .…”