Arylsulfatase A Overexpressing Human iPSC-derived Neural Cells Reduce CNS Sulfatide Storage in a Mouse Model of Metachromatic Leukodystrophy

Abstract

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder resulting from a functional deficiency of arylsulfatase A (ARSA), an enzyme that catalyzes desulfation of 3-O-sulfogalactosylceramide (sulfatide). Lack of active ARSA leads to the accumulation of sulfatide in oligodendrocytes, Schwann cells and some neurons and triggers progressive demyelination, the neuropathological hallmark of MLD. Several therapeutic approaches have been explored, including enzyme replacement, autologous hematopoietic stem cell-based gene therapy, intracerebral gene therapy or cell-based gene delivery into the central nervous system (CNS). However, long-term treatment of the blood-brain-barrier protected CNS remains challenging. Here we used MLD patient-derived induced pluripotent stem cells (iPSCs) to generate long-term self-renewing neuroepithelial stem cells and astroglial progenitors for cell-based ARSA replacement. Following transplantation of ARSA-overexpressing precursors into ARSA-deficient mice we observed a significant reduction of sulfatide storage up to a distance of 300 µm from grafted cells. Our data indicate that neural precursors generated via reprogramming from MLD patients can be engineered to ameliorate sulfatide accumulation and may thus serve as autologous cell-based vehicle for continuous ARSA supply in MLD-affected brain tissue.

Glial progenitor cell-based treatment of the childhood leukodystrophies

“…Intracerebral cell grafts of murine neural precursors overexpressing arylsulfatase A resulted in sulfatide reduction and modest improvement of a murine ARSA-deficient model (Givogri et al, 2006; Kawabata et al, 2006). Similar results were subsequently obtained using neural progenitors derived from ESCs and iPSCs (Doerr et al, 2015; Klein et al, 2006). Although the donor cells were capable of in vivo oligodendroglial and astroglial differentiation, they did so at low frequency, possibly accounting for the relatively modest treatment-associated suppression of demyelination with disease progression.…”

Whole-brain 3D mapping of human neural transplant innervation

“…Lt-NES cells represent a stable neural stem cell population, which can be extensively propagated while maintaining a stable neurogenic potential yielding fully functional neurons both in vitro and following transplantation into the rodent brain1617. Due to their robust proliferation and differentiation potential, lt-NES cells lend themselves particularly well to genetic modification and have been successfully used for lineage tracing, disease modelling and cell-mediated gene transfer181920. They exhibit a posterior phenotype with an anterior hindbrain identity, and mostly give rise to GABAergic interneurons as well as glutamatergic neurons, a differentiation pattern maintained after transplantation into neonatal and adult hosts1621.…”

“…To facilitate fast enrichment of lentivirally transduced cells, the expression cassette was transferred to the pLVXTP backbone which contains a puromycin resistance gene (Clonetech) by opening it using XhoI and MluI digestion and ligation with the PCR amplified (forward primer 5′-GCAGCAGTCGACACTGCAGAGGGCCCTGC-3′; reverse primer 5′-GCAGCAACGCGTTTGTCTGAGGTGTGACTGGAAAACC-3′) and SalI / MluI digested tracing cassette. Lentiviral particles were generated and concentrated using the polyethylene glycol method as described previously2043. Briefly, 293FT cells (Life Technologies) were transfected with the lentiviral plasmids pLVX-SynHTB or pLentiWE-Ef1α-mRFP1 and the packaging plasmid psPAX2 and the envelope plasmid pMD2.G.…”

Ehmt2/G9a controls placental vascular maturation by activating the Notch pathway

“…Our RNAseq data revealed that the G9a mutant labyrinth overexpresses secreted protein-encoding genes, including secreted modular calcium-binding protein 1 (Smoc1) and arylsulfatase family member 1 (Arsi). Smoc1 is expressed in endothelial cells and positively regulates cell proliferation (Awwad et al, 2015), and Arsi is secreted from proliferating long-term self-renewing neuroepithelial-like stem cells (Doerr et al, 2015). Thus, placental endothelial cells might limit trophoblast proliferation via G9a-mediated repression of secreted activators of cell proliferation.…”