Mucopolysaccharidosis type II (MPSII) is a rare X-linked lysosomal storage disorder caused by mutations in the iduronate-2-sulfatase (IDS) gene (IDS, Xq28). MPSII is characterized by skeletal deformities, hearing loss, airway obstruction, hepatosplenomegaly, cardiac valvular disease, and progressive neurological impairment. At the cellular level, IDS deficiency leads to lysosomal storage of glycosaminoglycans (GAGs), dominated by accumulation of dermatan and heparan sulfates. Human induced pluripotent stem cells (iPSC) represent an alternative system that complements the available MPSII murine model. Herein we report on the reprogramming of peripheral white blood cells from male and female MPSII patients into iPSC using a non-integrating protocol based on the Sendai virus vector system. We differentiated the iPSC lines into IDS deficient and GAG accumulating β-Tubulin III+ neurons, GFAP+ astrocytes, and CNPase+ oligodendrocytes. The lysosomal system in these cells displayed structural abnormalities reminiscent of those previously found in patient tissues and murine IDS deficient neuronal stem cells. Furthermore, quantitative determination of GAGs revealed a moderate increase in GAG levels in IDS deficient neurons and glia. We also tested the effects of recombinant IDS and found that the exogenous enzyme was internalized from the culture media and partially decreased the intracellular GAG levels in iPSC-derived neural cells; however, it failed to completely prevent accumulation of GAGs. In summary, we demonstrate that this human iPSC based model expresses the cellular and biochemical features of MPSII, and thus represents a useful experimental tool for further pathogenesis studies as well as therapy development and testing.

• MeSH
• Astrocytes enzymology   pathology   MeSH
• Cell Lineage MeSH
• Phenotype MeSH
• Glycosaminoglycans metabolism   MeSH
• Iduronate Sulfatase genetics   metabolism   MeSH
• Induced Pluripotent Stem Cells enzymology   pathology   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Lysosomes enzymology   pathology   MeSH
• Mucopolysaccharidosis II enzymology   genetics   pathology   MeSH
• Neural Stem Cells enzymology   pathology   MeSH
• Neurogenesis * MeSH
• Neuroglia enzymology   pathology   MeSH
• Neurons enzymology   pathology   MeSH
• Oligodendroglia enzymology   pathology   MeSH
• Oligodendrocyte Precursor Cells enzymology   pathology   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glycosaminoglycans MeSH
• Iduronate Sulfatase MeSH

We examined the differentiation potential of murine neural stem cells (NSCs) grown in vitro and transplanted into intact and irradiated recipients. NSCs were isolated from neonatal Balb/c mice using the neurosphere assay. On in vitro differentiation assays, NSCs produced beta-III tubulin(+) neurons, glial fibrillary acidic protein (GFAP(+)) astrocytes, and O4(+) oligodendrocytes. After neural grafting to histocompatible adult mice, NSCs gave rise to neuronal and glial cells. When cells were transplanted in the form of solid neurospheres, they reached terminal differentiation and spatial arrangements that mimicked the three-dimensional organization of nervous tissue. To create conditions that would allow us to assess the potential for generation of nonneural cells, NSCs were intravenously injected into irradiated mice. Transplantation of NSCs stimulated hematopoiesis because the number of colony-forming units of granulocyte-monocyte lineage (CFU-GM) colonies isolated from the spleen and bone marrow of transplanted mice was greater than that from irradiated, nontransplanted animals. Moreover, transplanted cells tagged with beta-galactosidase were identified in the thymus of animals grafted with labelled NSCs. NSCs harvested from the neurosphere assay produced viable and transplantable cells. In vitro differentiation assays and neural grafting confirmed the multipotency of NSCs and their commitment to generate neuronal and glial cells. Following intravenous injection of NSCs, the transplanted cells colonized hematopoietic and lymphatic organs, facilitating hematopoiesis in irradiated animals.

• MeSH
• Astrocytes cytology   MeSH
• Cell Differentiation MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Neurons cytology   MeSH
• Animals, Newborn MeSH
• Oligodendroglia cytology   MeSH
• Transplantation, Isogeneic MeSH
• Stem Cell Transplantation * MeSH
• Brain Tissue Transplantation methods   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential that can give rise to neurons and glia in vivo and in vitro. The aim of this study was to transplant NSCs as whole neurospheres into intact brain and assess the fate and phenotype of their progeny generated in vivo. We isolated NSCs from E14 foetal rat forebrains and cultured them in basic fibroblast and epidermal growth factor-supplemented serum-free medium in the form of neurospheres in vitro. Neurospheres were transplanted into the intact brains of 2 Wistar rats and after a period of 3 weeks, grafted brains were examined immunohistochemically. Neurospheres formed solid grafts that were found in the lateral ventricle and in the velum interpositum under the hippocampus. The majority of cells in the transplanted tissue were identified as beta-III-tubulin(+), NeuN(+), PanNF(+) and synaptophysin(+) neurons and were accumulated throughout the graft centre. GFAP(+) astrocytes were scattered throughout the entire graft and astrocyte processes delimited the outer and perivascular surfaces. A great number of NG2(+) oligodendrocyte precursors was detected. Nestin(+) endothelial cells were found to line capillaries growing in the transplant. These data indicate that nestin(+) NSCs prevailing in neurospheres differentiate following transplantation into nestin(-) neuronal and glial cells which confirms the multipotency of NSCs. Three weeks posttransplantation neuronal and astrocyte cells reached terminal differentiation (formation of synaptic vesicles and superficial and perivascular limiting membranes) while elements of oligodendroglial cell lineage remained immature. Grafting stem cells as non-dissociated neurospheres provide cells with favourable conditions which facilitate cell survival, proliferation and differentiation. However, in the intact brain, grafted neurosphere cells were not found to integrate with the brain parenchyma and formed a compact structure demarcated from its surroundings.

• MeSH
• Astrocytes chemistry   cytology   MeSH
• Cell Differentiation MeSH
• Immunohistochemistry MeSH
• Rats MeSH
• Neurons chemistry   cytology   MeSH
• Oligodendroglia chemistry   cytology   MeSH
• Rats, Wistar MeSH
• Stem Cell Transplantation * MeSH
• Brain Tissue Transplantation * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH