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Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model
Published on: September 6, 2014
In vitro characterization of genetically modified embryonic stem cells as a therapy for murine mucopolysaccharidosis
Adeline A Lau1, Kim M Hemsley, Adrian Meedeniya
1Lysosomal Diseases Research Unit, Department of Genetic Medicine, Women's and Children's Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia. adelinelau@hotmail.com
Abstract:
The mucopolysaccharidoses (MPS) are lysosomal storage disorders resulting from the impaired catabolism of glycosaminoglycans (GAG). MPS type IIIA patients have dysfunctional sulfamidase enzyme leading to lysosomal storage of the GAG heparan sulfate, severe neurological symptoms including regression in learning, behavioural abnormalities, and premature death. We have engineered mouse D3 embryonic stem (ES) cells to over-express recombinant human sulfamidase. Human sulfamidase was correctly folded and secreted 2h post-labelling as determined by immunoprecipitation and SDS-PAGE analysis of transfected ES cells. Secreted human sulfamidase present in conditioned ES cell media was able to be taken up via mannose-6-phosphate-mediated endocytosis and restored sulfamidase enzyme activity in human MPS IIIA fibroblast cell lines. ES cells underwent directed differentiation to neural precursor populations and were capable of sustained human sulfamidase over-expression at all stages. Additionally, transfected and control cells were proliferative (Ki67+) and expressed several neural markers (nestin, MAP-2, and NF160) as determined by immunofluorescence. These findings suggest the possibility of ES cell-based therapy for the treatment of neurological pathology of MPS IIIA.
Insights
Engineered mouse stem cells to produce a functional enzyme deficient in Mucopolysaccharidoses type IIIA (MPS IIIA). These cells show potential for treating MPS IIIA neurological symptoms.
Area of Science:
- Biochemistry
- Genetics
- Stem Cell Biology
Background:
- Mucopolysaccharidoses (MPS) are lysosomal storage disorders caused by impaired glycosaminoglycan (GAG) breakdown.
- MPS type IIIA is characterized by a deficient sulfamidase enzyme, leading to heparan sulfate accumulation and severe neurological deficits.
Purpose of the Study:
- To engineer mouse embryonic stem (ES) cells for sustained expression of human sulfamidase.
- To evaluate the potential of these engineered ES cells for MPS type IIIA therapy.
Main Methods:
- Mouse D3 ES cells were genetically modified to over-express recombinant human sulfamidase.
- Sulfamidase expression, secretion, and cellular uptake were analyzed using immunoprecipitation, SDS-PAGE, and enzyme activity assays.
- Directed differentiation of ES cells into neural precursors was performed, and neural marker expression was assessed via immunofluorescence.
Main Results:
- Engineered ES cells successfully produced and secreted functional human sulfamidase.
- The secreted enzyme was taken up by MPS IIIA fibroblasts, restoring sulfamidase activity.
- Differentiated neural precursor cells maintained sulfamidase over-expression and expressed neural markers.
Conclusions:
- Engineered ES cells demonstrate the capacity for sustained sulfamidase production and secretion.
- These findings support the potential of ES cell-based therapy for addressing the neurological pathology in MPS type IIIA.
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