Combination of low calcium with Y-27632 rock inhibitor increases the proliferative capacity, expansion potential and lifespan of primary human keratinocytes while retaining their capacity to differentiate into stratified epidermis in a 3D skin model.

Human keratinocytes are difficult to isolate and have a limited lifespan. Traditionally, immortalised keratinocyte cell lines are used in vitro due to their ability to bypass senescence and survive indefinitely. However these cells do not fully retain their ability to differentiate in vitro and they are unable to form a normal stratum corneum in organotypic culture. Here we aimed to generate a pool of phenotypically similar keratinocytes from human donors that could be used in monolayer culture, without a fibroblast feeder layer, and in 3D human skin equivalent models. Primary human neonatal epidermal keratinocytes (HEKn) were cultured in low calcium, (0.07 mM) media, +/-10 µM Y-27632 ROCK inhibitor (HEKn-CaY). mRNA and protein was extracted and expression of differentiation markers Keratin 14 (K14), Keratin 10 (K10) and Involucrin (Inv) assessed by qRT-PCR and Western blotting. The differentiation potential of the HEKn-CaY cultures was assessed by increasing calcium levels and removing the Y-27632 for 72 hrs prior to assessment of K14, K10 and Inv. The ability of the HEKn-CaY, to form a stratified epithelium was assessed using a human skin equivalent (HSE) model in the absence of Y-27632. Increased proliferative capacity, expansion potential and lifespan of HEKn was observed with the combination of low calcium and 10 µM ROCK inhibitor Y-27632. The removal of Y-27632 and the addition of high calcium to induce differentiation allowed the cells to behave as primary keratinocytes even after extended serial passaging. Prolonged lifespan HEK-CaYs were capable of forming an organised stratified epidermis in 3D HSE cultures, demonstrating their ability to fully stratify and retain their original, primary characteristics. In conclusion, the use of 0.07 mM Calcium and 10 µM Y-27632 in HEKn monocultures provides the opportunity to culture primary human keratinocytes without a cell feeder layer for extended periods of culture whilst retaining their ability to differentiate and form a stratified epithelium.

Screening patients referred to a metabolic clinic for lysosomal storage disorders.

BACKGROUND: Lysosomal protein profiling is being developed as a high throughput method to screen populations for lysosomal storage disorders (LSD). DESIGN: 1415 blood spots from patients referred to a metabolic clinic for LSD were screened using a single multiplex assay for 14 proteins in a dried blood spot. RESULTS: All patients with Pompe disease, metachromatic leukodystrophy, and mucopolysaccharidosis (MPS) type I, IIIA, IIIB and VI were identified by reduced lysosomal protein. Five samples were identified as possible pseudo-arylsulfatase A deficiency; four were confirmed. One multiple sulfatase deficiency patient was identified with multiple reduced sulfatase proteins. There were 10 MPS II patients identified with reduced iduronate 2-sulfatase, and one MPS II patient with iduronate 2-sulfatase in the unaffected range. For Fabry disease, 10 male patients were identified with reduced α-galactosidase and 2/6 female Fabry heterozygotes returned α-galactosidase concentrations in the male Fabry range. All 10 mucolipidosis II/III patients were identified with multiple raised proteins. For 79 blood spots with chitotriosidase >3.4mg/l, a follow-up one-plex chitotriosidase assay enabled identification of all nine Gaucher patients. CONCLUSION: This study demonstrates the sensitivity and specificity of this technology to accurately identify 99% of LSD patients, with the exception of one MPS II false negative.

Effect of cisternal sulfamidase delivery in MPS IIIA Huntaway dogs--a proof of principle study.

Mucopolysaccharidosis type IIIA (MPS IIIA) results from lack of functional sulfamidase (SGSH), a lysosomal enzyme. Its substrate, heparan sulfate, and other secondarily-stored compounds subsequently accumulate primarily within the central nervous system (CNS), resulting in progressive mental deterioration and early death. Presently there is no treatment. As a potential therapeutic strategy, recombinant human sulfamidase (rhSGSH) was administered into the CSF (via the cerebellomedullary cistern) of three adult MPS IIIA dogs either twice with a 4 day interval, or weekly for up to 4 weeks. The dogs were euthanased 24 h post-injection along with one untreated unaffected and two MPS IIIA controls. We have examined the three dimensional pattern of distribution of enzyme in the CNS and its ability to reduce primary substrate storage. High concentrations of rhSGSH protein, with up to 39-fold normal enzyme activity levels were detected within widespread areas of the CNS. RhSGSH protein was also detectable by immunohistochemistry in neurons and glia in all three enzyme-treated dogs. In both weekly-treated dogs, relative levels of a heparan sulfate-derived disaccharide, measured using tandem mass spectrometry, were lower in many brain regions when compared to untreated MPS IIIA controls. A moderately severe meningitis was also present as well as antibodies to rhSGSH in CSF/plasma. These findings demonstrate proof of principle that MPS IIIA can be treated by intracisternal enzyme replacement warranting further experiments in animals tolerant to rhSGSH. This enzyme delivery method may represent a means of treating neuropathology in MPS IIIA and other lysosomal storage disorders affecting the CNS.

Stabilising normal and mis-sense variant alpha-glucosidase.

alpha-Glucosidase (EC 3.2.1.3) is a lysosomal enzyme that hydrolyses alpha-1,4- and alpha-1,6-linkages of glycogen to produce free glucose. A deficiency in alpha-glucosidase activity results in glycogen storage disorder type II (GSD II), also called Pompe disease. Here, d-glucose was shown to be a competitive inhibitor of alpha-glucosidase and when added to culture medium at 6.0 g/L increased the production of this protein by CHO-K1 expression cells and stabilised the enzyme activity. D-Glucose also prevented alpha-glucosidase aggregation/precipitation and increased protein yield in a modified purification scheme. In fibroblast cells, from adult-onset GSD II patients, D-glucose increased the residual level of alpha-glucosidase activity, suggesting that a structural analogue of d-glucose may be used for enzyme enhancement therapy.

Newborn screening for lysosomal storage disorders.

Lysosomal storage disorders (LSD) are chronic progressive diseases that have a devastating impact on the patient and family. Most patients are clinically normal at birth but develop symptoms early in childhood. Despite no curative treatment, a number of therapeutic options are available to improve quality of life. To achieve this, there is a pressing need for newborn screening to identify affected individuals early, before the onset of severe irreversible pathology. We have developed a multiplexed immune-quantification assay of 11 different lysosomal proteins for the identification of individuals with an LSD and evaluated this assay in a retrospective study using blood-spots from; newborns subsequently diagnosed with an LSD (n=19, six different LSD), individuals sampled after diagnosis of an LSD (n=92, 11 different LSD), newborn controls (n=433), and adult controls (n=200). All patients with mucopolysaccharidosis type I (MPS I), MPS II, MPS IIIA, MPS VI, metachromatic leukodystrophy, Niemann-Pick disease type A/B, and multiple sulfatase deficiency could be identified by reduced enzyme levels compared to controls. All mucolipidosis type II/III patients were identified by the elevation of several lysosomal enzymes, above the control range. Most Fabry, Pompe, and Gaucher disease patients were identified from either single protein differences or profiles of multiple protein markers. Newborn screening for multiple LSD is achievable using multiplexed immune-quantification of a panel of lysosomal proteins. With further validation, this method could be readily incorporated into existing screening laboratories and will have a substantial impact on patient management and counseling of families.