Relative acidic compartment volume as a lysosomal storage disorder–associated biomarker

Lysosomal storage disorders (LSDs) occur at a frequency of 1 in every 5,000 live births and are a common cause of pediatric neurodegenerative disease. The relatively small number of patients with LSDs and lack of validated biomarkers are substantial challenges for clinical trial design. Here, we evaluated the use of a commercially available fluorescent probe, Lysotracker, that can be used to measure the relative acidic compartment volume of circulating B cells as a potentially universal biomarker for LSDs. We validated this metric in a mouse model of the LSD Niemann-Pick type C1 disease (NPC1) and in a prospective 5-year international study of NPC patients. Pediatric NPC subjects had elevated acidic compartment volume that correlated with age-adjusted clinical severity and was reduced in response to therapy with miglustat, a European Medicines Agency–approved drug that has been shown to reduce NPC1-associated neuropathology. Measurement of relative acidic compartment volume was also useful for monitoring therapeutic responses of an NPC2 patient after bone marrow transplantation. Furthermore, this metric identified a potential adverse event in NPC1 patients receiving i.v. cyclodextrin therapy. Our data indicate that relative acidic compartment volume may be a useful biomarker to aid diagnosis, clinical monitoring, and evaluation of therapeutic responses in patients with lysosomal disorders

Directed differentiation of embryonic stem cells using a bead-based combinatorial screening method

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported

Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin

Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the HEXB gene, which encodes the β-subunit of β-hexosaminidase. G(M2) ganglioside fails to be degraded and accumulates within lysosomes in cells of the periphery and the central nervous system (CNS). There are currently no therapies for the glycosphingolipid lysosomal storage diseases that involve CNS pathology, including the G(M2) gangliosidoses. One strategy for treating this and related diseases is substrate deprivation. This would utilize an inhibitor of glycosphingolipid biosynthesis to balance synthesis with the impaired rate of catabolism, thus preventing storage. One such inhibitor is N- butyldeoxynojirimycin, which currently is in clinical trials for the potential treatment of type 1 Gaucher disease, a related disease that involves glycosphingolipid storage in peripheral tissues, but not in the CNS. In this study, we have evaluated whether this drug also could be applied to the treatment of diseases with CNS storage and pathology. We therefore have treated a mouse model of Sandhoff disease with the inhibitor N- butyldeoxynojirimycin. The treated mice have delayed symptom onset, reduced storage in the brain and peripheral tissues, and increased life expectancy. Substrate deprivation therefore offers a potentially general therapy for this family of lysosomal storage diseases, including those with CNS disease.</p

An inducible mouse model of late onset Tay-Sachs disease

Mouse models of the GM2 gangliosidoses, Tay–Sachs and Sandhoff disease, are null for the hexosaminidase ? and ? subunits respectively. The Sandhoff (Hexb?/?) mouse has severe neurological disease and mimics the human infantile onset variant. However, the Tay–Sachs (Hexa?/?) mouse model lacks an overt phenotype as mice can partially bypass the blocked catabolic pathway and escape disease. We have investigated whether a subset of Tay–Sachs mice develop late onset disease. We have found that not, vert, similar65% of the mice develop one or more clinical signs of the disease within their natural life span (n = 52, P &lt; 0.0001). However, 100% of female mice with repeat breeding histories developed late onset disease at an earlier age (n = 21, P &lt; 0.0001) and displayed all clinical features. Repeat breeding of a large cohort of female Tay–Sachs mice confirmed that pregnancy induces late onset Tay–Sachs disease. Onset of symptoms correlated with reduced up-regulation of hexosaminidase B, a component of the bypass pathway

Mouse and human TH positive neuronal screens.

<p><b>(a)</b> Fluorescence micrographs showing hits bearing (i) mouse and (ii) human TH+ cells amongst negative beads. Scale bars  =  100 µm. <b>(b)</b> Schematic diagram illustrating an overlay of all protocols deconvoluted from screens for TH+ neurons generated using (i) mES and (ii) hES cells. Histograms representing each cell culture medium are proportional to the number of hits generated (written at the bottom of each bar). The opacity of the linkage lines is proportional to the number of hits generated by specific media combinations - the darkest line in each plot corresponds to (i) 21 and (ii) 12 beads. <b>(c)</b> Microgrographs showing immunofluorescence images of differentiated ES cells in monolayer cultures. Differentiation protocols shown for mES cells are <b>(i)</b> 5929 and <b>(ii)</b> 4671, stained for TH (red), β-III tubulin (green) and DAPI (blue). Differentiation protocol shown for hES cells is 8976 (iii and iv) stained for TH (red), β-III tubulin (green) and DAPI (blue); except in (iv) where FOXA2 is stained blue. Scale bars  =  100 µm. Representative photographs from a field of view are shown. Monolayer differentiations were repeated at least twice for the protocols shown. <b>(d)</b> Efficiency of (i) mES (ii) hES differentiation to TH+ neurons on PTC5000 beads measured by COPAS. Experiments were performed in duplicate wells (containing 4000 beads/well) and the proportion of beads bearing TH+ neurons in each well is plotted separately (light and dark histograms) and ranked according to the average proportion of positive beads. The bead number(s) and the corresponding deconvoluted protocol are listed below the histogram.</p

Analysis of Cluster A protocol derived phagocytes following further maturation in MethoCult or on OP9 cells supplemented with IL7.

<p><b>(a)</b> Dot plots illustrating flow cytometry analysis of cells obtained after maturation in IL7 supplemented MethoCult for 2 weeks, showing the appearance of cells co-expressing B220/CD43 and B220/CD19; <b>(b)</b> Histogram showing the proportions of different cell populations present in MethoCult or OP9 derived cultures, measured by flow cytometry analysis. Blue bars represent cells taken off beads on D15 (prior to maturation), red bars represent cell isolated on D15 and cultured for a further 2 weeks on OP9 feeder layers and green bars represent cells isolated on D15 and cultured in MethoCult for 2 weeks. The experiment was carried out twice and in duplicate the bars show the average percentage of cells, the error bars show the standard deviation. There is a statistically significant difference between the cells taken from the beads at D15 and those cultured on MethoCult for 2 weeks for markers CD43 and CD19 *p<0.05.</p

Mouse phagocytic screen.

<p><b>(a)</b> Schematic diagram illustrating design of the CombiCult screen. Ten different cell culture media were tested in each of four stages of differentiation by split-pool passaging cells seeded onto beads in differentiation media spiked with tags, on day 1 (D1), D2, D5 and D7. On D14 beads were assayed to identify beads bearing phagocytes or neuroectodermal precursors (‘hits’). A total of 300,000 beads were used in the experiment to test 10,000 protocols so that on average each protocol was sampled by 30 beads. <b>(b)</b> Fluorescence micrographs showing hits bearing (i) red fluorescent phagocytic cells or (ii) GFP-positive neuroectoderm cells amongst negative beads. Scale bars  =  100 µm. <b>(c)</b> Cummulative large particle flow sorter dot plots, showing parameters used to sort hits. (i) Gating of monomeric beads (gate 1) from higher order agreggates. (ii) Sorting of monomeric beads with high red (gate 2) and green (gate 3) fluorescent signal. <b>(d)</b> Micrograph images of sorted hits: (i) bead 31 (phagocyte), (ii) bead 131 (phagocyte), (iii) bead 76 (neuroectoderm), (iv) bead 34 (neuroectoderm) and (v) bead 34 (phagocyte and neuroectoderm). Scale bars  =  100 µm. <b>(e)</b> Deconvolution of cell culture history of bead 31 (pictured in d(i) above; one of a triple hit from the phagocyte screen) by FACS analysis of bound tags. A set of 30 unique tags distinguishable by size (small, medium, large) and fluorescence intensity (ten levels for each size set) was used to spike cell culture media in the first three stages of differentiation. The figure shows magenta peaks corresponding to quantitation of a reference sample of the 30 tags used to calibrate the flow cytometer (histograms and tag numbers shown in magenta) in order to set gates. Quantitation of tags released from bead 31 is superimposed in black, showing detection of tags SR10, MR1 and LR8, corresponding to media 1.10, 2.1 and 3.8. <b>(f)</b> Schematic diagram illustrating an overlay of all protocols deconvoluted from phagocyte hits. The height of boxes representing each cell culture medium is proportional to the number of hits generated by that medium (written at the bottom of each box). The opacity of the linkage lines is proportional to the number of hits generated by specific media combinations - the darkest line corresponds to 21 hits. <b>(g)</b> Hierarchical clustering analysis of 92 unique protocols derived from the phagocyte screen, showing protocol clusters A and B. Each node at the bottom of the dendrogram (leaf node) corresponds to a hit bead. The associated protocol is denoted by the column of four colours directly below the node, specifying the media sampled in splits 1–4. The legend at the bottom of the figure specifies the colour used to denote the cell culture media in each split. <b>(h)</b> Similarity matrix comprising a pair-wise comparison of all protocols. Each column and each row corresponds to a protocol. The brightness of each cell in the matrix is proportional to the number of identical cell culture media shared by the two protocols. The brightest cell corresponds to identical protocols, while a black cell corresponds to two protocols with no common media. The diagonal row of cells (from the top left to bottom right) corresponds to protocols being compared to themselves. Protocol families with high internal homology appear as bright red squares, e.g. Clusters A and B marked on the diagram, with Cluster A protocols being more conserved. <b>(i)</b> Efficiency of phagocyte and/or neuroectoderm cell generation from mES cells on PTC5000 beads using a selection of protocols discovered by CombiCult. Coloured bars represent the number of fluorescent colonies per square centimeter (64 fields of view). Red bars correspond to phagocytic colonies, green bars correspond to GFP-expressing (neuroectodermal) colonies, orange bars correspond to beads with both phagocytic and neuroectodermal colonies, black bars correspond to negative protocols. Hit bead numbers and protocols listed below the histograms are coloured to show whether they were derived from phagocyte- (red), neuroectoderm- (green) or phagocyte/neuroectoderm- (orange) bearing hits, whereas black corresponds to two negative protocols – i.e. protocols which did not return any hits, these represent a range of 12 protocols tested, all but one (shown) had 0 colonies. Each bar represents the average of 2 wells (each one containing 4000 beads) and the graph is representative of 3 separate experiments. Protocols with a statistically significant difference (p>0.05) to the negative control are marked with an asterisk (*).</p

Defective iron homeostasis and hematological abnormalities in Niemann-Pick disease type C1 [version 2; peer review: 2 approved]

Background: Niemann-Pick disease type C1 (NPC1) is a neurodegenerative lysosomal storage disorder characterized by the accumulation of multiple lipids in the late endosome/lysosomal system and reduced acidic store calcium. The lysosomal system regulates key aspects of iron homeostasis, which prompted us to investigate whether there are hematological abnormalities and iron metabolism defects in NPC1. Methods: Iron-related hematological parameters, systemic and tissue metal ion and relevant hormonal and proteins levels, expression of specific pro-inflammatory mediators and erythrophagocytosis were evaluated in an authentic mouse model and in a large cohort of NPC patients. Results: Significant changes in mean corpuscular volume and corpuscular hemoglobin were detected in Npc1-/- mice from an early age. Hematocrit, red cell distribution width and hemoglobin changes were observed in late-stage disease animals. Systemic iron deficiency, increased circulating hepcidin, decreased ferritin and abnormal pro-inflammatory cytokine levels were also found. Furthermore, there is evidence of defective erythrophagocytosis in Npc1-/- mice and in an in vitro NPC1 cellular model. Comparable hematological changes, including low normal serum iron and transferrin saturation and low cerebrospinal fluid ferritin were confirmed in NPC1 patients. Conclusions: These data suggest loss of iron homeostasis and hematological abnormalities in NPC1 may contribute to the pathophysiology of this disease