Supplemental Figure 1. Quantification of proliferation in thyroid of WT, Ctns -/- and grafted Ctns -/- mice. Cells immunolabeled for the proliferation marker (Ki-67) were counted in sections (n=3 WT, n=4 Ctns -/-, n=4 grafted Ctns -/- mice); 7 representative fields spanning each entire thyroid and corresponding to 2.6 10 5 µm 2 were analyzed. Notice significant decrease of proliferation in thyrocytes of grafted compared to non-grafted Ctns -/- mice.* p<0.05 for comparison of thyrocytes. Supplemental Figure 2. Electron microscopy. Upper panel. Two low-magnification views of grafted Ctns -/- thyroid at 8 months / 6 months post-transplant. For comparison with non-grafted Ctns -/- thyroid, see (Gaide Chevronnay et al. Endocrinology 2015). At (a), three preserved columnar thyrocytes (activated) with well-defined apical junctions (yellow arrowheads). CD, colloid droplet; asterisk, cluster of normal lysosomes. At (b), cluster of thyrocytes with pale chromatin (suggesting proliferation). Double asterisk denotes a huge apical vesicle filled with finely granular material, of comparable structure as colloid droplet identified at (a). At lower left, # indicates a cell body with electron-dense elongated nucleus, infiltrated within basement lamina boundaries (red dotted line) and extending a cone penetrating between thyrocytes (yellow arrow), suggesting an HSC with projecting radial TNT. Lower panel, lysosomes. c,d. Two thyrocytes in non-grafted Ctns -/- mice with crystal-bearing lysosomes of normal size (~400nm sectioned diameter) within preserved cytoplasm. e. Grafted Ctns -/- thyrocyte showing a cluster of normal lysosomes (~500nm sectioned diameter, arrowhead points to single limiting membrane with pale halo wrapping homogenous electron-dense matrix). Note the vicinity with longitudinal sections of TNTs (yellow arrows); filamentous core is best seen at double arrow.
Supplemental Figure 3. Inserted WT-eGFP-HSC emits thin and thick cytoplasmic extensions, allowing contacting several thyrocytes. 3-D reconstruction of z-stack series images (0.23 µm increment, 5 µm total thickness) after double immunolabelling for laminin (red, basement lamina) and GFP (green, HSCs). This inserted HSC with dendritic shape emits both thin (white arrows) and thick (yellow arrows) cytoplasmic extensions. Supplemental Figure 4. Fine structure of basal lamina. Grazing views in WT (a,d), Ctns -/- (b,e), and grafted Ctns -/- (c,f). Lower panels are enlargements of areas boxed above. Notice homogenous structure and regular circular orientation of laminin in WT thyroid, contrasting with scalloped contour and enclosures/cavities in Ctns -/-. HSCs grafting largely preserves circularity and homogeneity of follicle basement lamina, except at small discontinuities frequently occupied by HSC projections. Supplemental Figure 5. Follicle inserted GFP + -WT HSCs do not transdifferentiate into thyrocytes. A. GFP + -WT HSCs insert between follicular basement and thyrocytes. 3-D reconstruction of z-stack series images (0.23 µm increment, 5 µm total thickness) obtained after triple immunolabelling for E-cadherin (white, thyrocytes baso-lateral membrane), laminin (red, basement lamina) and GFP (green, HSCs). DsRed signal is not shown here, for clarity. A HSC has passed through the follicle basement lamina and is intertwined within the thyrocyte monolayer (at least two thyrocytes are contacted, one by the cell body at left, and the second by a large extension (yellow arrowhead) B. Follicle-inserted HSC do not form tight junction with thyrocytes. Triple immunostaining for E-cadherin (red), ZO-1 (white, thyrocyte tight junctions) and GFP (green, HSCs). The boxed HSC is inserted into the thyrocyte monolayer and appears circumscribed by E-cadherin but does not engage into apical tight junction, nor reaches the
lumen. C. Follicle-inserted HSC do not transdifferentiate in thyrocytes. Triple immunolabelling for E-cadherin (white), Nkx2.1 (red, thyroid-specific transcription factor) and GFP (green, HSCs). All nuclei are further stained with DAPI (blue). In contrast to thyrocyte nuclei (white arrowhead), the nucleus of inserted HSCs (red arrowhead) is not labelled for Nkx2.1. Boxed areas at B and C are split below into black/white images for the three indicated channels. Supplemental Figure 6. Endogenous macrophage recruitment in Ctns -/- thyroid correlates with thyrocyte proliferation and structural alterations. Time-course of endogenous macrophage infiltration was analyzed by immunolabelling (F4/80, red) and compared to proliferative repair (Ki-67, green) in thyroid of WT or Ctns -/- mice at 3, 6 and 9 months. Macrophages invade the cystinotic tissue between 6 and 9 months, concomitant with structural tissue alterations (E-cadherin; white) and increased cell proliferation (Ki67; green). As in Ctns -/- kidney (Gaide Chevronnay et al. JASN 2014), notice silent phase for at least 3 months. Starting around 6 months and maximal at ~9 months of age, endogenous, cystinosin-deficient macrophages insert within cystinotic follicles (red arrowheads, best seen in enlarged box), exactly as GFP + -WT HSCs bearing normal cystinosin, but fail to provide protection. Supplemental Figure 7. Pattern of GFP + -WT HSC infiltration in the thyroid of grafted Ctns -/- mice. (a) Immunolabelling of grafted WT-eGFP-HSCs (GFP, green) in one representative mouse out of 5 reveals the pattern of infiltrating HSCs in the entire cystinotic thyroid. Thyroid full view was obtained using MosaiX module of AxioVision Software allowing tilling and stitching of images captured across the thyroid section. Enlargement (b) shows individual HSCs
(white arrowheads), inserted within the epithelium (E-cadherin, white); enlargement (c) shows 3 foci of HSC clusters mainly in the interstitium of the center of the gland. Supplemental Figure 8. In contrast to kidney, only few WT-eGFP-HSCs grafted in the thyroid express F4/80 differentiation marker. Multiplex fluorescence labeling for GFP (green, HSC), F4/80 (red, macrophages) and E-cadherin (white, thyrocyte basolateral membrane) or by LT-lectin (white, kidney proximal tubule cells apical membrane). In contrast to endogenous macrophages, few WT-eGFP-HSCs grafted in the thyroid express detectable F4/80 marker. In kidney, both endogenous macrophages and grafted HSCs show strong comparable F4/80 signal. Boxed fields point to cells clearly immunolabelled for F4/80, enlarged in the insets as black/white.
Proliferative events 250 200 150 100 50 * * * thyrocytes, * p <0.05 interstitium, NS HSC 0 WT Ctns -/- grafted Ctns -/- Supplemental Figure 1
a b colloid ** CD * # capillary 2 µm c e 200 nm d 200 nm 500 nm Supplemental Figure 2 2 µm
laminin, WT-eGFP-HSCs lumen 5µm Supplemental Figure 3
laminin, WT-eGFP-HSCs, Hoechst a. WT b. Ctns -/- c. grafted Ctns -/- 10µm d. WT e. Ctns -/- f. grafted Ctns -/- 5µm Supplemental Figure 4
A E-cadherin, laminin, WT-eGFP-HSCs a lumen 5µm B E-cadherin, ZO-1, WT-EGFP-HSCs lumen 5µm E-cadherin GFP ZO-1 C E-cadherin, Nkx2.1, WT-eGFP-HSCs, DAPI GFP TTF1 DAPI 5µm Supplemental Figure 5
E-cadherin, Ki-67, F4/80 3 months 6 months 9 months WT 20µm Ctns -/- 20µm Supplemental Figure 6
E-cadherin, WT-eGFP-HSCs a b c 100µm b c 50µm 50µm Supplemental Figure 7
A. Thyroid E-cadherin, F4/80, WT-eGFP-HSCs a. Ctns -/- b. grafted Ctns -/- 20µm 20µm B. Kidney LT-lectin, F4/80, WT-eGFP-HSCs a. Ctns -/- b. grafted Ctns -/- 20µm 20µm Supplemental Figure 8