Brief reports: Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes

Abstract

Despite controversies on the potential of hematopoietic stem cells (HSCs) to promote tissue repair, we previously showed that HSC transplantation could correct cystinosis, a multisystemic lysosomal storage disease, caused by a defective lysosomal membrane cystine transporter, cystinosin (CTNS gene). Addressing the cellular mechanisms, we here report vesicular cross-correction after HSC differentiation into macrophages. Upon coculture with cystinotic fibroblasts, macrophages produced tunneling nanotubes (TNTs) allowing transfer of cystinosin-bearing lysosomes into Ctns-deficient cells, which exploited the same route to retrogradely transfer cystine-loaded lysosomes to macrophages, providing a bidirectional correction mechanism. TNT formation was enhanced by contact with diseased cells. In vivo, HSCs grafted to cystinotic kidneys also generated nanotubular extensions resembling invadopodia that crossed the dense basement membranes and delivered cystinosin into diseased proximal tubular cells. This is the first report of correction of a genetic lysosomal defect by bidirectional vesicular exchange via TNTs and suggests broader potential for HSC transplantation for other disorders due to defective vesicular proteins.

Keywords: Cystinosis; Hematopoietic stem cells; Lysosomal cross-correction; Macrophages; Tubular basement membrane; Tunneling nanotubes.

Fig. 1. TNT-mediated transfer of cystinosin is the preferred mode of cross-correction

(A, B) Histograms representing percent decrease in cystine content in DsRed-Ctns^−/− fibroblasts (recipient cells) when plated together with (A: contact co-culture assays) or separated by 1-µm pore transwell filters from (B: transwell assays) either eGFP-MSCs or eGFP-macrophages (donor cells) (n=4 replicates for each). Values are means ± standard deviations. *, p<0.05; **, p<0.01; ***, p<0.005. (C) Confocal image of TNTs (arrowheads) extended from eGFP-macrophages to DsRed-Ctns^−/− fibroblasts. (D) Scanning electron micrograph showing showing two thin TNTs (arrowheads) and one thick (arrow) TNT bridging a primary macrophage (M) and a Ctns^−/− fibroblast (F). The connection (box) is enlarged in the inset. (E) Transmission electron micrograph showing a thick TNT containing various organelles, including lysosomes (L), along microtubules (arrowheads). (F) Representative frames from a confocal imaging movie (Video 1) showing migration of cystinosin-eGFP-containing vesicles via TNTs from a CTNS-eGFP-expressing macrophage towards DsRed-Ctns^−/− fibroblasts (arrowheads). (G) Representative frames from a confocal imaging movie (Video 2A) showing bulk fission of a dilated tip of a TNT, delivering a pool of cystinosin-eGFP-containing vesicles to a DsRed-Ctns^−/− fibroblast (arrowhead). (H) Individual vesicles (arrows) were observed in the cytoplasm of the recipient fibroblasts, probably originating from further dissociation of the internalized pools (arrowheads; Video 2B). (I, J) Ctns^−/− cells enhance TNT formation, not elongation. Length of TNTs derived from eGFP-IC21 macrophages (I), and average number of TNTs extended by eGFP-IC21 and CTNS-eGFP-IC21 (J) macrophages, estimated after 3 days of co-culture with either DsRed-WT or -Ctns^−/− fibroblasts (n=4 replicates). Values are means ± standard errors of the mean. *, p<0.05; **, p<0.01. Bars: (C) 30 µm; (D) 10 µm; (D, inset) 1 µm; (E) 500 nm; (F – H) 20 µm.

Fig. 2. Lysosomal trafficking within the TNTs is bidirectional

(A) Representative frame from a confocal imaging movie (Video 3) of Lamp1-DsRed-expresssing Ctns^−/− fibroblasts co-cultured with CTNS-eGFP-expressing macrophages showing eGFP- and DsRed-positive lysosomes trafficking via the same TNT. (B) The green arrow marks the trajectory towards the Ctns^−/− fibroblast of a selected cystinosin-eGFP-containing lysosome (arrowhead) at the two indicated time intervals. (C) The red arrow marks the trajectory towards the macrophage of a selected Lamp1-DsRed-containing lysosome (arrowhead) at the two indicated time intervals. Consistent with previous studies [41], abnormally large lysosomes were observed in Ctns^−/− fibroblasts. Bar: 10 µm.

Fig. 3. TNT-mediated cystinosin transfer in vivo, study of the kidney

(A) Z-stack projections over 7 µm of confocal optical sections from an 8 month-old Ctns^−/− mouse kidney, transplanted at 2 months with eGFP-expressing WT HSC. Green, immunolabeled eGFP; red, laminin immunolabeling provides a grazing view of basement lamina; blue, PTC brush border–specific sugars labeling by Lotus Tetragonolobus (LT)-lectin defines PTCs (lumen, #). (a) eGFP-expressing HSC-derived cells display numerous tortuous extensions, which frequently appose onto the TBM of PTCs and show enlarged tips. Arrowheads indicate TBM crossing. Arrow indicates local lack of LT labeling of the PTC epithelium due to dedifferentiation or shedding (for 3D rotation, see Video 4 A). (b) At left, multiple extensions entering TBM of a PTC; mottled aspect of BM can be seen. At right, extension crossing TBM of another tubular section not labeled by LT (^). (c) Transferred material appearing as eGFP-expressing green structures become clustered in PTC apical cytoplasm (arrows) (Video 4 B). (B – D) Z-stack projections of kidneys obtained from Ctns^−/− mice transplanted with DsRed-Ctns^−/− HSCs (control, B) or DsRed-Ctns^−/− HSCs lentivirally transduced to express cystinosin-eGFP (C, D), after labeling for F-actin (phalloidin, magenta, C) or megalin immunolabelling to identify the brush border of proximal tubules (magenta, D). Engrafted DsRed-expressing bone marrow-derived macrophages surrounding kidney tubules contain eGFP-positive discrete vesicles (arrowheads; C and D) or enlarged structures (arrow; C), probably due to clustering and/or fusion of lysosomes due to CTNS over-expression, as previously described [8, 41]. Note the abundance of discrete cystinosin-eGFP-containing vesicles in the cytoplasm of transduced PTCs (C, D). Since not all DsRed-HSCs were transduced, some do not express CTNS-eGFP. Nuclei are stained in blue (DAPI). Bars: (A) 5 µm; (B – D) 10 µm.