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Stem Cell Res Ther
2024 Nov 13;151:421. doi: 10.1186/s13287-024-04050-2.
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Immunomodulatory role of Xenopus tropicalis immature Sertoli cells in tadpolemuscle regeneration via macrophage response modulation.
Zhao Q
,
Mertová I
,
Wróblová A
,
Žabková S
,
Tlapáková T
,
Krylov V
.
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BACKGROUND: Regenerative medicine and transplantation science continuously seek methods to circumvent immune-mediated rejection and promote tissue regeneration. Sertoli cells, with their inherent immunoprotective properties, emerge as pivotal players in this quest. However, whether Sertoli cells can play immunomodulatory role in tadpoletail regeneration and can thus benefit the regeneration process are needed to be discovered.
METHODS: Immature Sertoli cells from Xenopus tropicalis (XtiSCs) were transplanted into X. tropicalis tadpoles, followed by the amputation of the final third of their tails. We assessed the migration of XtiSCs, tail regeneration length, muscle degradation and growth, and macrophage counts across various regions including the entire tail, tailtrunk, injection site, and regeneration site. The interactions between XtiSCs and macrophages were examined using a confocal microscope. To deplete macrophages, clodronate liposomes were administered prior to the transplantation of XtiSCs, while the administration of control liposomes acted as a negative control. Student's t-test was used to compare the effects of XtiSCs injection to those of a 2/3PBS injection across groups with no liposomes, control liposomes, and clodronate liposomes.
RESULTS: XtiSCs have excellent viability after transplantation to tadpoletail and remarkable homing capabilities to the regeneration site after tail amputation. XtiSCs injection increased macrophage numbers at 3 days post-amputation and 5 days post-amputation in the tailtrunk, specifically at the injection site and at the regenerated tail, in a macrophage depleted environment (clodronate-liposome injection). What's more, XtiSCs injection decreased muscle fibers degradation significantly at 1 day post-amputation and facilitated new muscle growth significantly at 3 days post-amputation. In addition, whole-mount immunostaining showed that some XtiSCs co-localized with macrophages. And we observed potential mitochondria transport from XtiSCs to macrophages using MitoTracker staining in tadpoletail.
CONCLUSIONS: Our study delineates the novel role of XtiSCs in facilitating muscle regeneration post tadpoletail amputation, underscoring a unique interaction with macrophages that is crucial for regenerative success. This study not only highlights the therapeutic potential of Sertoli cells in regenerative medicine but also opens avenues for clinical translation, offering insights into immunoregulatory strategies that could enhance tissue regeneration and transplant acceptance.
Fig. 1. XtiSCs can migrate to the regenerated tail. (A) A1: XtiSCs microinjection to upper tail region of X. tropicalis tadpoles. A2-A6: Representative pictures of XtiSCs migration. A2: immediately after injection A3: 1 day post-amputation (1dpa) A4: 3 days post-amputation (3dpa) A5: 5 days post-amputation (5dpa) A6: 7 days post-amputation (7dpa). Red: Katushka RFP-XtiSCs, scale bar: 500 μm. (B) Tail amputation trigers XtiSCs migration from the injection area mostly to the injury site if compared with non-amputation control. Fisher’s exact test, ****P < 0.0001. Samples were obtained from three biological replicates: Control n = 71; Tail amputation n = 135
Fig. 2. XtiSCs induced macrophage enrichment in the tail trunk of clodronate-depleted tadpoles. (A) Experimental setting for macrophage depletion and XtiSCs injection. Briefly, two dosages of clodronate liposomes or control liposomes were injected into the tadpole heart vein area in two consecutive days, and the XtiSCs or 2/3PBS (control) were injected to the upper tail tissue region the next day after 2nd dose injection. One day after, tail amputation was performed. Samples were collected and the tail regeneration was analyzed at different time points: 1dpa, 3dpa, 5dpa and 7dpa. (B) Clodronate liposomes efficiently decrease macrophage numbers in the whole tail at 1 day post injection(dpi), 3 days post injection (3dpi) and 5 days post injection (5dpi). The macrophage numbers in the tail trunk were calculated using ImageJ (detailed in Materials and Methods) and the final result was adjusted to total area of the tail trunk. Statistical significance was calculated via GraphPad using student t-test, *P < 0.05, **P < 0.01, ****P < 0.0001, n.s., non-significant. Data were obtained from ≥ 7 tadpoles for each group from 3 biological replicates, with 7 days post injection (7dpi) samples an exception: 7dpi Uninjected n = 4; 7dpi Control lipo (Control liposomes-injected) n = 2; 7dpi Clodronate lipo (Clodronate liposomes-injected) n = 4. (C) Representative pictures of isolectin B4 stained macrophages in uninjected, control liposomes-injected and clodronate liposomes-injected tadpoles. Blue: DAPI, green: Isolectin B4. C1, C2: Uninjected group, 5 days after 2nd dose injection (5dpi); C3, C4: Control liposomes-injected group, 5 days after 2nd dose injection (5dpi); C5,C6: Clodronate liposomes-injected group, 5 days after 2nd injection (5dpi). Scale bar:100 μm. (D) Macrophage numbers/area after XtiSCs injection in the tail trunk in control liposomes-injected and clodronate liposomes-injected tadpoles. Statistical significance was calculated via GraphPad and student t-test was used for comparing between different treatments in each group, *P < 0.05, n.s., non-significant. Data were obtained from ≥ 6 tadpoles for each group from 3 biological replicates
Fig. 3. XtiSCs increased macrophage numbers at both the injection site and the regenerated tail. The macrophage numbers/area in the injection site (A) and the regenerated tail (B) were analyzed and data was shown in graphs. Statistical analysis was done with Student’s t test using GraphPad. *P < 0.05, **P < 0.01, n.s., non-significant. Data of (A) were obtained from ≥ 7 tadpoles and data of (B) were obtained from ≥ 9 tadpoles for each group from 3 biological replicates
Fig. 4. XtiSCs stabilize injured muscle fibers and enhance muscle fiber growth. (A) The area of undegraded muscle fibers was measured at 1dpa, and the final result was presented as remaining muscle fiber area from last intact somite/total area between last complete somite and the amputation plane. Data obtained from ≥ 3 biological replicates with n ≥ 14 in each group. (B) Representative pictures of the preserved muscle fibers (stained by 12/101, DSHB) at 1dpa in uninjected group, control liposomes-injected group and clodronate liposomes-injected group. Blue dashed lines delineate the amputation plane; yellow dashed lines delineate the remaining muscle fibers between last complete somite and the amputation plane; red dashed lines delineate the total area between last complete somite and the amputation plane. Scale bar:100 μm. Green: muscle fibers (C) The muscle fiber growth was measured at 3dpa and 5dpa. The muscle fiber growth was counted as a ratio between new muscle fibers area and total area encompassing new muscle fibers up to the most distant notochord (fin is not included). Data obtained from ≥ 3 biological replicates with n ≥ 12 in each group. (D) Representative pictures of the muscle fibers (stained by 12/101, DSHB) growth at 3dpa. The yellow dashed lines delineate the muscle fiber growth area, and the red dashed lines delineate the total regenerated area. Scale bar:100 μm. Green: muscle fibers. Statistical analysis was done using Student t-test, *P < 0.05, ***P < 0.001, n.s., non-significant
Fig. 5. XtiSCs and XtiSCs’ mitochondria directly interact with macrophages. Co-localization of XtiSCs and macrophages at the injection site, with mitochondrial signals from XtiSCs observed in macrophages in the proximity to the regenerated tail and injection area (A-C) The co-localization of XtiSCs and isolectin B4 labeled macrophages was detected by whole mount immunostaining and via confocal microscope using z-stack scanning. The co-localization pictures are presented as sum slices (Z projection type) and then 3 representative z-stack single slices. Red: XtiSCs with Katushka RFP. Green: Isolectin B4 stained macrophages. Blue: DAPI. Scale bar:100 μm. A1-A4: Uninjected tadpoles injected with XtiSCs, 3dpa. D1: sum slices; D2-D4: slice 9, 18, 23 of Z stack images. B1-B4: Control liposomes-injected tadpoles injected with XtiSCs, 1dpa. B1: sum slices; B2-B4: slice 5, 9, 12 of Z stack images. C1-C4: Clodronate liposomes-injected tadpoles injected with XtiSCs, 3dpa. C1: sum slices; C2-C4: slice 10, 17, 20 of Z stack images. (D): Co-localization of XtiSCs’ mitochondria and macrophages were detected near regenerated tail and at injection area at 1 dpa. The overlap in single slice was enlarged to the larger dashed box and displayed in the same picture. Red: XtiSCs’ mitochondria (labeled by MitoTracker® Red) Green: Isolectin B4 stained macrophages. Blue: DAPI. Scale bar:100 μm, except in D6, D8 scale bar:10 μm. View 1, 2, 3 are different parts from one tadpole, View 1 uses 10x magnification, View 2 uses 63x magnification, and View 3 uses 63x magnification. Sum slices in D1, D5, D7 means the Z projection type and D2-D4, D6,D8 are single slices from Z-stack image
