Regeneration of Thyroid Glands in the Spleen Restores Homeostasis in Thyroidectomy Mice

Abstract Surgical removal of the thyroid gland (TG) for treating thyroid disorders leaves the patients on lifelong hormone replacement that partially compensates the physiological needs, but regenerating TG is challenging. Here, an approach is reported to regenerate TG within the spleen for fully restoring the thyroid's functions in mice, by transplanting thyroid tissue blocks to the spleen. Within 48 h, the transplanted tissue efficiently revascularizes, forming thyroid follicles similar to the native gland after 4 weeks. Structurally, the ectopically generated thyroid integrates with the surrounding splenic tissue while maintaining its integrity, separate from the lymphatic tissue. Functionally, it fully restores the native functions of the TG in hormone regulation in response to physiological stimuli, outperforming the established method of oral levothyroxine therapy in maintaining systemic homeostasis. The study demonstrates the full restoration of thyroid functions post‐thyroidectomy by intrasplenic TG regeneration, providing fresh insights for designing novel therapies for thyroid‐related disorders.


Supplementary Figures
Regenerated parathyroid specimens were harvested from mice 4 weeks after intra-splenic transplantation (ST) of parathyroid glands of the same genotype for immunohistochemical staining.C,D) Serum hormone recovery in intrasplenic transplantation of frozen-thawed thyroid (1C1ST) and unfrozen thyroid (ST) mice.Serum was collected from mice after thyroidectomy, followed by intra-splenic transplantation of frozen-thawed thyroid and unfrozen thyroid at 4 and 16 weeks for determination of C) T3 and D) T4 concentrations by Electrochemiluminescence (ECL) technology, with N mice as the control.n=5.E) Major steps in spleen translocation.In Figure 1, an incision is made at the black dashed position; in     Spleen specimens were harvested from mice with intra-splenic regenerated thyroid at 2 to 16 weeks.C) H&E staining of the larynx (the spot where the thyroid gland used to be) of normal and total thyroidectomy mice.

Figure S1 .
Figure S1.Thyroid Transplantation into Translocated Mouse Spleens.A) Size distribution of chopped thyroid tissue.Thyroid suspensions of 0.2mm, 0.5mm and 0.7mm sizes were prepared, and the size of 20 of these tissues were randomly counted.n=20.B) Immunohistochemical staining for parathyroid.Immunohistochemical staining for parathyroid markers PTH and CaSR and negative control.

Figure 2 ,
Figure 2, the vessels are labeled with black dashed lines to mark the vessel position; in Figure 4, the black dashed lines mark the spleen position.F,G) F) Morphology of the spleen after transplantation without (upper) or with (lower) a transplanted thyroid and G) mean spleen weight in ST and normal mice at week 4. n=15.Specimens were harvested from the spleens of mice 4 weeks after intra-splenic transplantation of the same genotype of the thyroid (ST), with N mice as the control.H) Proportions of different cell populations in normal or thyroid-transplanted spleens 4 weeks after the transplantation.n=5.I) The Gating strategy for flow cytometry.T cell and B cell, NK cell, macrophage, fibroblast, and endothelial cell.Data are presented as means ± SEM.Statistical analyses were performed using two-way ANOVA followed by Tukey's multiple comparisons tests (C and D) and Student's t-test (G).PTH, parathormone; CaSR, calcium-sensing receptor.ANOVA; analysis of variance, 1C1 ST denotes mice transplanted with frozen-thawed thyroid glands.

Figure S2 .
Figure S2.Analysis of Thyroid Autotransplantation and Allotransplantation into Mouse Spleens.Spleen specimens were harvested 4 weeks after intra-splenic transplantation from mice with the same genotype of thyroid (ST), and splenocytes were subjected to flow cytometry to determine cell proportions, with normal spleens (N) as a control.A,B) A) ImmuCC estimates the composition of immune cells from spleen expression profiles, and B) calculates the immune infiltration score.n=3.Splenic sequencing data of N and ST were used to predict their immune cell infiltration by uploading to the ImmuCC web server.C) PCA analysis of N-spleen (control), ST-spleen, and thyroid.n=3.D) Spearman correlation analysis of gene expression profiles in the N-spleen, ST-spleen, and thyroid.n=3.E) KEGG analysis of upregulated enriched genes showing enrichment in the thyroid hormone synthesis pathway.Genome-wide microarray mapping of the spleen (ST-spleen) 4 weeks after thyroid transplantation, with thyroid and normal spleen (N-spleen) as controls.F) H&E staining of thyroid grafts transplanted into the spleens (ICR ST).The black arrows indicate the follicular structure and the black dashed curves outline the ST tissue.Thyroid glands of ICR mice were intra-splenic transplanted in C57 mice, and spleen specimens were harvested at 3, 10, and 30 posts after transplantation and subjected to H&E staining.G,H) Serum cytokine concentrations of G) TNF-α and H) INF-γ in mice transplanted with thyroid glands from donors with different genotypes, with N mice as the control.n=3.Thyroid glands of ICR mice were intra-splenic transplanted in C57 mice, and serum samples were collected at 1, 3, 5, and 7 days after transplantation.TNF-α and IFN-γ concentrations were measured by ELISA.Data are presented as means ± SEM.Statistical analyses were performed using Student's t-test (B, G, H).ANOVA; analysis of variance, ICR ST denotes mice transplanted with thyroid glands from donors with different genotypes.

Figure S3 .
Figure S3.Regeneration of Transplanted Thyroid Tissue in the Spleen.A) quantification statistics for the co-localization of PCNA with GFP.Proliferation of thyroid tissue was assessed by calculating the percentage of PCNA-positive nuclei in the nuclei of visible cells in each GFP + follicle.B) Immunofluorescence staining of EMCN and PCNA expression in the intra-splenic regenerated thyroid tissue at weeks 4 to 16. C) Representative immunofluorescence images for Cyclin D1 in intra-splenic regenerated thyroid tissue (ST).Co-localization of Cyclin D1 and donor-sourced GFP-labeled thyroid was detected by immunofluorescence staining of splenic tissue sections from mice intrasplenic transplanted at 2 to 16 weeks.D,E) The proliferative activity of intra-splenic thyroid after the inhibition of IGF-1 by IGF-1R inhibitor (AXL1717) was evaluated by PCNA D) immunofluorescence staining (co-staining with GFP to identify thyroid tissues from GFP transgenic mice) and E) quantification statistics for the co-localization of PCNA with GFP.Data are presented as

Figure S4 .
Figure S4.Independence of the thyroid gland in the spleen.A) Representative images of immunofluorescence for T cells (CD3 + ), B cells (B220 + ), and macrophages (F4/80 + ) in the intra-splenic regenerated thyroid tissue (ST) spleens.Expression of immune cell markers in the spleen was determined by immunofluorescence staining of splenic tissue sections from mice with intrasplenic transplantation of the same genotype of thyroid at 2 to 16 weeks.B) Fluorescence statistics of the immunofluorescence staining for T cells, macrophages, and B cells in the intra-splenic regenerated thyroid tissue (ST) spleens.n=5.Data statistics were performed using Image-Pro Plus version 6.0.C) Serum cytokine concentrations of TNF-α and INF-γ in mice transplanted with thyroid glands.n=5.Serum samples were collected from mice intrasplenically transplanted with the same genotype of thyroid at 7 days after transplantation, and TNF-α and IFN-γ concentrations were measured by ELISA.D) Representative images of immunofluorescence for collagen type I (COL Ⅰ) and collagen type IV (COL Ⅳ) in the intra-splenic

Figure S5 .
Figure S5.Representative images of PAS and immunofluorescence of thyroid markers.A) PAS staining labels thyroglobulin in the intra-splenic regenerated thyroid tissue (ST), showing the overall (upper) and local (lower) staining.The black dashed curves outline the ST tissue.Spleen specimens were harvested from mice with intra-splenic regenerated thyroid at 2 to 16 weeks.B) Expression of specific thyroid markers Tg, NKX2-1, PAX8, and NIS in the intra-splenic regenerated thyroid tissue (ST) by immunofluorescence staining of splenic tissue sections.The white dashed curves outline the ST tissue.

Figure S6 .
Figure S6.The Effects of the Intra-splenic Thyroids and L-T4 Treatment on Serum Hormone Level and Functional Gene Expression Profile in Liver, Heart, and Brain in the Hypothyroid Mice.The mice were divided into four groups: N mice (normal healthy mice), ST mice (hypothyroid mice receiving intra-splenic thyroid regeneration treatment), TH mice (mice with total thyroidectomy), and L-T4 mice (hypothyroid mice receiving oral L-T4 treatment).All measurements were performed 16 weeks after

Figure S7 .
Figure S7.Gene regulation of intra-splenic regenerated thyroid in the liver, heart, and brain.The mice were divided into four groups: N mice (normal healthy mice), ST mice (hypothyroid mice receiving intra-splenic thyroid regeneration treatment), TH mice (mice with total thyroidectomy), and L-T4 mice

Figure S8 .
Figure S8.Physiological homeostasis of mice with a second thyroid gland; Related to Figure 5. A) Serum hormone levels in N mice and overloaded thyroid mice (NT mice) after thyrotropin-releasing hormone (TRH) stimulation.n=5.Serum samples were collected from mice with intra-splenic regenerated thyroid without thyroidectomy at 16 weeks post-transplantation, and T4 concentrations were