Mesenchymal thymic niche cells enable regeneration of the adult thymus and T cell immunity

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Data availability

scRNA-seq data generated for this work have been deposited into the Gene Expression Omnibus database (GSE206459). Three-photon fluorescence microscopy images have been deposited into Zenodo: https://doi.org/10.5281/zenodo.15278001 (ref. ⁸⁶). All other data, reagents and protocols will be made available upon reasonable request to the corresponding authors. Source data are provided with this paper.

Code availability

All code for the scRNA-seq analysis is available at https://github.com/kharchenkolab/thymus-mesenchyme. The code for image analysis and quantification of the three-photon fluorescence microscopy is available at https://github.com/LinLabWellmanCenterForPhotomedicine/Three-photon-Mesenchymal-thymic-niche.

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Acknowledgements

We thank O. J. Benavidez from the Division of Pediatric-Congenital Cardiology at Massachusetts General Hospital for providing human thymic tissue. We were supported with expert technical assistance by the HSCI-CRM Flow Cytometry facility and the CRM Multiphoton Microscopy Core at Massachusetts General Hospital and the Bauer Core Facility at Harvard University. We thank Y. Kfoury for sharing her Cas9–GFP bone marrow stroma. We acknowledge S. Voinea for providing assistance with coding. We thank I. Adameyko for assistance in identifying neural crest progenitors in the dataset. K.G. and N.B. were supported by the Swedish Research Council. N.S. was a recipient of the AACR-Millennium Fellowship in Prostate Cancer Research. S.I. was supported by the Ministry of Science and Higher Education of the Russian Federation (grant number 075-15-2020-784). D.T.S. was supported by the Gerald and Darlene Jordan Professor Chair, Tracey and Craig A. Huff, the Harvard Stem Cell Institute, and PO1 HL131477, PO1 HL183483 and U19 HL156247. C.P.L. was supported by R01HL166266, R01NS127808, RC2DK131963 and P01HL142494. K.M. was a recipient of the Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship.

Author information

Author notes

Peter V. Kharchenko

Present address: Altos Labs, San Diego, CA, USA

Authors and Affiliations

Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA

Karin Gustafsson, Kameron A. Kooshesh, Ninib Baryawno, Anna Kiem, Nicolas Severe, Ting Zhao, Elizabeth W. Scadden, Hayalneh Gessessew, Konstantinos D. Kokkaliaris & David T. Scadden
Harvard Stem Cell Institute, Cambridge, MA, USA

Karin Gustafsson, Kameron A. Kooshesh, Ninib Baryawno, Anna Kiem, Nicolas Severe, Ting Zhao, Elizabeth W. Scadden, Hayalneh Gessessew, Konstantinos D. Kokkaliaris, Peter V. Kharchenko & David T. Scadden
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA

Karin Gustafsson, Kameron A. Kooshesh, Ninib Baryawno, Anna Kiem, Nicolas Severe, Ting Zhao, Elizabeth W. Scadden, Hayalneh Gessessew, Konstantinos D. Kokkaliaris & David T. Scadden
Research Institute of Personalized Medicine, National Center for Personalized Medicine of Endocrine Diseases, The National Medical Research Center for Endocrinology, Moscow, Russia

Sergey Isaev
Department of Neuroimmunology, Center for Brain Research, Medical University Vienna, Vienna, Austria

Sergey Isaev
Wellman Center for Photomedicine and Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

Kamdin Mirsanaye & Charles P. Lin
Dr. Senckenberg Institute of Pathology, University Hospital Frankfurt, Frankfurt, Germany

Johanna Hofmann & Konstantinos D. Kokkaliaris
Frankfurt Cancer Institute, Goethe University Frankfurt, Frankfurt, Germany

Johanna Hofmann & Konstantinos D. Kokkaliaris
Department 15, Biosciences, Goethe University Frankfurt am Main, Frankfurt am Main, Germany

Johanna Hofmann
Childhood Cancer Research Unit, Department of Children’s and Women’s Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden

Ninib Baryawno
Department of Bioengineering, NSF-CREST Center for Cellular and Biomolecular Machines, and Health Science Research Institute, University of California, Merced, Merced, CA, USA

Joel A. Spencer, Christian Burns & Kumaran Akilan
Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA

Nikolaos Barkas & Peter V. Kharchenko
German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Quantitative Spatial Cancer Biology Laboratory and German Cancer Research Center (DKFZ), Heidelberg, Germany

Konstantinos D. Kokkaliaris

Authors

Karin Gustafsson
Sergey Isaev
Kamdin Mirsanaye
Johanna Hofmann
Kameron A. Kooshesh
Ninib Baryawno
Anna Kiem
Nicolas Severe
Ting Zhao
Elizabeth W. Scadden
Joel A. Spencer
Christian Burns
Kumaran Akilan
Nikolaos Barkas
Hayalneh Gessessew
Konstantinos D. Kokkaliaris
Charles P. Lin
Peter V. Kharchenko
David T. Scadden

Contributions

K.G., N.B. and D.T.S. conceived the study. K.G. designed and performed most of the experiments, with technical assistance from and discussions with N.B., N.S., E.W.S., A.K., J.A.S., C.B., K.A., N.B., T.Z. and H.G. S.I. and P.V.K. performed the scRNA-seq analysis. K.A.K. designed and performed the CRISPR knockout studies. K.D.K. and J.H. developed and performed the three-dimensional tissue-wide confocal thymus imaging. C.P.L. and K.M. developed and performed the three-photon microscopy. K.G., S.I., N.B., P.V.K., C.P.L. and D.T.S. interpreted the data and wrote the paper. All authors read, edited and approved the paper.

Corresponding authors

Correspondence to
Karin Gustafsson or David T. Scadden.

Ethics declarations

Competing interests

D.T.S. is a director and shareholder of Agios Therapeutics and Editas Medicines; a shareholder and founder of Fate Therapeutics and Stratus Therapeutics; a director, founder and shareholder of Lightning Biotechnology; a director of Sonata Theraepeutics; and a consultant for Simcere Zaiming and VCanBio. D.T.S. and K.G. are inventors of patent US20220143099A1. P.V.K. serves on the scientific advisory board for Celsius Therapeutics, Inc. and Biomage, Inc. The other authors declare no competing interests.

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Peer review information

Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Flow sorting strategy and scRNA-seq overview of human and mouse thymic stroma.

(A) Gating strategy for flow cytometric sorting of human thymus stromal cells (CD45-CD235-Lineage-CD4-CD8-). Percentages refer to percent of parent gate. (B) Gating strategy for flow cytometric sorting of mouse thymus stromal cells (CD45-Ter119-). Percentages refer to percent of parent gate. (C) Distribution of number of expressed genes and number of UMIs per cell in the human and mouse data sets. (D) UMAP embedding of human thymus cell populations including CD3E + CD4 + CD8B + PTPRC+ hematopoietic cells with detailed annotation. (n = 3, total number of cells=21034) Three independent experiments. (E) Expression of marker genes for human hematopoietic cells visualized on joint embedding. All CD3E + CD4 + CD8B + PTPRC+ cells were excluded from future analysis. (F) UMAP embedding of mouse thymus cell populations including Cd3e + Cd4 + Cd8a+ Ptprc+ hematopoietic cells with detailed annotation. (n = 4, total number of cells=13952) Four independent experiments. (G) Expression of marker genes for mouse hematopoietic cells visualized on joint embedding. All Cd3e + Cd4 + Cd8a+ Ptprc+ cells were excluded from future analysis.

Extended Data Fig. 2 Marker and differential gene expression of thymic stromal subsets in human and mouse.

(A) The top most differentially expressed genes in each major human stromal cell subset shown as a dot plot. (B) Expression of marker genes for each major human stromal cell subset shown as a dot plot. (C) The top most differentially expressed genes in each major mouse stromal cell subset shown as a dot plot. (D) Expression of marker genes for each major mouse stromal cell subset shown as a dot plot. (E) Genes defining neural crest-like (NC) cells in mouse tissue shown as a dot plot. (F) Gating strategy for human thymic epithelial subsets. Only live, singlet cells are present in the data presented and percentages are of parent gate. cTECs are defined as EpCam+ Pdpn+CD49f + CD200-, immature mTECs as EpCam+ Pdpn+CD49f-CD200 + HLA-DR^Low and mature mTECs as EpCam+ Pdpn+CD49f-CD200 + HLA-DR^Hi. (G) Representative histograms showing flow cytometric analysis of IL15, CCL19 and FLT3LG in human ECs, MCs and TECs. The various stromal cell populations were gated in accordance with the strategy presented in Extended Data Fig. 2F.

Extended Data Fig. 3 Gene expression programs delineate three ThyMC subsets in human and mouse.

(A) Dot plots displaying genes differentially expressed across human and mouse thymic ThyMC subsets. (B) Expression of marker genes CD248, PENK, COL15A1, POSTN that define each ThyMC subtype in human (left) and mouse (right). Presented as UMAP embeddings and bar graphs displaying expression in each cell type. Statistical significance is based on a two-sided DESeq2-based Wald test with Benjamini-Hochberg FDR control of these comparisons. (human n = 3, mouse n = 4, mean ± SEM). Human samples: CD248 CD248+ ThyMC vs. POSTN+ ThyMC **** p = 1.6×10⁻¹⁶. CD248+ ThyMC vs. COL15A1+ ThyMC **** p = 5.5×10⁻⁷. COL15A1 COL15A1+ ThyMC vs. POSTN+ ThyMC **** p = 3.2×10⁻¹⁶. COL15A1+ ThyMC vs. CD248+ ThyMC **** p = 1.1×10⁻¹⁶. POSTN POSTN+ ThyMC vs. COL15A1+ ThyMC **** p = 4.6×10⁻¹³. Mouse samples: Cd248 Cd248+ ThyMC vs. Postn+ ThyMC **** p = 7.9×10⁻³⁶. Cd248+ ThyMC vs. Penk+ ThyMC **** p = 1.9×10⁻¹⁷. Penk Penk+ ThyMC vs. Cd248+ ThyMC *** p = 3.3×10⁻⁴. Penk+ ThyMC vs. Postn+ ThyMC * p = 1.9×10⁻². Postn Postn+ ThyMC vs. Penk+ ThyMC **** p = 2.7×10⁻⁵. Postn+ ThyMC vs. Cd248+ ThyMC **** p = 1.9×10⁻¹¹. (C) Normalized enrichment score for GSEA pathways specific to mouse Postn+ ThyMC presented as UMAP. (D) Expression of Ccl19, Flt3l, and Il15 across ThyMC subsets and other stromal cell types. (n = 4, mean ± SEM). Statistical significance is based on a two-sided DESeq2-based Wald test with Benjamini-Hochberg FDR control of these comparisons. Ccl19: Postn+ ThyMC vs. EC **** 1.49×10⁻⁸, Postn+ ThyMC vs. Cd248+ ThyMC **** p = 7.2×10⁻¹⁰, Postn+ ThyMC vs. vSMC **** p = 6.8×10⁻⁸, Postn+ ThyMC vs. PC ** p = 6.9×10⁻³, Postn+ ThyMC vs. Lrrn4+ **** p = 5.8×10⁻⁶, Postn+ ThyMC vs. Tuft cells **** p = 1.5×10⁻⁸. Flt3l: Postn+ ThyMC vs. EC **** p = 1.1×10⁻⁵, Postn+ ThyMC vs. Cd248+ ThyMC **** p = 2.6×10⁻⁶, Postn+ ThyMC vs. Penk+ ThyMC ** p = 6.9×10⁻³, Postn+ ThyMC vs. vSMC **** p = 5.6×10⁻⁶, Postn+ ThyMC vs. PC ** p = 5.0×10⁻³, Postn+ ThyMC vs. Lrrn4+ ** p = 2.4×10⁻³, Postn+ ThyMC vs. cTEC/mTECi **** p = 7.6×10⁻⁸, Postn+ ThyMC vs. mTECm ****p = 3.8×10⁻⁴, Postn+ ThyMC vs. Tuft cells ****p = 3.5×10⁻⁵. Il15 Postn+ ThyMC vs. Cd248+ ThyMC **** p = 1.1×10⁻⁹, Postn+ ThyMC vs. Penk+ ThyMC **** p = 3.7×10⁻⁵, Postn+ ThyMC vs. vSMC *** p = 9.3×10⁻⁴, Postn+ ThyMC vs. PC * p = 1.2×10⁻², Postn+ ThyMC vs. Lrrn4+ *** p = 2.6×10⁻³, Postn+ ThyMC vs. cTEC/mTECi *** p = 4.3×10⁻⁴. Postn+ ThyMC vs. Tuft cells ****p = 1.6×10⁻⁴.

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Extended Data Fig. 4 Cross-dataset integration confirms conserved ThyMC subsets in human and mouse.

(A) UMAP representation of conos joint embedding showing overlap between our human samples (colored dots) and a publicly available human dataset (gray dots). ThyMCs colored by origin (left). Overlays of individual population markers (right) indicate transcriptionally similar populations between the two sample sets. (B) UMAP of a joint conos embedding with our mouse dataset (blue dots) and publicly available mouse samples (orange dots) demonstrating significant overlap in identified populations (left panel). Annotation of CD248+ ThyMCs, Penk+ ThyMCs and Postn+ ThyMCs on joint UMAP representation of our mouse cells and a publicly available dataset (right panel). (C) UMAPs showing the expression of marker genes Cd248, Penk, and Postn that define each ThyMC subtype presented as joint conos embeddings with our dataset and publicly available mouse samples. (D) Conos joint embedding UMAP showing clustering of ThyMCs from a publicly available mouse dataset (orange dots) and our mouse samples (blue dots) (left panel). Annotation of CD248+ ThyMCs, Penk+ ThyMCs and Postn+ ThyMCs on joint UMAP representation of our mouse cells and a publicly available dataset (middle panel). Joint UMAP showing the annotation of ThyMCs identified by Handel et. al (right panel). (E) UMAPs showing the expression of marker genes Cd248, Penk, and Postn that define each ThyMC subtype presented as joint conos embeddings with our dataset and the mouse dataset published by Handel et al.

Extended Data Fig. 5 Validation of mouse ThyMC markers by flow cytometry and fibroblast colony-forming capacity.

(A) UMAP embedding of mouse thymus cell populations including Cd3e + Cd4 + Cd8b+ Ptprc+ hematopoietic cells with detailed annotation (left). ThyMC marker genes Cd99l2, Itgb5, Pdgfra and Cd248 presented as UMAP graphs (right). (n = 4) Four independent experiments. (B) FACS plot validating the presence of both CD99l2 and Itgb5 at the protein level on non-hematopoietic, non-epithelial, non-endothelial thymic cells. (C) Flow cytometric plots demonstrating the overlap between CD99l2 and Itgb5 and other cell type defining markers. Labels to the right of the flow plots refer to parent gate and the percentages are of the parent gate. (D) Image of fixed and Giemsa stained CD45-Ter119-CD31- EpCam- CD99l2+ Itgb5+ ThyMCs grown in aMEM with 20% FBS under hypoxic conditions for 6 days (n = 3). Three independent experiments. (E) Bar graphs displaying colony forming ability of individually sorted CD45-Ter119-CD31- EpCam- CD99l2+ Itgb5+ and CD45-Ter119-CD31- EpCam- CD99l2- Itgb5- cells grown of 7 days in aMEM with 20% FBS under hypoxic conditions. (F) Bar graph representation of bone marrow and thymic ThyMCs fibroblast colony forming units (CFU-F) after 6 days of hypoxic culture in aMEM with 20% FBS (n = 6 per group, mean ± SEM). Two independent experiments. (G) Flow cytometric analysis confirmation at the protein level of CD248 and Pdgfra. (H) Validation of increased Postn expression by qPCR on sorted CD248+ ThyMC compared to CD248- ThyMC. Statistical significance was calculated by a two-sided, unpaired student’s t-test. n = 4, mean ± SEM, from two independent experiments. CD248+ ThyMC vs. CD248- ThyMC * p = 4.8×10⁻².

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Extended Data Fig. 6 Marker validation of Penk+ and Postn+ ThyMCs.

(A) Gating strategy for FACS analysis of mouse thymus tdTomato+ (Penk + ) ThyMCs in Penk-Cre-tdT mice. Percentages refer to percent of parent gate. (B) FACS plots showing overlap between tdTomato+ cells and thymic epithelium (EpCam), endothelium (CD31), hematopoietic cells (CD45), and myeloid cells (F4/80, CD11b) in Penk-Cre-tdT mice. (C) Representative histograms from flow cytometric analysis of intracellular Cre protein staining in hematopoietic cells (CD45) and ThyMC (CD248-). (D) Bar graphs showing Postn and Ccl19 expression as determined by qPCR on sorted tdTomato+ (Penk ThyMC) and tdTomato- (Postn+ ThyMC) cells from Penk-Cre-tdT mice (n = 4, mean ± SEM). Two independent experiments. (E) Gating strategy for flow cytometric analysis of mouse thymus tdTomato+ (Postn + ) ThyMCs after in vivo administration of 4-hydroxytamoxifen in Postn-CreERT-tdT mice. Percentages refer to percent of parent gate. (F) Flow cytometric analysis of overlap between tdTomato+ cells and thymic epithelium (EpCam), endothelium (CD31), and hematopoietic cells (CD45) in Postn-CreER-tdT mice. (G) Bar graphs showing Postn and Ccl19 expression as determined by qPCR on sorted tdTomato+ (Postn+ ThyMC) and tdTomato- (Penk+ ThyMC) cells from 4-hydroxytamoxifen induced Postn-CreERT-tdT mice (n = 4, mean ± SEM). Two independent experiments. (H) Detailed ThyMC annotation presented as ThyMC specific UMAP embedding (left). UMAP showing expression of marker genes DPP4 (Dpp4), PDPN (Pdpn), LTBR (Ltbr), S100A4 (S100a4) in human (top) and mouse (bottom). (I) Representative histogram showing staining of S100A4 and Ltbr on thymic ThyMCs from Penk-Cre-tdT mice. (J) Bar graphs showing mean fluorescent intensity (MFI) on CD248+ ThyMC, Penk+ ThyMC and Postn+ ThyMC as determined by flow cytometry in Penk-Cre-tdT mice (n = 3, mean ± SEM). Two independent experiments. (K) FACS plots showing staining for DPP4 and Pdpn on CD248+ ThyMC, Penk+ ThyMC and Postn+ ThyMC from Penk-Cre-tdT mice. (L) Bar graphs showing the percentage of phenotypic cortical fibroblasts (cFib; DPP4+ Pdpn + ) and medullary fibroblasts (mFib; Dpp4- Pdpn + ) within CD248+ ThyMC, Penk+ ThyMC and Postn+ ThyMC subsets from Penk-Cre-tdT mice. (n = 3, mean ± SEM). Two independent experiments. Statistical significance is based on beta regression with Benjamini-Hochberg FDR control of these comparisons. Statistical significance is using a two-sided Beta regression-based Wald test with Benjamini-Hochberg FDR control of these comparisons. CD248+ ThyMC: cFib vs. mFib **** p = 3.8×10⁻¹³⁶. Penk+ ThyMC: cFib vs. mFib **** p = 2.1×10⁻⁶⁵. Postn+ ThyMC: cFib vs. mFib p = 0.21.

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Extended Data Fig. 7 Thymus stroma compositional changes with transplantation, IL7R KO, and aging.

(A) UMAP embedding showing stromal cell (Cd3e-, Cd4-, Cd8a-, Ptprc-) compositional differences between Control (n = 4, total number of stromal cells=5451), Transplantation (n = 3, total number of stromal cells=8057), IL7RKO (n = 4, total number of stromal cells=4551) and Aging (n = 4, total number of stromal cells=16178) samples. (B) Bar graphs comparing proportional shifts of different stromal cell subsets between Control, Transplantation, IL7RKO and Aging samples as determined by scRNAseq. (Control n = 4, Transplantation n = 3, IL7RKO n = 4, Aging n = 4, mean ± SEM). NCs were excluded from this analysis due to the populations small size (6.5 cells per sample in Controls). Statistical significance is using a two-sided Beta regression-based Wald test with Benjamini-Hochberg FDR control of these comparisons. EC: Control vs. Aged ** p = 7.1×10⁻³. PC: Control vs. IL7R KO ** p = 3.1×10⁻³, Control vs. Aged ** p = 1.7×10⁻³. vSMC: Control vs. Aged ** p = 6.0×10⁻³. (C) Dot plot showing expression of neural crest-like (NC) cell marker genes (rows) across different stress states (columns). Total number of NCs per condition noted in parenthesis. (D) Expression of marker genes defining mouse osteogenic smooth muscle cells (oSMC) presented as dot plot. (E) Bar graphs showing the difference in proportions within the MC population of Penk+ ThyMCs in Control, Transplantation, and Aging samples as determined by scRNAseq. (Control n = 4, Transplantation n = 3, Aging n = 4, mean ± SEM). Statistical significance is using a two-sided Beta regression-based Wald test with Benjamini-Hochberg FDR control of these comparisons. Control vs. Transplant **** p = 2.0×10⁻²⁸, Control vs. Aged **** p = 3.0×10⁻⁸. (F) Bar graphs showing Postn and Ccl19 expression as determined by qPCR on sorted CD248- ThyMC 2 months and 22-27 months old mice (n = 4, mean ± SEM). Statistical significance was calculated by unpaired two-tailed student’s t-test. Postn: Control vs. Aged **p = 9.9×10⁻³. Ccl19: Control vs. Aged ** p = 8.4×10⁻³.

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Extended Data Fig. 8 ThyMC stress responses and Penk+ ThyMC ablation.

(A) Heatmap displaying gene set enrichment analysis (GESA) of differentially expressed genes between Transplantation (n = 3) and Control (n = 4) samples, as well as Aging (n = 4) and Control (n = 4) samples across different mouse ThyMC subtypes. Significance level marked as dot corresponds to FDR between 0.05 and 0.25 following official GSEA User Guide. (B) Heatmap showing the expression of Ccl19, Flt3l and Il15 in different ThyMC subsets, across different stress states. (Control n = 4, Transplantation n = 3, Aging n = 4). (C) Bar graphs showing the results of ELISA measurements of Ccl19 and Flt3l protein levels in whole thymic tissue in Control (n = 5) and Transplant (n = 6) samples. (Mean ± SEM) Statistical significance was calculated by unpaired two-tailed student’s t-test. Ccl19: Control vs. Transplant * p = 4.3×10⁻². Flt3l: Control vs. Transplant ** p = 7.2×10⁻³. (D) Representative flow plots showing the percent of tdTomato labeled MC in Penk-Cre-tdT/iDTR and Penk-Cre-tdT mice respectively after diphtheria toxin mediated depletion. The analyzed cells are CD248- MC, gated on as described in Extended Data Fig. 6A. Percentages refer to percent of parent gate. (E) Bar graphs showing FACS quantification of stromal cell subsets in Control (iDTR Ctrl n = 6) and Penk-Cre-tdT/iDTR (Penk iDTR n = 7) mice 6 days post-bone marrow transplantation and diphtheria toxin injection. (Mean ± SEM) Statistical significance was calculated by unpaired two-tailed student’s t-test. Two independent experiments. CD248- ThyMC: iDTR Ctrl vs. Penk-iDTR **p = 4.9×10⁻³, CD248+ ThyMC: iDTR Ctrl vs. Penk-iDTR *p = 4.0×10⁻². (F) Flow cytometric analysis of thymocyte subsets Control (iDTR Ctrl n = 7) and Penk-Cre-tdT/iDTR (Postn iDTA n = 7) mice 6 days post-bone marrow transplantation and diphtheria toxin injection. (Mean ± SEM) Statistical significance was calculated by unpaired two-tailed student’s t-test. Two independent experiments. (G) Total number of thymocytes in Control (iDTR Ctrl n = 6) and Penk-Cre-tdT/iDTR (Postn iDTA n = 7) mice 6 days post-bone marrow transplantation and diphtheria toxin injection presented as bar graphs. (Mean ± SEM). (H) Representative flow plots showing the changes in ThyMC subsets in Control, PostnCreER-tdT/iDTA and PenkCre-tdT/iDTR mice. The analyzed cells are gated on as described in Extended Data Fig. 6A. Percentages refer to percent of parent gate. (I) Representative flow plots showing the changes in thymic progenitors in in Control, PostnCreER-tdT/iDTA and PenkCre-tdT/iDTR mice. The analyzed cells are gated on live singlet CD4- and CD8- thymocytes, except for the ETP gate which is set on DN1 thymocytes. Percentages refer to percent of parent gate.

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Extended Data Fig. 9 Postn+ ThyMC ablation and Ccr7 expression.

(A) Representative flow plots showing the extent of tdTomato depletion ThyMC in Postn-CreERT-tdT/iDTA and Postn-CreERT-tdT samples respectively after 4-HT induction. The analyzed cells are CD248- ThyMC, gated on as described in Extended Data Fig. 6E. Percentages refer to percent of parent gate. (B) Bar graphs showing the flow cytometric quantification of stromal cell subsets in Control (iDTA Ctrl n = 4) and Postn-CreER-tdT/iDTA (Postn iDTA n = 6) mice 6 days post-bone marrow transplantation and 4-HT induction. (Mean ± SEM) Statistical significance was calculated by two-tailed student’s t-test. Two independent experiments. CD248- ThyMC: iDTA Ctrl vs. Postn-iDTA *p = 1.3×10⁻². CD248+ ThyMC: iDTA Ctrl vs. Postn-iDTA ***p = 7.1×10⁻⁴. (C) FACS analysis of thymocyte subsets Control (iDTA Ctrl n = 4) and Postn-CreER-tdT/iDTA (Postn iDTA n = 6) mice 6 days post-bone marrow transplantation and 4-HT induction. (Mean ± SEM) Statistical significance was calculated by unpaired two-tailed student’s t-test. Two independent experiments. (D) Total number of thymocytes in subsets Control (iDTA Ctrl n = 4) and Postn-CreER-tdT/iDTA (Postn iDTA n = 6) mice 6 days post-bone marrow transplantation and 4-HT induction presented as bar graphs. (Mean ± SEM). (E) Analysis of DN1, DP, SP CD4, SP CD8 and Penk+ ThyMCs by flow cytometry, presented as absolute numbers per thymus in Penk-Cre-tdT mice Day 0-Day 14 following sublethal irradiation. (Day 0 = 5, Day 1 = 3, Day 3 = 4, Day 7 = 3, Day10 = 3, Day14 = 4) Two independent experiments. (Mean ± SEM) Statistical significance was calculated using unpaired two-tailed student’s t-test. DN1: Day 0 vs. Day 14 *** p = 6.5×10⁻⁴. Dashed lines indicates baseline levels of parameter measured. (F) Scatter plot showing correlation between Postn+ ThyMC (x-axis) and ETP numbers (y-axis, intercept: 2235.21, slope 17.05, Gray=95 CI based on 1000 bootstarp replicates for regression prediction boundaries) (left), ETP numbers (x-axis) and Ccl19 protein levels (y-axis, intercept: 112.14, slope 0.12, Gray=95 CI based on 1000 bootstarp replicates for regression prediction boundaries) (middle) and Postn+ ThyMC numbers (x-axis) and Ccl19 protein levels (y-axis, intercept: 105.89, slope 0.01, Gray=95 CI based on 1000 bootstarp replicates for regression prediction boundaries) (right). Statistical significance was calculated using a two-sided Pearson’s correlation test. (G) Dot plots showing the transcript levels of CCR7 across human and mouse thymus cell populations. (H) Bar graphs showing protein levels of Ccr7 across mouse thymus hematopoietic and stromal cell subsets as determined by flow cytometric analysis (n = 3, mean ± SEM). Two independent experiments. (I) Representative histogram showing flow cytometric analysis of Ccr7 expression on early thymic progenitor (ETP) cells. Representative histograms from flow cytometric quantification of Ccr7 protein levels on various hematopoietic and stromal cell populations from mouse thymus. (J) Representative histograms from flow cytometric quantification of Ccr7 protein levels on various hematopoietic and stromal cell populations from mouse thymus.

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Extended Data Fig. 10 Detection and quantification of ThyMCs and thymic compartments after intrathymic transfer.

(A) Representative flow plots showing the presence of GFP+ Penk+ ThyMCs and tdTomato+ Postn+ ThyMCs in the thymi of bone marrow transplantation recipients 6 days post-intrathymic injection of 5 000 cells. (B) Flow cytometric analysis of intrathymically injected GFP+ (Penk + ) ThyMC or tdTomato+ (Postn + ) ThyMCs 6 days ThyMCs 6 days after injection and bone marrow transplantation, presented as absolute numbers per thymus. (Dose of ThyMC 4000-8000) (Sham=9, Penk+ ThyMC=14, Postn+ ThyMCs=10, mean ± SEM) Three independent experiments. (C) Total number of thymocytes in recipients of Sham, GFP+ (Penk + ) ThyMC or tdTomato+ (Postn + ) ThyMCs 6 days after intrathymic injections and bone marrow transplantation,. (Dose of ThyMC 4000-8000) (Sham=9, Penk+ ThyMC=14, Postn+ ThyMCs=10, mean ± SEM) Three independent experiments. (D) FACS quantification of thymus endothelium 6 days after intrathymic injection of Sham, GFP+ (Penk + ) ThyMC or tdTomato+ (Postn + ) ThyMCs displayed as bar graphs. (Dose of ThyMC 4000-8000) (Sham=9, Penk+ ThyMC=14, Postn+ ThyMCs=10, mean ± SEM) Three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis. Sham vs. Postn+ ThyMC ** p = 1.3×10⁻³; Penk+ ThyMC vs. Postn+ ThyMC ** p = 7.7×10⁻³. (E) Assessment of thymus epithelium and ThyMCs 6 days after intrathymic injection of Sham, GFP+ (Penk + ) ThyMC or tdTomato+ (Postn + ) ThyMCs displayed as bar graphs. (Dose of ThyMC 4000-8000) (Sham=9, Penk+ ThyMC=14, Postn+ ThyMCs=10, mean ± SEM) Three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis. (F) Flow cytometric plots showing GFP labeled cells 6 days after adoptive transfer of 2000-4000 CD8 + T cells or CD248- ThyMCs. (G) Flow cytometric quantification of adoptively transferred GFP + CD248- ThyMCs 1-16 weeks following intrathymic injection and HSCT. (Day 6 = 10, Week 4 = 5, Week 8 = 5, Week 16 = 8, mean ± SEM) Five independent experiments. (H) 3D confocal microscopy images of thymic tissue 6 days following intrathymic injection of GFP + CD248- ThyMCs into bone marrow transplantation recipients. Tissue was stained with DAPI (white; cell nuclei), GFP (green; ThyMC or autofluorescent adipocytes) and BODIPY (red; adipocytes). Due to the autofluorescence of adipocytes and the fact that these cells increase in frequency following radiation conditioning, BODIPY staining was carried out to confirm GFP signal. (I) Bar graphs displaying the absolute number of thymocytes in thymi from recipients of intrathymic injections of Sham, CD8 + T cells and CD248- ThyMCs, 6 days after injections and bone marrow transplantation. (Dose of CD8 + T cells and CD248- ThyMCs: 2000-4000) (Sham n = 4, CD8 + T cells n = 6, CD248- ThyMC n = 8, mean ± SEM) Two independent experiments. (J) Bar graphs displaying the flow cytometric quantification of endothelial cells, 6 days following an intrathymic sham injection or adoptive transfer of CD8 + T cells and CD248- ThyMCs. (Dose of CD8 + T cells and CD248- ThyMCs: 2000-4000) (Sham n = 4, CD8 + T cells n = 6, CD248- ThyMC n = 8, mean ± SEM) Two independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis. Sham vs. CD248- ThyMC * p = 2.1×10⁻²; CD8 + T cells vs. CD248- * p = 4.3×10⁻². (K) FACS analysis of thymic epithelial cells and ThyMCs 6 days following an intrathymic sham injection or adoptive transfer of CD8 + T cells and CD248- ThyMCs. (Dose of CD8 + T cells and CD248- ThyMCs: 2000-4000) (Sham n = 4, CD8 + T cells n = 6, CD248- ThyMC n = 8 mean ± SEM) Two independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis.

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Gustafsson, K., Isaev, S., Mirsanaye, K. et al. Mesenchymal thymic niche cells enable regeneration of the adult thymus and T cell immunity.
Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02864-w

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Received: 25 June 2023
Accepted: 12 September 2025
Published: 30 October 2025
DOI: https://doi.org/10.1038/s41587-025-02864-w

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Extended Data Fig. 1 Flow sorting strategy and scRNA-seq overview of human and mouse thymic stroma.

Extended Data Fig. 2 Marker and differential gene expression of thymic stromal subsets in human and mouse.

Extended Data Fig. 3 Gene expression programs delineate three ThyMC subsets in human and mouse.

Extended Data Fig. 4 Cross-dataset integration confirms conserved ThyMC subsets in human and mouse.

Extended Data Fig. 5 Validation of mouse ThyMC markers by flow cytometry and fibroblast colony-forming capacity.

Extended Data Fig. 6 Marker validation of Penk+ and Postn+ ThyMCs.

Extended Data Fig. 7 Thymus stroma compositional changes with transplantation, IL7R KO, and aging.

Extended Data Fig. 8 ThyMC stress responses and Penk+ ThyMC ablation.

Extended Data Fig. 9 Postn+ ThyMC ablation and Ccr7 expression.

Extended Data Fig. 10 Detection and quantification of ThyMCs and thymic compartments after intrathymic transfer.

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