Thymus Extracellular Matrix-Derived Scaffolds Support Graft-Resident Thymopoiesis and Long-Term In Vitro Culture of Adult Thymic Epithelial Cells

Abstract

The thymus provides the physiological microenvironment critical for the development of T lymphocytes, the cells that orchestrate the adaptive immune system to generate an antigen-specific response. A diverse population of stroma cells provides surface-bound and soluble molecules that orchestrate the intrathymic maturation and selection of developing T cells. Forming an intricate 3D architecture, thymic epithelial cells (TEC) represent the most abundant and important constituent of the thymic stroma. Effective models for in and ex vivo use of adult TEC are still wanting, limiting the engineering of functional thymic organoids and the understanding of the development of a competent immune system. Here a 3D scaffold is developed based on decellularized thymic tissue capable of supporting in vitro and in vivo thymopoiesis by both fetal and adult TEC. For the first time, direct evidences of feasibility for sustained graft-resident T-cell development using adult TEC as input are provided. Moreover, the scaffold supports prolonged in vitro culture of adult TEC, with a retained expression of the master regulator Foxn1. The success of engineering a thymic scaffold that sustains adult TEC function provides unprecedented opportunities to investigate thymus development and physiology and to design and implement novel strategies for thymus replacement therapies.

Keywords: 3D scaffolds; decellularization; thymic epithelial cells; thymus engineering.

Figure 1

Decellularization of murine thymus in perfusion bioreactor allows efficient removal of cells and nuclear matter, while preserving extracellular matrix proteins. A) Murine thymi were decellularized in a perfusion bioreactor. B) Photographs and representative images of hematoxylin and eosin‐stained sections of thymic lobes before and after the decellularization process. Scale bar: 500 µm. C) Quantification of DNA content by Picogreen assay in native (N) and decellularized (D) thymi. D) Immunofluorescence staining for collagen IV, fibronectin, and laminin in native and decellularized tissue (green). Nuclei were stained by DAPI (blue).

Figure 2

Use of decellularized murine thymus for the production of 3D porous scaffolds with preserved ECM composition. A) Schematic representation of the 3D scaffold production process. B) Scanning electron microscopy images of longitudinal and transversal sections of the 3D thymus scaffold (TS). C) Immunofluorescence staining for collagen IV, fibronectin, and laminin in TS. Scale bar: 50 µm. D) Quantification of collagen IV, laminin, and fibronectin in native thymus (N) and 3D thymus scaffold (TS). Data are normalized over the total protein content.

Figure 3

Physical characterization of 3D thymus scaffold (TS) in comparison to native (N) and decellularized (D) thymus: A) swelling ratio over time and B) water content at equilibrium.

Figure 4

Nanomechanical characterization of native thymus (N), decellularized tissue (D), and 3D thymus scaffold (TS). A) Schematic representation of the atomic force microscopy working principle (modified with permission from Asgeirsson et al.).^{[ 35 ]} B) Comparison of backward elastic modulus. C–F) Representative force maps and G–J) elastic modulus distribution from each group, including a soft and a stiff TS.

Figure 5

TS seeded with fetal E14.5 stroma cells support the development of E14.5 CD4^negCD8^neg DN thymocytes to the CD4^posCD8^pos DP stage in vitro. Single‐cell suspensions obtained from collagenase digested fetal E14.5 thymic lobes were seeded on TS or mixed with decellularized tissue that has not been subjected to freeze/dry cycles. Reaggregate thymic organ cultures (RTOC) without TS were set up as positive controls. A) Macroscopic appearance of organoids after 5 d culture on floating filters and B) flow cytometric analysis of CD4 and CD8 expression. Data for RTOC and TS are representative of two independent experiments.

Figure 6

TS seeded with fetal TEC and grafted into athymic recipient mice support continuous de novo development of functionally competent thymocytes that repopulate the peripheral lymphoid organs. A) Schematic setup of in vivo TS grafting using congenic athymic nude mice. B) Kidney of host showing the successful establishment of thymic organoid 8 weeks after transplantation in one of three mice. C) Flow cytometric analysis of host‐derived T‐cell developmental stages within the graft. Blue arrow gating shows the maturational progression of early thymic progenitors, red arrow gating details CD4 SP differentiation stages. D) Immunohistological analysis of graft section detailing cytokeratin 8 (red) and cytokeratin 14 (gray) expression combined with nuclear DAPI stain (blue). E) Lymph nodes of grafted or control nude mice were stained for donor (CD45.1^pos) and host (CD45.1^neg) derived T cells. Naïve CD8 (gray arrow gating) and CD4 T cells (green arrow gating) were quantified and CD4 T cells were additionally analyzed for the presence of regulatory cells. F) Host derived CD4 T cell sort strategy and purities of conventional (CD4_conv) and regulatory (CD4_reg) cells. G) In vitro proliferation of CD3 stimulated CD4_conv in the absence or presence of CD4_reg added in a 1:2 ratio after 3 d of culture. Numbers indicate frequencies of divided cells with standard deviation (n = 4 w/o CD4_reg, n = 3 with CD4_reg).

Figure 7

TS seeded with FACS sorted adult or embryonic TEC support limited thymocyte development from the DN to the DP stage in vitro. A) Schematic setup of TS cultures with FACS sorted CD45^negEpCAM^pos adult TEC and TCR^neg DN thymocytes. Phenotypes before and after sorting are shown. B) Analysis of CD4 and CD8 expression after 5 d of culture in adult TEC seeded TS. Gating strategies and control thymus stain are shown. C) Schematic setup and outcome of TS cultures comparing the developmental progression of sorted DN thymocytes seeded on TS with either FACS sorted adult or fetal TEC.

Figure 8

TS seeded with adult TEC enriched cell suspensions and grafted into athymic recipient mice support de novo development and export of host‐derived T cells. A) Schematic in vivo setup of TS seeded with TEC enriched cell suspension isolated from CD45.1 adult mice thymic and grafted into CD45.2 congenic athymic nude mice. Phenotype of CD45 depleted, TEC enriched cell suspension is displayed. B) Kidneys of host mice showing graft remnants (outlined) and their thymocyte phenotype. C) T cell composition within the lymph nodes of the hosts harboring grafts with or without resident thymocytes as shown in B. D) Representative flow cytometric and E) quantitative analysis of host and donor derived T cell subsets 8 weeks after grafting TEC enriched adult thymus digests seeded on TS compared to non‐grafted control animals. Data represent two independent experiments with B) 1 out of 2 and D,E) 0 out of 4 successful reconstitutions.

Figure 9

TS support long‐term in vitro maintenance of adult TEC. A) Frequencies of EpCAM^pos cells derived from TEC enriched cell suspensions after 4 and 10 d of culture in CnT‐57 medium. B) Confocal images of TEC enriched GFP+ cells cultured in vitro onto 3D thymus scaffolds at different time‐points after seeding.

Figure 10

TS support prolonged in vitro culture of adult TEC expressing characteristic markers. Immunofluorescence analysis for the expression of cytokeratin 8 (K8, cortical TEC marker, red) and cytokeratin 14 (K14, medullary TEC marker, white), and of FoxN1 (transcription factor, pink) by adult TEC after 30 d of in vitro culture onto TS. Nuclei were stained by DAPI (blue). Scale bars: 50 µm.