Differentiation of Pluripotent Stem Cells Into Thymic Epithelial Cells and Generation of Thymic Organoids: Applications for Therapeutic Strategies Against APECED

Abstract

The thymus is a primary lymphoid organ essential for the induction of central immune tolerance. Maturing T cells undergo several steps of expansion and selection mediated by thymic epithelial cells (TECs). In APECED and other congenital pathologies, a deficiency in genes that regulate TEC development or their ability to select non auto-reactive thymocytes results in a defective immune balance, and consequently in a general autoimmune syndrome. Restoration of thymic function is thus crucial for the emergence of curative treatments. The last decade has seen remarkable progress in both gene editing and pluripotent stem cell differentiation, with the emergence of CRISPR-based gene correction, the trivialization of reprogramming of somatic cells to induced pluripotent stem cells (iPSc) and their subsequent differentiation into multiple cellular fates. The combination of these two approaches has paved the way to the generation of genetically corrected thymic organoids and their use to control thymic genetic pathologies affecting self-tolerance. Here we review the recent advances in differentiation of iPSc into TECs and the ability of the latter to support a proper and efficient maturation of thymocytes into functional and non-autoreactive T cells. A special focus is given on thymus organogenesis and pathway modulation during iPSc differentiation, on the impact of the 2/3D structure on the generated TECs, and on perspectives for therapeutic strategies in APECED based on patient-derived iPSc corrected for AIRE gene mutations.

Keywords: APECED; IPSC; differentiation; thymic epithelial cells (TEC); thymus; tolerance.

Figure 1

cTEC and mTEC orchestrate thymocyte maturation and selection. Early thymic progenitors (ETP) originating from the bone marrow enter the thymus and are attracted to the cortical region by chimiokines expressed by cTECs, also inducing growth and T lineage commitment by contact with NOTCH ligands. After TCR rearrangement CD4/CD8 DP thymocytes enter positive selection of the functional TCRs mediated by cTEC expressing the thymoproteasome. Resulting SP T cells are attracted to the medulla where thymocytes with auto-reactive TCRs are negatively selected by mature mTECs presenting the self-antigen repertoire under AIRE regulation. Thus, T cells with functional TCRs, able to recognize foreign antigens but tolerant to the self, are generated by the thymus.

Figure 2

Genetic and molecular networks regulating the thymus organogenesis. The thymus is a definitive endoderm derivative. The main steps of organogenesis of the pharyngeal apparatus and thymus formation are described, with time scales in mouse and human, key markers genes and signalling molecules. First, gradients of cytokines and small molecules pattern the endoderm, resulting in an anterior domain between D24-D26 in human. Further patterning results in formation of the pharyngeal domain and its segmentation in pharyngeal arches at 6 weeks. The 3PP forms under singling involving retinoic acid (RA), Hedgehog (SHH), NOTCH and fibroblast growth factors (FGF). 3PP gives rise to the thymic primordium expressing FOXN1 by week 7. By week 8, the thymic primordium migrates under TGFβ signaling to its final position and TEC mature in cTEC and mTEC.

Figure 3

A synthesis of pluripotent stem cells thymic differentiation strategies. A decade of advances in thymic differentiation resulted in perfected protocols allowing production of mature mTEC by mimicking the thymic organogenesis in vitro. For each reference, cell type used, pathway-modulating molecules timings and concentrations are indicated, as well as results in terms of nature of the obtained cells and their ability to induce T cell maturation. ActivinA (ActA), Fibroblast growth factors 7,8,10 (FGF7-8-10), Bone Morphogenetic Protein 4 (BMP4), LiCl (Lithium Chloride), Retinoic acid (RA), Cyclopamine (CPM, hedgehog inhibitor), Sonic Hedgehog (SHH), LY364947 (TGFβ inhibitor), SB43 (TGFβ inhibitor), Wingless family member 3 (WNT3), IWR1 (WNT inhibitor), Epidermal Growth Factor (EGF), CHIR99 (WNT3 agonist), LDN19 (BMP inhibitor), Noggin (NOG), SANT1 (Hedgehog inhibitor), Smoothened Agonist (SAG, Hedgehog agonist).

Figure 4

Potential cellular therapy strategies for treating APECED with induced TECs to restore thymic functionality. (A) Fibroblasts from the patient can be reprogrammed to iPSc and the defective gene corrected with gene editing tools. Differentiation of these iPSc into thymic epithelial cells, in combination with T cell progenitors differentiated from the same iPSc or directly purified from patient blood, would allow generation of artificial thymic organoids. These organoids could be either directly transplanted or used to generate competent T cells ex vivo to reconstitute the patient immune system. In this scenario T cell progenitors could be purified from patient blood or differentiated from patient iPSc. (B) Gene editing of a deficient gene from a patient can rely on CRISPR/Cas9 and be combined with reprogramming steps for the obtention of iPSc.