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.

Combinatory differentiation of human induced pluripotent stem cells generates thymic epithelium that supports thymic crosstalk and directs dendritic- and CD4/CD8 T-cell full development

“…Lastly, the effect of BMP, FGF10, EGF, IGF1 and RANKL modulation on TEP fate induction between D11 and D13 was investigated ( Figure 1B ). These pathways were selected based on studies of thymic organogenesis regulation (Balciunaite et al, 2002; Barbarulo et al, 2016; Bleul and Boehm, 2005; Davenport et al, 2016; Provin and Giraud, 2022) and previous thymic differentiation of hESc (Parent et al, 2013; Sun et al, 2013). A Plackett-Burman combinatorial screening design was performed for each of the three transitions by testing multiple combinations of factors with two modalities of concentration ( Supp Figure 1, D ).…”

“…Mimicking thymus organogenesis in vitro to generate TECs from embryonic (ESc) and induced pluripotent stem cells (iPSc) has been a long-standing objective over the last decade, using both reprogramming and mainly direct differentiation approach mainly (Bredenkamp et al, 2014; Chhatta et al, 2021; Gras-Peña et al, 2022; Parent et al, 2013; Ramos et al, 2022; Soh et al, 2014; Sun et al, 2013). However, the complexity of the regulation of the differentiation process and our limited comprehension of in vivo mechanisms hindered the achievement of this goal (Provin and Giraud, 2022). Indeed, thymic differentiation often yields immature and non-functional cells with a progenitor (TEP) identity and requires grafting in vivo to complete the maturation (Parent et al, 2013; Ramos et al, 2022; Sun et al, 2013).…”

Induced Pluripotent Stem Cells: Advances and Applications in Regenerative Medicine

“…The thymus plays a significant role in the establishment of immunological selftolerance and is required for the generation of T cell-mediated immunity. Thyroid progenitors, thymic epithelial cells, and thymic organoids derived from iPSCs can completely regenerate the thymus in vivo and demonstrate the potential for regenerative therapy in patients with immunodeficiency and hypothyroidism [146]. Despite the progress in generating iPSC-derived thymic cells, a poor understanding of thymus biology impedes the clinical translation of these cells.…”

Stem cell-based multi-tissue platforms to model human autoimmune diabetes

“…In vitro differentiation methods have been developed to produce thymic epithelial progenitors (TEPs) from human embryonic stem cells (hESCs) [ [110] , [111] , [112] , [113] , [114] ] and iPSCs [ 113 , [115] , [116] , [117] ] (SC-TEPs). These protocols recapitulate the multi-step development of the thymus and can generate immature SC-TEPs within ∼2 weeks (reviewed in [ 118 , 119 ]). However, these protocols have variable and relatively low efficiency, likely due to low expression of the key thymic transcription factor FOXN1.…”