Although many cell lineages generated from human pluripotent stem cells express appropriate marker genes, very few have been shown to give rise to fully differentiated functional cells. In this study, we present a method to differentiate hESCs into TEPs that mature into functional TECs when transplanted in vivo (). Our study demonstrates that the success of coaxing hESCs into functional TEPs depends on the accurate in vitro recapitulation of in vivo embryonic signaling events, including early specification of anterior-posterior and dorsal-ventral cell identities that are permissive for complete thymus development. Although it has been reported previously that anteriorization of DE can be achieved by blocking TGFβ signaling either alone (Mou et al., 2012
) or in conjugation with BMP inhibition (Green et al., 2011
; Longmire et al., 2012
), we show that activation of RA signaling is also required for accurate anterior-posterior patterning of the foregut prior to thymic specification. Indeed, expression of the transcription factor HOXA3, a member of the Hox family that specifies positional identity along the anterior-posterior axis in the developing embryo, was not induced in the absence of RA. Consistent with previous studies showing that Hoxa3 is essential for thymic specification in vivo (Manley and Capecchi, 1995
), our results strongly suggest that in vitro specification of hESCs to the thymic lineage also requires culture conditions that induce appropriate levels of expression of this transcription factor. Even though significant induction of FOXN1
expression was observed with this method, the levels of expression were lower than what is found in control thymic tissue. This is most likely due to the generation of heterogeneous cultures, as suggested by the quantification of HOXA3+
cells, which represent approximately 14% of the cells. Future studies will focus on defining conditions that enhance efficiency of differentiation to the thymic lineage as well as improving the purity of the cells by isolating them using cell surface markers yet to be identified.
Model for the Generation and Maturation of hESC-Derived TEPs in Thymus-Deficient Nude Mice
Importantly, our data reveal that transplantation of in-vitro-generated TEPs in athymic mice leads to the upregulation of multiple genes critical for TEC function. Indeed, hESC-derived TECs expressed the chemokines/cytokines CCL25
, and SCF
, which are normally produced by TECs to attract hematopoietic progenitors to the thymus and promote lymphoid progenitor development (Calderón and Boehm, 2012
). In addition, hESC-derived TECs expressed Delta-like 4 (DLL4
), a Notch ligand that induces the commitment of hematopoietic progenitors to the T cell lineage. The presence of this crucial molecule combined with the appropriate chemotactic cues thus most likely helped promote the homing and development of DP T cell progenitors in TEP grafts.
Furthermore, once T cell progenitors present in the thymus have rearranged the TCR gene and successfully progressed to the DP stage, their survival as well as their commitment to the CD4+ or CD8+ lineages depends on interaction with MHC-self-peptide complexes displayed on TECs. In addition to MHC class I molecules, which are expressed ubiquitously on most cells, the upregulation of MHC class II molecules by TEPs was likely to be critical for their ability to support positive selection of developing CD4+ T cells. The capacity of hESC-derived TECs to successfully regulate the transition of DP to SP T cells was confirmed by the presence of an increased number of diverse and responsive SP T cells in the peripheral blood and spleen of TEP-grafted mice. However, even though a significant amount of CD4+ SP T cells were generated in TEP-grafted mice, the efficiency was reduced when compared to HFT grafts, most likely due to lower MHC class II expression in hESC-derived TECs.
Importantly, T cells from TEP-grafted mice were responsive to direct TCR activation and stimulation by allogeneic antigen-presenting cells (APCs) in vitro. They could also mediate rejection of allogeneic skin grafts in vivo, further confirming their functionality. However, the response to allogeneic stimulator cells of CD4+ T cells from TEP-grafted mice was lower than what was observed in WT and HFT mice, suggesting that this subset of T cells was not able to mount efficient responses against alloantigens. Further studies will be required to address this issue in more detail. Interestingly, TECs also promoted the generation of Tregs, a subset of T cells that are crucial for the maintenance of immune tolerance through the inhibition of self-reactive T cells in the periphery. Our data thus clearly demonstrate that hESC-derived TEPs transplanted in athymic mice acquire characteristics of mature TECs that allow them to support multistage T cell development.
The ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications, including enabling modeling of human immune diseases using patient-specific induced pluripotent stem cell (iPSC) lines. It will also make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function. Importantly, since the thymus has the unique capacity to modify tolerance in an antigen-specific manner, transplantation of TEPs has the potential to promote graft-specific immune tolerance without the need for sustained immunosuppression (Nobori et al., 2006
). By allowing the generation of a T cell repertoire tolerant to stem cell self antigens, cotransplantation of stem-cell-derived TECs could prevent immune rejection of other transplants derived from the same cell line, potentially having a major impact on the effectiveness of stem-cell-based therapies. This may be a particularly attractive approach in autoimmune disease settings such as type 1 diabetes, where both β cell replacement and immunomodulation are likely required for a successful outcome.