Identification of fetal thymic epithelial stem/progenitor cells

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The debate regarding the existence and potential identity of thymic epithelial stem or progenitor cells (TESC, TEPC) has been complicated to some degree by a largely semantic confusion arising from assumptions that fetal and postnatal progenitors should have the same properties. Therefore, we will first describe current understanding of progenitor cells in the fetal thymus, and then separately discuss evidence for a postnatal TESC. 

During fetal development, the thymus arises from the endoderm of the third pharyngeal pouches (1). These bilateral structures form at around embryonic day 9.0 (E9.0) in the mouse, and it has been unequivocally demonstrated from E9.0, the third pharyngeal pouches contain some cells specified to the TE lineage (1). In terminology often applied to developing organs, these E9.0 third pharyngeal pouch cells can therefore be described as 'founder cells' for the thymic epithelial lineage - in other words, the earliest cell type which will adopt thymic epithelial but not other fates (80). While it has not been formally proven that these cells are irreversibly committed to TE fate, this is the earliest stage at which a 'TE committed' cell type has been demonstrated.

The first genetic evidence for a TEPC phenotype was provided by a study addressing the nature of the defect in nude mice, that suggested that in the absence of Foxn1, TEC lineage cells undergo maturational arrest and persist as progenitors, marked by the two antibodies MTS20 and MTS24 (81). Ontogenic analysis demonstrated that the proportion of MTS20⁺24⁺ epithelial cells is highest in the early thymus primordium, decreasing to less than 1% in the postnatal thymus (82), consistent with the expression profile expected of markers of fetal tissue progenitor cells.  The protein bound by MTS20 and MTS24 was subsequently identified as Plet-1 (83), a membrane-associated protein uniformly expressed in the third pharyngeal pouch endoderm from its formation at E9 until primordium formation at E11.5 (82-84). The functional capacity of isolated MTS20⁺24⁺ cells and MTS20^-24^- cells in the fetal thymus was assessed using ectopic transplantation. These studies showed that until at least E15.5, transplantation of limiting numbers of MTS20⁺24⁺ cells under the kidney capsule was sufficient to establish a completely functional thymus, while the MTS20^-24^- population was unable to do so (82, 85), demonstrating that a potent TEPC activity resided in the MTS20⁺24⁺ population. This conclusion was subsequently challenged by a study showing that at E15.5, both the MTS24⁺ and MTS24^- TEC compartments could form a functional thymus upon transplantation. However, this study both used large cell numbers for transplantation, and lacked analysis of the input population and therefore could not determine precursor:progeny relationships (86). As none of these experiments contained clonal analyses, they were unable to determine whether the MT20⁺24⁺ population is heterogeneous with respect to progenitor function. Therefore, while the Plet1⁺ population itself may be functionally heterogeneous, and it is probable that some Plet1^- intermediate progenitor cell types may exist in the fetal thymus at later stages, the earliest currently identified founder cells for the TEC lineage are Plet-1⁺ third pharyngeal pouch cells. Our unpublished observations further establish that diminishing Plet1 expression correlates with acquisition of differentiation markers during thymus ontogeny (Nowell and Blackburn, unpublished).

Two studies subsequently addressed the potency of TEPC at a clonal level. The hypothesis that TEPC are maintained in a state of maturational arrest in the absence of Foxn1 was tested and confirmed by an elegant study in which postnatal clonal reactivation of a conditional null allele of Foxn1 was shown to result in generation of functional thymus tissue containing organized cortical and medullary regions (87). This established unequivocally that in the absence of Foxn1, some persisting TECs have bi-potent progenitor activity. However, since the block in TE development in Foxn1^-/- thymi occurs in fetal development shortly after formation of the thymus primordium, these data demonstrated the existence of fetal TEPC, but did not address TEPC potential in the postnatal thymus. A second study used injection of single E12.5 TEC into fetal thymic lobes to demonstrate the existence of a bipotent TEPC that could contribute to both cortical and medullary TEC compartments (88).

Current evidence supports the existence of cortical and medullary sub-lineage specific progenitor cells from relatively early in organogenesis. Analysis of allophenic chimeras indicated the presence of medullary TEPC, as in this study no direct lineal relationship could be found between individual medullary islets and the surrounding cortical areas at least in early development (89). In this study, the mTEPC activity was shown to persist until at least E15.5, but the immunophenotype of the mTEPC was not determined. This issue was addressed in a later study, which identified the Cldn3,4^hi, UEA1^hi subpopulation of fetal TEC as progenitors for the Aire⁺ subpopulation of mTEC (90). Additionally, evidence suggests the existence in fetal thymus development of a cortical sub-lineage specific progenitor, characterized by expression of CD205 (91). A remaining question is the timing of emergence of the sub-lineage progenitors during organogenesis, and the extent to which they persist in the late fetal and postnatal organ.