In addition to the identification of strategies that can boost the recovery of residual thymic functionality, substantial progress has also been made over the past years to the engineering of a transplantable artificial thymus, Chaudhry et al (2017). Immense effort has been invested in the past decades in order to characterize and rebuild in vitro the complex 3D structure that confers the thymus its specialized microenvironment. A particularly important area of investigation is the identification of biomaterials that can reproduce the 3D artificial matrix able to support cell-to-cell interactions. However, while the proof of concept has been demonstrated that 3D matrices seeded with thymic stromal cells can partially support T cell development from precursor hematopoietic cells, Pinto et al (2013) the field has been limited by a lack of a sustaining source of epithelial cells to seed.
However, recent studies have used several approaches that could overcome this barrier, including 1) identifying endogenous thymic epithelial progenitor cells (TEPC), 2) driving differentiation of embryonic stem cells or iPS cells into TEPC, and 3) transdifferentiation of other cell lineages into TEC-like, T cell supporting cells. In history, there are countless attempts that has been made for thymus correction defects, manipulation of the thymus, either in vitro or in vivo and it didn’t failed to prove to be challenging. This is mainly attributed to the unique architecture of the thymic stroma that is essential for the maturation, survival, and function of the Thymic Epithetial Cell (TECs).
Unlike epithelial cells of other visceral organs, which form a two-dimensional (2-D) sheet-like structure on the basement membrane to create borders within and between organs, TECs form a sponge-like three-dimensional (3-D) network that is essential for their function. TECs cultured on irradiated 3T3 feeders (a 2-D environment) are unable to support T-cell differentiation from lymphocyte progenitors, but start to express markers of terminally differentiated epithelial cells. Recently, TEC stem cells derived from early embryos were shown to differentiate into skin cells when cultured in 2-D environment. Indeed, the expression of key genes for the specification and proliferation of TECs are shown to be dependent on the 3-D organization of the thymic stroma, further indicating that the unique microenvironment of the thymus is essential to maintain the unique property of TECs to support T lymphopoiesis (Fan et al., 2015).For many years, essential progress has been made to re-evaluate the thymic microenvironment. Matrigel and other collagen-based synthetic matrices were shown to be able to support limited differentiation of lymphocyte progenitors into T-cells, Tajima et al.
(2015). The artificial 3-d matrix has been used to culture the TECs and are viable and can support even not in full the thymocyte development. previously, Pinto et al.
(2015). developed a coculture system, in which mTECs were layered on top of a 3-D artificial matrix embedded with human skin-derived dermal fibroblasts. Under such conditions, mTECs can retain some of their key features (e.g., expression of FoxN1, Aire, and tissue-specific antigens).In a similar approach, Chung et al.
(2015). mixed TECs and thymic mesenchyme, both isolated from postnatal human thymus, with CD34+ cells from cord blood to form implantable thymic units. The thymic microenvironments of these thymic reaggregates can support thymopoiesis in vitro and are able to generate a complex T-cell repertoire when transplanted in nonobese diabetes (NOD).scid gamma humanized mice in vivo. However, to date, none of these approaches has been able to fully recapitulate the function of a thymus.
Lately, in the “cell-scaffold” technology there are significant advances that has been made. With the use of tissue decellularization methods. In this technique, all cells are removed from the organ, leaving the extracellular matrix intact. It has been reported that decellularization of the thymus, followed by reconstitution with thymic stromal cells and lineage negative BM progenitors, led to formation a functional thymus when transplanted into the kidney capsule of nude mice, Fan et al (2015). Allowing the the clearance of the cellular constituent of any organ of any scale, whith the use of a detergent-perfusion based, while retaining its original 3-D architecture and extracellular matrix (ECM) components.
Repopulating the decellularized natural scaffolds with tissue-residing mature cells or progenitor/stem cells can promote its recellularization and partially recover organ function.To date, these “cell-scaffolds” have been primarily applied to manufacture and implant relatively simple organs, such as tissue engineered vascular grafts and skin, with some success. Repopulating the decellularized natural scaffolds with tissue-residing mature cells or progenitor/stem cells can promote its recellularization and partially recover organ function. To date, these “cell-scaffolds” have been primarily applied to manufacture and implant relatively simple organs, such as tissue engineered vascular grafts and skin, with some success ,Goh et al. (2015) regeneration of complex organs such as liver, heart, lung, and kidney has also been attempted in animal models. Although limited, encouraging functional regeneration of the engineered organs was observed.
Furthermore, a successful clinical implantation of reconstructed decellularized trachea underlines the clinical potential of this technology. (Fan et al., 2015). The result of the investigation of the bioengineering thymus organoids with the decelluliarized thymus scaffolds has led to allow the removal off all the cellular elements of a mouse thymus while maintaining all the major ECM components. The used of decellularizing treatment has made the thymic stromal ECM largely intact, this is revealed through the use of scanning electron microscopy (SEM) analysis with the acellular thymic scaffolds’ cross-section image.
Bioengineered thymus can support T lymphopoiesis in vivo. The capability of the bioengineered thymus to support effective thymocyte development and maturation in vivo was examined with transplantation experiments. Thymus organoids reconstructed with mixtures of TSCs and Lin– BM progenitors at 1 : 1 ratio, both harvested from B6.
CD45.1 mice, were transplanted underneath the kidney capsules of B6.nude athymic recipients (designated as Tot.B6.nude for thymus organoid transplanted B6.nude mice hereinafter). Homing of hematopoietic progenitors to the thymus is an intermittent, gated process, alternating between ~1 week of receptive period and ~3 weeks of refractory period. The complement of BM progenitors was used to ensure the continuity of cross talk between TECs and the developing thymocytes that is essential for the survival of TECs, at the early post-transplantation stage.
The origins of the T-cells in the periphery were identified by FCM analysis of the CD45 congenic markers (i.e., CD45.1 and CD45.2 for donor and recipient origins, respectively).
(Banerjee et al., 2015).Effective cellular and humoral adaptive immunity mediated by T-cells matured in bioengineered thymus organoidsProliferation under various stimuli has been widely used as a tool to assess the functionality of T lymphocytes. To demonstrate that T-cells derived from the reconstructed thymus organoids are functionally competent, the authors labeled them with carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated them with anti-CD3 antibodies.
Similar to T-cells of naive B6 mice, a significant percentage of T-cells underwent division, as indicated by dilution of CFSE signals To further test the function of T-cells derived from the reconstructed thymi, the authors performed mixed leukocyte reaction experiments to evaluate their responses to alloantigens. Proliferation responses similar to those of wild-type B6 mouse were observed, indicating that these T-cells were capable to react to alloantigens .Overall, these results demonstrated that T-cells matured in the transplanted thymus organoids were capable to response to TCR stimulation. (Bertera et al., 2015)Induction of allo-skin tolerance with bioengineered thymus organoids constructed with TECs of F1 hybrid of donor and recipient miceAchieving donor-specific immune unresponsiveness, without the need for pharmacologic immunosuppression, remains a major goal of transplantation immunological research.
To prove the principle that transplantation of bioengineered thymus organoids expressing both donor and recipient major histocompatibility complexes (MHCs) can establish central tolerance to donor antigens, the authors reconstructed the acellular thymus scaffolds with TSCs harvested from the F1 offspring (B6.H-2b/g7) of a cross between B6 (H-2b) and B6.H-2g7 congenic mice, and transplanted them to the B6.nude recipients (H-2b).
The B6.H-2g7 mouse is a congenic line in which a 19 cM segment of Chr 17 including the MHC of the B6 mouse (of the H-2b haplotype) was replaced with that of the nonobese diabetes mouse line (of the H-2g7 haplotype). It was established through multiple rounds of backcrossing of the NODxB6 F1 mice to the parental B6 line. Once a substantial population of T-cells became detectable (12–16 weeks) in the peripheral bloods of the recipients, skin grafts harvested from both the syngeneic B6 (H-2b) and the allogeneic B6.
H-2g7 congenic mice were transplanted on their backs. To demonstrate that the thymus organoid transplanted recipients retained their capabilities to reject third-party alloantigens, skin grafts harvested from CBA/J (H-2k) mice were also transplanted .Successful engraftments of skin transplants from both the syngeneic B6 and the allogeneic B6.H-2g7 mice were observed, whereas the third-party CBA/J skin grafts were rejected within 2–3 weeks. Immune unresponsiveness to H-2g7 alloantigens in the recipients was further demonstrated in mixed leukocyte reaction assays. Overall, these results suggested that transplantation of bioengineered thymus organoids coexpressing both syngeneic and allogeneic MHCs can effectively establish donor-specific immune tolerance. (Rudert et al.
, 2015).Induction of allo-skin tolerance with bioengineered thymus organoids constructed with mixture of TECs from both the donor and the recipientAlthough reconstructing thymus organoids with TECs coexpressing both donor- and recipient-MHCs can effectively induce tolerance to donor MHC-expressing grafts, it is not clinically feasible to transfer donor MHC genes to the recipient’s TECs at such high efficiency (100% in the case of F1 TECs), using currently available gene engineering techniques. Moreover, epitopes derived from mismatched genes other than the MHCs in the allogeneic donor organ(s) can also contribute to its rejection.
A possible way to overcome these obstacles is to incorporate the donor TECs in the thymus organoids, together with the recipient’s TECs. To test this hypothesis, the authors performed the experiments schematically illustrated. TECs harvested from B6 (H-2b) and CBA/J (H-2k) were mixed at 1 : 1 ratio and coinjected with B6 BM progenitors to the decellularized thymus scaffolds. B6.nude mice reconstructed with the thymus organoids were challenged with allogeneic CBA/J skins, as well as skin grafts from the third-party Balb/C mice (H-2d). Prolonged survival of the CBA/J skin allografts was observed.
Consistently, results of mixed leukocyte reaction experiments revealed that when challenged with CBA/J APCs, the levels of T-cell proliferation were significant lower than those stimulated with Balb/C APCs. These findings further suggested that including donor TECs in the reconstruction of thymus organoids might be a clinical applicable way to induce donor-specific immune tolerance. (Trucco et al., 2015).