In lineages into TEC-like, T cell supporting cells.

In addition to theidentification of strategies that can boost the recovery of residual thymicfunctionality, substantial progress has also been made over the past years tothe engineering of a transplantable artificial thymus, Chaudhry et al (2017).

Immense effort has been invested in the past decades in order to characterizeand rebuild in vitro the complex 3D structure that confers the thymus itsspecialized microenvironment. A particularly important area of investigation isthe identification of biomaterials that can reproduce the 3D artificial matrixable to support cell-to-cell interactions. However, while the proof of concepthas been demonstrated that 3D matrices seeded with thymic stromal cells canpartially support T cell development from precursor hematopoietic cells, Pintoet al (2013)  the field has been limitedby a lack of a sustaining source of epithelial cells to seed.

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However, recentstudies have used several approaches that could overcome this barrier, including1) identifying endogenous thymic epithelial progenitor cells (TEPC), 2) drivingdifferentiation of embryonic stem cells or iPS cells into TEPC, and 3)transdifferentiation of other cell lineages into TEC-like, T cell supportingcells. Inhistory, there are countless attempts that has been made for thymus correctiondefects, manipulation of the thymus, either invitro or in vivo and it didn’tfailed to prove to be challenging. Thisis mainly attributed to the unique architecture of the thymic stroma that isessential for the maturation, survival, and function of the Thymic EpithetialCell (TECs). Unlike epithelial cells of other visceral organs, which form atwo-dimensional (2-D) sheet-like structure on the basement membrane to createborders within and between organs, TECs form a sponge-likethree-dimensional (3-D) network that is essential for their function.

TECscultured on irradiated 3T3 feeders (a 2-D environment) are unable to supportT-cell differentiation from lymphocyte progenitors, but start to expressmarkers of terminally differentiated epithelial cells. Recently, TEC stemcells derived from early embryos were shown to differentiate into skin cellswhen cultured in 2-D environment. Indeed, the expression of key genes forthe specification and proliferation of TECs are shown to be dependent on the3-D organization of the thymic stroma, further indicating that the uniquemicroenvironment of the thymus is essential to maintain the unique property ofTECs to support T lymphopoiesis (Fan et al., 2015).For many years, essential progress has been madeto re-evaluate the thymic microenvironment. Matrigel and other collagen-basedsynthetic matrices were shown to be able to support limited differentiation oflymphocyte progenitors into T-cells, Tajima et al. (2015).

The artificial 3-dmatrix has been used to culture the TECs and are viable and can support evennot in full the thymocyte development. previously, Pinto etal. (2015).

developed acoculture system, in which mTECs were layered on top of a 3-D artificial matrixembedded 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+ cellsfrom cord blood to form implantable thymic units. The thymicmicroenvironments of these thymic reaggregates can support thymopoiesis in vitro and are ableto generate a complex T-cell repertoire when transplanted in nonobese diabetes(NOD).scid gamma humanized mice invivo. However, to date, none of these approaches has been ableto fully recapitulate the function of a thymus.

Lately, in the “cell-scaffold” technology there aresignificant advances that has been made. With the use of  tissue decellularization methods. In thistechnique, all cells are removed from the organ, leaving the extracellularmatrix intact. It has been reported that decellularization of the thymus,followed by reconstitution with thymic stromal cells and lineage negative BMprogenitors, led to formation a functional thymus when transplanted into thekidney capsule of nude mice, Fan et al (2015).

Allowing the theclearance of the cellular constituent of any organ of any scale, whith the useof a detergent-perfusion based, whileretaining its original 3-D architecture and extracellular matrix (ECM)components. Repopulating the decellularized natural scaffolds withtissue-residing mature cells or progenitor/stem cells can promote itsrecellularization and partially recover organ function.To date, these”cell-scaffolds” have been primarily applied to manufacture and implantrelatively simple organs, such as tissue engineered vascular grafts and skin,with some success. Repopulating the decellularized natural scaffolds withtissue-residing mature cells or progenitor/stem cells can promote itsrecellularization and partially recover organ function. To date, these”cell-scaffolds” have been primarily applied to manufacture and implantrelatively simple organs, such as tissue engineered vascular grafts and skin,with some success ,Goh et al. (2015) regeneration of complex organs such asliver, heart, lung, and kidney has also been attempted in animalmodels. Although limited, encouraging functional regeneration of theengineered organs was observed. Furthermore, a successful clinical implantationof reconstructed decellularized trachea underlines the clinical potential ofthis technology.

(Fan et al., 2015). The result of the investigation of thebioengineering thymus organoids with the decelluliarized thymus scaffolds hasled to allow the removal off all the cellular elements of a mouse thymus whilemaintaining all the major ECM components.

The used of decellularizing treatmenthas made the thymic stromal ECM largely intact, this is revealed through theuse of scanning electron microscopy (SEM) analysis with the acellular thymicscaffolds’ cross-section image. Bioengineeredthymus can support T lymphopoiesis in vivo. The capability of the bioengineered thymus to supporteffective thymocyte development and maturation in vivo was examined withtransplantation experiments.

Thymus organoids reconstructed with mixtures ofTSCs and Lin– BM progenitors at 1 : 1 ratio, both harvestedfrom B6.CD45.1 mice, were transplanted underneath the kidney capsules ofB6.nude athymic recipients (designated as Tot.B6.nude for thymus organoidtransplanted B6.

nude mice hereinafter). Homing of hematopoietic progenitors tothe thymus is an intermittent, gated process, alternating between ~1 week ofreceptive period and ~3 weeks of refractory period. The complement of BMprogenitors was used to ensure the continuity of cross talk between TECs andthe developing thymocytes that is essential for the survival of TECs, at theearly post-transplantation stage. The origins of the T-cells in the peripherywere identified by FCM analysis of the CD45 congenic markers (i.e., CD45.

1 and CD45.2for donor and recipient origins, respectively). (Banerjee et al.

, 2015).Effective cellular and humoral adaptive immunity mediated by T-cellsmatured in bioengineered thymus organoids. Proliferation under various stimuli has been widely used as a tool toassess the functionality of T lymphocytes. To demonstrate that T-cells derivedfrom the reconstructed thymus organoids are functionally competent, the authorslabeled them with carboxyfluorescein diacetate succinimidyl ester (CFSE) andstimulated them with anti-CD3 antibodies. Similar to T-cells of naive B6 mice,a significant percentage of T-cells underwent division, as indicated by dilutionof CFSE signals To further test the function of T-cells derived from thereconstructed thymi, the authors performed mixed leukocyte reaction experimentsto evaluate their responses to alloantigens. Proliferation responses similar tothose of wild-type B6 mouse were observed, indicating that these T-cells werecapable to react to alloantigens .Overall, these results demonstrated thatT-cells matured in the transplanted thymus organoids were capable to responseto TCR stimulation.

(Bertera et al., 2015)Recently,functional thymus organoids have been successfully constructed by repopulatingdecellularized thymus scaffolds with TSCs (including TECs, thymic fibroblastsas well as endothelial cells) isolated from young adult mice, in conjunctionwith bone marrow progenitors.The microenvironments of the thymus scaffolds cansupport the survival and function of adult TECs in vitro, without changingtheir unique molecular properties. When transplanted into T-cell deficientathymic nude mice, the bioengineered thymus organoid can effectively attractthe homing of LPs from the host’s bone marrow and supports the generation of acomplex T-cell repertoire. B-cells in the treated mice can undergo affinitymaturation and class switching upon immunization with model antigens,indicating assistance from helper T-cells.

When challenged with allogeneic skingrafts, the treated mice can effectively mobilize cytotoxic T-cells for rapidrejection. In addition, nude mice transplanted with thymus organoidsconstructed with donor MHC-expressing TECs display donor-specific tolerance toskin grafts but can promptly reject third-party allogeneic skin grafts. Thisstudy shows that the bioengineering approach to regenerate thymus can not onlygenerate a functional T-cell compartment, but also serve as an immunomodulatingtool to modify the identity of “immunological self” and introducedonor-specific immune tolerance (Tajima et al., 2016)Thereis a big challenge facing thymus bioengineering is the limited number of TECsthat can be harvested from the adult thymus, together with our incapability toefficiently expand them ex vivo to combatthis shortage. For reasons largely unknown, the total numbers of TECs start todecrease at very young age (about 4-weeks postnatal in mouse and 1-year afterbirth in human), and such declines accelerate at puberty. Recently, a number ofstudies have shown independently that the numbers of clonogenic units, whichpresumably represent the proliferative thymic epithelial progenitor cells(TEPCs), drop drastically within the first week after birth, Okabe et al. (2015) How to prevent such early loss of TEPCs and expand them either in vitro or in vivo remains achallenging task.

Whilethymus bioengineering is still at its infancy and more research is needed tofurther advance the technology for clinical application, preclinical studieshave clearly demonstrated the proof-of-principle that it is an effectiveapproach to rejuvenate the function of the adaptive immune system. Recentadvances in stem cell research and regenerative medicine make it possible torepair and/or regenerate various tissues and/or organs in human bodies in the foreseeablefuture. However, immune incompatibility remains as one of the major obstaclesto render the artificial organoids as integral parts of the hosts. Thymusbioengineering is a promising approach to modulate the adaptive immunity of thepatient and achieve immunosuppression-free tissue/organ replacement.