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

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.

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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 organoids. Proliferation 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)

Recently,
functional thymus organoids have been successfully constructed by repopulating
decellularized thymus scaffolds with TSCs (including TECs, thymic fibroblasts
as well as endothelial cells) isolated from young adult mice, in conjunction
with bone marrow progenitors.The microenvironments of the thymus scaffolds can
support the survival and function of adult TECs in vitro, without changing
their unique molecular properties. When transplanted into T-cell deficient
athymic nude mice, the bioengineered thymus organoid can effectively attract
the homing of LPs from the host’s bone marrow and supports the generation of a
complex T-cell repertoire. B-cells in the treated mice can undergo affinity
maturation and class switching upon immunization with model antigens,
indicating assistance from helper T-cells. When challenged with allogeneic skin
grafts, the treated mice can effectively mobilize cytotoxic T-cells for rapid
rejection. In addition, nude mice transplanted with thymus organoids
constructed with donor MHC-expressing TECs display donor-specific tolerance to
skin grafts but can promptly reject third-party allogeneic skin grafts. This
study shows that the bioengineering approach to regenerate thymus can not only
generate a functional T-cell compartment, but also serve as an immunomodulating
tool to modify the identity of “immunological self” and introduce
donor-specific immune tolerance (Tajima et al., 2016)

There
is a big challenge facing thymus bioengineering is the limited number of TECs
that can be harvested from the adult thymus, together with our incapability to
efficiently expand them ex vivo to combat
this shortage. For reasons largely unknown, the total numbers of TECs start to
decrease at very young age (about 4-weeks postnatal in mouse and 1-year after
birth in human), and such declines accelerate at puberty. Recently, a number of
studies have shown independently that the numbers of clonogenic units, which
presumably 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 a
challenging task.

While
thymus bioengineering is still at its infancy and more research is needed to
further advance the technology for clinical application, preclinical studies
have clearly demonstrated the proof-of-principle that it is an effective
approach to rejuvenate the function of the adaptive immune system. Recent
advances in stem cell research and regenerative medicine make it possible to
repair and/or regenerate various tissues and/or organs in human bodies in the foreseeable
future. However, immune incompatibility remains as one of the major obstacles
to render the artificial organoids as integral parts of the hosts. Thymus
bioengineering is a promising approach to modulate the adaptive immunity of the
patient and achieve immunosuppression-free tissue/organ replacement.