Development of novel in vitro culturing systems topromote thymopoiesis

While the murine thymocyte development has been extensively outlined and mod-
eled, the human thymus has been a challenging organ to study due to a variety of
factors. One major reason for the lagging human research is the lack of in vitro plat-
forms and systems in which to study human T cell development. This work seeks to
add clarity to human thymopoiesis through the development of novel in vitro plat-
forms on which to study human T cell maturation in environments that replicate
the unique physiology of the thymus. These studies include examinations into both
the process of T cell maturation as well as the function and development of thymic
epithelial cells (TECs), the main environmental support cell in the thymus. Here, we
generated TECs from induced pluripotent stem cells (iPSCs) that displayed markers
of different critical thymus lineages as well as functionality in vitro. We were also
able to develop progenitor T cells (Pro-T) from human umbilical cord blood within
a 14-day period. Both the TECs and Pro-T cells were combined in a decellularized
murine scaffold to generate thymic organoids. In vitro analyses revealed that these organoids preferentially produced CD4 T cells, which has previously been difficult
to demonstrate. Additional functional analyses showed that T cells generated from
these organoids released cytokines in response to stimulation, specifically interferon
gamma. The organoid function was further interrogated through the generation of a
novel humanized mouse model, which supported various hematopoietic lineage cells, including a functional and diverse T cell compartment that was capable of TCR rearrangement, cytokine production, and teratoma response and clearance. To improve clinical applications including T cell development for disease treatments, another in-vitro platform was innovated that removed the use of any mouse components, which are found in the current leading human T cell generation platforms. Described here is the first Artificial Thymic Aggregate (ATAgg) system containing strictly human thymic and progenitor cells of interest. This model has shown that cross talk between Pro-T cells and TECs helps facilitate co-maturation events. This model also demonstrates promising CD4 T cell development, which may allow directed T cell development. These novel in vitro human thymopoiesis platforms offer new insights and innovative approaches to help better understand the difficult arena of human thymus biology, while also uncovering potential avenues for pre-clinical and clinical applications within them.