Poster CC11 - Increasing manufacturing throughput of induced pluripotent stem cell derived thymus epithelial progenitor cells.

Introduction: The human thymus is of increasing interest due to its role in T cell selection and unique architecture. The thymus possesses a fascinating combination of mechanical and structural properties that correspond to its function, making it the subject of recent efforts in mechanobiology, organoid generation, and bioprinting. The thymus is composed of two distinct stromal compartments comprised of thymic epithelial cells (TECs). In the cortical layer, cortical thymic epithelial cells (cTECs) participate in the process of positive selection which prunes a population of T cell progenitors (Pro Ts) to those that can mount an immune response against foreign invaders. In the thymic medulla, medullary thymic epithelial cells (mTECs) promote negative selection, which removes T cells that are autoreactive.
Recapitulation of the human thymus is of interest not only for increasing understanding of T cell selection and its relationship with thymus architecture, but also for rejuvenating its function. Here, thymic epithelial organoids were generated from induced pluripotent stem cells (iPSCs) and used to tissue engineer a functional thymus. iPSCs were used as they are readily renewable and can be differentiated into any tissue type. iPSCs differentiated into thymic epithelial progenitor cells (TEPCs) in a three-dimensional alginate encapsulation platform, which contributes necessary microphysiological cues. These differentiated cells were incorporated into a decellularized mouse scaffold to tissue-engineer an artificial thymus (iPSC-thymus) and utilized for further T-cell development and selection. Finally, we provide an avenue for scaling of this technology for use in a clinical setting through the incorporation of automatic bioprinting.

Materials and

Methods: Manual encapsulation of iPSCs was completed by suspending cells in 1.45% alginate and adding dropwise to 100 mM CaCl2 solution. iPSCs were cultured for five days to form aggregates and differentiated for 11 days to TEPC status. Single-cell RNA sequencing and RT-qPCR were performed to characterize iPSC differentiation. To test functionality of iPSC-TEPCs in vitro, decellularized mice thymus scaffolds were generated. TEPCs were combined with Pro T cells derived from CD34+ hematopoietic stem/progenitor cells (HSPCs) and were embedded into the scaffold. The tissue-engineered iPSC-thymus was cultured for one week and assessed with flow cytometry. To assess in vivo functionality, the TEPC seeded scaffolds were cultured for 5-7 days before implantation underneath the kidney capsule of CD34+ HSPC-engrafted immunodeficient NSG mice (designated as hu.THOR for humanized mice with thymus organoid).
To increase throughout, a bioprinting system was developed to simulate the formation of TEPCs through the printing of biopolymer beads. The bioink is extruded through a syringe pump, where an air jacket controls bead sphericity at the outlet (Figure 1A). An average capsule diameter of 500 m could be produced with a cell-seeding density of 1 million cells: 1mL alginate, crosslinking concentration of 200 mM CaCl2, air flow speed of 5 L/min, and an alginate concentration of 2.0% w/v (Figure 1B). Printed single cells were cultured for 1 day to form 40 μm aggregates, and then differentiated for 11 days to TEPC status. Following differentiation cells were decapsulated and prepared for genetic and phenotypic expression as in the manual encapsulation conditions.

Results, Conclusions, and Discussions: Single cell RNA sequencing analysis of manually encapsulated cells demonstrated the presence of three distinct cell populations pointing to the presence of cTEC and mTEC lineages in addition to TEPCs. This was not observed for cells that were cultured in a 2D setting demonstrating the necessity of alginate encapsulation to generate both mTEC and cTEC lineages (Figure 1C). When these cells were used to conduct in vitro functionality assessments, evidence of mature T cell populations was obtained from flow cytometry data and RNA sequencing. When iPSC/Pro T cell organoids are used to repopulate a decellularized thymus scaffold in vitro, CD3+CD4+CD8+ double positive, CD3+CD4+CD8− and CD3+CD4−CD8+ single positive cells were recovered. Hu.THOR mice develop a diverse and functional CD45+CD3+ T cell compartment, including both CD4+ and CD8+ subsets after 18 weeks of iPSC-thymus engraftment, indicting the functionality of the engrafted iPSC-TEPCs [1].
Automatic bioprinting of capsules with embedded iPSCs is high throughput, enabling production of 12,000 capsules after 10 minutes of printing. Phenotypic analysis of these cells indicated 87% expression of EpCAM, a key TEPC marker (Figure 1D). This is comparable to percentages obtained when using manual encapsulation. Additionally, KRT8, MHCII, CD205, and DLL4 populations were identified, indicating the presence of distinct TEPC lineages (cTEC vs. mTEC). Transplantation studies to test in vivo functionality of printed iPSCs are ongoing.
These results demonstrate that TEPCs generated from iPSCs differentiated in a three-dimensional alginate platform can be used to promote T cell selection in vitro and in vivo. When the automatic printing method was used it produced populations of iPSC-TEPCs that were phenotypically similar to TEPCs generated from manual encapsulation. Furthermore, the number of capsules produced using this method is greatly increased compared to manual encapsulation. This work expands the use of TEPC/Pro T cell organoids in research and clinical applications harnesses the potential to manufacturing these organoids at scale for future translational studies.

[1] Zeleniak, A., Wiegand, C., Liu, W. et al. De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. Nat Methods 19, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-3

Acknowledgements (Optional): We would like to acknowledge Sophie Kopyar, Julia Gibson, Arielle Recchione, Haonan Guan and Carl Engman for providing technical support to this work.