Human stem cell-derived thymic epithelial cells enhance human T-cell development in a xenogeneic thymus - 19/11/21

Doi : 10.1016/j.jaci.2021.09.038

Rafael Gras-Peña, PhD ^a,^b,^⁎^⁎ , Nichole M. Danzl, PhD ^b,^{∗
These authors contributed equally to this work.}, Mohsen Khosravi-Maharlooei, MD ^b,^{∗
These authors contributed equally to this work.}, Sean R. Campbell, MD, PhD ^b, Amanda E. Ruiz, BA ^b, Christopher A. Parks, PhD ^b, William Meng Suen Savage, BA ^b, Markus A. Holzl, PhD ^b, Debanjana Chatterjee, PhD ^b, Megan Sykes, MD ^b,^c,^⁎
^a Columbia Center for Human Development, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY
^b Columbia Center for Translational Immunology, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY
^c Department of Surgery and Department of Microbiology and Immunology, Columbia University, New York, NY

^∗Corresponding author: Rafael Gras-Peña, PhD, Columbia Center for Human Development, Department of Medicine, College of Physicians and Surgeons, Columbia University, 650 West 168th Street, Black Building, New York, NY 10032.Columbia Center for Human DevelopmentDepartment of MedicineCollege of Physicians and SurgeonsColumbia University650 West 168th Street, Black BuildingNew YorkNY10032^∗∗Megan Sykes, MD, Columbia Center for Translational Immunology, Department of Medicine, College of Physicians and Surgeons, Columbia University, 650 West 168^th Street, Black Building 1512, Mailbox 127, New York, NY 10032.Columbia Center for Translational ImmunologyDepartment of MedicineCollege of Physicians and SurgeonsColumbia University650 West 168^th StreetBlack Building 1512Mailbox 127New YorkNY10032

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Abstract

Background

Generation of thymic tissue from pluripotent stem cells would provide therapies for acquired and congenital thymic insufficiency states.

Objectives

This study aimed to generate human thymic epithelial progenitors from human embryonic stem cells (hES-TEPs) and to assess their thymopoietic function in vivo.

Methods

This study differentiated hES-TEPs by mimicking developmental queues with FGF8, retinoic acid, SHH, Noggin, and BMP4. Their function was assessed in reaggregate cellular grafts under the kidney capsule and in hybrid thymi by incorporating them into swine thymus (SwTHY) grafts implanted under the kidney capsules of immunodeficient mice that received human hematopoietic stem and progenitor cells (hHSPCs) intravenously.

Results

Cultured hES-TEPs expressed FOXN1 and formed colonies expressing EPCAM and both cortical and medullary thymic epithelial cell markers. In thymectomized immunodeficient mice receiving hHSPCs, hES-TEPs mixed with human thymic mesenchymal cells supported human T-cell development. Hypothesizing that support from non–epithelial thymic cells might allow long-term function of hES-TEPs, the investigators injected them into SwTHY tissue, which supports human thymopoiesis in NOD severe combined immunodeficiency IL2Rγ^null mice receiving hHSPCs. hES-TEPs integrated into SwTHY grafts, enhanced human thymopoiesis, and increased peripheral CD4⁺ naive T-cell reconstitution.

Conclusions

This study has developed and demonstrated in vivo thymopoietic function of hES-TEPs generated with a novel differentiation protocol. The SwTHY hybrid thymus model demonstrates beneficial effects on human thymocyte development of hES-TEPs maturing in the context of a supportive thymic structure.

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Graphical abstract

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Key words : Embryonic stem cell differentiation, TEC, third pharyngeal pouch, thymus, xenotransplantation

Abbreviations used : 3^rdPP, AFE, APC, cTEC, d0, DE, DP, E1, hES, hES-TEP, hHSPC, HuTHY, K5, NS, NSG, PE, PP, PSC, RA, SP, SwTHY, TEC, TEP, TMC, Treg, YM155

Plan

Methods

Quantitative real-time PCR

Immunohistochemistry and immunofluorescence

Animals and human tissues

Human immune system mice

Flow cytometry

High-throughput CDR3β TCR sequencing

Statistics

Results

Direct differentiation of hESs to AFE

Differentiating AFE toward 3^rdPP-biased PE

Distalization of 3^rdPP from 3^rdPP-biased PE

Optimizing hES-TEP culture to avoid teratomas

Functional thymic organ formation from hES-TEP/TMCs

A novel strategy for testing hES-TEPs: Evidence that they integrate into SwTHY grafts

hES-TEP injection into SwTHY improves human thymopoiesis

Discussion

This research was supported by the National Institutes of Health (NIH) National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) award UC4DK104207 and NIH National Institute of Allergy and Infectious Diseases (NIAID) P01AI045897. Research reported in this publication was performed in the CCTI Flow Cytometry Core and Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources, supported in part by the Office of the Director, NIH under award S10RR027050 and in part through Center grant P30CA013696. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. N.M.D. was supported by NIH NIDDK F32DK095539, Naomi Berrie Center Fellowship, and American Diabetes Association (ADA) grant 7-11-MN-62. M.K.M. was supported by an ADA Postdoctoral Fellowship and also a Columbia University Naomi Berrie Diabetes Center Russell Berrie Foundation Fellowship.

Disclosure of potential conflict of interest: The authors declare that the work described here is the subject of 2 pending US patent applications—62/827,383 and PCT/US19/51865. Patent 62/827,383 is a licensed patent, and M. Sykes, R. Gras-Peña, N.M. Danzi, and M. Khosravi-Maharlooei would receive licensing revenue distribution as inventors if revenues are collected. The authors declare that they have no relevant conflicts of interest.