Generation of Artificial Thymic Organoids from Human and Murine Hematopoietic Stem and Progenitor Cells.
Curr Protoc
; 2(4): e403, 2022 Apr.
Article
em En
| MEDLINE
| ID: mdl-35384408
ABSTRACT
The generation of T cells is a complex, carefully orchestrated process that occurs in the thymus. The ability to mimic T cell differentiation in vitro has opened up avenues to better understand different stages of thymopoiesis but has also enabled the in vitro production of mature T cells suitable for immunotherapy. Among existing protocols, the artificial thymic organoid (ATO) system has been shown to be the most efficient at producing mature conventional T cells. In this serum-free model, human or murine hematopoietic stem and progenitor cells (HSPCs) are combined with a murine stromal cell line expressing a Notch ligand in a 3D cell aggregate. In ATOs, although only simple medium changes are required throughout the cultures, HSPCs differentiate into T cells with kinetics and phenotypes similar to those of endogenous thymopoiesis. This article describes protocols for the generation of ATOs from human and murine HSPCs. © 2022 Wiley Periodicals LLC. Basic Protocol 1 Expansion and preparation of MS5-hDLL4 or MS5-mDLL4 cells Basic Protocol 2 Isolation of human hematopoietic stem and progenitor cells (HSPCs; CD34+ cells) Support Protocol 1 Transduction of human HSPCs (CD34+ cells) Basic Protocol 3 Production of thymic progenitors and mature T cells from human HSPCs in artificial thymic organoids (ATOs) Support Protocol 2 Phenotype analysis of human ATO cells by flow cytometry Basic Protocol 4 Isolation of murine HSPCs (Lin- Sca1+ cKit+; LSK) and hematopoietic stem cells (LSK CD150+ CD48-) Basic Protocol 5 Production of thymic progenitors and mature T cells from murine HSPCs in ATOs Support Protocol 3 Phenotype analysis of murine ATO cells by flow cytometry Alternate Protocol Generation of ATOs from single HSPCs.
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Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Células-Tronco Hematopoéticas
/
Organoides
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Curr Protoc
Ano de publicação:
2022
Tipo de documento:
Article