Background

The thymus is a glandular organ that is essential for the formation of the adaptive immune system by educating developing T cells. The thymus is most active during childhood and involutes around the time of adolescence, resulting in a severe reduction or absence of naive T-cell output. The ability to generate a patient-derived human thymus would provide an attractive research platform and enable the development of novel cell therapies.

Objectives

This study sought to systematically evaluate signaling pathways to develop a refined direct differentiation protocol that generates patient-derived thymic epithelial progenitor cells from multiple induced pluripotent stem cells (iPSCs) that can further differentiate into functional patient-derived thymic epithelial cells on transplantation into athymic nude mice.

Methods

Directed differentiation of iPSC generated TEPs that were transplanted into nude mice. Between 14 and 19 weeks posttransplantation, grafts were removed and analyzed by flow cytometry, quantitative PCR, bulk RNA sequencing, and single-cell RNA sequencing for markers of thymic-cell and T-cell development.

Results

A direct differentiation protocol that allows the generation of patient-derived thymic epithelial progenitor cells from multiple iPSC lines is described. On transplantation into athymic nude mice, patient-derived thymic epithelial progenitor cells further differentiate into functional patient-derived thymic epithelial cells that can facilitate the development of T cells. Single-cell RNA sequencing analysis of iPSC-derived grafts shows characteristic thymic subpopulations and patient-derived thymic epithelial cell populations that are indistinguishable from TECs present in primary neonatal thymus tissue.

Conclusions

These findings provide important insights and resources for researchers focusing on human thymus biology.

中文翻译：

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从诱导多能干细胞生成功能性人胸腺细胞

背景

胸腺是一种腺体器官，通过培养发育中的 T 细胞对适应性免疫系统的形成至关重要。胸腺在儿童时期最为活跃，并在青春期左右退化，导致幼稚 T 细胞输出严重减少或缺失。产生源自患者的人胸腺的能力将提供一个有吸引力的研究平台，并使新型细胞疗法的开发成为可能。

目标

本研究旨在系统地评估信号通路，以开发一种精细的直接分化方案，该方案从多种诱导多能干细胞 (iPSC) 中生成患者来源的胸腺上皮祖细胞，这些细胞在移植到无胸腺裸体中后可进一步分化为功能性患者来源的胸腺上皮细胞老鼠。

方法

iPSC 的定向分化产生了被移植到裸鼠体内的 TEP。移植后 14 至 19 周，取出移植物并通过流式细胞术、定量 PCR、大量 RNA 测序和单细胞 RNA 测序分析胸腺细胞和 T 细胞发育的标志物。

结果

描述了一种直接分化协议，允许从多个 iPSC 系生成患者衍生的胸腺上皮祖细胞。移植到无胸腺裸鼠后，患者来源的胸腺上皮祖细胞进一步分化为功能性患者来源的胸腺上皮细胞，可促进 T 细胞的发育。iPSC 衍生移植物的单细胞 RNA 测序分析显示特征性胸腺亚群和患者衍生的胸腺上皮细胞群与原代新生儿胸腺组织中存在的 TEC 无法区分。

结论

这些发现为专注于人类胸腺生物学的研究人员提供了重要的见解和资源。