In Vivo Generation of Lung and Thyroid Tissues from Embryonic Stem Cells Using Blastocyst Complementation
In Vivo Generation of Lung and Thyroid Tissues from Embryonic Stem Cells Using Blastocyst Complementation
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DOI：
10.1164/rccm.201909-1836oc
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发表时间：
2021-02-15
影响因子：
24.7
通讯作者：
Kalinichenko, Vladimir V.
中科院分区：
文献类型：
--
作者：
Wen, Bingqiang;Li, Enhong;Kalinichenko, Vladimir V.
基本原理：从诱导多能干细胞或胚胎干细胞衍生的细胞再生和替换肺细胞或组织代表了未来对危及生命的肺部疾病的治疗，但受到生产能够维持肺功能的高度分化细胞的技术挑战的限制。含有功能性肺组织的气道、肺泡、脉管系统和基质从未通过胚胎干细胞（ESC）或诱导多能干细胞的定向分化产生。我们试图通过胚胎互补产生从支气管到肺泡的所有肺组织成分。目的：确定胚胎干细胞是否能够在Nkx 2 -1（-/-）小鼠肺发育不全胚胎中产生肺组织。方法：使用囊胚互补从正常小鼠胚胎干细胞和Nkx 2 -1（-/-）胚胎中产生嵌合体。Nkx 2 -1（-/-）嵌合体进行了检查，使用免疫染色，透射电子显微镜，荧光激活细胞分选仪分析，和单细胞RNA sequencing. Measures和主要结果：虽然外周肺和甲状腺组织是完全缺乏Nkx 2 -1基因缺失的胚胎，肺和甲状腺结构在Nkx 2 -1（-/-）嵌合体ESC互补后恢复。Nkx 2 -1（-/-）嵌合体的恢复肺中的呼吸道上皮细胞谱系几乎完全来自ESC，而内皮细胞、免疫细胞和基质细胞是嵌合体。胚胎干细胞衍生的细胞从多个呼吸系统细胞系高度分化和内源性细胞的基础上，形态，超微结构，基因表达的签名，和细胞表面蛋白用于识别细胞类型的荧光激活细胞sorter.Conclusions：肺和甲状腺组织中产生的胚胎干细胞囊胚互补在体内。Nkx 2 -1（-/-）嵌合体可用作ESC衍生祖细胞的体内分化和功能研究的“生物反应器”。
Rationale: The regeneration and replacement of lung cells or tissues from induced pluripotent stem cell- or embryonic stem cell-derived cells represent future therapies for life-threatening pulmonary disorders but are limited by technical challenges to produce highly differentiated cells able to maintain lung function. Functional lung tissue-containing airways, alveoli, vasculature, and stroma have never been produced via directed differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells. We sought to produce all tissue components of the lung from bronchi to alveoli by embryo complementation.Objectives: To determine whether ESCs are capable of generating lung tissue in Nkx2-1(-/-) mouse embryos with lung agenesis.Methods: Blastocyst complementation was used to produce chimeras from normal mouse ESCs and Nkx2-1(-/-) embryos, which lack pulmonary tissues. Nkx2-1(-/-) chimeras were examined using immunostaining, transmission electronic microscopy, fluorescence-activated cell sorter analysis, and single-cell RNA sequencing.Measurements and Main Results: Although peripheral pulmonary and thyroid tissues are entirely lacking in Nkx2-1 gene-deleted embryos, pulmonary and thyroid structures in Nkx2-1(-/-) chimeras were restored after ESC complementation. Respiratory epithelial cell lineages in restored lungs of Nkx2-1(-/-) chimeras were derived almost entirely from ESCs, whereas endothelial, immune, and stromal cells were mosaic. ESC-derived cells from multiple respiratory cell lineages were highly differentiated and indistinguishable from endogenous cells based on morphology, ultrastructure, gene expression signatures, and cell surface proteins used to identify cell types by fluorescence-activated cell sorter.Conclusions: Lung and thyroid tissues were generated in vivo from ESCs by blastocyst complementation. Nkx2-1(-/-) chimeras can be used as "bioreactors" for in vivo differentiation and functional studies of ESC-derived progenitor cells.