间质和生物物理成分对远端肺类器官分化的影响。

Influence of mesenchymal and biophysical components on distal lung organoid differentiation.

机构信息

Translational Medicine Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, 686 Bay Street, Toronto, ON, M5G 0A4, Canada.

Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada.

出版信息

Stem Cell Res Ther. 2024 Sep 2;15(1):273. doi: 10.1186/s13287-024-03890-2.

DOI:10.1186/s13287-024-03890-2

PMID:39218985

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11367854/

Abstract

BACKGROUND

Chronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation.

METHODS

Human PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6), E-cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, surfactant proteins B (SFTPB) and C (SFTPC).

RESULTS

We observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1 lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1 lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking in situ fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation.

CONCLUSION

Our data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro.

摘要

背景

早产儿慢性肺病，又称支气管肺发育不良（BPD），目前缺乏有效的治疗方法，这凸显了临床前测试系统的必要性，该系统应反映人类病理学，以确定因果途径并测试新型化合物。源自人类多能干细胞（hPSC）的肺泡类器官是研究 BPD 等远端气道疾病的有前途的测试平台，但目前的方案无法准确复制天然肺的远端生态位环境。在此，我们研究了肺泡的细胞成分和胎儿呼吸运动对 hPSC 衍生的肺泡类器官形成的贡献。

方法

在 2D 培养中将人 PSCs 分化为肺祖细胞（LPC），然后在去除共同发育的中胚层细胞前后将其进一步分化为肺泡类器官。还在 Transwell®共培养物中分化了带有和不带有人类胎儿肺成纤维细胞的 LPC。使用 Flexcell®系统对形成的类器官进行阶段性机械应变。通过流式细胞术、免疫荧光和 qPCR 评估类器官和 Transwell®培养物中的分化，以评估肺上皮和肺泡分化标志物，包括 GATA 结合蛋白 6（GATA 6）、E-钙黏蛋白（CDH1）、NK2 同源盒 1（NKX2-1）、HT2-280、表面活性剂蛋白 B（SFTPB）和 C（SFTPC）。

结果

我们观察到共同发育的间充质祖细胞促进 hPSC 衍生的肺类器官中的 II 型肺泡上皮细胞（AEC2）分化。这种对 AEC2 分化的间质作用通过与人类胚胎肺成纤维细胞共培养 hPSC-NKX2-1 肺祖细胞得到证实。这种刺激作用不需要成纤维细胞和 NKX2-1 肺祖细胞之间的直接接触。此外，我们证明了 hPSC 衍生的肺类器官的阶段性机械变形，模拟原位胎儿呼吸运动，增加了 AEC2 的分化，而不影响近端上皮的分化。