新型高通量筛选报告称，苯并(a)芘会推翻小鼠滋养层干细胞的多能性，诱导 SAPK 活性、HAND1 和分化的滋养层巨细胞。

Novel high throughput screen reports that benzo(a)pyrene overrides mouse trophoblast stem cell multipotency, inducing SAPK activity, HAND1 and differentiated trophoblast giant cells.

机构信息

Department of Oncology, Wayne State University School of Medicine and Karmanos Cancer Institute, Detroit, MI, USA.

CS Mott Center/WSU Ob/gyn Department, USA; Reproductive Stress Inc, Grosse Pointe Farms, MI, USA.

出版信息

Placenta. 2024 Jul;152:72-85. doi: 10.1016/j.placenta.2023.12.020. Epub 2024 Jan 19.

DOI:10.1016/j.placenta.2023.12.020

PMID:38245404

Abstract

INTRODUCTION

Cultured mouse trophoblast stem cells (mTSC) maintain proliferation/normal stemness (NS) under FGF4, which when removed, causes normal differentiation (ND). Hypoxic, or hyperosmotic stress forces trophoblast giant cells (TGC) differentiate. Hypoxic, hyperosmotic, and genotoxic benzo(a)pyrene (BaP), which is found in tobacco smoke, force down-regulation of inhibitor of differentiation (Id)2, enabling TGC differentiation. Hypoxic and hyperosmotic stress induce TGC by SAPK-dependent HAND1 increase. Here we test whether BaP forces mTSC-to-TGC while inducing SAPK and HAND1.

METHODS

Hand1 and SAPK activity were assayed by immunoblot, mTSC-to-TGC growth and differentiation were assayed at Tfinal after 72hr exposure of BaP, NS, ND, Retinoic acid (RA), or sorbitol. Nuclear-stained cells were micrographed automatically by a live imager, and assayed by ImageJ/FIJI, Biotek Gen 5, AIVIA proprietary artificial intelligence (AI) software or open source, CellPose artificial intelligence/AI software.

RESULTS

BaP (0.05-1μM) activated SAPK and HAND1 without diminishing growth. TSC-to-TGC differentiation was assayed with increasingly accuracy for 2-4 N cycling nuclei and >4 N differentiating TGC nuclei, using ImageJ/FIJI, Gen 5, AIVIA, or CellPose AI software. The AIVIA and Cellpose AI software matches human accuracy. The lowest BaP effects on SAPK activation/HAND1 increase are >10-fold more sensitive than similar effects for mESC. RA induces 44-47% 1st lineage TGC differentiation, but the same RA dose induces only 1% 1st lineage mESC differentiation.

DISCUSSION

First, these pilot data suggest that mTSC can be used in high throughput screens (HTS) to predict toxicant exposures that force TGC differentiation. Second, mTSC differentiated more cells than mESC for similar stress exposures, Third, open source AI can replace human micrograph quantitation and enable a miscarriage-predicting HTS.

摘要

简介

在 FGF4 的存在下，培养的小鼠滋养层干细胞（mTSC）保持增殖/正常干细胞特性（NS），而当 FGF4 被去除时，会导致正常分化（ND）。低氧或高渗应激迫使滋养层巨细胞（TGC）分化。低氧、高渗和遗传毒性苯并（a）芘（BaP），存在于烟草烟雾中，下调分化抑制因子（Id）2，使 TGC 分化。低氧和高渗应激通过 SAPK 依赖性 HAND1 增加诱导 TGC。在这里，我们测试 BaP 是否在诱导 SAPK 和 HAND1 的同时迫使 mTSC 向 TGC 分化。

方法

通过免疫印迹法检测 HAND1 和 SAPK 的活性，在 BaP、NS、ND、视黄酸（RA）或山梨醇暴露 72 小时后检测 mTSC 向 TGC 的生长和分化。通过活成像仪自动对核染色细胞进行显微拍摄，并使用 ImageJ/FIJI、Biotek Gen 5、AIVIA 专有的人工智能（AI）软件或开源的 CellPose AI 软件进行分析。

结果

BaP（0.05-1μM）激活 SAPK 和 HAND1，而不减少生长。使用 ImageJ/FIJI、Gen 5、AIVIA 或 CellPose AI 软件，对 2-4 个 N 循环核和>4 个 N 分化的 TGC 核进行越来越准确的 TSC 向 TGC 分化检测。AIVIA 和 CellPose AI 软件与人的准确性相匹配。BaP 对 SAPK 激活/HAND1 增加的最低影响比 mESC 类似影响敏感 10 倍以上。RA 诱导 44-47%的第一谱系 TGC 分化，但相同剂量的 RA 仅诱导 1%的第一谱系 mESC 分化。