转录组分析揭示了 GDF6 介导的人脂肪干细胞向髓核细胞分化过程中的关键早期反应基因

IF 3.4 3区 医学 Q1 ORTHOPEDICS JOR Spine Pub Date : 2024-01-19 DOI:10.1002/jsp2.1315
Hamish T. J. Gilbert, Francis E. J. Wignall, Leo Zeef, Judith A. Hoyland, Stephen M. Richardson
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引用次数: 0

摘要

背景 基于干细胞的疗法有望成为修复退化椎间盘的一种手段,通常与细胞一起使用的生长因子有助于引导细胞向髓核(NP)样表型分化。我们之前通过评估末期分化参数,证明了脂肪源性干细胞(ASC)与GDF6的分化是生成NP样细胞的最佳选择。在此,我们对ASCs对GDF6刺激的反应进行了时间分辨转录组学表征,以了解向NP样细胞分化的早期驱动因素。 方法 用重组人 GDF6 处理人 ASCs 2、6 和 12 小时。使用 RNA 测序和详细的生物信息学分析来评估早期分化过程中的不同基因表达、基因本体(GO)和转录因子参与情况。定量聚合酶链反应(qPCR)用于验证 RNA 测序结果,抑制剂用于检测 Smad 和 Erk 信号通路,以及鉴定主要和次要反应基因。 结果 ASCs 对 GDF6 刺激的转录组反应具有时间分辨性和高度结构性,"细胞分化"、"发育过程 "和 "对刺激的反应 "被确定为关键生物过程 GO 术语。转录因子 ERG1 被确定为关键的早期反应基因。分化基因的时间聚类分析确定了对 NP 细胞分化的正向调控,以及对成骨和成脂的抑制。观察到 Smad 和 Erk 信号在调控 GDF6 诱导的早期基因表达反应中的作用,并确定了一级和二级反应基因。 结论 本研究发现了一种多因素的早期基因应答,这种应答有助于细胞系的承诺,同时还发现了一些潜在的有用的早期标志物,这些标志物可促进 ASCs 向 NP 细胞的分化。详细了解GDF6刺激ASCs的分子反应过程,对于开发高效、有效的细胞疗法治疗椎间盘变性相关背痛非常重要。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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Transcriptomic profiling reveals key early response genes during GDF6-mediated differentiation of human adipose-derived stem cells to nucleus pulposus cells

Background

Stem cell-based therapies show promise as a means of repairing the degenerate intervertebral disc, with growth factors often used alongside cells to help direct differentiation toward a nucleus pulposus (NP)-like phenotype. We previously demonstrated adipose-derived stem cell (ASC) differentiation with GDF6 as optimal for generating NP-like cells through evaluating end-stage differentiation parameters. Here we conducted a time-resolved transcriptomic characterization of ASCs response to GDF6 stimulation to understand the early drivers of differentiation to NP-like cells.

Methods

Human ASCs were treated with recombinant human GDF6 for 2, 6, and 12 h. RNA sequencing and detailed bioinformatic analysis were used to assess differential gene expression, gene ontology (GO), and transcription factor involvement during early differentiation. Quantitative polymerase chain reaction (qPCR) was used to validate RNA sequencing findings and inhibitors used to interrogate Smad and Erk signaling pathways, as well as identify primary and secondary response genes.

Results

The transcriptomic response of ASCs to GDF6 stimulation was time-resolved and highly structured, with “cell differentiation” “developmental processes,” and “response to stimulus” identified as key biological process GO terms. The transcription factor ERG1 was identified as a key early response gene. Temporal cluster analysis of differentiation genes identified positive regulation NP cell differentiation, as well as inhibition of osteogenesis and adipogenesis. A role for Smad and Erk signaling in the regulation of GDF6-induced early gene expression response was observed and both primary and secondary response genes were identified.

Conclusions

This study identifies a multifactorial early gene response that contributes to lineage commitment, with the identification of a number of potentially useful early markers of differentiation of ASCs to NP cells. This detailed insight into the molecular processes in response to GDF6 stimulation of ASCs is important for the development of an efficient and efficacious cell-based therapy for intervertebral disc degeneration-associated back pain.

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来源期刊
JOR Spine
JOR Spine ORTHOPEDICS-
CiteScore
6.40
自引率
18.90%
发文量
42
审稿时长
10 weeks
期刊最新文献
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