发育形态原引导人类诱导多能干细胞形成环状纤维样细胞表型。

IF 3.4 3区 医学 Q1 ORTHOPEDICS JOR Spine Pub Date : 2024-01-25 DOI:10.1002/jsp2.1313
Ana P. Peredo, Tonia K. Tsinman, Edward D. Bonnevie, Xi Jiang, Harvey E. Smith, Sarah E. Gullbrand, Nathaniel A. Dyment, Robert L. Mauck
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引用次数: 0

摘要

导言:由于内源性纤维环(AF)细胞无法对损伤做出反应并驱动组织再生,因此治疗椎间盘突出症的干预措施仍然很少。与软骨等其他骨科组织不同,将外源性细胞输送到椎间盘环损伤部位的技术仍不发达,这主要是由于缺乏理想的细胞来源以及细胞分离的侵入性。人类诱导多能干细胞(iPSC)可利用生化因子分化为特定的细胞命运,因此是细胞疗法的宝贵工具。虽然已开发出软骨和纤维结缔组织(如肌腱)的分化方案,但调节软骨和纤维结缔组织分化的信号还不明确、肌腱)的分化方案,但调节人类 iPSCs 向房颤命运诱导和分化的信号仍然未知。方法:用各种发育信号组合处理 iPSC 衍生的硬骨细胞,包括转化生长因子 beta 3(TGF-β3)、结缔组织生长因子(CTGF)、血小板衍生生长因子 BB(PDGF-BB)、胰岛素样生长因子 1(IGF-1)或刺猬通路激活剂 Purmorphamine,并评估主要 AF 相关 ECM 基因的基因表达变化。通过使用三种不同的 iPSC 株系和评估上调的相关 ECM 蛋白的生成情况,进一步验证了表现最佳的组合疗法。为了对每种因子组合引起的转录组变化进行更广泛的分析,并将处理过的细胞的基因图谱与成熟的人类房颤细胞进行比较,应用了96.96 Fluidigm基因表达阵列,并采用主成分分析法确定了每个细胞群和处理组与原生房颤细胞相比的转录特征:结果:TGF-β3 与 PDGF-BB、CTGF 或 IGF-1 结合使用可诱导 iPSC 衍生的硬骨体细胞中的关键 AF ECM 基因上调。特别是,与其他处理组相比,TGF-β3 与 PDGF-BB 联合处理 14 天可显著增加胶原蛋白 II 和凝集素的基因表达,并增加胶原蛋白 I 和弹性蛋白的蛋白质沉积。对AF细胞或SCL细胞分别表达的独特高表达基因的评估显示,在添加TGF-β3和PDGF-BB 14天后,AF细胞的基因特征发生了转变:这些发现代表了一种引导人类诱导多能干细胞向AF样命运转变的初步方法,可用于细胞递送策略。
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Developmental morphogens direct human induced pluripotent stem cells toward an annulus fibrosus-like cell phenotype

Introduction

Therapeutic interventions for intervertebral disc herniation remain scarce due to the inability of endogenous annulus fibrosus (AF) cells to respond to injury and drive tissue regeneration. Unlike other orthopedic tissues, such as cartilage, delivery of exogenous cells to the site of annular injury remains underdeveloped, largely due to a lack of an ideal cell source and the invasive nature of cell isolation. Human induced pluripotent stem cells (iPSCs) can be differentiated to specific cell fates using biochemical factors and are, therefore, an invaluable tool for cell therapy approaches. While differentiation protocols have been developed for cartilage and fibrous connective tissues (e.g., tendon), the signals that regulate the induction and differentiation of human iPSCs toward the AF fate remain unknown.

Methods

iPSC-derived sclerotome cells were treated with various combinations of developmental signals including transforming growth factor beta 3 (TGF-β3), connective tissue growth factor (CTGF), platelet derived growth factor BB (PDGF-BB), insulin-like growth factor 1 (IGF-1), or the Hedgehog pathway activator, Purmorphamine, and gene expression changes in major AF-associated ECM genes were assessed. The top performing combination treatments were further validated by using three distinct iPSC lines and by assessing the production of upregulated ECM proteins of interest. To conduct a broader analysis of the transcriptomic shifts elicited by each factor combination, and to compare genetic profiles of treated cells to mature human AF cells, a 96.96 Fluidigm gene expression array was applied, and principal component analysis was employed to identify the transcriptional signatures of each cell population and treatment group in comparison to native AF cells.

Results

TGF-β3, in combination with PDGF-BB, CTGF, or IGF-1, induced an upregulation of key AF ECM genes in iPSC-derived sclerotome cells. In particular, treatment with a combination of TGF-β3 with PDGF-BB for 14 days significantly increased gene expression of collagen II and aggrecan and increased protein deposition of collagen I and elastin compared to other treatment groups. Assessment of genes uniquely highly expressed by AF cells or SCL cells, respectively, revealed a shift toward the genetic profile of AF cells with the addition of TGF-β3 and PDGF-BB for 14 days.

Discussion

These findings represent an initial approach to guide human induced pluripotent stem cells toward an AF-like fate for cellular delivery strategies.

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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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