肺泡骨源性间充质基质细胞自发形成的球体通过调节低氧诱导因子维持干细胞的多能性。
Spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells maintain pluripotency of stem cells by regulating hypoxia-inducible factors.
机构信息
Shanghai University of Medicine and Health Sciences Affiliated Zhoupu Hospital, Shanghai, China, 201318.
The College of Medical Technology, Shanghai University of Medicine and Health Sciences, Shanghai, China, 201318.
出版信息
Biol Res. 2023 Apr 5;56(1):17. doi: 10.1186/s40659-023-00421-w.
BACKGROUND
Spontaneous spheroid culture is a novel three-dimensional (3D) culture strategy for the rapid and efficient selection of progenitor cells. The objectives of this study are to investigate the pluripotency and differentiation capability of spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells (AB-MSCs); compare the advantages of spontaneous spheroids to those of mechanical spheroids; and explore the mechanisms of stemness enhancement during spheroid formation from two-dimensional (2D) cultured cells.
METHODS
AB-MSCs were isolated from the alveolar bones of C57BL/6 J mice. Spontaneous spheroids formed in low-adherence specific culture plates. The stemness, proliferation, and multi-differentiation capacities of spheroids and monolayer cultures were investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, alkaline phosphatase (ALP) activity, and oil-red O staining. The pluripotency difference between the spontaneous and mechanical spheroids was analyzed using RT-qPCR. Hypoxia-inducible factor (HIFs) inhibition experiments were performed to explore the mechanisms of stemness maintenance in AB-MSC spheroids.
RESULTS
AB-MSCs successfully formed spontaneous spheroids after 24 h. AB-MSC spheroids were positive for MSC markers and pluripotency markers (Oct4, KLF4, Sox2, and cMyc). Spheroids showed higher Ki67 expression and lower Caspase3 expression at 24 h. Under the corresponding conditions, the spheroids were successfully differentiated into osteogenic and adipogenic lineages. AB-MSC spheroids can induce neural-like cells after neurogenic differentiation. Higher expression of osteogenic markers, adipogenic markers, and neurogenic markers (NF-M, NeuN, and GFAP) was found in spheroids than in the monolayer. Spontaneous spheroids exhibited higher stemness than mechanical spheroids did. HIF-1α and HIF-2α were remarkably upregulated in spheroids. After HIF-1/2α-specific inhibition, spheroid formation was significantly reduced. Moreover, the expression of the pluripotency genes was suppressed.
CONCLUSIONS
Spontaneous spheroids from AB-MSCs enhance stemness and pluripotency. HIF-1/2α plays an important role in the stemness regulation of spheroids. AB-MSC spheroids exhibit excellent multi-differentiation capability, which may be a potent therapy for craniomaxillofacial tissue regeneration.
背景
自发球状体培养是一种新的三维（3D）培养策略，可快速有效地选择祖细胞。本研究的目的是研究肺泡骨源性间充质基质细胞（AB-MSCs）自发球状体的多能性和分化能力；比较自发球状体与机械球状体的优势；并探讨从二维（2D）培养细胞形成球状体过程中增强干细胞特性的机制。
方法
从 C57BL/6J 小鼠的牙槽骨中分离 AB-MSCs。自发球状体在低附着专用培养板中形成。通过逆转录定量聚合酶链反应（RT-qPCR）、免疫荧光、碱性磷酸酶（ALP）活性和油红 O 染色，研究球状体和单层培养物的干细胞特性、增殖和多向分化能力。通过 RT-qPCR 分析自发球状体和机械球状体的多能性差异。进行缺氧诱导因子（HIFs）抑制实验，以探讨 AB-MSC 球状体中维持干细胞特性的机制。
结果
AB-MSCs 在 24 小时后成功形成自发球状体。AB-MSC 球状体呈间充质干细胞标志物和多能性标志物（Oct4、KLF4、Sox2 和 cMyc）阳性。球状体在 24 小时时 Ki67 表达较高，Caspase3 表达较低。在相应条件下，球状体可成功分化为成骨和成脂谱系。AB-MSC 球状体在神经发生分化后可诱导神经样细胞。在球状体中发现成骨标志物、成脂标志物和神经发生标志物（NF-M、NeuN 和 GFAP）的表达高于单层。自发球状体的干细胞特性高于机械球状体。球状体中 HIF-1α 和 HIF-2α 显著上调。HIF-1/2α 特异性抑制后，球状体形成明显减少。此外，多能性基因的表达受到抑制。
结论
AB-MSCs 自发球状体增强了干细胞特性和多能性。HIF-1/2α 在球状体干细胞特性调节中起重要作用。AB-MSC 球状体表现出优异的多向分化能力，可能是颅面组织再生的有效治疗方法。
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