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Abstract：
BACKGROUND: Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs).
METHODS: We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively.
RESULTS: iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and a PDGF receptor-beta inhibitor impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive mediators.
CONCLUSIONS: iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.
METHODS: We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively.
RESULTS: iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and a PDGF receptor-beta inhibitor impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive mediators.
CONCLUSIONS: iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.
摘要：
背景：周细胞是位于毛细血管上的多功能收缩细胞。周细胞是脑血流量和血脑屏障功能的关键调节因子,和周细胞功能障碍可能有助于人类神经系统疾病的病理生理学，包括阿尔茨海默病，多发性硬化症,和中风。诱导多能干细胞（iPSC）衍生的周细胞（iPericytes）是血管研究的有前途的工具。然而,尚不清楚iPericytes在功能上如何与原发性人类脑血管周细胞（HBVP）进行比较。
方法：我们使用已建立的方案将iPSC分化为中胚层或神经c谱系的iPericytes。我们比较了iPericyte和HBVP的形态，通过qPCR和批量RNA测序定量基因表达，并通过免疫细胞化学可视化周细胞蛋白标记。确定神经cipericytes的基因表达是否，中胚层ipericytes或HBVPs与其体外功能特性相关，我们量化了暴露于关键周细胞丝裂原后的EdU掺入，血小板衍生生长因子（PDGF）-BB和，收缩和松弛响应血管收缩内皮素-1或血管扩张剂腺苷，分别。
结果：iPericytes在形态上与HBVP相似，并表达了规范的周细胞标记。然而,与HBVPs相比，ipericytes有1864个差异表达基因，而神经c和中胚层ipericytes之间有797个基因差异表达。与HBVPs响应PDGF-BB信号的能力一致，PDGF-BB增强，PDGF受体β抑制剂损害了周细胞增殖。内皮素-1的给药导致周细胞收缩，腺苷导致周细胞松弛，与HBVPs引起的反应相似。我们确定神经cipericytes不太容易受到PDGFRβ抑制，但对血管收缩介质反应最强烈。
结论：iPericytes表达周细胞相关基因和蛋白质，在暴露于关键的内源性丝裂原或血管活性介质时表现出适当的生理反应。因此,功能性周细胞的产生将适用于未来的研究，探索神经系统疾病中的周细胞功能或功能障碍。
方法：我们使用已建立的方案将iPSC分化为中胚层或神经c谱系的iPericytes。我们比较了iPericyte和HBVP的形态，通过qPCR和批量RNA测序定量基因表达，并通过免疫细胞化学可视化周细胞蛋白标记。确定神经cipericytes的基因表达是否，中胚层ipericytes或HBVPs与其体外功能特性相关，我们量化了暴露于关键周细胞丝裂原后的EdU掺入，血小板衍生生长因子（PDGF）-BB和，收缩和松弛响应血管收缩内皮素-1或血管扩张剂腺苷，分别。
结果：iPericytes在形态上与HBVP相似，并表达了规范的周细胞标记。然而,与HBVPs相比，ipericytes有1864个差异表达基因，而神经c和中胚层ipericytes之间有797个基因差异表达。与HBVPs响应PDGF-BB信号的能力一致，PDGF-BB增强，PDGF受体β抑制剂损害了周细胞增殖。内皮素-1的给药导致周细胞收缩，腺苷导致周细胞松弛，与HBVPs引起的反应相似。我们确定神经cipericytes不太容易受到PDGFRβ抑制，但对血管收缩介质反应最强烈。
结论：iPericytes表达周细胞相关基因和蛋白质，在暴露于关键的内源性丝裂原或血管活性介质时表现出适当的生理反应。因此,功能性周细胞的产生将适用于未来的研究，探索神经系统疾病中的周细胞功能或功能障碍。
