Supporting data for “Conversion of stem cells from apical papilla into endothelial cells by small molecules and growth factors”.

Incorporating assembled microvascular networks into the bioengineered dental pulp constructs can significantly enhance functional blood circulation and tissue survival after transplantation. Endothelial cells (ECs), the essential cellular building blocks of vascular tissue, play an important role in the process of pre-vascularization. However, obtaining sufficient ECs from a suitable source for translational application is challenging. Dental stem cells (DSCs), which exhibit a robust proliferative ability and immunocompatibility because of their autologous origin, could be a promising alternative cell source for the derivation of endothelial lineages. Under specific culture conditions, DSCs differentiate into osteo/odontogenic, adipogenic, chondrogenic, and neurogenic cell lineages. DSCs also possess the potential to differentiate into endothelial-like cells.Recently, a new strategy has been developed to directly reprogram one cell type towards another targeted cell type using small molecule compounds modulating epigenetic status and signaling pathways. Human fibroblasts have been chemically reprogrammed into neuronal cells, Schwann cells and cardiomyocyte-like cells by different small molecules combinations. This study aimed to explore whether stem cells from apical papilla (SCAP) could be reprogrammed into endothelial cells (ECs) using the same strategy.We developed a set of small molecules and growth factors, including VPA, CHIR99021 Repsox, Forskolin, Y-27632, VEGF, BMP-4 and 8-Br-3,5-cAMP, to differentiate SCAP into endothelial linages. The expression level of endothelial specific genes and proteins were assessed by RT-PCR, western blotting, flow cytometry, and immunofluorescence after chemical induction of SCAP. The in vitro functions of SCAP-derived chemical-induced endothelial cells (SCAP-ECs) were evaluated by tube-like structure formation assay, acetylated low-density lipoprotein (ac-LDL) uptake and NO secretion detection. The proliferation and migration ability of SCAP-ECs were evaluated by CCK8 and Transwell assay. LPS stimulation was used to mimic inflammatory environment for demonstrating the ability of SCAP-ECs to express adhesion molecules. The in vivo Matrigel plug angiogenesis assay was performed to assess the function of SCAP-ECs in generating vascular structures using the immune-deficient mouse model.SCAP-ECs expressed up-regulated endothelial specific genes and proteins, displayed endothelial transcriptional networks, exhibited the ability to form functional tubular-like structures, uptake ac-LDL, secret NO in vitro, and contributed to generate blood vessels in vivo. The SCAP-ECs could also express adhesion molecules in the pro-inflammatory environment and have a similar migration ability like HUVECs. The results indicate that SCAP-ECs could be one promising cell source for vascular engineering and treatment of ischemic diseases.

将组装的微血管网络整合入生物工程化的牙髓构建中，能够显著增强移植后的功能血液循环和组织存活率。内皮细胞（ECs），作为血管组织的根本细胞构建单元，在血管发生过程中扮演着至关重要的角色。然而，从适宜的来源获取足够的ECs以应用于转化医学面临着巨大挑战。牙源干细胞（DSCs），因其自体来源而表现出强大的增殖能力和免疫相容性，可能成为获取内皮细胞谱系的潜在细胞来源。在特定的培养条件下，DSCs能够分化为骨/牙源性、脂肪源性、软骨源性和神经源性细胞谱系。DSCs还具备分化为类似内皮细胞的潜力。近期，一种新的策略被开发出来，通过调节表观遗传状态和信号通路的小分子化合物，直接将一种细胞类型重编程为另一种目标细胞类型。人类成纤维细胞通过不同的小分子组合已被化学重编程为神经元细胞、施万细胞和类似心肌细胞。本研究旨在探究来自牙尖乳头（SCAP）的干细胞是否能够采用相同策略重编程为内皮细胞（ECs）。我们开发了一套包括VPA、CHIR99021、Repsox、Forskolin、Y-27632、VEGF、BMP-4和8-Br-3,5-cAMP在内的小分子和生长因子，以分化SCAP为内皮谱系。通过逆转录聚合酶链反应（RT-PCR）、蛋白质印迹、流式细胞术和免疫荧光技术，在化学诱导SCAP后评估了内皮特异性基因和蛋白的表达水平。通过管状结构形成实验、乙酰化低密度脂蛋白（ac-LDL）摄取和一氧化氮（NO）分泌检测，评估了SCAP来源的化学诱导内皮细胞（SCAP-ECs）的体外功能。通过CCK8和Transwell实验评估了SCAP-ECs的增殖和迁移能力。使用脂多糖（LPS）刺激模拟炎症环境，以展示SCAP-ECs在促炎症环境中表达粘附分子的能力。通过免疫缺陷小鼠模型进行的Matrigel植入血管生成实验，用于评估SCAP-ECs在生成血管结构中的功能。SCAP-ECs表达上调的内皮特异性基因和蛋白，展现出内皮转录网络，表现出形成功能性管状结构的潜力，在体外摄取ac-LDL，分泌NO，并在体内有助于血管生成。SCAP-ECs在促炎症环境中也能表达粘附分子，并具有与HUVECs相似的迁移能力。结果表明，SCAP-ECs可能是血管工程和缺血性疾病治疗的潜在细胞来源。