Laboratory of Cell Biology, Department of Biotechnology and Life Sciences, University of Insubria, 21100, Varese, Italy.
Laboratory of Immunology and General Pathology, Department of Biotechnology and Life Sciences, University of Insubria, 21100, Varese, Italy.
J Biomed Sci. 2024 Nov 4;31(1):99. doi: 10.1186/s12929-024-01087-6.
Cell therapy has emerged as a revolutionary tool to repair damaged tissues by restoration of an adequate vasculature. Dental Pulp stem cells (DPSC), due to their easy biological access, ex vivo properties, and ability to support angiogenesis have been largely explored in regenerative medicine.
Here, we tested the capability of Dental Pulp Stem Cell-Conditioned medium (DPSC-CM), produced in normoxic (DPSC-CM Normox) or hypoxic (DPSC-CM Hypox) conditions, to support angiogenesis via their soluble factors. CMs were characterized by a secretome protein array, then used for in vivo and in vitro experiments. In in vivo experiments, DPSC-CMs were associated to an Ultimatrix sponge and injected in nude mice. After excision, Ultimatrix were assayed by immunohistochemistry, electron microscopy and flow cytometry, to evaluate the presence of endothelial, stromal, and immune cells. For in vitro procedures, DPSC-CMs were used on human umbilical-vein endothelial cells (HUVECs), to test their effects on cell adhesion, migration, tube formation, and on their capability to recruit human CD14 monocytes.
We found that DPSC-CM Hypox exert stronger pro-angiogenic activities, compared with DPSC-CM Normox, by increasing the frequency of CD31 endothelial cells, the number of vessels and hemoglobin content in the Ultimatrix sponges. We observed that Utimatrix sponges associated with DPSC-CM Hypox or DPSC-CM Normox shared similar capability to recruit CD45 stromal cells, CD45 leukocytes, F4/80 macrophages, CD80 M1-macrophages and CD206 M2-macropages. We also observed that DPSC-CM Hypox and DPSC-CM Normox have similar capabilities to support HUVEC adhesion, migration, induction of a pro-angiogenic gene signature and the generation of capillary-like structures, together with the ability to recruit human CD14 monocytes.
Our results provide evidence that DPSCs-CM, produced under hypoxic conditions, can be proposed as a tool able to support angiogenesis via macrophage polarization, suggesting its use to overcome the issues and restrictions associated with the use of staminal cells.
细胞疗法已成为通过恢复适当的脉管系统来修复受损组织的革命性工具。牙髓干细胞（DPSC）由于其易于进行生物学操作、体外特性以及支持血管生成的能力，已在再生医学中得到广泛探索。
在这里，我们测试了牙髓干细胞条件培养基（DPSC-CM）在常氧（DPSC-CM 常氧）或低氧（DPSC-CM 低氧）条件下通过其可溶性因子支持血管生成的能力。通过分泌组蛋白芯片对 CM 进行了表征，然后将其用于体内和体外实验。在体内实验中，将 DPSC-CM 与 Ultimatrix 海绵结合并注射到裸鼠中。切除后，通过免疫组织化学、电子显微镜和流式细胞术检测 Ultimatrix，以评估内皮细胞、基质细胞和免疫细胞的存在。对于体外程序，将 DPSC-CM 用于人脐静脉内皮细胞（HUVEC），以测试其对细胞黏附、迁移、管状结构形成以及招募人 CD14 单核细胞的能力的影响。
我们发现与 DPSC-CM 常氧相比，DPSC-CM 低氧具有更强的促血管生成活性，通过增加 Ultimatrix 海绵中 CD31 内皮细胞的频率、血管数量和血红蛋白含量来实现。我们观察到与 DPSC-CM 低氧或 DPSC-CM 常氧相关的 Ultimatrix 海绵具有招募 CD45 基质细胞、CD45 白细胞、F4/80 巨噬细胞、CD80 M1 巨噬细胞和 CD206 M2 巨噬细胞的相似能力。我们还观察到 DPSC-CM 低氧和 DPSC-CM 常氧具有相似的能力来支持 HUVEC 黏附、迁移、诱导促血管生成基因特征的表达以及生成毛细血管样结构，以及招募人 CD14 单核细胞的能力。
我们的研究结果提供了证据表明，在低氧条件下产生的 DPSCs-CM 可以作为一种通过巨噬细胞极化来支持血管生成的工具，这表明它可用于克服与使用干细胞相关的问题和限制。
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