Dual function of PDGFRalpha positive progenitor cells during regeneration and revascularization stimuli

These data show that PDGFRa+ cells are stromal cells and promote increased vessel stability after hind limb ischemia. On the contrary, differentiated cells increased vessel permeability. The overall analysis described a dual function of PDGFRa+ cells in tissue revascularization. Folder Flow cytometry and sorting of PDGFRα+GFP+ cells: In this folder there is an example of flow cytometry for sorting PDGFRα+ cells (n=3). Adductor skeletal muscles were isolated from 3 month-old male WT control mice and left un-labeled (also without DAPI) to set gates for sorting, or labeled with anti-PDGFRα APC-conjugated antibody and DAPI to sort for GFP+PDGFRα+ co-positive cells from PdgfrαH2B-eGfp mice. The Hoechst Blue channel distinguishes DAPI(-) live cells from dead (DAPI+) cells. Taking the live DAPI(-) population, FITC was used to specify for PdgfrαH2B-eGfp+ cells. In the final panel, the FITC (x axis) and APC (y axis) specifies for cells double positive for GFP and APC (after labeling with anti-PDGFRα APC-conjugated antibody). Sorted live GFP+PDGFRα+ co-positive cells were processed for further analyses (RNAseq and adoptive transfer experiments). Folder evans blue dye to measure vessel permeability: Vessel permeability was assessed as described previously (Radu and Chernoff, 2013). Briefly, 200 μL of 1% Evans blue dye (Sigma Aldrich, Inc.) diluted in 1x PBS was injected via tail vein. The adductor muscle was isolated 30 minutes after injection and weighed. Dissected tissues were left for 36 hours in 500 μL of formamide at 55°C prior to spectrophotometric measurements (Abs=610nm). The two excel files in this folder represent the raw data and the detailed calculations of the vessel permeability in nude mice injected with undifferentiated and differentiated myofibroblast-like PDGFRα+ cells before and after hind limb ischemia. Contralateral skeletal muscles without hind limb ischemia and injection were used as controls. Folder Flow cytometry of PDGRα cells stromal markers with and without ischemia: This analysis was performed to characterize the expression of stromal markers in skeletal muscle PDGFRα+ cells. Flow cytometric analysis was performed in murine skeletal muscle obtained from WT mice at 10-12 weeks of age. PDGFRα+ cells were gated first as DAPI(-), then as PDGFRα+ cells (APC antibody) and finally with antibodies PE-conjugated antibodies for stromal markers (CD73, CD105, CD29, and PDGFRb). The experiment was done in not injured and injured (hind limb ischemia) mice. Folder Flow cytometry of isotype control for stromal markers with and without ischemia: This folder contains samples labeled with isotype control for the previous experiment. This analysis was also perfomed in uninjured and injured tissues.本研究数据显示，血小板源性生长因子受体α阳性细胞（PDGFRa+）属于基质细胞，可在后肢缺血术后提升血管稳定性；与之相反，分化后的细胞会增强血管通透性。整体分析揭示了PDGFRa+细胞在组织血管重建过程中的双重功能。 文件夹：PDGFRα+GFP+细胞的流式细胞术分选 本文件夹包含PDGFRα+细胞分选的流式细胞术示例（n=3）。实验中，从3月龄雄性野生型（WT）对照小鼠体内分离内收骨骼肌，一组不进行标记（同时不添加4',6-二脒基-2-苯基吲哚（DAPI））以设置分选门限；另一组使用抗PDGFRα别藻蓝蛋白（APC）偶联抗体与DAPI进行标记，用于从PdgfrαH2B-eGfp小鼠体内分选GFP+PDGFRα+双阳性细胞。赫斯特蓝（Hoechst Blue）通道可区分DAPI阴性的活细胞与DAPI阳性的死细胞。选取DAPI阴性的活细胞群后，通过异硫氰酸荧光素（FITC）标记以鉴定PdgfrαH2B-eGfp+细胞。最终结果图中，以FITC为x轴、APC为y轴，可鉴定经抗PDGFRα APC偶联抗体标记后的GFP与APC双阳性细胞。分选得到的活GFP+PDGFRα+双阳性细胞将用于后续分析（RNA测序（RNAseq）及过继转移实验）。 文件夹：伊文思蓝染料（Evans blue dye）检测血管通透性 按照既往研究方法（Radu与Chernoff，2013）评估血管通透性。具体操作如下：将200 μL 1%伊文思蓝染料（Sigma Aldrich, Inc.）用1×磷酸盐缓冲液（PBS）稀释后，经尾静脉注射。注射30分钟后分离内收骨骼肌并称重。将解剖得到的组织置于500 μL甲酰胺中，于55℃孵育36小时，随后进行分光光度检测（吸光度波长610nm）。本文件夹内的两个Excel文件分别记录了后肢缺血前后，注射未分化及分化型肌成纤维细胞样PDGFRα+细胞的裸鼠的血管通透性原始数据与详细计算结果。以未经历后肢缺血且未进行注射的对侧骨骼肌作为对照。 文件夹：缺血与非缺血状态下PDGFRα细胞的基质标志物流式细胞术分析 本分析旨在鉴定骨骼肌PDGFRα+细胞的基质标志物表达特征。实验采用10~12周龄野生型（WT）小鼠的骨骼肌样本进行流式细胞术检测。分选步骤依次为：先筛选DAPI阴性细胞，再鉴定PDGFRα+细胞（使用APC标记的抗体），最后通过藻红蛋白（PE）偶联的基质标志物抗体（CD73、CD105、CD29及PDGFRb）进行标记。实验分别在未损伤及损伤（后肢缺血）的小鼠组织中完成。 文件夹：缺血与非缺血状态下基质标志物同型对照的流式细胞术分析 本文件夹包含上一实验中使用同型对照标记的样本，分析同样在未损伤及损伤的小鼠组织中完成。