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.

本数据集相关数据显示，PDGFRα+细胞属于基质细胞（stromal cells），可提升后肢缺血后的血管稳定性；与之相对，分化后的细胞则会升高血管通透性。整体分析揭示了PDGFRα+细胞在组织血管重建过程中的双重功能。

【流式细胞术分选PDGFRα+GFP+细胞】
本文件夹包含PDGFRα+细胞分选的流式细胞术（flow cytometry）示例（样本量n=3）。实验中，从3月龄雄性野生型（WT）对照小鼠中分离内收肌，一组未进行标记（亦未使用DAPI染色）以设置分选门限；另一组采用APC偶联抗PDGFRα抗体与DAPI进行标记，以从PdgfrαH2B-eGfp小鼠中分选GFP+PDGFRα+双阳性细胞。Hoechst蓝通道可区分DAPI阴性的活细胞与DAPI阳性的死细胞。选取活的DAPI阴性细胞群后，使用FITC标记以识别PdgfrαH2B-eGfp+细胞。最终面板中，以FITC为横轴、APC为纵轴，可筛选出经APC偶联抗PDGFRα抗体标记后的GFP与APC双阳性细胞。分选得到的活的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）。

【伊文思蓝染料检测血管通透性】
血管通透性检测参照此前报道的方法（Radu与Chernoff，2013）开展。具体操作如下：将200 μL 1%伊文思蓝染料（Evans blue dye，Sigma Aldrich公司产品）用1×PBS稀释后，经尾静脉注射。注射30分钟后分离内收肌并称重。将解剖获取的组织置于500 μL甲酰胺中，于55℃孵育36小时后进行分光光度检测（吸光度波长610nm）。本文件夹内包含两份Excel文件，分别为后肢缺血前后，向裸小鼠注射未分化及分化的成纤维细胞样PDGFRα+细胞后，血管通透性的原始数据与详细计算结果。未进行后肢缺血造模与注射的对侧骨骼肌作为对照。

【缺血与非缺血状态下PDGFRα细胞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。实验使用10~12周龄野生型小鼠的骨骼肌样本。首先以DAPI阴性设门筛选活细胞，再圈选PDGFRα+细胞（APC抗体标记），最后使用PE偶联的基质标志物抗体（CD73、CD105、CD29及PDGFRb）完成标记分析。实验分别在未损伤及损伤（后肢缺血）的小鼠中进行。

【缺血与非缺血状态下基质标志物同型对照流式细胞术】
本文件夹包含前述实验所用的同型对照（isotype control）标记样本，分析同样在未损伤与损伤组织中开展。

搜集汇总

背景与挑战

背景概述

该数据集研究了PDGFRα阳性祖细胞在组织再生和血管再刺激中的双重功能，通过流式细胞术分选、血管通透性测量和基质标志物分析等实验，发现PDGFRα+细胞作为基质细胞能增强血管稳定性，而分化细胞则增加血管通透性，为理解血管再生机制提供了关键数据。