Additional file 3 of Autologous non-invasively derived stem cells mitochondria transfer shows therapeutic advantages in human embryo quality rescue

Additional file 3: Fig. S1. The isolation and culture process of primary USC (A), GC (B), BMSC (C) and ADSC (D). Scale bars, 50 μm. Fig. S2. Identification of GC-specific surface marker FSHR. A Negative control. B Primary GC were positive for FSHR expression (stain brown). Scale bars, 50 μm. Fig. S3. USC tri-lineage differentiation in vitro. A Oil red O staining indicated lipid droplet formation after 21 days of in vitro adipogenic differentiation of USC. Scale bars, 50 μm. B, C In vitro osteogenic differentiation of USC after 14 days was identified by Alizarin red staining (B) and ALP staining (C) to indicate calcium nodule formation. Scale bars, 250 μm. D–F After 28 days of in vitro chondrogenic differentiation of USC, Toluidine blue (D), Safranin-O (E) and Masson’s trichrome (F) stainings revealed the presence of collagen and glycosaminoglycan (GAG) in the extracellular matrix. Scale bars, 50 μm. Fig. S4. The positive expressions of MSC surface specific markers in USC (A), BMSC (B) and ADSC (C). Black peaks represented isotype controls and green peaks represented various markers. All primary cells showed CD105 (+), CD73 (+), CD44 (+), HLA-DR (-), CD34 (-), CD45 (-). Fig. S5. Whole mitochondrial genome sequencing of 2 pairs of young and old USC. A Mitochondrial Circos map of YOUNG USC 1. B Mitochondrial Circos map of YOUNG USC 2. C Mitochondrial Circos map of OLD USC 1. D Mitochondrial Circos map of OLD USC 2. According to the structure of mitochondria, the Circos drawing is carried out according to the statistical depth. The Circos diagram has a total of 4 circles from the outside to the inside. The first ring is the mitochondrial H strand; the second ring is the mitochondrial L strand; in the third ring, the blue part is the coverage depth of each site of the mitochondria; the fourth ring is the position scale of the mitochondria. Fig. S6. Representative results of embryo ploidy by SurePlex WGA. A Representative result of euploid blastocyst from the Mito-ICSI group. B Representative result of segmental aneuploidy (≥ 10 Mbp) with deletion of ch.22q11.1-q13.33. C Representative result of complex aneuploidy (≥ 3 variations) with deletion of ch.4p16.3-q35.2, mosaicism at ch.6 and ch.14. D Representative result of mosaicism with both the partial duplication and deletion of multiple chromosomes. The black arrows represent the site of abnormal chromosomes. WGA, whole genome amplification Table S1. Comparison of USC mitochondrial genome sequencing between the young and old populations. Table S2. Clinical characteristics of IVF/ICSI donors of immature oocytes.

附加文件3：补充图S1。原代子宫基质细胞（Uterine Stromal Cells, USC）、粒层细胞（Granulosa Cells, GC）、骨髓间充质干细胞（Bone Marrow Mesenchymal Stem Cells, BMSC）以及脂肪来源间充质干细胞（Adipose-derived Mesenchymal Stem Cells, ADSC）的分离与培养流程（A：USC；B：GC；C：BMSC；D：ADSC）。比例尺：50 μm。
补充图S2。粒层细胞特异性表面标志物FSHR的鉴定。A：阴性对照；B：原代粒层细胞FSHR表达呈阳性（染色呈棕黄色）。比例尺：50 μm。
补充图S3。原代子宫基质细胞（USC）体外三系分化鉴定。A：USC体外成脂分化21天后，油红O染色可见脂滴形成。比例尺：50 μm。B、C：USC体外成骨分化14天后，分别通过茜素红染色（B）与碱性磷酸酶（Alkaline Phosphatase, ALP）染色（C）显示钙结节形成。比例尺：250 μm。D~F：USC体外成软骨分化28天后，甲苯胺蓝（D）、番红O（E）以及马松三色染色（F）均可显示细胞外基质中胶原与糖胺聚糖（Glycosaminoglycan, GAG）的存在。比例尺：50 μm。
补充图S4。子宫基质细胞（USC）、骨髓间充质干细胞（BMSC）以及脂肪来源间充质干细胞（ADSC）的间充质干细胞表面特异性标志物阳性表达情况。黑色峰为同型对照，绿色峰为各标志物染色结果。所有原代细胞均呈CD105(+)、CD73(+)、CD44(+)、HLA-DR(-)、CD34(-)、CD45(-)。
补充图S5。2对年轻与年老子宫基质细胞（USC）的全线粒体基因组测序结果。A：年轻USC 1的线粒体Circos图；B：年轻USC 2的线粒体Circos图；C：年老USC 1的线粒体Circos图；D：年老USC 2的线粒体Circos图。本研究根据线粒体结构，以统计测序深度绘制Circos图。该Circos图自外向内共包含4个环：第一环为线粒体H链；第二环为线粒体L链；第三环中蓝色区域代表线粒体各位点的测序覆盖深度；第四环为线粒体的位置刻度。
补充图S6。采用SurePlex全基因组扩增（Whole Genome Amplification, WGA）进行胚胎倍性检测的代表性结果。A：线粒体-卵胞浆内单精子注射（Mito-ICSI）组整倍体囊胚的代表性结果；B：片段型非整倍体（≥10 Mbp），即22号染色体q11.1-q13.33区域缺失的代表性结果；C：复合型非整倍体（≥3种变异），即4号染色体p16.3-q35.2区域缺失、6号与14号染色体存在嵌合现象的代表性结果；D：多条染色体同时存在部分重复与缺失的嵌合现象代表性结果。黑色箭头指向染色体异常位点。WGA：全基因组扩增。
表S1。年轻与年老子宫基质细胞（USC）线粒体基因组测序结果对比。
表S2。未成熟卵母细胞体外受精/卵胞浆内单精子注射（IVF/ICSI）供者的临床特征。