The online version of this article (doi:10.1186/s12958-017-0235-8) contains supplementary material, which is available to authorized users.
We have recently reported that human bone marrow-derived mesenchymal stem cells (MSCs) facilitate angiogenesis and prevent follicle loss in xenografted human ovarian tissues. However, the mechanism underlying this effect remains to be elucidated. Thus, determining the paracrine profiles and identifying the key secreted factors in MSCs co-transplanted with ovarian grafts are essential for the future application of MSCs.
In this study, we used cytokine microarrays to identify differentially expressed proteins associated with angiogenesis in frozen-thawed ovarian tissues co-transplanted with MSCs. The function of specific secreted factors in MSCs co-transplanted with human ovarian tissues was studied via targeted blockade with short-hairpin RNAi and the use of monoclonal neutralizing antibodies.
Our results showed that angiogenin (ANG) was one of the most robustly up-regulated proteins (among 42 protein we screened, 37 proteins were up-regulated). Notably, the targeted depletion of ANG with short-hairpin RNAi (shANG) or the addition of anti-ANG monoclonal neutralizing antibodies (ANG Ab) significantly reversed the MSC-stimulated angiogenesis, increased follicle numbers and protective effect on follicle apoptosis.
Our results indicate that ANG plays a critical role in regulating angiogenesis and follicle survival in xenografted human ovarian tissues. Our findings provide important insights into the molecular mechanism by which MSCs promote angiogenesis and follicle survival in transplanted ovarian tissues, thus providing a theoretical basis for their further application.
Additional file 1: Figure S1. Isolation of MSCs from human bone marrow tissues by density gradient centrifugation. (JPG 727 kb)12958_2017_235_MOESM1_ESM.jpg
Additional file 2: Figure S2. Identification of MSCs by Flow Cytometry. (A) Representative histogram of FACS results showing the MSCs surface marker profile. The blue peak indicated the specific antibodies: CD34, CD45, CD19, negative cocktail (including CD44, CD90 and CD105). The red peak represented the isotope antibodies. (B) Positive expression of CD34, CD45, CD19 and negative cocktail (including CD44, CD90 and CD105) in five individuals as examined by flow cytometry was expressed as mean ± SD. The proportion of cells expressing CD44, CD90, CD105 and negative cocktail, which were analyzed from 5 independent samples, were 96.6% ± 2.1%, 96.4% ± 2.2%, 97.2% ± 2.0% and 2.9% ± 0.6%. (JPG 418 kb)12958_2017_235_MOESM2_ESM.jpg
Additional file 3: Figure S3. Representative images showing expression of CD90 and CD105 in ovarian sections after co-transplantation of MSCs in both high and low magnification. MSC, mesenchymal stem cells. Scale bar = 200 μm in A and C, Scale bar = 50 μm in B and D. (JPG 453 kb)12958_2017_235_MOESM3_ESM.jpg
Additional file 4: Figure S4. The MSC clones stably knocking down ANG were identified and verified on qPCR and ELISA analysis. A) Results of quantitative PCR showed a significant knock-down of ANG mRNA expression in the shANG transfected MSCs (n = 3). B) Secreted ANG protein level in the shANG and shCTRL transfected MSCs groups were determined by ELISA (n = 3). Data are shown as means ± SEM of triplicates in a representative experiment. MSC, mesenchymal stem cells; ANG, angiogenin; shANG, ANG specific short hairpin RNA, shCTRL: control short hairpin RNA, * P < 0.01. (TIF 7881 kb)12958_2017_235_MOESM4_ESM.tif
Additional file 5: Figure S5. Representative images showing triple staining of Ki67, DAPI and CD31 in ovarian graft with or without co-transplantation of MSCs. Vasculature is shown in red, cell nuclei are shown in blue and Ki67 positive nuclei are shown in green. Scale bar = 50 μm. (JPG 299 kb)12958_2017_235_MOESM5_ESM.jpg
Additional file 6: Figure S6. Representative images showing HE staining of ovarian sections in ovarian graft with or without co-transplantation of MSCs in both high and low magnification. MSC, mesenchymal stem cells. Scale bar = 200 μm in A and C, Scale bar = 50 μm in B and D. (JPG 507 kb)12958_2017_235_MOESM6_ESM.jpg
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- Mesenchymal stem cell-derived angiogenin promotes primodial follicle survival and angiogenesis in transplanted human ovarian tissue
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