In vitro evidence that platelet-rich plasma stimulates cellular processes involved in endometrial regeneration

Abstract

Purpose: The study aims to test the hypothesis that platelet-rich plasma (PRP) stimulates cellular processes involved in endometrial regeneration relevant to clinical management of poor endometrial growth or intrauterine scarring.

Methods: Human endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSC), and Ishikawa endometrial adenocarcinoma cells (IC) were cultured with/without 5% activated (a) PRP, non-activated (na) PRP, aPPP (platelet-poor-plasma), and naPPP. Treatment effects were evaluated with cell proliferation (WST-1), wound healing, and chemotaxis Transwell migration assays. Mesenchymal-to-epithelial transition (MET) was evaluated by cytokeratin and vimentin expression. Differential gene expression of various markers was analyzed by multiplex Q-PCR.

Results: Activated PRP enhanced migration of all cell types, compared to naPRP, aPPP, naPPP, and vehicle controls, in a time-dependent manner (p < 0.05). The WST-1 assay showed increased stromal and mesenchymal cell proliferation by aPRP vs. naPRP, aPPP, and naPPP (p < 0.05), while IC proliferation was enhanced by aPRP and aPPP (p < 0.05). There was no evidence of MET. Expressions of MMP1, MMP3, MMP7, and MMP26 were increased by aPRP (p < 0.05) in eMSC and eSF. Transcripts for inflammation markers/chemokines were upregulated by aPRP vs. aPPP (p < 0.05) in eMSC and eSF. No difference in estrogen or progesterone receptor mRNAs was observed.

Conclusions: This is the first study evaluating the effect of PRP on different human endometrial cells involved in tissue regeneration. These data provide an initial ex vivo proof of principle for autologous PRP to promote endometrial regeneration in clinical situations with compromised endometrial growth and scarring.

Keywords: Endometrium; Platelet-rich plasma; Proliferation; Regeneration; Stem cells.

Fig. 1

Flow diagram of experimental design. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-poor plasma, naPPP non-activated platelet-poor plasma

Fig. 2

Time-dependent proliferative effect of PRP and PPP on endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSCs), and Ishikawa cells assayed with the WST-1 assay. Absorbance values indicate relative cell proliferation. Data represent the mean ± SD. Asterisk indicates the significance at p < 0.05 compared with the control group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-poor plasma, naPPP non-activated platelet-poor plasma

Fig. 3

a Wound-healing assays for endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSCs), and Ishikawa cells. Graphs show the percentage of covered surface (n = 3 images per well, in duplicate or triplicate, n = 3 for each cell type) for the control and treatment groups. Data represent the mean ± SD. Statistical significance accepted at p ≤ 0.05. Significant difference between different time points within the same treatment groups indicated by the same letter. Asterisk indicates significant difference compared to the respective time point in the aPRP group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-poor plasma, naPPP non-activated platelet-poor plasma. b Transwell migration assays for endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSCs), and Ishikawa cells at different time points. Graphs show the average number of migrated cells (n = 3 images per insert, in duplicate or triplicate, n = 3 for each cell type) for the control and treatment groups. Data represent the mean ± SD. Statistical significance accepted at p ≤ 0.05. Significant difference between different time points within the same treatment group is indicated by the same letter. Asterisk indicates significant difference compared to the respective time point in the aPRP group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-poor plasma, naPPP non-activated platelet-poor plasma. The y axis range is not unified so that the smaller-scale changes remain apparent to the reader

Fig. 3

a Wound-healing assays for endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSCs), and Ishikawa cells. Graphs show the percentage of covered surface (n = 3 images per well, in duplicate or triplicate, n = 3 for each cell type) for the control and treatment groups. Data represent the mean ± SD. Statistical significance accepted at p ≤ 0.05. Significant difference between different time points within the same treatment groups indicated by the same letter. Asterisk indicates significant difference compared to the respective time point in the aPRP group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-poor plasma, naPPP non-activated platelet-poor plasma. b Transwell migration assays for endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSCs), and Ishikawa cells at different time points. Graphs show the average number of migrated cells (n = 3 images per insert, in duplicate or triplicate, n = 3 for each cell type) for the control and treatment groups. Data represent the mean ± SD. Statistical significance accepted at p ≤ 0.05. Significant difference between different time points within the same treatment group is indicated by the same letter. Asterisk indicates significant difference compared to the respective time point in the aPRP group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-poor plasma, naPPP non-activated platelet-poor plasma. The y axis range is not unified so that the smaller-scale changes remain apparent to the reader

Fig. 4

mRNA expression of a MMPs (MMP 1, 3, 7, 26); b transgelin; c interleukins (IL1A, IL1B) and receptor IL1R2, and chemokines (CCL5, CCL7, and CXCL13); and d EGFR, FGFR2, PDGFRB, in endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSCs) upon treatment with PRP or PPP, normalized to vehicle controls. Statistical significance accepted at p ≤ 0.05. Data represent the mean ± SD. Asterisk indicates significant difference compared to the aPRP group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-poor plasma, naPPP non-activated platelet-poor plasma. The y axis range is not unified so that the smaller-scale changes remain apparent to the reader

Fig. 4

mRNA expression of a MMPs (MMP 1, 3, 7, 26); b transgelin; c interleukins (IL1A, IL1B) and receptor IL1R2, and chemokines (CCL5, CCL7, and CXCL13); and d EGFR, FGFR2, PDGFRB, in endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSCs) upon treatment with PRP or PPP, normalized to vehicle controls. Statistical significance accepted at p ≤ 0.05. Data represent the mean ± SD. Asterisk indicates significant difference compared to the aPRP group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-poor plasma, naPPP non-activated platelet-poor plasma. The y axis range is not unified so that the smaller-scale changes remain apparent to the reader

Fig. 5

a Immunofluorescent analysis of vimentin (Vim) and cytokeratin 18 (KRT18) protein in human endometrial mesenchymal stem cells (eMSC) in control cultures and cultures exposed to 5% PRP or PPP. Magnification, ×200. Negative controls are presented as inserts for vimentin and cytokeratin 18, respectively. No difference was observed between the groups. Data on endometrial stromal fibroblasts (eSF) or bone marrow-derived mesenchymal stem cells (BM-MSCs) not shown. b mRNA expression of cytokeratin 7 (KRT7) in eMSC, eSF, and BM-MSC and vimentin (VIM) in eSF. Significance accepted at p ≤ 0.05. Data represent the mean ± SD. Vimentin mRNA data for eMSC or BM-MSC not shown (no significant difference). aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-poor plasma, naPPP non-activated platelet-poor plasma