Nanoparticle labeling identifies slow cycling human endometrial stromal cells

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Abstract

Introduction. Evidence suggests that the human endometrium contains stem or progenitor cells that are responsible for its remarkable regenerative capability. A common property of somatic stem cells is their quiescent state. It remains unclear whether slow-cycling cells exist in the human endometrium. We hypothesized that the human endometrium contains a subset of slow-cycling cells with somatic stem cell properties. Here, we established an in vitro stem cell assay to isolate human endometrial-derived mesenchymal stem-like cells (eMSC). Methods. Single-cell stromal cultures were initially labeled with fluorescent nanoparticles and a small population of fluorescent persistent cells (FPC) remained after culture of 21 days. Two populations of stromal cells, namely FPC and non-FPC were sorted. Results: Quantitative analysis of functional assays demonstrated that the FPC had higher colony forming ability, underwent more rounds of self-renewal and had greater enrichment of phenotypically defined prospective eMSC markers: CD146 + /CD140b + and W5C5 + than the non-FPC. They also differentiate into multiple mesenchymal lineages and the expression of lineage specific markers was lower than that of non-FPC. The FPC exhibit low proliferation activities. A proliferation dynamics study revealed that more FPC had a prolonged G 1 phase. Conclusions: With this study we present an efficient method to label and isolate slow-proliferating cells obtained from human endometrial stromal cultures without genetic modifications. The FPC population could be easily maintained in vitro and are of interest for tissue-repair and engineering perspectives. In summary, nanoparticle labeling is a promising tool for the identification of putative somatic stem or progenitor cells when their surface markers are undefined. © 2014 Xiang et al.; licensee BioMed Central Ltd.

Figures

  • Figure 1 Nanoparticle-labeled endometrial stromal cells. Qtracker® label of endometrial stromal cells. Quantitation of nanoparticle-labeled endometrial stromal cells (A) with different duration of chase (n = 4 per time point) and (B) at different menstrual phases (n = 3, proliferative, white; n = 3, secretory, black). Fluorescence expressing cells are reported as means ± SEM of the percentage of total stromal cells seeded. Day 1 post-labeled stromal cells stained with DAPI nuclei stain (blue) indicating the nanoparticles (red) are located in the cytoplasm (C). Representative phase contrast images of nanoparticle-labeled stromal cells at different days in culture (D – H). Nanoparticle-labeled cells (arrows) detected on day 1 (D) and then among the unlabeled stromal cells on day 3 (E), day 6 (F) and day 15 (G). Fluorescent signal retained at day 21 (H) (E - H). Negative controls of unlabeled stromal cells are shown in the insets. Representative photographs of post-labeled day 21 endometrial stromal cells after FACS analysis as FPC (I) and non-FPC (J) population in culture for five days. Culture dish displaying distribution of stromal colony forming units (CFU) (K) after 15 days of culture. Morphology of large (L) and small (M) CFU. Scale bars = 50 μm (C, I, J) and 100 μm (D - H, L - M). DAPI, 4',6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; SEM, standard error of the mean.
  • Figure 2 Clonogenicity and self- renewal ability of stromal FPC and non-FPC post-labeled day 15 and 21. (A – D) Cloning efficiency of endometrial stromal cells post-labeled with nanoparticles at day 15 (white bars) and 21 (grey bars). Cloning efficiency of large CFU (A) FPC and (B) non-FPC. Cloning efficiency of small CFU (C) FPC and (D) non-FPC. (E – H) Self-renewal activity of endometrial stromal cells post-labeled with nanoparticles at day 15 (white bars) and 21 (grey bars) using serial cloning assay. Large CFU self-renewal ability of (E) FPC and (F) non-FPC. Small CFU self-renewal ability of (G) FPC and (H) non-FPC Results reported as means ± SEM, n = 4, *, a, bP <0.05. CFU, colony-forming units; FPC, fluorescent persistent cells; SEM, standard error of the mean.
  • Figure 3 Serial passage, clonogenicity and phenotyping of endometrial FPC and non-FPC post labeled day 21. (A) Rate of serial passage is shown for both large and small CFU of FPC (white) and non-FPC (black). (B) Percentage of large and small CFU at each passage of serial cloning for FPC (white) and non-FPC (black). Percentage of (C) CD146+/CD140b+ and (D) W5C5+ cells from FPC and non-FPC populations. Bar represents the mean. Representative dot-plots for co-staining of CD146/CD140b and single staining of W5C5. Single parameter histograms for individual markers CD146-FITC, CD140b-PE and W5C5-PE. Grey line indicates background fluorescence with isotype matched IgG control Results are reported as mean ± SEM, n = 7, *P < 0.05, **P < 0.01, ***P < 0.001. CFU, colony-forming units; FPC, fluorescent persistent cells; SEM, standard error of the mean.
  • Table 1 Total cell output from human stromal FPC and non-FPC CFU
  • Figure 4 The cell proliferation activity (A - E) and G1-phase length (F - H) photographs of (A) unselected stromal cells, (B) FPC and (C) non-FPC morpholog lected endometrial stromal cells (grey), clonally derived FPC (white) and non-FPC ( NF cell proliferation assay. The absorbance unit (AU) was measured at a waveleng were obtained from the same patient. Results are reported as means ± SEM, n = 3 FPC and non-FPC populations followed by time-lapse microscopy within a period (n= 20 cells), (G) FPC (n= 20 cells) and (H) non-FPC (n= 18 cells) from three patie
  • Figure 5 Differentiation potential of human endometrial stromal FPC and non-FPC into mesenchymal lineages in vitro. Myogenic differentiation (A) with αSMA (brown) staining on cells clonally derived from stromal large CFU of FPC and non-FPC. Protein expression and quantification of αSMA. Relative gene expression level of ACTA by real time PCR. Osteogenic differentiation (B) with osteopontin (brown) staining on cells clonally derived from stromal large CFU of FPC and non-FPC. Protein bands and quantification expression of osteopontin. Relative gene expression level of CBFA1 by real-time PCR. Chondrogenic differentiation (C) with safranin-O (red) histochemical staining on cells clonally derived from stromal large CFU of FPC and non-FPC. Immunofluorescent staining with DAPI (blue) and collagen II (pink) on stromal large CFU of FPC and non-FPC. Micromass structure depicted from FPC chondrogenic induced cells. Protein bands and quantification expression of collagen II. Relative gene expression level of COL2A1 by real-time PCR. mRNA expression levels were normalized to 18S. Expression of the control was set as one. Control cells stained for lineage markers shown in inset and western blots are unselected stromal cells grown in culture medium with fetal bovine serum for four weeks. Scale bar: 200 μm, including inset. Results shown from a single sample representative of three patients. Results are reported as means ± SEM for western blotting (n = 3) and for real-time PCR (n = 6), *P < 0.05, **P < 0.01. αSMA (ACTA), alpha smooth muscle actin; CBFA1: core binding factor 1; CFU, colony-forming unit; COL2A1: collagen type II alpha 1; FPC, fluorescent persistent cells; SEM, standard error of the mean.
  • Figure 6 Expression of pluripotent and self-renewal genes. The relative gene expression levels of (A) NANOG, (B) SOX2, (C) OCT-4 and (D) BMI-1 of passage 1 and 2 (P1, P2) FPC (white bars) and non-FPC (black bars) in comparison to human embryonic stem cells (hESC, grey bars) as positive control. Results are reported as means ± SEM, n = 3. FPC, fluorescent persistent cells; SEM, standard error of the mean.

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Xiang, L., Chan, R. W. S., Ng, E. H. Y., & Yeung, W. S. B. (2014). Nanoparticle labeling identifies slow cycling human endometrial stromal cells. Stem Cell Research and Therapy, 5(4). https://doi.org/10.1186/scrt473

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