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Nanoparticle labeling identifies slow cycling human endometrial stromal cells.

Xiang L, Chan RW, Ng EH, Yeung WS - Stem Cell Res Ther (2014)

Bottom Line: It remains unclear whether slow-cycling cells exist in the human endometrium.They also differentiate into multiple mesenchymal lineages and the expression of lineage specific markers was lower than that of non-FPC.In summary, nanoparticle labeling is a promising tool for the identification of putative somatic stem or progenitor cells when their surface markers are undefined.

View Article: PubMed Central - PubMed

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 G1 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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Both populations of stromal cells showed strong protein expression of the early smooth muscle cell marker (Figure 5A) αSMA in immunohistochemical staining and in western blotting when cultured in the myogenic inducing medium. Quantitative analysis revealed a significantly higher mRNA expression of αSMA (ACTA) in the FPC than in the non-FPC (P < 0.05, Figure 5A).


Nanoparticle labeling identifies slow cycling human endometrial stromal cells.

Xiang L, Chan RW, Ng EH, Yeung WS - Stem Cell Res Ther (2014)

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.
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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.
Mentions: Both populations of stromal cells showed strong protein expression of the early smooth muscle cell marker (Figure 5A) αSMA in immunohistochemical staining and in western blotting when cultured in the myogenic inducing medium. Quantitative analysis revealed a significantly higher mRNA expression of αSMA (ACTA) in the FPC than in the non-FPC (P < 0.05, Figure 5A).

Bottom Line: It remains unclear whether slow-cycling cells exist in the human endometrium.They also differentiate into multiple mesenchymal lineages and the expression of lineage specific markers was lower than that of non-FPC.In summary, nanoparticle labeling is a promising tool for the identification of putative somatic stem or progenitor cells when their surface markers are undefined.

View Article: PubMed Central - PubMed

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 G1 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.

No MeSH data available.


Related in: MedlinePlus