The bone marrow-derived human mesenchymal stem cell: potential progenitor of the endometrial stromal fibroblast

Abstract

The cellular sources that contribute to the renewal of human endometrium are largely unknown. It has been suggested that endometrial stem cells originate from bone marrow-derived mesenchymal stem cells (MSC), with subsequent development into endometrial stromal fibroblasts (hESF). We hypothesized that if bone marrow-derived MSC contribute to endometrial regeneration and are progenitors of hESF, their treatment with agents known to regulate hESF differentiation could promote their differentiation down the stromal fibroblast lineage. To this end, we treated bone marrow-derived MSC with estradiol and progesterone, bone morphogenetic protein 2 (BMP2), and activators of the protein kinase A (PKA) pathway and investigated specific markers of hESF differentiation (decidualization). Furthermore, we investigated the transcriptome of these cells in response to cAMP and compared this to the transcriptome of hESF decidualized in response to activation of the PKA pathway. The data support the idea that MSC can be differentiated down the hESF pathway, as evidenced by changes in cell shape and common expression of decidual markers and other genes important in hESF differentiation and function, and that bone marrow-derived MSC may be a source of endometrial stem/progenitor cells. In addition, we identified MSC-specific markers that distinguish them from other fibroblasts and, in particular, from hESF, which is of biologic relevance and practical value to the field of endometrial stem cell research.

FIG. 1.

Morphological transformation of MSC. A) MSC cultured for 14 days, vehicle control. B) MSC treated with 1 mM 8-Br-cAMP for 14 days. C) Human endometrial stromal fibroblasts (hESF) cultured for 4 days, vehicle control. D) hESF treated with 8-Br-cAMP for 4 days. Magnification ×40.

FIG. 2.

A) PRL and IGFBP1 mRNA levels in MSC treated with 1 mM 8-Br-cAMP for 14 days expressed as fold-change compared to 14 days, vehicle control. Time course of PRL and IGFBP1 protein expression in conditioned media from MSC treated with 1 mM 8-Br-cAMP were normalized to total RNA levels (n = 3). B) Expression of PRL, IGFBP1, and STC1 mRNA and/or protein in HDFn (human dermal fibroblasts, neonatal) treated with 1 mM cAMP for 14 days. PRL, IGFBP1, and STC1 mRNA levels are expressed as fold-change to time control (vehicle control at 3, 7, 14, and 18 days) normalized to t = 0. Note the difference in scale between A and B. PRL protein secretion at different culture time points was normalized to total RNA. No IGFBP1 secretion by HDFn occurred with or without cAMP treatment. Asterisk indicates statistically significant difference compared to vehicle control at each time point (P ≤ 0.05). Error bars represent the SEM.

FIG. 3.

A) LH receptor (LHCGR), FSH receptor (FSHR), estrogen receptors α and β (ESR1 and ESR2), progesterone receptors A and B (PGRA and PGRB, respectively), and androgen receptor (AR) mRNA expression in MSC treated with 1 mM 8-Br-cAMP for 14 days or 50 nM FSH for 14 days expressed as fold-change compared to 14 days, vehicle control. B) Messenger RNA expression for steroidogenic enzymes STAR, CYP11A1, and CYP19A1 in MSC treated with 1 mM 8-Br-cAMP for 14 days expressed as fold-change compared to 14 days, vehicle control. Error bars represent the SEM. *P ≤ 0.05.

FIG. 4.

A) Hierarchical clustering analysis of MSC at t = 0 (T=0), MSC treated with vehicle for 14 days (T=14d), and MSC treated with 8-Br-cAMP for 14 days (T=14d+cAMP) are also shown. B) Dendogram of hierarchical clustering analysis of MSC at t = 0 (t-0) and 14 days after vehicle (14d veh ctr) or 8-Br-cAMP (14d cAMP) treatment. C) PCA of MSC at t = 0 and 14 days after vehicle or 8-Br-cAMP treatment.

FIG. 5.

A) Real-time RT-PCR validation of selected genes in MSC treated for 14 days with cAMP versus vehicle control. Correspondent microarray data for stanniocalcin 1 (STC1), spermine oxidase (SMOX), spondin 1 (SPON1), and IGF-binding protein 5 (IGFBP5) are presented. Data are presented as fold-change. Error bars represent the SEM. The left y-axis addresses quantitative PCR results; the right y-axis addresses microarray data. B) Venn diagram of common and unique genes expressed in cAMP-treated MSC and hESF compared to their respective controls.

FIG. 6.

Relative expression of ENPP2, EFEMP1, ALDH1A1, and CNTN1 mRNA in MSC (t = 0 [t-0], 14-day control [14d control], and 14 days treated with cAMP [14d cAMP]), HDFn (t-0, 14d control, and 14d cAMP), and hESF (t-0 and 14d control). Inserts indicate the fold-change (FC) expression of genes in the microarray data set in the present study. The y-axis is presented as a log scale in the two graphs on the left. An asterisk indicates a statistically significant difference (P ≤ 0.05) compared to t-0 control for each gene and each cell type; a number symbol indicates a statistically significant difference (P ≤ 0.05) between vehicle control and cAMP-treated cells. Error bars represent the SEM.