progenitor cell-like characteristics

Reproductive BioMedicine Online (2010) 21, 783– 793

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ARTICLE

Side population cells in the human decidua of early pregnancy exhibit stem/progenitor cell-like characteristics Chun Guo a, Huili Zhu a, Wei Huang Yaofang Liu a, Aixiang Tan a

a,*

, Shengfu Li b, Wenwen Qu a,

a

Department of Reproductive Endocrinology, West China Second University Hospital, Sichuan University, Chengdu 610041, PR China; b Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, PR China * Corresponding author. E-mail address: [email protected] (W Huang). Dr Wei Huang received her Masters degree in 1985 and PhD in 1992 from West China Medical University, Chengdu Sichuan, China, and has worked as attending doctor and associate professor there since then. In 2004, she became full professor of obstetrics and gynaecology, becoming involved in teaching, clinical work and basic science. Her research focuses on human decidual stem cells, proliferation and interaction at the maternal–fatal interface, endometriosis and female infertility.

Abstract It is proposed that human decidua contains a population of stem cells that are responsible for the proliferation ability

during the process of embryo implantation and placenta formation and that factors in the crosstalk between the decidua and chorion may mediate decidual stem cell differentiation. This study analysed the phenotype of side population (SP) cells and investigated the clonogenicity and differentiation ability of SP cells in human decidua of early pregnancy. Serum-free culture-conditioned media of human decidua and chorion were obtained from decidua and chorion explant culture. Decidual SP cells were isolated by fluorescence-activated cell sorting. Different inducing media were added and the functional differentiation of decidual SP cells was examined. Decidual SP cells were negative for the mature decidual cell marker CD13 and prolactin and negative for CD34 and CD45 expression. Decidual SP cells formed clones after culture in colony-forming medium and they could form clones again. Differentiated cells expressing CD13 and prolactin were observed and stroma-like structures expressing CD13 were obtained. These results indicate that decidual SP cells are enriched for stem cell activity. Oestradiol, progesterone and factors in culture-conditioned media of human decidua and chorion induced their proliferation and differentiation. RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: cell differentiation, decidua, side population cells, stem cells

1472-6483/$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2010.07.010

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Introduction The human endometrium is characterized by a constant and rapid process of cell proliferation, differentiation and breakdown as part of each menstrual cycle throughout the reproductive period (Ferenczy et al., 1979). After shedding the endometrial lining, the endometrium proliferates dramatically and completely regenerates within 2 weeks (McLennan and Rydell, 1965). It has been confirmed that the human endometrium contains a population of stem/progenitor cells that is responsible for its remarkable regeneration ability (Padykula, 1991). Recently, evidence based on stem cell functional assays demonstrated that 0.22 ± 0.07% of endometrial epithelial cells and 1.25 ± 0.18% of stromal cells formed colonies when cultured in vitro, which confirmed the existence of putative endometrial epithelial and stromal stem cells (Chan et al., 2004; Gargett, 2006). It is also possible that stem cells in decidua tissues that come from secretory endometrium are responsible for their remarkable regeneration ability. Over the course of pregnancy, the human uterus undergoes a 500–1000-fold increase in volume and a 24-fold increase in weight. The decidua increases noticeably too. The origin of these new decidual cells, however, is unclear. They may arise from cell proliferation of existing decidual cells inside tissues or may result from the transformation of stem cells. Strakova et al. (2008) reported human uterine fibroblasts isolated from the decidua parietalis to be multipotent, forming colonies when plated at low densities and differentiating into osteoblasts, adipocytes and chondrocytes with differentiation-inducing media. It has been suggested that decidua tissue should contain adult stem cells, which can transform to mature cells in certain conditions, such as pregnancy. Stem cell subpopulations, known as side population (SP) cells, have been isolated in many mammalian tissues, including human tissues such as bone marrow, liver, skeletal muscle, mammary gland, brain and endometrium using the fluorescent dye Hoechst 33342 efflux phenomenon. In many cases, this cell population has been shown to contain apparently multipotent stem cells (Challen and Little, 2006). Some researchers have reported the existence of SP cells in human endometrium that show the characteristics of stem cells (Kato et al., 2007; Tsuji et al., 2008). A previous study has also demonstrated that SP cells exist in decidual tissues of human early pregnancy and that sorted decidual SP cells can proliferate and form clones gradually within 1 month when cultured on collagen-coated plates in conditioned medium (Qu et al., 2008). But the phenotype and the differentiation ability of decidual SP cells have not been determined yet. This study isolated and analysed the phenotype of SP cells in the human decidua of early pregnancy and investigated the clonogenicity and differentiation ability of decidual SP cells with various treatments.

Materials and methods Human decidua and chorion tissue collection Decidua and chorion tissues were obtained from 22 pregnant women aged 20–32 years from 6 to 10 weeks of gestation

C Guo et al. undergoing elective termination of pregnancy. All samples were collected from normal pregnancy without any pregnancy-related disorders or any medicine usage within 3 months. The study was approved by the ethical committee of West China Second University Hospital of Sichuan University and informed consent was obtained from each woman. Collected tissues were put into ice-cold calcium- and magnesium-free Hanks’ balanced salt solution (D-Hank’s) for decidua explant or decidual cell culture.

Decidua-conditioned media and chorionconditioned media Decidua and chorion tissues were prepared using a method based on that of Wright et al. (2006), with minor modifications. Firstly, chorion and decidua tissues were isolated, washed several times in D-Hank’s and carefully dissected to 2 mm pieces, then placed in a six-well plate, approximately 100 mg tissue/well. Decidua and chorion explants were cultured at 37C (5% CO2, 95% O2) for 1 h before adding 3 ml of 1:1 serum-free Dulbecco’s modified Eagle’s media (DMEM) and F12 culture medium (Gibco, USA) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 10 nmol/l oestradiol (Sigma, USA) and 1 lmol/l progesterone (Sigma) (Omigbodun et al., 1997; Popovici et al., 2000). Cultures were maintained in standard culture conditions for a further 48 h, and then decidua-conditioned media and chorion-conditioned media (DCM and CCM, respectively) were harvested by centrifugation at 350g for 5 min to separate tissue, filtrated through a 0.22 lm filter and stored at 80C until use.

Preparation of human decidual cells Decidual tissue was minced to 1 mm pieces and digested with 1 mg/ml collagenase IV (Sigma, USA) and 0.05% trypsin in serum-free DMEM for 30 min incubation on a rotator at 37C. Enzymes were inactivated with 10% fetal bovine serum (FBS), cells were collected through a 74 lm mesh filter, then plated in 25 cm2 cell culture flasks at a density of 2 · 106/ml and cultured in 10% FBS DMEM supplemented with 100 lg/ml streptomycin, 100 U/ml penicillin and 0.08 IU/ml insulin.

Flow cytometry for SP cells Decidual cells maintained for 24–48 h were digested with 0.25% trypsin-EDTA, resuspended in DMEM containing 2% FBS at a density of 1 · 106 cells/ml and incubated with 2.5 lg/ml Hoechst 33342 (Fluka, USA) for 90 min at 37C. To choose verapamil-sensitive SP cells, another tube of the cells was preincubated with 100 lg/ml verapamil (Shanghai Harvest Pharmaceutical, China) for 10 min before the addition of Hoechst 33342 dye. After incubation, cells were centrifuged at 400g for 5 min, resuspended in ice-cold D-Hank’s containing 2% FBS and strained through a 74 lm mesh and kept at 4C until analysis. Cells stained with Hoechst 33342 dye were further stained with antibodies CD13, CD34 and CD45 (BD Pharmingen, USA), then analysed or sorted on a cell sorter (FACS Aria I; BD, USA). Emission was at 407 nm (violet laser diode), Hoechst blue and red

Side population cells in the human decidua exhibit stem cell characteristics fluorescence emissions were collected using a combination of 440-nm pass and 675-nm pass filters. Then cells were shown in a Hoechst blue versus Hoechst red dot plot and were analysed with FlowJo software.

Immunocytochemistry and fluorescent immunophenotyping Cultured cells on cover slips were immunostained for mouse anti-human E-cadherin monoclonal antibody, mouse antihuman vimentin monoclonal antibody and rabbit anti-human prolactin (PRL) polyclonal antibody (all 1:200; Dako). Cultured cells were seeded onto collagen-coated coverslips. After 48 h of culture, decidual cells were fixed in acetone for 15 min at 4C and endogenous peroxidase activity was quenched in 3% hydrogen peroxide in methanol for 15 min at room temperature. Primary antibodies were applied to the sections in a moist chamber for overnight incubation at 4C. The secondary antibody for immunostaining was Envision/horseradish peroxidase (anti-mouse, Dako) for E-cadherin and vimentin and Envision/horseradish peroxidase (anti-rabbit, Dako) for PRL, at 37C for 20 min. Subsequent steps were carried out according to the manufacturer’s instructions. The reaction was visualized using diaminobenzidine, the cover slips were dehydrated through graded alcohols and then mounted using synthetic resin. Negative controls were isotype-matched irrelevant immunoglobulin G (IgG). For fluorescent immunocytochemistry, cell smears were fixed in 4% paraformaldehyde for 15 min at room temperature, washed in phosphate-buffered saline and permeabilized for 5 min in 0.1% Triton X-100 phosphate-buffered saline. Primary antibodies were incubated overnight at 4C with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or goat anti-rabbit IgG (Zhong shan Golden bridge Biotechnology, China) at a concentration of 1:50 served as the secondary antibody. Cells were counterstained using the nuclear dye (4,6-diamidino-2-phenylindole) (Sigma). SP cells and non-SP (NSP) cells were stained with phycoerythrin (PE)-CD13 (BD Pharmingen), after Hoechst 33342 sorting, the expression of CD13 in SP cells and NSP cells was obtained and red fluorescence of CD13 positive cells was detected under fluorescence microscope.

Clonogenicity and differentiation of decidual SP cells To test the clonogenicity of decidual SP cells, the sorted cells were plated at a density of 300 cells/cm2 on cell culture plates precoated with collagen (Rat tail collagen, type I; BD Pharmingen) in colony-forming medium (CFM), including DMEM containing 10% FBS, 100 ng/ml interleukin (IL)-6, 100 ng/ml stem cell factor (SCF) and 10 ng/ml thrombopoietin (TPO) (all from R and D Systems, USA), as previously described (Kato et al., 2007). When the cells formed colonies finally, they were digested with 0.25% trypsin-EDTA and reseeded in conditioned medium to investigate whether they had the ability to form colonies again. The cells were then digested again and reseeded in medium of 10% FBS DMEM/F12 for 20 days to determine the ability for natural differentiation.

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The sorted cells seeded at the same density were incubated in different media as below in triplicate: group I: sorted SP cells in CFM; group II: sorted NSP cells in the same density in DMEM/F12 and 10% FBS; the other four groups of sorted SP cells cultured in medium containing DMEM/F12 and 10% FBS plus: group III: 50% CCM; group IV: 50% DCM; group V: 10 nmol/l oestradiol + 10 lmol/l progesterone; and group VI: 50% (CCM + DCM). The medium was replaced every 5–7 days.

Results Surface markers of decidual cells in early human pregnancy The expression of E-cadherin, vimentin and PRL in decidual primary cells and cells of first passage were analysed by immunolabelling. The results showed that some of the decidual cells can express vimentin and PRL, while few of them express E-cadherin, indicating that these cells are decidual stromal cells (Figure 1).

Phenotype of decidual SP cells in early human pregnancy It has been demonstrated that the efficiency of efflux of the dye Hoechst 33342 accounts for the uniquely low fluorescence of the SP population (Goodell et al., 1996) and this ability was later shown to be due to expression of the ATP-binding cassette (ABC)-dependent transporter ABCG2/Bcrp1 (Zhou et al., 2001). The SP cell population would diminish remarkably because of increased staining by treatment with verapamil, an inhibitor of ABCG2, indicating that this population truly consisted of SP cells. Therefore, SP cells were sorted from decidual cells using Hoechst staining based on the studies by Qu et al. (2008) and Goodell et al. (1996; Figure 2). After addition of verapamil, the percentage of the P3 area decreased from 0.6% to 0.2%, indicating that 0.4% cells were real SP cells (Figure 2A and B). CD34 and CD45, markers of haematopoietic cells, showed negative expression in decidual SP cells (Figure 2C and D), indicating these decidual SP cells were not of haematopoietic origin. PE-CD13 was added to decidual cells after Hoechst 33342 staining (Figure 3A). The percentage of CD13-positive cells in SP cells (Figure 3B) was much lower than in NSP cells (Figure 3C; 8.8% ± 4.13% and 95.46% ± 3.61%, respectively; Figure 3D, three independent repeats), demonstrating that SP cells were immature decidual cells. In contrast to the large NSP cells, sorted SP cells were visualized as small, round and bright cells under phase-contrast microscopy (Figure 4A and E). The immunophenotype result showed that decidual SP cells were negative for the mature decidual cell marker CD13 (Figure 4F) and PRL (Figure 4H), but were positive for the vimentin (Figure 4G), demonstrating that decidual SP cells may be of mesenchyme origin.

In-vitro growth and differentiation of decidual SP cells The sorted SP cells were seeded at clonal density on cell-culture plates pre-coated with collagen in CFM. Decidual SP cells

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Figure 1 Surface markers of decidual cells of human first-trimester pregnancy for (A–D) primary decidual cells and (E–H) cells of first passage. (A) Control; (B and F) E-cadherin expression; (C and G) vimentin expression; and (D and H) PRL expression. Bars = 100 lm.

showed clonogenic growth and formed clusters after at least 21 days of culture (Figure 5A and B). NSP cells in the same condition were used as a control group and showed no clone appearance in long-time culture in vitro. To further investigate whether the decidual SP cells were progenitor cells or stem cells, clusters were digested and reseeded in CFM. New clones formed again after another 28 days of culture, although the number of these clones was much less than that of primary culture (Figure 5C). However, without IL-6, SCF and TPO in the medium, clones of decidual SP cells were not formed after 3 weeks of culture (Figure 5D). In all media, decidual SP cells grew very slowly in the first week. However, after 2 weeks of incubation, the differentiation speed varied noticeably among groups. The

highest cell density was obtained in group VI (50% (CCM + DCM)), then group IV (50% DCM) and group III (50% CCM), group V (10 nM oestradiol and 10 lmol/l progesterone) was the lowest; cells in group I (CFM) formed clones after 3 weeks (Figure 6). These differentiated cells were stained by PRL and CD13. PRL (Figure 6) and CD13 (Figure 7A, from group IV) were expressed in differentiated cells in groups II–VI after 21 days of culture, though the staining intensity varied among groups. Decidual SP cell clones in group I were negative for PRL and CD13 expression. The decidual SP cells maintained in medium with DCM (group IV) formed a multilayer structure in the core of the adherent cell layer, with more than 20 cells were aggregated in one of the spindle-like structures, and the whole

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Figure 2 Sorting of SP cells from decidual cells. Decidual cells were digested and labelled with Hoechst 33342 in the presence or absence of verapamil and at least 1 · 106 cells were analysed by flow cytometry. Populations of decidual cells were separated using a two-window discrimination of red and blue fluorescence emissions along the abscissa and ordinate axis respectively. (A and B) On the fluorescence profile, whole decidual cells could be divided into two distinct major populations (annotated P3 and P4). The SP cells exhibited low Hoechst staining correlating with the fluorescence feature of an active Hoechst efflux (A; 0.6% P3) and disappeared in the presence of verapamil (B; 0.2% P3), therefore the percentage of real SP cells is 0.4%. (C) Phycoerythrin-CD34 (anti-human) and (D) Allophycocyanin-CD45 (anti-human) were added to decidual cells after Hoechst staining, their prevalence in SP cells (C; P2) were analysed (D) Q1 and Q4 represent the percentage of CD34 and CD45 positive cells respectively, and Q2 represents the percentage of both CD34 and CD45 positive cells, while Q3 represents both CD34 and CD45 negative cells. SP cells were mainly detected in Q3 and were negative for both CD34 and CD45.

core had a meshwork appearance consistent with characteristics of decidual stromal cells. PRL expression was detected by immunocytochemistry in the meshwork-like core (Figure 7B). If the SP cells were putative stromal stem cells, they could differentiate into stroma-like structures under certain conditions. Indeed, when decidual SP cells formed clones after 21 days of culture, they were digested with trypsin and reseeded on collagen-coated cell culture plates in CFM for another 28 days until colonies formed again. Then

the cells were digested and the medium was replaced by DMEM/F12 and 10% FBS; 20 days later, stroma-like tissues were found and CD13 was expressed (Figure 7C and D).

Discussion It has been suggested that the human decidua of first-trimester pregnancy contains a population of stem/progenitor cells that are responsible for their remarkable regeneration

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Figure 3 Phycoerythrin-CD13 staining of SP cells and NSP cells after Hoechst sorting. (A) After 48 h of culture in vitro, primary decidual cells were stained with Hoechst 33342 and subsequently PE-CD13 (anti-human); SP-gated and NSP-gated cells are indicated as P2 and P3. (B and C) Percentage of CD13-positive cells from P2 and P3 are indicated as P4 and P5. (D) 8.8% ± 4.13% and 95.46% ± 3.61% of CD13-positive cells were detected in SP cells and NSP cells, respectively. Results are from experiments performed in triplicate.

ability during embryo implantation. The data reported in the present study is a strong argument in support of the existence of decidual stem cells. Stem cells are characterized by the ability of self-renewal and the ability of daughter cells to differentiate into more specific phenotypes. SP cells are stem cell subpopulations identified with the fluorescent dye Hoechst 33342 in many mammals, including humans (Challen and Little, 2006). In 2007, Kato firstly reported the presence of SP cells in the normal human endometrium, which can generate daughter cells, form colonies and form either glandular- or stroma-like structures (Kato et al., 2007). The current study first isolated these stem-like or progenitor cells, the decidual SP cells, from human decidua and found that they survived and proliferated on cell-culture plates pre-coated with collagen under CFM sup-

plemented with IL-6, TPO and SCF. When the clones formed, they were digested and reseeded in CFM and clones formed again. This result demonstrated the self-renewal property of decidual SP cells, which is fundamental for the stem cells. In support of the current results, Gargett et al. (2009) reported that single endometrium stromal or epithelial cells can form colonies and can be further serially cloned more than three times and underwent more than 30 population doublings in a number of consecutive clonings. Although the exact origin of the decidua cell is still uncertain, decidual cells express CD13, vimentin and PRL, as shown in previous reports (Garcı´a-Pacheco et al., 2001; Imai et al., 1992; Reis et al., 2002) and the current study. In the meantime, the decidual SP cells are positive for vimentin but negative for CD34 and CD45, indicating they

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Figure 4 Immunophenotypes in human decidua of first-trimester pregnancy of (A–D) NSP cells and (E–H) SP cells. (A and E) Phase-contrast microscopy; (B and F) phycoerythrin (PE)-CD13 staining; (C and G) vimentin staining; and (D and H) NSP cells were positive for prolactin (PRL) staining. Bars = 100 lm (A–D), 50 lm (E), 20 lm (F–H).

are of non-haematopoietic cell origin. Li et al. (2005) reported that mesenchymal stem cells derived from human placenta express vimentin as well as CD29, CD44, CD105 and CD166 and about 60% of skin-derived mesenchymal stem cells expressed vimentin (Yang et al., 2005). Therefore, this study proposed that decidual SP cells may be part of mesenchymal stem cells, though vimentin is not a specific marker of mesenchymal stem cell. In support of this argument, Gargett et al. (2009) reported that endometrium stromal clones expressed mesenchymal stem cell markers CD29, CD44, CD73, CD90, CD105, CD140B and CD146 but not endothelial or haemopoietic markers CD31, CD34 and CD45. Qu et al. (2008) reported that 31.4% of decidual cells can express CD44 under fluorescent microscope or flow cytome-

try, which is thought to be a marker of mesenchymal stem cells. In coincidence with these results, Dimitrov et al. (2010) have recently reported that human decidua contains a population of multipotent mesenchymal stem cells, which were demonstrated to express CD29, CD73 and CD90. Further detailed and rigorous phenotyping is required to obtain a definitive answer on this point. CD13, also known as aminopeptidase N and myelomonocytic antigen, is a typical mesenchymal stem cell marker and is expressed in decidual stromal cells as well as endometrial stromal cells (Garcı´a-Pacheco et al., 2001). PRL was produced by human endometrial stromal cells undergoing decidualization during the secretory phase of the menstrual cycle. If pregnancy occurs, these decidual cells

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Figure 5 Clonogenicity of decidual SP cells in in-vitro culture. (A and B) Clones (arrows) were formed after 21 days of culture in CFM. (C) Formation of clones (arrow) 28 days after digestion and reseeding in CFM. (D) No clones were formed after 21 days of culture in media without IL-6, SCF and TPO. Bars = 100 lm (A), 50 lm (B), 50 lm (C), 100 lm (D).

continue to produce PRL (Reis et al., 2002). Therefore, PRL is considered to be a well-characterized marker of endometrial stromal cell differentiation and a well-established end-point of decidualization (Christian et al., 2001). This study found that sorted decidual SP cells do not express CD13 and PRL, suggesting that these decidual SP cells represent an immature or undifferentiated population. Interestingly, CD13 and PRL can be further induced in in-vitro culture with different conditioned medium in these decidual SP cells. The expression of CD13 and PRL means that mature and functional decidual cells are derived from the incubation of decidual SP cells in vitro. In agreement with the current result, Dimitrov et al. (2010) reported that the stem cells in decidua could be induced to produce PRL in medium added with 10 lmol/l medroxyprogesterone acetate, 10 nmol/l oestradiol, and 0.5 mmol/l 8-bromo-cyclic-adenosine monophosphate too. In order to explore the contribution of different factors involved in decidual SP cell differentiation, this study used different inducing media in the experimental design: the decidual SP cells were incubated in DCM, CCM, both DCM and CCM or oestradiol and progesterone only. These media were used to attempt to mimic the in-vivo environment. When the SP cells were induced in medium with oestradiol and progesterone only, differentiated cells of varied morphology were obtained. The differentiation speed differed among all groups, the fastest was in DCM and CCM together, the next in DCM, the slowest in medium supplemented with oestradiol and progesterone only. These data support the notion that there exists a molecular dialogue between the decidual SP cells and secreted products from human chorion and/or decidua that induces the differentiation of these decidual SP cells. Hess et al. (2007) found that decidual stromal cell respond to paracrine signals from the trophoblast

through the amplification of immune and angiogenic modulators, demonstrating the impact of paracrine signals on decidual stromal cells. Keskin et al. (2007) found that Lin( ) CD34(+) CD45(+) haematopoietic stem/progenitor cells could be isolated from decidual tissue and that these progenitors produced natural killer cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor, supporting the important function of paracrine factors on decidual stem cell differentiation. The differentiated cells differed in morphology and the stain intensity of PRL varied among groups. This may be because of the progesterone added to medium in each group in order to mimic the endocrine environment in vivo. Other studies have reported the increase of PRL in endometrium cells in the secretory phase in response to ovarian hormone stimulation. The product of PRL increased significantly after a decidualization stimulus of cAMP plus medroxyprogesterone acetate under in-vitro conditions (Mak et al., 2002). In another in-vitro decidualization model using oestradiol and progesterone, endometrium SP cells were reported to secrete PRL (Tsuji et al., 2008). The current study showed that decidual SP cells may respond to progesterone in conjunction with other factors such as cytokines or chemokines in CCM and DCM in the medium in a paracrine manner. To explore the different inducing contribution among groups, other more specific differentiated cell markers could be used in the following-up studies. Of major interest is the identity of the products in conditioned medium that elicit the responses in decidual SP cells. An adequately developed decidua is important for pregnancy maintenance. If a technique of using stem cells to regenerate or repair the decidua is established, it will be clinically significant for the treatment of pregnancy related disorders. In the current study, the differentiated daughter

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Figure 6 Growth of decidual SP cells in different conditioned medium (A) at 1 week, (B) at 3 weeks and with (C) prolactin (PRL) staining. PRL-positive cells were found in group II (NSP cells cultured in DMEM/F12 and 10% FBS) and groups III–VI (DMEM/F12 and 10% FBS plus: 50% CCM (group III), 50% DCM (group IV), 10 nmol/l oestradiol and 10 lmol/l progesterone (group V) or 50% (CCM + DCM) (group VI)); stain intensity varied among different groups. Bars = 100 lm (1 and 3 weeks), 50 lm (PRL staining).

cells formed colonies and finally formed stroma-like structures expressing CD13. Further studies testing the function of the structures will clarify the similarities and differences in biology between the structure differentiated in vitro and the decidua in vivo.

The origin of the human decidual stromal cells remains unknown. It is so far not clear whether the same population of stromal stem cells is isolated from early decidua or the endometrium. It is reasonable to assume that it is one cell population that is under a different control by hormonal,

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Figure 7 (A) Expression of CD13 in differentiated cells from groups II–VI by fluorescent immunophenotyping. CD13 was expressed in each group. (B) Group IV, from medium with DCM, forming a multilayered structure, which looked like a meshwork and was consistent with characteristics of decidual stromal cells. Prolactin (PRL) was positively expressed. (C and D) When decidual SP cells formed clones after 21 days of culture, they were digested and reseeded in CFM for another 28 days until colonies formed again, then the cells were digested and the medium was replaced by DMEM/F12 and 10% FBS for 20 days: stroma-like tissues were found and CD13 was expressed again. Bars = 20 lm (A, C, D), 50 lm (B).

paracrine and autocrine factors and the fact that stem cells are in different conditions in the internal environment may account for the difference of the results (Kyurkchiev et al., 2010). Further detailed studies are needed to clarify the possible origin of the stromal cells in human endometrium and decidua as well as their precise functions in the reproductive process (Kyurkchiev et al., 2010). Such knowledge will help us understanding the role of stem cells in the pathogenesis of gynaecological diseases and pregnancy-related disorders associated with abnormal endometrial regeneration and decidual development and may pave the ground for alternative therapy in these diseases.

Acknowledgements The authors wish to thank all staff of the study centre’s operation room for their assistance of tissue collection during this study. They thank Wentong Meng and Jilong Gou for technical assistance on flow cytometry and Li Li for her help on immunohistochemistry. The authors are indebted to Mr Xu Wenming for his advice on the manuscript. Author contributions: Chun Guo, Huili Zhu, Wei Huang designed experiment; Chun Guo, Shengfu Li, Wenwen Qu, Yaofang Liu, Aixiang Tan performed experiments; Chun Guo, Huili Zhu, Wei Huang wrote the paper.

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