Human decidual stromal cells express {alpha}-smooth muscle actin and show ultrastructural similarities with myofibroblasts

C. Oliver, M.J. Montes, J.A. Galindo, C. Ruiz and E.G. Olivares1

Unidad de Inmunología, Instituto de Biotecnología, Universidad de Granada, E-18012 Granada, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous reports in human and mouse material demonstrated that decidual stromal cells expressed antigens associated with haematopoietic cells, exerted immune functions, and originated from bone marrow. These findings suggested that these cells belonged to the haematopoietic lineage. We purified and expanded in culture precursors of human decidual stromal cells, and found in electron microscopic images that the ultrastructure of these cells was similar to that of myofibroblasts, which are of mesenchymal origin. The relationship between these two types of cell was confirmed by the detection (by flow cytometry) in the decidual precursors of {alpha}-smooth muscle actin, a contractile microfilament expressed solely by smooth muscle cells, myofibroblasts and related cells. This filament was also detected in decidual stromal cells decidualized in vitro by the effect of progesterone. We also found vimentin in decidual precursors and decidualized cells. This intermediate filament has been previously reported to be expressed by all decidual stromal cells and also by myofibroblasts. Desmin, another intermediate filament expressed by myofibroblasts, was not detected in the decidual precursors; however, this filament was observed in decidualized cells. The expression of {alpha}-smooth muscle actin by decidual stromal cells was also found by immunostaining in cryostat sections of early decidua. Our results suggest that decidual stromal cells are related to myofibroblasts.

Key words: decidual stromal cell/desmin/myofibroblast/{gamma}-smooth muscle actin/vimentin


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Decidual stromal cells (DSC), the major cellular component of the human decidua, originate from the proliferation and differentiation (decidualization) of a fibroblast-like stromal cell precursor (preDSC) detected in the endometrium (Richards et al., 1995Go). During the luteal phase of the menstrual cycle, or if pregnancy takes place, preDSC are induced to decidualize by progesterone (Bulmer and Peel, 1974Go). Decidualized cells (dDSC) become rounder, express desmin in their cytoplasm, and secrete prolactin (Riddick and Kusmik, 1977Go; Glasser and Julian, 1986Go; Tabanelli et al., 1992Go). Although the function and cell lineage of DSC are not fully understood, they have classically been considered fibroblastic cells with a nutritional and an endocrine role to support pregnancy (Riddick and Kusmik, 1977Go). Nevertheless, several reports have demonstrated that human and murine DSC are also involved in different immune functions (Tabibzadeh et al., 1989Go; Dudley et al., 1993Go; Montes et al., 1995Go; Olivares et al., 1997Go; Ruiz et al., 1997Go), and inflammatory cytokines such as interleukin (IL)-1 and tumour necrosis factor (TNF){alpha} inhibit decidualization (Kariya et al., 1991Go; Jikihara and Handwerger, 1994Go). Furthermore, several groups have shown that most human DSC express CD10 and CD13 (Imai et al., 1992Go; Montes et al., 1996Go); both these antigens are peptidases associated with myelomonocytic cells. Our group reported that CD10+ CD13+ human preDSC also expressed other antigens associated with haematopoietic cells (Montes et al., 1996Go; Olivares et al., 1997Go). Moreover, Lysiak and Lala (1992) showed that certain mouse DSC and endometrial stromal cells (ESC) were actually of bone marrow origin, suggesting that these cells should be included in the haematopoietic cell lineage (Lysiak and Lala, 1992Go). To determine the cell lineage to which DSC belong, we studied the ultrastructural morphology of these cells, and found that they show similarities with myofibroblasts, cells of mesenchymal origin that exhibit a fibroblastic appearance and some structural characteristics of muscle cells (Schürch et al., 1992Go). Because DSC have been widely reported to express vimentin and desmin (Glasser and Julian, 1986Go; Tabanelli et al., 1992Go), two cytoskeletal proteins also detected in myofibroblast, we studied the expression by DSC of {alpha}-smooth muscle actin ({alpha}-SM actin), a contractile microfilament which is also marker of myofibroblasts (Darby et al., 1990Go, Foo et al., 1992Go) to analyse further the relation between these two types of cells.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tissues
Ten samples from elective vaginal terminations of first trimester pregnancy (6–11 weeks) from healthy patients aged 20–30 years were used. The specimens were obtained at the Clínica El Sur (Málaga) and Gineclínica (Granada). Informed consent was obtained from each patient. This study was approved by the Comité Etico y de Investigación of the Hospital Universitario de San Cecilio, Granada.

Isolation and culture of decidual stromal cells
Samples of decidua from different patients were not mixed, to avoid alterations in the DSC phenotype resulting from allogeneic reaction and secretion of cytokines by lymphocytes that initially contaminate DSC cultures. Tissues were extensively washed in RPMI 1640 medium with 100 IU/ml penicillin and 50 µg/ml gentamicin, and the decidua was carefully freed from the trophoblast. It was then washed in Ca2+, Mg2+-free phosphate-buffered saline (PBS), minced, and left in a solution of 0.25% trypsin (Sigma, St Louis, MO, USA) and 0.02% EDTA (Merck, Darmstadt, Germany) for 15 min at 37°C. The enzymatic reaction was stopped by adding cold RPMI with 20% fetal calf serum (FCS) (Flow Laboratories, Irvine, UK); the suspension was filtered through gauze and centrifuged at 425 g for 7 min. The supernatant was discarded and the cell pellet was suspended in RPMI and centrifuged on Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden) for 20 min at 600 g. Cells were collected from the interface, suspended in RPMI and washed. This suspension, containing mainly DSC and leukocytes, was incubated in culture flasks for 1 h in complete RPMI with 10% FCS to allow macrophages and granulocytes to adhere to the flask. The supernatant was incubated overnight so that DSC adhered to the flask. Lymphocytes in the supernatant were then discarded, leaving a highly purified population of DSC free of granulocytes and macrophages. Proliferating DSC were expanded in culture for several passages; the medium was changed twice a week. In this work DSC maintained in culture were studied approximately 4–6 weeks after primary culture. During this period, proliferating DSC overgrew other possible contaminating cells. The purity of the culture was confirmed by the proportion of cells expressing both CD10 and CD13 and lacking CD14 and CD15 (Imai et al., 1992Go; Montes et al., 1996Go). These cells express antigens associated with haematopoietic cells and have been previously identified as preDSC (Montes et al., 1996Go; Olivares et al., 1997Go).

Progesterone treatment
Decidual stromal cells isolated and maintained in culture as indicated above were cultured for 15 days in the presence of 100 nmol/l progesterone (preg-4-ene-3,20-dione, Sigma). This treatment led to decidualization, as shown by changes in cell morphology, expression of desmin, and expression or secretion of prolactin (Riddick and Kusmik, 1977Go; Glasser and Julian, 1986Go; Tabanelli et al., 1992Go; Olivares et al., 1997Go).

Electron microscopy
Decidual stromal cells were fixed in 1% glutaraldehyde in 0.1 mol/l sodium cacodylate/0.1% sucrose buffer. Cells were washed in the same buffer and postfixed in 2% osmium tetroxide, dehydrated through a graded acetone series, and embedded in Epon 812. Thin sections were cut on a Reichert-Jung Ultracut E ultramicrotome, stained with lead citrate, and visualized and photographed in a transmission electron microscope (10C; Carl Zeiss, Inc., Oberkochen, Germany).

Monoclonal antibodies
The monoclonal antibodies (mAb) used in this study were anti-desmin (Eurodiagnostica, Apeldoorn, Holland); CAM 5.2 (cytokeratin) (Becton Dickinson, San Jose, CA, USA); OKM13 (CD13), OKM14 (CD14), OKM15 (CD15), anti-CD45 and OKDR (HLA-DR) (Ortho Diagnostic Systems, Beerse, Belgium); anti-human Thy-1, anti-vimentin (Serotec, Oxford, UK); BU38 (CD23) (The Binding Site; Birmingham, UK); anti-{alpha}-SM actin and anti-human follicular dendritic cells (HJ2) (Sigma); anti-human dendritic reticulum cells (DRC-1, CD21L) and anti-human common acute lymphoblastic leukaemia antigen (CD10) (DAKO, Glostrup, Denmark).

Flow cytometric analysis
Decidual stromal cells were washed and suspended in PBS at 106 cells/ml. Aliquots of 100 µl of the cell suspension were incubated with 10 µl of the appropriate mAb for 30 min at 4°C in the dark. Cells were washed, suspended in 1 ml PBS and immediately analysed in a flow cytometer (Ortho-Cytoron, Ortho Diagnostic Systems, Raritan, NJ, USA). As a negative control, cells were stained with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labelled mouse IgG or IgM. The percentage of cells that were antibody-positive was calculated by comparison with the appropriate control. For double labelling, we followed the same procedure except that a second mAb with a different fluorescent marker from the first mAb was also added. For intracytoplasmic staining of cytoskeletal filaments, DSC were fixed with 4% paraformaldehyde for 20 min at 4 °C, and permeabilized with cold acetone for 10 min before the mAb was added.

Immunostaining
For immunostaining, cryostatic sections (5 µm) of early human decidua were fixed with acetone and labelled by an indirect immunoperoxidase method. Briefly, samples were rehydrated in PBS and incubated with hydrogen peroxide and AB human serum to block endogenous peroxidase and Fc receptors respectively. The samples were then incubated for 30 min at room temperature in a humid chamber with an appropriately diluted mAb. Normal mouse serum or an irrelevant mAb as negative controls were substituted for the first antibody. After three brief washings with PBS, samples were overlaid with peroxidase-conjugated goat antimouse IgG (Bio-Rad, Richmond, CA, USA) and diluted 1:100 in 1% PBS-BSA, and the reaction was developed with 0.5 mg/ml diaminobenzidine (Sigma) containing 0.01% hydrogen peroxide. The reaction was stopped after 5–10 min by washing in excess PBS. Samples were counterstained with Mayer's haematoxylin.


    Results
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 Materials and methods
 Results
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 References
 
Flow cytometric study of surface antigens of cultured precursors of decidual stromal cells
In the absence of progesterone in the culture medium, preDSC multiply (Montes et al., 1996Go; Olivares et al., 1997Go). These cells were analysed by flow cytometry (Figure 1Go). More than 95% of the cultured preDSC expressed both CD10 and CD13; however, CD14 and CD15 were not detected, and CD45 was expressed only weakly if at all. These results confirm the purity of the culture and the absence of contaminant cells such as macrophages and granulocytes. Cultured preDSC were positive for DRC-1 and HJ2, antigens specific of follicular dendritic cells (FDC) (Naiem et al., 1983Go; Butch et al., 1995Go). CD23 and HLA-DR antigens, also expressed by FDC (Schriever and Nadler, 1992Go), were detected on preDSC. Thy-1, which some authors consider a marker of DSC (Fernández-Shaw et al., 1992Go), was also found on cultured preDSC (Figure 1Go).



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Figure 1. Flow cytometric analysis of surface antigens on cultured precursors of decidual stromal cells. Double labelling (a); single labelling (b).

 
Ultrastructure of decidual stromal cells
PreDSC purified and expanded in culture were studied by electron microscopy (Figure 2Go). The cells were fusiform and irregular in shape (Figure 2aGo), and had cytoplasmic extensions, microvilli (Figure 2bGo) and pinocytotic vesicles (Figure 2cGo). Within the cytoplasm, preDSC revealed a well developed rough endoplasmic reticulum (Figure 2dGo) and Golgi areas (Figure 2eGo). Mitochondria were observed in a moderate amount (Figures 2c, e, fGo) and some lipid droplets were also noted (Figure 2fGo). PreDSC contained numerous bundles of microfilaments usually arranged parallel to the long axis of the cell, among which numerous dense bodies were interspersed (Figures 2a, b, dGo). The nucleus was indented, an ultrastructural feature that has been associated with cellular contraction in several systems (Franke and Schinko, 1969Go), and nucleoli were conspicuous (Figure 2aGo). Taken together, these morphological features, especially the abundant microfilaments of the cytoplasm, were fully compatible with those reported for myofibroblasts (Schürch et al., 1992Go).



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Figure 2. (AF) Transmission electron micrographs of cultured precursors of decidual stromal cells. The cytoplasm contained a well developed rough endoplasmic reticulum (RER) and many microfilaments (MF). N = nucleus; NU = nucleolus; CE = cytoplasmic extensions; M = mitochondrion; PV = pinocytotic vesicles; G = Golgi apparatus; LD = lipid droplets; DB = dense bodies.

 
Under the effect of the progesterone, preDSC underwent differentiation (decidualization) into dDSC in vivo and in vitro (Bulmer and Peel, 1974Go; Riddick and Kusmik, 1977Go; Glasser and Julian, 1986Go; Tabanelli et al., 1992Go). When DSC were cultured with progesterone they exhibited a denser cytoplasm with larger numbers of ribosomes, residual bodies, lysosomes and microfilaments than DSC cultured without progesterone (Figure 3Go).



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Figure 3. (A, B) Transmission electron micrographs of decidual stromal cells cultured with progesterone. MF = microfilaments; R = ribosomes; LY = lysosome.

 
Flow cytometric study of cytoskeletal filaments of cultured decidual stromal cells
As previously reported (Glasser and Julian, 1986Go), DSC cultured with and without progesterone expressed vimentin and were negative for cytokeratin (Figure 4Go). Desmin was absent or weakly expressed in DSC cultured without progesterone but was clearly detected in DSC treated with this hormone. Alpha-SM-actin was detected in DSC cultured with and without progesterone (Figure 4Go).



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Figure 4. Flow cytometric analysis of cytoskeletal filaments of decidual stromal cells cultured without (P–) and with progesterone (P+).

 
Expression of {alpha}-SM-actin in sections of early decidua
The monoclonal antibody anti-Thy-1 labelled DSC around vessels (preDSC) and in the rest of the decidua (dDSC) (Figure 5bGo). Alpha-SM-actin was strongly expressed by cells located around the decidual vessels, mainly by vascular smooth muscle cells. Decidual stromal cells were also positive for this antigen (Figures 5c and dGo).



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Figure 5. Immunoperoxidase staining of sections of early human decidua with (A) negative control ; (B) anti-human Thy-1; (C) and (D) anti-{alpha}-SM actin. D = decidual stromal cells; V = vascular or perivascular staining.

 

    Discussion
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 Materials and methods
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 References
 
`Stroma' is an old histological term coined to represent the `non-essential' part of organs and tissues (normally connective tissue). In contrast, the term `parenchyma' (normally epithelial tissue) identifies the functional parts. Although both terms are still in use, we now know that in many instances the stroma may be as important functionally as the parenchyma. For example, in decidua the stroma is composed mainly of leukocytes and DSC, and many reports have demonstrated the involvement of decidual leukocytes in immunological interrelationships between mother and fetus (Loke and King, 1995Go). However, because of the fusiform morphology of DSC along with their ability to produce extracellular matrix proteins and capacity for self-renewal, these cells have classically been considered fibroblast-like cells, hence stromal cells (Oliveira et al., 1991Go). The relationship between DSC and fibroblasts is also supported by recent evidence that dermal fibroblasts secrete prolactin (Richards and Hartman, 1996Go), an activity previously reported in dDSC (Riddick and Kusmik, 1977Go; Tabanelli et al., 1992Go). Furthermore, Thy-1, an antigen expressed by fibroblasts (Linge et al., 1989Go), is also detected in all DSC (Figures 1 and 5GoGo) (Fernández-Shaw et al., 1992Go).

Nevertheless, on the basis of the many immune activities reported for ESC and DSC (Tabibzadeh et al., 1989Go; Dudley et al., 1993Go; Montes et al., 1995Go; Olivares et al., 1997Go; Ruiz et al., 1997Go) and in view of their possible bone marrow origin (Lysiak and Lala, 1992Go), some authors have proposed that DSC belong to the haematopoietic lineage (Lysiak and Lala, 1992Go; Montes et al., 1996Go). We previously demonstrated that human preDSC expressed antigens of myelomonocytic and B cell lineages, class II-HLA antigens, and low or undetectable levels of CD45 (Montes et al., 1996Go), an antigenic profile that recalls that of the FDC (Schriever and Nadler, 1992Go). The relationship between preDSC and FDC is further supported by the fact that preDSC express the FDC specific antigens DRC-1 and HJ2 (Figure 1Go). Although FDC are dendritic cells with a clear immune function (antigen presentation to B lymphocytes in the lymphoid follicle), their spindle-shaped morphology, capacity for local self-renewal and failure to display the leukocyte common antigen (CD45) have led some authors to assign them to the mesenchymal rather than to the haematopoietic lineage (Schriever and Nadler, 1992Go). The fact that preDSC also exhibited these latter mesenchymal features (Montes et al., 1996Go; Olivares et al., 1997Go), together with the expression of {alpha}-SM-actin by DSC (Figures 4 and 5GoGo), a microfilament detected only in some cells of mesenchymal origin (Darby et al., 1990Go), strongly support a mesenchymal lineage for these cells. Desmin, another cytoskeletal filament expressed by dDSC (Glasser and Julian, 1986Go; Tabanelli et al., 1992Go) (Figure 4Go), is also found only in certain mesenchymal cells. The expression of vimetin (V) and {alpha}-SM-actin (A) (VA phenotype) by preDSC, and of vimetin, {alpha}-SM-actin and desmin (D) (VAD phenotype) by dDSC (Figure 4Go) relates these cells to myofibroblasts (cells which exhibit the VA and VAD phenotypes), to vascular smooth muscle cells, and to pericytes (both of which show the VAD phenotype) (Schürch et al., 1992Go). Furthermore, preDSC are located around the vessels (Ferenczy and Guralnick, 1983Go), which is also where pericytes are typically found (Schürch et al., 1992Go). Myofibroblasts, vascular smooth muscle cells and pericytes are probably related in their origin or differentiation, and they may even correspond to cellular isoforms (Schürch et al., 1992Go). Thus, the cytoskeletal filament phenotype of DSC (Figure 4Go) and morphological similarities between preDSC and myofibroblasts reported in this work (Figure 2Go) strongly suggest that DSC are fibroblast-like cells that may also belong to the myofibroblast family. Recent observations have shown that FDC also express {alpha}-SM-actin (C.Oliver and E.G.Olivares, unpublished results), which indicates that these cells may also belong to this family.

The immunological activities and expression of haematopoietic antigens by the DSC do not contradict their possible mesenchymal origin, since it has been widely reported that fibroblasts and myofibroblasts express antigens associated with haematopoietic cells (Dongari-Bagtzoglou et al., 1997Go; Pechhold et al., 1997Go; Sempowski et al., 1997Go), secrete cytokines (Dongari-Bagtzoglou et al., 1997Go; Sempowski et al., 1997Go), costimulate T lymphocyte proliferation (Roberts et al., 1997Go; Sempowski et al., 1997Go) and also appear to be involved in transplant rejection (Pedagogos et al., 1997Go). The possible bone marrow origin of the DSC (Lysiak and Lala, 1992Go) is not incompatible with a mesenchymal origin, since stromal stem cells, which give rise to the progenitors of different mesenchymal cell types, were recently identified in bone marrow (Simmons and Torok-Storb, 1991Go).

Members of the myofibroblast family exhibit contractile functions (for example, wound retraction for myofibroblasts or modulation of blood flow for pericytes) together with immune activities (Pedagogos et al., 1997Go; Roberts et al., 1997Go). In the case of the DSC, although many immune functions have been observed in these cells (Tabibzadeh et al., 1989Go; Dudley et al., 1993Go; Montes et al., 1995Go; Olivares et al., 1997Go; Ruiz et al., 1997Go); their contractile activity remains to be established. Nevertheless, the expression of {alpha}-SM-actin and the ultrastructural observation of abundant microfilaments in the cytoplasm of both pre and dDSC (Figures 1–5GoGoGoGoGo) strongly suggest that these cells are contractile. The perivascular location of preDSC suggests that they may also play a role in blood flow regulation, while contractile DSC may collaborate in the expulsion of the trophoblast during abortion, or of the endometrium during menstruation. The immunological activities of DSC are predominantly associated with preDSC, whereas these activities are down-modulated in dDSC (Montes et al., 1995Go; Ruiz et al., 1997Go). The fact that a cytokine such as transforming growth factor-ß1 stimulates cell contractility (Moulin et al., 1998Go) and blocks decidualization (Jikihara and Handwerger, 1994Go; Kubota et al., 1997Go) suggests that contractile activities may be carried out mainly by preDSC. Therefore, these cells may simultaneously perform contractile and immune functions in the elimination of trophoblast or endometrium. Nevertheless, further studies of the contractility of DSC will be needed to test this hypothesis.


    Acknowledgments
 
We are grateful to Dr S.Jordán and Dr C.Sánchez from the Clínica el Sur (Málaga) and Dr A.Stolzenburg from Gineclínica (Granada) for providing us with decidual specimens. We are also grateful to Dr M.Cámara from the Departamento de Anatomía Patológica, Universidad de Granada, for his histological advice, and Dr M.A.Abaurrea from the Departamento de Biología Celular, Universidad de Granada for her help with the electron microscopy figures. We thank K.Shashok for improving use of English in the manuscript. This work was supported by grant 91/0711 from Fondo de Investigaciones Sanitarias.


    Notes
 
1 To whom correspondence should be addressed at: Unidad de Inmunología, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Granada, 18012-Granada, Spain Back


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 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on September 15, 1998; accepted on February 10, 1999.