Molecular Human Reproduction, Vol. 7, No. 12, 1151-1157,
December 2001
© 2001 European Society of Human Reproduction and Embryology
Uterine physiology |
Human decidual stromal cells express CD34 and STRO-1 and are related to bone marrow stromal precursors
1 Unidad de Inmunología y Biología Molecular, Hospital do Meixoeiro, Vigo and 2 Unidad de Inmunología, Facultad de Medicina, Universidad de Granada, Spain
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Decidual stromal cells (DSC) are the main cellular component of the human decidua, but thus far their ascription to a given cell lineage is uncertain. In previous studies, these cells have been isolated and maintained in culture, and their antigen phenotype has been analysed to determine their affiliation. However, the presence in the culture medium of high proportions of fetal calf serum (FCS) may inhibit the expression of some surface antigens. In the present study, we show by flow cytometry that CD34 is rapidly down-regulated in human DSC cultured in RPMI 1640 with 20% FCS. For this reason, we used fibroblast medium, which contains only a small proportion (2%) of FCS, to isolate and culture these cells. Under these conditions DSC exhibited a stable antigen phenotype highly similar to that of these cells in vivo. Flow cytometry results confirmed that DSC cultured in fibroblast medium expressed CD34 protein, and reverse transcription–polymerase chain reaction findings showed that they have CD34 mRNA. Decidual stromal cells were also positive for STRO-1, an antigen that identifies stromal precursors of the bone marrow which also expresses CD34. The expression of CD10, CD13, alkaline phosphatase and
-smooth muscle actin by DSC, and the absence of expression of CD14 and CD45, further confirmed their relationship with the stromal precursors. CD34/decidual stromal cells/fetal calf serum/STRO-1/stromal precursors
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Decidual tissue, the maternal component of the maternal–fetal interface, is composed predominantly of typical stromal-type cells as well as glandular cells and leukocytes (Bulmer, 1995
). Decidual stromal cells (DSC) constitute a distinctive cell class that appears in the endometrium of mammalian uteri during pregnancy, usually after implantation of the blastocyst. Although their function, cell lineage and origin are not fully understood, DSC have classically been considered as fibroblastic cells with a nutritional and endocrine role in pregnancy (Riddick and Kusmik, 1977). Nevertheless, several reports have demonstrated that human and murine DSC or their endometrial counterpart, endometrial stromal cells (ESC), are also involved in different immune functions such as the production of cytokines (Dudley et al., 1993b; Montes et al., 1995; Nasu et al., 1999; Iwabe et al., 2000), antigen presentation (Olivares et al., 1997) and phagocytosis (Ruiz et al., 1997). Furthermore, inflammatory and Th1 cytokines enhance some of these functions (Dudley et al., 1993a; Ruiz et al., 1997; Arima et al., 2000), while inhibiting the process of DSC differentiation (decidualization) (Kariya et al., 1991; Jikihara and Handwerger, 1994; Kanda et al., 1999). On the other hand, these immune functions decrease significantly when DSC or ESC are cultured with progesterone to induce decidualization (Kariya et al., 1991; Montes et al., 1995; Ruiz et al., 1997; Arici et al., 1999). Previous studies with human material have demonstrated that DSC express antigens associated with haematopoietic cells (Imai et al., 1992; Montes et al., 1996; Olivares et al., 1997). This finding, together with the immune functions summarized above, has led some authors to propose that DSC might be true immune cells (Lysiak and Lala, 1992). Nevertheless, the ultimate precursor of these cells has not yet been determined unequivocally. Lysiak and Lala showed that certain mouse DSC and ESC are actually of bone marrow origin (Lysiak and Lala, 1992); however, some mesenchymal (non-haematopoietic) cell characteristics exhibited by human DSC contradict their possible haematopoietic adscription (Oliver et al., 1999).
To study DSC, cell purification is necessary because the human decidua contains high proportions of leukocytes (Bulmer, 1995) which may contaminate the DSC preparations and lead to spurious conclusions (Ruiz et al., 1997). We have previously obtained pure DSC populations in culture (Montes et al., 1995, 1996; Olivares et al., 1997; Ruiz et al., 1997; Oliver et al., 1999), which has allowed us to perform phenotypic and functional studies. Nevertheless, high proportions of fetal calf serum (FCS) in the culture medium may inhibit the expression of some surface antigens (Tsunoda et al., 1990). Therefore in this work, we isolated, purified and cultured DSC with fibroblast medium, which contains a low proportion of FCS, to further study the antigen phenotype of these cells. With this method, we isolated DSC with a stable antigen phenotype, similar to that of the bone marrow stromal precursors (Simmons and Torok-Storb, 1991a,b).
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Materials and methods |
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Tissues
Eighteen 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.
Fibroblast medium
According to the information provided by the manufacturer (Sigma, St Louis, MO, USA), fibroblast medium contains Fibroblast Basal Medium (a modified version of the culture medium MCDB 105) supplemented with 2% FCS and unspecified amounts of basic fibroblast growth factor, heparin, epidermal growth factor and hydrocortisone.
Isolation and culture of DSC
Decidual tissues were examined histologically to exclude the presence of infection or inflammatory infiltration. Samples of decidua from different patients were not mixed, to avoid the induction of cytokine secretion as a result of an allogeneic reaction of leukocytes that initially contaminate DSC cultures. Tissues were extensively washed in phosphate-buffered saline solution (PBS) and the decidua was carefully freed from the trophoblast. Decidual fragments were finely minced between two scalpels in a small volume of RPMI 1640 medium with 100 IU/ml penicillin and 50 mg/ml gentamicin, and put in a solution of 0.5% trypsin and 0.2% EDTA (Sigma) for 15 min at 37°C. The reaction was stopped by adding cold RPMI with 20% FCS (Gibco, Paisley, UK) and the suspension was filtered through gauze and centrifuged at x425 g for 10 min. The supernatant was discarded and the cell pellet was suspended in RPMI and centrifuged on Ficoll-Paque (Pharmacia LKB, Uppsala, Sweden) for 20 min at x600 g. Cells were collected from the interface, suspended in PBS 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, granulocytes and gland cells to adhere to the flask. The supernatant, containing DSC and lymphocytes, was washed and incubated either in fibroblast medium with 100 IU/ml penicillin and 50 mg/ml gentamicin, or in complete RPMI medium with 20% FCS. After overnight incubation so that DSC adhered to the flask, lymphocytes in the supernatant were then discarded, leaving the adherent cells, which were mainly DSC. The corresponding culture medium was then replaced. Both types of culture medium were changed twice a week. Proliferating DSC overgrew other possible contaminant cells, thus further guaranteeing the purity of the cultures. Cell lines were first studied when they covered the whole surface of the 25 cm2 culture flask. Supernatants from confluent cultures were collected, concentrated x10 by a Minicon concentrator (Amicon, Beverly, MA, USA) and analysed for the presence of prolactin by using an electrochemiluminescence immunoassay (Roche Diagnostics, Indianapolis, IN, USA).
Isolation of fresh decidual stromal cells
The decidua was washed in Ca2+, Mg2+-free PBS and minced between two scalpels in a small volume of RPMI 1640 with 10% FCS. The cell suspension was filtered through sterile gauze, washed by centrifugation and suspended in culture medium. This suspension was centrifuged at x650 g for 30 min over a discontinuous gradient of 20 and 30% Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden). Decidual stromal cells were collected from the 20/30% interphase and washed in PBS.
Monoclonal antibodies
The monoclonal antibodies (mAb) used in this study are shown in Table I.
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Flow cytometry
Decidual stromal cells were detached from the culture flask by treatment with 0.04% EDTA at 37°C. Cells were centrifuged, the supernatant was discarded and the pellet was suspended in PBS at 1x106 cells/ml. For direct staining, 100 µl of the cell suspension was incubated with 10 µl of the appropriate monoclonal antibody 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). To identify dead cells we incubated DSC with propidium iodide (Sigma). The percentage of cells that were antibody-positive was calculated by comparison with the appropriate isotype control (Table I
). 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 indirect labelling, fluorescein isothiocyanate (FITC)-labelled goat anti-mouse immunoglobulin was added after the first mAb. For intracytoplasmic labelling, 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.
Polymerase chain reaction (PCR) primers
Primers used in this study are shown in Table II. They were designed according to sequences available from Genbank (http://www2.ncbi.nlm.nih.gov/cgi-bin/genbank) and synthesized by Genset (Paris, France). In order to prevent the amplification of contaminant genomic DNA, sense and antisense primers were designed, when possible, from sequences located far apart on different exons, and tested in PCR reactions with the RNA used for cDNA synthesis.
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Reverse transcription (RT)–PCR
Total RNA from cells was extracted by the UltraspecTM RNA isolation method according to the manufacturer's protocol (Biotecx Laboratories Inc., Houston, TX, USA). A single strand cDNA copy was made from total RNA using random hexamers (Pharmacia Biotech, Uppsala, Sweden) and M-MLV H minus RNase reverse transcriptase (Promega, Madison, WI, USA). After heating to 65°C for 5 min and quickly cooling to 4°C in a thermal cycler (Geneamp PCR System 9600, Perkin-Elmer, Cetus, Norwalk, CT, USA) for denaturation, reverse transcription was performed for 1 h at 37°C. Starting with cDNA equivalent to 75 ng RNA, amplification was carried out in a total volume of 12.5 µl of the amplification mix, 10 mmol/l Tris-Cl (pH 8.4), 50 mmol/l KCl, 2 mmol/l MgCl2, 0.01% gelatine, 0.2 mmol/l dNTPs, 5% glycerol, 0.25 mmol/l of each primer and 0.02 IU/ml Taq DNA Polymerase (Promega). After incubation for 5 min at 96°C, each cycle consisted of 94°C for 30 s, 57°C for 30 s and 72°C for 30 s, for a total of 32 cycles. A total of 1 µl of the first round product was used for the second 32-cycle round. The PCR products were size-separated on ethidium bromide-stained 2% Agarose gels, and a 100 bp DNA ladder was included in each run.
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Down-regulation of CD34 by decidual stromal cells cultured with RPMI 1640 with 20% FCS
Figure 1
shows data from DSC lines cultured with 20% FCS. Although the proportion of CD10-positive cells was constant, the proportion of CD34-positive cells decreased steadily with time. After 8–10 weeks of culture, CD34 was practically absent in most DSC lines. These results explain why we previously reported that CD34 was not expressed by DSC in culture (Montes et al., 1996); in earlier work all DSC lines were studied much later (after 12 weeks of culture) than in the present work.
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) and CD34 (•) in four different lines of decidual stromal cells cultured with RPMI 1640 containing 20% fetal calf serum.Antigen phenotype of decidual stromal cells cultured in fibroblast medium
Cells lines were maintained in fibroblast medium for 2–4 weeks after the primary culture (see Materials and methods), a shorter period than earlier cultures with RPMI 1640 with 20% FCS (Montes et al., 1996
). In fibroblast medium, DSC lacked CD14, CD15 and CD45, whereas they expressed CD34 and STRO-1, an antigen detected in stromal precursors of the bone marrow (Simmons and Torok-Storb, 1991a). Other antigens detected in the DSC were CD10, CD13, CD21, CD23, CD80, CD86, HLA-DR, HJ2, -smooth muscle actin (ASMA), and alkaline phosphatase (ALP) (Table III, Figure 2). The antigen phenotype of DSC cultured in fibroblast medium was equivalent to that reported previously by us in DSC cultured with RPMI 1640 and 20% FCS (Montes et al., 1996; Olivares et al., 1997; Oliver et al., 1999). The exceptions were CD34, which was reported to be negative in cultures containing RPMI 1640 and 20% FCS (Montes et al., 1996), and ALP and STRO-1, which were not previously studied. DSC proliferated in fibroblast medium for ~8–12 weeks; during this period, this antigen phenotype was observed to be stable, and no down-regulation of CD34 or any other antigen was observed.
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Detection of CD34 mRNA in DSC cultured in fibroblast medium
The expression of CD34 was confirmed by RT–PCR. CD34 mRNA, together with CD10 and CD13 (antigens that are expressed by most DSC, Table III
) mRNAs, were detected in DSC cultured in fibroblast medium. Because prolactin is secreted by DSC only when they are cultured with progesterone (Tabanelli et al., 1992), and our cultures lacked this hormone, we did not detect prolactin in the supernatants (results not shown). Nevertheless, small amounts of prolactin mRNA were detected in some lines (Figure 3).
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Expression of CD34 and STRO-1 by fresh DSC
To confirm that the expression of CD34 was not due to an artefact of the culture conditions, we tested this antigen by flow cytometry in fresh DSC. The absence of contaminant leukocytes was confirmed by the lack of CD45-, CD14- or CD15-positive cells in the fresh DSC suspension. Decidual stromal cells were identified by the expression of CD10 and we observed that significant proportions of the fresh CD10-positive DSC also expressed CD34 and STRO-1 (Figure 4
). These data, together with those reported previously for fresh DSC (Montes et al., 1996), confirmed that the phenotype of DSC in fibroblast medium is equivalent to that of DSC in vivo.
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Discussion |
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Although DSC are the main cellular component of the decidua, other elements such as epithelial cells or leukocytes are also abundant in this tissue (Bulmer, 1995
). When fresh DSC or ESC have been obtained by differential sedimentation (Kariya et al., 1991; Imai et al., 1992), differential adhesion (Shiokawa et al., 1996; Iwabe et al., 2000), binding to monoclonal antibodies against antigens expressed by haematopoietic cells (Fernández-Shaw et al., 1992) or separation on Percoll gradients (Imai et al., 1995), leukocytes have been frequent contaminants of fresh cell preparations (Ruiz et al., 1997). This may have led to misinterpretation of experimental results, especially with regard to the expression by DSC of antigens normally associated with leukocytes, or to the immunological functions of DSC. In decidual sections, DSC are usually identified by their morphology; however, sometimes it is difficult to distinguish them from other large cells such as macrophages. In culture, DSC proliferate and overwhelm other non-proliferating contaminant cells, so that pure preparations of DSC can be obtained (Montes et al., 1995, 1996; Olivares et al., 1997; Ruiz et al., 1997; Oliver et al., 1999). However, the high concentration of FCS necessary to maintain DSC in culture may inhibit the expression of some antigens (Tsunoda et al., 1990). In this study, we found that CD34 was rapidly down-regulated by DSC in culture with 20% FCS and this explains why this antigen was previously reported to be not expressed by these cells in long-term cultures with a high proportion of FCS (Montes et al., 1996). The expression of CD34 and other antigens by DSC cultured in fibroblast medium was found to be stable, and their antigen phenotype was equivalent to that of the DSC in vivo (Montes et al., 1996). These findings, together with the fact that DSC lines are obtained earlier in fibroblast medium than in other culture media, makes fibroblast medium a suitable option for the study of DSC.
The findings that DSC and ESC in mice originate from the bone marrow (Lysiak and Lala, 1992) and exhibit several immune functions (Dudley et al., 1993a,b; Montes et al., 1995; Olivares et al., 1997; Ruiz et al., 1997; Nasu et al., 1999; Iwabe et al., 2000) suggest that DSC may be ascribed to the haemapoietic lineage. The expression by DSC of CD34, an antigen detected on the precursors of haemopoietic cells (Civin et al., 1984), and of other antigens associated with haematopoietic cells (CD10, CD13, CD21, CD23, CD80, CD86 and HLA-DR) supports this possibility. Nevertheless, the findings that DSC lack CD45, a marker of the haematopoietic lineage, and that they express antigens associated with mesenchymal (non-haemapoietic) cells, such as ASMA and ALP, contradict this ascription. The expression by DSC of STRO-1, an antigen that identifies stromal precursors of the bone marrow (Simmons and Torok-Storb, 1991a), relates these two types of cell. Like DSC, these stromal precursors express CD34 and also rapidly lose this antigen in cultures containing a high proportion of FCS (Simmons and Torok-Storb, 1991b). Furthermore, DSC and the stromal precursors both express CD10, CD13, ALP and ASMA and both lack the expression of CD14 and CD45 (Simmons and Torok-Storb, 1991a). The relationship between DSC and the stromal cell precursors of the bone marrow also reconciles the apparent contradiction between the bone marrow origin of DSC (Lysiak and Lala, 1992) and their mesenchymal (non-haematopoietic) characteristics (Oliver et al., 1999). Studies in mice have suggested that precursor cells of DSC can migrate from their origin (yolk sac and/or bone marrow) to the uterus any time between embryonic life and the onset of reproductive life, and that beyond this period they locally self-renew (Lysiak and Lala, 1992). Decidual stromal cells have also been related with follicular dendritic cells (FDC), cells of the lymphoid follicle which prevent B cells from undergoing apoptosis and which are involved in antigen presentation to B cells in the secondary response (Lindhout et al., 1993). Decidual stromal cells express FDC antigens (CD21, CD23, DRC-1, HJ2) and, like FDC, also lack CD45 (Montes et al., 1996; Oliver et al., 1999). Follicular dendritic cells and DSC may therefore belong to the same cell family (Oliver et al., 1999), and may have a common bone marrow stromal cell precursor.
After primary culture, obviously only DSC with the capacity to proliferate are positively selected. In the absence of progesterone in the culture medium, these cells correspond to precursors of the DSC, i.e. predecidualized cells (Glasser and Julian, 1986; Tabanelli et al., 1992; Montes et al., 1996). In fact, like predecidualized cells, DSC cultured in fibroblast medium did not secrete prolactin (results not shown). By analogy with the developmentally regulated expression of CD34 by primitive haematopoietic cells (Strauss et al., 1986), CD34 may be considered a marker of cell precursors (haematopoietic, stromal or decidual) which is lost as cells differentiate. Nevertheless, serial experiments with progesterone-decidualized cells need to be carried out to confirm this possibility in DSC. CD34, expressed by haematopoietic stem cells and by stromal cells, is probably involved in the adhesion and interaction between these two types of cell in the regulation of haematopoesis (Healy et al., 1995). Decidual stromal cells that express CD34 may have an equivalent function in decidua. Normal human decidua contains a high proportion of leukocytes that play a role in the maternal–fetal inter-relationships (Bulmer, 1995) and differentiate in situ as pregnancy progresses (Mincheva-Nilsson et al., 1997). The possibility that DSC are involved in this differentiation is supported by the findings of King et al. who showed that DSC influence proliferation and survival of decidual NK cell through cell-to-cell contact (King et al., 1999). It will be interesting to determine whether CD34 mediates in the interaction between DSC and decidual NK cells.
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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 thank K.Shashok for improving the use of English in the manuscript. This work was supported by grants from Fondo de Investigaciones Sanitarias.
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3 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. E-mail: engarcia{at}ugr.es
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Submitted on April 23, 2001; accepted on September 26, 2001.
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