Comparing to bone marrow mesenchymal stem cells (MSCs), placenta-derived MSCs have the advantages of adequate sources, low immunogenicity, little risk of viral contamination, and no ethical controversy, and thus possess a better prospect for clinical application. Placental tissue not only includes chorionic and amniotic, but also contains decidua basalis which locate in the maternal placenta surface. The biological characteristics of MSCs isolated from decidua basalis have not been well studied. This study was aimed to investigate the biologic characteristics of placenta decidua basalis-derived MSC from placenta decidua basalis (DB) by enzymatic digestion. Short tandem repeats (STR) test was used to identify the cells derived from the maternal placenta surface. Growth rate of decidua basalis mesenchymal stem cells (DB-MSC) was measured by MTT. Cell cycle and cell phenotype were detected by flow cytometry. Inducing differentiation was used to evaluate multipotency of DB-MSC. For testing the immunosuppression of DB-MSC, they were co-cultured with peripheral blood mononuclear cells (PBMNC) stimulated by phytohemagglutinin (PHA) and then IFN-γ in the co-cultured media was quantified by ELISA. The results showed that the cells were derived from the maternal placenta by STR analysis. DB-MSC showed typical fibroblast morphology in the culture and were positive for the MSC surface markers: CD90, CD73, CD105, CD44 and negative for CD45, CD11b, and CD34. DB-MSC underwent osteogenic, adipogenic and chondrogenic differentiation in inducing medium. DB-MSC could inhibit the secretion of IFN-γ by PBMNC. It is concluded that the cells are isolated from placenta decidua basalis and possess the basic characteristics of MSC. DB-MSC can be an important maternal autologous MSC and may be a safe and effective treatment for immune system diseases, which makes the DB-MSC as an important source of autologous MSC from mother. DB-MSC can be safely for the treatment of the mother's immune system diseases.
Cell Differentiation ; Cells, Cultured ; Decidua ; cytology ; Female ; Flow Cytometry ; Humans ; Mesenchymal Stromal Cells ; cytology ; Placenta ; cytology ; Pregnancy

OBJECTIVETo develop a convenient method for isolation and purification of human extravillous cytotrophoblasts (EVCTs) and decidual stromal cells (DSCs) and establish a co-culture system.

METHODSThe DSCs were digested with trypsin and purified by Percoll gradient. The EVCTs were digested with trypsin and purified by BSA gradient. Immunohischemistry and immunofluorescent study are performed to characterize these isolated cells. The EVCTs and DSCs were placed in Matrigel-coated Transwell upper and lower chamber, respectively, to study the invasive ability of the EVCTs.

RESULTSImmunohischemistry revealed that the purity of EVCTs and DSC exceeded 95%. Cultured EVCTs retained their capacity to invade Matrigel-coated Transwell filters with the invasion index of 3.22-/+0.04.

CONCLUSIONThis co-culture model established by isolating highly purified EVCTs and DSCs in vitro can be useful for studying the trophoblast invasion mechanisms.

Cell Communication ; physiology ; Cells, Cultured ; Chorion ; cytology ; Coculture Techniques ; Decidua ; cytology ; Female ; Humans ; Models, Biological ; Stromal Cells ; cytology ; Trophoblasts ; cytology ; physiology

OBJECTIVETo investigate the biological features of human decidua basalis-derived mesenchymal stem cells (PDB-MSCs) in vitro and identify their capacity of multilineage differentiation.

METHODSPDB-MSCs were harvested from the decidua basalis of term placental by enzymatic digestion and density gradient centrifugation, and the growth characteristics and morphological changes of the MSCs were observed by inverted microscope. The proliferative ability of the cells was assessed by Cell Counting Kit-8. The cell cycle and expressions of the surface markers (CD29, CD44, CD73, CD90, CD34, CD45, and CD14) of the MSCs were identified by flow cytometry. Multilineage differentiation capacity of the cells was tested by inducing their differentiation toward osteoblasts, adipocytes and chondroblasts in vitro.

RESULTSMSCs isolated from human decidua basalis of term placental exhibited a morphology similar to that of bone marrow-derived MSCs, and grew into colonies in in vitro culture, where the cells proliferated rapidly after passage with a cell doubling time of 2.21∓0.21 days. More than 70% of the cells stayed in the resting stage (G(0)/G(1)) and showed positivity for CD29, CD44, CD73 and CD90, but not for CD14, CD34 or CD45. After induction, the cells showed positive results of alizarin red staining, oil red O staining and Alcian blue staining.

CONCLUSIONHuman decidua basalis contains a rich source of MSCs, which can be easily isolated and cultured without affecting their capacity of multilineage differentiation. The PDB-MSCs may have the potential as a novel source of stem cells.

Cell Differentiation ; physiology ; Cell Separation ; Cells, Cultured ; Decidua ; cytology ; Female ; Humans ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; cytology ; Placenta ; cytology ; Pregnancy

OBJECTIVETo study the expression of matrix metalloproteinases (MMPs ) in the decidual stromal cells (DSCs) and extravillous cytotrophoblasts (EVCT) in human early pregnancy and explore the change of MMPs in endometrial stromal cell (ESC) decidualization and its impact on implantation and placentation.

METHODSThe decidua and villi from 5 women with early pregnancy and mid-secretory endometrium from 5 normal women were collected and cultured in vitro, and the supernatants of the culture media were collected after 48 hours of incubation. The expression of the MMPs in the ESCs, DSCs and EVCTs was detected using Luminex xMAP system simultaneously and the difference in MMPs expression and their correlations were analyzed with SPSS10.0 software.

RESULTSThe MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, and MMP-9) were expressed in ESCs, DSCs and EVCTs, while MMP-12 was not found in ESCs and MMP-13 not in DSCs. Expressions of MMP-8, MMP-12, and MMP-13 were lowered. Compared with the ESCs, DSCs and EVCTs showed significantly lowered expressions of MMP-1, MMP-3, and MMP-7 (P<0.05), whereas expression of MMP-2 and MMP-9 increased significantly, and the high expressions of MMP-1, MMP-3, and MMP-7 was especially obvious in the DSCs. The expressions of MMP-1, MMP-3, and MMP-7, however, were significantly decreased in the EVCTs in comparison with the DSCs. Significant correlations were noted between MMP-1, MMP-3, and MMP-7, and MMP-2 was closely correlated with MMP-9. MMP-8 was significantly lower and MMP-12 and MMP-13 showed no obvious variation in the cell culture.

CONCLUSIONMMPs are secreted by ESCs, DSCs and EVCTs. Diverse MMPs play an important role in proliferation and differentiation of the ESC to affect embryo implantation and placentation. All MMPs establish a balance to co-regulate the process of pregnancy.

Adult ; Cells, Cultured ; Decidua ; cytology ; enzymology ; Endometrium ; cytology ; enzymology ; Female ; Humans ; Isoenzymes ; metabolism ; Matrix Metalloproteinases ; metabolism ; Pregnancy ; Pregnancy Trimester, First ; Stromal Cells ; cytology ; enzymology ; Trophoblasts ; cytology ; enzymology

Human placental decidua basalis-mesenchymal stem cells (PDB-MSCs) are multipotent cells from the human term placenta, which are ethically conducive, easily accessible and high-yielding source. PDB-MSCs can differentiate into adipogenic, osteogenic and neurogenic cells under appropriate conditions, which may be an attractive and alternative source of seed cells for tissue engineering. To investigate the effect of hypoxia (1% O2) on human PDB-MSCs and the expression of cytokine, PDB-MSCs were isolated from human placenta by density gradient centrifugation and cultured in the Dulbecco's modified Eagle's medium-high glucose (DMEM-HG) containing 10% fetal bovine serum (FBS), and the fifth passage of PDB-MSCs were taken. PDB-MSCs were divided into 4 groups according to the concentrations of O2 and FBS: 20% O2, 10% FBS; 20% O2, 0% FBS; 1% O2, 10% FBS; 1% O2, 0% FBS. The proliferation and apoptosis of PDB-MSCs were detected by MTT and flow cytometric analysis at the time points of 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h, respectively. Vascular endothelial growth factor (VEGF) released from PDB-MSCs was detected by enzyme-linked immunosorbent assay (ELISA) at the same time points. The results showed that hypoxia enhanced the proliferation of PDB-MSCs at 12 h under the condition of 10% FBS, while at 24 h under the condition of 0% FBS (P<0.01, n=3). In normoxia, the cells cultured in 10% FBS displayed a significant proliferation compared to those cultured in 0% FBS. However, in hypoxia, the number of cells cultured in 0% FBS (serum deprivation) increased significantly compared to that cultured in 10% FBS at 24 h and 96 h respectively (P<0.05, P<0.01, n=3). With the flow cytometric analysis of cell apoptosis under the condition of hypoxia and serum deprivation, we found that hypoxia and serum deprivation did not induce PDB-MSCs apoptosis (P>0.05, n=3). This conclusion may relate to the expression of VEGF which needs further research. In conclusion, the results obtained indicate that PDB-MSCs are able to bear hypoxia and serum deprivation, suggesting that PDB-MSCs can be used as seed cells for ischemia related tissue engineering.
Apoptosis ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Decidua ; cytology ; Female ; Humans ; Mesenchymal Stromal Cells ; cytology ; Placenta ; cytology ; Pregnancy ; Tissue Engineering ; Vascular Endothelial Growth Factor A ; metabolism

To investigate the effect of placental isoferritin (PLF) on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell, 8-cell and morula (P>0.05). PLF at the doses of 10 and 100 U/mL significantly enhanced more embryos development to the blastocyst and hatching blastocyst (P<0.05). PLF at the dose of 1000 U/mL depressed more embryos development from 2-cell to hatching blastocyst, meanwhile such phenomena as cell degeneration and irregular cleavage were observed in part of embryos, but there was no significant difference in statistics (P>0.05). It was concluded that PLF at the concentration of 10-100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantation blastocysts.
Coculture Techniques ; Decidua/*cytology ; Embryo, Mammalian/*cytology ; Embryo, Mammalian/drug effects ; Embryo, Mammalian/embryology ; Embryonic Development/*drug effects ; Ferritins/isolation & purification ; Ferritins/*pharmacology ; Placenta/*chemistry ; Tissue Culture Techniques

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in the endometrium during the menstrual cycle and in early pregnancy. Specificity of integrins is known to be different in human endometrial stromal cells and decidual cells. These shifts of integrins suggested to play an important role in embryo implantation and can be modulated by progesterone, cAMP derivatives, and cytokines. The mechanisms of decidualization and its precise physiological role are still not clearly understood and in vitro systems could provide an alternative that overcomes limitations of studying such complex biological phenomena in vivo at the time of implantation. This study was undertaken to establish an in vitro model system for human decidualization using 8-bromo-cAMP and to investigate the characteristics of stromal integrin expression in vitro by 8-Br-cAMP. Endometrial stromal cells were isolated and cultured, and then were induced to decidualize by 0.5 mM 8-Br-cAMP for 15 days. Immunofluorescence staining and flow cytometric analyses of the integrin subunits (alpha1, alpha4, alpha5, alpha6, beta1 and alpha v beta3) were performed at day 9. In the presence of 8-Br-cAMP, the staining intensity of alpha v beta3 was significantly higher than control and measurements for alpha1, alpha4, alpha5, alpha6, and beta1 were similar. Immunofluorescent localization of the integrins reflected the differences obtained from the flow cytometric analyses described above. In summary, the expression of alpha;avbeta;b3 integrin increased in stromal cells in vitro decidualized by 8-Br-cAMP and this up-regulation of alpha v beta3 integrin expression during decidualization might influence on human implantation.
8-Bromo Cyclic Adenosine Monophosphate/*pharmacology ; Cell Size ; Cells, Cultured ; Decidua/*cytology/drug effects/metabolism ; Female ; Flow Cytometry ; Human ; Integrins/*analysis/metabolism ; Prolactin/analysis ; Stromal Cells/cytology/*drug effects/*metabolism

OBJECTIVETo study the effect of exogenous cytokine-stimulated decidual cells on IgG secretion of B lymphocytes and investigate the features of local immunological microenvironment of the decidua.

METHODSExogenous cytokines interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-6 and epidermal growth factor (EGF) were added separately in cultured decidual cells, and the supernatant of the culture medium was prepared for stimulating human peripheral blood lymphocytes. IgG secretion of the B cells was measured by radio-immunological method.

RESULTThe decidual cells of normal early pregnancy stimulated B lymphocyte IgG secretion, and the supernatant of exogenous cytokine-stimulated decidual cells had the same effect, which, however, was depended not on the concentration and category of the cytokines, but only on the time of treatment.

CONCLUSIONThe exogenous cytokines can increase the humoral immunity in the decidual immunological microenvironment, but such effect might result from a self-regulatory mechanism of the local immunological microenvironment of the decidua, which can be fundamental for maintenance of normal pregnancy.

Adult ; B-Lymphocytes ; cytology ; drug effects ; metabolism ; Cells, Cultured ; Culture Media, Conditioned ; pharmacology ; Cytokines ; pharmacology ; Decidua ; cytology ; drug effects ; metabolism ; Female ; Humans ; Immunoglobulin G ; biosynthesis ; Interferon-gamma ; pharmacology ; Interleukin-2 ; pharmacology ; Pregnancy ; Pregnancy Trimester, First

OBJECTIVETo investigate the relationship between the expression of uncoupling protein 2 (UCP2) and ratio of interleukin-10/interferon-gamma (IL10/IFNgamma) in the macrophages from patients with unexplained recurrent spontaneous abortion (URSA).

METHODSTwelve women undergoing selective termination of normal early pregnancy (control) and 11 having URSA were included in this study. Magnetic cell sorting (MACS) was used to isolate the macrophages in the decidua, and the expression of UCP2 was detected with flow cytometry. Cytokine (IL10 and IFNgamma) secretion by the macrophages was detected by enzyme-linked immunosorbent spot-forming (ELISPOT) cell assay.

RESULTSCompared to the control group, the women with URSA showed significantly decreased expression of UCP2 on decidual macrophage (136-/+25 vs 201-/+31, P<0.01), and the expression of UCP2 was positively correlated to the ratio of IL10/IFNgamma(r=0.73, P<0.01).

CONCLUSIONUCP2 may play an important role in the regulation of macrophage activity and cytokine secretion to contribute to spontaneous abortion.

Abortion, Habitual ; metabolism ; Adult ; Cytokines ; metabolism ; Decidua ; cytology ; metabolism ; Female ; Humans ; Interferon-gamma ; secretion ; Interleukin-10 ; secretion ; Ion Channels ; metabolism ; Macrophage Activation ; Mitochondrial Proteins ; metabolism ; Pregnancy ; Uncoupling Protein 2

OBJECTIVE: To observe the expression of progesterone receptor (PR), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX-2) induced by lipopolysaccharide (LPS) or Toll-like receptor 4 antagonist (TLR4 mAb) in decidual cells in vitro, and then to explore the effect of LPS and its antagonist on PR of decidual cells and the relation between PR and inflammatory cytokines. METHODS: We isolated and cultured human decidua of early abortion in the sterile state. When the cells passaged to the 4th generation, the cells were randomly divided into 6 pore plates: A control group was added the culture medium alone; experimental group I was added 100 ng/mL of LPS; experimental group II was add 1 μg/mL of TLR4 mAb; experimental group III was added 3 μg/ mL of TLR4 mAb; experimental group IV was added 1 μg/mL of TLR4 mAb pretreatment for 24 h, and then 100 ng/mL LPS; and experimental group V was added 3 μg/mL of TLR4 mAb pretreatment for 24 h, and then 100 ng/mL LPS for 24 h culture. Subsequently, HE staining and immunofluorescence were used to observe the morphology and identify the purity of decidual cells in the 6 groups. The levels of mRNA expression of PR, IL-1β, and COX-2 were detected by reverse transcription PCR (RT-PCR). RESULTS: LPS reduced the mRNA expression of PR (P<0.05), increased the mRNA expression of IL-1β and COX-2 (P<0.05). TLR4 mAb increased the mRNA expression of PR (P<0.05) and reduced the mRNA expression of IL-1β (P<0.05) after LPS-stimulated decidual cells. High concentrations of TLR4 mAb reduced the mRNA expression of COX-2 (P<0.05) after LPS stimulated decidual cells. CONCLUSION The mRNA expression of PR is reduced, and the mRNA expressions of IL-1β and COX-2 are increased after LPS-stimulated decidual cells in vitro. TLR4 mAb antagonize the role of LPS on PR, IL-1β, and COX-2.
Adult ; Cells, Cultured ; Cyclooxygenase 2 ; genetics ; metabolism ; Decidua ; cytology ; metabolism ; Female ; Humans ; Interleukin-1beta ; genetics ; metabolism ; Lipopolysaccharides ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Receptors, Progesterone ; genetics ; metabolism ; Toll-Like Receptor 4 ; antagonists & inhibitors ; Young Adult