OBJECTIVETo investigate the possibility of placenta mesenchymal stem cells (PMSCs) differentiation into dermal fibroblast, and the potency of PMSCs used in cutaneous wound healing and stored as seed cells.

METHODSEnzyme digestion method was used to obtain PMSCs, and PMSCs were amplified after culture in vitro. Flow cytometry assay, osteogenic and adipogenic differentiation were done for MSCs identification. The induction medium composed of DMEM/F12 + 50 microg/ml VC + 100 ng/ml connective tissue growth factor (CTGF) was added into the 24-well plate for 16 days induction period. Pictures were taken to record morphologic change. Immunofluorescence tests were performed to detect Vimentin, FSP-1, collagen I , collagen III, desmin and laminin expression before and after induction. At the same time osteogenic and adipogenic differentiation were used to assay the differentiation ability change after induction. The induced dermal fibroblasts were frozen in liquid nitrogen and recovery and trypan blue was used to detect cell viability.

RESULTSAfter CTGF induction, PMSCs got obvious fibroblasts morphology, the protein level of Vimentin, FSP-1, collagen I, collagen III and Laminin increased, PMSCs started to express Desmin, the dermal fibroblasts specific proteins, and osteogenic and adipogenic differentiation ability was diminished. PMSCs were successfully induced into dermal fibroblasts, and these induced cells could get a high cell viability ( more than 90% ) after recovery.

CONCLUSIONSPMSCs could be induced into dermal fibroblasts by CTGF in vitro. PMSCs have the potential application in skin wound healing, and can be used as seed cells of dermal fibroblasts.

Cell Differentiation ; drug effects ; Cells, Cultured ; Connective Tissue Growth Factor ; pharmacology ; Female ; Fibroblasts ; cytology ; drug effects ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Placenta ; cytology ; Pregnancy

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

OBJECTIVETo observe the effect of Bushen Yiqi Huoxue Recipe (BYHR, a Chinese medical prescription for reinforcing Shen, replenishing qi and promoting blood circulation) on placental trophoblast apoptosis in fetal growth restricted (FGR) pregnant rat for the sake of explore its mechanism of action in treating FGR.

METHODSFGR animal model was established by passive smoking, 32 pregnant rats were divided into four groups at random: the normal group, the model group, the Chinese medicine (CM) group (model rats treated by BYHR) and the Western medicine (WM) group (model rats treated by arginine). The histological change of placenta was examined with HE stain, the trophoblast apoptosis was detected by TUNEL and RT-PCR, and the expressions of Bcl-2 and Bax in the placenta were detected by immunohistochemical method.

RESULTSBlood stasis and villous ischemia were seen in placenta of FGR model rats. Placental microcirculation was significantly improved in the CM group, but in the WM group only partially improved. The median apoptotic index of syncytial trophoblast cells in the four groups, in normal, model, CM, and WM order, was 45%, 75%, 57% and 70%, as compared with the model group, it was much lower in the normal group and the CM group (P < 0.01), but a similar level was shown in the WM group. No significant difference in mRNA and protein expressions of Bcl-2 and Bax was found among the four groups.

CONCLUSIONBYHR can improve the placental microcirculation in FGR rats to prevent excessive apoptosis through a mechanism other than the classical Bax/Bcl-2 apoptotic pathway, which needs further exploring.

Animals ; Apoptosis ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Fetal Growth Retardation ; prevention & control ; Microcirculation ; drug effects ; Placenta ; blood supply ; cytology ; Pregnancy ; Rats ; Trophoblasts ; cytology

OBJECTIVE: To determine the apoptosis in placenta tissues of patients with hypertensive disorder complicating pregnancy and its relationship with Bcl-2, TGFbeta1, and to explore the etiology of hypertensive disorder complicating pregnancy. METHODS: Forty-five placenta samples were obtained from pregnancies with hypertensive disorder (15 gestational hypertension, 15 mild preeclampsia, and 15 severe preeclampsia) and 45 normal placenta tissues were enrolled from the third-trimester pregnancies. Immunohistochemistry (SP method) was used to study the expression of Bcl-2 and TGFbeta1 in human trophoblasts. Terminal deoxynucleotidyl transferase-dUTP nick end-labeling (TUNEL) was used to quantify the incidence of apoptosis in human trophoblasts. RESULTS: The apoptosis rate and TGFbeta1 expression in hypertensive disorder complicating pregnancy group was higher than that in the control group, but the Bcl-2 expression was significantly lower than the control group (all Ps<0.01). With the aggravation of this illness, the apoptosis rate and TGFbeta1 expression in the gestational hypertension group, mild preeclampsia group, and severe preeclampsia tended to be increasing, but the Bcl-2 expression was decreasing (P<0.001). The apoptosis of placenta villi and TGFbeta1 expression were positively correlated in the severe preeclampsia group and mild preeclampsia group,but the apoptosis of placenta villi and Bcl-2 were negatively correlated (all Ps<0.05). TGFbeta1 and Bcl-2 expressions in the severe preeclampsia group and mild preeclampsia group were negatively correlated (P<0.05). CONCLUSION Apoptosis of the placental trophoblasts of pregnancies with hypertensive disorder is evidently enhanced. The TGFbeta1 expression increases and the Bcl-2 expression decreases. The imbalance between TGFbeta1 and Bcl-2 expression may induce the hypertensive disorder.
Adult ; Apoptosis ; Female ; Humans ; Hypertension, Pregnancy-Induced ; metabolism ; Placenta ; cytology ; metabolism ; Pregnancy ; Pregnancy Trimester, Third ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo construct adeno-associated virus (AAV) expression system for transforming growth factor beta3 (TGFbeta3 ) and detect its biological effect on proteoglycan synthesis of the earlier and later dedifferentiated rabbit lumbar disc nucleus pulpous (NP) cells, which was compared with that of adenovirus (AV) expression system for TGFbeta1.

METHODSTGFbeta3 gene was obtained using PCR. Its upstream contained restriction enzyme site Kpn I, and its downstream contained restriction enzyme site Sal I. Using the restriction enzyme sites of PCR product of TGFbeta3 and the corresponding multiple cloning site (MCS) in plasmid AAV, TGFbeta3 was subcloned into AAV. The recombinant plasmid AAV-TGFbeta3 was transfected into H293 cells with Lipofectamine 2000, and the expression of TGFbeta3 gene was detected using immunofluorescent analysis. After AAV-TGFbeta3 virus particle with infectious activity was packaged, TGFbeta3 expression in NP cells was detected by immunoblotting, and its biological effect on proteoglycan synthesis was detected by antonopulos method and compared with that of AV-TGFbeta1 in the earlier and later dedifferentiated NP cells.

RESULTSFor the earlier dedifferentiated NP cells, AAV-TGFbeta3 slowly and stably enhanced proteoglycan synthesis, but AV-TGFbeta1 rapidly and transiently enhanced its synthesis. For the later dedifferentiated NP cells, AAV-TGFbeta3 stably enhanced proteoglycan synthesis, but AV-TGFbeta1 inhibited its synthesis.

CONCLUSIONAAV expression system can mediate TGFbeta3 gene to be expressed stably, and AAV-TGFbeta3 can enhance proteoglycan synthesis of the earlier and later dedifferentiated NP cells.

Animals ; Cell Line ; DNA, Recombinant ; genetics ; Dependovirus ; genetics ; metabolism ; Female ; Humans ; Intervertebral Disc ; cytology ; Placenta ; cytology ; Plasmids ; genetics ; Polymerase Chain Reaction ; Pregnancy ; Proteoglycans ; biosynthesis ; Rabbits ; Transforming Growth Factor beta1 ; genetics ; Transforming Growth Factor beta3 ; genetics ; Viral Proteins ; biosynthesis

OBJECTIVETo investigate the molecular mechanisms of effective ingredients of Astragalus-Salvia compound (ASC) in increasing placental blood supply, to provide thought for establishing an effective prevention and treatment of insufficient placental blood supply caused complications of pregnancy.

METHODSThe effective ingredients of ASC was isolated and abstracted and the rat model of inhibited nitric oxide synthesis was established by using L-Arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor. The mRNA expression of vascular endothelial growth factor (VEGF) in placental trophocyte and primordial micrangial endothelial cell was determined by using in situ hybridization at 12th day of pregnancy.

RESULTSThe VEGF mRNA expression was significantly lower in nitric oxide synthesis inhibited model rats than that in rats non-modeled, or in model rats treated by ASC abstracts or nitroglycerin (P < 0.05, P < 0.01).

CONCLUSIONEffective ingredients of ASC may improve VEGF mRNA expression in placental trophocyte and vascular endothelial cell in early pregnancy, and thus be favorable for placental vascular net formation, trophocyte infiltration and mother-placental circulation constitution for increasing placental blood supply to prevent and treat the insufficient placental blood supply caused diseases such as pregnancy-induced hypertension syndrome.

Animals ; Astragalus membranaceus ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; cytology ; Female ; Male ; Nitric Oxide ; biosynthesis ; Placenta ; cytology ; metabolism ; Pregnancy ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Salvia miltiorrhiza ; chemistry ; Trophoblasts ; metabolism ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

OBJECTIVE: To compare the mRNA level of cell proliferation-related genes Twist1, SIRT1, FGF2 and TGF-β3 in placenta mesenchymal stem cells (PA-MSCs), umbilical cord mensenchymals (UC-MSCs) and dental pulp mesenchymal stem cells (DP-MSCs). METHODS: The morphology of various passages of PA-MSCs, UC-MSCs and DP-MSCs were observed by microscopy. Proliferation and promoting ability of the three cell lines were detected with the MTT method. Real-time PCR (RT-PCR) was used to determine the mRNA levels of Twist1, SIRT1, FGF2, TGF-β3. RESULTS: The morphology of UC-MSCs and DP-MSCs was different from that of PA-MSCs. Proliferation ability and promoting ability of the PA-MSCs was superior to that of UC-MSCs and DP-MSCs. In PA-MSCs, expression level of Twist1 and TGF-β3 was the highest and FGF2 was the lowest. SIRT1 was highly expressed in UC-MSCs. With the cell subcultured, different expression levels of Twist1, SIRT1, FGF2, TGF-β3 was observed in PA-MSCs, UC-MSCs and DP-MSCs. CONCLUSION Up-regulated expression of the Twist1, SIRT1 and TGF-β3 genes can promote proliferation of PA-MSCs, UC-MSCs and DP-MSCs, whilst TGF-β3 may inhibit these. The regulatory effect of Twist1, SIRT1, FGF2 and TGF-β3 genes on PA-MSCs, UC-MSCs and DP-MSCs are different.
Cell Differentiation ; Cell Proliferation/genetics* ; Cells, Cultured ; Dental Pulp/cytology* ; Female ; Fibroblast Growth Factor 2/genetics* ; Humans ; Mesenchymal Stem Cells/cytology* ; Nuclear Proteins/genetics* ; Placenta/cytology* ; Pregnancy ; Sirtuin 1/genetics* ; Transforming Growth Factor beta3/genetics* ; Twist-Related Protein 1/genetics* ; Umbilical Cord/cytology*

This study was done to evaluate the stemness of human mesenchymal stem cells (hMSCs) derived from placenta according to the development stage and to compare the results to those from adult bone marrow (BM). Based on the source of hMSCs, three groups were defined: group I included term placentas, group II included first-trimester placentas, and group III included adult BM samples. The stemness was evaluated by the proliferation capacity, immunophenotypic expression, mesoderm differentiation, expression of pluripotency markers including telomerase activity. The cumulative population doubling, indicating the proliferation capacity, was significantly higher in group II (P<0.001, 31.7+/-5.8 vs. 15.7+/-6.2 with group I, 9.2+/-4.9 with group III). The pattern of immunophenotypic expression and mesoderm differentiation into adipocytes and osteocytes were similar in all three groups. The expression of pluripotency markers including ALP, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and telomerase were strongly positive in group II, but very faint positive in the other groups. In conclusions, hMSCs from placentas have different characteristics according to their developmental stage and express mesenchymal stemness potentials similar to those from adult human BMs.
Antigens, Surface/metabolism ; Bone Marrow Cells/*cytology/metabolism ; Cell Proliferation ; Female ; Humans ; Immunophenotyping ; Mesenchymal Stem Cells/*cytology/metabolism ; Mesoderm/cytology ; Octamer Transcription Factor-3/metabolism ; Placenta/*cytology/growth & development ; Pregnancy ; Pregnancy Trimester, First ; Proteoglycans/metabolism ; Stage-Specific Embryonic Antigens/metabolism ; Telomerase/metabolism

OBJECTIVETo isolate and culture human placenta derived adherent cells (hPDAC) and assay their hematopoietic growth factor expression.

METHODSBy enzyme digestion, hPDAC were isolated from human placenta tissue and cultured, and their biological characteristics were studied. The hematopoietic growth factor (HGF) mRNA expression of hPDAC was assayed by RT-PCR.

RESULTShPDAC was successfully isolated from human placenta tissue, which was further confirmed as mesenchymal stem cell-like cells. HGF including SCF, FL, G-CSF, GM-CSF, M-CSF and IL-6 were expressed in hPDAC.

CONCLUSIONhPDAC could be used as feeder layer for umbilical cord blood CD(34)(+) cells ex vivo expansion.

Cells, Cultured ; Female ; Gene Expression ; Granulocyte Colony-Stimulating Factor ; genetics ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Hematopoietic Cell Growth Factors ; genetics ; Humans ; Interleukin-6 ; genetics ; Macrophage Colony-Stimulating Factor ; genetics ; Membrane Proteins ; genetics ; Placenta ; cytology ; metabolism ; Pregnancy ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; genetics