Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation

Human placenta-derived mononuclear cells （MNC） were isolated by a Percoll density gradient and cultured in mesenchymal stem cell （MSC） maintenance medium. The homogenous layer of adherent cells exhibited a typical fibroblastlike morphology, a large expansive potential, and cell cycle characteristics including a subset of quiescent cells. In vitro differentiation assays showed the tripotential differentiation capacity of these cells toward adipogenic, osteogenic and chondrogenic lineages. Flow cytometry analyses and immunocytochemistry stain showed that placental MSC was a homogeneous cell population devoid of hematopoietic cells, which uniformly expressed CD29, CD44, CD73, CD105, CD166, laminin, fibronectin and vimentin while being negative for expression of CD31, CD34, CD45 and m-smooth muscle actin. Most importantly, immuno-phenotypic analyses demonstrated that these cells expressed class Ⅰ major histocompatibility complex （MHC-I）, but they did not express MHC-Ⅱ molecules. Additionally these cells could suppress umbilical cord blood （UCB） lymphocytes proliferation induced by cellular or nonspecific mitogenic stimuli. This strongly implies that they may have potential application in allograft transplantation. Since placenta and UCB are homogeneous, the MSC derived from human placenta can be transplanted combined with hematopoietic stem cells （HSC） from UCB to reduce the potential graft-versus-host disease （GVHD） in recipients.

Effect of vitamin E and human placenta cysteine peptidase inhibitor on expression of cathepsins B and L in implanted hepatoma Morris 5123 tumor model in Wistar rats

AIM: To examine the effectiveness of human placental inhibitors, by injecting vitamin E to rats with transplanted Morris-5123 hepatoma, on the expression of cathepsins B and L in tumor, liver, lung and blood sera after transplantation of Morris 5123 hepatoma. METHODS: Animals were divided into 10 groups receiving three different concentrations of vitamin E and inhibitors along or in combination and compared with negative control (healthy rats) and positive control (tumor rats). Effectiveness of treatment was evaluated with regard to survival time, tumor response and determination of the activities of proteolytic enzymes and their inhibitors using flurogenic substrates. RESULTS: Cathepsins B and L activities were elevated by 16-fold in comparison with negative control tissues, and their endogenous inhibitor activity decreased by 1.2-fold before treatment. In several cases, tumors completely disappeared following vitamin E plus human placental cyteine protease inhibitor (CPI) compared with controls. The number of complete tumor responses was higher when 20 m/kg vitamin E plus 400 μg of CPI was used, i.e. 7/10 rats survived more than two mo. Cathepsins B and L were expressed significantly in tumor, liver, lung tissues and sera in parallel to the increasing of the endogenous inhibitor activity compared with the controls after treatment(P<0.0001) CONCLUSION: The data indicate formation of metastasis significantly reduced in treated rats, which might provide a therapeutic basis for anti-cancer therapy.

Neural differentiation of human placenta-derived mesenchymal stem cells following neural cell co-culture

We induced human placenta-derived mesenchymal stem cells （hPMSCs） to differentiate into neural cells by adding chemical reagents, despite the fact that toxic chemicals induce cell shrinkage or cytoskeletal formation, which does not represent a proper cell differentiation process. The present study established a co-culture system with hPMSCs and neural cells and analyzed the influence of neural cells on hPMSC differentiation in a co-culture system, hPMSCs were isolated and purified from human full-term placenta using collagenase digestion. Fetal neural cells were co-cultured with hPMSCs for 48 hours using the Transwell co-culture system, hPMSCs co-cultured with neural cells exhibited a slender morphology with a filament. After 96 hours, hPMSCs expressed neuron-specific enolase, which suggested that co-culture of hPMSCs and neural cells induced neural differentiation of hPMSCs.

ENZYME-LINKED IMMUNOSORBENT ASSAY OF HUMAN PLACENTA TYPE GLUTATHIONE S-TRANSFERASE AND ITS APPLICATION IN THE DIAGNOSIS OF HEPATOCARCINOMA

GST-π was purified from human placenta and its antiserum was raised in rabbits. The antibody IgC was purified and degraded into Fab' fragment which was conjugated with horseradish peroxidase (HRP) using N-succinimidyl-4-(N-maleimido-methyl) cyclo-hexane-1-carboxylate (SMCC) as crosslinking reagent to produce Fab'-HRP conjugate. A sandwich ELISA was established for the microquantitative determination of GST-π. The sensitivity was 11 pg/tube, which was far more sensitive than the radioimmunoassay so far reported. Using this method, the serum GST-π of 41 cases normal adult was found to be 1.06±0.94 ng/ml. The upper limit of the normal value was 2.6 ng/ml. In 30 cases of primary hepatocarcinoma, the level of serum GST-π was 24.4± 17.4 ng/ml, which was 23 times higher than the normal average value (P<0.01). The positive rate was 90%. In contrast, serum GST-π in 25 cases of chronic hepatitis was determined to be 1.74±1.16 ng/ml, which was not significantly different from the normal value (P>0.05). The pseudo-positive rate was 12.0%.

ANALYSIS OF HUMAN PLACENTA BY ^(31)P NUCLEAR MAGNETIC RESONANCE AND THIN-LAYER CHROMATOGRAPHY SCANNING COMBINED WITH THE CORRECTIVE METHOD OF ABSORBANCE PROPORTIONAL COEFFICIENT

Five phospholipids in human placenta were determined by phosphorus 31 nuclear magnetic resonance(^(31)P NMR)spectroscopy and thin-layer chromatography(TLC) scanning combined with the corrective method of absorbance proportional coefficient. The NMR spectrometer used this investigation was a Bruker AM-500 spectrometer operating at 202.4 MHz for ^(31)P chemical shifts are relative to 85% phosphoric acid. TIC was carried out by silica gel H plate developed in chloroform-methanol-glacial acetic acid-ethanol-water(25:4:6:2:0.5),with Vaskovsky reagent as colour -developing agent of phospholipids.

Uric acid promotes neuronal differentiation of human placenta-derived mesenchymal stem cells in a time- and concentration-dependent manner

Uric acid is an important, naturally occurring serum antioxidant. The present study investigates the use of uric acid for promoting proliferation and neuronal differentiation of mesenchymal stem cells derived from human placenta tissue. Human placenta-derived mesenchymal stem cells were pre-induced in the presence of either 0, 0.2, 0.4 or 0.8 mM uric acid in combination with 1 mM β-mercaptoethanol for 24 hours, followed by exposure to identical uric acid concentrations and 5 mM β-mercaptoethanol for 6 and 10 hours. Cells developed a neuronal-like morphology, with formation of interconnected process extensions, typical of neural cells. Immunocytochemistry and immunofluorescence staining showed neuron specific enolase positive cells were present in each group except the control group. A greater number of neuron specific enolase positive cells were observed in 0.8 mM uric acid in combination with 5 mM β-mercaptoethanol at 10 hours. After 24 hours of induction, Nissl bodies were detected in the cytoplasm of all differentiated cell groups except the control group and Nissl body numbers were greatest in human placenta-derived mesenchymal stem cells grown in the presence of 0.8 mM uric acid and 5 mM β-mercaptoethanol. These results suggest uric acid accelerates differentiation of human placenta-derived mesenchymal stem cells into neuronal-like cells in a time-and concentration-dependent manner.

Improvement of Atrophic Acne Scar and Skin Complexity by Combination of Aqueous Human Placenta Extract and Mesenchymal Stem Cell Mesotherapy

Atrophic scars, a permanent complication of severe acne, have negative effect on psychology in adolescent. The treatment of atrophic scar is depended on types of scar and it is difficult to improve by a single treatment. Mesenchymal stem cell is a scientific approval for surgery scar treatment and wound healing. We present a case report of female presented with atrophic acne scar distributed on both cheeks. The case aims to prove that the combination of MSCs and aqueous human placenta extract (RGF&#174) contained bioactive therapeutic molecules obviously promoted the improvement of skin scar to reach the optimal outcomes. We first found that MSCs-contained human placenta extract solution combination subcision improves the atrophic acne scar and skin complexity by enhancement of skin cell regeneration.

Case Report: Improvement of Long Lasting Hyperpigmentation by Aqueous Human Placenta Extract (Reju Growth Factor, RGF&#174) Treatment

Hyperpigmentation is a common skin problem in a woman. Prolonging topical use of skin whitening may cause hyperpigmentation such as ochronosis whose condition is a challenge for treatment. An aqueous human placenta extract (RGF&#174) contains bioactive therapeutic molecules. There is evidence of human placenta extract showing that melanin synthesis is inhibited by placenta extract in melanocytes. We first reported the case of the hyperpigmentation improvement following face skin mesotherapy human placenta extract treatment.

The Effect of Ethanol on the Transport of a Neutral Amino Acid in the Perfused Whole Human Placenta

The effect of ethanol on the transport of amino acids across the human placenta was studied in the dual perfusion apparatus using a non-metabolizable α-amino isobutyric acid (AIB). Results were obtained for thirty intact whole human placentas in the absence (control group) and presence (ethanol group) of ethanol (500 - 1000 mg/dL). Experimental determinations of AIB transport at AIB concentrations of 5 - 100 mg/l, measured radioactively using (1&#8722;14 C-) AIB, were compared with a dual-active transport model. The diffusion coefficients of AIB were found to be (3.7 × 10&#8722;9 cm2/s) in the absence of ethanol and (2.3 × 10&#8722;9 cm2/s) in the presence of ethanol with no statistical difference (P = 0.25). The ratio of the fetal to maternal perfusate concentrations in the absence of ethanol (1.44) was statistically significant (P = 0.016) from the ratio in the presence of ethanol (1.20), which may indicate that active transport in the human placenta is inhibited by the presence of ethanol. The placental uptake from the maternal circulation was 2.6 (control) and 2.5 (ethanol) times greater than the uptake from the total circulation. The relative contribution of the diffusive transport to the net placental uptake of AIB from both the maternal and fetal circulations was less than that of active transport regardless of the presence of ethanol: control (38%) and ethanol (35%). It appears that the placental tissue plays the role of a mediator to maintain a fetal concentration higher than the maternal one by either enhancing the transfer from the maternal to the placental tissue or impairing the transfer in the opposite direction.

Hypoxia Downregulates the Angiogenesis in Human Placenta via Notch1 Signaling Pathway

Placentation, which is critical for maternal-fetal exchange of nutrients and gases, is a complicated process comprising stepwise vasculogenesis and angiogenesis. Hypoxia caused by impaired trophoblast invasion may cause various angiogenic abnormalities in human placenta. The Notch1 signaling pathway plays an important role in the regulation of angiogenesis. The angiogenesis of human umbilical vein endothelial cells（HUVECs） under normal/hypoxic conditions and the m RNA/protein level of Notch1/Dell4/Jagged1 were investigated in this study. The effects of DAPT/JAG-1 on the migration of HUVECs were also assessed by cell wound healing assay, so as to discover the possible role of notch1 signaling pathway in the angiogenesis of human placenta. The results showed that angiogenic ability of HUVECs was seriously reduced under hypoxic conditions. The m RNA and protein levels of Notch1/Dell4/Jagged1 were decreased in the hypoxic group compared to the control one. In addition, the migration capability of HUVECs was significantly obstructed when treated with DAPT and under hopoxic condition, but promoted when treated with JAG-1. The above results demonstrate that hypoxia downregulates the angiogenesis in human placenta via Notch1 signaling pathway.

Immunohistochemical study of substance P receptor (SPR) in early human placenta

We sought to determine whether substance P receptors (SPR) exist in human placenta and if do their cellular localization in placental villi, and to supply morphological evidence for the functional significance of SP in placental and fetal development. Methods: Immunohistochemical ABC method was used in the experiment. Results: Both syncytiotrophoblasts and cytotrophoblasts, stromal cells, capillary endothelium, lymphocytes in capillary cavity, and all blood islet in cells early human placenta showed SPR immunoreactivity in cytoplasm but with negative nuclei. Conclusion: SP produced by placental villi mediated by SPR might be responsible for the synthesis and release of placental hormone, the development of capillaries and the regulation of microcirculation in placental villi and fetal immune function.

Development of matrix solid-phase dispersion method for the extraction of short-chain chlorinated paraffins in human placenta

Chlorinated paraffins（SCCPs） are widely used worldwide, and they can be released into the environment during their production, transport, usage and disposal, which pose potential risks for human health. In this work, an efficient, reliable and rapid pretreatment method based on matrix solid-phase dispersion（MSPD） was developed for the analysis of short-chain CPs（SCCPs） in human placenta by gas chromatograph-electron capture negative ion low-resolution mass spectrometry（GC-ECNI-LRMS） and gas chromatography–quadrupole time-of-flight mass spectrometry（GC–QTOF-HRMS）. The MSPD-relevant parameters including dispersing sorbent,sample-to-sorbent mass ratio, and elution solvent were optimized using the orthogonal test.Silica gel was found to be the optimal dispersing sorbent among the selected matrices. Under the optimal conditions, 44% acidic silica gel can be used as the co-sorbent to remove lipid and eluted by the mixture of hexane and dichloromethane（7：3, V/V）. The spiked recoveries of the optimized method were 77.4% and 91.4% for analyzing SCCPs in human placenta by GC-ECNI-LRMS and GC–QTOF-HRMS, and the corresponding relative standard deviations were10.2% and 5.6%, respectively. The method detection limit for the total SCCPs was 36.8 ng/g（dry weight, dw） and 19.2 ng/g（dw） as measured by GC-ECNI-LRMS and GC–QTOF-HRMS,respectively. The concentrations of SCCPs in four human placentas were in the range of

Expression of two alternative splicing isoforms of fragile X gene in human placenta

Fragile X (Fra (X)) syndrome is the most frequent cause of inherited mentalretardation, and it is associated with the fragile site at Xq27.3. In 1991, the FMR1(fragile X mental retardation 1) gene was cloned in the vicinity of this site. The muationand abnormal expression of FMR1 are the direct causes of Fra(X) syndrome. The

Construction of tissue-engineered cartilage using human placenta-derived stem cells

Human placenta-derived stem cells (hPDSCs) were isolated by trypsinization and further induced into cartilage cells in vitro.The engineered cartilage was constructed by combining hPDSCs with collagen sponge and the cartilage formation was observed by implantation into nude mice.Results showed that hPDSCs featured mesenchymal stem cells and maintained proliferation in vitro for over 30 passages while remaining undifferentiated.All results indicated that hPDSCs have the potential to differentiate into functional cartilage cells in vitro when combined with collagen sponge,which provided experimental evidence for prospective clinical application.

Localization of somatostatin mRNA in early human placental villi by in situ hybridization histochemistry

Previous immunohistochemical studies showed that the human placenta contained notonly many regulatory peptides, but also 5-HT and dopamine (DA) classicalneurotransmitters. These results made us suggest the following hypothesis: the human placentamight be a complex endocrine organ. In order to confirm this hypothesis, it must beunderstood whether these regulatory peptides and classical neurotransmitters can be pro-

Immunohistochemical and in situ hybridized histochemical studies of SP and SPmRNA in early human placenta

Previous studies have demonstrated that the human placental villi contain neuropeptideY (NPY), somatostatin (SS) and β-endorphin (β-EP) neuropeptidies, suggesting that theneuropeptides might play an important role in development of the placenta and feta. SP isa neuropeptide and was discovered earliest. It functions importantly in regulating bloodcirculation. But up to now, SP existing in human placenta has never been reported. Inthis experiment, we used immunohistochemical method and in situ hybridization at

Isolation and partial molecular characterization of heat-stable growth factor from human placenta

A heat\|stable growth factor has been purified to homogeneity from human placenta. Purification procedure involved extraction with acidic medium, precipitation using ethanol, DE\|52 ion\|exchange chromatography, and reversed\|phase HPLC. The purified preparation has been named human placental growth factor\|1 (HPGF\|1), which simulates the proliferation of some cell lines, such as human amniotic cell, mouse marrow tumor cell, and mouse fibroblast cell. The growth factor has an approximate molecular weight of 1 900. It is a glycopeptide which contains a polypeptide chain consisting of 10 amino acid residues with molecular weight of 1 195. The N\|terminal residue of the polypeptide chain part is phenylalanine. The molecular weight of non\|peptide part is 682. The isoelectric point of the growth factor is pI 6.8.

STUDY ON REPRODUCTIVE ENDOCRINOLOGY OF HUMAN PLACENTA (Ⅲ)——HORMONAL REGULATION OF PROGESTERONE PRODUCTION BY TROPHOBLAST TISSUE OF FIRST TRIMESTER

The effect of hormones on progesterone secretion by 6-8 week human trophoblast tissue cultured in serum-free medium has been investigated. GnRH at low concentration (10-10-10-8 mol/L) stimulated progesterone secretion, while high dose (10-6-10-5 mol/L) produced inhibitory effect. The progesterone secretion could be significantly decreased by addition of anti-hCG antiserum or monoclonal anti-hCG IgG in a dose- and time-dependent manner. Various concentrations of TRH, PGE2, PGF2α, testosterone and estradiol were found to be ineffective. These data indicate clearly that progesterone production by human trophoblast tissue at early gestation stage is under the modulation of GnRH and hCG.

Localization and quantitative analysis of interleukin-6 in human placenta

The localization of IL6 at light microscopic levels and its quantitative analysis in human placentas were studied by using immunohistochemistry. Both syncytiotrophoblast and cytotrophoblast in placental villi, white blood cells in villose capillary cavity stromal cells and capillary endothelium present IL6 immunoreactivity in cytoplasm. Using a quantitative immunohistochemical method, the amounts of IL6 in placenta were low at the 6th week of gestation, increased saliently and peaked at the 7th, then reduced gradually during the rest of the gestation period and maintained the same level as that in the 6th week of gestation. This changing trend was paralleled with that of GnRH in our previous studies. These results revealed that IL6 could be produced by the human placenta and may take part in the regulation of placental hormone releasing.

Microscopic and ultramicroscopic localizations and quantitative analysis of 5-HT receptors in human placentas

The localizations of 5-hydroxytryptamine receptor (5-HTR) at light and electron microscopic levels and its quantitative analysis in human placentas were studied by using immunohistochemistry and in situ hybridization. Both syncytiotrophoblast and cytotrophoblast in placental villi and fetal white blood cells in villose capillary cavity showed 5-HT receptor immunoreactivity, with 5-HT 1A receptor mRNA hybridized signal detected in cytoplasm. But the stromal cells and capillary endothelium in placental villi showed 5-HT receptor immunoreactivity in cytoplasm, without 5_HT\-1A receptor mRNA hybridized signal detected. This suggested that two layers of trophoblast cells may produce 5-HT 1 and 5-HT 2 receptors, that the stromal cells and capillary endothelium in placental villi may only produce 5-HT 2 receptor. By immunohistochemistry at electron microscopic level, the small flattened vesicles and large dense cored vesicle within trophoblast cells showed 5-HT receptor immunoreactivity. This suggested that it may be the result of 5-HT receptors internalization and transportion. Using a quantitative immunohistochemical method, the contents of 5-HT receptor in placenta were higher during the 6th week of gestation, and decreased in 7th and 8th, reoccurred the second peak in the 9th, reduced gradually during the 10th, 20th and 40th of the gestation period. These changes paralleled the contents of 5-HT in the authors’ studies, reflecting that 5-HT may be one of the most important bioactive substances in placental self-regulation.