Establishment of an untransfected human corneal epithelial cell line and its biocompatibility with denuded amniotic membrane

AIM: To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM). METHODS: The torn HCEP pieces were primarily cultured in DMEM/F12 media (pH 7.2) supplemented with 20% fetal bovine serum and other necessary factors, yielding an HCEP cell line which was its growth performance, chromosome morphology, tumorigenicity and expression of marker proteins analyzed. In addition, the biocompatibility of HCEP cells with dAM was evaluated through histological and immunocytochemistry analyses and with light, electron and slit-lamp microscopies. RESULTS: HCEP cells proliferated to confluence in 3 weeks, which have been subcultured to passage 160. A continuous untransfected HCEP cell line, designated as utHCEPC01, was established with a population doubling time of 45.42 hours as was determined at passage 100. The cells retained HCEP cell properties as were approved by chromosomal morphology and the expression of keratin 3. They, with no tumorigenicity, formed a multilayer epithelium-like structure on dAMs through proliferation and differentiation during air-liquid interface culture, maintained expression of marker proteins including keratin 3 and integrin p 1 and attached tightly to dAMs. The reconstructed HCEP was highly transparent and morphologically and structurally similar to the original. CONCLUSION: An untransfected and non-tumorigenic HCEP cell line was established in this study. The cells maintained expression of marker proteins. The cell line was biocompatible with dAM. It holds the potential of being used for in vitro reconstruction of tissue-engineered HCEP, promising for the treatment of diseases caused by corneal epithelial disorders.

Human amniotic epithelial cell transplantation for the repair of injured brachial plexus nerve: evaluation of nerve viscoelastic properties

The transplantation of embryonic stem cells can effectively improve the creeping strength of nerves near an injury site in animals. Amniotic epithelial cells have similar biological properties as em-bryonic stem cells; therefore, we hypothesized that transplantation of amniotic epithelial cells can repair peripheral nerve injury and recover the creeping strength of the brachial plexus nerve. In the present study, a brachial plexus injury model was established in rabbits using the C6root avulsion method. A suspension of human amniotic epithelial cells was repeatedly injected over an area 4.0 mm lateral to the cephal and caudal ends of the C6 brachial plexus injury site （1 × 106 cells/mL, 3μL/injection, 25 injections） immediately after the injury. The results showed that the decrease in stress and increase in strain at 7,200 seconds in the injured rabbit C6 brachial plexus nerve were mitigated by the cell transplantation, restoring the viscoelastic stress relaxation and creep properties of the brachial plexus nerve. The forepaw functions were also signiifcantly improved at 26 weeks after injury. These data indicate that transplantation of human amniotic epithelial cells can effec-tively restore the mechanical properties of the brachial plexus nerve after injury in rabbits and that viscoelasticity may be an important index for the evaluation of brachial plexus injury in animals.

Monoamine alterations and rotational asymmetry in a rat model of Parkinson's disease following lateral ventricle transplantation of human amniotic epithelial cells

BACKGROUND： Human amniotic epithelial cells （HAECs） can differentiate into neurons, astrocytes and oligodendrocytes. They biologically secrete many active neurotrophins and have the capacity to metabolize dopamine enzymes. These features underlie a theoretical basis for the treatment of Parkinson＇s disease （PD）. OBJECTIVE： To investigate the survival and differentiation of transplanted HAECs in the lateral ventricle of PD model rats, and to explore its effect on circling behavior, as well as levels of dopamine （DA）, the metabolite homovanillic acid, dihydroxyphenyl acetic acid, 5-hydroxyindoleacetic acid, and 5-hydroxytryptamine in the striatum. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, and Shanghai Celstar Institute of Biotechnology from May 2007 to December 2008. MATERIALS： HAECs were derived from the placental chorion following caesarean delivery at the Shanghai International Matemal and Child Health Hospital. 6-hydroxydopamine （6-OHDA）, and mouse anti-human Vimentin monoclonal antibody were purchased from Sigma, USA; mouse anti-human nestin and tyrosine hydroxylase （TH） monoclonal antibodies were purchased from Chemicon, USA. METHODS： A total of 114 healthy, adult, Sprague Dawley rats were randomly assigned to two groups： PD model [n = 90, stereotactic microinjection of 2 μL 6-OHDA （3.5 μg/uL） into the striatum] and control （n = 24, no treatment）. The 51 successful PD model rats were randomly divided into 3 subgroups （n = 17）： HAEC, PBS, and model. The HAEC and PBS groups were respectively injected with 10 μL PBS solution containing 1 × 10^5/mL HAECs or 10 pL PBS into the lateral ventricle. The model group was not treated. MAIN OUTCOME MEASURES： TH protein expression in the striatum was evaluated by immunohistochemistry 5 weeks after HAEC transplantation. At 10 weeks, HAEC survival in the lateral ventricle was investigated by immunofluorescent staining; differentiation of HAECs in the lateral and third ventricles was examined by TH immunohistochemistry; concentrations of DA, homovanillic acid, dihydroxyphenyl acetic acid, 5-hydroxyindoleacetic acid, and 5-hydroxytryptamine in the striatum, as well as DA concentration in the cerebrospinal fluid, were measured with high-performance liquid chromatography-electrochemical detection. Circling behavior of PD model rats was consecutively observed for 10 weeks following intraperitoneal injection of amphetamine 1 week after successful model establishment. RESULTS： tn the HAEC group, the number of TH-positive cells significantly increased in the striatum, and circling behavior significantly decreased, compared with the PBS and model groups （P 〈 0.01）. In addition, monoamine concentrations in the striatum, as well as DA concentrations in the cerebrospinal fluid, significantly increased, compared with the PBS group （P 〈 0.05-0.01）. Moreover, a large number of nestin-, vimentin-, and TH-positive cells were observed in the lateral and third ventricles following HAEC injection.CONCLUSION： HAECs survived for 10 weeks with no overgrowth following transplantation into the lateral ventricle of PD model rats. Moreover, the cells differentiated into dopaminergic neurons, which increased DA secretion. HAEC transplantation improved cycling behavior in PD model rats.

Human amniotic epithelial cells express specific markers of nerve cells and migrate along the nerve fibers in the corpus callosum

Human amniotic epithelial cells were isolated from a piece of fresh amnion. Using immunocytochemical methods, we investigated the expression of neuronal phenotypes （microtubule-associated protein-2, glial fibrillary acidic protein and nestin） in human amniotic epithelial cells. The conditioned medium of human amniotic epithelial cells promoted the growth and proliferation of rat glial cells cultured in vitro, and this effect was dose-dependent. Human amniotic epithelial cells were further transplanted into the corpus striatum of healthy adult rats and the grafted cells could integrate with the host and migrate 1 2 mm along the nerve fibers in corpus callosum. Our experimental findings indicate that human amniotic epithelial cells may be a new kind of seed cells for use in neurograft.

Transplantation of human amniotic epithelial cells repairs brachial plexus injury:pathological and biomechanical analyses

A brachial plexus injury model was established in rabbits by stretching the C6 nerve root. Imme- diately after the stretching, a suspension of human amniotic epithelial cells was injected into the injured brachial plexus. The results of tensile mechanical testing of the brachial plexus showed that the tensile elastic limit strain, elastic limit stress, maximum stress, and maximum strain of the injured brachial plexuses were significantly increased at 24 weeks after the injection. The treatment clearly improved the pathological morphology of the injured brachial plexus nerve, as seen by hematoxylin eosin staining, and the functions of the rabbit forepaw were restored. These data indicate that the injection of human amniotic epithelial cells contributed to the repair of brachial plexus injury, and that this technique may transform into current clinical treatment strategies.

Differentiation of human amniotic epithelial cells into corneal epithelial-like cells in vitro

·AIM: To explore the feasibility that human amniotic epithelial cells (hAECs) have the potential to differentiate into corneal epithelial -like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells (S-ihCECs). ·METHODS: hAECs were isolated by enzyme digestion, and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA -DR. Recovered and cultured S -ihCECs, immunocytochemistry was used to detect the expression of CK3/12. The proliferation of S - ihCECs handled by different concentrations of mitomycin was detected by CCK -8. The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK -8. After filtered out the optimal conditions, we collected S-ihCECs culture media for 5 days, then prepared conditioned medium to incubate hAECs, inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs. Quantitative real -time reverse transcription -polymerase chain reaction (QRT - PCR) was carried out to evaluate the expression of Oct - 4, NANOG, PAX6, and CK12 in the differentiation period. Immunocytochemistry and western bloting were used to detect the expression of CK3/12. ·RESULTS: The culture media collected every 12h, from 20μg/mL mitomycin pretreatment S -ihCECs could significantly promote the proliferation of hAECs. In the period of differentiation, the morphology of differentiated hAECs was obviously different compared with the control group, and the distinctive CK3/12 for corneal epithelial cells was detected.·CONCLUSION: This study showed that hAECs can differentiate into corneal epithelial -like cells by replication of the corneal epithelial microenvironment, using the culture media collected from S -ihCECs, and it is possible that S -ihCECs culture media could be used in corneal tissue engineering. ·

Cytoprotective effect of amniotic membrane extracts on human corneal epithelial cells exposed to benzalkonium chloride in vitro

Background:The goal was to explore the protective effect and potential mechanism of amniotic membrane extracts(AME)on the ocular surface exposed to benzalkonium chloride(BAC).Methods:The human corneal epithelial cell(HCEC)line SD-HCEC1s was cultured in 5 groups:normal control(NC),NC+AME,BAC,BAC+NC,and BAC+AME.Cell viability analysis,flow cytometry analysis,real-time polymerase chain reaction(PCR),and western blot were employed to measure changes in cell function.Matrix metalloproteinases(MMPs)and inflammatory cytokines were assayed by enzyme-linked immunosorbent assay(ELISA)and activity assays.Results:Real-time PCR and western blot analysis demonstrated that the expressional level of caspase-8 was increased while the levels of Muc1,Muc4,and Muc16 were decreased after treatment with 0.02%BAC for 1 h.When the SD-HCEC1s were withdrawn from the BAC and switched to media containing 10%AME for 2 days,the expression level of capsase-8 was decreased while the levels of Muc1,Muc4,and Muc16 were increased.Real-time PCR and ELISA demonstrated that the mRNA and protein levels of MMP-1,MMP-3,MMP-13,CXCL1,interleukin(IL)-1β,IL-6,and tumor necrosis factor-alpha(TNF-α)were significantly increased after treatment with 0.02%BAC,whereas those of MMP-8 were decreased.When the 0.02%BAC was withdrawn and the SD-HCEC1s were cultured in 10%AME,the mRNA and protein levels of MMP-1,MMP-3,MMP-13,CXCL1,IL-1β,IL-6,and TNF-αwere decreased,while those of MMP-8 were increased.MMP-8 activity assays confirmed that IL-1βand TNF-αdownregulated the protein levels of MMP-8.Conclusions:AME protects SD-HCEC1s when stressed in BAC via upregulation of MMP-8 and downregulation of IL-1βand TNF-α.AME may have the potential functions to be employed as a topical adjunctive therapy in eyes chronically exposed to BAC.

In vitro reconstruction and characterization of tissue-engineered human corneal epithelium with seeder cells from an untransfected human corneal epithelial cell line

AIM: To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium (TE-HCEP) with seeder cells from an untransfected HCEP cell line. METHODS: The TE-HCEPs were reconstructed in vitro with seeder cells from an untransfected HCEP cell line, and scaffold carriers of denuded amniotic membrane (dAM) in air-liquid interface culture for 3, 5, 7 and 9 days, respectively. The specimens were examined with hematoxylin-eosin (HE) staining of paraffin-section, immunocytochemical staining, scanning and transmission electron microscopy. RESULTS: During in vitro reconstruction of TE-HCEP, HCEP cells formed a 3-4, 6-7 and 8-10 layers of an HCEP-like structure on dAMs in air-liquid interface culture for 3, 5 and 7 days, respectively. But the cells deceased to 5-6 layers and the structure of straified epithelium became loose at day 9. And the cells maintained positive expression of marker proteins (keratin 3 and keratin 12), cell-junction proteins (zonula occludens-1, E-cadherin, connexin 43 and integrin beta 1) and membrane transport protein of Na+-K+ ATPase. The HCEP cells in TE-HCEP were rich in microvilli on apical surface and established numerous cell-cell and cell-dAM junctions at day 5. CONCLUSION: The morphology and structure of the reconstructed TE-HCEP were similar to those of HCEP in vivo. The HCEP cells in the reconstructed TE-HCEP maintained the properties of HCEP cells, including abilities of forming intercellular and cell-extracellular matrix junctions and abilities of performing membrane transportation. The untransfected HCEP cells and dAMs could promisingly be used in reconstruction HCEP equivalent for clinical corneal epithelium transplantation.

Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment

BACKGROUND To solve the problem of liver transplantation donor insufficiency,an alternative cell transplantation therapy was investigated.We focused on amniotic epithelial cells(AECs)as a cell source because,unlike induced pluripotent stem cells,they are cost-effective and non-tumorigenic.The utilization of AECs in regenerative medicine,however,is in its infancy.A general profile for AECs has not been comprehensively analyzed.Moreover,no hepatic differentiation protocol for AECs has yet been established.To this end,we independently compiled human AEC libraries,purified amniotic stem cells(ASCs),and co-cultured them with mesenchymal stem cells(MSCs)and human umbilical vein endothelial cell(HUVECs)in a 3D system which induces functional hepatic organoids.AIM To characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells METHODS AECs,MSCs,and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients.Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry,Alkaline phosphatase(AP)staining,and flow cytometry.An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay.AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics.The 2D and 3D culture were compared by relative gene expression using several differentiation protocols.ASCs,MSCs,and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging,periodic acid Schiff,and an indocyanine green(ICG)test.RESULTS AECs have certain stemness markers such as EPCAM,SSEA4,and E-cadherin.One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers.Moreover,it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage.Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells.TJP1,associated with epidermal growth factor receptor,and MET,associated with hepatocyte growth factor receptor,were upregulated and may be important for hepatic differentiation.In conventional flat culture,the cells turned unviable and did not readily differentiate into hepatocytes.In 3D culture,however,hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol.Furthermore,the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity,hepatic-like glycogen storage,and ICG absorption/elimination.CONCLUSION Human amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness.Under a 3D co-culture system,functional hepatic organoids were generated in a multicellular microenvironment.

Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models

AIM: To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS: TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS: After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575 mu m in thickness during the monitoring period. A 45 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION: The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.

Transplantation of human amniotic epithelial cells improves hindlimb function in rats with spinal cord injury

Background Human amniotic epithelial cells （HAECs）, which have several characteristics similar to stem cells, therefore could possibly be used in cell therapy without creating legal or ethical problems. In this study, we transplanted HEACs into the injured spinal cord of rats to investigate if the cells can improve the rats＇ hindlimb motor function. Methods HAECs were obtained from a piece of fresh amnion, labeled with Hoechst33342, and transplanted into the site of complete midthoracic spinal transections in adult rats. The rats （n=21） were randomly divided into three groups： Sham-operation group （n=7）, cells-graft group （n=7）, and PBS group （n=7）. One rat of each group was killed for histological analysis at the second week after the transplantation. The other six rats of each group were killed for histological analysis after an 8-week behavioral testing. Hindlimb motor function was assessed by using the open-field BBB scoring system. Survival rate of the graft cells was observed at second and eighth weeks after the transplantation. We also detected the myelin sheath fibers around the lesions and the size of the axotomized red nucleus. A one-way ANOVA was used to compare the means among the groups. The significance level was set at P〈0.05. Results The graft HAECs survived for a long time （8 weeks） and integrated into the host spinal cord without immune rejection. Compared with the control group, HAECs can promote the regeneration and sprouting of the axons, improve the hindlimb motor function of the rats （BBB score： cells-graft group 9.0 ± 0.89 vs PBS group 3.7± 1.03, P〈0.01）, and inhibit the atrophy of axotomized red nucleus [cells-graft group （526.47±148.42）μm^2 vs PBS group （473.69±164.73） μm^2, P〈0.01]. Conclusion Transplantation of HAECs can improve the hindlimb motor function of rats with spinal cord injury.

Exosomes derived from human amniotic epithelial cells accelerate diabetic wound healing via PI3K-AKT-mTOR-mediated promotion in angiogenesis and fibroblast function

Background:Diabetic wounds are one of the most common and serious complications of diabetes mellitus,characterized by the dysfunction of wound-healing-related cells in quantity and quality.Our previous studies revealed that human amniotic epithelial cells(hAECs)could promote diabetic wound healing by paracrine action.Interestingly,numerous studies demonstrated that exosomes derived from stem cells are the critical paracrine vehicles for stem cell therapy.However,whether exosomes derived from hAECs(hAECs-Exos)mediate the effects of hAECs on diabetic wound healing remains unclear.This study aimed to investigate the biological effects of hAECs-Exos on diabetic wound healing and preliminarily elucidate the underlying mechanism.Methods:hAECs-Exos were isolated by ultracentrifugation and identified by transmission electron microscopy,dynamic light scattering and flow cytometry.A series of in vitro functional analyses were performed to assess the regulatory effects of hAECs-Exos on human fibroblasts(HFBs)and human umbilical vein endothelial cells(HUVECs)in a high-glycemic microenvironment.Highthroughput sequencing and bioinformatics analyses were conducted to speculate the related mechanisms of actions of hAECs-Exos on HFBs and HUVECs.Subsequently,the role of the candidate signaling pathway of hAECs-Exos in regulating the function of HUVECs and HFBs,as well as in diabetic wound healing,was assessed.Results:hAECs-Exos presented a cup-or sphere-shaped morphology with a mean diameter of 105.89±10.36 nm,were positive for CD63 and TSG101 and could be internalized by HFBs and HUVECs.After that,hAECs-Exos not only significantly promoted the proliferation and migration of HFBs,but also facilitated the angiogenic activity of HUVECs in vitro.High-throughput sequencing revealed enriched miRNAs of hAECs-Exos involved in wound healing.Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses have shown that the target genes of the top 15 miRNAs were highly enriched in the PI3K-AKT pathway.Further functional studies demonstrated that the PI3K-AKT-mTOR pathway was necessary for the induced biological effects of hAECs-Exos on HFBs and HUVECs,as well as on wound healing,in diabetic mice.Conclusions:Our findings demonstrated that hAECs-Exos represent a promising,novel strategy for diabetic wound healing by promoting angiogenesis and fibroblast function via activation of the PI3K-AKT-mTOR pathway.

Intracerebroventricular transplantation of human amniotic epithelial cells ameliorates spatial memory deficit in the doubly transgenic mice coexpressing APPswe and PS1△E9.deleted genes

Background Human amniotic epithelial cells （HAECs）, which have characteristics of both embryonic and pluripotent stem cells, are therefore a candidate in cell therapy without creating legal or ethical problems. In the present study, we aimed to investigate the effects of intracerebroventricular transplantation of HAECs on doubly transgenic mice of Alzheimer＇s disease （AD） coexpressing presenilin-1 （PS1） and mutant Sweden amyloid precursor protein （APPswe） genes. Methods The offspring mice genotypes were detected using PCR identification of APPswe and PS1 gene. The doubly transgenic （TG） mice （n=20） and wild-type （WT） mice （n=20） were randomly divided into two groups respectively： the transplantation group treated with HAECs and the control group with phosphate buffered saline. Six radial arm water maze test was used to assess the spatial memory in the TG and WT mice. Amyloid plaques and neurofibrillary tangles were analyzed using congo red and acid-silver methenamine staining respectively. was used to track the survival of HAECs. Immunohistochemistry was used octamer-binding protein 4 （Oct-4） and Nanog in the HAECs. High performance measure acetylcholine in hippocampus. The density of cholinergic neurons in hippocampus was measured using acetylcholinesterase staining. Immunofluorescence cytochemistry to determine the expression of quid chromatography was used to basal forebrain and nerve fibers in Results Amyloid deposition occurred in hippocampus and frontal cortex in the double TG mice aged 8 months, but not in WT mice. The results also showed that transplanted HAECs can survive for at least 8 weeks and migrate to the third ventricle without immune rejection. The graft HAECs can also express the specific marker Oct-4 and Nanog of stem cell. Compared with the control group, transplantation of HAECs can not only significantly improve the spatial memory of the TG mice, but also increase acetylcholine concentration and the number of hippocampal cholinergic neurites. Conclusions These results demonstrate that intracerebroventricular transplantation of HAECs can improve the spatial memory of the double TG mice. The higher content of acetylcholine in hippocampus released by more survived cholinergic neurites is one of the causes of this improvement.

Ureaplasma urealyticum-derived lipid-associated membrane proteins introduce IL-6, IL-8, and TNF-α cytokines into human amniotic epithelial cells via Toll-like receptor 2

Objective： The purpose of this study was to determine the role of Ureaplasma urealyticum-derived lipidassociated membrane proteins （LAMPs） in the host innate immune system, specifically their effect on Toll-like receptors （TLRs）. Methods： LAMPs were derived from U. urea/yticum strains, and human amniotic epithelial cells （HAECs） were isolated from healthy full-term placentas. Cytokine concentrations were determined by enzyme-linked immunosorbent assay （ELISA） and TLR2 mRNA by real-time PCR. Expression of TLR2 was confirmed by Western blotting and immunohistochemistry. Results： LAMPs induced HAECs to produce inflammatory cytokines interleukin （IL）-6, IL-8, and tumor necrosis factor （TNF）-α. Cytokine production was reduced after blocking TLR2 using TLR2 inhibitor （anti-hTLR2-IgA）. Conclusions： LAMPs isolated from U. urealyticum induced TLR2-dependent up-regulation of inflammatory genes and cytokines in HAECs.

Therapeutic effect of human amniotic epithelial cell transplantation into the lateral ventricle of hemiparkinsonian rats

Background Human amniotic epithelial cells （HAECs） are able to secrete biologically active neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3, both of which exhibit trophic activities on dopamine neurons. Previous study showed that when human amniotic epithelial cells were transplanted into the striatum of 6-hydroxydopamine （6-OHDA）-induced Parkinson disease rats, the cells could survive and exert functional effects. The purpose of this study was to investigate the survival and the differentiation of human amniotic epithelial cells after being transplanted into the lateral ventricle of Parkinson＇s disease （PD） rats, and to investigate the effects of grafts on healing PD in models. Methods The Parkinson＇s model was made with stereotactic microinjection of 6-hydroxydopamine （6-OHDA） into the striatum of a rat. The PD models were divided into two groups： the HAECs group and the normal saline （NS） group. Some untreated rats were taken as the control. The rotational asymmetry induced by apomorphine of the HAECs group and the NS group were measured post cell transplantation. The expression of nestin and vimentin in grafts were determined by immunohistology. Ten weeks after transplantation the density of tyrosine hydroxylase positive cells in the substantia nigra of the HAECs group, NS group and the untreated group was determined. The differentiation of grafts was determined by TH immunohistology. High performance liquid chromatography （HPLC） was used to determine monoamine neurotransmitter levels in the striatum. Results The rotational asymmetry induced by apomorphine of the HAECs group was ameliorated significantly compared to the NS group two weeks after transplantation （P 〈0.01）. The grafts expressed nestin and vimentin five weeks after transplantation. TH immunohistochemistry indicated that the TH positive cells in the substantia nigra of the HAECs group increased significantly compared to the NS group （P 〈0.01）. Tyrosine hydroxylase （TH） positive cells in the substantia nigra of the HAEC group and the NS group were decreased compared to the untreated group （P 〈0.01）. Dopamine and DOPAC levels in the striatum of the HAECs group increased significantly compared to the NS group （P 〈0.05）. Homovanillic acid （HVA） levels in the striatum of the HAECs group increased significantly compared to the NS group （P 〈0.01）. In addition dopamine, DOPAC, and HVA levels in the striatum and dopamine levels in the cerebrospinal fluid of the HAECs group and the NS group were decreased compared to the untreated group （P 〈0.05）. Conclusions Human amniotic epithelial cells could be used to ameliorate the rotational asymmetry induced by apomorphine of the PD models. This could have been due to the increased content of dopamine and its metabolic products, DOPAC and HVA, in the striatum in the PD models.

Therapeutic effects of human amniotic epithelial cell transplantation on double-transgenic mice co-expressing APPswe and PS1ΔE9-deleted genes

Human amniotic epithelial cells (HAECs), which exhibit characteristics of embryonic and pluripotent stem cells, could be utilized for cell therapy without legal or ethical problems. Double-transgenic (TG) mice (n=20) and wild-type (WT) mice (n=20) were randomly assigned to two groups, respectively. The transplantation group was treated with HAECs and the control group with PBS. A six-radial arm water maze was used to assess spatial memory. Immunofluorescence was utilized to track HAEC survival. Immunohistochemistry was used to determine octamer-binding protein 4 (oct-4) and nanog expression in the HAECs. High-performance liquid chromatography (HPLC) was used to measure acetylcholine levels in the hippocampus. The density of cholinergic neurons in the basal forebrain and nerve fibers in the hippocampus was measured following acetylcholinesterase staining. Results showed that transplanted HAECs survived for at least eight weeks and migrated to the third ventricle without immune rejection. Graft HAECs also expressed the specific stem cell markers oct-4 and nanog. Compared with the control group, HAEC transplantation significantly ameliorated spatial memory deficits in TG mice, as well as increased acetylcholine levels and the number of hippocampal cholinergic neurites. Intracerebroventricular HAEC transplantation improved spatial memory in double-TG mice, and results suggested that increased acetylcholine levels in the hippocampus, released by surviving cholinergic neurites, were responsible for this improvement.

复方丹参注射液对人羊膜上皮细胞水通道蛋白3表达的影响

目的研究复方丹参注射液对人羊膜上皮细胞(human amniotic epithelial cells,hAECs)水通道蛋白3(aquaporin 3,AQP3)表达的影响,探讨复方丹参注射液治疗羊水量过少的机制。方法选取无并发症的8例羊水过少和8名羊水量正常的足月产妇的hAECs进行原代培养,采用RT-PCR和免疫印迹Western blot技术,检测复方丹参注射液不同浓度(0.000、0.001、0.010、0.020、0.060、0.100mg/mL)、不同作用时间(0、6、12、24、48h)作用前后,两组hAECs中AQP3 mRNA和蛋白的表达。结果 (1)羊水过少组hAECs中AQP3的表达较羊水正常组下调(P<0.05)。(2)复方丹参注射液浓度为0.010mg/mL时,两组hAECs中AQP3的表达均达到峰值,与其他浓度作用结果比较,差异有统计学意义(P<0.05)。(3)0.010mg/mL浓度的复方丹参注射液作用12h,两组hAECs中AQP3的表达均达到峰值,与其他作用时间比较,差异有统计学意义(P<0.05)。(4)0.010mg/mL浓度的复方丹参注射液作用12h后,两组hAECs中AQP3的表达均上调,但羊水过少组中AQP3的表达上调较羊水正常组明显(P<0.05)。结论复方丹参注射液可调节hAECs中AQP3的表达,羊水过少时复方丹参注射液对hAECs中AQP3表达的调节作用更加明显。

双室共培养中人羊膜上皮细胞诱导分化为腺泡细胞样细胞研究

目的:研究人羊膜上皮细胞(hAECs)体外诱导分化为唾液腺腺泡细胞样细胞的可能性。方法:分离hAECs并体外培养、鉴定。与8 d龄SD大鼠下颌下腺细胞(SMGCs)在双室共培养系统中共培养。免疫细胞化学、实时荧光定量RT-PCR等方法检测α-淀粉酶及其mRNA的表达。结果:双室共培养1、2周,hAECs中α-淀粉酶mRNA的表达量分别为共培养前的3.38倍及6.6倍。结论:hAECs具有向唾液腺腺泡细胞样细胞分化的能力。

人羊膜上皮干细胞对皮肤增生性瘢痕形成的作用

目的探讨人羊膜上皮细胞的干细胞对皮肤增生性瘢痕形成的作用。方法从足月分娩的人胎盘中剥离羊膜,采用胰蛋白酶消化法分离人羊膜上皮干细胞(human amniotic epithelial stem cells,hAECs),并在体外使用含表皮生长因子的LG-DMEM培养基进行培养,利用免疫荧光染色、流式细胞术和定向诱导等鉴定人羊膜上皮细胞的干细胞特性。构建兔耳全层皮肤缺损模型,左耳创面注射磷酸盐缓冲液(PBS)作为对照组,右耳创面注射羊膜上皮细胞悬液为实验组。结果实验中hAECs表达干细胞标志物SSEA-4和OCT-4,并表达间充质干细胞表面标记物,具有多向分化潜能。1个月后,兔子右耳瘢痕比左耳瘢痕薄,HE染色示:移植干细胞组有效的抑制了增生性瘢痕的形成,纤维化面积较小。在激光共聚焦细胞仪下可发现移植的hAESCs存活。结论人羊膜上皮细胞可以促进创面愈合,减少炎症反应,有效抑制兔耳创面瘢痕的形成。这可能成为瘢痕预防和治疗的新手段和切入点。

人羊膜上皮细胞干细胞特性及向胰岛素分泌细胞分化的研究

目的建立人羊膜上皮细胞(hAECs)的分离及培养方法,探讨其向胰岛素分泌细胞分化的能力。方法观察不同培养方法对hAECs生长倍增时间的影响以优化其培养体系;通过细胞免疫荧光法检测不同培养代数细胞多能干细胞的表面标记;通过体外添加诱导因子的方法,探讨诱导hAECs向胰岛素分泌细胞分化的潜能。结果在10 ng表皮生长因子培养下,hAECs可长期传代。hAECs表达胚胎干细胞表面标记如阶段特异性胚胎抗原4等;采用悬浮培养法诱导分化,可检测到胰岛发育、胰岛功能基因如胰腺十二指肠同源盒、神经源素3、同源异形盒转录因子6、胰岛素、胰高糖素的表达,免疫组化检测细胞可以表达胰岛素,并且随着外界葡萄糖浓度升高,胰岛素分泌显著增加。结论建立了人羊膜上皮细胞的分离和培养方法(不添加动物血清);在体外可诱导分化为胰岛素分泌细胞。