Human amniotic epithelial cells combined with silk fibroin scaffold in the repair of spinal cord injury

Treatment and functional reconstruction after central nervous system injury is a major medical and social challenge. An increasing number of researchers are attempting to use neural stem cells combined with artificial scaffold materials, such as fibroin, for nerve repair. However, such approaches are challenged by ethical and practical issues. Amniotic tissue, a clinical waste product, is abundant, and amniotic epithe- lial cells are pluripotent, have low immunogenicity, and are not the subject of ethical debate. We hypothesized that amniotic epithelial cells combined with silk fibroin scaffolds would be conducive to the repair of spinal cord injury. To test this, we isolated and cultured amniotic epithelial cells, and constructed complexes of these cells and silk fibroin scaffolds. Implantation of the cell-scaffold complex into a rat model of spinal cord injury resulted in a smaller glial scar in the damaged cord tissue than in model rats that received a blank scaffold, or amniotic epithelial cells alone. In addition to a milder local immunological reaction, the rats showed less inflammatory cell infiltration at the trans- plant site, milder host-versus-graft reaction, and a marked improvement in motor function. These findings confirm that the transplantation of amniotic epithelial ceils combined with silk fibroin scaffold can promote the repair of spinal cord injury. Silk fibroin scaffold can provide a good nerve regeneration microenvironment for amniotic epithelial cells.

In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma

AIM:To reconstruct the lamellar cornea using human amniotic epithelial(HAE) cells and rabbit cornea stroma in vitro using tissue engineering technology.·METHODS:Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells,and the cells were cultured to proliferate.Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets.The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days,then transferred to an air-liquid interface environment to culture for 2weeks.Tissue engineered lamellar cornea(TELC)morphology was observed by Hematoxylin-eosin(HE)staining;its ultrastructure was observed by transmission electron microscopy(TEM) and scanning electron microscopy(SEM);corneal epithelial cell-specific keratin3 and keratin 12 were detected with immunofluorescence microscopy.·RESULTS:HAE cells grew on the rabbit corneal stroma,forming a monolayer after 1-2 days.About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation,a result similar to normal corneal epithelium.Rabbit corneal stromal cells were significantly reduced after one week,then almost completely disappeared after 2 weeks.TEM showed desmosomes between the epithelial cells;hemidesmosomes formed between the epithelial cells and the basement membrane.SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli.The tissue-engineered cornea expressed keratin 3 and keratin 12,as detected by immunofluorescence assay.·CONCLUSION:Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma.

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.

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.

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.

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. ·

Stem cell properties and neural differentiation of sheep amniotic epithelial cells

This study was designed to verify the stem cell properties of sheep amniotic epithelial cells and their capacity for neural differentiation. Immunofluorescence microscopy and reverse transcription-PCR revealed that the sheep amniotic epithelial cells were positive for the embryonic stem cell marker proteins SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, and the totipotency-associated genes Oct-4, Sox-2 and Rex-1, but negative for Nanog. Amniotic epithelial cells expressed β-Ⅲ-tubulin, glial fibrillary acidic protein, nestin and microtubule-associated protein-2 at 28 days after induction with serum-free neurobasal-A medium containing B-27. Thus, sheep amniotic epithelial cells could differentiate into neurons expressing β-Ⅲ-tubulin and microtubule-associated protein-2, and glial-like cells expressing glial fibrillary acidic protein, under specific conditions.

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.

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.

Secretion of nerve growth factor,brain-derived neurotrophic factor,and glial cell-line derived neurotrophic factor in co-culture of four cell types in cerebrospinal fluid-containing medium

The present study co-cultured human embryonic olfactory ensheathJng cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells in complete culture medium- containing cerebrospinal fluid. Enzyme linked immunosorbent assay was used to detect nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor secretion in the supernatant of co-cultured cells. Results showed that the number of all cell types reached a peak at 7-10 days, and the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor peaked at 9 days. Levels of secreted nerve growth factor were four-fold higher than brain-derived neurotrophic factor, which was three-fold higher than glial cell line-derived neurotrophic factor. Increasing concentrations of cerebrospinal fluid （10%, 20% and 30%） in the growth medium caused a decrease of neurotrophic factor secretion Results indicated co-culture of human embryonic olfactory ensheathing cells, human Schwann cells human amniotic epithelial cells and human vascular endothelial cells improved the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. The reduction of cerebrospinal fluid extravasation at the transplant site after spinal cord injury is beneficial for the survival and secretion of neurotrophic factors from transplanted cells.

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.