Inhibition of retinal ischemia-reperfusion injury in rats by inhalation of low-concentration hydrogen gas.

PURPOSE: To investigate the inhibitory effect of hydrogen gas inhalation on retinal ischemia reperfusion (I/R) injury using a rat model. METHODS: Six-week-old male Sprague-Dawley rats were used. A 27G needle connected by a tube to a saline bottle placed 200 cm above the eye was inserted into the anterior eye chamber to create a rat retinal I/R model. In the ischemia-plus-hydrogen-gas group (H2( +) group), the ischemia time was set to 90 min, and 1.8% hydrogen was added to the air delivered by the anesthesia mask simultaneously with the start of ischemia. In the non-hydrogen-treatment ischemia group (H2( -) group), I/R injury was created similarly, but only air was inhaled. ERGs were measured; after removal of the eyes, the retina was examined for histological, immunostaining, and molecular biological analyses. RESULTS: The mean thickness of the inner retinal layer in the H2( +) group was 107.2 ± 16.0 µm (n = 5), significantly greater than that in the H2( -) group (60.8 ± 6.7 µm). Immunostaining for Iba1 in the H2( -) group showed increased numbers of microglia and microglial infiltration into the subretinal space, while there was no increase in microglia in the H2( +) group. B-wave amplitudes in the H2( +) group were significantly higher than in the H2( -) group. In the membrane antibody array, levels of interleukin-6, monocyte chemotactic protein 1, and tumor necrosis factor alpha were significantly lower in the H2( +) group than in the H2( -) group. CONCLUSION: Inhalation of 1.8% hydrogen gas inhibited the induction of inflammation, morphological/structural changes, and glial cell increase caused by retinal I/R injury.

Experiences and care needs of post-discharge patients who underwent cardiac surgery: A qualitative study.

AIM: This study clarifies the physical, psychological, and social forms of distress in, and care needs of, cardiac surgery patients, including optimal times for supporting them in their post-hospital discharge daily lives. METHODS: Semi-structured qualitative interviews were conducted. Participants included 12 adults (11 male and one female, mean age = 66.5 years) who had undergone cardiac surgery, experienced intensive care, and received outpatient care at the first post-discharge visit (around 2 ~ 3 weeks after discharge), around 3 months after discharge, and between 3 months and 1 year after discharge. Verbatim transcripts were analyzed based on similarities and differences for codes based on assessment items, and subcategories and categories were generated. RESULTS: After surgery, patients experienced physical, psychological, and social distress. First, they experienced physical pain shortly after discharge. Moreover, as they recovered at home, a gap between their sense of their recovery and the perceptions of those around them about their recovery often persisted, which led to psychological and social distress. Patients gained a sense of safety through "assurance of physical recovery" and security through "shared subjective distress." CONCLUSIONS: Post-cardiac surgery patients seek reassurance and safety by sharing experiences owing to daily life distress. Our findings could help provide better support to meet the care needs of such patients.

Hyperdry Human Amniotic Membrane as a Protective Dressing for Open Wounds With Exposed Bowel in Mice.

INTRODUCTION: An enteroatmospheric fistula forms when the exposed bowel is perforated with chronic enteric fistula formation. Currently, there is no established preventative method for this condition. Hyperdry (HD) amniotic membrane (AM) can promote early granulation tissue formation on the exposed viscera and is suitable for dressing intractable wounds as it possesses anti-inflammatory, antibacterial, and immunomodulatory properties. This study investigated whether HD-AM promotes early formation of blood vessel-containing granulation tissue for enteroatmospheric fistula treatment. METHODS: An experimental animal model of an open wound with exposed bowel was developed. A 15 × 20 mm wound was prepared on the abdomen of Institute of Cancer Research mice, and the HD-AM was placed. The mice were assigned to one of the following groups: HD-AM group, in which the stromal layer of the HD-AM was placed in contact with the exposed bowel; HD-AM UD group, in which the epithelial layer of the HD-AM was placed in contact with the exposed bowel; and the HD-AM (-) or control group, in which the HD-AM was not used. RESULTS: On postoperative days 7 and 14, granulation tissue thickness significantly increased in the HD-AM and HD-AM UD groups compared with that in the HD-AM (-) group. Macrophages accumulated in the HD-AM epithelium only in the HD-AM group. During HD-AM contact, a subset of invading macrophages switched from M1 to M2 phenotype. CONCLUSIONS: HD-AM is a practical wound dressing with its scaffolding function, regulation of TGF ß-1 and C-X-C motif chemokine 5 (CXCL-5), and ability to induce M1-to-M2 macrophage conversion.

[Autoimmune hemolytic anemia complicated with acute kidney injury and tubulopathy due to hemoglobin casts].

A 62-year-old male patient was admitted for close monitoring of anemia (hemoglobin level, 8.2 g/dl). Hemolytic anemia was observed; however, the direct antiglobulin test (DAT) result (standard tube method) was negative. Nevertheless, autoimmune hemolytic anemia (AIHA) was still suspected; therefore, a DAT (Colum method) and quantifying levels of red-blood-cell bound immunoglobulin G were performed, resulting in a definite diagnosis of warm AIHA. The patient also had an acute kidney injury (AKI) from the time of admission, which was poorly improved by supplemental fluids therapy alone. Therefore, renal biopsy was performed. Renal biopsy revealed acute tubular injury due to hemoglobin columns, and a diagnosed AKI caused by hemolysis due to AIHA. Following the definitive diagnosis of AIHA, the patient was treated with prednisolone, and after approximately 2 weeks, the anemia and nephropathy completely improved, which is maintained to this day. We report this case as a rare case of AKI induced by hemolysis of AIHA and a successful case of renal salvage by early administration of steroid.

Clinical Application of Hyperdry Amniotic Membrane in Cleft Palate Repair.

OBJECTIVE: To examine the safety and efficacy of hyperdry amniotic membrane (HDAM) for wound closure after palatoplasty in cleft palate patients. METHODS: HDAMs were prepared by washing and drying under infrared rays and microwaves at temperatures less than 60°C using a hyperdrying device. A total of 16 cleft palate patients (8 males, 8 females), aged 1 to 3 years (mean age 1 year 9 months), received one-stage pushback palatoplasty. The remaining raw wound after surgery was covered by an HDAM and a plastic cover plate. The cover plate was removed 1 week after surgery and parameters including temperature, feeding, allergic reactions, postoperative bleeding, re-epithelialization, wound dehiscence, and infection were monitored during the follow-up period of 31.2 months. RESULTS: All patients could adequately ingest at 5 days postoperation and after removal of the cover plate. None of the patients had a persistent fever or allergic reactions. Ingestion was feasible immediately in all patients, and no postoperative bleeding was observed during ingestion. No secondary hemorrhages were observed during follow-up. No postoperative wound dehiscence on the midline of the palate was observed. No infections were observed after the removal of the cover plate. No patients suffered from severe scar formation or contracture of the wound in the follow-up period. Hemorrhage, undue epithelialization, and scar contracture did not occur in any patient. The mean evaluation score was 7.75 points. CONCLUSION: HDAM can be used safely and effectively for wound closure following palatoplasty in cleft palate infants. Future studies testing the safety of patient's own amnion for palatoplasty, are required.

Distribution of amniotic stem cells in human term amnion membrane.

Amnion membrane studies related to miscarriage have been conducted in the field of obstetrics and gynecology. However, the distribution of stem cells within the amnion and the differences in the properties of each type of stem cells are still not well understood. We address this gap in knowledge in the present study where we morphologically classified the amnion membrane, and we clarified the distribution of stem cells here to identify functionally different amniotic membrane-derived stem cells. The amnion can be divided into a site that is continuous with the umbilical cord (region A), a site that adheres to the placenta (region B), and a site that is located opposite the placenta (region C). We found that human amnion epithelial stem cells (HAECs) that strongly express stem cell markers were abundant in area A. HAEC not only expressesed stem cell-specific surface markers TRA-1-60, Tra-1-81, SSEA4, SSEA3, but was also OCT-3/4 positive and had alkaline phosphatase activity. Human amniotic mesenchymal stem cells expressed KLF-A, OCTA, Oct3/4, c-MYC and Sox2 which is transcription factor. Especially, in regions A and B they have expressed CD73, and the higher expression of BCRP which is drug excretion transporter protein than the other parts. These data suggest that different types of stem cells may have existed in different area. The understanding the relation with characteristics of the stem cells in each area and function would allow for the efficient harvest of suitable HAE and HAM stem cells as using tool for regenerative medicine.

Effect of hyperdry amniotic membrane in preventing tendon adhesion in a rabbit model.

BACKGROUND: No anti-adhesive materials are currently in clinical use for orthopaedic surgery. We developed a hyperdry amniotic membrane (HD-AM) for easy storage and transplantation as amniotic membrane. The purpose of this study was to examine the application of HD-AM to reduce peritendinous adhesions without impairing tendon healing. METHODS: We randomly divided 3 digits (2nd, 3rd, and 4th digits) from each rabbit into three groups: a tendon repair group; a tendon repair with HD-AM group (HD-AM group); and a control group (cast only). The effects of HD-AM on peritendinous adhesions and tendon healing were examined using microscopic, histological, and mechanical analyses in a rabbit flexor digitorum profundus tendon model. RESULTS: Adhesions on macroscopic evaluation of the tendon repair site were significantly smaller in the HD-AM group than in the tendon repair group. Little adhesion formation or foreign body reactions were seen by on histologic evaluation in the HD-AM group. Range of motion following tendon repair was significantly better in the HD-AM group than in the tendon repair group. Maximal tensile strength required to pull the tendon from the site of adhesion was significantly smaller in the HD-AM group than in the tendon repair group. As for tendon repair site, no significant difference was seen between the tendon repair and HD-AM groups. CONCLUSIONS: HD-AM prevented peritendinous adhesion macroscopically, pathologically, and mechanically without impairing the sutured tendon. HD-AM has already been clinically applied in neurosurgery, ophthalmology, and otolaryngology, and clinical application as an anti-adhesive materials may be achieved in the future.

Identification of Chemo and Radio-Resistant Sub-Population of Stem Cells in Human Cervical Cancer HeLa Cells.

BACKGROUND: Cervical cancer ranks the second female malignancy after breast cancer. Cancer stem cells (CSCs) are hard to be eradicated, so can recur. We aim to isolate and characterize CSCs from HeLa cells. METHODS: These cells express clusters of differentiation (CDs), 44 and 24, to be sorted by fluorescence-activated cell sorting (FACS). RESULTS: CD44+CD24+ cells showed potential to form spheres, tumorigenicity, stemness genes and higher resistance to cisplatin, X-ray. CONCLUSION: CD44+CD24+ HeLa cells hold characteristics of CSCs, in vitro, in vivo studies, suggesting that targeting may lead to screening of new anti-cancer therapies.

Preconditioning of Adipose-Derived Mesenchymal Stem-Like Cells with Eugenol Potentiates Their Migration and Proliferation In Vitro and Therapeutic Abilities in Rat Hepatic Fibrosis.

Mesenchymal stem cells (MSCs) have considerable therapeutic abilities in various disorders, including hepatic fibrosis. They may be affected with different culture conditions. This study investigated, on molecular basics, the effect of pretreatment with eugenol on the characteristics of adipose tissue-derived MSCs (ASCs) in vitro and the implication of eugenol preconditioning on the in vivo therapeutic abilities of ASCs against CCl4-induced hepatic fibrosis in rats. The effect of eugenol on ASCs was assessed using viability, scratch migration and sphere formation assays. Expressions of genes and proteins were estimated by immunofluorescence or qRT-PCR. For the in vivo investigations, rats were divided into four groups: the normal control group, fibrotic (CCl4) group, CCl4+ASCs group and CCl4 + eugenol-preconditioned ASCs (CCl4+E-ASCs) group. Eugenol affected the viability of ASCs in a concentration- and time-dependent manner. Eugenol improved their self-renewal, proliferation and migration abilities and significantly increased their expression of c-Met, reduced expression 1 (Rex1), octamer-binding transcription factor 4 (Oct4) and nanog genes. Furthermore, E-ASCs showed more of a homing ability than ASCs and improved the serum levels of ALT, AST, albumin, total bilirubin and hyaluronic acid more efficient than ASCs in treating CCl4-induced hepatic fibrosis, which was confirmed with histopathology. More interestingly, compared to the CCl4+ASCs group, CCl4+E-ASCs group showed a lower expression of inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), cluster of differentiation 163 (CD163) and tumor necrosis factor-α (TNF-α) genes and higher expression of matrix metalloproteinase (MMP)-9 and MMP-13 genes. This study, for the first time, revealed that eugenol significantly improved the self-renewal, migration and proliferation characteristics of ASCs, in vitro. In addition, we demonstrated that eugenol-preconditioning significantly enhanced the therapeutic abilities of the injected ASCs against CCl4-induced hepatic fibrosis.

AT-MSCs Antifibrotic Activity is Improved by Eugenol through Modulation of TGF-ß/Smad Signaling Pathway in Rats.

For hepatic failure, stem cell transplantation has been chosen as an alternative therapy, especially for mesenchymal stem cells (MSCs). The aim of this study was to investigate the effect of eugenol (EUG) on the in vivo antifibrotic activity of adipose tissue-derived MSCs (AT-MSCs) and the underlying mechanism. After characterization of MSCs, rats were divided into five groups, Group 1 (normal control), Group 2 (CCl4), Group 3 (CCl4 + AT-MSCs), Group 4 (CCl4 + EUG) and Group 5 (CCl4 + AT-MSCs + EUG). Biochemical and histopathological investigations were performed. Furthermore, expression of type 1 collagen, α-SMA, TGF-ß1, Smad3 and P-Smad3 was estimated. Compared to the single treatment with AT-MSCs, the combination treatment of the fibrotic rats with AT-MSCs and EUG significantly improved the plasma fibrinogen concentration, IL-10 level and proliferating cell nuclear antigen expression, and also significantly decreased the serum levels of liver enzymes, IL-6, IL-1ß, TNF-α, type III collagen, hyaluronic acid, hydroxyproline and the TGF-ß growth factor. Furthermore, the combination treatment significantly decreased the hepatic expression of fibrotic markers genes (Type 1 collagen and α-SMA) and proteins (α-SMA, TGF-ß1 and phospho-Smad3) more than the treatment with AT-MSCs alone. We demonstrated that the combination treatment with EUG and AT-MSCs strongly inhibited the advancement of CCl4-induced hepatic fibrosis, compared with AT-MSCs alone, through TGF-ß/Smad pathway inhibition. This approach is completely novel, so more investigations are necessary to improve our perception of the underlying molecular mechanisms accountable for the effects of EUG on the antifibrotic potential of AT-MSCs.

Periostin expression and characters of human adipose tissue-derived mesenchymal stromal cells were aberrantly affected by in vitro cultivation.

BACKGROUND: Human adipose tissue-derived mesenchymal stromal cells (AD-MSCs) have been under focus in regenerative medicine since their discovery as a suitable source of MSCs. AD-MSCs are heterogeneous cells and exhibit variations in population doubling time, morphology and proliferative capacity. This study investigated if human AD-MSCs are developing, during in vitro long-term cultivation, in an unwanted or aberrant way. METHODS: This study monitored AD-MSCs during their in vitro culture till the tenth passage investigating proliferation kinetics, DNA index and surface markers expression. Also, periostin gene expression was examined. RESULTS: The proliferation capacity and colony forming unit were decreased after passage 6 and the population doubling time was increased. Flow cytometric analysis revealed that newly cultivated population strongly expressed MSCs markers, furthermore, reduction of CD105 expression appeared in passage 5 onwards, the later was associated with significant increase in expression of CD34 (a hematopoietic cell marker). Also, reduction of CD73 and CD90 expression was observed from passage 8. Furthermore, during the first six passages, periostin expression was significantly unchanged, with significant upregulation in late passages. CONCLUSIONS: Long-term cultivation of human AD-MSCs changed their characters in an aberrant way and the first four passages might be the most appropriate passages for therapy. More investigation and understanding of these variations are needed to help in standardizing the expansion of MSCs-based therapies.

Differential response of immortalized human amnion mesenchymal and epithelial cells against oxidative stress.

Cells are equipped with various antioxidant defense factors to antagonize insults from reactive oxygen species (ROS), thus the antioxidant capacity has been characterized by a variety of cellular responses during the pathophysiological processes. Amniotic cells have been extensively applied in clinical practice for burn treatment, corneal repair, and tissue regeneration. However, the antioxidative properties of amniotic cells have not yet been fully understood. Therefore, the current study was aimed to observe the response of amniotic cells against ROS stimuli, and to investigate the underlying molecular mechanisms. The immortalized human amniotic mesenchymal cells (iHAMs) and immortalized human amniotic epithelial cells (iHAEs) were used. The human skin fibroblast (HSF) was used as a control cell line. Changes in intracellular ROS generation, cell viability, and cellular morphology were investigated to reveal the response of amniotic cells against oxidative stresses induced by x-rays and hydrogen peroxide. In addition, expression of apoptosis-related proteins and response to antioxidative stress was also examined. The intracellular ROS level and cell apoptosis in iHAMs was remarkably increased. iHAEs showed relatively high resistance to ROS stimulation, which can be attributed to the high SOD2 expression and up-regulation of Nrf2, HO-1 after x-rays exposure. In contrast, iHAMs were found sensitive to oxidative damage. Expression of caspase-3, caspase-8 and BAX was increased, whereas down-regulation of Bcl-xL, Nrf2, HO-1, and TrxR-1. Taken together, findings have highlighted the characterization of response of amniotic derived epithelial and mesenchymal cells to oxidative stress. In physiological processes, iHAMs may play an important role to maintain the homeostasis of the pregnancy environment. However, under oxidative stimulations, iHAEs provides protection against oxidative damage in amnion tissue.

Isolation of Stem-Like Cancer Cells in Primary Endometrial Cancer Using Cell Surface Markers CD133 and CXCR4.

Endometrial cancer (EC) is the most common familiar gynecologic malignant tumor identified in the female reproductive system and has been increasing yearly. In this study, we will identify the surface markers and stem cell markers related with cancer stem cells (CSCs) of EC. Tissue samples were obtained from endometrial cancer patients during surgical procedures. Single cells were isolated from the tissues for culturing, transfection into nude mice, and histopathology analysis. RT-PCR demonstrated that the cultured cells strongly expressed stemness-related genes, such as c-Myc, Sox-2, Nanog, Oct 4A, ABCG2, BMI-1, CK-18, Nestin and ß-actin. The expression of surface markers CD24, CD133, CD47, CD29, CD44, CXCR4, SSEA3 and SSEA4, CD24, and CD133 and chemokine markers such as CXCR4 were measured by flow cytometry. Then the double percentage of CD133+CXCR4+ cells constituted 7.2% and 9.3% in EC cells originated from two different patients, respectively. The CD133+CXCR4+ primary endometrial cancer cells grew faster, exhibited high expression of mRNA of stemness-related genes, produced more spheres, and had higher clonogenic ability than other subpopulations. They are also more resistant to anti-cancer drugs than other subpopulations. These findings indicate that CD133+CXCR4+ cells may possess some characteristics of CSCs in primary endometrial cancer. These cell surface markers may be useful for the development of drugs against CSC molecular targets or as a predictive marker for poor prognosis in primary endometrial cancer.

Quantification of autonomic nervous activity by heart rate variability and approximate entropy in high ultrafiltration rate during hemodialysis.

BACKGROUND: Few studies have focused on the imbalance of the autonomic nervous system in ultrafiltration rate (UFR) subjects without blood pressure variation during maintenance hemodialysis (HD), although the role of autonomic nervous system activation during HD has been proposed to be an important factor for the maintenance of blood pressure. METHODS: Variations over time in autonomic nervous activity due to differences in UFR were evaluated by measuring heart rate variability (HRV) and approximate entropy (ApEn) in 35 HD patients without blood pressure variations during HD session. The subjects were divided into 3 groups, those with UFR <10 ml/h/kg; ≥10 ml/h/kg but ≤15 ml/h/kg; and >15 ml/h/kg, and Holter ECG was recorded continuously during HD session using frequency analysis of RR intervals. High frequency (HF) and low frequency (LF) spectral components are found to be representative of the parasympathetic nervous system and sympathovagal balance, respectively, with the ratio of LF to HF of HRV providing a measure of sympathetic nervous system. RESULTS: In subjects with UFR >15 ml/h/kg, HF components were significantly lower, and LF/HF and ApEn values were significantly higher, in the latter half of an HD session than before starting HD. CONCLUSION: Removing water from these subjects would promote sustained sympathetic nervous overactivity. These findings indicate that the UFR during HD needs to be set at ≤15 ml/h/kg.

A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair: In Vitro and In Vivo Studies.

OBJECTIVE: Extracellular matrix (ECM) derived from human amniotic mesenchymal cells (HAMs) has various biological activities. In this study, we developed a novel HAM-derived ECM-coated polylactic-co-glycolic acid (ECM-PLGA) scaffold, examined its property on mesenchymal cells, and investigated its potential as a cell-free scaffold for cartilage repair. MATERIALS AND METHODS: ECM-PLGA scaffolds were developed by inoculating HAM on a PLGA. After decellularization by irradiation, accumulated ECM was examined. Exogenous cell growth and differentiation of rat mesenchymal stem cells (MSCs) on the ECM-PLGA were analyzed in vitro by cell attachment/proliferation assay and reverse transcription-polymerase chain reaction. The cell-free ECM-PLGA scaffolds were implanted into osteochondral defects in the trochlear groove of rat knees. After 4, 12, or 24 weeks, the animals were sacrificed and the harvested tissues were examined histologically. RESULTS: The ECM-PLGA contained ECM that mimicked natural amniotic stroma that contains type I collagen, fibronectin, hyaluronic acid, and chondroitin sulfates. The ECM-PLGA showed excellent properties of cell attachment and proliferation. MSCs inoculated on the ECM-PLGA scaffold showed accelerated type II collagen mRNA expression after 3 weeks in culture. The ECM-PLGA implanted into an osteochondral defect in rat knees induced gradual tissue regeneration and resulted in hyaline cartilage repair, which was better than that in the empty control group. CONCLUSION: These in vitro and in vivo experiments show that the cell-free scaffold composed of HAM-derived ECM and PLGA provides a favorable growth environment for MSCs and facilitates the cartilage repair process. The ECM-PLGA may become a "ready-made" biomaterial for cartilage repair therapy.

Construction and characterization of human oral mucosa equivalent using hyper-dry amniotic membrane as a matrix.

BACKGROUND: Human amniotic membrane(HAM) as a graft material has been used in various fields. Hyper-dry amniotic membrane (HD-AM) is a novel dried amniotic membrane that is easy to handle and can be preserved at room temperature without time limitation. The purpose of this study was to investigate the useful properties of HD-AM in reconstruction of the oral mucosa. METHODS: Human oral keratinocytes were isolated and seeded on HD-AM in serum-free culture system. Oral mucosa equivalent (OME) was developed and transplanted onto full-thickness wound on athymic mice. The wound healing was analyzed and the OME both before and after transplantation was analyzed with hematoxylin-eosin staining and immunohistochemical staining for Cytokines 10 (CK10), Cytokines 16 (CK16), and Ivolucrin (IVL). RESULTS: Oral keratinocytes spread and proliferated well on HD-AM. Two weeks after air-lifting, OME had formed with good differentiation and morphology. We confirmed immunohistochemically that the expression of CK10 was positive in all suprabasal layers, as was CK16 in the upper layers, while IVL was present in all cell layers. Three weeks after transplantation to athymic mice, the newly generated tissue had survived well with the smallest contraction. The epithelial cells of newly generated tissue expressed CK10 throughout in all suprabasal layers, IVL was mainly in the granular layer, and CK16 positive cells were observed in all spinous layer and granular layer but were not expressed in the mouse skin, all of which were similar to native gingival mucosa. CONCLUSIONS: The OME with HD-AM as a matrix revealed a good morphology and stable wound healing. This study demonstrates that HD-AM is a useful and feasible biomaterial for oral mucosa reconstruction.

Use of hyperdry amniotic membrane in operations for cleft palate: a study in rats.

The growth of maxillary bone and the development of dentition are often impaired in patients who have had pushback operations for repair of a cleft palate. There has been considerable discussion about the most suitable technique or material used in such repairs to resolve the problem. Hyperdry amniotic membrane, a new preservable material derived from human amnion, has recently been introduced in several procedures. We have evaluated its use during pushback surgery in animal studies to try to correct the inhibition of growth and development of the maxilla. Mucosal defects were created in 3-week-old rats, and then covered with hyperdry amniotic membrane or not. Healing was assessed by histological and morphological examination at 1 week and 7 weeks postoperatively. In the group treated with hyperdry amniotic membrane, submucosal tissue was reconstructed successfully during the early postoperative period. Lateral palatal growth was not inhibited as much, and medial inclination of the teeth was less, after a period of growth using this material. The results suggest that hyperdry amniotic membrane is a suitable new dressing material for use in the treatment of cleft palate.

Human Amnion-Derived Stem Cells Have Immunosuppressive Properties on NK Cells and Monocytes.

Human amnion-derived cells are considered to be a promising alternative cell source for their potential clinical use in tissue engineering and regenerative medicine because of their proliferation and differentiation ability. The cells can easily be obtained from human amnion, offering a potential source without medical intervention. It has been proven that human amnion-derived cells express immunosuppressive factors CD59 and HLA-G, implying that they may have an immunosuppressive function. To assess the immunosuppressive activity, we investigated the effect of human amnion-derived cells on NK cell and monocyte function. Amnion-derived cells inhibited the cytotoxicity of NK cells to K562 cells. The inhibition depended on the NK/amnion-derived cell ratio. The inhibition of NK cytotoxicity was recovered by continuous culturing without amnion-derived cells. The inhibition of NK cytotoxicity was related to the downregulation of the expression of the activated NK receptors and the production of IFN-γ, as well as the upregulation of the expression of IL-10 and PGE2 in human amnion-derived cells. The addition of antibody to IL-10 or PGE2 inhibitor tended to increase NK cytotoxicity. IL-10 and PGE2 might be involved in the immunosuppressive activity of amniotic cells toward NK cells. Amniotic cells also suppressed the activity of cytokine production in monocytes analyzed with TNF-α and IL-6. These data suggested that amniotic cells have immunosuppressive activity.

Characterization of amniotic stem cells.

The amnion membrane is developed from embryo-derived cells, and amniotic cells have been shown to exhibit multidifferentiation potential. These cells represent a desirable source for stem cells for a variety of reasons. However, to date very few molecular analyses of amnion-derived cells have been reported, and efficient markers for isolating the stem cells remain unclear. This paper assesses the characterization of amnion-derived cells as stem cells by examining stemness marker expressions for amnion-derived epithelial cells and mesenchymal cells by flow cytometry, immunocytochemistry, and quantitative PCR. Flow cytometry revealed that amnion epithelial cells expressed CD133, CD 271, and TRA-1-60, whereas mecenchymal cells expressed CD44, CD73, CD90, and CD105. Immunohistochemistry showed that both cells expressed the stemness markers Oct3/4, Sox2, Klf4, and SSEA4. Stemness genes' expression in amnion epithelial cells, mesenchymal cells, fibroblast, bone marrow-derived mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) was compared by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Amnion-derived epithelial cells and mesenchymal cells expressed Oct3/4, Nanog, and Klf4 more than bone marrow-derived MSCs. The sorted TRA1-60-positive cells expressed Oct3/4, Nanog, and Klf4 more than unsorted cells or TRA1-60-negative cells. TRA1-60 can be a marker for isolating amnion epithelial stem cells.