Investigating the regenerative effects of folic acid on human amniotic epithelial stem cells and amniotic pore culture technique (APCT) model in vitro using an integrated pharmacological-bioinformatic approach.

INTRODUCTION: Disruption of fetal membranes before the onset of labor is referred to as premature rupture of membranes (PROM). Lack of maternal folic acid (FA) supplementation reportedly leads to PROM. However, there is a lack of information on the location of FA receptors in the amniotic tissue. Additionally, the regulatory role and potential molecular targets of FA in PROM in vitro have rarely been investigated. METHODS: The three FA receptors (folate receptor α isoform [FRα], transporter of reduced folate [RFC], and proton-coupled folate transporter [PCFT]) in human amniotic epithelial stem cells (hAESCs) and amniotic tissue were localized using immunohistochemistry and immunocytochemistry staining. Effect and mechanism analyses of FA were performed in hAESCs and amniotic pore culture technique (APCT) models. An integrated pharmacological-bioinformatics approach was utilized to explore the potential targets of FA for the treatment of PROM. RESULTS: The three FA receptors were widely expressed in human amniotic tissue, especially in the hAESC cytoplasm. FA stimulated the amnion regeneration in the in vitro APCT model. This mimics the PROM status, in which cystathionine-ß-synthase, an FA metabolite enzyme, may play an important role. The top ten hub targets (STAT1, mTOR, PIK3R1, PTPN11, PDGFRB, ABL1, CXCR4, NFKB1, HDAC1, and HDAC2) of FA for preventing PROM were identified using an integrated pharmacological-bioinformatic approach. DISCUSSION: FRα, RFC, and PCFT are widely expressed in human amniotic tissue and hAESCs. FA aids the healing of ruptured membrane.

Effect of Human Amniotic Epithelial Stem Cell Transplantation on Preterm Premature Rupture of Fetal Membrane Using the Amniotic Pore Culture Technique in vitro.

OBJECTIVES: The objective of this study was to evaluate the efficacy of cell therapy using human amniotic epithelial stem cells (hAESCs) for the treatment of premature rupture of membranes (PROM) in vitro. DESIGN: Using the amniotic pore culture technique (APCT), we mimicked the environment of PROM in vitro, thus enabling the observation of the healing process of hAESC-treated amniotic membranes. MATERIALS: Amniotic membrane samples were collected from placentas of pregnant women who underwent elective cesarean sections. APCT model and isolated hAESCs were used in this study. All patients who participated in this study provided their written informed consent prior to the commencement of the study. SETTINGS: To create the APCT model in vitro, isolated amniotic membranes were punched to create 5 mm diameter circles and re-punched to form a 1-mm pore at the center. Membranes were cultured in α-minimal essential medium, and the hAESCs were collected and cultured as well. Subsequently, the APCT models were divided into two groups: hAESC treated and control. METHODS: Within the culture period, pore sizes were calculated to evaluate the degree of tissue regeneration in both groups. We then evaluated the histology, cell density, and epithelial thickness of the regenerated tissues. Statistical analyses were performed using SPSS software ver. 20.0 (IBM, Armonk, NY, USA) with repeated-measures one-way analysis of variance or paired samples t test. The significance level was set at p < 0.05. RESULTS: As per the evaluation of the APCT model in vitro, the pore size in the hAESC-treated group reduced by 62.2% on day 6 (62.2 ± 0.19, n = 24), whereas in the control group, it shrank by only 36.8% (p < 0.05) (36.8 ± 0.19, n = 24). Furthermore, the epithelial thickness in the amniotic epithelial stem cell-treated group (10.08 ± 1.26 µm, n = 8) was significantly higher than that in the control group (5.87 ± 0.94 µm, n = 8). Cell density in the regenerated tissue in the amniotic epithelial stem cell-treated group (57 ± 2.77, n = 8) was significantly higher than that in the control group (49 ± 2.23, n = 8). LIMITATIONS: In this study, we did not explore the molecular mechanisms by which hAESCs participate in membrane healing in the APCT model. Although our results showed a significant difference, this difference was not too obvious. Therefore, further research on the mechanisms of hAESCs is needed, with more amniotic tissues and APCT samples being tested. CONCLUSIONS: We developed an APCT model to investigate the PROM conditions in vitro. By implanting donor hAESCs in the pores of the APCT model, we observed that hAESCs seeding accelerated pore healing in vitro. Thus, hAESCs may be a valuable source of cells for cell therapies in regenerative medicine.

PD-L1 Expression Correlated with Clinicopathological Factors and Akt/Stat3 Pathway in Oral SCC.

Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule that inhibits immune responses. The physiological and prognostic role of the PD-L1 signaling pathway in the oral maxillofacial region is unclear. This study aimed to investigate the role of PD-L1 in the progression of oral squamous cell carcinoma (OSCC). Furthermore, clinicopathological factors related to PD-L1 expression were examined in patients with OSCC through immunohistochemistry (IHC) of tissue sections and through an in vitro study in OSCC cells. The medical records, radiographic findings, and mortality referrals of 81 patients obtained from the National Statistical Office were reviewed. IHC was performed on tissue specimens of these patients to determine the expression levels of PD-L1, which showed significant statistical differences based on age, tumor size, TNM stage, cervical lymph node metastasis, and locoregional recurrence. Patients with a high PD-L1 expression had significantly poorer survival rates. Multivariate analysis using the Cox proportional model confirmed the high relative risk ratio for high PD-L1 expression, TNM stage, and neck node metastasis, all of which were significantly associated with a poor prognosis in patients with OSCC. The in vitro study showed that SAS and YD38 cells transfected with PD-L1 siRNA had significantly increased apoptosis, reduced proliferative capacity, and tumorigenicity.

Spontaneous healing of human amnion in the premature rupture of membrane model.

INTRODUCTION: This study aimed to explore the spontaneous healing of ruptured fetal membranes experimentally in the prelabor rupture of membrane model using the amnion pore culture technique. METHODS: The human amniotic membrane was separated from the post-delivery term placenta in women with normal pregnancies who delivered by scheduled unlabored cesarean section and stained immunohistochemically with primary antibodies against SSEA-4, OCT-3/4, and TRA-1-60. The characteristics of the cultured amniotic epithelial cells were examined by fluorescence-activated cell sorting analysis. Amniotic membranes with perforations that were 1, 2, and 3 mm in diameter were cultured in αMEM containing 10% heat-inactivated FBS, 1% penicillin-streptomycin, and 10 ng/mL EGF at 37 °C in a humidified incubator with 5% CO2. Next, the pore sizes were calculated to evaluate the healing process. RESULTS: The amniotic membrane stained positive for CD49d and pluripotent stem cell markers such as SSEA-4, TRA 1-60, and OCT-4 in the stromal and epithelial cell layers. In the flow cytometry analyses, the extracted amniotic epithelial stem cells were observed to express indicator markers for stem cells such as SSEA-4, OCT-4, SOX-2, and Nanog. In the amnion pore culture technique model, the 1-mm pores healed completely, whereas the 2- and 3-mm pores did not heal substantially. DISCUSSION: The amnion pore culture technique was useful for demonstrating the natural healing process of the human amniotic membrane. Stem cells in the human amnion might facilitate the resealing of small pores in the amniotic membrane, as observed in this model.

Mandibular intraosseous squamous cell carcinoma lesion associated with odontogenic keratocyst: a case report.

Squamous cell carcinoma (SCC) is the most common malignant tumor in the oral cavity, and it accounts for about 90% of all oral cancers. Several risk factors for oral SCC have been identified; however, SCC associated with odontogenic keratocysts have rarely been reported. The present study describes the case of a 36-year-old man with SCC of the right ramus of the mandible, which was initially diagnosed as a benign odontogenic cyst. He underwent enucleation at another hospital followed by segmental mandibulectomy and fibular free flap reconstruction at our institution. In this case, we introduce a patient with oral cancer associated with odontogenic cyst on the mandible and report a satisfactory outcome with wide resection and immediate free flap reconstruction.

The impact factors on 5-year survival rate in patients operated with oral cancer.

OBJECTIVES: The purpose of this study is to analyze clinical impact factors on the survival rate, and to acquire basic clinical data for the diagnosis of oral cancer, for a determination of the treatment plan with long-term survival in oral cancer patients. MATERIALS AND METHODS: Through a retrospective review of the medical records, the factors for long-term survival rate were analyzed. Thirty-seven patients, among patient database with oral cancer treated in the Department of Oral and Maxillofacial Surgery at Pusan National University Hospital within a period from March 1998 to March 2008, were selected within the study criteria and were followed-up for more than 5 years. The analyzed factors were gender, age, drinking, smoking, primary tumor site, type of cancer, TNM stage, recurrence of affected region, and metastasis of cervical lymph node. The 5-year survival rate on the impact factors was calculated statistically using the Kaplan-Meier method. RESULTS: By classification of clinical TNM at the 1st visit, there were 11 (29.7%) cases for stage I, 11 (29.7%) cases for stage II, 3 (8.1%) cases for stage III, and 12 (32.5%) cases for stage IV. The 5-year survival rate of total oral cancer patients after the operation were 75.7%, pathological TNM stage related 5-year survival rate were as follows: stage I 90.0%, stage II 81.8%, stage III 100% and stage IV 45.5%; in which the survival rate difference by each stage was significantly observed. The recurrence of cervical lymph node was the significant impact factor for the survival rate, because only 30.0% the survival rate in recurrent cases existed. During the follow-up, there were 15 (40.5%) patients with confirmed recurrence, and the 5-year survival rate of these patients was decreased as 46.7%. CONCLUSION: The classification of clinical and pathological TNM stage, local recurrence after surgery, and metastasis of cervical lymph node after surgery were analyzed as the 3 most significant factors.

Human feeder cells can support the undifferentiated growth of human and mouse embryonic stem cells using their own basic fibroblast growth factors.

In the culture system using human feeder cells, the mechanism through which these cells support undifferentiated growth of embryonic stem cells (ESCs) has not been well investigated. Here, we explored the mechanisms of 3 kinds of human feeder cells, including human placental cells from the chorionic plate, human bone marrow stromal cells, and human foreskin fibroblasts. First, we determined that undifferentiated growth of 2 kinds each of human (H1 and HSF6) and mouse (D3 and CE3) ESCs was possible in all human feeder cell types tested (human placental cells, human bone marrow stromal cells, and human foreskin fibroblasts), without the need for exogenous cytokine supplementation including basic fibroblast growth factor (bFGF) and leukemia inhibitory factor. We then prepared their corresponding endogenous bFGF-knockout feeders using siRNA and tried to maintain human and mouse ESCs in their undifferentiated state; however, neither human nor mouse ESCs could be maintained in bFGF-knockout human feeder cells. The expressions of stemness markers such as Oct-4 and Nanog were significantly decreased in the bFGF-knockout group compared with those in the controls, and differentiation had already occurred, despite the undifferentiated morphologic appearance of the ESCs. In conclusion, human feeder cells are able to support the undifferentiated growth of human and mouse ESCs via bFGF synthesis. Further, a bFGF-dependent pathway might be crucial for maintaining the undifferentiated characteristics of mouse and human ESCs.

The efficacy of human placenta as a source of the universal feeder in human and mouse pluripotent stem cell culture.

The use of a mouse embryonic fibroblast (MEF) feeder for culture of embryonic stem cells (ESCs) is a widely accepted method, regardless of the ESCs' origin and type. In this study, we performed the undifferentiated propagation of human ES cell lines (hESCs, H1, and HSF6) and mouse ES cell lines (mESCs, D3, and CE3), which were previously maintained on an MEF feeder, using human placenta-derived fibroblast-like cell (HPC) feeders originated from chorionic villi of women who had undergone therapeutic abortion due to known maternal disease that is aggravated by pregnancy. Moreover, we tried to introduce the HPC feeder for the establishment of inducible pluripotent stem cells (iPSCs) from human placental mesenchymal stem cells (MSCs). On the HPC feeder we were able to propagate ESCs and iPSCs colonies as an undifferentiated state up to the 50th passage and 20th passage, respectively. Maintenance of undifferentiated ESCs was identified by the expression of ALP, SSEA-1, SSEA-4, TRA-81, TRA-60, Oct-4, Nanog, or Rex-1. Also, addition of leukemia inhibitory factor was not required for undifferentiated propagation of mESCs on the HPC feeder. The efficiency and expression of three germ layer markers of embryoid bodies (EBs) from ESCs were satisfactory in both the MEF and HPC group. EBs formed from iPSCs were scant, and differentiation to the three germ layers was identifiable by reverst transcription-polymerase chain reactio (RT-PCR) only in the HPC group. In conclusion, the HPC feeder can efficiently support the undifferentiated propagation of hESCs, mESCs, and iPSCs, suggesting that human placenta may be a useful source of universal feeder cells for hESC, mESC, and iPSC culture.

Undifferentiated propagation of the human embryonic stem cell lines, H1 and HSF6, on human placenta-derived feeder cells without basic fibroblast growth factor supplementation.

In order for human embryonic stem cells (hESCs) to be cultured on mouse embryonic fibroblast (MEFs) feeder cells, continuous basic fibroblast growth factor (bFGF) supplementation is required. However, the role of bFGF in a culture system using human-derived feeder cells has not been evaluated until now. In this study, we propagated the widely used hESC lines, H1 and HSF6, on human placenta-derived feeder cells (HPCs) without exogenous bFGF supplementation, and were able to propagate hESCs on HPC feeders up to 50 passages. The absence of bFGF in culture media did not interrupt the undifferentiated propagation and the expression of pluripotent stem cell markers ALP, SSEA-4, TRA-60, Oct-4, Nanog, and Rex-1, as well as the formation of embryoid bodies (EBs) and their differentiation potential. In contrast, hESCs cocultured with MEF feeders could not propagate and form EBs without exogenous bFGF supplementation. Expression of bFGF and the activation of the ERK1/2-c-Fos/c-Jun pathway, which is known as the signaling pathway of bFGF, were identifiable not only in hESCs cultured in bFGF-containing media regardless of feeder cell type, but also in hESCs cocultured with HPC feeder cells in media without bFGF. These findings may support the hypothesis that HPC feeder cells enhance endogenous bFGF production and activation of the ERK1/2-c-Fos/c-Jun pathway, which suggests that HPCs have an additional advantage in their hESC propagation compared with MEF.

Human placenta-derived feeders support prolonged undifferentiated propagation of a human embryonic stem cell line, SNUhES3: comparison with human bone marrow-derived feeders.

Co-culture of human embryonic stem (ES) cells on mouse fibroblast feeders is the commonly used method for in vitro expansion of human ES cells in an undifferentiated state. However, it has potential risks of pathogen transmission from animals; thus, human cell-derived feeders have been employed to minimize this problem. In this study, we compared human placenta-derived feeders with bone marrow to demonstrate its effectiveness as feeders for in vitro long-term culture of human ES cells. We cultured a human ES cell line, SNUhES3, on human placenta-derived mesenchymal stem cell feeders and compared their culture efficiency with human bone marrow-derived feeders and control group (mouse fibroblast feeders, STO). The mean number of human ES cell colonies was 166 +/- 35 in the placenta feeders; this was significantly higher than bone marrow-derived feeders (87 +/- 16, p < 0.05). We could propagate the culture of SNUhES3 on the placenta feeders past the 50th week similar to control group. During the culture, the maintenance of undifferentiated state of SNUhES3 was demonstrated by the expression of SSEA-4, TRA-1-81, TRA-1-60, and Oct-4. However, we failed to propagate the culture of human ES cells on the human bone marrow-derived feeders past the 5th week. The efficiency of embryoid body formation was similar between placenta and control group, indicating the preservation of differentiation ability. Thus, placenta-derived feeders are more efficient for the long-term in vitro culture of human ES cells than bone marrow-derived feeders suggesting the possible role of placenta as a source for human cell-derived feeders.