Chika Koike

Isolation and characterization of human amniotic mesenchymal stem cells and their chondrogenic differentiation.

Background Freshly isolated human amniotic mesenchymal (fHAM) cells contain somatic stem cells possessing proliferative ability and pluripotency, including a chondrogenic lineage. However, little is known about the biology of amnion-derived mesenchymal stem cells (MSCs) because fHAM cells can barely survive to expand under culture conditions in vitro for a long time. Methods In this study, we separated fHAM cells and seeded them to isolate MSCs and analyze its character. In addition, suitable chondrogenic growth factor was determined by pellet culture, and their viability under xenogenic environment was examined by transplantation into rabbit knee joints. Results We succeeded in purifying proliferative subpopulations of fHAM cells, which could continue to proliferate more than 50 cumulative population doubling levels, and designated them as HAM&agr; cells. Flow cytometry analysis revealed that they were positive for MSC markers (CD44, CD73, CD90, and CD105) and negative for hematopoietic cell markers (CD34, CD14, and CD45) and major histocompatibility complex class II antigen (human leukocyte antigen–DR). The expression of various stem-cell markers such as OCT3/4, C-MYC, SOX2, NANOG, CD44, SSEA-3, and SSEA-4 was also proved by immunocytochemical staining. Pellet culture using chondrogenic medium supplemented with transforming growth factor &bgr;3, transforming growth factor &bgr;3 plus bone morphogenetic protein (BMP)-2, or BMP-2 implied that supplementation of BMP-2 alone most effectively induced chondrogenesis in vitro. Xenotransplantation of HAM&agr; cells achieved 8-week survival in vivo. Conclusions These results suggest that HAM&agr; cells correspond to MSCs that are highly proliferative and multipotent. Their chondrogenic potential and low immunogenicity indicate that HAM&agr; cells could be an allotransplantable cell resource for cartilage repair.

CD44 and SSEA-4 positive cells in an oral cancer cell line HSC-4 possess cancer stem-like cell characteristics

BACKGROUND Cancer may be derived from cancer stem-like cells (CSCs), which are tumor-initiating cells that have properties similar to those of stem cells. Identification and isolation of CSCs needs to be improved further. MATERIALS AND METHODS CSCs markers were examined in human oral cancer cell lines by flow cytometry. The stem cell properties of subpopulations expressing different markers were assessed further by in vitro sphere formation assays, expression of stemness genes, drug resistance assays, and the ability to form tumors in nude mice. RESULTS We demonstrated that CSCs could be isolated by the cell surface markers CD44 and stage-specific embryonic antigen-4 (SSEA-4). CD44+SSEA-4+ cells exhibited cancer stem-like properties, including extensive self-renewal into the bulk of cancer cells. In vivo xenograft experiments indicated that CD44+SSEA-4+ cells exhibit the highest tumorigenic capacity compared with the remaining subpopulations and parental cells. Double-positive cells for CD44 and SSEA-4 exhibited preferential expression of some stemness genes and were more resistant to the anticancer drugs, cisplatin and 5-fluorouracil (5-FU). In addition, cells expressing CD44 and SSEA-4 were detected in all tumor specimens analyzed, while coexpression of CD44 and SSEA-4 was not detectable in normal oral mucosa. CONCLUSION Our findings suggest that CD44+SSEA-4+ cells exhibit the characteristics of CSCs in oral squamous cell carcinoma and provide a target for the development of more effective therapies.

Clinical application of a hyperdry amniotic membrane on surgical defects of the oral mucosa.

PURPOSE The aim of this study was to evaluate the usefulness of a hyperdry amniotic membrane (AM), a novel preservable human amnion, as a wound-dressing material for surgical defects of the oral mucosa. MATERIALS AND METHODS A hyperdry AM was used in the treatment of 10 patients who had developed secondary defects in the tongue and buccal mucosa after the surgical removal of cancerous or precancerous lesions. The effectiveness of the hyperdry AM was assessed by scoring its operability during the surgical procedure and by the hemostatic status, pain relief, feeding situation, epithelialization, and scar contracture in the postoperative period. Its usefulness was evaluated by considering its effectiveness and safety based on the absence of wound infection and graft rejection. RESULTS The membrane was found to be easy to handle as an oral-dressing material. It adhered well to the bare connective and muscular tissues. One lingual case showed slight postoperative bleeding, which astriction then stopped. No remarkable adverse effects were observed in the process of wound healing. The average score of the patients was 11.2 points (10 to 13 points) in the present evaluation, with 14 being the highest possible score. CONCLUSIONS This study showed the clinical usefulness of the hyperdry AM as an intraoral wound-dressing material. Although the number of cases was small, the results suggested that the hyperdry AM is biologically acceptable to oral wounds and could be a suitable clinical alternative for the repair of the oral mucosa.

Alpha 1 antitrypsin activity is decreased in human amnion in premature rupture of the fetal membranes

Preterm premature rupture of the membranes (PPROM) has been considered to be closely associated with chorioamnionitis. However, the detailed mechanism is not well understood. Alpha 1 antitrypsin (AAT) was reported to decrease in concentration in amniotic fluid obtained from patients with PPROM. However, the origin of AAT in amniotic fluid has not been clarified. In this study, we assessed the expression and localization of AAT in human amnion, as well as its biological activity in cases with PROM. Human amniotic epithelial (hAE) cells expressed AAT. After stimulation with oncostatin M (OSM), interleukin-6 (IL-6) or tumor necrotic factor alpha (TNF alpha), hAE cells increased the expression of AAT, while the expression of MMP9 was reduced by OSM and induced by TNF alpha. Oxidized AAT (inactivated form) was detected in the amnion with PPROM and TPROM, but not in specimens without PROM. Moreover, AAT activity was decreased in amnions from cases with PROM, regardless of gestational age. Thus, the results showed that AAT in the amnion may function as a protective shield at inflammatory sites, and not as it loses it inhibitory activity in cases with PROM, possibly by oxidation, suggesting that its imbalance contributes to PROM.

Hyperdry human amniotic membrane is useful material for tissue engineering: Physical, morphological properties, and safety as the new biological material

Human amniotic membrane (AM) has been used widely as graft biomaterial for a variety of clinical applications. But, there are some persistent problems related to the preparation, storage, and sterilization. To resolve these problems, we developed hyperdry AM (HD-AM) using far-infrared rays, depression of air, and microwaves and then sterilized by γ-ray irradiation. To elucidate the benefit of HD-AM as biological materials, compare with the physical and histological properties of HD-AM with a freeze-dried AM (FD-AM) as typical freeze-dried methods, evaluate the safety of HD-AM in vivo experiment used nude mice, and demonstrate the feasibility of HD-AM transplant in pterygium. The water permeability and the sieving coefficient of HD-AM were significantly lower than that of FD-AM. HD-AM has kept the morphological structure of epithelium and connective tissues. At 18 months after transplanted, single and multilayers of HD-AM in the intraperitoneal cavity was degraded without any infiltrated cells. For clinical treatment, recurrence of pterygium and regrowth of the subconjunctival fibrosis were not observed during the 6-month follow-up periods after the surgery. It was proposed that HD-AM was a safe and effective new biological material for clinical use including treatment for recurrent pterygium.

Intraoral application of hyperdry amniotic membrane to surgically exposed bone surface

Hyperdry amniotic membrane, a novel preservable material derived from the human amnion, has been introduced clinically in ophthalmology and other fields. This membrane is available as a wound dressing material for surgical wounds of the tongue and buccal mucosa but has not been used on wounds of the alveolar mucosa. This paper reports 2 cases in which intraoral alveolar wounds with bone exposure were successfully treated with the use of hyperdry amniotic membrane: a 74-year-old woman with gingival leukoplakia of the edentulous mandible, and a 43-year-old man who underwent vestibuloplasty of the reconstructed mandible. The results indicate that the hyperdry amniotic membrane is a useful dressing material, not only for soft tissue wounds, but also for exposed bone in the oral cavity.

Establishment and characterization of immortalized human amniotic epithelial cells.

Human amniotic epithelial cells (HAEs) have a low immunogenic profile and possess potent immunosuppressive properties. HAEs also have several characteristics similar to stem cells, and they are discarded after parturition. Thus, they could potentially be used in cell therapy with fewer ethical problems. HAEs have a short life, so our aim is to establish and characterize immortalized human amniotic epithelial cells (iHAEs). HAEs were introduced with viral oncogenes E6/E7 and with human telomerase reverse transcriptase (hTERT) to create iHAEs. These iHAEs have proliferated around 200 population doublings (PDs) for at least 12 months. High expression of stem cell markers (Oct 3/4, Nanog, Sox2, Klf4) and epithelial markers (CK5, CK18) were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). These iHAEs were expanded in ultra-low-attachment dishes to form spheroids similarly to epithelial stem/precursor cells. High expression of mesenchymal (CD44, CD73, CD90, CD105) and somatic (CD24, CD29, CD271, Nestin) stem cell markers was detected by flow cytometry. The iHAEs showed adipogenic, osteogenic, neuronal, and cardiac differentiation abilities. In conclusion, the immortalization of HAEs with the characteristics of stem cells has been established, allowing these iHAEs to become useful for cell therapy and regenerative medicine.

Selective isolation of nanog-positive human amniotic mesenchymal cells and differentiation into cardiomyocytes.

Adult cardiomyocytes have little ability to regenerate, thus cardiac regeneration therapy represents a potential method for treating severe heart failure. Human amniotic mesenchymal cells (hAMCs) have the potential to be a useful cell source for cardiac regeneration therapy. We attempted to isolate stem cells from hAMCs and differentiate them into cardiomyocytes. Nanog promoter-Cre plasmid and cytomegalovirus (CMV) promoter-loxP-STOP-loxP-Red-puro(r) plasmid were co-transfected into immortalized hAMCs (iHAMs). Nanog-positive iHAMs were treated with 5-azacytidine (5-aza), trichostatin A (TA), activin A (AA), and bone morphogenetic protein-4 (BMP-4), or co-cultured with murine fetal cardiomyocytes for cardiomyocytes differentiation. Isolated Nanog-positive iHAMs were analyzed by quantitative RT-PCR and immunofluorescent staining before and after differentiation. Expression of Nanog, Oct3/4, Sox2, and Klf4 was significantly higher in Nanog-positive than in Nanog-negative iHAMs. Nanog-positive iHAMs were stained for Nanog and Oct3/4 in the nucleus. Nanog-positive iHAMs treated with 5-aza expressed Nkx2.5, GATA-4, human atrial natriuretic peptide (hANP), cardiac troponin T (cTnT), myocin light chain (Mlc)-2a, Mlc-2v, β-myosin heavy chain (β-MHC), hyperpolarization-activated cyclic nucleotide gated channels (HCN)-4, and inwardly rectifying potassium channels (Kir)-2.1. Although Nanog-positive iHAMs treated with TA, AA, or BMP-4 expressed several cardiac markers, no contraction was observed. Co-cultured Nanog-positive iHAMs with murine fetal cardiomyocytes spontaneously contracted in a synchronized manner and expressed the cardiac markers. In conclusion, Nanog-positive hAMCs with characteristics of stem cells were isolated and differentiated into cardiomyocyte-like cells, suggesting that these isolated hAMCs could be a useful cell source for cardiac regeneration therapy.

Application of 2-octyl-cyanoacrylate for corneal perforation and glaucoma filtering bleb leak.

Background This paper reports on the efficacy of a tissue adhesive, 2-octyl-cyanoacrylate, in the treatment of corneal perforation and glaucoma filtering bleb leak. Methods Two eyes from two patients with corneal perforation or laceration and two eyes from two patients with bleb leak were included. The patients underwent application of 2-octyl-cyanoacrylate onto the perforated or leaking site, and a hydrogel contact lens was applied as a bandage. We also evaluated the in vitro cell toxicity of 2-octyl-cyanoacrylate in HeLa cells. Results The two cases of corneal perforation were repaired within 22 days with one application of the tissue adhesive. The two cases of bleb leak were repaired with 2–4 applications of the tissue adhesive over 134 (range 17–134) days). There were no recurrences or adverse effects during a mean follow-up period of 12.7 months. In vitro testing revealed that 2-octyl-cyanoacrylate was markedly toxic to HeLa cells. Conclusion Four patients with corneal perforation or bleb leak were successfully managed using 2-octyl-cyanoacrylate tissue adhesive. This simple and easy surgical technique may become an alternative therapeutic option for corneal perforation or bleb leak, although several applications of this tissue adhesive may be required. Although 2-octyl-cyanoacrylate was toxic to HeLa cells, no adverse clinical effects were noted using this adhesive.

Effect of Exogenous Oct4 Overexpression on Cardiomyocyte Differentiation of Human Amniotic Mesenchymal Cells

Regenerative therapy is a new strategy for the end-stage heart failure; however, the ideal cell source has not yet been established for this therapy. We expected that the amnion might be an ideal cell source for cardiac regenerative therapy and that the differentiation potency of the human amnion mesenchymal cells (hAMCs) could be improved by overexpression of Oct4, a key factor that maintains the undifferentiated state. A plasmid vector was made by insertion of the Oct4 open reading frame (ORF) under control of a cytomegalovirus (CMV) promoter (pCMV-hOct4) and transfected into hAMCs by electroporation. The optimum induction time was investigated by comparing the quantity of stem cell-specific mRNAs, cardiac-specific mRNAs, and cardiac-specific proteins with time. hAMCs already expressed cardiac-specific proteins such as Nkx2.5 and Connexin43. After pCMV-hOct4 transfection, endogenous Oct4 mRNA and other stem cell markers showed a transient increase. With 5-azacytidine treatment, quantities of the cardiac-specific mRNAs, such as GATA4 and myosin light-chain-2v (Mlc-2v), were increased significantly. After Oct4 overexpression, the highest expression of cardiac-specific mRNAs and stem cell makers was seen at almost the same time. Furthermore, more mature myocardial contraction proteins were observed when hAMCs were induced at specific optimal times after gene transfection. In conclusion, hAMCs were activated to an undifferentiated state by overexpression of Oct4, and their cardiac differentiation potency was improved. Thus, the single-time transfection of the Oct4 expression vector may be a useful strategy for effective cell therapy. The use of cryopreserved hAMCs in cell therapy still requires more investigation.