Glandular and squamous atypia and intraepithelial lesions in atrophic cervicovaginal smears. One institution's experience

OBJECTIVE: To review the cytologic features and follow-up histologic findings in atrophic cervicovaginal smears with the diagnoses of glandular or squamous atypia or intraepithelial lesion. STUDY DESIGN: A total of 228 cases were included in the study. The selection criteria included: age > 48 years and a diagnosis of either atypical glandular cells (AGC) (51 cases), cellular changes suggestive of human papillomavirus (HPV) infection (S/O HPV, 97 cases), low grade squamous intraepithelial lesion (LSIL) (60 cases) or high grade squamous intraepithelial lesion (HSIL) (20 cases). Follow-up biopsy information was available for 103 cases (45%). RESULTS: From the AGC group, 35 (69%) cases had tissue studies; 14 (40%) cases showed glandular lesions; 5 (14%) showed squamous intraepithelial lesion (SIL) and atypical cells. Follow-up information was available for 32 (33%) cases classified as S/O HPV; significant lesions (glandular/squamous) were found in 11 (34%). In the LSIL category, 22 (37%) cases had follow-up; 16 (73%) showed SIL. In the HSIL category, 14 cases (70%) underwent biopsy, and all showed SIL (four LSIL and nine HSIL) or squamous cell carcinoma. CONCLUSION: Even though atrophy-related epithelial changes often pose diagnostic difficulties in the interpretation of postmenopausal smears, application of reproducible and established cytologic criteria in diagnosing SIL and/or glandular lesions can improve diagnostic accuracy and result in selection of patients for follow-up tissue studies.     

Target specificity and cell surface structures involved in the human cytolytic T lymphocyte response to endothelial cells

Two long-term cytolytic T lymphocyte (CTL) lines derived from the peripheral blood lymphocytes (PBL) of a single donor were analyzed for target specificity and involvement of cell surface molecules in CTL-target interactions. One line, AH2, was generated after stimulation with B lymphoblastoid cells. Cytolysis by these cells was restricted to targets expressing the appropriate HLA-A2 specificity and was blocked by mAb recognizing CD2, CD3, CD8, LFA-1, and LFA-3. The second line, AE1, was generated after stimulation with cultured endothelial cells derived from human newborn preputial microvessels. These CTL lysed all human target cells tested, except autologous cells and the Class I negative cell line Daudi. In addition, mAb specific for CD2, CD3, and CD8 did not affect cytolysis. Anti-LFA-1 and -LFA-3 mAb blocked cytolysis of B lymphoblastoid targets but not endothelial targets. These results indicate that some CTL utilize as yet uncharacterized cell surface structures for CTL-target interactions.     

Higher expression and activity of metalloproteinases in human cervical carcinoma cell lines is associated with HPV presence.

Matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in 3 human invasive cervical carcinoma cell lines. Two HPV16-positive cell lines (SiHa and CaSki) and an HPV-negative cell line (C33A) were cultured either onto a type-I collagen gel, Matrigel, or plastic, to recreate their three-dimensional growth environment and evaluate the expression of these genes using quantitative real-time PCR. We also analyzed the gelatinolytic activity of MMP-2 and MMP-9 by zymography. We found that HPV (human papillomavirus)-positive cell lines express higher levels of MMP-2, MT1-MMP, and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with higher pro-MMP-2 activity. Furthermore, Matrigel substrate influences MMP-2 expression in both SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP-2, MT1-MMP, and TIMP-2, and that pro-MMP-2 activity is higher in HPV-positive than in HPV-negative cells.     

Structural characterization of SIL, a gene frequently disrupted in T-cell acute lymphoblastic leukemia.

The SIL (SCL interrupting locus) gene was initially discovered at the site of a genomic rearrangement in a T-cell acute lymphoblastic leukemia cell line. This rearrangement, which occurs in a remarkably site-specific fashion, is present in the leukemic cells of 16 to 26% of patients with T-cell acute lymphoblastic leukemia. We have now cloned a normal SIL cDNA from a cell line which does not carry the rearrangement. The SIL cDNA has a long open reading frame of 1,287 amino acids, with a predicted molecular size of 143 kDa. The predicted protein is not homologous with any previously described protein; however, a potential eukaryotic topoisomerase I active site was identified. Cross-species hybridization using a SIL cDNA probe indicated that the SIL gene was conserved in mammals. A survey of human and murine cell lines and tissues demonstrated SIL mRNA to be ubiquitously expressed, at low levels, in hematopoietic cell lines and tissues. With the exception of 11.5-day-old mouse embryos, SIL mRNA was not detected in nonhematopoietic tissues. The genomic structure of SIL was also analyzed. The gene consists of 18 exons distributed over 70 kb, with the 5' portion of the gene demonstrating alternate exon utilization.     

Atypical glandular cells of undetermined significance on cervical smears. A study with cytohistologic correlation.

OBJECTIVE: The incidence of endocervical adenocarcinoma has increased steadily over the past two decades. Since the Bethesda System was introduced, the diagnosis of atypical glandular cells of undetermined significance (AGUS) has also risen and now accounts for 0.46-1.83% of all cervical (Pap) smears. The purpose of this study was to evaluate the significance of a diagnosis of AGUS using cytohistologic correlation. STUDY DESIGN: A retrospective review of archival material from 1993 through 1996 identified 64 patients who had smears diagnosed as AGUS and had a subsequent surgical biopsy. The smears were reviewed and cytologic features analyzed and correlated with the histologic diagnosis. RESULTS: On biopsy, 3 (5%) of the 64 cases showed endocervical adenocarcinoma in situ (AIS) (1 case with invasive adenocarcinoma also), 14 (22%) had a benign glandular lesion (endocervical polyp, tubal metaplasia, microglandular hyperplasia, reactive changes), 35 (54%) had squamous intraepithelial lesion (SIL) (15 diagnosed on the original smear), and 12 (19%) had no abnormality. Among the cytologic criteria evaluated, feathering (P = .01), palisading (P < .001) and chromatin clearing (P = .002) were shown to have a significant association with the histopathologic diagnosis of AIS/adenocarcinoma. These features were also useful in distinguishing AIS/adenocarcinoma from SIL and benign glandular changes from AIS/adenocarcinoma but not benign/reactive glandular changes from SIL. CONCLUSION: A diagnosis of AGUS correlated with a clinically significant lesion in the majority of cases. Squamous dysplasia (SIL) was the most common lesion identified. The presence of feathering, nuclear palisading and chromatin clearing increased the likelihood of a histologic diagnosis of AIS/adenocarcinoma.     

Estimating the sensitivity of cervical cytology: errors of interpretation and test limitations

The objective of this study was to estimate: (i) the sensitivity of cytologists in recognizing abnormal smears; (ii) the sensitivity of cervical cytology as a method of detecting abnormal smears among those obtained in the presence of cervical intraepithelial neoplasia (CIN). Study subjects were 61 women with a histologically confirmed CIN identified through colpohistological and cytologic screening. For objective (i) new smears were taken from study subjects just before treatment, mixed with routine preparations, interpreted by unaware cytologists and then blindly reviewed by a group of three expert supervisors, who reached a consensus diagnosis. Cytologists classified as positive for squamous intraepithelial lesion (SIL) 30 of the 34 smears judged as positive by supervisors (100% of smears classified as high-grade and 67% of smears classified as low-grade SIL by the supervisors). Our approach, based on creating a set of smears with a high a priori probability of being positive, proved to be an efficient way of estimating errors of interpretation. For objective (ii), smears taken at the moment of diagnosis, just before biopsy, were also reviewed by the same supervisors. These CIN cases were identified among asymptomatic women independently of cytological findings and results are therefore not subject to verification bias. Among the 33 histological CINII/III, four (12%) smears had no atypical cells (three negatives and one unsatisfactory) at review. The same proportion was 26% (four negatives and one unsatisfactory) among the 19 histological CINI. No significant differences in smear content were found between the seven ‘false negatives’ and a sample of ‘true positives’ and ‘true negatives’ for a number of formal adequacy criteria (including presence of endocervical cells). Strong differences were found between positive smears taken just before biopsy and those taken just before treatment (in 11 women the first smear only was positive, while the opposite was never observed), suggesting an effect of punch biopsy in removing lesions.     

Performance of an imaging system vs. manual screening in the detection of squamous intraepithelial lesions of the uterine cervix

OBJECTIVE: To compare the ThinPrep Imaging System (Cytyc Corp., Boxborough Massachusetts, U.S.A) to manual screening in the detection of cervical squamous epithelial lesion (SIL). STUDY DESIGN: A total of 27,525 manually screened ThinPrep Pap tests were compared with 27,725 imaged ThinPrep Pap tests for: (1) diagnostic rates of atypical squamous cells of undetermined significance; atypical squamous cells of undetermined significance, cannot rule out high grade SIL (ASC-H); low grade SIL and high grade SIL (HSIL); (2) ASC/SIL ratio; (3) high-risk HPV positivity for ASC; and (4) biopsy follow-up for ASC-H and HSIL. RESULTS: There were significant increases in the percentage of cytologic diagnoses in all categories with the imager. The ASC/SIL ratios of both groups were comparable. There was a significant decrease in HPV positivity in the imager group of ASC. Biopsy results confirmed a significant increase in the detection of HSIL in both the ASC-H and HSIL groups of the imaged cohort. CONCLUSION: The ThinPrep Imaging System is significantly better than manual screening in the detection of cervical SIL.     

Accuracy of liquid-based Pap tests: comparison of concurrent liquid-based tests and cervical biopsies on 782 women with previously abnormal Pap smears

OBJECTIVE: To evaluate the diagnostic performance of a liquid-based Pap test, the ThinPrep Pap test (TP) (Cytyc Corp., Boxborough, Massachusetts, U.S.A.), by comparing concurrent TP and cervical biopsy results on 782 patients who were referred for colposcopy because of previously abnormal conventional Pap smears (CPs). STUDY DESIGN: The ability of TP diagnoses of atypical cells of undetermined significance (ASC-US) and squamous intraepithelial lesions (SILs) to predict biopsy diagnoses of cervical intraepithelial neoplasia (CIN) was analyzed using chi2 and McNemar tests. RESULTS: The rate of agreement between diagnoses of SIL by TP and CIN by biopsy was 74.7%. ASC-US accounted for 16.0% of TP diagnoses. ASC-US had biopsy diagnoses of CIN 1 in 60% and CIN 2/3 in 12.8% of cases. For TP diagnosis of low grade SIL, biopsy diagnoses of CIN 2/3 were found in 13.5% of cases. For TP diagnoses of ASC-US and higher, the proportions of TP and cervical biopsies in comparable diagnostic categories were statistically significant (p < 0.001), with TP having sensitivity of 89.4% and positive predictive value of 89.7% for the detection of CIN. The false positive rate for TP was 8.1%, but rescreening confirmed the presence of abnormal cells in 51 of 63 (81.0%) cases of ASC-US or higher having negative biopsies. TP had a false negative rate of 8.3% and negative predictive value of 61.3%. Rescreening showed that most (77.6%) of the false negative TP specimens failed to have abnormal cells on the slides. CONCLUSION: For patients having previously detected cervical abnormalities by CP, concurrent TP demonstrated the following: (1) that it has high diagnostic accuracy for SIL, (2) that ASC-US was diagnostically equivalent to LSIL, and (3) that false negative TP for SIL can be attributed primarily to sampling rather than cytotechnologists' screening errors.     

Altered expression of keratin and vimentin in human retinal pigment epithelial cells in vivo and in vitro.

Actively proliferating human retinal pigment epithelial (RPE) cells grown in tissue culture possess keratin-containing intermediate filaments that react with a combination of AE1 and AE3 anti-keratin monoclonal antibodies. Antibody reactivity is lost, however, from RPE cells as the cell population ceases to proliferate when it approaches confluence and attains morphological characteristics more similar to those in vivo. In contrast, clone 8.13 anti-keratin antibody stains all cells in the culture at all stages of the growth cycle and cell densities. These findings were reflected in vivo using retinal pigment epithelium taken directly from the eye. Normal non-proliferating RPE cells bound 8.13 antibody to cytoskeletal structures, as judged by indirect immunofluorescence, but did not bind AE1/AE3 antibodies. However, proliferating dedifferentiated RPE cells from the vitreous humor of patients with proliferative vitreoretinopathy possess filaments that bind both AE1/AE3 and 8.13 antibodies. Thus it appears that structures detected by AE1/AE3 antibodies only occur in actively growing RPE cells in vitro and in vivo. Keratins produced by RPE cells were identified using Western blotting. Species with molecular masses of 54 (keratin 7), 52 (keratin 8), 42 (keratin 18), and 40 (keratin 19) kiloDaltons were the most abundant in proliferating cultured cells, but cells isolated directly from the eye were found to lack keratin 7 and 19. Keratin 19 was, however, observed in proliferating RPE cells from some patients with proliferative vitreoretinopathy. The latter findings explain the differential staining observed with AE1/AE3 antibodies in cells in culture and isolated directly from the eye since these antibodies interact primarily with keratin 19 which is absent from non-proliferating RPE cells. In contrast to the presence of keratin-containing intermediate filaments in human RPE cells in vivo, there are apparently no detectable vimentin-containing cytoskeletal structures. However, all RPE cells cultured in vitro develop filaments composed of vimentin which persist in cells that have reached confluence.     

Targeting of Drugs to Solid Tumors Using Anti-Her2 Immunoliposomes

Abstract

Cancer therapy would clearly benefit from a carrier system capable of intracellular delivery of systemically administered drugs to cancer cells in solid tumors. Sterically stabilized immunoliposomes specific to the cells expressing HER2 protooncogene (anti-HER2 SIL), were designed by conjugating Fab’ fragments of a recombinant humanized anti-HER2 MAb to the distal termini of poly(ethylene glycol) chains on the surface of unilamellar liposomes (size 90–100 nm) of phosphatidylcholine, cholesterol, and poly (ethylene glycol)—derivatized phosphatidylethanolamine. Anti-HER2 SIL avidly and specifically bound to cultured HER2-overexpressing cancer cells (8,000–23,000 vesicles per cell) and became endocytosed (k_e = 0.022–0.033 min.^?1) via the coated pit pathway. Anti-HER2 SIL showed prolonged circulation lifetime in rats (blood MRT approx. 24 hours) and significantly increased antitumor activity of encapsulated doxorubicin against HER2-overexpressing human breast cancer xenografts in nude mice. Although the accumulation of anti-HER2 SIL in HER2-overexpressing tumor xenografts was not increased over that of non-targeted sterically stabilized liposomes (SL), microscopic examination revealed abundance of anti-HER2 SIL in the interstitial spaces, as well as within the cytoplasm of cancer cells, while identical liposomes lacking anti-HER2 Fab’ were located predominantly within tumor-resident macrophages. Anti-HER2 SIL, a targeted vehicle capable of in vivo intracellular delivery of substances to HER2-overexpressing solid cancers, enhances the potential for tumor targeting and opens new avenues for better treatment of cancer.     

Humoral immune response to melanoma-associated membrane antigen and fetal brain antigen demonstrated by indirect membrane immunofluorescence. I

Summary A melanoma-associated membrane antigen and a fetal brain antigen were identified on the surface of a human melanoma cell line by indirect membrane immunofluorescence techniques. The target melanoma cells were grown in gamma globulin-depleted human serum. Sera from melanoma patients were used as the source of antimelanoma antibodies. To remove alloantibodies, the allogeneic sera were preabsorbed with cultured lymphoblastoid cells derived from the peripheral lymphocytes of the donor of the target cell line. To further define the antigen responsible for antibody activity, sequential absorption tests were performed with fetal brain cells, cultured sarcomas, and breast carcinomas. Some antibody activity was removed by fetal brain tissues. Further absorption with fetal brain or the cultured sarcoma or breast carcinoma did not remove additional activity. However, antibody activity was completely removed by either cultured or biopsy-derived melanoma cells. A serum autochthonous to the target cell line was also tested. The antibody titer of the serum was completely removed by absorption with either autochthonous biopsied tumor or an allogeneic melanoma cell line, but not with the normal tissues. Thus it appeared that sera from melanoma patients contained antibody to both a melanoma-associated membrane antigen and a fetal brain antigen.     

兔角膜缘干细胞体外分离、培养和生物学鉴定的实验研究

通过体外培养兔角膜缘干细胞,观察其生物学特性,建立兔角膜缘干细胞的体外培养方法。方法0．25%胰蛋白酶消化角膜缘组织,用含15%胎牛血清的DMEM和F12(1:1)的培养液(DF)对兔角膜缘干细胞进行体外培养,形态学观察,培养的细胞早期使用AEl/AE3、晚期使用AE5角蛋白特异的单克隆抗体)作细胞免疫化学鉴定。结果:原代培养细胞48h后开始贴壁,部分细胞由圆形变为卵圆形或长梭形;10～14d形成单层,细胞呈圆形、卵圆形,类角膜上皮细胞;细胞传到第5代左右开始出现老化状态;免疫细胞化学染色:培养的细胞早期AEl/AE3呈阳性而少部分细胞AE5呈阳性,培养的细胞晚期AE5呈阳性。结论:本实验初步建立了一套兔角膜缘干细胞的体外培养方法。     

A lymphoblastoid cell line with high phagocytic activity established from peripheral blood of a patient with chronic myelocytic leukemia in blast crisis

A human hematopoietic cell line (K-23-M) was established from a patient with chronic myelocytic leukemia in blast crisis. Morphologically, the cultured cells were lymphoblastoid cells that produced IgA and were Epstein-Barr viral nuclear antigen positive. But they showed high phagocytic activity to glutaraldehyde-treated sheep red cells and had properties of a monocyte or macrophage that included surface Fc receptors, alpha-naphthyl butyrate esterase positivity blocked by NaF, migration in soft agar and the ability to attach to a glass surface. Lysozyme secretion was absent, and chromosomes were diploid and Ph1 negative. This cell line is unique in that it has strong phagocytic activity. Its existence shows that lymphoblastoid cell line may be a more important cell line for the study of human hematopoietic cells than previously has been believed.     

Body cavity fluid can induce epithelial and mesothelial differentiation from CD34 positive peripheral blood stem cells in vitro

H. Kataoka, T. K. Kobayashi, S. Amano, E. Yamada, M. Ishida, R. Kushima and H. Okabe Body cavity fluid can induce epithelial and mesothelial differentiation from CD34 positive peripheral blood stem cells in vitro Objective: Primary culture of CD34 positive stem cells collected from human peripheral blood was performed with and without supplementation with concentrated ascitic fluid; morphological and immunocytochemical pictures of cultured cells were taken chronologically and compared. Methods: CD34‐positive stem cells collected from peripheral blood were cultured for 1, 24 and 48 hours. Concentrated ascitic fluid was added to the plates for the 24‐and 48‐hour cultures. For immunocytochemical studies, CD34, AE1/AE3, Ber‐Ep4 (EA), EMA, EGFR, CD31, CA125 and D2‐40 monoclonal antibodies were used. Results: After culture, small round cells with naked nuclei began to enlarge and to exhibit various changes in the cytoplasm and nucleus. Supplementation with concentrated body cavity fluid enhanced these changes. CD34‐positive cells with small round cell features were detected 1 hour after culture and these had no epithelial or mesothelial markers. After 24 hours, CD34‐positive cells had disappeared and cells weakly positive for EGFR, EMA, CA125 and D2‐40 were detected. Cells with strong and moderate positive reactions for EGFR, AE1/AE3, EA, EMA, D2‐40 and CA125 were detected after 48 hours. Supplementation with concentrated body cavity fluid increased the intensity and number of positive cells for these markers compared with the control group. The positive reaction, not only for the epithelial markers such as EGFR and AE1/AE3, but also for mesothelial markers such as CA125 and D2‐40, was found to be increased in small numbers of cells in direct proportion to the duration of the primary culture of the peripheral blood cells. CD31, characteristically expressed in endothelial cells, was negative in the cultured cells. Conclusion: Supplementation of peripheral blood CD34‐positive stem cells with body cavity fluid in vitro enhanced their differentiation toward cells of an epithelial or mesothelial phenotype, concomitant with loss of immunoreactivity for CD34. It is assumed that the routine cytological observation of cells obtained from body cavity fluid might cause possible cytomorphological and immunophenotypical changes due to the action of the growth factors contained in the body cavity fluid.     

Outcomes of women with atypical squamous cells of undetermined significance and high-risk human papillomavirus DNA

OBJECTIVE: To study the risk of high-grade squamous intraepithelial lesion (HSIL) and cervical intraepithelial neoplasia (CIN) 2 or 3 in women with human papillomavirus (HPV)-positive atypical squamous cells of undetermined significance (ASCUS) cytology over a 2-year period using the 2001 Bethesda System and ThinPrep Paps. STUDY DESIGN: In 2002, 846 patients with ThinPrep cervical cytology having an ASCUS interpretation and positive for high-risk HPV DNA were identified. A cohort of 514 (60.8%) patients with follow-up by repeat cytology, cervical biopsy or both was included in the study. Patient age was 12-81 years, with a median of 25 years. RESULTS: There were 291 women (56.6%) with negative status by cytology, HPV testing or biopsy with a median interval of 8.5 months, and an additional 174 patients (33.9%) had persistent ASCUS, positive HPV DNA or low-grade SIL/CIN 1. Finally, 49 patients (9.5%) had CIN 2 or 3, with a median interval of 8.5 months. CONCLUSION: Our study suggests that HSIL or CIN 2 or 3 will be detected in 1 in 10 women with HPV-positive index ASCUS cervical cytology at initial colposcopy or within a 2-year follow-up period.     

Eliminating the diagnosis atypical squamous cells of undetermined significance: impact on the accuracy of the Papanicolaou test

OBJECTIVE: To assess eliminating the diagnosis "atypical squamous cells of undetermined significance" (ASCUS) from the Bethesda System for Reporting Cervical/Vaginal Cytological Diagnoses and analyze its impact on the sensitivity and positive predictive value of Pap smears. STUDY DESIGN: A total of 166 previously diagnosed ASCUS cases with follow-up biopsy results available were prospectively downgraded to within normal limits/benign cellular changes or upgraded to specific squamous intraepithelial lesions (SILs) or the malignant category. These review cytodiagnoses were compared with the histologic outcome. The impact on the sensitivity and positive predictive value of Pap smears was also assessed. RESULTS: Though there was a decrease in the sensitivity of the Pap smear from 100% to 76.3% for SIL overall and from 100% to 80% for high grade SIL (HSIL) alone, there was an improvement in the positive predictive value of diagnosing SIL from 46% to 85% and from 6% to 15% for HSIL alone. CONCLUSION: The ASCUS diagnosis can be minimized to a great extent, if not eliminated completely. The "ASCUS-favor reactive" group can be eliminated, while the diagnoses "ASCUS favor SIL" and "ASCUS-not otherwise specified" should be used sparingly.     

Establishment and characterization of a novel uterine carcinosarcoma cell line,TU-ECS-1, with mutations of <Emphasis Type="Italic">TP53</Emphasis> and <Emphasis Type="Italic">KRAS</Emphasis>

A new human uterine carcinosarcoma (UCS) cell line, TU-ECS-1, was established and characterized. The morphological appearance of the cultured cells was an insular of epithelial-like cells arranged in the form of a jigsaw puzzle and mesenchymal-like cells with a spindle-shaped or fibroblast-like morphology. A relatively high proliferation rate was observed with a doubling time of 18.2 h. The chromosome number ranged from 44 to 49 and had an extra chromosome 12 (trisomy 12). The respective half-maximal inhibitory concentrations of cisplatin, paclitaxel, and doxorubicin were 2.9 µM, 154 nM, and 219 ng/mL, respectively. Mutational analysis revealed that TU-ECS-1 cells have mutations of TP53 in exons 4, 6, and 8 and of KRAS at codon 12 (G12D) in exon 2, which is a mutation hot spot on this gene. Western blot analysis showed that p53 protein was overexpressed in TU-ECS-1 cells. Immunostaining of the cultured cells and in vivo tumors showed that the TU-ECS-1 cells and xenografts were positive for epithelial marker cytokeratin AE1/3 and mesenchymal marker vimentin. These results suggested that TU-ECS-1 cells might have both epithelial and mesenchymal characteristics. This cell line may be useful to study the carcinogenesis of UCS and contribute to the development of novel treatment strategies.     

Amnion Epithelial Cells of Buffalo (Bubalus bubalis) Term Placenta Expressed Embryonic Stem Cells Markers and Differentiated into Cells of Neurogenic Lineage In Vitro

Aim of the present study was the isolation, culture, and characterization of amniotic membrane-derived epithelial cells (AE) from term placenta collected postpartum in buffalo. We found that cultured cells were of polygonal in shape, resistance to trypsin digestion and expressed cytokeratin-18 indicating that they were of epithelial origin. These cells have negative expression of mesenchymal stem cell markers (CD29, CD44, and CD105) and positive for pluripotency marker (OCT4) genes indicated that cultured cells were not contaminated with mesenchymal stem cells. Immunofluorescence staining with pluripotent stem cell surface markers, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81 indicated that these cells may retain pluripotent stem cell characteristics even after long period of differentiation. Differentiation potential of these cells was determined by their potential to differentiate into cells of neurogenic lineages using retinoic acid. In conclusion, we demonstrate that AE cells expressed pluripotent stem cell markers and have propensity to differentiate into cells of neurogenic lineage upon directed differentiation in vitro.