Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line

Purpose

Mouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC propagation and derivation. Such xeno-free systems may prove essential for the establishment of clinical grade human ESC lines suitable for therapeutic application.

Methods

A novel line of human endometrial cells were seeded in a 6-well dish. Filter inserts containing mouse ESCs were placed on these wells and passaged 2–3 times per week. Inner cell masses derived from mouse blastocysts were also cultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells.

Results

ESC colonies retained their stem cell morphology and attributes for over 120 days in culture and 44 passages to date. Inner cell mass derived ESC cultures were maintained in a pluripotent state for 45 days, through 6 passages with retention of all stem cell characteristics. The stem cell colonies expressed stem cell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology within the colonies and evidence of ESC differentiation.

Conclusions

Human feeder layers offer a simple path away from the use of MEF feeder cells or MEF conditioned medium for ESC culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent contamination of stem cell preparations with feeder cells during passaging.     

人子宫内膜腺癌细胞原代培养方法及细胞系鉴定

目的优化人子宫内膜癌细胞原代培养方法,建立体外培养细胞系。方法将辽宁医学院附属第一医院2009年10月至11月收治的3例子宫内膜癌患者手术切除的子宫内膜腺癌组织采用酶消化法制成单细胞悬液,用含血清DMEM/F-12培养基进行原代和传代培养,通过自然纯化、冻存及低血清培养法纯化腺上皮癌细胞。在倒置显微镜下观察细胞形态,行HE染色,免疫荧光染色对细胞进行鉴定,绘制生长曲线。结果 2例子宫内膜腺癌体外成功建系,传代数扩展至50～70代。细胞形态符合上皮癌细胞系特征,细胞角蛋白表达阳性,细胞生长曲线为S形。结论应用酶消化法原代培养子宫内膜腺癌细胞,并采用自然纯化、冻存及低血清培养法纯化腺上皮癌细胞简单、易行、高效。此2株细胞系可望作为子宫内膜腺癌研究的实验模型。     

Ultrastructure of endometrial epithelial cells in a three-dimensional cell culture system for human implantation studies

Purpose A three-dimensional cell culture system imitating normal uterine endometrium has previously been established. To what degree do cultured epithelial cells retain their morphological characteristics as compared to in vivomaterial obtained simultaneously from the same tissue donor.Results We found a high degree of similarity between the in vivoand in vitrosituations. The present culture system furthermore imitates the day-to-day morphology of the cycle.Conclusions This indicates, that a correct timing of the biopsy tissue is important for future human implantation studies.Presented at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, April 3–7, 1995, Vienna, Austria.     

Development and integration of an extended embryo culture program.

OBJECTIVE: To study and evaluate a sequential, extended embryo culture system. DESIGN: Prospective study. SETTING: University-affiliated IVF clinic. PATIENT(S): All couples who were treated between October 1997 and July 1998. INTERVENTION(S): A standard human tubal fluid plus 10% serum substitute supplement (SSS) culture medium was used. The embryos were transferred to extended culture medium (S2 or G2) on day 3. MAIN OUTCOME MEASURE(S): Blastocyst formation and implantation and pregnancy rates. RESULT(S): Forty percent of the 20 donated cryopreserved embryos progressed to the blastocyst stage by day 6. Clinically, 7 (5.6%) of the 125 cycles did not result in a transfer. Blastocyst formation rates ranged from 33%-63% in the five study groups. Implantation rates ranged from 15%-52% and pregnancy rates ranged from 37%-75%. CONCLUSION(S): Extended culture to day 5 or 6 results in acceptable blastocyst formation rates, implantation rates, and pregnancy rates.     

Molecular genetic characterization of tamoxifen-associated endometrial cancer

OBJECTIVES: The non-steroidal, selective estrogen receptor modulator tamoxifen is currently the most extensively used hormonal agent for the prevention and treatment of estrogen receptor-positive breast cancer. Epidemiologic studies and clinical trials have shown an increased risk of endometrial cancer with tamoxifen exposure; however, few studies have examined these tumors on a molecular level. We sought to elucidate the molecular genetic alterations found in tamoxifen-associated endometrial cancer. METHODS: Twenty-nine breast cancer patients with a history of tamoxifen use who subsequently developed endometrial cancer were retrospectively identified and matched for endometrial histologic subtype and grade to 29 endometrial cancers from breast cancer patients never exposed to tamoxifen. Endometrial tumor tissue was microdissected and genomic DNA extracted for each case. Direct DNA sequencing of the most commonly mutated genes in sporadic endometrial cancer, PTEN, K-RAS, TP53, and CTNNB1, was performed in addition to microsatellite instability (MI) studies. Fisher's Exact Test was utilized for statistical analyses. RESULTS: Of 29 tamoxifen-associated cancers, 10 (34.5%) contained PTEN mutations compared to 13 (44.8%) of the non-tamoxifen-associated cancers (P = 0.59). All PTEN mutations were found in tumors with endometrioid histology, reflecting what is seen in sporadic endometrial cancers. Mutations of K-RAS, TP53, and microsatellite instability were present in similar frequencies between the two breast cancer groups, and moreover, these were similar to mutational frequencies found in sporadic endometrial cancer. CONCLUSION: Tamoxifen and non-tamoxifen-associated endometrial carcinomas arising in women with breast cancer contain similar genetic alterations to those of sporadic endometrial carcinomas.     

Serum and tissue angiogenin in patients with endometrial hyperplasia

ObjectiveTo evaluate angiogenin levels in both tissue and serum of patients with endometrial hyperplasia with and without atypia.MethodsSixty women were classified according to the histopathological findings of endometrium into three groups. The control group consisted of 20 women with normal non-hyper plastic endometrium. The second group included 20 women diagnosed as complex endometrial hyperplasia without atypia. The third group included 20 women diagnosed as complex endometrial hyperplasia with atypia. Serum and tissue angiogenin were measured by enzyme immunoassay (EIA) technique and confirmed in tissues with Western Blotting (WB) technique.ResultsThere was a statistically significant increase in serum and tissue angiogenin levels of endometrial hyperplasia groups compared to those of control group (P<0.001). Serum and tissue angiogenin levels were with a statistically significant higher (P<0.001) in group III compared to group II. The sensitivity of serum angiogenin to detect the potential possibility of endometrial hyperplasia with atypia in endometrial hyperplasia patients was 100%, specificity 85%, positive predictive value 86.9%, negative predictive value 100%, positive likelihood ratio 6.6%, negative likelihood ratio 0% and accuracy 91.7%.ConclusionElevated levels of serum angiogenin in endometrial hyperplasia could assist in determining which patients are at high risk for atypical change requiring aggressive treatment.     

Detection of Chlamydia trachomatis in formalin-fixed endometrial tissue

Formalin-fixed tissue biopsies from 29 women with the histological diagnosis of severe endometritis were examined for chlamydial antigen by fluorescein-conjugated monoclonal chlamydial antibodies (SYVA) technique; 15 (52%) were positive. In nine patients also samples for isolation of Chlamydia trachomatis by cell-culture technique were obtained peroperatively; seven were culture-positive. In all these patients fluorescent chlamydial antigen was detected, whereas the two culture-negative patients had no such antigen. The technique presented makes it possible to examine series of formalin-fixed endometrical biopsies to evaluate the significance of C. trachomatis being etiologic agent in endometritis in females.