Modulation of glioma cell invasion and motility by adenoviral gene transfer of PAI-1

A number of studies have emphasized the role of PAI-1 as an important regulator of tumor cell invasion and metastasis. The hallmark of primary tumors of the central nervous system and glioblastomas in particular is the diffuse invasion into the normal brain tissue. Since PAI-1 is expressed in such tumors, we studied the effect of adenoviral-mediated transfer of the PAI-1 gene in regulating the in vitro invasiveness of D54Mg glioma cells into Matrigel, and into fetal rat brain aggregates. Treatment of D54Mg cells with 50 MOI (multiplicity of infection) of the replication defective vector AdCMVPAI-1 increased PAI-1 expression 23-fold compared to control vectors, and the invasion through Matrigel was reduced by 67%. The motility of the cells was reduced by 58% compared to controls (indicating that inhibition of motility was the principal effect of PAI-1 in these cells). The ability of D54Mg tumor spheroids to invade fetal rat brain aggregates was not reduced by the PAI-1 gene transfer. The results show that overexpression of PAI-1 can inhibit glioma cell motility and invasion through extracellular matrix (ECM) components, like laminin and collagen, but does not inhibit tumor cell invasion in a three-dimensional invasion assay, simulating normal brain tissue having a different ECM and interstitial composition. The different results obtained in the two invasion assays reflect the complex biological effects of the uPA/PAI-1 system, and questions a simplistic view of PAI-1 as an inhibitor of brain tumor invasion. This revised version was published online in July 2006 with corrections to the Cover Date.     

骨髓间充质干细胞和软骨细胞共培养复合同种异体脱钙骨基质修复关节软骨全层缺损

目的:探讨自体骨髓间充质干细胞(BMSCs)与软骨细胞共培养复合同种异体脱钙骨基质(DBM)修复关节软骨缺损的可行性,评价修复效果,为优化种子细胞源提供依据.方法:取浓度为5×109/L的第二代BMSCs和软骨细胞,按2:1比例混匀共培养作为种子细胞.DBM与共培养细胞复合植入修复为实验组(A组),单纯材料DBM组(B组)和不处理组(C组)作为实验对照组.移植8和16 wk后经大体观察、组织学评分和免疫组化染色评价缺损修复.结果:共培养的软骨细胞基质合成丰富,细胞增殖快,共培养5～7 d两种细胞比例达1:1以上.A组缺损修复组织呈软骨样,表面光滑平坦,与周围软骨整合的软骨细胞更为成熟.B组和C组的修复组织呈纤维组织.组织学评分表明A组优于B,C两组,差异具有统计学意义(P＜0.01),B组与C组差异无统计学意义(P＞0.05).免疫组化染色显示A组修复组织的细胞为透明软骨样细胞,柱状排列,Ⅱ型胶原染色阳性,与周围软骨及软骨下骨整合良好.结论:自体BMSCs与软骨细胞共培养,BMSCs能增强软骨细胞的增殖,促进软骨细胞基质合成,缩短软骨细胞培养时间和减少传代次数,可节省大量的软骨细胞,与DBM复合后能有效修复关节软骨缺损.     

Improvement in blastocyst hatching of mouse embryos cocultured with human placental cells

Purpose Although the hatching of embryos is an important phenomenon, the mechanism of hatching remains controversial. Therefore, we attempted to develop a new coculture system with human placental cells to investigate the hatching of mouse embryos.Results In our new system there was no difference in development from the two-cell stage to blastocysts between embryos cultured with a T6 medium and embryos cocultured with human placental cells at 1 × 10⁵, 5 × 10⁵, and 1 × 10⁶ cells/ml. However, the hatching-rate cell number increased significantly in embryos cocultured with placental cells compared to embryos cultured without placental cells. [³H]Thymidine uptake did not show any significant difference from the beginning of in vitro culture to the hatching stage between the coculture group and the control group. Nevertheless, the [³H]uridine uptake was significantly different in the two groups, measuring 2167 ± 532 cpm/10 embryos in the coculture group and 804 ± 86 cpm/10 embryos in the control group at 114 hr after human chorionic gonadotropin injection (P < 0.01).Conclusion These results therefore seem to indicate that the hatching of blastocysts depends on the protein synthesis of the embryos and not on DNA duplications.     

Endometrial secretory proteins enhance early embryo development

Purpose To evaluate the role of endometrial stromal cells and their secretory proteins in early embryo development, two-celled CB6F1 mouse embryos were cultured alone or cocultured with human endometrial stromal cells in various culture conditions.Results The percentage of embryo blastocyst formation, hatching, and outgrowth was significantly greater in (1) coculture with endometrial stromal cells than in a cell-free control when both coculture and control were carried out in protein-free medium or in RPMI 1640 plus 10% fetal calf serum; (2) coculture with hormone (i.e., progesterone plus relaxin)-treated cells than in coculture with hormone-nontreated cells; and (3) media supplemented with isolated endometrial secretory proteins than in media supplemented with BSA (0.35%). Embryo development was not found to be significantly different in coculture and in media supplemented with endometrial secretory protein.Conclusion Our data provides credence to the theory that endometrial stromal cells enhance embryo development by secreting specific proteins that are beneficial to embryo growth in vitro.Presented at the 48th Annual Meeting of the American Fertility Society, New Orleans, Louisiana, October 31–November 5, 1992.     

Coculture of mouse embryos with cryopreserved human oviduct epithelial cells

Purpose The effect of coculturing mouse embryos with cryopreserved human oviduct epithelial cells was investigated. The cryopreserved cells in Cellbanker were thawed and cultured in Richard D. Goldsby culture medium to establish monolayers. Two-cell-stage mouse embryos were cultured alone (control group) or cocultured with monolayers established from cryopreserved cells (cryopreserved coculture group) or from fresh cells (fresh coculture group). The rates of embryo development and the qualities of the blastocysts in the three groups were compared.Results The two coculture groups had significantly higher blastocyst development rates (cryopreserved coculture group, 81.6%; fresh coculture group, 82.2%) than the control group (63.1%). The two coculture groups had significantly more blastomeres (cryopreserved coculture group, 108.3 ± 25.9; fresh coculture group, 108.4 ± 25.1) than the control group (87.7 ± 31.9).Conclusion The method of cryopreservation of human oviduct epithelial cells using Cellbanker is simpler than conventional cryopreservation methods. These cryopreserved human oviduct epithelial cells may provide a constant supply of cells for coculture for in vitro fertilization and embryo transfer.     

Cytogenetic and cryobiology of human cocultured embryos: A 3-year experience

Purpose Coculture, which allows good-quality human blastocysts with good yields to be obtained, has been designed mainly to select the best embryos for transfers.Methods In a first attempt during coculture, we have studied by fluorescent in situhybridization the chromosomic content of the in vitroblocked embryos, using centromeric probes for chromosomes 1, 12, and 18. Close to 37% of the arrested embryos show aneuploidymosaicism.Results Freezing cocultured blastocysts gives good recovery rates after transfer (83%). The ongoing pregnancy rates per transfer (19%) are high, and the implantation rate per embryo is 13%. This compares favorably with freezing at an early stage.Conclusions We observed that the quality of the endometrium is always the limiting step, as first of all we observed wide variations according to the hormonal preparation of the patients. Moreover the implantation per embryo in the pregnant patients is very high (57%), indicating that most of the losses are directly related to the receptivity of the endometrium.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.     

Coculture cells that express leukemia inhibitory factor (LIF) enhance mouse blastocyst developmentin vitro

Purpose Leukemia inhibitory factor is a cytokine that plays an important role in implantation and enhances mouse preimplantation embryo development in vitro.Since leukemia inhibitory factor enhances early embryo development, we tested the hypothesis that coculture cells that express leukemia inhibitory factor would enhance mouse blastocyst development in vitro.In this study, Northern analysis for leukemia inhibitory factor was performed on total RNA extracted from Vero cells, human embryonic, fibroblasts, and human placental fibroblasts.Methods Two-cell mouse embryos were cultured to blastocyst in Ham's F-10 with 15% human cord serum (control) or with the coculture cells. Northern analysis demonstrated expression of LIF mRNA in Vero cells and embryonic fibroblasts but not in placental fibroblasts. Development to blastocyst was significantly enhanced in two-cell embryos cultured with Vero cells (86%, 140/163) and embryonic fibroblasts (83%, 118/142) when compared to controls (71%, 67/94, P<0.05) or placental fibroblasts (71%, 95/134), P<0.05).Conclusion These data suggest that coculture cells that express leukemia inhibitory factor may be superior to nonleukemia inhibitory factor expressing cells for early preimplantation embryo development.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.     

人分泌早期子宫内膜共培养体系对早期鼠胚凋亡的影响

目的研究人分泌早期子宫内膜共培养体系对早期鼠胚凋亡的影响。方法将2一细胞期鼠胚与子宫内膜(A冻融、B传代)共培养以及单一培养液培养(C组)。72h后对桑椹期鼠胚用TUNNEL法进行凋亡检测。结果共培养组鼠胚的凋亡率明显低于单一培养组，且不受内膜细胞传代及冻融的影响；共培养组优质胚胎数均高于对照组，而共培养组之间无明显差异。结论共培养人分泌早期子宫内膜能减少早期鼠胚的凋亡，提高胚胎体外发育的质量，延长体外培养时间。     

Effect of Vero Cell Coculture on the Development of Frozen–Thawed Two-Cell Mouse Embryos

Purpose: Our purpose was to evaluate the beneficial effects of long-term coculture of Vero cells on the development of frozen–thawed two-cell mouse embryos. Methods: Two-cell mouse embryos were frozen slowly with 1,2-propandiol and sucrose as cryoprotectants and thawed rapidly, followed by stepwise dilution. Vero cells were cultured in drops of RPMI 1640 to establish monolayers. Frozen–thawed embryos were cultured alone (control) or cocultured with Vero cells. The rate of development in both groups was compared. Results: After 4 days of culture, significantly more embryos in coculture were developed to expanded blastocysts (61 vs 37% for controls; P 0.0001). In addition, on the fifth day of cultivation, more embryos in coculture showed the potential of hatching from the zona pellucida (26 vs 7% in controls; P 0.0001). The rate of degeneration in coculture was also much lower than in controls (6 and 15%, respectively). Conclusions: Coculture of cryopreserved preimplantation-stage embryos with Vero cells seems to be a useful tool to eliminate the postthaw deleterious effect of freezing and also to obtain better-quality embryos appropriate for transfer.     

Effects of human follicular fluid on spermatozoa that have been cocultured with human oviductal cells

Objective: To investigate the sequential effects of human oviductal cells and human follicular fluid (hFF) on various sperm functions.Design: Laboratory experimental study.Setting: University gynecology unit.Patient(s): Fallopian tubes were from patients undergoing tubal ligation or hysterectomy. Semen was from men attending the subfertility clinics.Intervention(s): Spermatozoa were treated with [1] 6 hours in Earle’s balanced salt solution (EBSS-BSA; control); [2] 5 hours in EBSS-BSA and 1 hour with hFF (hFF); [3] 5 hours with oviductal cells and 1 hour in EBSS-BSA (coculture); and [4] 5 hours with oviductal cells and 1 hour with hFF (sequential).Main Outcome Measure(s): Motility, acrosome reaction, zona binding, and oocyte fusion.Result(s): Groups II and III spermatozoa had similar motility and were better than that of group I. Group IV displayed higher motility parameters than the other groups. Human follicular fluid induced acrosome reaction. The incidence of acrosome reaction in group IV was significantly lower than that in group II. Group III did not affect the acrosome reaction. Spermatozoa in groups II–IV had lower zona binding capacity than those in group I. Human follicular fluid stimulated oocyte penetration, whereas oviductal cells suppressed this effect of hFF.Conclusion(s): Oviductal cells maintained the fertilizing capacity of spermatozoa, whereas hFF facilitated the fertilization process of oviductal spermatozoa.