In vitro development of human primordial follicles to preantral stage after vitrification.

PURPOSE: The aim was to culture primordial follicles in vitro to reach preantral stage in vitrified human ovarian tissue. METHODS: Ovarian tissue samples were obtained from six women. Tissue strips were vitrified by infiltration with a cryoprotectant followed by mounting on a stainless steel carrier. After culturing for 7 days the morphology and developmental stages of follicles enclosed in fresh and vitrified groups were analyzed. RESULTS: High proportion of viable follicles in vitrified ovarian strips was obtained. After culturing for 7 days the percentage of secondary and preantral follicles increased significantly (P < 0.05) whereas primordial and transitory follicles showed a significant decrease (P < 0.05) compared to their respective counterparts at day 0 of culture. CONCLUSIONS: Vitrification of ovarian strips with an improved carrier device and culturing of follicles in ovarian strips after warming yielded developed follicles with high viability and morphological integrity that may be suitable for use in fertility preservation among cancer patients.

Development of 4-cell mouse embryos after re-vitrification.

This paper reports studies on the effects of re-vitrification by the CPS (Closed Pulled Straw) method on the development of 4-cell stage mouse embryos. The procedure involved culturing 2-cell mouse embryos in G-1 medium until the 4-cell stage followed by the division of the normal 4-cell stage embryos into a control group (non-vitrified) and two experimental subgroups (vitrified and re-vitrified). Embryos in the vitrified subgroup were cryopreserved by the CPS vitrification method. In the second experimental subgroup (re-vitrified), embryos that were already vitrified were warmed and cryopreserved again by the same method. There was no significant reduction in the rate of blastocyst formation after vitrification and re-vitrification. However, re-vitrification reduced the total cell number, ICM (inner cell mass) percent and blastocyst diameter (P<0.05). These results showed that vitrification and re-vitrification by the CPS method did not negatively affect the development of vitrified-warmed 4-cell mouse embryos, whereas re-vitrification significantly reduced both the cell number and diameter of blastocysts.

Comparison of gene expression profiles in erythroid-like cells derived from mouse embryonic stem cells differentiated in simple and co-culture systems.

The feeder layer and the presence of specific growth factors are thought to induce the differentiation of embryonic stem cells (ESCs) in culture. The aim of this study was to evaluate the effect of erythropoietin (EPO) on the differentiation of ESCs into erythroid colonies in simple and co-culture systems. Embryoid bodies were dissociated and replated in semisolid medium in simple culture or in a co-culture system with bone-marrow stromal cells (BMSCs), both in the presence or absence of EPO. Colony assays, benzidine staining, and ultrastructural studies were carried out until day 10 of culture. Expression of the epsilon globin, betaH1 globin, runt-related transcription factor 1 (RUNX1), betamajor globin, and erythropoietin receptor (EPOR) genes was evaluated using semi-quantitative RT-PCR. A comparison with the corresponding controls showed that colony size increased in both systems (P

Effect of basic fibroblast growth factor on cardiomyocyte differentiation from mouse embryonic stem cells.

OBJECTIVE: To investigate the effect of basic fibroblast growth factor (bFGF) on the differentiation of embryonic stem cells (ESCs) into early cardiomyocytes. METHODS: Embryoid bodies (EBs) were produced from mouse ESC line (Royan B1) in hanging drops and cultured for 5 days as suspension. During the first 2 days of suspension, the EBs of the experimental group were treated with 10 ng/ml of bFGF and subsequently plated onto gelatin-coated tissue culture dishes (day 7). The differentiated cells were evaluated pharmacologically, by immunocytochemistry, and so forth. The study was carried out in the Department of Stem Cells, Royan Institute, Tehran, Iran in 2005. RESULTS: The beating frequency in the bFGF treated EBs was less than that in the control group. In addition, the beating in the EBs of the experimental group, treated with isoprenaline and phenylephrine, was only more than 7+3 days in comparison to the control group. The response of the EBs to carbachol was more in the bFGF group than 7+14 days. In all the stages of development, the beating cells in the EBs of both groups expressed beta-actinin, myosin light chain isoform 2V, cardiac alpha-myosin heavy chain (alpha-MHC), and cardiac beta-myosin heavy chain (beta-MHC). Nonetheless, during 7+3 days, the last 2 genes were more advanced in the bFGF group. The atrial natriuretic factor was also expressed at a late stage in both groups. CONCLUSION: Basic fibroblast growth factor can only promote the early maturation of ESC-derived cardiomyocytes in terms of chronotropic characteristics and expression of cardiac alpha-MHC and beta-MHC.

Generation of new human embryonic stem cell lines with diploid and triploid karyotypes.

Human pluripotent embryonic stem cells (hESC) have great promise for research into human developmental biology and the development of cell therapies for the treatment of diseases. To meet the increased demand for characterized hESC lines, we present the derivation and characterization of five hESC lines on mouse embryonic fibroblast cells. Our stem cell lines are characterized by morphology, long-term expansion, and expression profiles of a number of specific markers, including TRA-1-60, TRA-1-81, alkaline phosphatase, connexin 43, OCT-4, NANOG, CXCR4, NODAL, LEFTY2, THY-1, TDGF1, PAX6, FOXD3, SOX2, EPHA2, FGF4, TAL1, AC133 and REX-1. The pluripotency of the cell line was confirmed by spontaneous differentiation under in vitro conditions. Whereas all of the cell lines expressed all the characteristics of undifferentiated pluripotent hESC, two of the cell lines carried a triploid karyotype.