Behrouz Aflatoonian

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

In vitro post-meiotic germ cell development from human embryonic stem cells

BACKGROUND Investigating the mechanisms of human primordial germ cell (PGC) and gamete development are important for understanding the causes of infertility and effects of environmental chemicals on reproductive development. However, there are practical and ethical difficulties associated with obtaining human tissue in early development. The aim of this study was to investigate whether human embryonic stem cell-hESC-generated germ cells could provide an in vitro model of gamete development. METHOD Human ESCs were differentiated as embryoid bodies (EBs) in vitro. Gene and protein marker expression profiles of EBs in different periods of culture were analysed by quantitative polymerase chain reaction (Q-PCR) and immunolocalization to monitor germ cell development. Secretion of hormones involved in germ cell maturation was measured, to detect the existence of a germ cell niche within EBs. RESULTS Q-PCR revealed gene expression profiles consistent with PGC formation and germ cell development. A small population of post-meiotic spermatid cells were identified using sperm-specific antibodies (Protamine 1 and 1.97). Although gene expression profiles characteristic of oocyte development and follicle-like structures were detected, a committed oocyte with extra-cellular zona pellucida was not recognized with zona pellucida-specific monoclonal antibody. CONCLUSIONS hESCs can form PGCs and post-meiotic spermatids in vitro, however, there remains doubt about oocyte development. Levels of steroid hormones produced by EBs were significant when compared with known values for a similar quantity of human testis, suggesting that hESC may intrinsically create a favourable hormonal niche for spermatogenesis.

Germ cells from mouse and human embryonic stem cells

Mammalian gametes are derived from a founder population of primordial germ cells (PGCs) that are determined early in embryogenesis and set aside for unique development. Understanding the mechanisms of PGC determination and differentiation is important for elucidating causes of infertility and how endocrine disrupting chemicals may potentially increase susceptibility to congenital reproductive abnormalities and conditions such as testicular cancer in adulthood (testicular dysgenesis syndrome). Primordial germ cells are closely related to embryonic stem cells (ESCs) and embryonic germ (EG) cells and comparisons between these cell types are providing new information about pluripotency and epigenetic processes. Murine ESCs can differentiate to PGCs, gametes and even blastocysts - recently live mouse pups were born from sperm generated from mESCs. Although investigations are still preliminary, human embryonic stem cells (hESCs) apparently display a similar developmental capacity to generate PGCs and immature gametes. Exactly how such gamete-like cells are generated during stem cell culture remains unclear especially as in vitro conditions are ill-defined. The findings are discussed in relation to the mechanisms of human PGC and gamete development and the biotechnology of hESCs and hEG cells.

Stem cells for reproductive medicine.

The generation of various pluripotent stem cell lines provides a new route to investigate developmental process of germ cell and embryo development, which until now was difficult to access in the human. In the future these cells may be used for new therapies in reproductive medicine. This brief review outlines the development of germ cells and their pluripotent capabilities, how embryonic and germline stem cells can mimic developmental processes in vitro and generate gamete and trophoblast phenotypes for research and potential treatments.

Coronary stents seeded with human trophoblastic endovascular progenitor cells show accelerated strut coverage without excessive neointimal proliferation in a porcine model.

AIMS The success of percutaneous coronary intervention (PCI) has been limited by restenosis and stent thrombosis. Delayed or incomplete endothelial regeneration is believed to be a key factor responsible for these events. Developing a stent with an accelerated healing profile may be of benefit. We aimed to evaluate the feasibility and safety of seeding a bare metal stent (BMS) with human trophoblastic endovascular progenitor cells (hTEC) derived from human embryonic stem cells. A porcine coronary artery model was used to compare the rate and extent of endothelial regeneration and the degree of neointimal proliferation. Characterisation of hTEC confirmed a mixed progenitor and endothelial cell phenotype. The biodistribution and fate of hTEC were studied using radiolabelled 111Indium oxine and fluorescent in situ hybridisation. Scanning electron microscopy showed earlier endothelial coverage in hTEC-seeded stents as compared to similar BMS. hTEC-seeded BMS achieved complete stent coverage in three days. Quantitative coronary angiography, intravascular ultrasound assessment and histomorphometry showed no difference in neointimal hyperplasia between hTEC-seeded and control BMS. hTEC seeding of coronary stents is a novel and safe approach to accelerate endothelial regeneration without increasing neointimal proliferation.

Pluripotency and differentiation of cells from human testicular sperm extraction: An investigation of cell stemness.

Human male germ‐line stem cells (hmGSCs) and human testis‐derived embryonic stem cell‐like (htESC‐like) cells are claimed to be in vitro pluripotent counterparts of spermatogonial stem cells (SSCs), but the origin and pluripotency of human testis‐derived cell cultures are still under debate. The aim of this study was to generate putative pluripotent stem cells in vitro from human testicular sperm‐extracted (TESE) samples of infertile men, and to assess their pluripotency and capacity to differentiate. TESE samples were minced, enzymatically disaggregated and dispersed into single‐cell or cluster suspensions, and then cultured. Initially, cell clusters resembled those described for hmGSCs and htESC‐like cells, and were positive for markers such as OCT4/POU5F1, NANOG, and TRA‐2‐54. Prolonged propagation of cell clusters expressing pluripotency markers did not thrive; instead, the cells that emerged possessed characteristics of mesenchymal stromal cells (MSCs) such as STRO‐1, CD105/EGLN1, CD13/ANPEP, SOX9, vimentin, and fibronectin. KIT, SOX2, and CD44 were not expressed by these MSCs. The multipotential differentiation capacity of these cells was confirmed using Oil Red‐O and Alizarin Red staining after induction with specific culture conditions. It is therefore concluded that pluripotent stem cells could not be derived using the conditions previously reported to be successful for TESE samples. Mol. Reprod. Dev. 83: 312–323, 2016.

152 Stem Cell Coated Metallic Coronary Stents Show Accelerated Strut Coverage without Excessive Neointimal Proliferation in a Porcine Model

Introduction The success of percutaneous coronary intervention (PCI) has been limited by restenosis and stent thrombosis. Delayed or incomplete endothelial regeneration is believed to be a key factor responsible for these events. Developing a stent with an accelerated healing profile may be of benefit. We aimed to evaluate the feasibility and safety of seeding a bare metal stent (BMS) with human trophoblastic endovascular progenitor cells (hTEC) derived from human embryonic stem cells. Methods hTEC were derived by the terminal differentiation of trophoblast stem cells while the latter were derived as distinct trophoblast cell lines from human embryonic stem cells. BMS were seeded with hTEC by co-culturing for 3 days. The biodistribution and fate of hTEC were studied using radiolabeled 111Indium oxine and fluorescent in-situ hybridisation. A porcine coronary artery model was used to compare the rate and extent of endothelial regeneration and the degree of neointimal proliferation. Results Characterisation of hTEC confirmed a mixed progenitor and endothelial cell phenotype. For the stents seeded with 111Indium-labelled hTEC, the radioactivity measured over the explanted stented LAD was 77,642 cpm at 1 h, 20,048 cpm at 1 day and 2,323 cpm at 7 days, with no significant radioactivity detected at any of the distal sites including blood vessels, heart, lung, spleen, liver and kidneys. Scanning electron microscopy showed earlier endothelial coverage in hTEC-seeded stents as compared to similar BMS (Figure 1). Abstract 152 Figure 1 hTEC-seeded BMS achieved complete stent coverage in 3 days (Figure 2). Quantitative coronary angiography, intravascular ultrasound assessment and histomorphometry showed no difference in neointimal hyperplasia between hTEC-seeded and control BMS (Figure 2). Abstract 152 Figure 2 Conclusion hTEC seeding of coronary stents is a novel and safe approach to accelerate endothelial regeneration without increasing neointimal proliferation.

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma

Background: Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named “hTERT messenger ribonucleic acid (mRNA)” the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. Materials and Methods: U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0– 160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control– untreated group. Results: The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent (P < 0.05) and not in time-dependent manner (P > 0.05), in vitro. Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. Conclusion: With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM.