1. Short report of visiting research course By: Kambiz Hassanzadeh Supervisor: Professor Sandra Ceccatelli Karolinska Institute, Stockholm 2009

2. I was involved in two projects 1)Evaluation the effect of perflurooctanesulfonic acide (PFOS) on proliferation and differentiation of cortical neural stem cells isolated from embryo of mice. 2) Effect of gender and age difference on the reactivity of human neurospheres to different toxicant stimulus. I was involved in two projects 1)Evaluation the effect of perflurooctanesulfonic acide (PFOS) on proliferation and differentiation of cortical neural stem cells isolated from embryo of mice. 2) Effect of gender and age difference on the reactivity of human neurospheres to different toxicant stimulus.

3. Techniques In order to be involved in cell culture projects, I learned the different techniques which are needed for working with cells such as (seeding, splitting, harvesting, freezing, …..). Also I learned the test of viability of cells (Trypan blue), immunocytochemistry and histochemical methods to investigate the proliferation and differentiation of cortical neural stem cell pathways. Additionally I learned gene expression assay & analysis using Real-Time PCR method.

4. Project 1Methods: 1) Preparation of cortical neural stem cells from embryo of mice 2) Exposing the cells to different doses of PFOS (12.5, 25, 50 100 nM) 3) Harvesting the cells and Trypan blue test 4) Evaluation of different factors which are involved in proliferation and differentiation using immunocytochemistry ( Nestin, SOX2, Tuj1, Ki 67, BDNF, GFAP, TrkB, ….).

5. Project 2 method: Cell culture: Cryopreserved human neural spheres were cultured at 37°C and 5% CO2 as a single cell suspension culture in proliferation medium consisting of Dulbecco ’ s modified Eagle medium (DMEM) and Hams F12 (3:1) supplemented with B27, 20 ng/mL epidermal growth factor, and 20 ng/mL recombinant human fibroblast growth factor. Single cell suspension was prepared for each experiment. Differentiation was initiated by growth factor withdrawal in differentiation medium [DMEM and Hams F12 (3:1) supplemented with N2 supplement and plated onto poly-d- lysine/laminin – coated cover glasses. Cell culture: Cryopreserved human neural spheres were cultured at 37°C and 5% CO2 as a single cell suspension culture in proliferation medium consisting of Dulbecco ’ s modified Eagle medium (DMEM) and Hams F12 (3:1) supplemented with B27, 20 ng/mL epidermal growth factor, and 20 ng/mL recombinant human fibroblast growth factor. Single cell suspension was prepared for each experiment. Differentiation was initiated by growth factor withdrawal in differentiation medium [DMEM and Hams F12 (3:1) supplemented with N2 supplement and plated onto poly-d- lysine/laminin – coated cover glasses.

6. Chemical exposure: The cells were exposed to: 1) MeHg (2.5, 10, 25, 100 nM) 2) H 2 O 2 (5, 25, 50, 100 μM) 3) Staurosporin (5, 10, 15 nM) in proliferation and differentiation medium.

7. Immunohistochemistry: Proliferating or differentiating single cell suspension was fixed in 4% paraformaldehyde for 30 min. After washing the cells in phosphate-buffered saline (PBS), they were incubated with primary antibody for evaluation of differentiation overnight at 4°C. Afterward, cell suspension was incubated with secondary antibody for 1 hour and then nuclei were counterstained with Hoechst. Antibodies for staining was β(III)tubulin (1:200; Sigma Aldrich) which is a marker for neurons

8. Individuals Quality Developmental stage (Gestational Week) GenderIndividualsIdentificationNumber Very good in proliferation 16♂503 8.5♀585:2 Very week in proliferation 6♂557:4 Good in proliferation 9.5♀ 9 1/2 Week in proliferation 8♀603:3

9. Study Design for Immunocytochemistry 1. H 2 O 2 ± GF 5, 25, 50, 100 µ M Apoptosis: 24hrs in culture Exposure Fixing and Staining Apoptosis: 24hrs in culture Exposure Fixing and Staining Differentiation: Exposure 4 days in culture Fixing and Staining Differentiation: Exposure 4 days in culture Fixing and Staining 2. MeHg ± GF 2.5, 10, 25, 100nM Apoptosis & Differentiation: Apoptosis & Differentiation: Exposure 4 Days in culture Fixing and Staining 3. Staurosporine ± GF 5, 10, 25nM Apoptosis Apoptosis 24hrs in culture Exposure Fixing and Staining 24hrs

10. Gene expression analysis In order to find the possible mechanisms we examined the bellow genes involving in differentiating pathways. NOS3, VEGF, ENO2, Hes family genes, Jag1, Notch1, Mash1, DKK1, SOX2, PAX6, ….. Method: Cells in culture Exposure to H 2 O 2 or MeHg for 4, 24, 48 hrs Harvesting 24 hrs

11. Conclusion Our results showed that there are differences in susceptibility of different individuals to toxicant stimulus based on their sex and age. This study is continuing now and for confirming, some other individuals are being studied by our colleagues in lab.

12. Meetings & Seminars Also I have attended in group meetings and journal clubs as well as seminars organised by Karolinska Institute.