Short report of visiting Short report of visiting research courseresearch course

By: By: Kambiz HassanzadehKambiz Hassanzadeh

Supervisor:Supervisor: Professor Sandra Professor Sandra CeccatelliCeccatelli

Karolinska Institute, Stockholm Karolinska Institute, Stockholm 20092009

I was involved in two projectsI was involved in two projects

1)Evaluation the effect of perflurooctanesulfonic 1)Evaluation the effect of perflurooctanesulfonic acide (PFOS) on proliferation and differentiation acide (PFOS) on proliferation and differentiation of cortical neural stem cells isolated from of cortical neural stem cells isolated from embryo of mice.embryo of mice.

2) Effect of gender and age difference on the 2) Effect of gender and age difference on the reactivity of human neurospheres to different reactivity of human neurospheres to different

toxicant stimulus.toxicant stimulus.

TechniquesTechniquesIn order to be involved in cell culture In order to be involved in cell culture

projects, I learned the different projects, I learned the different techniques which are needed for techniques which are needed for working with cells such as (seeding, working with cells such as (seeding, splitting, harvesting, freezing,…..). splitting, harvesting, freezing,…..).

Also I learned the test of viability of cells Also I learned the test of viability of cells (Trypan blue), immunocytochemistry (Trypan blue), immunocytochemistry and histochemical methods to and histochemical methods to investigate the proliferation and investigate the proliferation and differentiation of cortical neural stem differentiation of cortical neural stem cell pathways. cell pathways.

Additionally I learned gene expression Additionally I learned gene expression assay & analysis using Real-Time PCR assay & analysis using Real-Time PCR method.method.

Project 1MethodsProject 1Methods::

1) Preparation of cortical neural stem cells 1) Preparation of cortical neural stem cells from embryo of micefrom embryo of mice

2) Exposing the cells to different doses of 2) Exposing the cells to different doses of PFOS (12.5, 25, 50 100 nM)PFOS (12.5, 25, 50 100 nM)

3) Harvesting the cells and Trypan blue test3) Harvesting the cells and Trypan blue test

4) Evaluation of different factors which are 4) Evaluation of different factors which are involved in proliferation and differentiation involved in proliferation and differentiation using immunocytochemistry ( Nestin, using immunocytochemistry ( Nestin, SOX2, Tuj1, Ki 67, BDNF, GFAP, TrkB, ….). SOX2, Tuj1, Ki 67, BDNF, GFAP, TrkB, ….).

Project 2 methodProject 2 method::

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

Chemical exposureChemical exposure::

The cells were exposed to:The cells were exposed to:

1) MeHg (2.5, 10, 25, 100  nM) 1) MeHg (2.5, 10, 25, 100  nM)

2) H2) H22OO22 (5, 25, 50, 100  μM) (5, 25, 50, 100  μM)

3) Staurosporin (5, 10, 15 nM) 3) Staurosporin (5, 10, 15 nM)

in proliferation and differentiation in proliferation and differentiation medium.medium.

ImmunohistochemistryImmunohistochemistry::

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

IndividualsIndividualsIndividualsIndividuals

IdentificationIdentification

NumberNumber

GenderGenderDevelopmental Developmental

stagestage ((Gestational Gestational

Week)Week)

QualityQuality

503503♂♂1616Very good in Very good in proliferationproliferation

585:2585:2♀♀8.58.5Very good in Very good in proliferationproliferation

557:4557:4♂♂66Very week in Very week in proliferationproliferation

9 1/29 1/2♀♀9.59.5Good in proliferationGood in proliferation

603:3603:3♀♀88Week in proliferation Week in proliferation

Study Design for Study Design for ImmunocytochemistryImmunocytochemistry

1. H1. H22OO2 2 ± GF 5, 25, 50, 100µM± GF 5, 25, 50, 100µM

Apoptosis: Apoptosis: 24hrs in culture Exposure 24hrs in culture Exposure Fixing and StainingFixing and Staining

Differentiation:Differentiation: Exposure 4 days in culture Exposure 4 days in culture Fixing and Staining Fixing and Staining

2. MeHg ± GF 2.5, 10, 25, 100nM2. MeHg ± GF 2.5, 10, 25, 100nM Apoptosis & Differentiation:Apoptosis & Differentiation:

Exposure 4 Days in culture Fixing and StainingExposure 4 Days in culture Fixing and Staining

3. 3. Staurosporine Staurosporine ± GF 5, 10, 25nM± GF 5, 10, 25nM ApoptosisApoptosis

24hrs in culture Exposure Fixing and Staining24hrs in culture Exposure Fixing and Staining

24hrs

24hrs

Gene expression analysisGene expression analysis

In order to find the possible mechanisms In order to find the possible mechanisms we examined the bellow genes involving we examined the bellow genes involving in differentiating pathways. in differentiating pathways.

NOS3, VEGF, ENO2, Hes family genes, NOS3, VEGF, ENO2, Hes family genes, Jag1, Notch1, Mash1, DKK1, SOX2, Jag1, Notch1, Mash1, DKK1, SOX2, PAX6,…..PAX6,…..

Method:Method:

Cells in culture Exposure to HCells in culture Exposure to H22OO2 2 or or MeHg for 4, 24, 48 hrs HarvestingMeHg for 4, 24, 48 hrs Harvesting

24 hrs

ConclusionConclusion

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

Meetings & SeminarsMeetings & Seminars

Also I have attended in group meetings Also I have attended in group meetings and journal clubs as well as seminars and journal clubs as well as seminars

organised by Karolinska Institute.organised by Karolinska Institute.