THE PROMISE OF STEM CELL RESEARCHTHE PROMISE OF STEM CELL RESEARCH

Marisa BowersMarisa Bowers

Donghong ZhaoDonghong Zhao

Karen S. Aboody, M.D. et al.Karen S. Aboody, M.D. et al.

Assistant Professor

Divisions of Hematology/HCT & Neurosciences

City of Hope National Cancer Center & Beckman Research Institute

COH Summer Student Program

June 26, 2005

MBowers/Aboody Lab 2005

Outline

•Background Information/Definitions

•Neural Stem Cell Migration

•Migration Assays

•Migration Data and Results

•Future Experiments

MBowers/Aboody Lab 2005

Background – Stem Cells

•What is a Stem Cell?

-undifferentiated cell has potential to differentiate into some/all cell types in body

•What is a Neural Stem Cell?

-differentiate into neuron, astrocyte or oligdendrocyte

•What are the potential therapeutic benefits of stem cells?

-exogenous stem cells repair damaged/diseased tissue

-deliver therapeutic agents to tumors

-prevent/treat birth defects in womb

MBowers/Aboody Lab 2005

Background - Glioblastoma

•What is Glioblastoma?

-Type of human brain cancer

-Highly invasive: metastasizes quickly throughout brain, surgeons remove main tumor mass, missing metastases

-Expected life-span of a patient with glioma is 3-6 months

-No cure

MBowers/Aboody Lab 2005

Past and Present Glioma Research

•Previous research: drug delivery

-through gene therapy

-problem with drug delivery: no movement of cells within tissue, does not target metastases

-blood-brain barrier prohibits entry of some therapeutic agents into brain

•Potential Solution: neural stem cells as vehicles

-targeted delivery of therapeutic agents

MBowers/Aboody Lab 2005

NSC’s Directed Migration to Tumor Cells

Cancer cell Stem cell

MBowers/Aboody Lab 2005

Why does Directed Migration of NSC’s to Tumor Cells Occur?

•Theories:

-tumor cells excrete cytokines, attract stem cells

-pockets of hypoxia exist within tumor mass, draws stem cells

-genes expressed in cells different in hypoxic (1%O2) and normoxic (16%O2) conditions

•My Project: Determine factors involved in NSC migration to tumor

•Hypothesis: one or more of the following cytokines effects NSC migration under hypoxic conditions

-SDF-1/CXCR-4

-HGF/C-Met

-SCF/CKit

-VEGF/VEGFR

-EGF/EGFR

-IL8/IL8R

signaling pathways Stat3, RaS and Wnt are downstream of genes code for cytokines, pathway effected by cytokine expression

Tumor Cell NSC

MBowers/Aboody Lab 2005

•Initial Tests: Boyden Chamber Migration Assays

MBowers/Aboody Lab 2005

•Variable Conditions:

-Upper Chamber:

-NSC grown at 16%O2

-NSC grown at 16% then incubated 24 hours at 1%O2

-Lower Chamber:

-tumor CM from tumor cells incubated 48 hours under 16%O2

-tumor CM from tumor cells incubated 48 hours under 1%O2

-media containing different levels of serum

Boyden Chamber Migration Set-up

MBowers/Aboody Lab 2005

•Controls:

-DMEM/RPMI

-5%BSA

-10%FBS

•Tumor CM

-16%O2 U251

-1%O2 U251

-16%O2 U251 +SDF-1

-16%O2 SKNAS

-1%O2 SKNAS

-16%O2 SKNAS +SDF-1

Lower Wells

Boyden Chamber Set-Up

16%O2 F3.C1 1%O2 F3.C1

Upper Wells

MBowers/Aboody Lab 2005

U251 Graph

U251CMcell number-4data

0

1000

2000

3000

400016%F3C1 on 16%plate1%F3C1 on 16%plate16%F3C1 on 1%Plate1%F3C1 on 1%plate

CM

ce

ll n

um

be

r

16%O2 Plate

1%O2 Plate

MBowers/Aboody Lab 2005

SKNAS Graph

SKNAS CM -cell number-4data

0

500

1000

1500

200016%F3C1 on 16%plate1%F3C1 on 16%plate16%F3C1 on 1%plate1%F3C1 on 1%plate

CM

ce

ll n

um

be

r

16%O2 Plate1%O2 Plate

MBowers/Aboody Lab 2005

Migration Results

•Effect of SDF-1 on Migration

-no effect on migration

-seen repeatedly in assays

-either not involved in migration or levels of SDF-1 high prior to migration, additional SDF-1 no effect

•Effect of VEGFR on Migration

-normoxic and hypoxic plates: blocked/hindered migration

-may be migration inducing factor

MBowers/Aboody Lab 2005

Future Experiments

•Changes for future experiments:

-culture stem cells in 1%O2 for less than 24 hours

-run many tests to find good time span

-real time-PCR on SC cultured for different amounts time

-use U251 and Brain cell line 5904, not SKNAS

-SKNAS: lower response to F3.C1 SC

•Repeat Boyden Chamber

-making changes

-compare migration results

MBowers/Aboody Lab 2005

Next Step

•Extract stem cell RNA and perform real time-PCR

- Use mRNA expression to determine regulation of candidate ‘homing’ genes during migration

MBowers/Aboody Lab 2005

•Use female immune compromised nude mice

-inject mice with:

-‘normal’ human brain tumor or

- HIF siRNA brain tumor: HIF expression blocked

-decrease cytokine expression

- both groups will receive injection of stem cells

In Vivo Model

MBowers/Aboody Lab 2005

•harvest mice, remove tumor tissue for RNA extraction and real time-PCR

-determine gene expression of tumor tissue and NSC

-expect: gene expression to be different in normal and HIF siRNA tumors

-use immnuohistochemical staining compare distribution stem cells in brains

-expect: more stem cells in normal tumor than HIF siRNA tumor

Data Analysis

MBowers/Aboody Lab 2005MBowers/Aboody Lab 2005

Thank You

Dr. Karen Aboody

Donghong Zhao, PhD

The Aboody Lab

The Barish Lab

The Glackin/Flannigan Labs

Queenie Du and the Summer Student Program