Lecture 4 - Using Human Fetal Neural Stem Cells to Understand Glioblastoma Initiation

glioblastoma

most common malignant brain tumor in adults, growth is sustained by stem like cells and the cell of origin is unclear

cell of origin

tumor initiating cell, normal cell that has 1st promoting cancer mutation

cancer stem cell

tumor propagating cell, cellular subset in the tumor sustaining its growth

stochastic model

all isolated tumor cells have the capacity to differentiate indefinitely and form new tumors, all cells can be cancerous but only some will contribute to tumor formation in response to some stimuli

cancer stem cell model

only cancer stem cells have the ability to form tumors, initiate or regrow a tumor

cancer stem cell + cell of origin same identity

normal stem cells could have oncogenic transformation into cancer stem cells

cancer stem cell + cell of origin are different

cancer stem cell could come from a more differentiated cell that gets access through stem programs then that would make them different

radial glial neural stem cell

responsible for generating main functional units of the main vertebrate system

progenitor cells

neuronal progenitor, astrocytic progenitor and oligodendrocyte progenitor

• limited self renewal potential

terminally differentiated cells

neurons, astrocytes and oligodendrocytes

self renewal

can divide to create indefinitely more identical undifferentiated cells

multipotentiality

can be triggered to differentiate, change stem cell identity and become tissue specific with a specialized function

CNS

parts of the nervous system encased in bone

• brain + spinal cord

CNS main components

the cerebrum, cerebellum and brain stem

cerebral cortex

outermost layer of the brain and half of its whole mass

gyri

ridges of the cerebral cortex

sulcus

grooves of the cerebral cortex

lobes of the brain

frontal (higher level processing), parietal (tactile), temporal (auditory) and occipital (visual)

neocortex

90% of the brain (new), synonymous with the cerebral cortex

allocortex

10% of the brain (primitive

what are the two hemispheres of the brain connected with?

the corpus callosum

dendrite

receives chemical messages from other cells

axon

long projection of a neuron that conducts electrical impulses (action potentials) away from the cell body transmitting information to different neurons, muscles and glands

gray matter

consists of cell bodies and dendrites (non myelinated)

white matter

consists of bundles of axons (myelinated)

CNS glial cell types

astrocytes, oligodendrocytes, microgila and ependymal cells

PNS glial cell types

satellite cells and Schwann cells

astrocytes

most numerous glial cell type in the brain and regulates the chemical content of the extracellular fluid tightly controlled concentration of chemicals that could interfere with function like potassium

oligodendrocytes

provides the myelin sheath and nodes of Ranvier

myelin sheath

provides layers of membrane that insulin axons

node of ranvier

region of axonal membrane that is not insulated by myelin

what cells make up the outer subventricular zone?

oRG (outer radial glia), intermediate progenitor cells and transit amplifying progenitor cells

inside out mechanism of cortex formation

early neurons populate the deeper layers (vRG) and later ones are at the upper layers/surface (oRG)

intermediate progenitor cell

produced by vRG and oRG and proliferate to produce neurons and rise up the cortical plate

oRG role in rodent brains

said to be responsible for increase in cortex size and complexity due to more stem and progenitor cells and more self renewal and proliferation is greater (transit amplification)

human cerebral cortex formation

minimal proliferation during adulthood but massive proliferation during development creating a landscape for cancer acquisition

• cells w/rapid self renewal and proliferation more likely to acquire cancer mutations

what similarity do oRG and primary GBM cells share?

they have a similar transcriptional profile

LIFR

leukemia inhibitory factor receptor, strong correlation with signatures of stemness and invasion in GBM cells

ITGB5 and PTPRZ1

novel oRG markers related to ECM formation, stem cell maintenance and cell migration

what might cause the reactivation of normal developmental programs in adult GBM tumors?

• if some ORG persists into the adult brain

• if a downstream cell type dedifferentiates to become oRG like via reprogramming to embryonic state to drive tumor initiation or maintenance

what are the project aims?

• in depth in vitro characterization of vRG and oRG to investigate their similarities and differences

• comparison of vRG and oRG propensity to initiate GBM by evaluating cancer properties/outputs following mutational burden

project hypothesis

oRG are the most susceptible stem cell population in the brain to oncogenic transformation into GBM

how were vRG and oRG isolated from the developing human cerebral cortex?

via fluorescence-activated cell sorting (FACS)

how was the regional identity of the sorted samples verified?

via immunochemistry (IHC)

how was the growth of vRG and oRG maintained overtime?

via cell culture

passaging

when there is no more room to grow in the culture dish they have to be transferred to a new growth medium in order to have room for additional growth

immunostaining

general term that refers to the use of any antibody based method to detect a specific protein of interest in a sample

antigen

ay substance/molecule that causes your immune system to produce antibodies against it

what are the two types of immunostaining?

immunohistochemistry and immunocytochemistry

immunohistochemistry (IHC)

detection of antigens in cells within intact tissue section (in situ), advantageous to determine localization and distribution of antigens within histological context/whole tissue context (god for drug development and diagnosis)

immunocytochemistry (ICC)

detection of antigens in cultured cells

chromogenic reporters

enzyme and a chromogenic substrate with enzyme-conjugated antibodies

what enzymes are used in chromogenic detection?

horseradish peroxidase (HRP) and alkaline phosphatase (AP)

what substrates are used for HRP?

3,3’ - diaminobenzidine (DAB, most common, brown to black) and amino acetyl carbazole (AEC, red)

what substrates are used for AP?

fast red (most common, red) and a combination of NBT and BCIP ( black to purple)

what is the advantage of using chromogenic detection?

cheaper cause imaging is possible just with a light microscope

fluorescent receptors

used fluorophore-conjugated antibodies (immunofluorescence)

fluorescence detection advantage

multiplex staining meaning that different colors are assigned to different antigens so multiple antigens can be visualized at once

hollow peak

excitation spectrum

solid peak

emission spectrum

when is vRG present?

when CD133 is high

when is oRG present?

when CD133 is low

how is vRG and oRg isolated by FACS?

a piece of cortex is received by pregnancy termination then dissociation and immunolabelling is done by single cell suspension and incubation using antibodies against cell surface markers then FACS is performed

immunohistochemical staining of SOX2 in the cerebral cortex

IHC is used to figure out if the sample is cortex or another part of the brain and a counter stain is used to detect the SOX2 marker which is found in the lower region

hematoxylin

counter stain used in IHC

HRP DAB

used for detection of the SOX2 marker shown by brown precipate

why is vRG more proliferative then oRG

because the cells have bigger sphere sizes indicating more proliferation compared to oRG

how is stem cell frequency accessed within each population

plate ever population as single cell then measure by counting number of spheres formed over the total number of single cells

secondary spheres

determine if the primary spheres can generate passages which is a good measure of true stem cells that posses long term self renewal ability

secondary passage results

found that vRG and oRG can passage but not IPCs although they can still proliferate but at a limited capacity

fold change between sphere formation

found that it was higher in oRG meaning that it is more potent at initiating subsequent spheres even though they are less proliferative

asymmetric divisions

associated with vRG

symmetric divisions

associated with oRG

what was the effect of the addition of growth factors on vRG?

found that there was an increase in proliferation

what was the effect of the addition of growth factors on oRG?

no effect on confluence proliferation

confluence

percentage of the surface factor growth area covering the plate

how does LIF addition differ between vRG and oRG?

more highly expressed in oRG because its addition alongside the standard increased proliferation compared to vRG so might indicate that it helps keep oRG in its undifferentiated state meaning its more vulnerable to gaining tumor-initiating mutations

EGF

epidermal growth factor

FGF

fibroblast growth factor

IGF

insulin like growth factor

VEGF

vascular endothelial growth facto

NGF

nerve growth factor

LIF

leukemia inhibitory factor

vRG passaging conclusions

makes fewer but larger spheres over passaging meaning it is more proliferative

oRG passaging conclusions

make more but same sized spheres over passaging meaning it has more potential for self renewal

org consists of…

neurons

vRG consists of…

astrocytes and stem cells (gliogenesis)

vRG heatmap

both neuronal and astrocytic markers co expressed showing multipotentiality (no commitment to a specific fate)

• begins neurogenesis first so might have quicker progression

oRG heatmap

more committed to producing neurons

heatmaps

quantifying expression of different markers by immunostaining at different time points

CRISPR

clustered regularly interspaced short palindromic repeats cutting target DNA sequence by a customizable guide sequence

Crisper associated (Cas) endonuclease

binds and cuts DNA

guide RNA (gRNA) sequence

directs the Cas protein to its target, discovered in bacterial immune systems where it cuts the DNA of bacteriophages disabling them

gRNA components

• scaffold: necessary for Cas binding

• spacer: defines the genomic target to be identified

p53 knockout effect on oRG vs vRG

no affect on vRG but in oRG there were significantly more spheres following the p53 knockout making it more proliferative and higher potential for self renewal

HSCs

have more protective mechanisms against DNA damage compared to downstream progenitor cells

how is apoptosis initiated

by activation of the unfolded protein response, this allows HSCs to maintain clonal integrity preventing propagation of damaged stem cells which is detrimental to downstream progenitor cells cause they have a high self renewal ability

etoposide

DNA damage agent that inhibits topoisomerase 2 producing double stranded breaks

topoisomerase 2

resolves tangles or supercoils in the DNA by repairing the double strand break

yH2AX

phosphorylated form of H2AX (histone); recruits DNA repair proteins