BIL368

Cellular and Molecular Neuroscience

Stem cells
Dr. Valerie Bracchi-Ricard
vbracchi@miamiproject.med.miami.edu

04-9-2009

Stem cells
I- Definition of stem cells

II- Neurogenesis in the adult brain

III-Transdifferentiation
IV- Therapeutic use of Stem Cells

What are stem cells?

Three important characteristics
1- Capable of dividing and renewing themselves
for long periods
2- Unspecialized
3- Pluripotent

Stem cell

Progenitor cells

Precursor cells

Differentiated
cells

Two kinds of stem cells

Embryonic stem cells
Adult stem cells

What are embryonic stem cells?

4-5 day old Blastocyst
Trophectoderm

Inner Cell Mass

(about 30 cells)

Blastocoel
Inner Cell Mass cell

Fertilized egg
ectoderm

endoderm
mesoderm
liver
skin
lungs
lining
nerves bones blood
of gut
eyes muscles

What are adult stem cells?

An adult stem cell is an unspecialized cell found
among differentiated cells in a tissue or organ, it
can renew itself, and
can differentiate to yield the major specialized cell
types of the tissue or organ.

Their primary roles:

To maintain and repair the tissue in which they are
found.

Hematopoietic Stem Cells

Bone marrow stromal cells (mesenchymal stem cells):

bone cells (osteocytes), cartilage cells (chondrocytes),
fat cells (adipocytes), and other kinds of connective
tissue cells such as those in tendons.
Epithelial stem cells in the lining of the digestive tract:
absorptive cells, goblet cells, Paneth cells, and
enteroendocrine cells.
Skin stem cells in the basal layer of the epidermis and at the
base of hair follicles:
keratinocytes, hair follicle and epidermis.

Neural Stem Cells

Potency: The range of commitment options available

to a cell.
Totipotent Sufficient to form entire organism
Fertilized egg
Pluripotent Able to form all the bodys lineages
Embryonic stem cells

Multipotent Can form multiple lineages that constitute

an entire tissue or tissues.
Haematopoietic stem cells
Oligopotent Able to form two or more lineages within
a tissue
Neural stem cell
Unipotent

Forms a single lineage.

Adult stem cells

Tissue biopsy, dissociation, culture in appropriate medium

Adult mouse neural stem cells

grown as neurospheres

I- Definition of stem cells

II- Neurogenesis in the adult brain

III-Transdifferentiation
IV- Therapeutic use of Stem Cells

Charles G. Gross (2000) Nature Reviews Neuroscience 1: 67-73.

Neurogenesis in the adult brain:

death of a dogma
Central dogma of neuroscience (for over 100 years):
Neurons are not added to the adult mammalian
brain.
Neurogenesis (the production of new neurons)
occurs only during development and stops before
puberty.

-Ming G-L and Song H (2005) Adult neurogenesis in the mammalian central nervous system.
Annu. Rev. Neurosci. 28: 223-250.

Neural Stem Cell

Historically (Wilhelmen His, 1889), it was believed that
neuroepithelial cells (NE) in the early neural tube produced two
separate pools of committed glial and neuronal progenitors.

after birth
NE

NE

Committed
Glial
Precursor

Committed
Neuronal
Precursor

Committed
Glial
Precursor

Committed
Neuronal
Precursor

Glia

Neurons

Glia

Neurons

(1950s) Tritiated thymidine autoradiography

[3H]-thymidine is incorporated into the DNA of
dividing cells. Therefore the progeny of cells that had
just divided could be labeled.
(1960s) Joseph Altman reported evidence for new
neurons in various structures in the young and adult
rat brain using thymidine autoradiography.
But still as late as 1970, an authoritative text book of
developmental neuroscience stated there is no
convincing evidence of neuron production in the
brains of adult mammals.

(1970-80s) Electron Microscopy (EM)

15 years after Altmans first report, Michael Kaplan
group showed that [3H]-thymidine labeled cells in the
dentate gyrus and olfactory bulb of adult rats have the
ultrastructural characteristics of neurons using EM
(dendrites, synapses) but not of astrocytes or
oligodendrocytes

But still Kaplans work had a small impact on the

dogma.

(1990s) Increasing evidence for CNS plasticity

-Introduction of the synthetic thymidine analogue BrdU

(5-bromo-3-deoxyuridine). Like thymidine, BrdU is
incorporated into the DNA of cells that divide. It can be
detected by immunocytochemistry.
-Introduction of cell-specific markers.

BrdU
CNP

BrdU
MAP2
(neuron)

(oligodendrocyte)
Frontal cortex
Dentate gyrus
Adult macaque Adult macaque

(image from E. Gould)

BrdU
b-tubulin III
(neuron)

BrdU
Fast Blue
(neuron)

Dentate gyrus
Adult rat

Parietal cortex
Adult macaque

Neurogenesis occurs in the mammalian adult brain

Subventricular Zone (SVZ)

Hippocampus

Neurogenesis in the subventricular zone (mouse)

E : ependymal cell
B neural stem cell
C neuronal progenitor
A neuroblast

BMP: Bone Morphogenic Protein

RMS: Rostral Migratory Stream

Alvarez-Buylla A. et al. (2001) Nature Reviews Neuroscience 2: 287-293.

neuroblast

Neuronal
progenitor

Neuroepithelial
cell
Neuroepithelium

Neural stem cell

Radial glia

Embryonic histogenesis

Astrocyte
Adult SVZ

Neurogenesis in the hippocampus

Dividing B cells generate immature D cells that divide and

mature into granule neurons G.
Alvarez-Buylla A. et al. (2002) 57: 751-758.

Neurogenesis in adult mammals :

A role in learning and memory?
1- Neurons are added to the hippocampus.

2-Decrease in proliferation of granule cells in the dentate

gyrus impairs hippocampal-dependent learning.
Acute/chronic stress, ageing, opiate administration
3-Increase in proliferation of hippocampal cells enhance
learning on hippocampal-dependent tasks.
Increased estrogen levels, environment complexity, wheel
running
4-To make new memories may need new neurons
5-New neurons are more plastic and less prone to GABAmediated inhibition than older cells

I- Definition of stem cells

II- Neurogenesis in the adult brain

III-Transdifferentiation
IV- Therapeutic use of Stem Cells

Transdifferentiation
The differentiation of a tissue-specific stem cell into
another type of cell as, for example, a bone marrow
stem cell differentiating into a neuron.
Neural stem cell

Oligodendrocyte Astrocyte Neuron

Hematopoietic stem cell

www.sciencemag.org SCIENCE VOL 290 (December 2000) 1779-1782

Homozygous for a mutation

in the PU.1 gene

XX

XY

Y-chromosome staining in the CNS

female

female

male

female

Neu N,
neurons
FITC-ISH,
Y-chromosome
DAPI,
cell nuclei

Priller J. et al. J. Cell Biol. (2001) 155: 733-738

GFP
GFP: Green Fluorescent Protein

Confocal microscopic analysis of (Bone-marrow)-derived

Purkinje cells

12 months after transplantation of BM cells from transgenic

mice that ubiquitously express GFP, donor-derived Purkinje
cells were visualized by confocal microscopy.

Priller J. et al. J. Cell Biol. (2001) 155: 733-738

Alvarez-Dolado M. et al. Nature (2003) 425: 968-973.

R26R transgenic line

Cre transgenic line

loxP
RTV

loxP
STOP

b-gal

+++
CMV
enhancer b-actin
promoter

Cre

Cre

+++
RTV

b-gal

RTV

Cre is a recombinase enzyme that recognizes specific sequences called

loxP sites.

RTV

R26R

Cell Fusion

R26R/
Cre

Cre

(a) b-gal+ Purkinje neuron in the cerebellum of a R26R mouse transplanted

with Cre+ bone marrow cells. (c) Electron microscopy of the bi-nucleated
fused Purkinje cell.
Alvarez-Dolado M. et al. Nature (2003) 425: 968-973.

IV- Therapeutic use of Stem Cells

Bone Marrow
Arthritis Transplant

Drug
Discovery

Diabetes

Parkinsons
disease
Spinal cord
injury

Heart
Failure

Osteoporosis and
Bone Fractures

Stem Cell Research

Therapeutic Opportunities and Challenges
Head Injury
1.5 Million Cases/Year 100,000 Deaths
5.3 Million Survivors Live with TBI
Societal Costs: $56.3 Billion/Year

Stroke/Cerebrovascular Disease
700,000 Cases/Year 2.4 Million Survivors
#3 Killer in U.S.
Societal Costs: $56.8 Billion/Year

Cardiovascular Disease/Cardiac Arrest

1.2 million Cases/Year
#1 Killer in U.S.
Societal Costs: $142.1 Billion/Year

Spinal Cord Injury

11,000 Cases/Year 250,000 Survivors
Societal Costs: $4 Billion/Year

Neurodegenerative Diseases
Alzheimers Disease 4.5 Million Living with the Disease/$100 Billion/year
Parkinsons Disease 1.5 Million Living with the Disease/$25 Billion/year
ALS 30,000 Living with the Disease/25
year life expectancy

Embryonic Stem Cells

How are the embryonic stem cells grown in the laboratory?

-Transfer of the inner cell mass into a culture dish that contains a
nutrient broth (culture medium).
-The culture dish is coated with mouse embryonic skin cells that have
been treated so they do not divide (feeder layer).
This provides:
1- A sticky surface to which the cells can attach.
2- Nutrients
-To generate cultures of specific types of differentiated cells, scientists
1- Change the chemical composition of the culture medium,
2- Alter the surface of the culture dish, or
3- Modify the cells by inserting specific genes.

Embryonic vs adult stem cells

In cell-based regenerative therapies
Embryonic
-Pluripotent (can become
all cell types of the body)

Adult
-Are limited to become
cell types of their tissue
of origin

-Large number of cells

can be relatively easily
grown in culture

-Rare in adult tissues

and difficult to expand in
vitro

-Possibility of transplant
rejection

-Use of patients own

stem cells would prevent
rejection by the immune
system

-Ethical issues

-No Ethical issues

Somatic Cell Nuclear Transfer (SCNT)

Wilmut I. et al. (1997) Viable offspring derived from fetal

and adult mammalian cells. Nature 385:810-813.

Dolly

SCNT or Therapeutic Cloning

Therapeutic cloning involves

removing the nucleus of an
unfertilized egg cell, replacing
it with the material from the
nucleus of a "somatic cell" (a
skin, heart, or nerve cell, for
example), and stimulating this
cell to begin dividing. Once
the cell begins dividing, stem
cells can be extracted 5-6 days
later and used for research.

magazine.wustl.edu/ Spring05/StevenTeitelbaum.htm

Application of Stem cells for neurological disorders

Stem cell
and
Spinal Cord Injury

Human embryonic stem cell-derived oligodendrocyte

progenitor cell transplants remyelinate and restore
locomotion after spinal cord injury.
Keirstead HS et al. (2005) J. Neuroscience 25: 4694-4705.
7 days p.i.

10 months p.i.

hESC-derived OPCs
Transplanted cells survived
Redistributed over short distances
Differentiated into oligodendrocytes

Enhanced remyelination
Improve locomotor ability

no enhanced remyelination
no locomotor recovery

hESC-derived OPCs : GRNOPC1 Investigational New Drug

January, 2009

First study of a human embryonic stem cell (hESC)-based

therapy in man. Geron plans to initiate a Phase I multi-center
trial that is designed to establish the safety of GRNOPC1 in
patients with "complete" American Spinal Injury Association
(ASIA) grade A subacute thoracic spinal cord injuries.

The ultimate goal for the use of GRNOPC1 is to achieve

restoration of spinal cord function by the injection of hESCderived oligodendrocyte progenitor cells directly into the lesion
site of the patient's injured spinal cord

Transplanted Embryonic Stem Cells Survive,

Differentiate and Promote Recovery in Injured Rat
Spinal Cord
McDonald JW et al. (1999) Nature Medicine 5:1410-1412.

Human Neural Stem Cells differentiate and promote

locomotor recovery in spinal cord-injured mice
Cummings BJ et al. (2005) PNAS 102:14069-14074.

Allodynia limits the usefulness of intraspinal neural

stem cell grafts; directed differentiation improves
outcome
Hofstetter CP et al. (2005) Nature Neurosci. 8: 346-353.

Stem cell and Stroke

Blockage of cerebral artery -> Focal ischemia -> Loss of neurons

and glial cells -> motor, sensory or cognitive impairments.

Transplantation of stem cells into injured brain

stroke-damaged striatum
Human fetal NS cells
Lindvall O. and Kokaia Z (2006) Stem cells for the treatment of neurological disorders.
Nature 441: 1094-1096.

Generation of striatal neurons from endogenous stem cells after stroke.

Figure 2. Possible Approaches to Stem Cell Therapy for Cerebral

Infarction
A, Organ-specific stem cells are harvested from the brain, expanded in
vitro, and reimplanted into the patient. New neurons may be derived from
the neural stem cells to replace those lost during infarction, enabling the
patient to regain lost neurologic function. B, Human embryonic stem cells
from allogeneic donors are reprogrammed in vitro into neural precursor cells
and then reimplanted into the patient. C, Somatic cells (eg, skin cells) are
obtained from the patient, and somatic nuclei are harvested and transferred
to enucleated human oocytes. A blastocyst is formed from the resulting cell.
Cells from the inner mass of the blastocyst are cultured and reprogrammed
in vitro to create neural precursor cells, which are then used to repopulate
the damaged tissue without risk of immunologic rejection. D, Bone marrow
stem cells are harvested from the patient, reprogrammed in vitro to become
neural precursor cells, and reimplanted into the patient to repopulate the
damaged area. E, Combination therapy in which bone marrow stem cells
are harvested, genetically altered through gene transduction, reprogrammed
in vitro to become neural precursor cells, and reimplanted into the patient.

Risks associated with stem cell transplantation

Neuronal progenitor

neuron
astrocyte

oligodendrocyte

Neural stem cell

Glial progenitor

neuroblastoma
neuron
astrocyte

oligodendrocyte
Maturation arrest

Glioblastoma