Psy 163594TK Adult Neural Stem Cells and Neurogenesis

1
Psy 163/594TK Adult Neural Stem Cells and
Neurogenesis

• Seminar Overview
• Everyone has heard something to the effect of
  "take care of your brain cells because you cannot
  grow new ones." During the last decade, this
  central belief in brain biology has been found to
  be less than completely true. The discovery that
  the adult brain has the capacity for producing
  new brain cells or "neurogenesis" has been
  verified in a wide range of species, including
  humans. The present seminar will discuss our
  current understanding of the extent and limits,
  the biology, function, and potential therapeutic
  uses of adult neurogenesis.

http//mentor.lscf.ucsb.edu/course/spring/psyc163t
k/
2
Seminar 1 Embryonic Adult Stem Cells.
3

• Development of Multi-cellular Organisms
• Comparative Embryology

Multi-cellular animals share a common
developmental pattern comprising proliferation
and differentiation (specialization) of
cells. Stem Cells are specialized to
self-renew and NOT differentiate. -important
developmentally for tissue generation (embryonic
gestational tissue-specific) -important in
adult for tissue turnover repair.
Start single cell
Finish billions of cells with hundreds of types.
4
Seminar 1 Outline

• Cell basics
• Cell proliferation
• Early development
• Embryonic Stem cells
• Adult stem cells
• Neural stem cells adult neurogenesis (next day)

5
1. Cell Basics
Cells are composed of distinct structures/compartm
ents containing specialized organelles. Making
new cells requires making copies of all
components of the cell.
Note the schematic is representative of a
relatively undifferentiated cell cellular
specialization involves development of distinct
anatomical structures necessary for specific
functions.
6
Cellular membrane -separates cell from outside
world
Lipids barrier to water Proteins
receptors channels pumps enzymes
An important aspect of the membrane is that it is
dynamic protein lipid components change and
move around critical in proliferation
adaptations.
7
Cytoskeleton -comprised of protein polymers to
gives cell shape again, highly dynamic.
8
Mitochondria -gives cell energy
9
Nucleus -stores information about making proteins
Nuclear Envelope separates from
soma Chromosomes store genetic info Nucleolus
production of RNA
10
Gene Expression -using info stored in nucleus

• Gene Expression
• Transcription
• Translation

1
2
11
Transcription factors control regulation of genes
(i.e. bind to promoters or repressor
regions). Extracellular signals ontribute to the
levels of transcription factors
12
Chromatin Modification
?Acetylation ?Methylation ?Trascription
?Acetylation ?Methylation ?Trascription
13
RNA Silencing
Mechanisms of miRNA-Mediated Gene Silencing (A)
Post-initiation mechanisms. MicroRNAs (miRNAs
red) repress translation of target mRNAs by
blocking translation elongation or by promoting
premature dissociation of ribosomes (ribosome
drop-off). (B) Cotranslational protein
degradation. This model proposes that translation
is not inhibited, but rather the nascent
polypeptide chain is degraded cotranslationally.
The putative protease is unknown. (CE)
Initiation mechanisms. MicroRNAs interfere with a
very early step of translation, prior to
elongation. (C) Argonaute proteins compete with
eIF4E for binding to the cap structure (cyan
dot). (D) Argonaute proteins recruit eIF6, which
prevents the large ribosomal subunit from joining
the small subunit. (E) Argonaute proteins prevent
the formation of the closed loop mRNA
configuration by an ill-defined mechanism that
includes deadenylation. (F) MicroRNA-mediated
mRNA decay. MicroRNAs trigger deadenylation and
subsequent decapping of the mRNA target. From
Eulalio et al 2008 Cell 132, 9-14.
14
2. Cell Proliferation
Multi-cellular life depends on the cells being
able to make copies of themselves through cell
division i.e. proliferate
15
Cell Proliferation

• Cell proliferation is comprised of several stages
  cell cycle.
• Mitosis is the stage of cell division 1 cell
  becomes 2.
• Interphase is the series of stage(s) between
  subsequent mitotic divisions involves
  preparation for mitosis.
• Go is exit from cell cycle important for
  differentiation potentially for understanding
  adult stem cells as well.

16
Mitosis- cell division.
Interphase
Anaphase
Telophase
Prophase
Metaphase

• Mitosis is the most obvious and dynamic stage of
  the cell cycle with easily observable events.

Like all good performances, mitosis requires
extensive preparation.
17
Interphase

• Interphase is divided into separate events
• Gap 1-preparation for doubling DNA
• S-replication of DNA
• Gap 2-preparation for mitosis.
• Transition between stages involves checkpoints
• Checkpoints serve as the regulatory controls for
  cell cycle

18
Checkpoint regulation of Cell Cycle

• The level of various cyclin proteins are the
  molecular basis of checkpoints.
• Cyclins active cyclin-dependent kinases (Cdks)
  which are enzymes/proteins which cause the
  initiation of cell cycle events.

19
Checkpoint regulation of Cell Cycle

• Cell cycle inhibition is mediated by a family of
  proteins which are CDK inhibitors.
• CDKIs mediate exit of cell cycle (to become
  differentiated cells e.g. neurons)
• Critical for prevention of cancers and other
  cellular pathologies.

20
3. Early Development Embryonic Stem Cells
(ESCs)
21
Early Development.
22
(No Transcript)
23
Early Development
24
Development of Lineage Layers
All nervous system tissues develop as part of the
ectoderm.
25
(No Transcript)
26

• Cells isolated from the inner cell mass of the
  blastocyst can be grown in petri dish under
  defined conditions (i.e. cultured).
• These cells can give rise to high numbers of
  cells via cell division.
• Some divisions produce identical cells that
  remain undiferentiated symmetric division
• Some divisions produce non-identical cells with
  one differentiating asymmetric division
• Undifferentiated cells are referred to as
  Embryonic stem cells (ESs).

27

• ICM derived ESs are cultured on a feeder layer
  (i.e. need other cells for characteristics this
  may suggest stemness is a niche involving
  cell-cell interaction rather than a
  cell-autonomous property).
• Cultures require the addition of specific factors
  to help cell survive, proliferate, and
  differentiate (or not). Note Serum is undefined
  bad for understanding the biology.
• Cells are grown until they fill dish
  (confluent) and then passaged.
• Also, need to demonstrate clonality i.e. a
  single cell ( feeders) have properties of
  stemness.

28
(No Transcript)
29
(No Transcript)
30

• Later in development pluripotent SCs can not be
  isolated.
• ESs have a transient existence.
• Multipotent SCs arise and remain for rest of
  life a.k.a Adult Stem Cells (next day)

31

• Basic idea of a stem cell is a cell that it can
  (1) proliferate to produce copies of itself
  self-renewal (2) produce multiple types of
  differenitated cells potency.
• Cells that can form all cells of developing fetus
  are Totipotent (includes extra-embryonic tissue
  cells that can form all cells of the embryo are
  called Pluripotent cells that can form all cells
  of a specific tissue are called Multipotent.

32
(No Transcript)
33
The human embryonic stem cells cultured at
UW-Madison have been observed to randomly
differentiate in culture into a variety of
different cell types, including (A) gut, (B)
neural cells, (C) bone marrow cells, (D)
cartilage, (E) muscle and (F) kidney cells.
Although such differentiation occurs
spontaneously under certain culture conditions,
scientists do not yet know how to direct the
development of embryonic stem cells into specific
cell types.
Note culture systems give rise to heterogeneity
with some cells not differentiating (i.e. remain
ES cells) but we have little idea of how this
works. A cell division that produces two
different daughter cells is called assymmetric
there is growing information of how this occurs
in some cell systems but a priori identification
of stem versus non-stem cell is not complete
34
(No Transcript)
35
(No Transcript)
36
Proof of ES Principle Transgenic Mice

• Making Transgenic Mice
• Embryonic stem cells are manipulated in vitro.
• ES cells are then implanted into pregnant female.
• F1 Offspring are bred.
• F2 Offspring are genotyped for presence of
  manipulated genes if positive then breed, if not
  then go back to 1.

37
Creation of Transgenic Mice
Manipulating ES cells

• ES cells are derived from embryo
• ES cells are manipulated in vitro
• Mutant ES cells are injected into blastocyst
  (embryo)
• Blastocyst containing mutant ES cells are
  implanted into pseudo-pregnant female.
• Blastocyst develops into Chimera animal which
  contains cells of different genotypes.

38
Breeding of Transgenics

• In order for it to work, need mutant ES cell to
  go germ line i.e. generate gonads so that
  mutant gametes are formed (if not time to cry).
• Chimeras (animals with cells of mixed genotypes)
  can be bred to each other or wild-type.
• Offspring will contain mutant gene and can be
  bred as homozygous mutant or heterozygous mutant
  (if transgenetic has breeding problems).

39
Types of Trangenics Knock-In
Knock-In - transgenic mouse expresses extra
gene e.g. fluorescent proteins (below),
pathological genes (expanded huntingtin),
over-expression of endogenous genes
40
ES cells discovered from clinical and basic
science with technologies enabling IVF and
transgenic animals.
41

• Cells isolated from the inner cell mass of the
  blastocyst can be grown in petri dish under
  defined conditions (i.e. cultured).
• These cells can give rise to high numbers of
  cells via cell division.
• Some divisions produce identical cells that
  remain undiferentiated symmetric division
• Some divisions produce non-identical cells with
  one differentiating asymmetric division
• Undifferentiated cells are referred to as human
  embryonic stem cells (hESCs or hESs).

42
(No Transcript)
43
Enter California Institute of Regenerative
Medicine-provides funding for human ES work which
can not be funded federallywill come back to
this in last class.
44
My bet for most feasible ESCs medical
useinsulin-producing cells in treatment of type
I diabetius.

• Pancreas and Diabetes
• Pancreas creates hormone called insulin which
  allows cells to take up glucose. Cells use
  glucose for metabolism.
• Diabetes (honey pee) results from insulin
  deficiency leading to high blood glucose which
  can damage a number of organs.

45
Some differentiated cells derived from ES cells
can produce insulin in response to the glucose
(i.e. act as a glucose receptor and provide
endocrine response). This is likely the most
promising/feasible area for employment of
ES-derived cells in regenerative medicine.
46
(No Transcript)
47
Embryonic SCs and Morality
48
INT 94JY Getting New Brain Cells Neurogenesis in
the Adult Brain Seminar 6 Adult Stem Cells.
49
Embryonic Stem Cells Development
Proof of Principle Basis of all transgenic
Technology
50
(No Transcript)
51
Provide pool of cells for adult tissue
repair/turnover.
Can be maintained in vitro and transplanted for
repair.
52
(No Transcript)
53
Discovery of Adult Stem cells

• Till McColluch, 1961.
• Studying recovery from radiation poisoning via
  bone marrow transplants.
• Found colonies of transplanted cells formed in
  the spleen.

54
Adult vs Embryonic SCs

• Proliferate
• Multipotent
• Replace cells during turnover
• Repair damage to tissue.
• Last life time.

• Proliferate
• Pluripotent
• Important for development of tissue lineages
• Transient existence during early embryogenesis

55
Defining characteristics of Adult Stem Cells

• Proliferate
• Multipotential
• Last life-time
• (extensive self-renewal)

56
(No Transcript)
57
Blood (hematopoietic) Stem Cells
Individual cells in bone marrow can repopulate
blood system. -proliferate, -multipotential,
-last lifetime. -Unique gene expression profile
Proof of Principle Cancer (leukemia) treament
58
(No Transcript)
59

• HSC found in
• Bone marrow
• Peripheral blood
• Umbilical cord

60
(No Transcript)
61
(No Transcript)
62
(No Transcript)
63
What about HSCs and repair of other tissue?
Several studies have reported that
transplantation of an adult stem cell from one
tissue results in production of new cells in a
different tissue (even across lineages).
Plasticity of adult stem cells remain
controversial.
64
Lineage Restriction, Plasticity,
Transdifferentiation
Transdifferentiation
De-differentiation
65
Transdifferentiation versus Fusion
Transdifferentiation
Fusion
Mechanism(s) of SC plasticity remains unclear.
Further, may or may not be able to occur at level
sufficient for biological or clinical relevance.
Nuclear Separation