Difference between revisions of "Talk:2009 Lecture 21"

From CellBiology
(Removing all content from page)
(4 intermediate revisions by the same user not shown)
Line 1: Line 1:
ANAT3231 Lecture 13�Stem Cells/Development
Dr Mark Hill
Room G20
Email: m.hill@unsw.edu.au
UNSW Copyright Notice
Textbook/Online References
Essential Cell Biology
Chapter19 Tissues p622-627
Molecular Biology of the Cell (3rd ed)
Chapter 19 Cellular Mechanisms of Development p1037-1039
Online References
UNSW Embryology
Molecular Biology of the Cell online
Molecular Cell Biology online. 4th ed. Lodish etal
Lecture- Development/Stem Cells
Establishment of order from 1 cell
stem cells
Developmental Models
Signals, position, time
Patterns, programs
3 Adult Cell Types (Proliferation)
Differentiated cells
no longer capable of cell division
Cardiac muscle cells, neurons
produced during embryonic development, differentiate, then retained throughout life of organism
Cells in G0 stage of cell cycle
resume proliferation when needed to replace cells
skin fibroblasts, smooth muscle cells, endothelial cells, epithelial cells of internal organs, such as the liver, pancreas, kidney, lung, prostate and breast
Stem Cells
differentiated cells have short life spans continually replaced
blood cells, epithelial cells of skin and digestive tract
fully differentiated cells do not proliferate
proliferation of less differentiated- stem cells
produce daughter cells that either differentiate or remain as stem cells
Blood Cells
All different types of blood cells develop from a pluripotent stem cell in bone marrow
Precursors of differentiated cells undergo several rounds of cell division as they mature
proliferation ceases at terminal stages of differentiation
Movie: Neural Stem Cell
PubMed- Stem Cells
Medline Search “stem cell”
2002 - 110,920
2004 - 128,485
2005 - 140,966
2006 - 154,176
NIH - Stem Cells
National Institute of Health (USA)
Stem Cells Report (2001)
Stem cells in Development
Why are they in the News?
Scientific and Ethical
Therapeutic uses
Issues relating to human cloning
Use of excess human eggs/sperm for research purposes
Availability of human stem cell lines
What can they be used for?
Generation of “knock out” mice
Studying regulation of cell differentiation in development
Therapeutic uses?
Genetic disease
FourHumanESCell Lines
Long Term ES Cell Culture
Human ES Cell Differentiation
Stem Cells in the Adult
Connective Tissue
Bone marrow
Blood Cells, Osteoclasts, blasts
Epidermis: Immortal Stem Cell
Stem cell
each generation at least 1 "immortal" stem cell
descendants present in patch in future
Other basal cells
leave basal layer and differentiate
Committed, born different
or may be stem cells
equivalent to immortal stem cell in character
mortal in sense that their progeny jostled out of basal layer and shed from skin
Amplifying Cells
Stem cells in many tissues divide only rarely
give rise to transit amplifying cells
daughters committed to differentiation that go through a limited series of more rapid divisions before completing the process.
each stem cell division gives rise in this way to eight terminally differentiated progeny
Stem Cell Production
Stem Cell Daughter Fates
Environmental asymmetry
daughters are initially similar
different pathways according to environmental influences that act on them after they are born
number of stem cells can be increased or reduced to fit niche available
Divisional asymmetry
stem cell has an internal asymmetry
divides in such a way two daughters are already have different determinants at time of their birth
Neural Stem Cells: Development
Neural Stem Cells: Adult
Neural Stem Cells: Adult
Purification of a pluripotent neural stem cell from the adult mouse brain.
Rietze et al. The Walter and Eliza Hall Institute of Medical Research
purified NSCs from the adult mouse brain by flow cytometry, and directly examined the cells' properties
Nature. 2001 Aug 16;412(6848):736-9.
Current research on stem cells
How to:
Maintain, store
Therapeutic uses
Growth of Embryonic Stem Cells
Mouse blastocyst-derived ES cell line D3
from American Type Culture Collection (ATCC)
Undifferentiated ES cells
maintained on gelatin-coated dishes
earlier studies, feeder layer
DMEM (dulbecco’s modified essential media) with
2 mM glutamine (essential amino acid)
0.001% beta-mercaptoethanol (reducing agent)
1x nonessential amino acids (amino acids for growth)
10% donor horse serum (source of growth factors etc)
human recombinant leukemia inhibitory factor (LIF) 2,000 units/ml
Stem Cell Markers
Every cell surface has specialized proteins (receptors) that can selectively bind or adhere to other “signalling” molecules (ligands)
Different types of receptors differ in structure and affinity for signalling molecules
Cells use these receptors and molecules that bind to them as a way of communicating with other cells and to carry out their proper functions in the body
Covered in Lecture 11, 12
Same cell surface receptors are stem cell markers
Stage-Specific Embryonic Antigen-1,4
Tumor Rejection Antigen (TRA-1-60)
Stem Cell Antigen 1 (Sca-1)
Marker Examples
Stage-Specific Embryonic Antigen-1 (SSEA-1)
role in cell adhesion, migration and differentiation
often differentially expressed during development
Stage-Specific Embryonic Antigen-4 (SSEA-4)
surface embryonic antigen of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES)
down-regulated following differentiation of human EC cells
Antigen not expressed on undifferentiated murine EC, ES and EG cells but up-regulated on differentiation of murine EC and ES cells
ES Therapeutics
Possible Therapeutic Uses
Parkinson’s, ALS, spinal cord injury……..
Cell Replacement
cell death, loss of function
where host-graft rejection normally requires substantial ongoing immunosuppression
Spinal cord and brain injury
Other Diseases
Diabetes, muscular dystrophies, cardiac, vital organs……
Stem Cells and Grafting
Nature paper showed possible therapeutic use of ES cells in grafting
host-graft rejection normally requires substantial ongoing immunosuppression
Pre-implantation stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning
Aug02 FANDRICH etal.
Stem Cell Experiments
2 labs in 1998 human embryonic stem cells Gearhart 1998, Thomson et al. 1998
inner cell masses of embryos not implanted into infertility patients
germ cells derived from spontaneously aborted fetuses
ES cells cultured in conditions to form glial stem cells
glial stem cells were transplanted into mice that had a genetic deficiency of glial function, and cured defect (Brüstle et al. 1999)
Neural stem cells derived from mouse ES cells
divide and differentiate into functional neurons when injected into a damaged rodent nervous system (McDonald et al. 1999)
Human Stem Cells
Human neural stem cells improve sensorimotor deficits in adult rat brain with experimental focal ischemia
Ischemic stroke caused by interruption of cerebral blood flow leads to brain damage with long-term sensorimotor deficits
intravenous injection
determine migration, differentiation and long-term viabilities of human NSCs in rat brain
Human NSCs were detected in lesion side and labelled with marker for neurons or astrocytes
Post-ischemic hemispheric atrophy noted but reduced in NSCs-ischemia group
Neural Therapeutic uses?
Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model
Implantation of fetal dopamine (DA) neurons can reduce parkinsonism in patients
current methods are rudimentary
lacking a reliable donor cell source
Transplanted ES cells can develop spontaneously into dopamine (DA) neurons
Such DA neurons can restore cerebral function and behavior in an animal model of Parkinson's disease
Björklund et al Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 4, 2344-2349, February 19, 2002
Parkinson Rat Model
Embryonic stem cell Transplant
transplanting low doses of undifferentiated mouse embryonic stem (ES) cells into rat striatum
results in a proliferation of ES cells into fully differentiated DA neurons
ES cell-derived DA neurons caused gradual and sustained behavioral restoration of DA-mediated motor asymmetry
Björklund et al Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 4, 2344-2349, February 19, 2002
Staining of a Graft
16 weeks after implantation of D3 ES cells into adult 6-OHDA lesioned striatum
TH-positive neurons were found within the graft (A and B, green)
All TH-positive profiles coexpressed the neuronal marker NeuN (A, red)
TH (B) also was coexpressed with DAT (C, red) and AADC (D, blue), shown by white triple labelling (E)
Rotation response to Amphetamine
6-OHDA-lesioned animals were selected for transplantation by quantification of rotational behaviour in response to amphetamine
response was examined post-transplantation at 5, 7, and 9 weeks
Animals with ES cell-derived DA neurons showed recovery over time from amphetamine-induced turning behavior
Developmental Models
What makes one cell different from the next?
Developmental Models
drosophila- fruit fly
c. elegans- worm
xenopus- frog
zebrafish- fish
chicken- bird
mouse- mammal
Background to Patterning
All cells contain the same genetic material and are initially equipotent
Development is about pattern formation
axis and tissue formation
Regulators of pattern - signaling
Contact (cell-cell, cell-ECM, surfaces)
Morphogen (soluble, modified)
“window”, undifferentiated/differentiated, stem
Genome modification, regulators
Drosophila Development Model
Position-Specific Patterning
Cascade of short range inductions
Secretion of long range signal
Long range secondary signal from primary inductive
spinal cord /neural crest, examples of complex structure generated by such a signalling process
Short Range Inductive Cascade
Long Range Signal
Long Range Secondary Signal from Primary Inductive

Latest revision as of 10:12, 2 June 2009