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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
 
clonal
 
stem cells
 
Developmental Models
 
Signals, position, time
 
differentiation
 
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)
 
http://stemcells.nih.gov/index.asp
 
Stem Cells Report (2001)
 
http://stemcells.nih.gov/info/scireport/
 
 
 
Stem cells in Development
 
Blastocyst
 
 
 
 
Chimeric Mouse
 
ES or teratocarcinoma
 
shows that stem cells can combine with cells of a normal blastocyst to form a healthy chimeric mouse
 
Historic References
 
Mouse
 
Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.
 
Martin GR. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634-8.
 
Characterization of a pluripotent stem cell line derived from a mouse embryo.
 
Wobus AM, Holzhausen H, Jakel P, Schoneich J. Exp Cell Res. 1984 May;152(1):212-9.
 
Transgenesis by means of blastocyst-derived embryonic stem cell lines
 
Proc Natl Acad Sci U S A. 1986 Dec;83(23):9065-9. Gossler A, Doetschman T, Korn R, Serfling E, Kemler R.
 
Historic References
 
Pig and Sheep
 
Derivation of pluripotent, embryonic cell lines from the pig and sheep.
 
Notarianni E, Galli C, Laurie S, Moor RM, Evans MJ. J Reprod Fertil Suppl. 1991;43:255-60.
 
Primate
 
Isolation of a primate embryonic stem cell line.
 
Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, Hearn JP. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7844-8.
 
Human
 
Embryonic stem cell lines derived from human blastocysts.
 
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Science. 1998 Nov 6;282(5391):1145-7.
 
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
 
Neurodegenerative
 
Injury
 
 
 
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
 
Epithelia
 
Gut
 
Skin
 
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:
 
Isolate
 
Grow
 
Maintain, store
 
Differentiate
 
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
 
Neural
 
Parkinson’s, ALS, spinal cord injury……..
 
Cell Replacement
 
cell death, loss of function
 
Grafting
 
where host-graft rejection normally requires substantial ongoing immunosuppression
 
Repair
 
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
 
Human
 
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
 
Mouse
 
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)
 
Time
 
“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