ANAT3231: lectures overview - … lectures overview ... Stem Cell Biology Stem Cell Technology...
Transcript of ANAT3231: lectures overview - … lectures overview ... Stem Cell Biology Stem Cell Technology...
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ANAT3231: lectures overview
Dr Annemiek Beverdam – School of Medical Sciences, UNSW Wallace Wurth Building Room 234 – [email protected]
Stem Cell Biology
Stem Cell Technology
Resources: http://php.med.unsw.edu.au/cell biology/
Essential Cell Biology – 3rd edition Alberts
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ANAT3231: lab next week
Dr Annemiek Beverdam – School of Medical Sciences, UNSW Wallace Wurth Building Room 234 – [email protected]
Journal club presentation
Student groups
Choose 2-3 articles and send me PDFs by Friday afternoon 5 pm
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ANAT3231: lectures overview
Dr Annemiek Beverdam – School of Medical Sciences, UNSW Wallace Wurth Building Room 234 – [email protected]
Stem Cell Biology
Tissue homeostasis and regeneration Stem cell biology Stem cell niches
Stem cell regulation Stem cells and cancer
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ANAT3231: lectures overview
Dr Annemiek Beverdam – School of Medical Sciences, UNSW Wallace Wurth Building Room 234 – [email protected]
Stem Cell Technology
Regenerative Medicine Stem Cell Sources
Future of Regenerative Medicine Knock-out Technology
CRISPR/CAS9 Genome Editing
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Regenerative medicine the clinical application of stem cells
"process of replacing or regenerating human cells, tissues or organs
to restore or establish normal function"
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Stem Cell Sources for Regenerative Medicine
Multipotent
Stem cells derived from embryos
Stem cells derived from adults
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Stem Cell Sources for Regenerative Medicine Waddington's model of epigenetic determination of development
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Embryonal Carcinoma Cells are pluripotent
1964 – Pierce and Kleinsmith isolate EC cells from teratocarcinomas Source: gametes
Pluripotent In vitro culture and expansion
Genetic abnormalities
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Embryonic Stem Cells are pluripotent
1981 – Martin Evans, Matthew Kaufman and Gail Martin
Pluripotent No genetic abnormalities
In vitro culture and expansion Ethical issues
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Adult stem cells
“An undifferentiated cell, found among differentiated cells in a tissue or organ that can renew itself and can differentiate to yield some or all of
the major specialized cell types of the tissue or organ”
- Bone marrow stem cells: haematopoietic stem cells - Neural stem cells - Intestinal stem cells - Skin stem cells - Umbilical cord stem cells: haematopoietic stem cells
No ethical issues Restricted plasticity Limited quantities Hard to identify
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Somatic Cell Nuclear Transfer John Gurdon, 1958
The developmental potential of nuclei of differentiated cells
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Somatic Cell Nuclear Transfer
“mature, differentiated cells can be reprogrammed to become pluripotent”
Pluripotent (totipotent?) Low success rate
Genetic/phenotypic abnormalities Ethical issues
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Reproductive/Therapeutic Cloning
Pluripotent (totipotent?) Low success rate
Genetic/phenotypic abnormalities Ethical issues
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Nuclear Reprogramming Induced pluripotency (iPS), Yamanaka, 2006
“mature, differentiated cells can be reprogrammed to become pluripotent”
Oct4 Sox2 c-Myc Klf4
2-3 weeks
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Nuclear Reprogramming Induced pluripotency (iPS), Yamanaka, 2006
“mature, differentiated cells can be reprogrammed to become pluripotent”
Oct4 Sox2 c-Myc Klf4
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Stimulus-Triggered Acquisition of Pluripotency (STAP) (Obokato 2014, controversial)
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Stem Cell Sources Embryonic vs Adult Stem Cells
iPS Cells
- Can generate any cell type - Easy to generate, maintain
and grow in lab - Perfect genetic match to
patient
- May retain age of parental cell
- Inheritance of mutations: teratomas
- May retain age of parental cell
- Inheritance of mutations: teratomas
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The Future of Regenerative Medicine
! ?
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?
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Future Stem Cell Technologies
1- how we can induce and maintain pluripotency? 2- how we can direct differentiation? 3- how we can cure diseased cells? 4- how we can repair mutations in cells?
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Future Stem Cell Technologies
How can we direct differentiation? - Uncontrolled differentiation
- Directed differentiation
? ?
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Future Stem Cell Technologies Directed differentiation of cardiomyocytes
Mummery et al., Circ Res 2012
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Future Stem Cell Technologies Directed differentiation of motor neurons
Dong et al., Nature 2014
Pluripotent stem cells
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Future Stem Cell Technologies Directed differentiation of pluripotent stem cells
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Future Stem Cell Technologies
How can we direct differentiation?
Directed differentiation: learn from developmental biology!
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Future Stem Cell Technologies
How can we cure disease?
Disease Modeling and Drug discovery
(personalized medicine)
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Future Stem Cell Technologies
How can we repair mutations in cells?
Gene Therapy:
Knock out technology
CRISPR/CAS9 genome editing
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Crossing over is a natural process that happens during meiosis
Knock out technology = directed homologous recombination in pluripotent ES cells
Knock out technology
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Endogenous gene
ATG
neoR
= regions of homologous DNA sequence
neoR
Targeting vector
Knock out allele
Homologous recombination
Knock out technology
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Knock out technology
1 Creating knockout DNA construct
3. Heterozygous mutant ES cells
2. Generating embryonic stem cells
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Chimeric mice
Genetic crosses to obtain Homozygous mutant mice
Heterozygous mutant Embryonic stem cells
Knock out technology
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Knock out technology Engineering of targeting vectors
Repair mutations
*
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Knock out technology Engineering of targeting vectors
Expressing multiple genes from same GM locus
Gene X
Gene X
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Knock out technology
Allows us to:
Study gene function in mice
Repair mutations or to express a second protein from a GM locus in ES cells
However:
Heterozygosity in recombined ES cells
(Low rates of homologous recombination)
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CRISPR/Cas9 Genome Engineering (Clustered Regularly Interspaced Short Palindromic Repeats)
Guide RNA and Cas9
http://www.youtube.com/watch?v=0dRT7slyGhs
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CRISPR/Cas9 Genome engineering Repair
Homology-directed repair: Provide donor template with homology arms
Gene mutation/correction/addition (Cas9 D10A mutant)
Non-homologous end joining: Small insertion/deletion
gene disruption (and occasional errors)
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CRISPR/Cas9 Genome engineering Applications in Stem Cells
ES cells iPS cells
Zygotes
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CRISPR/Cas9 Genome engineering Applications in regenerative medicine
http://www.youtube.com/watch?v=0dRT7slyGhs
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CRISPR/Cas9 Genome engineering Repair of Cystic Fibrosis Gene CFTR
(cystic fibrosis transmembrane conductor receptor)
Schwank et al., Cell Stem Cell 2013
Forskolin -> CFTR -> expansion
In vitro assay in intestinal organoids:
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Stem Cell and Gene Therapy
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ANAT3231: lectures overview
Dr Annemiek Beverdam – School of Medical Sciences, UNSW Wallace Wurth Building Room 234 – [email protected]
Stem Cell Technology
Regenerative Medicine Stem Cell Sources
Future of Regenerative Medicine Knock-out Technology
CRISPR/CAS9 Genome Editing