Chris O’Neill, Head Centre for Developmental & Regenerative Medicine
Kolling Institute for Medical Research [email protected]
99264870
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Stem cells • Indefinite proliferation • Capacity to differentiate into functional somatic cells (unipotent v multipotent v pluripotent) • Genetically and epigenetically stable • To be useful,
•Must be able to survive in recipients •need to be ability to control (direct) differentiation
Potential for stem cells in Medicine
Stem cells
Cell therapy. Eg insulin release, Nerve repair
Immunomodulation MSC are immunosuppressive, Eg Multiple sclerosis
Promote endogenous Repair. Secrete factors that promote capacity of resident cells to repair
Production of specialised cells For in vitro development and testing of new drugs
Production of specialised cells for toxicology studies
Reduce use of animals in medicine
Delivery of repaired genes
Recapitulating in the test tube embryonic development of particular cell types
Early embryo cells – Totipotent, Potential to form ALL the cells in the body
Later in development – pluripotent, Potential to form a range of cells
Progenitor cells – potential to form a particular specialised cell type
Formation of each of the 220 specialized cell types that form the body
GI epithelium Endocrine gland nerve muscle
endoderm mesoderm ectoderm
Placenta Trophectoderm Inner cell mass
epidermis Nails, teeth
Totipotency/Pluripotency
• Cell has capacity to form all lineages within the embryo
• Each cell in early embryo is totipotent • Differentiation leads to a restriction in
potency – pluripotent but not totipotent • Terminal Differentiation – potency
constrained to only one lineage
100,000 sperm
1 embryo (zygote)
48h
4-cell embryo
Blastocyst Attachment
Blastocyst Outgrowth
Trophoblast transformation
UTF1 - epiblast CDX2 - Trophectoderm/-blastGATA6 - Hypoblast (PE) Eomes - T. Giant Cells
5meC REMODELLING POST-IMPLANTATION
Pluripotent - epiblast Utf1
Endoderm – multipotent Gata6
IVF embryos are the source of embryonic stem cells
Keep in test tube (6 days)
Transfer to the uterus (8 weeks)
Collect the stem cells and culture on a ‘feeder layer’
Epigenetics of embryonic development Early embryo cells – Totipotent
Pluripotent progenitors
Multipotent Progenitor
Differentiated – 220 specialized cell types Blood gland nerve muscle
Trophectoderm – Cdx2, Eomes
Totipotent
Pluripotent Endoderm – Gata4, FoxA2, Sox7 Mesoderm – Gsc,Brachury,Mox1 Ectoderm – Lhx1, Elf5, Zbtb17
Differentiated – e.g pancreatic beta-cell, insulin production Pdx1, Pax4, Ngn3, MafA
Pluripotent Multipotent
Inner cell Mass –Utf1, Pou5f1, Sox2, Nanog, Klf4
Differentiation
• Spatiotemporal cues can lead to differences in gene expression
• If get different expression of homeobox genes – differentiation
• Homeobox genes code for the repetiore of gene expression to create a different cell type.
Positional information within the early embryo
• Initially cells have no means of determining their position within time and space
• As embryo becomes more complex – sub-populations have a different spatio-temporal ‘experience’
• This difference creates the opportunity for cells to receive different information and this provides the basis for differentiation
48h
4-cell embryo
2cell 4cell 8cell Morula Blastocyst
DNA
5meC
5hmC
merge
Epigenetics of embryonic development Early embryo cells – Totipotent
Pluripotent progenitors
Multipotent Progenitor
Differentiated – 220 specialized cell types Blood gland nerve muscle
Trophectoderm – Cdx2, Eomes
Totipotent
Pluripotent Endoderm – Gata4, FoxA2, Sox7 Mesoderm – Gsc,Brachury,Mox1 Ectoderm – Lhx1, Elf5, Zbtb17
Differentiated – e.g pancreatic beta-cell, insulin production Pdx1, Pax4, Ngn3, MafA
Pluripotent Multipotent
Inner cell mass –Utf1, Pou5f1, Sox2, Nanog, Klf4
Epigenetics • A mitotically stable/heritable alteration in
the capacity of a gene to be expressed, without any change in the underlying nucleotide sequence.
• Mechanisms influencing epigenetic control include: – Methylation of cytosine within DNA – Patterns of Histone modification – other chromatin molecule e.g. polycomb
proteins, and short RNAs
Epigenetics of embryonic development
Early embryo cells – Totipotent,
Pluripotent progenitors
Multipotent Progenitor
Differentiated – 220 specialized cell types Blood gland nerve muscle
http://www.nature.com/nrg/journal/ v3/n11/fig_tab/nrg933_F3.html
Epigenetic landscape CH Waddington
DNMT
…….CG…….. ……CG……
Provides Topological information on an otherwise bland landscape
Range of specific meC binding proteins, e.g. MBD1
Recruitment of tertiary factors, e.g. SETDB1
Creates higher order chromatin organisation – Heterochromatin
2-cell
4-cell
8-cell morula
Post-implantation
blastocyst
PN1~2 PN3 PN4 PN5
Relative methylation levels
High Low
blastocyst
Epigenetics • On/Off switch or rheostat
active
silent
High resistance to transcription
Low resistance to transcription
demethylated
Methylated
We inherit more than genes from our parents, for some genes we inherit their capacity to be expressed
Male Liger (male) Lion X (female) Tiger
Male Tigon (male) Tiger X (female) Lion
A party trick to demonstrate the power of epigenetics
male donkey X female horse
Mule
male horse X female donkey
Hinny
Gene Imprinting – a special form of epigenetic modification
– Monoallelic expression of some genes depending on the allele’s parent of origin -
Chromatin structure modifiers of gene expression
Chromatin structure modifiers of gene expression
Chromatin structure modifiers of gene expression
Type of modification
Histones H3K4 H3K9 H3K27 H3K79
monomethylation Activators Activators Activators Activators
trimethylations Activators Repressive Repressive repression, activation
acetylation
H3K9 H3K14
Activators Activators
other Prns
PolyComb Protein (PcG) Repressive
Trithorax-group Proteins (TrxG) Activators
Epigenetics • On/Off switch or rheostat
active
silent
High resistance to transcription
Low resistance to transcription
Low resistance to transcription
High resistance to transcription
Activator Chromatin proteins
Repressor Chromatin proteins
demethylated
Methylated
Potential for stem cells in Medicine
Stem cells
Cell therapy. Eg insulin release, Nerve repair
Immunomodulation MSC are immunosuppressive, Eg Multiple sclerosis
Promote endogenous Repair. Secrete factors that promote capacity of resident cells to repair
Production of specialised cells For in vitro development and testing of new drugs
Production of specialised cells for toxicology studies
Reduce use of animals in medicine
Delivery of repaired genes
Viacyte Inc, has created functional beta-cell progenitors from human ESC.
~ USD50 million dollars to date. Another USD100 million and 9-11 years to do safety and clinical testing before possible therapeutic roll out. First clinical testing to commence in 2014. Phase 1 – safety testing only.
Spinal injury clinical trial • Geron and early-stage study for stem cell
therapy on people recently suffering complete thoracic spinal cord injuries(& five other hESC products).
• GRNOPC1 hESCs derived oligodendrocyte progenitor cells (OPC)- neurotrophic actions.
• Initial clinical goals – improved bladder and bowel control. Major improvement in quality of life.
• Trial now cancelled – cost / benefit concerns
Making new eggs and sperm
Nature 2013, 500:392
Major problem with ESC – immunological rejection
• ESC come from a genetically unique individual embryo. Hence different MHC from host (recipient).
Solutions ? • Encapsulation (OK for hormone production, e.g.
insulin, but not for a structural commponent • build very large banks of ESC to cover all
the major MHC classes • Somatic cell nuclear transfer • reprogramme adult cells into embryonic
Historically
• Gonads a special place where cells kept their genetic integrity – totipotency
• Somatic cells - progressive selective silencing of genetic information, except as required for their particular lineage – pluripotency.
• Cloning (Dolly the sheep) – definitive proof that germline and somatic line are not structurally different, rather germline adapted to ensure efficiency of totipotency.
Cloning • Transfer of the nucleus from terminally
differentiated somatic cells to the replace the nucleus of a zygote – reprograms the nucleus to a totipotent state.
• Shows that the loss potency during differentiation is not irreversible.
• The environment of the oocyte/zygote can induce changes that reverse the changes to the genome that occur during differentiation.
• An understanding of how this is achieve has implications for ageing, cancer and developmental abnormalities.
• This are the real implications of development in cloning research.
Cell 2013, 153:1128-38
Epigenetics of embryonic development
Early embryo cells – Totipotent,
Pluripotent progenitors
Multipotent Progenitor
Differentiated – 220 specialized cell types Blood gland nerve muscle
http://www.nature.com/nrg/journal/ v3/n11/fig_tab/nrg933_F3.html
Epigenetic landscape CH Waddington
Induced Pluripotency
Early embryo cells – Totipotent,
Pluripotent progenitors
Multipotent Progenitor
Differentiated – 220 specialized cell types Blood gland nerve muscle
Pou5f1, Sox2, Nanog, Myc, Klf4
Totipotent
Pluripotent Endoderm – Gata4, FoxA2, Sox7 Mesoderm – Gsc,Brachury,Mox1 Ectoderm – Lhx1, Elf5, Zbtb17
Differentiated – e.g pancreatic beta-cell, insulin production Pdx1, Pax4, Ngn3, MafA
iPS cells
Induced pluripotency
• Forced expression of homeotic genes coding for ‘stemness’ can cause differentiated cells to be reprogrammed to embryonic cells Oct-4, Sox2 Nanog
• a gene that induces cell division – c-Myc • a gene that reduces cell death – Klf-4
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of
Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 2007; 131: 861-872.
iPS cells maintain some epigenetic memory of their
donor past
Incomplete reprogramming
Mechanism unknown •Failure to erasure some epigenetic information •An understanding of this required for •Further progress but also valuable for Developmental medicine
Challenges
• Development of personalised versus ‘off-the-shelf’ cell lines
• Adequate demonstration of safety – guard against rogue cells = cancer
• Demonstration of successful Business Model for high cost of development. Particularly for diseases of relatively low prevalence
Nobel Prize for Physiology and Medicine, 2012
John Gurdon Shinya Yamanaka First SCNT Frog (1962)
First iPS cells Mouse (2006)
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