Chris O’Neill, Head Centre for Developmental & Regenerative Medicine

Kolling Institute for Medical Research chris.oneill@sydney.edu.au

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)