Detailed revision notes from lecture course on stem cells taught by Dr Khaled, University of Cambridge, with additional reading and insights from key publications summarised.
Stem Cells in Health, Disease, Pharmacology and Regenerative Medicine
Walid Khaled wtk22@cam.ac.uk
Outline
Embryonic stem cells
• Origin of embryonic stem cells (ECSs)
• Regulation of lineage choice - epigenetics
• Derivation of ESCs – benefits of pharmacological approach
• Manipulating ESCs
Generation of pluripotent stem cells from differentiated adult lineages
• Cell fusion
• Somatic cell nuclear transfer
• Induced pluripotency (iPS)
• Pharmacological and regenerative applications of iPS cells
Adult stem cells
• Regulation of self-renewal versus differentiation - importance for tissue health
Stem cells in regenerative medicine and research
Reading List:
Stem Cells in the Embryo
Yeo & Ng 2013 Cell Res 23:20. The transcriptional regulation of pluripotency.
Wray et al 2010 Biochem Soc Trans 38:1027. The ground state of pluripotency.
Stem Cells and Epigenetics
*Christophersen N S , and Helin K J Exp Med 2010;207:2287-2295.
*Young 2011 Cell 144:940. Control of the embryonic stem cell state.
Hemberger et al 2009 Nat Rev Mol Cell Biol 10:526. Epigenetic dynamics of stem cells and
lineage commitment: digging Waddington’s canal
Culturing Embryonic Stem Cells Ex Vivo
*Nichols & Smith 2012 Cold Spring Harb Perspect Biol 4:a008128. Pluripotency in the
embryo and in culture.
Methods for Reprogramming to Pluripotency
*Yamanaka & Blau 2010 Nature 465:704. Nuclear reprogramming to a pluripotent state by
three approaches.
Epigenetic Mechanisms of Reprogramming
Plath & Lowry 2011 Nat Rev Genet 12:253. Progress in understanding reprogramming to the
induced pluripotent state.
*Orkin & Hochedlinger 2011 Cell 145: 835. Chromatin connections to pluripotency and
cellular reprogramming.
Drug Development, Toxicology and Disease Modelling
Jensen et al 2009 J Cell Physiol 219:513. Human embryonic stem cell technologies and drug
discovery.
,Yamanaka 2009 Cell 137:13. A fresh look at iPS cells
*Bellin et al 2012 Nat Rev Cell Mol Biol 13:713. Induced pluripotent stem cells: the new
patient?
Shi Y et. al Nature Reviews Drug Discovery 2017 Induced pluripotent stem cell technology: a
decade of progress.
Essays Questions:
Examples:
How can you make stem cells from skin cells?
Discuss the challenges to using iPSCs in the clinic.
2018 (paper 3) Discuss how induced pluripotent stem cells can be used for disease
modelling of, and drug discovery for, complex diseases.
2017 (paper 3) What is the impact of the niche and of intrinsic factors on developing
effective regenerative therapies?
2016 (paper 3) Compare and contrast the different methods for reprogramming somatic
cells to pluripotent stem cells
2015 (paper 4) Discuss how induced pluripotent stem cells (iPSCs) are transforming the field
of regenerative medicine.
2014 (paper 4) What genetic, epigenetic and signalling mechanisms are required for
pluripotency in embryonic stem cells? How do these relate to the induction of pluripotency
in differentiated cells?
2013 (paper 4) Discuss the mechanisms required to generate induced pluripotent stem cells
(iPSCs) from differentiated somatic cells. How can iPSCs enhance disease modelling, drug
discovery and regenerative therapies?
,Lecture 1: Stem cells
What is a stem cell?
• Stem cell is a cell that divides indefinitely in culture and is undifferentiated but can
generate differentiated daughter cells.
Embryonic stem cells (ESCs) are derived from inner cell mass of preimplantation blastocyst
• Spontaneously form 3 primary germ layers – ectoderm, mesoderm and endoderm in
culture
• When injected into immune-suppressed mice, form teratomas (benign tumours that
consist of differentiated cells from all 3 germ layers)
• When injected into host blastocysts, may generate all lineages (~210 different cell
types in mice)
Adult stem cells
• Undifferentiated cells (quite rare) found in most tissues that can self-renew and yield
specialised cell types of that tissue
• Internal repair system
• These cells are long-lived but not immortal
Definitions
• Totipotency = ability to give rise to all cell types of the body and all of the cell types
that make up extraembryonic tissues
• Pluripotency = ability to generate all of the cell types of the body, i.e. not extra-
embryonic tissues
• Multipotency = develop into more than one cell type of the body
• Unipotency = develop into one cell type of the body
Early embryonic development in the mouse
• At the late blastocyst stage, embryonic stem cells form, developmental potential of
the zygote diminishes as the embryo develops. Lose totipotency between day 0 and
day 4. Reside in inner cell mass. Three germ layers in the gastrula. Predetermination
for future differentiation. Embryonic stem cells in vivo are very transient. Only exist
for 1 day in this state from E4.5-E5.5.
• At the gastrula stage, ecotderm (external layer) differentiates into skin cells of
epidermis, neurons of the brain and pigment cells; germ cells – into sperm and egg
, (gender-based); mesoderm (middle layer) – into cardiac muscle, skeletal muscle,
tubule cells of the kidney, red blood cells, smooth muscle in the gut; endoderm
(internal layer) – lung cells, thyroid cells, pancreatic cells.
Establishing embryonic stem cell cultures (Evans et al, 1981)
• ESCs isolated for the 1st time in vitro from the inner cell mass of a late stage
blastocyst
• Managed to culture in vitro on irradiated mouse fibroblasts
• Can be propagated almost indefinitely and remain the characteristics of stem cells
• Tightly packed round-shaped colonies
How do cells repair their genome?
• Used in gene targeting but very low efficiency and ratio of HDR to NHEJ is very low (1
in 1000)
Gene targeting using embryonic stem cells
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