A5-A6

Question 1

There are two type of
‘Stem Cells’.
* Embryonic
* Adult
* Adult stem cells have
different plasticity /
programming ability.

Question 2

Stem cell – a relatively undifferentiated (primitive) cell
that can proliferate (symmetric division) or self-renew
and differentiate (asymmetric division) with one
daughter progressing along a lineage.
* Stem cells can be formed from non-renewing
progenitor cells.
* The fate of one stem cell daughter is usually a
terminally differentiated effector cell.

Question 3

Embryonic stem cells, (ESCs) were first isolated by culturing cells derived
from the inner cell mass (ICM) of blastocysts.
* It is still unclear if ICM cells are homogeneously pluripotent, and if ES cells
derive from a definable and functionally identical pluripotent ICM cell or
population of cells that exists within the blastocyst prior to ES cell isolation
and culture.
* ES cells injected in this manner do NOT form the entire organism but rather
mingle with the cells of the recipient blastocyst.

Question 4

Mouse embryonic fibroblasts (MEFS) are a
differentiated somatic cell lineage with a
‘traditional’ cell cycle.
* The cell cycle in ESCs is relatively rapid,
especially early in development.
* It seems that a lot of Cyclins/Cdks are
dispensable for the cell cycle.

Question 5

• Mouse embryonic stem cells (ESCs)
• Very little D-cyclin expression
• Cyclin E and Cyclin A are always expressed
• No CKIs
• RB always hyper-phosphorylated
• Very short G1

Question 6

Cdh1 – Activates the APC complex (APC/C)
* Emi1 (Early Mitotic Inhibitor 1) – Represses APC/C

Question 7

Human (hESCs) versus mouse (mESCs)
* Embryonic stem cells
* Cyclin B acts as a mitotic cyclin
* No D-cyclin expression
* Cyclin E and Cyclin A are always expressed in mouse ESCs but are
degraded during G2-M phase in hESCs
* Very short G1 in both hESCs and mESCs

Question 8

• Even between mammals, the unique aspects of the ESC cycle are not
  conserved.
• Cyclin B and Cyclin D periodicity are similar between ESCs and the
  somatic (MEF) cell cycle, but there are differences in the relative
  expression level

Question 9

Seeding – movement
of stem cells into the
Niche
* Niche – a specialized
microenvironment
that supports and
promotes Stem Cell
identity.
* Transit Amplifying
Progenitors – Lineage
committed cells that
retain an ability to go
through the cell cycle,
usually to increase
cell numbers before
terminal
deafferentation.

Question 10

Adult stem cells are quiescent,
primitive cells usually located in
specialized microenvironments
(niches) in the body.
* The relative molecular environment
promoting quiescence can be
relatively easy to change (shallow/
primed) or more difficult to change
(deep / dormant).
* Stem cell senescence will be covered
in depth in Lecture 8.

Question 11

• The niche provides signaling, contact
  and other factors that establish and
  maintain a stem cell state.
• The direction of mitosis is critical in
  terms of maintaining a sufficient stem
  cell population or in promoting
  differentiation of daughter cells.

Question 12

Differentiated cells in a niche can
induced toward a stem cell state.

Question 13

Differentiation may simply be a factor of
stem cells asymmetrically dividing out of
range of the niche microenvironment

Question 14

During G1, the chromatin region
where key developmental genes
are found is in a state that would
allow binding/activation by
master transcription factors.
* Cyclin B1 phosphorylates
Smad2/3 which keeps them in
the cytoplasm.
* Cyclin D1 directly represses
Endoderm/Neuroectoderm
genes (Research paper #3).

Question 15

• SRY (sex determining region Y)-box 2 (Sox2) – a transcription factor needed for maintaining self-renewal
  (pluripotency) of embryonic stem cells.
• RB Transcriptional Corepressor Like 1 (RBL1) – Regulates Notch signaling via HES1
• Hairy and Enhancer of Split-1 (HES1) – A transcription factor that interacts with NCID
• CCND1 (encodes Cyclin D), CCNE2(encodes Cyclin E2)

Question 16

The Hematopoietic stem cell (HSC)

Answer 16

• Originally isolated from bone marrow
• Self renewing, can be cultured in-vitro
• Multipotent
• Can develop into each the different blood cell lineages.
• Today – Commonly used to treat leukemia and similar cancers (for
  over 40 years)
• Autologous HSC transplantation is used to reconstitute
  hematopoiesis after myeloablation.
• The range of therapeutic applications of HSC-therapy has increased
  from hematological malignancies to immune deficiencies, solid
  tumours and auto-immune diseases.
• Until recently, HSCs were collected from bone marrow.
• Now, HSC can be obtained from peripheral blood from cytokinemobilized donors and embryonic umbilical cord blood
  precursors.

Question 17

While cells at the end of
the HSC lineage are
easily distinguishable by
morphology, cells in
earlier steps in the
lineage are
indistinguishable
* Cell surface markers are
proteins with
extracellular domains
that can be recognized
by specific (Cluster of
Differentiation)
antibodies that mark
cells for flow cytometry /
cell sorting.

Question 18

The niche is a combination of cellular, signaling,
inflammation, metabolic and ECM/physical
conditions.
* Stem cells must have the appropriate receptors,
CAMs etc. to respond to the niche.
* Niches for each stem cell population are different
and may contain some or all of these factors.
* Niches change over time. * Extrinsic signals must support a quiescent G
0
state.

Question 19

Thrombospondin
-1 (TSP
-1) receptor (CD47)
–
indicates self
-cells to macrophages
* Hedgehog
–
(Hh)
– a morphogen pathway like
Wnt.
* Chemokine
– small molecules that induce cell
movement.
* Fibronectin / Collagen
– structural components of
the ECM

Question 20

Stromal cells -
connective tissue
cells.
Osteoblast – cells
that secrete new
bone mineral matrix
Signalling in the HSC niche – Bone Marrow

Question 21

• Epidermis is composed of a 3D matrix of highly
  adherent cells, layered on top of a well-developed
  basement membrane.
• Impermeable (protective).
• Strongly adherent (cells act as a single
  physical unit).
• Epidermal cell layers are stratified with more
  primitive cells in the basal layer. Symmetric cell
  divisions (SCDs) replenish the basal layer, while
  asymmetric cell divisions (ACDs) drive epidermal
  stratification into the suprabasal layer

Question 22

Single progenitor (SP) model –
An epidermal stem cell (SC)
where cell-fate decisions are
stochastic to replenish SC
population or differentiate.
* SC-CP model - Rare slow-cycling
SCs give rise to committed
progenitor (CP) cells biased
towards differentiation
* 2×SC model - two populations of
SCs: one fast cycling and one
slow cycling. Each make
stochastic fate choices. Proposes
skin regions with fast-cycling SCs
with faster regeneration rates
compared ty regions with slowcycling SCs.

Question 23

ECM / physical stiffness is a
key part of the epidermal
stem cell niche

Answer 23

Niche factors are often specialized for a given
stem cell population. Wound healing, skin
rejuvenation directly responds to tension in
the epidermal layer.
* Wounding would cause areas of lower
tension leading to an increase in SCs
differentiating by asymmetric division
into the suprabasal layer.
* Taut/stiff epidermal layer is characteristic
of ‘normal’ epidermis promoting
replenishment of SC populations
(symmetric divisions in basal layer).
* Response to tissue stiffness / cell spreading is
regulated by the HIPPO pathway interacting
with Notch signalling between adjacent cells.

Question 24

Since cell differentiation states are simply a
product of a specific cell program, it should be
possible to reprogram any cell with an intact
genome to a pluripotent cell-like state.
* In 2006 Taklashai and Yamanaka identified the four
factors (Yamanaka factors) that when activated in a
fibroblast induced a pluripotent state.

Question 25

SNCT is how cloning is usually performed. Transfer to an enucleated oocyte (review Lecture 1) induces a stem cell state. * Fusion of a somatic and existing stem cell leads to a stem-cell state. * Explanted germ cells can be manipulated to undergo parthogenesis to form stem cells. * Retroviral transfection with transgenes expressing Yamanaka factors (or even just transfecting protein) to somatic cells causes transdifferentiation to an iPSC state)

Question 26

* Nanog - Tír na nÓg (Irish for "Land of the Young") – a transcription factor that helps expression of genes that induce a pluripotent state.

Question 27

* A major role of the niche is to promote a quiescent population of stem cells that will divide in a regulated fashion. * A change in niche signals converts it into an environment favoring cell proliferation and growth.

Question 28

Cytoablative chemotherapy of a primary tumour can induce additional programming changes that induce CSC evolution and metastasis.

Question 29

Identifying and studying CSCs is exceedingly difficult as they are present in a mixed population

Question 30

The most commonly used functional test to examine human CSCs in mice (xenografts)

Answer 30

Xenografted cells should be able to progressively grow a tumour with cells that resemble the original (tumour initiating cells). * Isolated cells can be diluted to determine frequency of CSCs in the original tumour.

Question 31

Symmetric versus asymmetric CSC division may affect the severity of cancer

Answer 31

* Disruption of asymmetric division may promote aggressive cancers. * In low-grade cancers, symmetric renewal and asymmetric divisions are fairly balanced, resulting in tumour heterogeneity and maintenance of cancer stem cells (CSCs). * In high-grade cancers, this balance may be shifted towards increased symmetric renewal, resulting in the expansion of the stem cell fraction.

Question 32

CSCs and EMT

Answer 32

EMT model of metastasis (top) - cancer cells lose epithelial cell traits and gain mesenchymal cell traits (dark blue) * Cells to detach from the primary tumour and invade surrounding tissue, intravasate and survive in circulation and, finally, extravasate and form a tumour at a distant site. * Several genes drive EMT, and their expression serves as a ‘marker’. * Some cancer stem cells (CSCs) (bottom) express the EMT gene signature.

Question 33

Tissue engineering / Regenerative Medicine

Answer 33

a) Traditional tissue-engineering seeds cells into engineered scaffolds and ex vivo. b) In situ tissue regeneration uses materials that promote the innate regenerative ability of the body. These include biochemical and biophysical cues to recruit endogenous cells / stem cells.

Question 34

Reprogramming gene expression

Answer 34

Gene expression to drive stem cell differentiation can be regulated by epigenetics, transcriptional control, RNA processing, the biophysical and biochemical microenvironment, and external stimuli. b) One new technique may be nanoparticle -based delivery of transcription factors (TFs) to reprogram cells from one cell type to another has the potential to remodel. c) The Waddington model for cellular reprogramming (we will expand on this in Lecture 7). Cells can be reprogrammed from one type to other by the expression of pioneer transcription factors.

Question 35

Organoids

Answer 35

* Advantages * It is now possible to study human cell derived organ -like structures in a laboratory setting * Some of these organoids may soon be suitable for transplantation into patients. * Disadvantages * These are not true ‘organs’ in most cases. * Organization is often too simple. E.g. no vascularization. * Cells in the center of the organoid are often necrotic due to lack of nutrients or oxygenation.