L16 stem cell

Class CELL BIOLOGY

Created Dec 11, 2020 1249 PM

Materials

Reviewed

Type

What is a Stem Cell ?

Stem Cell Potentials

hierarchy of cell potency

totipotent stems cells- (omnipotent) can give rise to any of the 220 cell types
found in an embryo as well as extra-embryonic cells (placenta)

pluripotent stem cells- can give rise to all cell types of the body (not placenta)
multipotent stem cells- can develop into a limited number of cell types in a
particular lineage
unipotent Precursor cells- tissue resident progenitor/stem cells, capacity to
give rise to one cell type only

L16 stem cell 1

 Mouse Embryonic Stem Cells

L16 stem cell 2

 You see here a picture of the blastocyts in Red ICM (which gives rise to the 3
germ layers , therefore all organs and tissues in our body), in green the
primitive endoderm in blue the surrounding trophectoderm that gives rise to
the placenta and extra-embryonic tissue.

How cell identity is established ?

L16 stem cell 3

 Unraveling control mechanisms of cell identity has direct clinical and
translational relevance
The identity of a cell is established by the interplay between intrinsic factors,
such as TF and epigenetic/chromatin remodeling factors, and extrinsic factors,
released from the surrounding microenvironment
For example, ESC identity requires the TFs OCT4 and NANOG and the addition
of LIF as soluble extrinsic factor to the culture medium

Pluripotent stem cell states in mouse and human

r, Human ESC were established in culture more recently

This is an overview of pluripotent stem cell states in mouse and human.
Both arise from ICM and share self-renewal and pluripotency properties.
Two distinct pluripotent stem cell states have been stably isolated from mouse
embryos:
embryonic stem cells ESCs) derived from the pre-implantation blastocyst are
considered to be in a “naive” pluripotent state, whereas epiblast stem cells
EpiSCs) derived from the post-implantation epiblast are in a “primed”
pluripotent state.
The naive state has an unbiased developmental potential (requires LIF, while
the primed state is slightly more advanced.
Human embryonic stem cells ESCs) and induced pluripotent stem cells (iPSCs)

L16 stem cell 4

 most closely correspond to primate post-implantation embryos /PRIMED state
and do not require LIF but FGF and ACTIVIN (as extrinsic factors)
Work is ongoing to establish human ESCs in a naive state that faithfully
resembles the human pre-implantation blastocyst.

Induced Pluripotent Stem (iPS) cells

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 mayour
specialised cell types of the tissue or organ

L16 stem cell 5

 Adult hematopoietic stem cells HSCs) are a perfect example of professional
ASC, residing primarily within bone marrow, they give rise to blood and immune
cells and are responsible for the constant renewal of blood—the production of
billions of new blood cells each day.

The Stem Cell niche concept

L16 stem cell 6

 HSC have a well-defined niche in the BM
The niche preserves the stem cells in an undifferentiated state
The niche can be cellular or non-cellular for example composed of ECM (eg. in
the case of muscle stem cells)
In the next two slides you will see additional examples of ASC in their niches.

The Epidermal Stem Cell niche

The epidermal stem cells reside in the hair follicle niche

The Intestinal Stem Cell niche

Or the GUT epith stem cell niche.

The small intestinal epithelium
displays an extremely short turnover
time of 5 days.
Actively proliferating Lgr5 intestinal
stem cells reside at the crypt base.
Their rapidly dividing, transit-
amplifying TA daughter cells
occupy the
remainder of the crypts and, upon
differentiation, move onto the flanks
of the villi to eventually die at the
villus tips.

Adult Stem cells

Professional and facultative stem cells
Another example of Professional ASC are the Satellite cells in the skeletal
muscles: a dedicated (professional) population of muscle stem cells. Under
normal conditions (in homeostasis), satellite cells are dormant, following
muscle damage, the stem cells begin both to self-renew and to give rise to a
series of intermediate progeny, which differentiates and form fully mature
muscle cells called myofibres, which contain multiple
nuclei.

L16 stem cell 7

 The Liver does not contain professional stem cells. Under homeostasis,
hepatocytes and bile-duct cells maintain their own populations by proliferation.
However, following damage, unipotent progenitors can be activated and
acquire a bi-potential progenitor state, from which they can self-renew and
give rise to both hepatocytes and duct cells.
Whether a bi-potent progenitor exists in homeostasis is yet to be confirmed >>
this is an example of facultative stem cells

Applications of Pluripotent Stem cells

L16 stem cell 8

 What can we do with pluripotent SC ESC or iPSC?
- Develop Approaches to cell- based therapy : for ex. differentiate ESC or iPSC
into the cell type we need (eg. insulin producing beta-cells and use the cells to
replace the lost cells or non-functional cells in diabetic individuals)
- For Disease modelling: for example combining CRISPRCas9 technology with
iPSCs to insert the gene mutations responsible for the disease
- drug discovery: using drug screening in iPSC-based disease models or
patientderived iPSCs
ALL THESE APPROACHES ARE BASED ON SUCCESSFUL DIRECTED
DIFFERENTIATION OF THE ESC OR iPSC into THE DESIDERED CELL TYPES –
SO THIS IS EXTREMELY IMPORTANT AND A CHALLENGE IN THE FIELD OF SC

Progress in therapies based on iPSCs

L16 stem cell 9

 Before moving into liver and pancreas, I just want to highlight a particular
advantage of using iPSC in Regenerative Medicine approaches compared to
human ESCs. The advantage is the possibility of establishing cells directly from
the patient which in turn will allow autologous iPSC based therapy, gene
editing using to enable repair of disease- causing genetic lesions and drug
screening

Intestine paradigm versus liver and pancreas

L16 stem cell 10

 THE LIVER AND PANCREAS ARE INTERESTING EXAMPLE TO BE DISCUSSED
BECAUSE the 2 tissues are OF HIGH INTEREST FOR CELL REPLACEMENT
THERAPIES/REGENERATIVE MEDICINE, BUT THEY LACK TYPICAL ADULT
STEM CELL COMPARTMENT.
This is completely DIFFERENT for example when COMPARED TO THE
INTESTINE : You have heard in the first part of the lecture about LGR5 cells
AS A PARADIGMATIC EXAMPLE OF ADULT STEM CELLS

Lgr5 organoids technology

L16 stem cell 11

 Lgr5 + cells were subsequently isolated form human intestinal biopsies, are the
ASC of the intestine also in human and can form organoids.

Like the liver, the pancreas is comprised of long-lived cell types

- LIVER IS HIGHLY REGENERATIVE WHILE THE PANCREAS BARELY
REGENERATES
- THEY ALL ORIGINATE FROM A COMMON ENDODERM PROGENITOR
CELLS DURING DEVELOPMENT LIVER AND PANCREAS THEY ARISE A
COMMON BP endoderm progenitor during embryonic development. THE 2 cell
types DIVERGE LATER AND CONTINUE SHARE SOME FEATURES IN ADULT
LIFE.

They are 2 KEY METABOLIC ORGANS

- LIVER (central organ for metabolism, for detoxification, and produce
fundamental enzymes, the serum albumin, growth factors, and bile acids).
- PANCREAS (is two organs in one: as you can see in this picture 2
compartments with 2 vital functions: endocrine compartment/islet control the
blood glucose metabolism; the exocrine/acinar and duct compartment control
the food digestion)

The 2 compartments of the pancreas are targets of still incurable diseases,

such as
Pancreatic cancer which affects the exocrine compartment and is one of the
cancer with worst prognosis.

L16 stem cell 12

 Diabetes which affect endo compartment. Diabetes is a global epidemic with
more than 400 individuals suffering of diabetes.
Importantly, stem cells and learning how to differentiate them into pancreatic
cells is of fundamental importance for developing a cure to diabetes.

Liver diseases

The Liver is also target of diseases: including genetic metabolic diseases,

cancer and hepatitis > these diseases when they become chronic they may
result into liver failure
Even if the liver has the ability to regenerate, proper regeneration is often
comprised in chronic conditions

How these different cell IDs are established? This is controlled by the interplay
between a series of Molecular Players that can be distinguished into intrinsic
factors TF, epigenetic modifiers) and extrinsic factors (which are the signaling
molecules released
by the surrounding tissues)
In the next slides I will discuss some of these molecular players, I chose some
specific examples.
Unravelling the mechanisms behind cell IDs has direct clinical relevance,
because we can reuse these same mechanism for example to differentiate a
SC into pancreatic cells and use it for cell replacement therapy of diabetes.

Pancreatic lineage fate decision

L16 stem cell 13

 Here you are looking at a schematics illustrating the main steps of the lineage
pancreatic fate restriction and you also recognize the cascade of pancreatic
TFs, which regulate this different steps and the lineage decision.
For example, once pancreatic fate is specified the pancreatic progenitors start
expressing PDX1 and later Endocrine progenitor cells turn on this factor called
NGN3 and this cell type will form the islet cells [these beautiful structures in
Green] that produce insulin.
Now this cascade of TF is initiated and regulated by the surrounding extrinsic
factors, signaling molecules, such as FGF10 that is fundamental for the growth
and expansion of the PP.

Different approaches for making pancreatic cells

L16 stem cell 14

 How this knowledge on cell ID can be used for making pancreatic cells?
Programming ESC into pancreatic cells Or using lineage reprograming.
In lineage reprogramming, adult differentiated cell types can be used as cellular
source and converted into pancreatic beta cells: starting from another
pancreatic cell type (intraorgan) or a cell type from another organ (interorgan
reprogramming)
The liver is a quite attractive source because of all the information I shared with
you during my talk:
Common origin (so in theory easy to convert)
Abundance of the cells
Easy access

Engineering pancreatic cells using a developmental logic

Because of this good level of conservation, the scientific field has started using
this knowledge of pa lineage development and differentiation to define
protocols for differentiating human ESC into pancreatic cell types.
First attempts started in early 2000 this was the first successful protocol
published
Recapitulate pancreatic development is the recipe for success

Regenerative Cell Therapies for Diabetes

L16 stem cell 15

 Having defined a road map for generating beta-cells in the dish > this has
opened up new avenues for cell therapies for curing diabetes
The current challenges and bottlenecks are the fact the obtained beta-cells are
not perfectly mature and functional as the endogenous counterpart and the
challenges of immunosuppressant and transplantation of stem-cell derived
beta-cells

Studying human pancreas development

L16 stem cell 16

 Besides the regenerative medicine applications, these differentiation protocols
have enabled us to develop model systems for studying human pancreatic
development by combining the differentiation protocol with CRISPRCas9 for
gene editing in human ES or iPS cells.
For example, in this paper the role of the TF NGN3 was finally elucidate in the
human cells.
These studies confirmed conservation between human and mouse pancreatic
development
Specifically NGN3 mutations, these 2 papers confirmed the notion that human
point mutations identified are hypomorphic and not complete loss of function,
thus allowing these patients to be born with a functional endocrine pancreas.
Valuable model system for studying human pancreatic developm

Sequential stages of liver formation

Now I go back to the liver and very briefly illustrate some of the main steps of
liver development and acquisition of hepatic cell ID
Hepatoblasts are bi-potent progenitor cells that can give rise to the 2 lineages

L16 stem cell 17

 that compose the liver parenchyma: the heps and bile duct
cells/cholangiocytes
The cascade of TFs controlling the different steps of specification and
differentiation has been also well figured out in the liver thanks to genetic
experiments in the mouse embryo.
Important TFs in conferring hep cell ID are the FOXAs, GATA4 and HNF4,
HNF1b.
Like what I showed you in the pancreas, signaling pathways (released from the
surrounding microenvironment, such as BMP, Wnt signaling) impact on the TF
cascade to control the lineage decision.

Cell-Based Regenerative Therapy for Liver Disease

There is also high demand of fully functional hepatocytes that can be

differentiated from stem cells to be used for transplantation in case of liver
failure for example.
The idea is the same as for pancreatic cell differentiation: the differentiation
protocol for generating hepatocytes has been worked out based on the
knowledge of liver development and hepatocyte differentiation. Similar
limitations as for the pancreas are:

L16 stem cell 18

 MATURITY of the cells and the abundance.
It is also possible to use hESC or iPSC-derived cells to generate organoids.

Generating iPSC–derived liver organoids

In this example from the Takebe lab. not only they made liver organoids from
pluripotent cells, but they mix different cell types (endothelial and
mesenchymal cells), as I mentioned earlier several times during my talk this is
what happens in normal organogenesis

Summary
• Fundamental properties of stem cells
• Different types of stem cells
• Potential applications of stem cells in regenerative medicine
• Intestine: adult stem cells & organoids
• Pancreas and liver: applying developmental biology
lessons to stem cell differentiation

L16 stem cell 19