Tuesday:

Monday:
Induced pluripotent stem
Neural Development
technology
The magic of the biology
The magic of applying our
behind the creation of the
knowledge to create the
human brain.
human brain in vitro (models
Stem Cells and induced
pluripotent stem cells
Sebastian Illes
Department of Physiology
Institute for Neuroscience and Physiology
Contact: sebastian.illes@neuro.gu.se

February, 2023
Overview
• Definition of stem cells
• Adult neurogenesis
• Exogeneous resources for human neural stem cells
• Embrynoic stem cells
• iPSC technology
• Direct reprogramming
• Potential medical application of iPSC technology
• Human iPSC brain cell in vitro models
• Our research in the field

Above listed sections are linked to the slides within this power point presentation. Each slide has
in the right upper corner an ”overview” box that is linked to this overview slide.
Exam questions can be answered solely based on the information the slides provide.
Slides will be uploaded after the lectures have been held.
 overview
Defining Stem Cells
Stem cells can:
• Maintain themselves over long a
period of time as unspecialized cells
(life-long for adult stem cells).
• Expand through symmetric division.
• Generate various specialized cell types
through asymmetric division.
• Def. symmetric division: Daughter
cells are equal. A stem cell give rise to
two stem cells.
• Def. asymmetric division: Daughter
cells are not equal. A stem cell give
rise to a stem cell and differentiated
cell
 overview
Regulation of tissue growth and differentiation
 overview
Definitions of Stem Cell Potency
• Totipotent:
Can generate all cell types of an organism,
including the extra-embryonic tissues (e.g.
zygotes)
• Pluripotent:
Can generate cell types from the 3 germ
layers (e.g. cells from the blastula's inner
mass)
• Multipotent:
Can generate several cell types (often
layer/organ specific e.g. mesenchymal stem
cells generate osteoblasts, chondrocytes, and
adipocytes)
• Bi-, Unipotent:
Concept still in evolution. Unclear how stem
cells with such a limited potency should be
distinguished from the precursor or
progenitor cells.
 overview
Endogenous human neural stem cells: Adult neurogenesis
• Detection of adult neurogenesis

Bromodeoxyuridine Thymidine
 overview
Neurogenesis in the adult human brain

svz

BrdU
Eriksson et al. 1998 Nature Medicine
Neurons
Astrocytes
 overview
Pioneer in neurogenesis in the adult human brain

1959-2007
 overview
Exogenous resources for human neural stem cells
• aborted fetal humans

• Teratoma-tissue derived neural stem cells

• Transdifferentiation (classical / reprogramming)

• embryonic stem cells / induced pluripotent stem cells

 overview
Historical achievements: embryonic stem cells
1981: Evans and Kaufmann. 1. mouse ESC
lines.
1995: Thomson et al. 1. primate ESC lines
1998: Thomson et al. 1. human ESC lines
 overview
Historical achievements: first neuron from embryonic stem cells

1984: Wobus. 1. microscopy image of

mouse ESC-derived neuronal like cells
1995: Bain et al. 1. systematic
characterisation incl. neuronal and
astrocyte marker stainings and patch-
clamp recordings from mouse ESC-derived
neurons
 overview
In vitro fertilization

http://www.meditourcz.com/wp-
content/uploads/2012/06/in-vitro-
fertilization.gif
 overview
In vivo development of a blastocyst
In vivo In vitro

Bradley et al. 2002, Nature Reviews Immunology

 overview
Required properties of embryonic stem cells
• Cells show high nuclear and
cytoplasmic ratio (More nuclear than
cytoplasm)
• Expression of Oct4 and Nanog, two
transcription factors regulating genes
involved in stem cells maintenance
and renewal, is critical.
• Normal karyogram
• Possible to freeze and thaw cells
• Can differentiate in cell types of the
three germ layers: ectoderm,
mesoderm and endoderm. Tested by
in vitro embryoid body and/or in vivo
teratoma formation
 overview
Morphology of embryonic stem cells (ESCs)
 overview
Fluorescent images of ESC-markers
 overview
Karyogram
• G-banding or Giemsa banding is a DNA
staining technique that is used to visually
investigate the structure of condensed
chromosomes within the nucleus of a cell.
The Giemsa staining technique is named after
the German chemist and bacteriologist
Gustav Giemsa, who invented the technique
in 1902 to detect malaria and Treponema
pallidum in blood smears.
• The basic principle of G-banding is that it is
specific to the phosphate groups of DNA and
attaches itself to regions of DNA where there
are high amounts of adenine-thymine
bonding, creating dark bands. Conversely, less
condensed chromatin, which tends to be rich
with guanine and cytosine, incorporates
lesser Giemsa stain and appears as light
bands. The result of G-banding creates a
karyogram (chromosome map), which can be
used to identify chromosomal aberrations
such as translocations and rearrangements
 overview
What is an embryoid body?
• Embryoid bodies (EBs) are three-
dimensional aggregates of pluripotent
stem cells generated by hang-drop or
cell culture plates with microgrooves.
• EBs are differentiation of human
embryonic stem cells into embryoid
bodies comprising the three
embryonic germ layers.

Machado et al. Cells 2021, 10(9), 2477

Cell 126, 663–676, August 25, 2006

 overview
Teratoma formation
• teratoma is a nonmalignant tumor comprised of a
disorganized mixture of cells and small foci of tissue
comprised of cells from all three of the embryonic
germ-layers. By definition, a cell is pluripotent if it can
differentiate into cells derived from all three of the
embryonic germ-layers: ectoderm, mesoderm, and
endoderm. In the teratoma assay, putative pluripotent
stem cells (PSCs) are implanted into an immune-
compromised mouse where they may proliferate and Kim et al. April 2014 Tissue Engineering and Regenerative Medicine 11(2):121-130
differentiate to form a teratoma. The PSCs grow at the
implantation site supported by a complex mixture of
factors from the local milieu, as well as circulating
factors that are vital components of normal
mammalian physiology. After a predetermined time of
6-12 weeks or when the tumor has reached sufficient
size, it is removed and subjected to histopathological
analysis. The teratoma may be further processed by
immunocytochemistry and gene expression profiling.
This chapter describes methods to generate
teratomas through the implantation of putative PSC
lines in the SCID mouse. Implantation at the following
sites is described: (1) intramuscular, (2) subcutaneous,
(3) under the testis capsule, and (4) under the kidney
capsule.

Cell 126, 663–676, August 25, 2006

 overview
2012 Nobel Prize for Physiology or Medicine

Sir John B. Gurdon Shinya Yamanaka

Nobel Prize motivation: “for the discovery that mature cells Nobel prize motivation: “for the discovery that mature
can be reprogrammed to become pluripotent” cells can be reprogrammed to become pluripotent”
 overview
Pioneer work of cellular reprogramming
John B. Shinya
Gurdon Yamanaka

In 1962, John Gurdon removed the nucleus of a fertilized egg cell from a frog
and replaced it with the nucleus of a cell taken from a tadpole's intestine. This
modified egg cell grew into a new frog, proving that the mature cell still
contained the genetic information needed to form all types of cells.

Pasque et al, Trends in genetics, 2011

22
 overview
"Dolly“: First born mammal achieved by nuclear transfer
• First mammals born out of a somatic nuclear transfer
(Campbell et al. 1996 – Wilmut et al. 1997).

• Process is highly inefficient. Dolly was the only lamb that

survived to adulthood from
• 277 eggs involved
• 29 embryos generated

• Dolly died prematurely at the age of 6 years from a lung

cancer.

• Some speculations concerning premature ageing have

been made, especially in respect to the short length of
Dolly's telomers (Shiels et al. 1999).

• Dolly is not a clone! Mitochondrial DNA comes from the

egg donor
Rodolfa, K. T. (2008). Inducing pluripotency.
Harvard Stem Cell Institute.
 overview
What are induced pluripotent stem cells?
John B. Shinya
Yamanaka factors
Gurdon Yamanaka
• Oct3/4
• Sox2
• Klf4
• c-Myc

In 2006, Shinya Yamanaka succeeded in identifying a

small number of genes within the genome of mice
that proved decisive in this process. When activated,
skin cells from mice could be reprogrammed to
immature stem cells, which, in turn, can grow into
Pasque et al, Trends in genetics, 2011
24 different types of cells within the body.
 overview
Pioneering work in iPSC technology

Mouse embryonic
fibroblasts
 overview
Further development of iPSC technology
 overview
Further development of iPSC technology
Genetic stability

overview
 overview
Common abnormal regions
 overview
Genetic (in)stability further readings
A critical review about the use of iPSC
for clinical purpose based on genetic
instability of iPSC:

https://www.mdpi.com/2077-
0383/8/3/288

And Chapter 2 in the following book:

https://link.springer.com/chapter/10.10
07/978-3-030-31206-0_2
Epigenetic (partly) reset
overview

The iPSCs epigenetic imprinting is

significantly influenced by the cell of
origin and therefore current iPSC lines
have more variability
 overview
Using iPSC-technology
for de- and redifferentiation and differentiation?
fibroblast neurons
(mesodermal origin) (ektodermal origin)

Dedifferentiation by Redifferentiation
reprogramming Neuronal
differentiation

pluripotent stem cells Neural differentiation neural stem cell

(multipotent)
 overview
Concept of direct reprogramming
Mesenchymal stem cells neurons
(mesodermal origin) (ektodermal origin)

Direct
Reprogramming

Differentiation
Direct
Reprogramming

neural stem cell

(multipotent)
 overview
Further readings about direct reprogramming
 overview
Potential medical application of iPSC technology
personalized autolog transplantation
personalized
medical treatment personalized
drug discovery & cell-replacement therapy
development

35
 overview

The origin of the brain

blastocyst

human brain

neural stem cells within the neural tube

 overview

Making neural tube like structures in vitro from iPSC

blastocyst
pluripotent stem cells

neural differentiation
in vitro

in vivo
in vitro
in vivo
neural stem cells within the neural tube
neural stem cells within the neural
rossettes (10 days in vitro)
 overview
The brain tissue in a culture dish

cortical structures

neural differentiation

Eiraku et al. 2008, Cell Stem Cells

in vivo

in vitro

in vivo

neural tube in a
culture dish
in vitro Gaspard, Vanderhaeghen. 2010, Frontiers in Neuros.
 overview
Applying knowledge about in vivo brain development on in vitro
human iPSC.
published 2009
In vivo

In vitro
 overview
Brain organoid technology
Published 2014
 overview
Brain organoid properties
 overview
Brain organoid properties
 overview
Brain organoid properties

Outer radial glial cells

 overview
Organoid: Definition
Human organoid or·gan·oid (ôr'gə-noid’)

Definition: A 3D in vitro model where free-floating

cell assemblies resemble cytoarchitecture and
function of human organs. Can be obtained from
iPSCs, ES cells or adult-derived stem cells.

Improved physiological relevance by:

➢ 3-D organisation of human cells
➢ permits complex cell-cell contact signaling by
extracellualr matrix and cell-adhesion
molecules Cerebral organoids free-floating in
➢ free-floating cell assembly embed in a liquid cell culture media
mileu Lancaster and Knoblich., Science, 2014
 overview
Organoid: Organ-like structure

Organoids resemble aspects of organ-like

cytoarchitecture by:

• containing multiple organ-specific cell types.

• cell types are located in specific regions

• complex cell processes occur, e.g. interkinetic

nuclear migration

• temporal emergence of organ-specific cells is

preserved, e.g. Lancaster
sequential corticogenesis, neuro-to-
and Knoblich., Science, 2014
glial switch
Cerebral organoid displaying
cells with spatial orientation and
differential marker expression
 overview
Challenges with the brain organoid in vitro approach
“Cheerio
Effect?”
Wang et al. 2022, Nature Comm.

1-month 5-month

Large amount of Merging can Be Observed Early

on in Culture in DIY matrix-free protocols
 overview
The brain comprise of organized brain regions…

human brain
 overview
Comparison of cytoarchitecture in vivo and brain organoids:
mouse

Hallmark of the developing in vivo mouse cortex (embryonic day 18):

• Comprise of three regions: ventricular zone, subventricular/intermediate
zone and cortical plate
• Neurons within the cortical plate have a clear layered organisation.
Flores et al. 2017, Stem Cell Reports
 overview
Comparison of cytoarchitecture in vivo and brain organoids:
mouse

Hallmark of the developing in vivo mouse cortex (embryonic day 18):

• Comprise of three regions: ventricular zone, subventricular/intermediate
zone and cortical plate
• Neurons within the cortical plate have a clear layered organisation.
Flores et al. 2017, Stem Cell Reports
 overview
Comparison of cytoarchitecture in vivo and brain organoids:
mouse
TBR1 SatB2

In postnatal day 7 mouse brains:

• Satb2 is expressed in layer II/III
• CTIP2 in layer V neocortical neurons and striatum
• Tbr1 in layer VI neocortical neurons
 overview
Comparison of cytoarchitecture in vivo and brain organoids:
human (gw23)

Saito et al. 2011, Cerebral Cortex, Neocortical Layer Formation of Human Developing Brains and Lissencephalies:
 overview
Comparison of cytoarchitecture in vivo and brain organoids:
human (gw37)

Saito et al. 2011, Cerebral Cortex, Neocortical Layer Formation of Human Developing Brains and Lissencephalies:
 overview
Comparison of cytoarchitecture in vivo and brain organoids:
human (gw37)

Saito et al. 2011, Cerebral Cortex, Neocortical Layer Formation of Human Developing Brains and Lissencephalies:
 overview
Which cytoarchitectural features are present in brain organoids?

5-month sNR-organoids:
Differentiatio • show a ventricular
n-zone zone,
(cortical plate) subventricular/interme
diate zone and cortical
plate
• Cortical plate neurons
SVZ-like do not have a layered
organisation

VZ-like
Wang et al. 2022, Nature Comm.
 overview
Which cytoarchitectural features are present in brain organoids?

Differentiation-
zone (cortical
plate)

SVZ-like

VZ-like
Wang et al. 2022, Nature Comm.
 overview
Challenges with the brain organoid in vitro approach
• Individual neural tube-like cell assemblies are in different developmental stages
and fuse together over time. ➔ high variability. To prevent this, single neural
rosette isolation is required
• Long-cultivation times (several months)
• Brain organoids show a ventricular zone, subventricular/intermediate zone and
cortical plate, however cortical plate neurons lack a clear layered organisation
(cytoarchitecture) as present within the mouse and human brain.
• “core” of dead cells within brain organoids (not presented)

Note, scientific challenges are only temporary and at one point will be solved.
 overview

If you are interested in doing a master thesis project?

Research group:
Functional human iPSC-brain cell in vitro models
We strive for the most functional human brain in vitro platform possible
to improve efficiency in CNS drug discovery and to enhance functional translatability of CNS disease in vitro models.

Sebastian Illes
Department of Physiology
Institute for Neuroscience and Physiology
Contact: sebastian.illes@neuro.gu.se