STEM CELL BIOLOGY &

TISSUE ENGINEERING
PROF EARL PRINSLOO
RHODES UNIVERSITY BIOTECHNOLOGY INNOVATION CENTRE
Topic 7
Induced Pluripotent Stem Cells (iPSC) pt II
Purpose
• Following the brief introduction of cancer
stem cells, I hope you have an idea of what
uncontrolled gene regulation can do to a cell.
• Now lets consider the genes used to
reprogramme iPSC, are they truly safe?
• Are there methods of making iPS cells safer?
• What can we use these safe iPSC for?
So in TOPIC 4 we saw that NANOG is so
important to pluripotency that it is considered
the master controller of pluripotency. But in
TOPIC 5 we got introduced to the concept of
iPSC, where NANOG is not required for
reprogramming BUT it is switched on by the
reprogramming factors

OCT 3/4, SOX2, KLF4 and C-MYC (also referred

to as OSKM or the Yamanaka Factors)
So lets look at the YAMANAKA FACTORS
• OCT3/4
• “Oct-4 is a homeodomain transcription factor of the POU family. It is critically
involved in the self-renewal of undifferentiated embryonic stem cells. As such, it is
frequently used as a marker for undifferentiated cells. Oct-4 expression must be
closely regulated; too much or too little will cause differentiation of the cells.”
• SOX2
• “SRY (sex determining region Y)-box 2, also known as SOX2, is a transcription factor
that is essential for maintaining self-renewal, or pluripotency, of undifferentiated
embryonic stem cells.”
• KLF4
• “Kruppel-like factor 4 is a member of the KLF family of zinc finger transcription
factors, KLF4 is involved in the regulation of proliferation, differentiation, apoptosis
and somatic cell reprogramming. In embryonic stem cells (ESCs), KLF4 has been
demonstrated to be a good indicator of stem-like capacity. It is suggested that the
same is true in mesenchymal stem cells (MSCs).”
• c-MYC
• c- Myc is a mostly constitutively active protooncogene that regulates global gene
expression. It is estimated that c-Myc regulates approximately 15% of all genes.

OSKM
Takahashi and Yamanaka Original iPSC

The team cloned the genes into retrovirus vectors that were then used to infect
the fibroblasts. The idea being that because the retrovirus reverse transcribes its
genome (RNA to DNA) and inserts that genome into the host fibroblast that
would ensure that their OSKM gene copies would be inserted and expressed
constitutively. Of course these genes were overexpressed in the fibroblasts.

So, questions regarding safety were raised by Yamanaka and we saw in TOPIC 5
that the team were very careful to test whether their cells were potential cancer
cells. The largest concern was c-Myc.
c-Myc

c-Myc as a reprogramming factor

• Myc is a universal gene upregulator

• c-Myc is a known proto-oncogene i.e. a gene that is known to regulate cellular
transformation if unregulated.

Would you want c-Myc to be continuously overexpressed in a cell?

*Hint – remember how retroviruses work?

Lets look at the alternatives that have been developed
to prevent potential overexpression
While other viral
vector systems have
been used, concerns
regarding use of
viruses is always an
issue. Transposons
have the potential of
homologous
recombination and
small molecules are
not that efficient.
small molecules are
drugs that may be able
to replace some of the
factors by switching on
endogenous genes
Lets look at the alternatives that have been developed
to prevent potential overexpression
The safest method is
simply to use
modified RNA. On its
own. Introduction of
the RNA into the
fibroblast would
result in translation
to protein. After a
while the RNA would
simply be degraded
by the host cell.
 ** This has been shown to evade the cells natural capacity to break down mRNA
Modified RNA
Before we continue, it should be remembered that RNA is far less stable than DNA, in fact
this can be viewed as a control mechanism. Imagine if the RNA was as stable as genomic
DNA we would always have genes being translated. Overexpressed genes can lead to errors
in signalling and this would result in cell death. Most likely evolution selected for this RNA
stability to ensure maintenance of a cell. That being said, nothing stops us from tinkering in
vitro

Messenger RNA can be modified to ensure stability. By increasing the half-life* of an mRNA
we can extend the potential time that the mRNA has to be translated. Or we can conversely
say we can delay the degradation/breakdown of the mRNA.

The RNA’s may be modified using a combination of pseudouridine, methylated bases or

by simply including a methylated guanine nucleotide “cap” at the 5’ end.**

*Half-life “the time required for a quantity to reduce to half of its initial value.”
 ** This has been shown to evade the cells natural capacity to break down mRNA
Modified RNA**
So in the case of producing iPS cells safely we can use these modified RNA molecules to
efficiently transfect fibroblasts (somatic cells) and grow the cells in vitro to allow for the
reprogramming using the OSKM factors. As a result of the fact that there is no integration
into the genome, there is no risk of the introduced gene being expressed once the cells
have been reprogrammed. This is critical to using the cells in clinical trials. We do not want
cells that have been derived from iPS cells to suddenly revert to cancer cells as a result of
integrated genes.
NO NUCLEAR
INTEGRATION

O iPSC
S
K
M
Ribosomal mediated translation
Lets look at Warren et al. 2010 who
modified the OSKM (or as they called
it KMOS) mRNA’s and transfected
keratinocytes*
In Figure 5E they show how on a
timeline, embryonic stem cell like
colonies form at least 10 days earlier
than retroviral transduction of OSKM.
If we look at their colony assay we
see how there are far fewer iPSC
colonies (less reddish brown colour)
in the virus transduced sample than
in the RNA transfected experiments.
They tracked iPSC colonies using a
biomarker TRA-1-60.
TRA-1-60 is a biomarker of
embryonic stem and germ cells *Keratinocytes – “Keratinocytes constitute 90% of the cells of the
epidermis, the outermost layer of the skin. Basal cells in the basal
layer (stratum basale) of the skin, are sometimes referred to as
basal keratinocytes”
 Naturally, truly safe is application in a
human
Before we continue, let us define the following concepts.

Autologous Transplantation – Cells derived from an individual are transplanted back to

the same individual

Allogeneic Transplantation – Cells derived from a donor where the human leukocyte
antigens (HLA*) are characterised. These cells can be used to transplant into a different
individual who is a HLA match.

Heterologous Transplantation – Cell transplants from a mixed population of donor cells

*”The human leukocyte antigen (HLA) system or complex is a group of related proteins that are encoded by the
major histocompatibility complex (MHC) gene complex in humans. These cell-surface proteins are responsible
for the regulation of the immune system.”
 iPSC Clinical Trial
Successful autologous iPSC derived cell transplant.
Trial received permission in 2014 in a 77 year old patient with age-
related macular degeneration. Lead Dr Masayo Takahashi.

iPS cells were differentiated into sheets of retinal pigment epithelium.

2019: It was reported that the sheet of cells had engrafted and
survived for 4 years, reversing the degeneration

https://www.sciencedirect.com/science/article/pii/S2468653019300909
IMPORTANT
AT NO POINT SHOULD iPS CELLS BE INJECTED DIRECTLY. THESE ARE
PLURIPOTENT STEM CELLS. THEY HAVE THE POTENTIAL OF FORMING
TERATOMAS IF NOT CONTROLLED.

SO IF iPSC THERAPIES ARE DEVELOPED THEY NEED TO BE

DIFFERENTIATED INTO SPECIFIC CELL TYPES FIRST. THOSE CELLS NEED
TO BE VERIFIED TO BE NOT iPS CELLS.

ONLY THEN CAN THEY BE USED FOR CELL REPLACEMENT THERAPY

An iPSC Bank?
Much like a blood bank…
“Banking on the future Yamanaka is establishing an iPS cell bank, which
depends on matching donors to recipients on the basis of three genes that
code for human leukocyte antigens (HLAs) — proteins on the cell surface that
are involved in triggering immune reactions. His iPS Cell Stock for Regenerative
Medicine currently has cell lines from just one donor. But by March 2018, he
and his colleagues hope to create HLA-characterized cell lines from 5-10
different donors, which should match 30–50% of Japan’s population.”

Great Idea – But would it work in other countries? Lets consider South Africa,
unfortunately little is known regarding HLA diversity in South Africa. Before
stem cell banks are to become accessible to all we need to understand this
diversity more.
Self-reflective exercise

• Do you understand why retroviruses are not

the ideal method of introducing
reprogramming genes into fibroblasts?