Primer for Reprogramming Lecture
Essential questions (R, B and V)
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What das Nuclear Reprogramming mean (definition)?
How can you reprogram a somatic cell towards pluripotency?
Does reprogramming occur in nature? If so, where?
What are the main differences between the reprogramming strategies?
Is an ES cell identical to an iPS cell?
What are the Yamanaka factors?
What happens with a somatic cell during reprogramming with regard of its
genome?
Key knowledge and skills will you acquire as a result of this lecture
Students will know:
 Key Terms: embryonic, and induced pluripotent stem cells, somatic nuclear
transfer, cell fusion, direct reprogramming, transcription factor, ectopic
expression, epigenome, epigenetic modification
 The history of nuclear reprogramming
 The Yamanaka factors and the basic function of OCT3/4 and SOX2
 The basic difference between iPS cells and ES cells
 The different nuclear reprogramming towards pluripotency strategies and
their differences
 The therapeutic potential of RiPSCs
 The basic molecular events during nuclear reprogramming
Students will be able to:
 Describe the potential and advantage of iPS cells in contrast to ES cells for
stem cell research (drug development, biology of pluripotency, disease
modeling)
 Describe the basic events that happen after forced expression of
transcription factors
 Highlight the different methods of reprogramming and the key factors that
lead to the transition for each strategy.
 Recognize the potential to study stem cell biology (factors necessary for
pluripotency, self-renewal, etc.) for each reprogramming method
As cells proceed in normal development from embryonic and fetal stages to an
adult state, they become increasingly committed to their differentiated state.
Hardly ever do cells reverse this process and go back from a differentiated state
to an embryonic one. The process of mammalian cell differentiation can be
described as a ball rolling down a hill with many valleys [The landscape model of
mammalian cell differentiation (modified from Keeton & Gould 1984)]. When the
ball is on tip of the hill, it can roll down through any valleys below; this represents
the process of a totipotent cell that can differentiate into any tissue of the body.
However, as the ball rolls passed an intersection, the available valleys for the ball
to roll down become limited. When the ball reaches the bottom of the hill, it can
no longer move to another valley or back to the tip of the hill. This model was
used to illustrate a totipotent cell choosing among different developmental paths;
when the cell’s fate is partially determined, its differentiation potential becomes
limited (slide 2).
In the current literature, the term “nuclear reprogramming” is defined as either
the switch of the gene expression state from one cell type to another or the
change of a differentiated, specialized cell into a developmentally more primitive
but more pluripotent state (slide 2). Until the early 1950s the validity of the dogma,
that the process of a cell, to differentiate and eventually commit to its fate, is
irreversible has not been questioned. However, early studies in frog cloning
starting in 1952 demonstrated that the process of cell differentiation could be fully
reversed without the alteration of the gene content (slide 4). Soon after this first
experimental evidence for reprogramming, a number of different techniques have
been developed over the years to induce reprogramming and pluripotency in
differentiated somatic cells including nuclear transfer, cell fusion,
reprogramming through cell extracts and direct reprogramming (slide 3). Latter
has drawn much attention and undergone dramatic advancement and
improvements since first described in 2006.
To produce pluripotent cells using nuclear transfer, the nucleus of a somatic cell
(e.g. fibroblast) is removed (slide 6). At the same time, the nucleus of an egg cell
(oocyte) is taken out and discarded. The nucleus of the somatic cell is then
inserted into the enucleated egg cell. After insertion into the egg, the somatic cell
nucleus is reprogrammed (i.e. has its gene expression altered, turning OFF
genes marking its differentiated state and turning ON genes that induce
pluripotency) by unknown factors present within the oocyte. The egg, now
containing the nuclear DNA of the somatic cell, is stimulated with electricity and
begins dividing. After several mitotic divisions in culture, a blastocyst may form
from which pluripotent stem cells can be derived. This process is very inefficient
in species where it does work, and as of this writing has never been
accomplished using human cells. The technique of transfering a nucleus from a
somatic cell into an oocyte is also referred to as cloning. Importantly, these NTES cells contain the same genetic material (DNA) as the original fibroblast cells
(i.e. those taken from the patient’s skin) and may in theory be used for
therapeutic purposes in a manner that would avoid immune system rejection. As
described above, such cells made from patients may be differentiated into
specific lineages (e.g. dopaminergic neurons) to enable the study of a particular
disease (e.g. Parkinson’s disease). Currently no human ES stem cell lines have
been derived using this method. This technique is not very widely studied due to:
1) the considerable difficulty, both practically and ethically, in obtaining human
eggs, and 2) the advent of iPS technology (see below).
Cell fusion describes a process of forcing several nuclei to share a common
cytoplasm, leading to multinucleated cells or hybrid cells (slide 8). In
multinucleated cells, so called heterokaryons, one cell type usually dominates
over the other, such that it imposes its state on another nucleus, where
eventually nuclear reprogramming occurs. Fusion of somatic cells with
pluripotent cells and initiating reprogramming is a direct and fast method (1-2
days).
Another method to generate pluripotent stem cells is through a technique called
direct or cellular reprogramming (slide 10). Induced pluripotent stem cells
(iPS cells) are reprogrammed cells made by a technique that “forces” expression
of pluripotency-related genes in a somatic differentiated cell (slide 18). iPS cells
are similar to ES cells in many respects, such as their pluripotency and matching
expression of genes and proteins (slide 18). iPS cells show genome-wide histone
methylation patterns characteristic of ES cells, identical morphology, expression
of key pluripotency markers and even reactivation of the inactive X chromosome
and restoration of telomerase activity. Despite the functional and molecular
similarity between ES and iPS cells (with the tetraploid (4N) complementation
assay being the most stringent), iPS cells are not identical to ES cells in terms of
global gene expression, epigenetic modifications and germ line transmissibility.
iPS cells were first produced in 2006 from mouse cells with the forced expression
of the Yamanaka factors and in 2007 from human cells (slide 10). This was an
incredibly important and monumental advance in biology, as it not only opened
the door to a much clearer understanding of how pluripotency is biologically
regulated but may also allow researchers to obtain pluripotent stem cells which
are a genetic match for patients, without the controversial use of human embryos.
iPS cells are typically derived by introducing specific pluripotent stem cellassociated genes into non-pluripotent cells such as adult fibroblasts, by way of a
viral vector in a process called transduction (slides 12). This is often achieved
using a type of virus called a retrovirus. Retroviruses have an RNA genome that
is converted to DNA before inserting at random locations into the host cell’s own
genome. The reprogramming genes include the master transcription factors
Oc3/4 and Sox2. The Oct3/4 and Sox2 transcriptional regulators and are
necessary to induce somatic cells into an embryonic state. Oct-4 plays a crucial
role in maintaining ES cell pluripotency, and is primarily found only in pluripotent
cells such as ES cells (slide 20). The absence of Oct-4 in blastomeres and ES
cells leads them to change into trophoblast cells. The Sox2 gene is also
associated with maintaining pluripotency (slide 21). Klf4 was initially required for
mouse iPS cell production but was later shown not to be required for human iPS
cells. The c-Myc proto-oncogene has been implicated in cancer. Using the "myc"
family of genes to induce iPS cells is troublesome because 25% of mice
transplanted with c-myc-induced iPS cells developed lethal tumors. Klf-4 and cMyc work by changing gene expression in differentiated cells, primarily by
pushing cells to proliferate, preventing cell death and making the genome more
responsive to changes in patterns of gene expression. Reprogramming factors
turn OFF genes that are active in differentiated cells and turn ON genes that
maintain pluripotency. Soon after, the work by the group has been developed
and optimized from various point of views, including the demonstration that the
usage of a different set of reprogramming factors, namely the "Thomson factors"
(OCT3/4, SOX2, NANOG and LIN28) also leads to the derivation of iPS cells in
humans. In ES cells, Nanog is also required for pluripotency. However, Nanog is
not necessary for iPS induction, although human iPS cells are often produced
using Nanog as one of the factors. Lin-28 is a regulator of a specific class of
factors known as microRNAs, that in turn regulate many oncogenes.
Several other genes such as hTERT and SV40 or the combination of the
Thomson and Yamanaka factors increase the production efficiency of iPS cells.
Though cellular reprogramming superficially appears “simple” considering that
very few genes are required to push mature cells “developmentally backwards”
into pluripotency, in reality the process is anything but simple when one
considers the many interactive regulatory networks affected by each of these
factors during the reprogramming process. Reprogramming usually takes
between 2-4 weeks after reprogramming genes are introduced to cells. Small
numbers of cells become morphologically and biochemically similar to pluripotent
stem cells, and are isolated through morphological selection (formation of
colonies), the presence of a genetic marker or antibiotic selection (slide 16).
A variety of innovative techniques have been shown to reprogram somatic cells
into pluripotent stem cells since 2006 including the use of viruses, proteins,
plasmids and mRNA molecules (slide 11, 17). In addition, different cell types
have been reprogrammed including skin fibroblasts, keratinocytes, and blood
cells (slide 22). Reprogramming adult cells to obtain iPS cells may pose
significant risks that could also limit their use in humans. If viruses are used to
alter the cells genetically, the expression of cancer-causing genes or oncogenes
may be activated after they are injected into organisms. In February 2008,
scientists developed a technique to remove these reprogramming genes after
inducing pluripotency, increasing the potential safety of iPS cells for the
treatment and study of human diseases. In April 2009, scientists produced
mouse iPS cells without any genetic alteration of adult cells. Here, a repeated
treatment of the cells with critical proteins was enough to induce pluripotency
(slide 13). The most recent development within this field comes from a research
group at Harvard University. The Rossi laboratory accomplished to reprogram
fibroblasts from different sources into iPS cells by transfecting them with
synthetic mRNAs, each encoding the different reprogramming factors (slide 15).
To date, this is the only study that demonstrates that this new methodology is a
robust and efficient way of generating iPSCs without compromising genomic
integrity. By such means, today’s breakthroughs are refined further to create
even more effective methods for eventual use in cellular therapies.
Little by little, the processes behind cellular reprogramming of somatic
differentiated cells into induced pluripotent stem cells are being discovered and
more detailed studies of the molecular mechanisms behind are being undertaken.
Nuclear reprogramming is an incredibly complex and dynamic process likely to
follow a specific set of sequential events involving epigenetic changes combined
with the conversion of an entire transcriptional network (slide 24).
An epigenome of a cell consists of a record of the chemical changes to the DNA
and histone proteins of an organism; these changes can be passed down to an
organism’s offspring. Changes to the epigenome can result in changes to the
structure of chromatin and changes to the function of the genome. Epigenetic
changes of the genome are controlled by DNA methylation, histone modifications,
and in a broader sense, small, noncoding RNAs. Both DNA methylation and
histone modifications affect the structure of the chromatin rather than the
sequence. Thus, these modifications can be erased and reestablished without
mutating the genome. DNA is methylated on the cytosines of CpG dinucleotides
by DNA methyltransferases (DNMTs). The extent of cytosine methylation,
predominantly on CpG islands of promoters, reflects the expression status of the
gene, with hypermethylation keeping the gene silent and hypomethylation
allowing high gene expression (slide 24).
In differentiated cells, many developmental and differentiation associated genes
are active and show enrichment for H3K4me3 and lack of DNA methylation.
Some early developmental genes have been silenced through polycombmediated H3K27me3 and all pluripotency associated genes show high levels of
DNA methylation. In pluripotent cells, these pluripotency-associated genes are
active and show H3K4me3 and lack of DNA methylation. Many of the
developmental genes show a ‘bivalent’ chromatin configuration and tissuespecific genes tend to be DNA methylated.
It is believed that the process of nuclear reprogramming may involve a sequence
of stochastic epigenetic events and involves sequential activation of pluripotency
markers (slide 25). In mice, alkaline phosphatase (AP) and SSEA1 positive cells
are already detected 3 and 9 days, respectively, after viral reprogramming factor
transduction, whereas GFP expressed from the endogenous Oct4 or Nanog loci
first appear only after 2 weeks. The virally transduced factors need to be
expressed for about 2 weeks to initiate the reprogramming process. Another
model suggests that reprogramming may be described as a two-stage process
(slide 25). In the first stage, exogenous Oct4 and Sox2 cause the downregulation
of lineage-associated genes and upregulation of a subset of ES cell-specific
genes. In addition, the first stage of reprogramming comprises widespread
epigenetic remodelling: epigenetic enzymes that are most likely activated by
reprogramming factors induce a global unfolding of chromatin and catalyze the
removal of repressive chromatin modifications from key pluripotency genes. This
allows these pluripotency genes to be targeted and activated by exogenous Oct4
and Sox2, resulting in the revival of the interconnected autoregulatory loop and
reactivation of the ES cell transcriptional network. At the same time, transgene
silencing that was initiated during the first reprogramming stage reaches
completion, leading the fully re-established pluripotent state to be independent
from continuous transgene expression
In all proposed models exogenous Oct4 and Sox2 resuscitate the interconnected
autoregulatory loop during reprogramming (slide 26). In infected fibroblasts,
endogenous Oct4, Sox2 and Nanog are reactivated by ectopic expression of
Oct4, Sox2 and other reprogramming factors. Progression of the reprogramming
process results in increased selfsustainability of the endogenous interconnected
loop. The interconnected loop is now resuscitated and is able to stably maintain
the pluripotent state.
Concept Mapping Terms
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Induced pluripotent stem cell
Nuclear transfer
Transcription factor
Direct reprogramming
Reprogramming factor
Epigenome
Transgene
Transcriptional Regulatory Circuitry
Yamanaka factors
Oncogene
Methylation
Readings, Videos, and Slide Presentations for Lecture Nuclear
Reprogramming
 Please watch a series of movies from the International Society for Stem Cells
(http://www.isscr.org/public/MakingSenseOfStemCells.htm) regarding human
embryonic stem cells, adult stem cells and their therapeutic use and cloning
and nuclear transfer.
References
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Nuclear Reprogramming and Stem Cells (Stem Cell Biology and
Regenerative Medicine) by Justin Ainscough, Shinya Yamanaka and
Takashi Tada (Hardcover - Sep 1, 2011)
Stem Cells: Nuclear Reprogramming and Therapeutic Applications,
Novartis Foundation Symposium (Novartis Foundation Symposia) by
Novartis Foundation (Hardcover - May 16, 2005)
Cellular Programming and Reprogramming: Methods and Protocols
(Methods in Molecular Biology) by Sheng Ding (Hardcover- Mar 25,
2010)
Engineering of Stem Cells (Advances in Biochemical Engineering
Biotechnology) by Ulrich Martin (Hardcover - Oct 27, 2009)
Stem cells, the molecular circuitry of pluripotency and nuclear
reprogramming by R. Jaenisch and R. Young (2008)
The molecular mechanism of induced pluripotency: a two-stage
switch by W. Scheper and S. Copray (2009)
Induced pluripotent stem cells: current progress and potential for
regenerative medicine by G. Amabile and A. Meissner (2009)
Nuclear reprogramming to a pluripotent state by three approaches by
S. Yamanaka and H.M. Blau
Totipotency, pluripotency and nuclear reprogramming by S. Mitalipov
and D. Wolf
Biological Science by Keeton W & Gould J (1984, New York: WW Norton
and Company, Inc.)