Nature Reports Stem Cells
Published online: 3 April 2008
What comes after iPS?
Thomas P. Zwaka1
Though applications of reprogrammed cells will be valuable, the
questions they engender will be just as important
"And start to be at the beginning.
Accept swift working of the plan:
Then, following eternal norms,
You move through multitudinous forms,
To reach at last the state of man."
—Johann Wolfgang von Goethe, Faust II, Act 2
It sounds like alchemy: cells within an organism are genetically
almost identical, yet they form cell types as disparate as pulsing
neurons, engulfing macrophages and enzyme-secreting villus cells.
Recently developed techniques appear able to prompt cells from a
terminally differentiated state into one in which they not only
divide indefinitely but can, in theory, become any cell type found in
adults. Last year's advances in generating such cells from mice and
humans have opened what could be a new era of pluripotent stem cell
biology.
It is difficult to underestimate these advances; they may stimulate a
paradigm shift away from the concept of the embryonic stem cells as
the sole source of pluripotent stem cells. Conceivably, in a hundred
years, the time between 1981 and 2007 will be considered the era of
the embryonic stem cell, which supplanted a previous era of
pluripotent stem cells isolated from tumours (that is, embryonic
carcinoma cells) and gradually gave way to an era of induced
pluripotent stem cells generated from differentiated precursors.
What, then, might an era of induced pluripotent stem cells yield to?
Though their applications will be valuable, I propose that the
questions induced pluripotent stem cells engender will prove just as
important as the cells themselves.
Asking embryonic stem cells for reprogramming secrets
The search for factors that reprogram differentiated cells has been
under way for at least two decades. Embryonic stem (ES) cells harbour
reprogramming elements that could be dysfunctional or absent in
healthy differentiated cells, so the field's initial focus on ES
cells seems logical. In early work, the introduction of two
transcription factors identified in ES cells hinted that somatic
cells could respond to pluripotency signals. When Nanog was expressed
in cells fused with ES cells, reprogramming was much more efficient.
Also, ectopic misexpression of Oct4 in adult mouse tissue caused
hyperproliferation1
Shinya Yamanaka from Kyoto University in Japan sought a core set of
factors that would initiate reprogramming in mouse cells. He and his
colleagues identified 24 factors essential to pluripotency in ES
cells and introduced them into mouse embryonic fibroblast cells using
genetically modified retroviruses2. Using a clever selection scheme,
they found that some of the somatic cells were in fact reprogrammed.
By a series of deductive steps, they narrowed the original set of
factors to four (Oct4, Sox2, Myc and Klf4). Nanog, surprisingly, was
absent.
Though remarkably similar to ES cells, the first-reported induced
pluripotent stem (iPS) cells were not identical to them either in
their gene-expression profile or in their behaviour. For instance,
when injected into preimplantation mouse embryos, they failed to
contribute to normal development and so failed the most rigorous test
for pluripotency. More detailed analysis implicated incomplete
reprogramming as the likely reason for this. Follow-up reports with
ever-improving screening techniques have yielded more ES cell–like
cells including mice in which reprogrammed cells contribute to every
sort of body cell3, 4, 5.
Do iPS cells make ES cells less useful?
Reprogramming somatic cells into embryonic-like stem cells by
expressing a defined set of transcription factors seems far simpler
and more efficient than generating ES cells, whether starting from
left-over preimplantation embryos or using somatic cell nuclear
transfer (SCNT). It requires only a small-scale laboratory effort.
There are, apart from those for standard tissue culturing, no
requirements for special techniques, animals, eggs or embryos to be
used in the process. The direct derivation of ES cells from
preimplantation embryos requires a high level of technical skill, a
large number of embryos and an array of special equipment.
Despite its simplicity, the four-factor method of reprogramming used
by Yamanaka is relatively inefficient: less than one percent of
treated fibroblasts acquire pluripotency. Also, in contrast to
reprogramming by SCNT, the acquisition of pluripotency requires
multiple days, and it is still unclear which sorts of cells can be
reprogrammed. Not only does SCNT currently yield higher-quality
pluripotent cells, it also possesses a significant advantage because
it does not use genomic alteration to introduce reprogramming
factors. Generating iPS cells requires the forced expression of
tumour-promoting factors. And mice derived from iPS cells appear more
prone to cancer than their normal counterparts.
Suppose, though, that iPS cells can be generated without genetic
manipulation. Many labs are pursuing this goal, and it could be
achieved soon. Does this mean that SCNT can be discarded, that it
should become a relic like mouth pipetting? Absolutely not!
Though pressure from religious and other groups might shift the field
away from ES cells and toward iPS cells regardless of scientific
merit, the scientific questions SCNT can address overlap only
marginally with those that can be tackled through direct
reprogramming. SCNT allows the study of how epigenetic and genetic
components contribute to the earliest steps of development.
Introducing pluripotency factors, on the other hand, does not produce
an embryo, nor does it allow one to study the events occurring as an
embryo forms and the embryonic epigenome and transcriptome become
established.
Applying iPS
Still, iPS cells promise several practical applications. Procedures
already validated for ES cells will investigate iPS cells' potential
to differentiate into functioning, specialized tissues. Working in
sickle-cell anaemia, Rudolf Jaenisch and co-workers have already
shown, in an elegant proof-of-concept study in the mouse, how
reprogramming, tissue-specific differentiation and gene therapy can
be used to cure inherited disorders6.
The clearest potential advantage of using this technology to
reprogram human cells will be using it to generate disease-specific
cells from many patients or to subject derivatives of reprogrammed
cells to diagnostic or pharmacological tests in an individualized
medicine approach. In contrast to the term 'therapeutic cloning'
coined for SCNT-derived applications, this would be 'therapeutic
reprogramming'
fields that will benefit most from these recent discoveries are
probably those that seem obvious now.
Indeed, the potential applications for iPS technology are endless. An
overlooked application of iPS cells would be using them to bypass the
difficulty of working with species for which establishing ES cells is
difficult or impossible. The ability to perform genetic manipulations
would help to engineer traits such as disease resistance or greater
muscle mass in domestic or threatened animals. Freezing batches of
iPS cells from endangered species may also help to preserve them.
Jumping from iPS to greater reprogramming knowledge and power
Although practical applications may be the easiest to explain, iPS
cells could become invaluable for studies that distinguish between
epigenetic and genetic effects. Both genetic mutations and epigenetic
dysregulation contribute to tumourigenesis, for example. iPS cells
could help establish the contributions of these aberrations. Though
SCNT-derived ES cells could also serve this purpose, the necessary
resources and techniques make using them in this way less practical.
Despite the remaining uncertainty concerning both iPS cells and
techniques to distinguish between epigenetic and genetic effects, iPS
cells can move the cancer field forward by allowing more types of
experiments.
Late in 2007, Yamanaka showed that the factors that reprogram mouse
cells can do the same in human cells. Reporting at the same time,
Jamie Thomson and colleagues from the University of Wisconsin-Madison
developed a protocol to reprogram human cells. He also used four
factors, but only two (Oct4 and Sox2) were the same as those used by
Yamanaka7. This highlights our poor understanding of reprogramming.
Why can different sets of genes have the same outcome?
The ultimate question in the field of iPS research is whether
identified sets of reprogramming factors truly represent a 'core'
regulatory circuit. Several lines of evidence indicate that they may
not. The recent successes in this area reasonably limited study only
to factors important to ES-cells' self-renewal; however, this
strategy omits candidate genes not exclusively active in ES cells,
and these may initiate reprogramming far more efficiently. Some of
the factors discovered so far could just be bystanders of
reprogramming; they could simply activate other genes that are
the 'real' reprogramming factors. It has already been shown that some
of these reprogramming factors are more powerful than others. Myc is
useful but ultimately dispensable. In the mouse, iPS cells that are
capable of contributing to the gamete are only obtained when a weak
selection marker (Fbx15) is exchanged for a more predictive one
(Nanog).
The ultimate question in the field of iPS research is whether
identified sets of reprogramming factors truly represent a 'core'
regulatory circuit.
Ultimately, reprogramming studies will allow us to ask questions such
as "What is a cellular state?" Until now, we have assumed that a
differentiating cell goes through distinct, defined steps to
terminally differentiate: that a synchronized cascade of specific
transcription and epigenetic factors moves a cell from one state to
another, and that the order of steps is critical for successful
differentiation. Both SCNT and other methods to induce pluripotency
suggest that these transitions are not as linear as we had thought.
However, the fact that cells can 'jump' from a state of terminal
differentiation to a flexible state indicates that the concept of
lineage linearity can be bypassed, at least artificially in the
laboratory. Nevertheless, genetic reprogramming seems to involve a
series of intermediate states in which cells' pluripotency machinery
reactivates over several cell divisions; in contrast, reprogramming
using SCNT may not be immediate, but it is sufficient to generate an
embryo. Not knowing why this is so is a fundamental problem of
biology.
Paradoxically, the success of techniques to induce pluripotency
indicate that converting one cell type into another may not be
necessary to generate pluripotent cells at all. Once we fully
understand how to establish the epigenetic state of one cell type, it
might be possible to impose that state on another cell type, for
example, to generate neurons or haematopoietic stem cells directly
from fibroblasts without first reprogramming the cells to a
pluripotent state. At present, only a few cell types can be
successfully differentiated in a culture dish; 'jumping' from one
cell type to another might expand this repertoire. Even if this most
extreme form of reprogramming does not work, it may also be possible
to program pluripotent cells to differentiate into neurons or
haematopoietic stem cells in vitro without mimicking development.
The discovery of reprogramming teaches us an important lesson —
cellular states can indeed change quite markedly within a narrow
window of time. Given that the number of factors that can accomplish
reprogramming is limited, it is almost certain that similar phenomena
occur when tumours form or adapt to new requirements (such as in
metastasis). Such uncontrolled reprogramming may not only promote
certain events in cancer development but may also be essential for
disease progression. Thus, a detailed knowledge of the events that
occur during reprogramming could help answer fundamental questions
about cancer stem cells.
Although it may be a decade or more before the current work on
pluripotency affects work in the clinic, the progress stimulated by
the pioneers in the field of reprogramming has moved cellular
reprogramming from the alchemist's shelf to the forefront of
research. Dust will not settle on this field for many years hence.
References (NOTE: LINKS LIVE AT NATURE URL BELOW]
Hochedlinger, K. et al. Ectopic expression of Oct-4 blocks progenitor-
cell differentiation and causes dysplasia in epithelial tissues. Cell
121, 465–477 (2005). | Article | PubMed | ISI | ChemPort |
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors.
Cell 126, 663–676 (2006). | Article | PubMed | ISI | ChemPort |
Okita, K., Ichisaka, T., & Yamanaka, S. Generation of germline-
competent induced pluripotent stem cells. Nature 448, 260–262 (2007).
| Article |
Maherali, N. et al. Directly reprogrammed fibroblasts show global
epigenetic remodeling and widespread tissue distribution. Cell Stem
Cell 1, 55–70. | Article | ChemPort |
Wernig, M. et al. In vitro reprogrammed fibroblasts have a similar
developmental potential as ES cells and an ES cell-like epigenetic
state. Nature doi: 10.1038/05944 (2007). | Article |
Hanna, J. et al. Treatment of sickle cell anemia mouse model with iPS
cells generated from autologous skin. Science 318, 1920–1923 (2007).
| Article | PubMed | ChemPort |
Yu, J. et al. Induced pluripotent stem cell lines derived from human
somatic cells. Science 318, 1917–1920 (2007). | Article | PubMed |
ChemPort |
Author affiliation
Thomas P. Zwaka is at the Center for Cell and Gene Therapy and the
Departments of Molecular and Cellular Biology and Human Genetics,
Baylor College of Medicine, Houston, Texas
e-mail: tpzwaka@bcm.
http://www.nature.
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