September 26, 2016 by Wallace Raven
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| Induced pluripotent stem cells that were reprogrammed from normal adult human tissue and have not yet been differentiated. Credit: Gladstone Institutes
Human
cortex grown in a petri dish. Eye diseases treated with retinal cells derived from
a patient's own skin cells. New drugs tested on human cells instead of animal
models.
Research
and emerging treatments with stem cells today can be traced to a startling
discovery 10 years ago when Shinya Yamanaka, MD, PhD, and his graduate student
Kazutoshi Takahashi, PhD, reported a way to reprogram adult mouse cells and
coax them back to their embryonic state – pluripotent stem cells.
A
year later, they accomplished the feat with human cells. For this
research coup and his leading role pioneering stem cell work, Yamanaka – who
holds academic appointments at Kyoto University and UC San Francisco – was the
co-recipient of the 2012 Nobel Prize
in Medicine or Physiology.
The
breakthrough provides a limitless supply of induced pluripotent stem cells
(iPSCs) that can then be directed down any developmental path to generate
specific types of adult cells,
from skin to heart to neuron, for use in basic research, drug discovery and
treating disease.
The
achievement opened up a practical way – and in some critical cases, the only
way – to directly study human "diseases in a dish," and track the
early stages of both healthy and abnormal development. It also allowed
researchers to screen new drugs directly in human cells rather than relying on
animal models, which more often than not fail to accurately predict a new
drug's effects on people.
The
dazzling iPSC breakthrough has spurred rapid progress in some areas and posed
major challenges in others. It has already proved a boon to basic research, but
applying the new technology to treat diseases remains daunting. Some types of
cells have proved difficult to reprogram, and even the protocols for doing so
are still in flux as this is still a very young field.
iPSCs
in Basic Biomedical Research
For
many basic biomedical scientists, the capability offered by iPSCs technology is
like a dream come true, says neuroscientist Arnold Kriegstein, MD, PhD,
director of UCSF's Eli and Edythe Broad Center of Regeneration Medicine and
Stem Cell Research.
"Induced
pluripotent stem
cells have given us a window into human development unlike anything
we had before," Kriegstein said. "I'm interested in the early
development of the brain's cortex. Of course, we've never had unrestricted
access to living human brain cells. Now we can take skin cells and grow human
cortex in a dish. It's a game-changer for discovery about early human
development."
Kriegstein
is enthusiastic about what researchers can learn from "organoids" – a
pea-sized stage of a developing organ derived from iPSCs. By this stage, cells
are already clumping together and starting to signal and differentiate into
what will become the adult organ.
A human cortex organoid that’s grown in a lab dish. Credit: Elizabeth DiLullo
"It's
a very close model of the real thing," Kriegstein says. "We have
recently discovered that even in this early stage, the organoids are able to
develop intrinsic organization, including a front-and-back orientation, and
different parts start to look like they do in the embryonic brain."
Some
scientific papers have suggested that organoids can model diseases found in
adulthood – even disorders of late adulthood such as Alzheimer's disease.
Even
though organoids can reveal developmental steps not seen before, Kriegstein
worries that some researchers are getting too far ahead of themselves.
"It's
an embryonic brain," he stresses. "The longest period of growth we
can model would be full fetal development. How likely is it that gene
expression, cell signaling and a myriad of other interactions at this organoid
stage could accurately represent the development of Alzheimer's disease, a
disease that affects people at 60 or 70?
"I
think we need to take some of these studies with a grain of salt. Stem cell
technology now is so variable that replication is difficult. We need to
establish protocols to reliably compare different methods and then use these
standardized methodologies to advance research and treatment. But I am 100
percent convinced that we will get there."
Building
on the Original Breakthrough
Yamanaka
currently directs the 500-person Center for iPS Cell Research and Application
at Kyoto University, runs a research lab at the Gladstone Institute for
Cardiovascular Disease in San Francisco, and serves as a professor of anatomy
at UCSF, and Takahashi is a visiting scientist at the Gladstone Institutes and
runs Yamanaka's lab there. Both have continued to build on their iPSC work, as
have other researchers.
In
their seminal work, Yamanaka and Takahashi had introduced four genetic factors
to prompt adult cells back to the pluripotent state. Soon after their iPSC
breakthrough, Sheng Ding, PhD, who has a lab at the Gladstone Institutes and is
a professor in UCSF's Department of Pharmaceutical Chemistry, began refining
the reprogramming cocktail.
Eventually,
Ding was able to substitute drug-like molecules for these gene transcription
factors, eliminating the risk of new genetic material altering the cells.
Today, labs around the world pursue and tout different chemical recipes, often
depending on the type of cell they are trying to reprogram.
Other
recent advances to induce pluripotency harness different kinds of proteins that
influence gene activity in the cell nucleus. Robert Blelloch, MD, PhD, a stem
cell scientist at UCSF's Broad Center, has shown that some small RNA molecules
called microRNAs promote adult cell "de-differentiation" and others
promote the reverse: ability of stem cells to differentiate into adult cells.
By tweaking microRNA activity, his lab has been able to improve reprogramming
yields a hundred-fold.
Shinya Yamanaka, MD, PhD, in his lab at the Gladstone Institutes. Credit: Chris Goodfellow/Gladstone Institutes
He
and colleagues have also become intrigued by the role of so-called epigenetic
factors – naturally occurring or introduced molecules that modify proteins in
the nucleus. Manipulation of these molecules too can affect the efficiency of
inducing pluripotent cells.
The
Promise of Treatments
Six
years after Yamanaka's iPSCs discovery, researchers in a very different field
developed a new gene-editing technology of unprecedented speed and precision,
known as CRISPR-Cas9. The potent new tool has revolutionized efforts to
"cut and paste" genes and has been very quickly adopted by thousands
of researchers in basic biology and drug development.
"CRISPR
has provided us with an extraordinary new capability," Kriegstein says.
"It allows us to tease apart the genetic causes or contributors to
developmental diseases. We can edit out mutations to determine if they are
critical to early developmental defects."
CRISPR's
speed and precision may some day allow stem cell researchers to reach their
most ambitious goal: Genetically abnormal cells from patients with inherited
diseases such as sickle cell anemia or Huntington's could be reprogrammed to
the pluripotent stem cell state; their genetic defects could be
"edited" in a petri dish before being differentiated into healthy
adult cells. These cells could then be transplanted into patients to restore
normal function.
While
that goal is still beyond reach, many early-stage clinical trials are underway
using induced iPSCs to treat diseases, from diabetes and heart disease to
Parkinson's.
One
trial has already treated its first patient. In 2014, Japanese scientists made
iPSCs from skin cells of a woman with macular degeneration and then
differentiated them into adult retinal cells. Surgeons transplanted
the retinal cells into her eyes in order to treat the disease – the first
patient treated using iPSCs.
Researchers
focused on the eye disease in part because differentiating stem cells into
retinal cells has proven to be fairly straightforward compared to many other
cell types, Kriegstein says. Also, it is relatively easy to transplant cells
into the eye.
Preparations
to treat a second patient using patient-derived cells were stopped because the
researchers detected a mutation in one of the genes in the iPS cells. No
reports had linked the gene to cancer, but they decided not to use the stem
cells to eliminate any risk.
The
success of treatments relies in part on stem cells' rapid rate of
proliferation. Hundreds of billions of cells may sometimes be needed for a
transplantation. But if just a few of the stem cells fail to differentiate into
the target adult cells, they may reproduce rampantly when transplanted and form
a tumor.
"It's
a two-edged sword," says Yamanaka. "In the pre-transplant stage, you
want stem cells that proliferate very rapidly. But after the transplant, if
there are only five or 10 cells that didn't differentiate into adult cells,
they can reproduce infinitely. They create a kind of residue of tumor."
Research
to ensure that all stem cells differentiate before transplantation is now one
of the main issues in this field, he says.
To
eliminate cancer risk, the researchers are now "deep sequencing" the
genetic makeup of each of the stem cell lines they might use. They have also
decided to use donor cell lines rather than the patient's own cells. This
avoids the very expensive prospect of having to carry out quality checks like
deep sequencing of each patient's own pluripotent cell lines.
Use
in Drug Screening
The
promise of treatment related to stem cells includes the promise of drug testing
in adult human iPSC-derived cells rather than using animal models.
One
recent example is the work of Catherine Mummery, a neurologist at Great
Britain's National Hospital for Neurology and Neurosurgery, who used
iPSC-derived adult human cardiomyocytes – heart cells that will beat in a petri
dish – to test two different commercially available drugs for cardiovascular
disease. She showed that each drug triggered the same kind of therapeutic
effect at one dose and the same type of toxicity at another dose that had been
found in patients.
"This
was impressive," Kriegstein says. "It was an early proof-of-principle
that drug testing in iPSC-derived adult human cells, rather than in animal
models, can provide reliable results – and results that are more directly
relevant to patients. Drug companies are starting to screen drugs in
iPSC-derived human cells and organs."
Other
Sources, Old and New
Research
using stem cells isn't limited to IPSCs. In some respects, embryonic stem
cells (ESCs) remain the gold standard. UCSF stem cell biologist
Susan Fisher, PhD, sees early-stage ESCs as a blank slate.
"They
have less of a history than iPS cells. They carry less baggage," she says.
But like Yamanaka and Kriegstein, she considers the field too young to declare
the superiority of one strategy over the other.
These cardiomyocytes were reprogrammed from normal adult human skin samples. Credit: Matt Spindler/Gladstone Institutes
A
strong endorsement for tapping ESCs in transplantation medicine came two years
ago when a Harvard team showed that cell lines derived from embryonic stem
cells could produce unlimited supplies of insulin-producing islet cells. Early
stage clinical trials are now underway, testing the safety and efficiency of
transplanting islet cells into patients to treat type 1 diabetes.
As
of 2014, hundreds of clinical trials are underway in many countries, primarily
testing safety and efficacy of treatments for diseases, from heart failure to
Parkinson's.
The
two well-known strategies to derive stem cells have recently been joined by a
third, called direct cellular reprogramming or transdifferentiation. In this
method, skin cells are turned directly into cells of the desired organ – brain,
heart, pancreas – without first being drawn all the way back to a fully
pluripotent stem cell stage. As a result, the method skirts the cancer risk
inherent in differentiating truly pluripotent cells.
This
year, Deepak Srivastava, MD – director of cardiovascular and stem cell research
at the Gladstone Institutes and a professor of Pediatrics and of Biochemistry
and Biophysics at UCSF – and a team led by Gladstone's Ding efficiently transformed
mouse skin cells into brain cells as well as beating heart cells
using a combination of chemicals. The approach could prove effective in efforts
to regenerate dying or diseased cells and tissues, Ding says.
Ethics
and Public Perception
As
the science progresses on many fronts, Yamanaka has become concerned that the
science has gotten far ahead of efforts to consider the ethics of some of the
research.
"When
we made iPS cells, our purpose was to overcome ethical issues of embryonic stem cells. Now we are
creating new ethical questions," Yamanaka says. "We can now make
sperm or eggs from iPS cells, at least in mice. We may be able to make human
organs in pigs and other animals by injecting human iPS cells into animal
embryos– creating so-called chimeras.
"The
speed of scientific progress is getting faster and faster, so if we discuss the
ethical issues slowly ... this is a big concern. I have been asking many
bioethicists to think about these issues more aggressively. Some think it's
science fiction. But it's not for the future. It's for ourselves."
At
the same time, he is concerned too about public perception that the rate of
progress may be slower than expected.
"I
am fascinated by how rapidly science is advancing. It's amazing. But for the
most part, developing new treatments – doing the science, testing the safety
and effectiveness of new therapies – takes a great deal of money and many
years," Yamanaka said.
"Developing
new treatments may take 10 years, 20 years, 30 years. That is what we have been
trying to say to our patients: 'We are making great progress, so do keep up
your hope. But it takes time.'"
More
information: Kazutoshi Takahashi et al. Induction of
Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by
Defined Factors, Cell (2006). DOI: 10.1016/j.cell.2006.07.024
Provided by: University of California, San Francisco
http://medicalxpress.com/news/2016-09-pluripotent-stem-cells10-years-breakthrough.html
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