You are on page 1of 9

Harvard and Brigham and Women’s Hospital disavow

the work by former faculty member Piero Anversa.


Oct 15, 2018
ASHLEY P. TAYLOR

ABOVE: © ISTOCK.COM, RAZVAN25

H arvard Medical School and Brigham and Women’s Hospital announced yesterday
(October 14) that following an internal investigation they recommend the retraction of 31
papers by a former faculty member, cardiac stem cell researcher Piero Anversa, as Retraction
Watch and STAT report. These studies “included falsified and/or fabricated data,” the two
institutions tell Retraction Watch and STAT, adding that they have “notified all relevant
journals.”

This is the latest in a series of black marks on research by Anversa, who claimed, as early
as 2003, to have identified adult stem cells—dubbed c-kit cells—that can regenerate cardiac
muscle. Efforts by other labs to reproduce Anversa’s findings and to use the cells to heal
damaged hearts have produced contradictoryand sometimes mysterious results.

See “Adult Cardiac Stem Cells Don’t Exist: Study”

In 2014, Anversa and coauthors had a paper retracted from the journal Circulation after
Harvard Medical School and Brigham and Women’s determined that data were
“compromised,” as the retraction notice states. That same year, the Lancet published an
“expression of concern” about the results of a clinical trialby Anversa, again after the
institutions contacted the journal to warn them that some of the data were under scrutiny in an
ongoing investigation.

Anversa’s lab at Brigham and Women’s closed in 2015. Last year, after disclosing to the
Department of Justice that Anversa had won federal research funding using fraudulent data,
the hospital agreed to pay the federal government $10 million to resolve the matter, The
Washington Post reports.

“There’s been grave damage done to the field, and potentially a generation of young
researchers who’ve come into the field of cardiac regeneration at a time that ideas that largely
derived from what appear to be fraudulent papers have held a lot of sway,” Jonathan Epstein,
a cardiologist at the University of Pennsylvania’s Perelman School of Medicine, tells
the Post.

See “More Doubt Cast Over Cardiac Stem Cells”

Researchers continue to conduct studies and publish papers based on Anversa’s fraudulent
publications, heart researcher Jeffery Molkentin of Cincinnati Children’s
tells STAT and Retraction Watch. “It’s just discouraging when you see these papers keep
popping up,” Molkentin says. “There are no stem cells in the heart. Quit trying to publish
those results.”
STAT and Retraction Watch report that “according to publications” Anversa “was most
recently affiliated with the Cardiocentro Ticino and University of Zurich,” but that an email
sent to his Cardiocentro Ticino email address came back to the sender.

Keywords:

cell & molecular biology

data manipulation

fraud

heart

misconduct

nutshell

cardiac

cardiac stem cell

research integrity

stem cell

2Speeding Up Stem Cell Growth


Scientists fiddle with formulas to boost the growth of their stem cell cultures.

Sep 1, 2018
AMBER DANCE
THEY’VE GOT THE BEAT: Heart muscle cells derived from human iPSCs with NME7AB in
the media form much more organized myofibrils, needed for contraction, compared to those raised in
fibroblast growth factor 2 (FGF2).
ANDREW STEWART, MINERVA BIOTECHNOLOGIES

C ell biologist Alicia Lyle hoped to use mouse mesenchymal stem cells to deliver molecular cargos to
tissues, and she also wanted to study how MSCs from different lines of knockout mice assemble into blood
vessels. But Lyle’s group, at Emory University in Atlanta, soon hit a snag: growing the cells took ages.

“Even with the tenderest of care, it was taking somewhere close to eight to twelve weeks to even reach a point . .
. to passage them,” recalls Lyle, referring to the point when cells crowd a dish and need to be split between
multiple culture flasks. And by passage seven or eight, Lyle’s cells began to senesce, losing their ability to either
maintain pluripotency or differentiate.

While mouse MSCs are particularly difficult to work with, Lyle’s complaints echo those heard across the stem
cell field. To both understand stem cells and use them to treat diseases, efficiency will be crucial. “We want to
harness that power of stem cells, we want to use it to treat patients, but then you are limited by how many you
can make,” laments Abba Zubair, medical director for transfusion medicine and the Human Cell Therapy
Laboratory at the Mayo Clinic in Jacksonville, Florida. No one wants to wait around for months while stem cells
grow, or discover that only a few have the potential to differentiate as desired.

And stem cell treatments will only become affordable and commonplace if the technical processes used to
develop them are efficient. For now, another type of cell therapy, the first approved CAR T-cell treatment for
cancer, Novartis’s Kymriah, costs $475,000 for a single dose, suggesting that stem cell–based treatments using
today’s protocols will be similarly complicated and expensive. Still, the potential demand for stem cells could
be quite high, both to directly treat patients and for research into future treatments. To screen 1 million drugs
would require on the order of 100 billion stem cells, as would building a human heart or liver from scratch.

We want to harness the power of stem cells . . . but you are limited by how
many you can make.
—Abba Zubair, Mayo Clinic

How can scientists speed things up? Every stem cell type, from embryonic to induced pluripotent to tissue-
specific, has its own particular requirements for healthy, optimal growth and differentiation. But there are a few
general tips that all stem cell researchers can employ. Some scientists obtain good results by trying different
growth factors. One particular recombinant growth factor, NME7AB, which supports cells in their most naïve,
pluripotent state, can streamline experiments. Another tactic is to develop stem cell-nurturing, three-dimensional
culture techniques, such as a new approach that encases cells in long, sausage-like tubes. And experimentalists
can also improve their stem cell quality and yields by combining more than one method.

Overall, what seems to work is to provide a cell culture setting that mimics the natural stem-cell niche as closely
as possible, which minimizes cell stress, advises Yuguo Lei, a biomedical engineer at the University of
Nebraska–Lincoln.

Embrace Naïveté
Induced pluripotent stem cells (iPSCs), made from a person’s blood or skin cells, have become tremendously
popular. But Cynthia Bamdad, CEO of Minerva Biotechnologies in Waltham, Massachusetts, says most iPSC
cultures are not as “stem” as they could be.

The stem cells in the inner cell mass of an embryo, known as naïve stem cells, are what many scientists would
like to imitate with iPSCs. They lack epigenetic marks that program cell fates, making them fully pluripotent. In
contrast, most iPSC cultures are primed stem cells. They represent a more mature embryonic tissue, the epiblast
that forms the external layer of an embryo. Primed iPSCs often contain epigenetic marks, though the genes
turned on and off are fairly random, says Bamdad.
That means not all cells in an iPSC culture are truly pluripotent; many are already on their way to one cell fate
or another. “It is difficult to make primed stem cells differentiate into mature, functional cell types that we need
for therapeutics,” says Bamdad.

The reason iPSCs tend to turn out primed instead of naïve, she adds, is that most protocols use media containing
fibroblast growth factor (FGF). “FGF, which is used to grow these cells during reprogramming and after,
prevents them from going all the way back to the earliest state that we call naïve.” Only about 15 percent of
iPSCs generated with FGF turn out to be “bona fide” iPSCs, says Bamdad.

Minerva’s AlphaSTEM cell culture products provide a more native environment, she says, because they use a
different growth factor, NME7 (Stem Cells, 34:847–59, 2016). NME7 is typically expressed in very early
embryos, so it better mimics the naïve stem cell niche. Using the recombinant NME7AB, Bamdad says, yields
better iPSCs. “By ‘better,’ we mean works every time . . . better functional profile, higher yields,” she says.
“They all can become any cell type.” With AlphaSTEM Naïve HPSC Medium ($295/500 mL), researchers can
reprogram the majority of cells in their cultures to a pluripotent state, she adds.

Other pro-naïve solutions exist, such as 2i and 5i media formulations. These rely on leukemia inhibitory factor
(LIF) from mice and other biochemical inhibitors. While these media do yield cells that are more naïve than
primed cells, Bamdad notes they can create instability in chromosome numbers, and she’s not sure how well
they mimic true embryonic stem cells.

Minerva researchers say naïve iPS cells could streamline cell production, improve gene-editing efficiency, and
boost the overall quality of therapeutic stem cells. “If you start with better cells, everything’s going to be
easier,” Bamdad says.

Add Dimensions
Another way to improve the stem cell culture environment is to consider its topo-graphy. Cells in the lab often
grow in monolayers on flat glass or plastic. All those plates require a lot of space, media, and time to care for
them; it’s highly inefficient. One solution is to grow cells in suspension, in large flasks or bioreactors. (See
“Easier Ways to Grow Stem Cells,” September 2017, The Scientist.)

But big bioreactors create problems, too, says Lei. At first, the stem cells begin to cluster together into little
spheroids. That’s good; stem cells don’t typically grow alone in situ. But in bioreactors, those spheroids
eventually grow and coalesce into big aggregates. Nutrients and oxygen can’t reach the cells in the center of the
aggregate, and wastes don’t easily diffuse out of the blob. “That makes the cell culture very inhomogeneous,”
says Lei. Aggregation can slow growth, induce apoptosis, or alter differentiation patterns.

Stirring the tank minimizes aggregation, but such agitation creates a shear force that can kill cells, especially
sensitive human stem cells. Lei’s lab has found that up to 40 percent of stem cells grown in spinning suspension
die every day.
TOTALLY TUBULAR: Growing human embryonic stem cells in hydrogel tubes protects them from
moving media and promotes health and higher yields.
LI ET AL., BIOFABRICATION, 10:025006, 2018, DOI:10.1088/1758-5090/AA6B5, CC BY 3.0

Lei sought a way to manage stem cell cluster size while protecting the fragile cells from shear forces. He
developed a hydrogel tube in which stem cells can grow (Biofabrication, 10:025006, 2018). The lab uses a
custom micro-extruder to make the tubes, which are up to 400 µm in diameter. As they extrude the alginate for
the tube through a cylindrical channel (shell flow), they simultaneously pump a cell suspension through its
center (core flow). These materials land in a calcium chloride buffer, which makes the alginate solidify into
tubes around a cell suspension core. With the AlgTubes, his cultures look like vats of udon noodle soup, Lei
says.

The tubes constrain the size of the cell aggregates so that nutrients and oxygen can still diffuse in, and wastes
can filter out. The hydrogel barrier insulates cells from shear forces in moving media.

Lei’s results indicate stem cells are much more comfortable in the tubes. Fewer than 10 percent die every day.
Yields are higher, with up to 500 million stem cells growing in every milliliter of media within the tubes,
compared to 2 million to 5 million per milliliter in suspension culture. Lei expects it will be possible to scale
that up. For example, one could make 10 billion cells with just two milliliters of space contained in tubes. The
cells in AlgTubes can be cultured for more than 50 days, can expand 1,000-fold in just 10 days, and can be
differentiated into a variety of cell types.

AlgTubes can last for months, and when the researchers want to get the cells back out, it’s easy. They use
EDTA to chelate the calcium ions in the hydrogel, and the tubes fall apart.

Lei can provide scientists with detailed instructions for making their own AlgTube extruder, or his group can
produce them for collaborators. He’s started a company, CellGro Technologies, to commercialize the extruder.

Synergize
If one method improves stem cell yields and quality, wouldn’t two be better? That’s what
Lyle tested with the mouse MSCs she isolated from bone marrow. A good protocol to grow
and maintain mouse MSCs would be a boon for research, she says, because scientists already
have so many genetically engineered mouse lines. Researchers could isolate MSCs from
those lines, and then determine how different genes are involved in differentiation, she says.

It doesn’t have to be a one-pronged approach. It can be multifactorial.


—Alicia Lyle, Emory University

Lyle read that basic fibroblast growth factor (bFGF) worked well for MSCs, likely due to its
pro-growth and pro-survival signaling. (FGF wouldn’t be a bad choice in Lyle’s case,
because she wasn’t trying to grow naïve cells.) She’d also seen reports that hypoxia would
help. That’s probably because oxygen levels in the bone marrow, where MSCs naturally
exist, are around 5 percent, much less than the atmospheric 21 percent that most stem cells
are cultured in.

The research team tested four treatments: normoxia and hypoxia, each with or without bFGF.
Then, when the cells covered much of the dish surface and were ready for their first passage,
the scientists measured the concentration of cells per square centimeter. With atmospheric
oxygen and no bFGF, the cells were ready to passage in 39 days, and there were about 6,600
cells per square centimeter. With bFGF, the cells achieved the required confluence within 27
days, and they crowded together at 43,000 per square centimeter. With hypoxia and no bFGF,
the cells were ready after 20 days, and their numbers rose to 99,000 per square centimeter.
But with both hypoxia and bFGF, the cells were confluent at 14 days, and the concentration
was the highest—220,000 per square centimeter (Sci Rep, 7:13334, 2017).

Image Container (remove this text once done styling the container)

TWO ARE BETTER THAN ONE: While both low oxygen (right column) and basic
FGF (bottom row) improve growth of mouse mesenchymal stem cells, the
combination of the two (bottom right) works the best.
CAROTI ET AL., SCI REP, 7:13334, 2017.

Overall, the MSCs in low-oxygen, bFGF conditions grew 2.8 times faster than under standard
protocols, and reached passage 3, when cells are typically ready to test, within one month
instead of the usual two or three. They were less likely to senesce, and retained their ability to
multiply or differentiate until at least passage 11.The results of the dual treatment are “crazy
good,” says Lyle, “I think because it’s closer to what their native niche is.”

Lyle has already used her protocol to generate MSCs from transgenic mice that make
luciferase or GFP. Scientists could then engineer these MSCs and implant them into mice
without the luminescent or fluorescent markers, and track where the glowing cells end up and
how long they survive.

And she’d be happy to add other treatments to improve yields and efficiency. “It doesn’t have
to be a one-pronged approach,” says Lyle. “It can be multifactorial.”

Indeed, Lyle adds, current stem cell culture protocols probably haven’t reached anywhere
near the optimal conditions for efficient growth and division. “There is still a lot of area for
improvement,” she says.

Keywords:

3-D cell culture

cell & molecular biology

cell aggregation

cell culture

stem cell

stem cell research

Human Skeletal Stem Cell Found

Researchers recovered the cells that give rise to


bone and cartilage from fetal and adult bone marrow
and also derived them from induced pluripotent
stem cells.
Sep 20, 2018
ABBY
9203115OLENA

ABOVE: This image shows tissue derived from a single human skeletal stem cell. Bone is in yellow,
blue indicates cartilage, and marrow is pictured in red.
CHAN AND LONGAKER ET AL.
T hree years after its discovery of skeletal stem cells in mice, the same research team has identified
the human version of this precursor to bone, cartilage, and stroma, the bone marrow’s support cells.
In a study published today (September 20) in Cell, the authors show that these skeletal stem cells are
both self-renewing and multipotent.

“For many years there’s been this debate about a true human skeletal stem cell. This study
unequivocally demonstrates that it’s there and that it is self-renewing,” says Richard Oreffo, a stem
cell biologist at the University of Southampton in the UK who did not participate in the work. “There’s
still a lot to do, but this is a tremendous step forward for the field.”

Michael Longaker, a craniofacial surgeon at Stanford University, tells The Scientist that one of the
problems he faces in the operating room is having enough bone to do the reconstructions his patients
need. In the search for a source of more tissue, he and colleagues identified the mouse skeletal stem
cell in 2015, and in the current study focused their attention on finding the human equivalent.

The researchers started with a human fetal femur and sorted the nonhematopoietic cells from blood
precursors. They then sequenced individual, nonblood cells’ RNA. Based on the expression profiles of
cells located in areas of active growth in the fetal bone, they found a suite of four proteins—PDPN,
CD146, CD73, and CD164—the presence or absence of which the team hypothesized could define
skeletal stem cells. None of these markers were shared with mouse skeletal stem cells.

To test their hypothesis, the authors used fluorescence-activated cell sorting to isolate groups of cells
positive for PDPN, CD73, and CD164 and negative for CD146. Both in culture and when transplanted
underneath the outer layer of the kidney in adult mice—a system that serves as a kind of in vivo
incubator—these cells were capable of regenerating themselves indefinitely and differentiating into
cartilage, bone, and stroma.

Notably, these cells do not become fat cells, which differentiates them from mesenchymal stem cells,
a term that some researchers have used interchangeably with skeletal stem cells in the past, but that
the authors write likely contain a mix of distinct stem cell types.

Isolating a skeletal stem cell in fetal bone is one thing, but the researchers wanted to check whether
they could gather these cells from more accessible sources. They determined that skeletal stem cells
were present in both damaged and undamaged adult human femurs. Plus, with the application of
certain factors, the scientists could derive human skeletal stem cells from blood cells and from
adipose stroma—the nonfat, nonvascular cells that physicians collect during liposuction.

A comparison of skeletal stem cells from each of these sources using single-cell RNA sequencing
confirmed that while they all had similar gene-expression profiles, the fetal and induced pluripotent
stem cell–derived cells were more like each other than they were to those from adult bone or adipose
stroma.

“This is a really valuable paper and a next step in the process of unraveling the stem and progenitor
populations that are present in bone marrow,” says George Muschler, an orthopedic surgeon at the
Cleveland Clinic who was not involved in the study. The authors have “added several new tools—
particularly this PDPN marker—to the set of markers that we as scientists can use to find cells of
interest,” he adds.

Linda Sandell, who studies osteoarthritis at Washington University in St. Louis and did not participate
in the work, hesitates at the designation of the cell identified in the study as a stem cell, given that it
can only give rise to three types of cells. Nonetheless, she says, by defining the markers for different
subpopulations of progenitor cells, the authors have provided a starting point to identify cells that only
become cartilage, a finding that could be a boon for treating diseases such as osteoarthritis, in which
the protective cartilage at the ends of bones wears down.
“If tissue engineering and regenerative medicine is going to go in the direction of growing and using
cells that can be expanded in the laboratory, these tools are really extraordinarily important in helping
people start from a controlled environment,” says Muschler. “Having better tools that narrow the range
and variation in the starting materials . . . could have important implications on cell manufacturing in
the future for therapies.”

Longaker agrees that the findings could open the door to regeneration of the aging skeleton. “In
plastic surgery we want to replace like with like, so if you have the skeletal stem cell or the bone-
forming progenitor, use that for bone and cartilage for cartilage,” he says.

C.K.F. Chan et al., “Identification of the human skeletal stem cell,” Cell, doi:10.1016/
j.cell.2018.07.029, 2018.

Keywords:
cell & molecular biology

skeleton
bone
human

stem cell
marrow
News

stem cells
cartilage
regenerative medicine

You might also like