Transplanted skin stem cells help blind mice see light

Transplants of skin cells that were turned into light-sensing eye cells enabled
blind mice to detect light, a new study says. In previous research with lab
animals, scientists have programmed stem cells created from blood or skin cells to
become photoreceptors and transplanted them into the back of the eye. Researchers
said this study shows that it's possible to skip the stem cell middle step and
directly reprogram skin cells into photoreceptors for transplantation into the
retina. The U.S. National Eye Institute-funded study was published April 15 in the
journal Nature. "This is the first study to show that direct, chemical
reprogramming can produce retinal-like cells, which gives us a new and faster
strategy for developing therapies for age-related macular degeneration and other
retinal disorders caused by the loss of photoreceptors," said Anand Swaroop, senior
investigator in the NEI Neurobiology, Neurodegeneration, and Repair Laboratory. "Of
immediate benefit will be the ability to quickly develop disease models so we can
study mechanisms of disease. The new strategy will also help us design better cell
replacement approaches," he said in an NEI news release. Induced pluripotent stem,
or iPS, cells that are developed in a lab from adult cells -- rather than fetal
tissue -- can be used to make nearly any type of replacement cell or tissue. But
iPS cell reprogramming can take six months before cells or tissues are ready for
transplantation. The direct reprogramming used in this study turned skin cells into
functional photoreceptors ready for transplantation in only 10 days. The
researchers used both mouse- and human-derived skin cells and transplanted them in
the eyes of blind mice. "Our technique goes directly from skin cell to
photoreceptor without the need for stem cells in between," said lead investigator
Dr. Sai Chavala, CEO and president of CIRC Therapeutics and the Center for Retina
Innovation, and a professor of surgery at the University of North Texas Health
Science Center. While results from animal studies are often not the same in humans,
the researchers are planning a clinical trial to test the therapy in people with
degenerative retinal diseases, such as retinitis pigmentosa. More information The
International Society for Stem Cell Research has more on stem cells. Copyright 2020
HealthDay. All rights reserved.

AAV-mediated cardiac gene transfer of wild-type desmin in mouse models for

recessive desminopathies

Animal procedures and study protocol Two-month-old homozygous DKO mice (B6J.129Sv-
Destm1Cba; http://www.informatics.jax.org/allele/MGI:2159584, breeding pairs of the
latter were received by courtesy from Denise Paulin, Université Pierre et Marie
Curie, Paris, France) [20] and homozygous R349P DKI mice (B6J.129Sv-Destm1.1Ccrs;
http://www.informatics.jax.org/allele/MGI:5708562) [21] were treated with AAV-DES
(AAV9-hTNT2-mDes, DKO n = 7, DKI n = 7) [23]; or NaCl (DKO n = 7, DKI n = 8).
Randomization was performed manually shuffling cards. Sample sizes were chosen
based on experiences from previous studies. A dose of 5 × 1012 vector genomes (VG)
was systemically administered through the tail vein, resulting in a mean body
weight (BW) adjusted dose of ~2.7 × 1014 VG/kg BW. WT littermates were used as
additional controls (WT n = 8). A week after injection, mice were subjected to a
daily 4-week program of controlled swimming exercise (see Fig. 1a) [24]. Fig. 1:
Study design. a  Eight-week-old desmin knockout mice (DKO, n = 14; AAV-DES, n = 7;
NaCl, n = 7) as well as homozygous R349P desmin knockin mice (DKI, n = 15; AAV-DES,
n = 7; NaCl, n = 8) were injected with a dose of 5 × 1012 of AAV9-hTNT2-mDES (AAV-
DES) or isotonic saline (NaCl). Eight untreated wild-type (WT) mice served as
controls. Swimming experiments were performed in all groups as indicated by the
gray rectangle. Mice were assessed by echocardiography (echo) prior, during and
after the swimming exercise at the indicated time points after starting swimming
exercise. Numbers on the right-hand and on the left-hand sides indicate the animal
numbers at the beginning and the end of the experiment, respectively. Time point of
death of animals that spontaneously died during the experiment are annotated by a
cross.  b  The AAV-DES vector genome consists of the DES cDNA under control of the
human troponin T2 ( hTNT2) promoter. Animals were fed ad libitum and housed in a
temperature- and humidity-controlled room in a specified pathogen-free environment
under 12:12 h light/dark cycles. Genotyping was performed as previously described
[21, 23]. All procedures involving the use and care of animals were performed
according to the Directive 2010/63/EU of the European Parliament and the German
animal protection code. Approval was granted by the local ethics review board
(Administrative Council [Regierungspräsidium] Karlsruhe, Germany, G-143/11). Left-
ventricular function was assessed using transthoracic echocardiography before
vector application, two weeks into swimming exercise, upon completion of the
swimming exercise, 6 months and 15 months after vector application. All
measurements were performed investigator blind. Mice were sacrificed by cervical
dislocation and weighed. Hearts were removed, washed in phosphate-buffered saline
(PBS), weighed and cut in half biventricularily along the SAX of the left
ventricle. One half was embedded in TissueTek (Sakura, Staufen, Germany) and frozen
on dry ice. The other half was further dissected. Atria and the right ventricle
were removed and snap-frozen. Additional samples from liver and soleus muscle were
taken. All measurements were performed by a blinded investigator. Generation of AAV
vectors Murine desmin cDNA was put under the control of the human troponin T
promoter (Fig. 1b) and packaged in AAV9 capsids by cotransfection of pds_hTNNT2-
mDES [22, 23] together with pDP9rs, a derivate from pDP2rs [25] with the AAV9 cap
gene from p5E18-VD2–9 [26] in HEK293T cells using polyethylenimine (Sigma Aldrich,
Taufkirchen, Germany). AAV was purified and titrated as previously described [27].
Swimming exercise protocol Endurance exercise was carried out as previously
described [24]. Mice were subjected to a step-up swimming protocol starting with
10 min twice daily increasing the duration by 10 min each day until an exercise
duration of 90 min twice daily was reached. The protocol was ended after 30 days.
Mice were closely observed at all times to avoid hypoxia. Transthoracic
echocardiography Mice were depilated using Veet Sensitive (Reckitt Benckiser, Hull,
UK). Long axis and short axis (SAX) B-mode cine loops were obtained. SAX M-mode
loops were taken mid-ventricularly at the papillary muscle level. Images were
obtained in hand restrained mice using the Vevo2100 system with a MS400 transducer.
Three consecutive M-mode measurements of the diastolic posterial wall thickness
(PWTd) were performed. Fractional shortening (FS) and left-ventricular enddiastolic
diameters were obtained measuring more than 6 consecutive heartbeats using the LV-
trace function on a SAX M-mode. Western blot Heart tissue stored at −80 °C was
thawed on ice and homogenized in lysis buffer (pH 6.8, 5 mM Tris, 10 %SDS, 0.2 M
DTT, 1 mM EDTA, 100 mM NaF, 50 mM beta-glycerophosphate, 2 mM Na3VO4, 1 mM PMSF,
complete mini protease inhibitor cocktail (Roche, Mannheim, Germany)). Three
hundred microliters of lysis buffer was used per 10 mg of cardiac tissue. Tissue
was homogenized using tissue lyser 2 (Qiagen, Hilden, Germany) and denatured by
incubation at 95 °C for 5 min, upon which samples were centrifuged for 10 min at
13,000 rpm. The supernatant was aliquoted and stored at −20 °C. Lysates were
adjusted to an identical total protein concentration after quantitative analysis of
Coomassie-stained SDS-polyacrylamide gels. For comparative quantitative blotting,
identical total protein amounts were loaded in all lanes. Gel-electrophoresis was
carried out using a 12% sodium dodecyl sulfate glycine gel at 80 V for 2.5–3.0 h.
Gels were blotted to nitrocellulose (Whatman Protran BA83; Sigma Aldrich) for 1.5 h
with 400 mA. Membranes were blocked with milk powder (5% in TBS-T) for 1 h on a
shaker and washed three times for 10 min with distilled water. Incubation with
primary antibodies for desmin (DE-U-10; diluted 1:5000, Merck, Darmstadt, Germany)
and GAPDH (Sigma Aldrich, 1:7500) solved in TBS-T was carried out overnight on a
shaker. Secondary antibodies goat anti-mouse-HRP (Santa Cruz Biotechnology, Dallas,
TX, USA) and goat-antirabbit-HRP (Santa Cruz Biotechnology) were applied for 1.5 h
on a shaker at a dilution of 1:5000. Membranes were washed thoroughly in TBS-T
after each antibody staining as well as after antibody stripping (Thermo
Scientific, Rockford, IL, USA) which was performed between desmin and GAPDH
staining. Blots were read out on a ChemiDoc XRX Imaging System (BioRad) applying
Pierce ECL2 western blotting substrate (Thermo Scientific) following the
manufacturer’s instructions. Expression levels were quantified using ImageJ (NIH,
Bethesda, NJ, USA). Immunofluorescence Frozen heart tissue embedded in optimal
cutting temperature compound (TissueTek O.C.T. Compound, Sakura Finetek, Neumatten,
Germany) and stored at −80 °C was cut in 9 µm slices using a Leica CM1950 cryostat
and put on microscope slides. Tissues were fixed in aceton in a −20 °C freezer for
10 min, slowly thawed for ~30 min at room temperature and blocked for 1 h 10% FCS,
1% GS in PBS in a wet chamber. Slides were incubated with primary antibody RB-
alpha-Desmin (Biozol, Eching, Germany, LS-B2264) diluted 1:75 in PBS and 10% FCS in
a wet chamber at 4 °C overnight. Slides were washed three times for 5 min in PBS.
Subsequently, the secondary antibody Alexa Fluor 488 goat anti-rabbit lgG (Thermo
Fischer, Dreieich, Germany) was applied for 1 h in a wet chamber. After washing the
slides three times for 5 min in PBS, cell nuclei were stained with DAPI 1:1000 in
PBS, upon which slides were washed again and mounted in Mowiol mounting medium.
Confocal microscopy was performed with a Zeiss LSM 780. Masson-Trichrome staining
Slides with heart tissue slices of 9 µm thickness were thawed at room temperature
and fixed in 10% formalin for 60 min and refixed in Bouin’s solution overnight to
intensify contrast and colors. The next morning, slides were washed under running
tap water (18–26 °C) and nuclei were stained for 5 min with Weigert’s Hematoxylin
and blued for 10 min under lukewarm running tap water to remove excess of
hematoxylin, followed by a short rinse in distilled water. Cell cytoplasm, muscle,
and collagen fibers were stained red for 5 min in Bieberich scarlet acid fuchsin
solution. Slides were rinsed three times in distilled water for 1 min and incubated
for 10 min with a combination of 1:1 phosphotungstic/phosphomolybdic acid solution,
to decolorize collagen fibers of Biebrich scarlet acid fuchsin solution and prepare
uptake of aniline blue. The excess solution was drained on a paper towel, upon
which aniline blue solution was applied for 15 min without rinsing to stain
collagen fibers. Finally, slides were washed three times in distilled water and
dehydrated with graded alcohols in ascending order (70, 90, 100% EtOH) ending with
Xylol for 5 min prior to mounting with Vitro-Clud mounting medium (Langenbrinck,
Emmendingen, Germany). Images were acquired on an Olympus BX 51 microscope with an
Olympus XC 30 camera using the cellSens software. Fibrous tissue was quantified
using ImageJ as previously described [23]. Statistics Statistics were performed
using GraphPad Prism 8 (GraphPad, San Diego, CA, USA). All data are expressed as
mean ± standard error of the mean. Unless stated otherwise, a one-way-ANOVA was
used to test for differences between groups. The Tukey’s honest significance test
was applied for p value adjustment. A p value below 0.05 was considered
significant.

With laboratories shut, coronavirus forces scientists to ‘stop cold’

Dr. Nader Pouratian implanted matchbook-sized devices into the brains of four blind
volunteers more than two years ago. Since then, the participants in the
neurosurgeon's pioneering study have visited his UCLA lab each week to let him hone
a system that could give them a rudimentary form of vision. Pouratian hoped to
expand his experiment next year to include dozens of people around the country, and
eventually make the treatment available to blind people everywhere. Then the
coronavirus came along. Now seven years of work have ground to a sudden halt.
Regular life will resume someday, but Pouratian's project may not. "Our first
concern obviously has to be the well-being of the people we work with," he said.
"But as a scientist, it is hard to just stop cold like this." Hardly a facet of
life remains untouched by the sweeping effort to slow the coronavirus' spread.
Researchers like Pouratian are facing more than lost income and social isolation.
The abrupt stoppage of a vast array of exploration and experimentation at
universities and other research institutions has left scientists wondering about
the discoveries that may never be made, the sick people who will miss the chance at
a breakthrough cure and the careers that may never be launched. The longer the
emergency measures remain in place, the more scientists stand to lose. Cell lines
specially engineered for particular experiments get older every day. Distant stars
cycle out of view of even the largest telescopes. Valuable data is lost before it
can be collected. When the orders came down to close their laboratories, scientists
scrambled to mothball their experiments in ways that would maximize their chances
of being revived. Those who need only a computer and an internet connection to run
simulations or crunch complex numbers continued their work from home. Now almost
everyone is barred from their labs. Exceptions have been granted to the relatively
few scientists who could show they'd lose irreplaceable work if their research
couldn't continue and those running clinical trials that provided critical care to
patients. The rest have relied on skeletal staffs to tend to the animals, cells and
expensive equipment that were suddenly abandoned. People who were investigating the
new coronavirus or possible treatments for COVID-19, the disease it causes, were
exempt from the shutdown orders. A hiatus of several weeks isn't likely to result
in irreparable harm, said Randy Katz, vice chancellor for research at UC Berkeley.
If the restrictions stay in place for months, however, losses will become
increasingly difficult to avoid. For example, mice that have been bred to have a
particular genetic condition or disease must begin an experiment at a particular
age, giving scientists a narrow window for conducting their experiments. "Animals
don't live forever," Katz said. California's stay-at-home order has complicated
efforts by Berkeley scientists to measure the state's snowpack at field stations in
the Sierra Nevada, Katz said. State officials rely on that data to determine how
much water will be available for drinking and for irrigating crops. "We obviously
need to go when there is snow," Katz said. "If we wait too long, the opportunity is
lost." UC Berkeley astronomer Alex Filippenko figured he was in the clear when he
got special dispensation for a last look at several celestial objects, including a
group of supernovas that could help scientists determine the current expansion rate
of the universe. He settled in at a remote observing room on campus that connected
to one of the 10-meter telescopes at the W.M. Keck Observatory on Hawaii's Mauna
Kea. But on the first night of his observing run, he got the news: The telescope
was shutting down. Filippenko scrambled to reprioritize. He dropped plans to watch
another type of supernova that could reveal more about how stars explode and the
chemical elements they forge. By the time he's able to look again, these stellar
phenomena will have long since faded away, he said. Caltech geobiologist Victoria
Orphan had recently returned from a research cruise with a trove of deep-sea
microbes when word came down that nonessential experiments would have to stop.
Orphan's work with extremophiles — organisms that survive under extreme conditions
like high pressure or a complete lack of sunlight — could shed light on the origins
of life on Earth and the potential for life on other worlds, among other things.
And because these microbes live in these extreme environments, they're not easy to
collect. Orphan didn't want to lose them. Handling the delicate microbes in the
oxygen-free conditions they're used to can be tricky, she said, so one of her grad
students wrote out detailed instructions for a lab manager who is still going on
campus to take care of the living things left behind. Some of the high-pressure
experiments she had in mind were moved to the back burner since they couldn't be
monitored from home, but if the microbes are still hearty when the stay-at-home
order ends, she'll give them a try then, she said. And Saul Villeda, a
neuroscientist and stem cell biologist at UC San Francisco, said his "head is still
kind of spinning" after he was told last month that all nonessential work would be
suspended within about 72 hours. "It was incredibly fast," he said. Villeda's
research on aging has turned up compounds in the blood of young mice that may help
reverse cognitive decline in older animals. When the shutdown order came, he
gathered his team on a video conference to make quick decisions no scientist ever
wants to consider: What tissues could they harvest? Which ones could be preserved,
and which would have to be discarded? "It was just this overwhelming feeling
because you're not sure how you're going to make it work," said Villeda, who
managed to freeze his most precious samples."And then, of course, you make it
work." Researchers running trials on experimental medical treatments have been
granted reprieves if participants in their studies are dependent on the treatments.
Pouratian's work at UCLA did not meet this high bar, so it had to shut down. The
system to create a type of artificial vision, which he helped a medical technology
company develop, requires subjects to wear special glasses with small embedded
video cameras that connect wirelessly to the brain implants. The implants then
translate the video footage into patterns of stimulation that the brain interprets
as flashes of light, allowing the person to discern motion such as whether someone
is approaching them or walking away. Much work remains to fully understand how a
brain's visual cortex and the device interact. After being designated a
"breakthrough device" by the Food and Drug Administration, the invention had been
on an expedited track toward regulatory approval for widespread use. But now
everything is on hold, and the company that produced the device is shutting down,
an economic casualty of of the pandemic. There is confusion about other sources of
funding as well. Government agencies and private groups dole out billions of
dollars in grant money each year — but it usually has to be spent during a limited
period of time and requires researchers to finish projects on a strict schedule.
Matthew Fenton, director of the extramural activities division for the National
Institute of Allergy and Infectious Diseases, said his agency has been fielding
urgent questions from anxious researchers who fear their end-of-year reports won't
show enough progress. "We assure them that that is not the case at all," Fenton
said. "We're there to support them as much as we can to get through this." Perhaps
the most vulnerable researchers are those just starting out in their careers who
don't have a track record of success to fall back on. For newly minted doctorates,
advancing up the scientific ranks is a challenge in normal times. Now many fear it
will be all but impossible as their laboratory work — and the coveted academic
publications that should follow — enters a sort of purgatory it may never escape.
Karla Satchell, a microbiologist studying cholera and cancers at Northwestern
University Feinberg School of Medicine, said she was particularly concerned for
three of the graduate students working in her lab. One of them was set to get his
doctorate this summer but can't complete experiments he needs for his thesis. "It's
really tough on these trainees," Satchell said. "You sort of get a momentum in
graduate school and you keep that momentum going." To be sure, many scientists
whose work is primarily computer-based have been able to continue analyzing their
data and running simulations as they shelter at home, albeit with more distractions
than usual. That's little consolation for people like Pouratian, who sees a
promising breakthrough possibly slip away. His thoughts keep returning to the four
volunteers who agreed to have brain surgery for the sake of his experiment. "They
did it not only for themselves, but because it might help others down the road," he
said. "And, now, they're just sitting at home." ——— ©2020 the Los Angeles Times
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