ISS Research to Advance Health on Earth

ISS Research to Advance Health on Earth
• DR. BESS DAWSON-HUGHES, SENIOR SCIENTIST AND
DIRECTOR, BONE METABOLISM LABORATORY, TUFTS
UNIVERSITY
• DR. MILLIE HUGHES-FULFORD, SCIENTIFIC ADVISOR TO
THE UNDER SECRETARY OF THE DEPARTMENT OF
VETERANS AFFAIRS, SAN FRANCISCO VA HEALTH CARE
SYSTEM
• ROSAMUND SMITH, RESEARCH FELLOW,
BIOTECHNOLOGY DISCOVERY RESEARCH, ELI LILLY AND
COMPANY
• DR. EUGENE BOLAND, CHIEF SCIENTIST, TECHSHOT, INC.
SPEAKERS:

Using ISS to Study T Cell Activation
Dr. Millie Hughes-Fulford
milliehf@gmail.com

Infection in Spaceflight
• Apollo 7 all three crew members had a
cold.
• Apollo 13, one of the crew members
became ill with a common bacteria
which usually does not affect people
with normal immune systems.
• In all, 15 of the 29 Apollo astronauts
had an infection either during flight or
within one week after flight.
(Biomedical Results of Apollo, Johnston, Dietlein, and Berry, NASA Washington, D.C. 1975
© 2016 Hughes-Fulford

Activation of T cells in the Immune System

The 3 Signals Required for Full T Cell Activation
Antigen/MHC
TCR/CD3
CD28
IL-2R
B7 (Accessory cell)
IL-2
(Autocrine
signal)
Anti-CD3
or Con A
anti CD28
TNFa
INFg
Chemokines/Cytokines
Inflammatory factors

Experiment Housed in ESA
Kubic On board incubator
with 1 g control
SpaceX3 launch Tcell Activation in Aging

Visualization of gene array chip

mRNA expression Heat Map from Activation of T cells in
Spaceflight

Immune Genes Regulated by Gravity

Are T cells in other mammals activated normally
in spaceflight?
• Test the effects of microgravity in vivo in spaceflight in the mouse
model on STS-131.

Experimental Design for C57BL/6 mouse STS-131
T cell activation Experiment
Flight Mice (n=8) 22.01 +/- 0.85g
Ground Mice (n=8) 22.04 +/- 0.94g
Spaceflight (n=8)
Ground (n-8)

mRNA Gene Array Heat Map analysis of Mouse T-cells
Gene expression 10 hours after landing

Gene expression of Mouse T cells after Flight using qRTPCR

Conclusions
• These data support a physiological dependence of the
immune system on gravity.
• These data also suggest that this might be a common
thread throughout the animal kingdom, since two
mammalian immune systems that developed on this
planet are dependent on gravity for full function.

2016 Hughes-Fulford
TRANSCRIPTION
TRANSLATION
TNFa
INFg
GMCSF
CCL3
• The messenger (mRNA) is
converted to protein through a
process called translation.
• It has long been known that the
translation step is regulated by
transcription factors in the
promoter regions, however new
data has proven that there is a
second level of regulation by
miRNA.

miRNA Regulates Protein Synthesis
• The first miRNA was found in nematode C. elegans
in 1993 they were found in humans 2004.
• miRNAs act as binary switches turning protein
translation on and off
• MicroRNAs (miRNA) are small non-coding RNAs
about 20 nucleotides in length

New mode of gene regulation: miRNA

Is miRNA Regulated in the
Human Immune System?
Our early ISS experiments was designed to analyze RNA
expression we had an unusual finding-
One miRNA was found to be differentially regulated.

Human T Cell Had Altered miR-21 in in Flight

ISS Experiment Results: Gene Expression of
miR-21 Protein Targets

Using microgravity, we discovered a new mechanism of
resolution of inflammation, this offers new insight on
the normal regulation of the immune response and a
new way to approach treating immune disease.

Biological Drugs Act on the Immune System
T cell
Macrophage
B-cell
INFg , TNFa
IL-2Ra IL-2
CD-20

Top Biotech Biological Drugs Targeting
Small Immune Molecules
Drug Company Action Disease treated
1. Enbrel (Amgen) TNF inhib RA
2. Rituxan (Genentech) a CD20 leukemia
3. Humira (Abbott) TNF inh RA
4. Remicade (J & J) a TNFa autoimmune dis
5. Gleevec (Novartis) T. K. inhibitor leukemia
6. Neulasta (Amgen) CSF stimulates marrow

miRNA Regulates Protein Synthesis
• Seed sequence in the small microRNA matches to the
3” end of the messenger RNA and blocks it translation.
• We are activity searching for the regulators of
inflammatory molecular regulators of

T cell Activation in Aging Experiment
SpaceX5 Launch
Experiment Housed in ESA
Kubic On board incubator
with 1 g control

Discovery of miRNAs downregulated
in 1-g Immune Activation are Missing
in SpaceX5 experiment

Final Remarks
• Using the microgravity of International Space Station
National Laboratory, we have discovered a method of
finding the pivotal points of miRNA regulation thereby
providing us with new pharmaceutical targets for
treatment of immune dysfunction such as immuno-
senescence in the elderly.
• We will seek out miRNAs that can ameliorate the
excessive immune response seen in autoimmune
diseases such as rheumatoid arthritis, multiple
sclerosis and Crohn’s diseases.

HUGHES-FULFORD LABORATORY
UCSF,NCIREANDVAMCSANFRANCISCO
:
NCC-2-1086,NAG-2-1286,NIH-1aAgingand CASISGrants
Zurich Laboratories
Augusto Cogoli
Isabelle Walters
Università diSassari
GraziaGalleri‡,
MariaAntonia Meloni‡,
ProtoPippia
Many Thanks tothe NIH, CASIS, NASA
and ESA Administration &Staff
Hughes-Fulford Laboratory
TaraCandelario
Emily Martinez
Tammy Chang,MD
Marlene Grenon, MD
JoeMessier
JesusAguago
JimBoonyaratanakornkit,Ph.D, MD
ChaiFei Li,DVM
Millie.Hughes-Fulford@ucsf.edu

Acknowledgements
• Lilly
Marty Cramer
Pam Mitchell
Ken Savin
• BioServe
• CASIS
• NASA
• Taconic

Eli Lilly and Company
We make medicines that help people live longer, healthier, more active lives
♦ Company founded May 10, 1876 by U.S. Civil War
veteran Colonel Eli Lilly
♦ Headquarters located in Indianapolis, Indiana, U.S.A.
♦ Approximately 40,000 employees worldwide
♦ Products marketed in 125 countries
♦ More than 8,000 employees engaged in research and
development, or 20% of total workforce
♦ Lilly products treat cancer, depression,
schizophrenia, diabetes, osteoporosis, psoriasis and
many other conditions
♦ Expertise in musculoskeletal conditions
• Osteoporosis, Muscle atrophy
• Preclinical and clinical measures of bone and
muscle
• Myostatin inhibition

Skeletal Muscle Atrophy occurs with Many
Diseases
• Over 124 conditions/diseases have been associated with skeletal muscle
wasting/atrophy
• Includes certain forms of cancer, ALS, muscular dystrophy, sarcopenia, COPD, critical illness
myopathy
• And of course astronauts in space
• Its not the size but the decrease in muscle function that is most critical to the patient
• Decreased muscle power, strength, endurance
• Decreased muscle function leads to diminished functional performance
• Decreased ability to climb stairs, withstand medical treatments, perform activities of daily
living
• Increased chance of falls, injuries from falls, fractures, hospitalizations, death
• Often leads to lack of independence
• Associated with poor prognosis and outcomes

Muscle Atrophy Represents a Large Unmet
Medical Need
• The only treatment currently for muscle atrophy is physical
therapy/exercise
• May not be sufficient
• Poor compliance with the ill/elderly
• Is it possible to develop a drug to combat muscle atrophy?
• Some drugs are working their way through clinical trials, but nothing
near FDA approval

Mouse Models of Muscle Atrophy
• Mechanisms of muscle wasting highly conserved between mice and man
– Similar cell signaling pathways and key genes are involved
• In drug discovery it is important to test compounds in mouse disease
models before progressing into the clinic
• Mouse models of muscle atrophy are not ideal
– Unloading models, eg hindlimb suspension, limb casting
– Tumor models
– Genetic models eg SOD1 transgenic mice, mdx mice
• In microgravity, muscles are ‘unloaded’ and evidence to date suggests they
undergo atrophy

Evidence for Muscle Wasting in Mice under
Conditions of Microgravity
Species
Number
/group
Age at
onset
Duration
Days
Included Measures Transporter Ref
Rat
Fisher 344
12 17 -5% BW; -24% gastroc Space shuttle
Lalini et al
2000
Mouse
C57Bl/6J
12 9 week 11
Decreased muscle CSA; -
5%BW from baseline; atrophy
soleus> gastroc
Space shuttle
(Amgen)
Harrison et al
2003
Mouse
C57Bl/6J
12 9 week 13
-8.5% BW; decreased CSA
particularly type II fibers (IIb,
IIx). Soleus -30%; gastroc -15%
wt.; masticater muscles not
affected
Space Shuttle
(Amgen)
Ferguson et al
2012; Sung et
al 2013;
Philippou et al
2015
RR-3
Mouse
Balb/c
10 12 week 42
Body composition,
grip strength
ISS Lilly

Rodent Research 3
• Goal: To determine if a novel investigational compound
is able to prevent the muscle wasting that is expected to
occur on the ISS
• Primary goal:
– Use the results to help in discovery research to find ways to
develop new medicines for patients in need with muscle
wasting conditions
• Secondary goals:
– Continue to learn how animal cellular functions are affected
by longer term microgravity

Unique Features of RR-3
• Novel investigational compound (myostatin inhibitor)
• New strain of mice, Balb/c
• 6 week experiment
• Measure muscle function with grip strength meter
• Anaesthesia followed by recovery on the mice to enable interim
body composition measures
• Body composition with DEXA Instrument (Bone Densitometer,
TechSHOT), 2nd experiment to use this capability

Experimental Design
Groups:
1. Baseline, n=10
2. Flight mice, control treated, n=10
3. Flight mice, drug treated, n=10
4. Ground control, control treated, n=10
5. Ground control, drug treated, n=10
Female Balb/c mice, approx 12 weeks of age at launch
Duration of experiment= 6 weeks
Ground controls offset 3 days from flight animals
Animals randomized on body weight to groups
Control and drugs delivered by sub-cutaneous injection at 0,2,4 weeks
Treated and control mice mixed in each habitat (3,2)
• Tail tattoo used for rapid identification of treatment (treated vs. control)
• Mice identified by implanted microchip

Pre-flight Adjustments
• Mice acclimatized to drinking apparatus and food bars 4 weeks
prior to launch
• Mice acclimatized to lack of bedding and wire floors 4 weeks prior to
launch
• Mice moved into Dragon habitat (10 mice/habitat) at L-24h

We have Lift-Off !
April 8, 2016

Life Onboard ISS
• Dragon docked 1 day 17 hours post-launch
• Mice moved into ISS habitat

Live Phase Measures: Grip Strength
Columbus Instruments, OH
Grip strength measured pre-flight, interim and
termination for both flight and ground control mice
• 4 measures taken
per mouse per time
point
• 2 middle values
averaged

Live Phase Measures: Body composition
• Body composition measured by DEXA (dual energy x-ray absorptiometry) at
interim (approx 4 weeks) and at termination (approx. 6 weeks)
– Lean mass, fat mass, bone mineral content and density
Bone scan of anesthetized mouse

Live Phase Measures:
Anaesthesia Recovery
• DEXA performed on anesthetized mice
• First time to perform anesthesia followed by recovery on
mice in space
• Mixture of ketamine/xylazine used, followed by
atipamezole to speed recovery
• Mice kept separately in newly designed hardware
(Anesthesia/Recovery System or ARS)

Termination Sampling
• Serum (cardiac puncture)
• Hindlimb put into formalin for later histology
measures
• Carcass frozen at -80oC
• Samples due back to Earth in late August on
SPX-9

Later Measures
• Muscle weights (including gastrocnemius, quadriceps, soleus)
• Brain weights
• Muscle hindlimb histology, myofiber cross-sectional area
• Muscle gene expression
• Compound exposure (serum)
• Bone parameters including biomechanics
• Other measures still to be determined
• Plan is to give unused tissues from control mice to other
spaceflight researchers

Myostatin - a regulator of
muscle mass
• Compound tested is a myostatin inhibitor
(LSN2478185, YN41)
– Myostatin is a growth factor that is a negative
regulator of muscle mass
– Has been shown to prevent muscle wasting in
terrestrial mouse models of muscle wasting (Smith et
al 2015; unpublished)
• A less specific inhibitor of myostatin, called ActRIIB-
Fc, was tested by Amgen on a Space Shuttle mission
(13 day flight)
– Was able to reduce or prevent muscle wasting
(Ferguson et al, 2012)
Smith et al 2015
Indicates significance relative to the control p< 0.05.
**

Preliminary Results from Live Phase Study
– Muscle Function
♦ Grip strength in ground controls relatively stable
• Increase at week 6 likely age-related increase in size
♦ Grip strength decreased in flight animals at week 4
• Recovered towards baseline by week 6
• Increase at week 6 likely due to age-related increase
in size
• Note different operator and orientation of grip
strength meter of baseline vs weeks 4 and 6
measures
♦ In both ground control and flight animals myostatin
inhibition significantly increased grip strength over
control animals at both weeks 4 and 6
• To baseline values in flight animals
• Above baseline values in ground controls
Effects of myostatin inhibition on
skeletal muscle function can occur
under conditions of microgravity
(*), refers to significance relative to its respective
control at each time point, ($), refers to significance
relative to the flight control group. All p values < 0.05.
*
*
$
*
$
*

– Lean Mass (DEXA)
♦ Increase in lean mass seen with myostatin inhibitor
compared to respective controls at both 4 and 6 weeks
♦ Ground and flight controls show increase in lean mass with
time
• Likely age related, as body weight increases with time for
ground controls
♦ Surprisingly, flight controls did not have decreased lean
mass compared to ground controls
• Previous shuttle missions had shown loss of body weight, lean
mass and individual hind limb muscles but these were earlier
time points
• Discordance with grip strength data
RR-3 Lean Mass (DXA)
Weeks
0 1 2 3 4 5 6 7
%ChangefromBaseline
-5
0
5
10
15
20
25
Flight Control
Flight YN41
Ground Control
Ground YN41
*
*
*
*
control at each time point, All p values < 0.05.
Effects of myostatin inhibition on skeletal muscle
mass can occur under conditions of microgravity

– Lean Mass (DEXA)
♦ Explanations for apparent lack of muscle wasting
• Are the ground controls doing poorly?
– Equivalent ground control experiment performed
independently at Lilly in regular housing and results show
ground controls performed as expected in terms of body
weight gain
• Wasting may have occurred earlier but mice
recovered/overcompensated?
• Fluid distribution in space affects DEXA readings?
• Greater levels of altered types of exercise by flight animals?
♦ Important to get muscle weights to validate DEXA results
♦ Determine if changes in lean mass are seen in all or subset of
muscles
control at each time point, All p values < 0.05.
Effects of myostatin inhibition on skeletal
muscle mass can occur under conditions of
microgravity
*
*
*
*

Next Steps
• Continue analysis of samples and data
• Use data to further understand effects of microgravity on skeletal muscle
• Use data to assist drug discovery efforts to find drugs to combat muscle-
wasting diseases

Space BioManufacturing
Techshot, Inc.
Eugene Boland, PhD – Chief Scientist

Mechanical Devices will Keep Us Alive:
Until We Can Print the Replacements

3D Printing and Biology Makes Headlines
Modern medicine: Lab-grown genitals,
spray-on skin ... - NBC News
3-D printed livers offer glimpse into the
future ... - NBC News
Father and Son Produce Leather via
Bioprinting - VegNews Magazine
Bioprinting cartilage into people
is doctor's goal
New bioprinting technique creates
thicker, healthier tissue
New Bioprinting Method Borrows Ancient
Technique
3D bioprinting could spark ethical debate
3D Printing Reshapes Healthcare
“ team of cardiovascular scientists has
announced it will be able to 3D print a
whole heart from the recipients' own cells
within a decade."

Reality of Organ Shortage*
• 120,206 People on transplant list in the USA
• 16,445 organs transplanted in first 6 months of 2016
• 7,764 organ donors in the first 6 months of 2016
• For 2016, 1 person is added to list every 10 minutes
• For 2016, 22 people die waiting each day
All Data current as of 2:30 pm EDT 07/12/2016 as reported by the
United Network of Organ Sharing (UNOS)

What if One Day?
• Personalized Medicine was more than a better prescription?
• Stem Cell Therapy moved from the Wall Street Journal and
USA Today to Main Street even if it had to go to space first?
• A child born with a congenital heart defect had only one
surgery then grew up to be the best third baseman on his/her
little league team instead on just watching sports?
• Third Party Payers realized it is cheaper to fix a problem right
the first time that prolong the wrong treatment?

What is Bioprinting?
Cells
Polymers
Image File
Image Processing
Additives
Organs
Tissues
3D
Constructs
Image Rendering
BioInk
Dispensing Pen
Transfer Language Bioprinter
Pre-Fabricated
Systems
Structural
Components

Development Team
•Project management (prime contractor), spaceflight hardware
development, integration and operation. Space medical solutions
provider. Tissue bioprinter production management/supervision.
•Pioneer in the Direct-Write 3D tissue printing field. Has
expertise in Bioink and currently has a patent protected delivery
system with the highest resolution and broadest dynamic range
in terms of bioink capability. Supplier of delivery systems and
control software integration partner.

Relevant Technologies
• Techshot
• ADSEP bioreactor (STS-95 heart patch)
• ACT2
• MVP
• Substage illuminator
• Electrospinning device
• n-Scrypt
• Bioassembly Tool
• Smart Pump
• Dispenser pens
• Catalog of 10,000+ printed materials

Relevant Sizes
Water Glucose Antibody Virus Bacteria Human Cell Period Heart
Nanometers
Nanotechnology
Range
Bioprinting
Range

What We Can Grow Today
Chang, et al. 2012, ATVB

Technology Milestones*
• Month 8/15 – Parabolic/suborbital flights to
test enabling technologies in microgravity.
Key parameters include viscosity, cavitation
potential / pressure gradient, cell viability,
density gradient and thermal gradients.
• Month 28 - ISS Technology Demonstration.
Print a microvascularized tissue patch and
return it for terrestrial evaluation.
• Month 36 - ISS Experiment. Print a beating
heart patch within bioreactor to mature the
tissue. Tissue to contain arteriole, capillary
bed and venule structures. Tissue will be
perfused to demonstrate the ability for use
as a transplant.
*no NASA or CASIS funding or commitments have been implied or received in these forward looking statements

ISS Research to Advance Health on Earth

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