Hepatocyte transplantation improves blood and brain Phe and neurotransmitter imbalances in a
mouse model of PKUKristen J. Skvorak, Ph.D.
Postdoctoral Fellow University of Pittsburgh
Mentors: Dr. Stephen Strom and Dr. Jerry Vockley
NPKUA ConferenceCherry Hill, NJ
July 26-29, 2012
OutlineA mouse model of PKU and the human diseaseCurrent TreatmentsLiver Transplant vs. Liver Cell Transplant
Cell transplant and metabolic diseaseTreatment StrategyResults
BloodBrain
SummaryHuman amnion epithelial stem cellsWhat’s next?Acknowledgements
A mouse model of PKU
Missense mutation results in complete inactivity of phenylalanine hydroxylase (PAH) enzyme
High levels of Phe in the blood, tissue, and brainHypopigmented (fur changes from black to light
brown)Slight delay in growth compared to healthy siblingsCognitive (memory, learning) impairedAdult female mice are fertile, but offspring suffer early
developmental defects similar to human maternal PKU
PKUenu2 mouse is a great model for human PKU.
Phe TyrosinePAH
+BH4
Dopamine HVADOPAC
3-MT
Current Treatments
Harding, C. , Clin. Genet., 2008 Aug;74(2):97-104
Dietary restriction Complicated, expensive Bad taste Non-compliance
BH4 (Kuvan™) supplementation Does not work for
everyoneLiver Cell Transplantation
(Tx) Healthy liver cells contain
100% functional PAH to boost enzyme levels thus reducing Phe levels
Benefits of Liver Cell Tx over Liver TxLess expensive (5-10% the cost of liver transplant)Less invasive, faster recoveryFewer incidents of serious complications or
surgical related deathsMultiple treatments are possible into one patientCells are harvested from donor livers rejected for
whole liver transplant Still limited by the availability of donor livers
Transplanted cells do not need to support all liver functions, only that of the missing enzyme (PAH) If cells fail, the patient would only revert to the condition
he/she was in before undergoing liver cell transplant
Liver Cell Tx and Metabolic DiseaseLiver cell transplant has cured many preclinical animal
models of metabolic disease Crigler-Najjar Maple Syrup Urine Disease (K. Skvorak) PKU (C. Harding) Glycogen storage disease Wilson’s Disease
Liver cell transplant has already been used clinicallyCrigler-NajjarGlycogen storage diseaseOrnithine Transcarbamylase (OTC) deficiencyFactor VII deficiencyBiliary AtresiaAdditional Urea Cycle disordersLiver failure
Treatment StrategyLiver cell transplant could increase enzyme activity
thus helping to improve patient symptoms<10% activity: more manageable disease; increased Phe
tolerance10-20% activity: potential cure (Harding & Gibson, 2010)
Treatment would be most beneficial at birthClinically relevantAvoid surgery – mouse livers are clearly visible through
skinBy weaning (21 days old) mice are already showing
symptoms of PKUNeonatal mouse livers are rapidly expanding
possible growth advantage for transplanted cells
Treatment Strategy
PKU mice (birth)
7 days
Liver Cell Tx
1 million cells transplanted directly into liver
(1 liver ~30 transplants)
Isolate mouse liver
cells
14 days 21 days
(normal diet)
Treatment Strategy
AA profiles (blood and brain)Neurotransmitter profiles
PKU mice (birth)
7 days
Liver Cell Tx
2 million cells transplanted into the spleen
(1 liver ~30 transplants)
Isolate mouse liver
cells
35 days14 days 21 days
(young adult)
28 days
(normal diet)
Results – Phe was reduced in blood after cell transplant
Females had almost double blood Phe levels compared to males.
Phe was reduced 18% in Rosa tx Females and 25% in C57 and AE tx Females.
Interestingly, human placental stem cells (AE) were just as effective as mouse liver cells.
A combination of early + late tx is most beneficial because it will maximize cells engrafted in the liver.
Transplanted CellsMouse liver cells: Rosa, C57Human stem cells from placenta: AE
Results – Phe was reduced in brain after cell transplant
Statistics* = p<0.05** = p<0.01*** = p<0.001
Phe was reduced ~50% in the brains of PKU mice tx with Rosa mouse liver cells.
Phe was reduced ~75% in the brains of PKU mice tx with C57 mouse liver cells.
Phe was normalized in the brains of PKU mice tx with human AE cells.
There was no difference between males and females.
Transplanted CellsMouse liver cells: Rosa, C57Human stem cells from placenta: AE
Results – Many other amino acids were normalized in brain
Statistics* = p<0.05** = p<0.01*** = p<0.001 High Phe concentrations in the brain disturb the healthy levels of
other important chemicals such as neurotransmitters, which are important brain messengers that carry information from one cell to another.
Neurotransmitter Pathways
Tryptophan and Serotonin were normal in the PKU mouse, but 5-HIAA was significantly reduced. 5-HIAA was not improved with cell transplant.
Metabolites along the Dopamine (Phe) pathway were improved after cell transplant.
Phe
Results – improvements in the Dopamine pathway after cell transplant
Sig
nifi
can
tly
Corr
ect
ed
Norm
ali
zed
Statistics* = p<0.05** = p<0.01*** = p<0.001
SummaryMy research involves testing cell therapy in a mouse
model of PKU that closely resembles the human disease.Cell therapy consists of a combination of several early
transplants immediately after birth and one transplant in older mice.
Blood Phe was improved 18-25% after cell transplantObserved difference between male and female
Unique to this mouse model , this has not been reported in the human disease
Brain Phe was improved 50-75% after mouse liver cell transplant while many other amino acids were normalized.
Brain Phe was normalized after human AE cell transplantMetabolites along the Dopamine pathway were either
normalized or significantly corrected after mouse liver cell transplant.
Placental Amnion Epithelial (AE) Stem Cells Amnion – thin membrane surrounding the
fetus during pregnancy Epithelium – cells that make up the outer
layer of the body
Acquired from human placenta following full term birth Plentiful – more than 1.2 million c-
sections/year in the US Easy to isolate, easy to maintain in culture Non-controversial source of stem cells
Not cord blood cells AE does not primarily function to produce blood
cellular components
Documented anti-fibrotic, anti-inflammatory, and anti-microbial characteristics
Can evade immune detection Freeze/thaw well
x100
Amnion
Chorion
Decidua
Placental Tissue
KJ Skvorak, et al. 2012
Liver analysisEnzyme activityAA and Neurotransmitters (Blood and Brain)Survival and Growth
1 week
hAE Tx
1 million cells/mouse transplanted directly
to liver (birth)
2 million cells/mouse transplanted directly
to liver (3+ weeks)
Long term study
14 weeks(100d)
5 weeks(35d)
3 weeks
MSUD mice • 16-fold increase BCAA/ala VS wildtype• 5-6% of normal BCKDH activity• Survive to ~3-4 weeks of age•Fed a Normal Protein Diet Throughout Study
Isolate human amnion epithelial
cells (hAEC)
Hepatocyte Tx in a mouse model of MSUD
Mol Ther. 2009; 17(7): 1266-73Biochim Biophys Acta. 2009;
1792(10):1004-10
Improvements in Growth and Survival
Growth was normalized in MSUD animals after AE cell transplant
All untreated MSUD animals consistently lost weight and died prior to 28 days.
Survival was significantly improved after AE cell transplant
100% survival at 35 days 82% survival at 100 days 0% survival post-28 days
in untreated animals
hAE Transplant doubled residual enzyme activity
BCKDH enzyme activity was increased from 6% to ~13% in AE transplanted animals.
6%
~13%
Amino Acid Improvements
At 100 days of age, hAE improved (in brain): leucine, isoleucine, and valine (the BCAA) by >60% BCAA/ Alanine ratio by >50% Alloisoleucine (biophysical marker of MSUD) >80% Normalized other Large Neutral Amino Acids and GABA (neurotransmitter) All improvements were also seen in blood, and at both timepoints at 35 days
hAE Cell Summary A mouse model of MSUD was partially corrected
after liver cell transplant (Skvorak et al. Mol Ther. 2009 17(7): 1266-73 and Biochim Biophys Acta. 2009; 1792(10):1004-10)
Further studies involving human AE cell transplant in this mouse model improved:Survival and growthBCKDH enzyme activityBCAA levels in blood and brainOther relevant amino acid levelsSome neurotransmitters (GABA, serotonin, dopamine
metabolites, serotonin and dopamine turnover)If clinical hepatocyte transplantation proves
successful for PKU, placental derived AE “stem” cells may provide an alternate source of cells for cell transplant to treat metabolic disease.
What’s next?Continue testing cell therapies in the PKU
mouseAcquire patient cells and make induced
pluripotent stem cells (iPSC)Correct the mutation(s) with molecular meansMay also provide alternate source of cells
Continue to work with the clinical hepatocyte (liver cell) transplant program at the Children’s Hospital of Pittsburgh
AcknowledgementsUniversity of Pittsburgh FundingStephen Strom NPKUARoberto GramignoliKen DorkoMarc HanselVeysel TahanJerry Vockley
Michigan Tech UniversityK. Michael Gibson
Baylor Research InstituteErland ArningTerry Bottiglieri
Karolinska InstitutetStockholm, Sweden
Thank you!
Questions?The Golden Triangle
Pittsburgh, PA
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