247 Embryonic Renal Progenitor Cells Activate the γ Catenin/TCF Signaling Pathway during Integration into Damaged Adult Renal Tubules Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © T[.]
Trang 1Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
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FETAL AND ADULT STEM CELLS
MicroRNA Card to analyze a focused set of genes and miRNA
expression patterns in iPSC clones generated using CytoTuneTM
iPS Sendai Reprogramming Kit and isolated using AP Live stain
Comprehensive analysis of the resulting can be utilized not only to
derive similaritites between the iPSC clones and control hESC lines
but also dissect subtle differences further evaluate their impact on
functionality and long term stability In addition, TaqMan® assays
can be extended to carry out additional analysis for the confi rmation
of cellular karyotype
Precise Genome Editing with rAAV
Rob Howes,1 Alex Rieck,1 Eric Rhodes.1
1 Horizon Discovery, Cambridge, United Kingdom.
The ability to edit the human genome in a targeted and specifi c
way in cell lines to introduce specifi c mutations relevant to human
diseases is essential in understanding disease biology Horizon
Discovery’s GENESIS gene-editing platform uses the unique
ability of targeted rAAV vectors to naturally stimulate and take
advantage of the high fi delity homologous recombination DNA repair
pathway in human cells to precisely engineer any DNA variation
or tag in any endogenous protein of choice rAAV has been used
in multiple cell types to introduce disease-associated mutations to
allow understanding of their phenotype in an isogenic background
It has also been used to correct existing mutations in cell lines
derived from patient samples, for example correction of a mutation
causing osteogenesis imperfecta in patient-derived iPSC’s Horizon
Discovery established its Centre of Excellence (COE) program in
2011 to allow rapid adoption of this technology within academia
The aim of this program is to give the global academic community
free access to Horizon’s GENESIS genome editing technology to
facilitate new discoveries in life science research using our novel
vector improvements and the depth and breadth of knowledge about
AAV There are many benefi ts to joining the COE program including
expert support, access to in-house optimised protocols and produced
reagents and technical improvements in rAAV gene targeting
Members also become part of the rAAV gene editing community
allowing collaboration and interaction with world-leaders in this
technology The program has developed rapidly since its launch
with over 30 centres and 120 projects, across a range of therapeutic
areas including oncology and regenerative medicine, and using over
25 different cell lines in various tissue types To demonstrate the
success of the program, we will describe in detail the generation and
characterisation of human isogenic cell lines involved in oncology and
cardiovascular disease We will present data detailing how the use of
these cell lines has led to a better understanding of these diseases We
will also describe how academic groups can access this precise and
fl exible technology through the COE program at no cost
of Egr-1 Promoter on Human Primary Tenocytes
Francisco Martinez-F,1,2 Araceli Barrera-Lopez,1 Hugo
Sandoval-Zamora,1 David T Curiel,3 Juan A Madinaveitia-V,1 Rebecca E
Franco-Bourland.4
1 Molecular Biotherapeutic Program, Skin & Tissue Bank,
National Institute of Rehabilitation Ministry of Health, Mexico
City, DF, Mexico; 2 Department of Pharmacology, School of
Medicine, National Univeristy of Mexico, Mexico City, DF,
Mexico; 3 Department of Radiation Oncology, School of Medicine,
University of Washington, St Louis, MO; 4 Department of
Biochemistry, National Institute of Rehabilitation, Mexico.
Introduction: Tenocytes are the principal cell population in human
tendon with high metabolic activity and synthesis of matrix protein
Egr-1 expression has been implicated in tendon differentiation,
synthesis and increase of collagen expression during tendon cell differentiation However, transcriptional regulation on the egr-1 promoter has not been described in adult human tenocytes Hereby,
we evaluate the effect of UV light on egr-1 promoter in human primary tenocytes transduced with adenoviral vector Adegr1-Luc Materials and methods: Cells and adenoviral vectors: No replicative recombinant adenovirus (AdEgr1-Luc) were packaged at large scale
in HEK-293 cells and purifi ed according to the current protocol of the Core Facility of PBM-INR, based on cesium chloride gradient protocol for in vivo application Viral Stock was titled by plaque assay and OD Human primary tenocytes (HPT) were obtained based on collagenase digestion protocol cells were cultured in DMEM/F12 media supplemented with 10% HI-FBS and antibiotics under standard culture conditions for 1 week and stored for experimental procedure 5x104 tenocytes were seeded/well After 12 hrs, cells were infected with AdEgr1-Luc at 50 MOI´s during two hrs in serum reduced media After infection, cells were keeped in 1% of FBS for 24 hours
at environment standard conditions for culture until exposition to UV light (15, 30, 60, 90 and 120 seg) Protein extraction was performed
at 2, 4 and 6 hrs based on Cell Glo Lysis Buffer (Promega corp.) and luciferasa activity was quantifi ed using a multidetector DTX-880 Results: Luciferase assays from proteins obtained of not transduced cells not shows luciferase activity (0.33-0.5 LC/s) UV exposition
of human tenocytes transduced with Adegr1-Luc induces positive activity of egr-1 promoter Luminescent activity was observed at two, 4 and 6 hrs (1,490 LC/S; 2,904.66 LC/s and 29,511.33 LC/s, respectively) Transcriptional activity induced by EGF (29,511.33 LC/s) was over the positive control with 10% of FBS (23,915.33 LC/s.) Conclusion UV light activates transcriptional activity of egr-1 promoter in human tenocytes However, this activity could
be a fi nal event of no genomic pathway, and could be discerned by other studies using proteomic tools This research project is granted
by the National Council of Science and Technology of México Grant FOSIS/CONACYT-Salud-2011-1-161624
Fetal and Adult Stem Cells
the β-Catenin/TCF Signaling Pathway during Integration into Damaged Adult Renal Tubules
Paul Goodyer,1 Zhao Zhang,1 Diana Iglesias.1
1 Pediatrics, McGill University, Montreal, QC, Canada.
The therapeutic potential of exogenously infused mesenchymal stem cells is limited to their salutary paracrine effects, since they are unable to differentiate toward an epithelial phenotype and are rarely integrated into damaged adult tissue In contrast, renal progenitor cells (RPC) isolated from the metanephric mesenchyme
of embryonic kidney are rapidly integrated into damaged proximal tubules following acute glycerol-induced kidney injury During nephrogenesis, CD24(+) RPC in the cap mesenchyme are induced to differentiate by Wnt9b signals arising from the ureteric bud (UB) We reasoned that activation of the canonical WNT/ -catenin signaling might be essential for successful integration and differentiation of exogenous stem cells into the damaged adult kidney To address this hypothesis, we fi rst isolated CD24(+) cells from embryonic E15 mouse kidney, labeled them with PKH26 Red and infused them into adult mice 3 and 4 days after glycerol-induced renal proximal tubular injury We observed wide CD24(+) cell integration into damaged tubules The red-labeled cells also showed epithelial polarization and staining for the proximal tubular cell marker lotus tetragonolobus agglutinin We then isolated embryonic CD24(+) cells from a mouse bearing a -catenin/TCF reporter transgene (RPCTCF) and showed that they activate the canonical WNT pathway in response
to co-culture with E15 UB cells or L-cells expressing WNT3a We next infused the CD24(+) RPCTCF into adult mice at the peak of
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FETAL AND ADULT STEM CELLS
glycerol-induced proximal tubular injury and tracked their WNT
signalling pathway activity with X-gal staining The exogenous
RPCTCF showed robust reporter activity at sites within the urinary
pole of Bowman’s capsule along the proximal tubule Cells with
robust reporter activity co-stained for both WNT4 and PCNA To
examine WNT/ -catenin pathway activation in endogenous tubular
cells, we also induced injury in adult mice bearing the -catenin/
TCF reporter and noted patchy but widespread reporter activity and
WNT4 expression among endogenous tubular cells Based on these
observations, we propose that infused embryonic RPC activate the
WNT -catenin/TCF pathway as they integrate, proliferate and
differentiate within the damaged tubule; this recapitulates the events
which occur during primary nephrogenesis and may be essential for
renal tubular regeneration
248 Preclinical Investigations of Therapeutic
Benefi ts and Biodistribution from Stress
Erythropoiesis on Minimal Dose of Lysosomal
Enzyme Required for Phenotypic Correction in
Mice with Hurler Syndrome
Jingfen Han,1 Mei Dai,2 Salim S Elamouri,1 Phuong Cao,1 Dao
Pan.1,2
1 Experimental Hematology and Cancer Biology, Cincinnati
Children’s Hospital Medical Center, Cincinnati, OH; 2 Department
of Pediatrics, University of Cincinnati School of Medicine,
Cincinnati, OH.
The benefi ts of reprogramming erythroid cells for high-level
lysosomal enzyme production with less risk of activating oncogenes
in hematopoietic stems cells (HSC) and their progeny have been
recently reported by our group However, it remains unclear what
is the minimal dose of alpha-L-iduronidase (IDUA), a lysosomal
enzyme defi cient in patients with Mucopolysaccharidosis type I (MPS
I), required for phenotypic correction in organs and if increasing
erythropoiesis can provide further therapeutic benefi ts We sought
to evaluate IDUA expression and potential therapeutic improvement
following stress erythropoiesis induced by repeated phlebotomy,
and determine the minimal transgene doses required for metabolic
correction in major organs, including liver, spleen, kidney, heart
and brain, in MPS I mice after HSC-mediated gene transfer with a
lentiviral vector expressing IDUA from an erythroid/megakaryocytic
promoter Based on transgene frequency determined by qPCR in bone
marrow, treated MPS I animals were divided into four groups as MPS/
GT 200%, MPS/GT 10%, MPS/GT 2%, and MPS/GT <1% Transient
reticulocytosis was verifi ed by up to 5-fold increase in the numbers
of reticulocytes using FACS analysis with Thiazole Orange-staining,
which peaked on day 7 and recovered to the normal levels on day
30 Plasma IDUA activities peaked at day 7 with 4.5-, 2-, 1.5-fold
increase in MPS/GT200%, MPS/GT10%, and MPS/GT2% groups,
respectively, and decreased to baselines by day 23 These changes
were also associated with glycosaminoglycan (GAG) accumulation
in urine, which steadily reduced to a plateau on day 16 until day 30
Complete systematic metabolic correction was observed in all four
peripheral organs by just 10% HSC gene transfer, using either urinary
or organ GAG levels as the effective biomarkers Stress erythropoiesis
further reduced the GAG levels signifi cantly in both liver and spleen
of MPS I with 2% HSC gene transfer Organ pathology evaluation
was conducted for cells with cytoplasmic vacuoles, the pathogenesis
feature of MPS I that represents distended lysosomes from which the
GAG content have been leached by fi xation We determined that 2%
HSC gene transfer was suffi cient to completely correct lysosomal
storage pathology in both Kupffer cells and hepatocytes of the liver
In spleen, the main responsive organ to stress erythropoiesis, complete
normalization of GAG levels and pathology are observed in MPS I
mice with <1% HSC gene transfer after stress erythropoiesis More
studies are underway to evaluate the threshold levels of HSC gene
transfer required for any detectable neurological benefi ts Our results demonstrate that long-term visceral phenotypic corrections could be further improved by stress erythropoiesis with complete normalization
in spleen of MPS I mice with minimum HSC gene transfer (<1%), thus provide valuable preclinical information that may lead to future clinical trial
Co-Culture in Delta-Mediated Expansion
Korashon L Watts,1 Colleen Delaney,1,2 Hans-Peter Kiem.1,2
1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2 Medicine, University of Washington, Seattle, WA.
Our laboratory has recently demonstrated rapid engraftment and transfusion independence in Macaca nemestrina subjects transplanted with cord blood (CB) cells expanded on Delta1-expressing OP9 cells (OP9-DL) Here, it was our goal to explore the role of co-culture
in Delta-mediated expansion, and to investigate whether improved engraftment kinetics result solely from Delta1 exposure or also from cell-to-cell interactions associated with co-culture Macaque
CB cells were collected via C-section Red blood cells were lysed and CD34+ cells were enriched using immunomagnetic column separation CD34+ cells were then cultured in Stemspan serum-free media supplemented with antibiotics and growth factors and cultured for 15 days on one of the following surfaces: (1) Delta1 protein (DLext-IgG)-coated plates, (2) OP9 cell feeder layers, (3) OP9-DL cell feeder layers, or (4) control tissue-culture plates Flow cytometry was used to evaluate the percentage of CD34+ hematopoietic stem cells and CD41+ megakaryocyte precursors every 3 to 4 days From these values, we then calculated overall expansion of CD34+ and CD41+ cells Comparative statistical analyses were used to assess the contribution of DLext-IgG versus OP9-DL to overall expansion,
as well as the value of OP9 co-culture to overall expansion Analysis
of CD34+ expansion kinetics indicated that optimal expansion occurred at day 12 for cells cultured on DLext-IgG, OP9 co-cultured cells, and control cells, while cells cultured on OP9-DL continued
to expand through day 15 Average fold expansion of CD34+ cells
at day 12 was as follows: 65.2±4.5 (DLext-IgG), 40.4±6.1 (OP9), 71.1±5.3 (OP9-DL), and 31.9±4.9 (control) CD41+ cells, by comparison, reached optimal expansion capacity at day 8 of culture; the average fold expansion of CD41+ cells at this time under each condition was as follows: 84.0±3.2 (DLext-IgG), 69.7±2.6 (OP9 co-culture), 88.7±3.1 (OP9-DL coculture), and 43.3±2.8 (control) Further analysis confi rmed that, though both DLext-IgG and OP9-DL facilitated CD41+ expansion, OP9-DL was the more effi cient of the two, resulting in higher expansion at each time point and signifi cantly higher expansion at days 8 and 12 (p=0.05 and p=0.02, respectively) Furthermore, we discovered that OP9 co-culture (even in the absence
of Delta1) facilitated signifi cantly greater CD41+ expansion than control conditions (p=0.03 at day 8; p=0.05 at day 12) However, OP9-DL resulted in signifi cantly higher CD41+ expansion at all time points (p=0.02) These data demonstrate that there is a functional role
of co-culture in Delta1-mediated expansion of macaque CB cells; additionally, we show that co-culture is essential to achieve optimal expansion of both CD34+ and CD41+ cells Therefore, we conclude that the improved platelet engraftment kinetics observed in our transplant animals can be attributed to the combination of co-culture and Delta1 exposure, since neither factor independently promoted expansion equivalent to that of OP9-DL This provides incentive to generate a source of clinically relevant Delta-expressing cells that can be used in clinical trials for CB expansion