660 Bowel/Bladder Sensation and Control in Patients with Spinal Cord Injury Treated with Human Embryonic Stem Cell Therapy Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American[.]
Trang 1Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
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657 Delivery of Genetically Engineered
Adipose-Derived Cell Sheets for Treatment of
Ischemic Disorders - Development of Application
in Animal Models
Pavel I Makarevich,1,3 Konstantin V Dergilev,1 Zoya I
Tsokolaeva,1 Anastasia Yu Efimenko,1,2 Evgeny V Gluhanuk,2 Julia
O Gallinger,2 Polina A Rodina,2 Stepan S Sarkisyan,2 Ksenia V
Kerova,2 Yu-Chen Hu,4 Yelena V Parfyonova.2,3
1 Laboratory of Regenerative Medicine, Lomonosov Moscow
State University, Moscow, Russian Federation; 2 Faculty of
Medicine, Lomonosov Moscow State University, Moscow, Russian
Federation; 3 Laboratory of Angiogenesis, Russian Cardiology
Research and Production Complex, Moscow, Russian Federation;
4 Department of Chemical Engineering, National Tsing Hua
University, Hsinchu, Taiwan.
Cell sheet (CS) approach is extensively developed in recent years
for a number of applications in regenerative medicine including
lesion treatment, tissue-engineered constructs and organoplastics In
this study we have targeted ischemic diseases - myocardial infarction
(MI) and peripheral artery disease (PAD) using CS from differetnt
sources transplanted as CS to the impaired region
We established a protocol for generation of CS from c-kit+ cardiac
primitive cells and cardiosphere cells for delivery to rats with induced
MI using clinical-grade fibrin glue for CS adhesion subepicardially
We found CS to be vascularized after transplantation and used
ultrasound assessment to detect improvement of left ventricle function
and significant positive changes of tissue status in histology studies:
capillary density and reduction of fibrosis) Moreover, we conducted
a comparative analysis of this approach vs CS from adipose-derived
stomal cells (ADSC) and VEGF-expressing ADSC The latter were
obtained via baculovirus transduction of CS, which enabled significant
production of human VEGF165 by murine cells and, thus, enhanced
their therapeutic potential
Our previous application of ADSC for therapeutic angiogenesis and
established methods for genetic modification of cells allowed us to
develop a protocol in limb ischemia model We have demonstrated,
that subcutaneous implantation of CS or VEGF-expressing CS from
mouse ADSC resulted in significant improvement of limb perfusion
with modified cells to be superior to mock-transduced Increased
blood flow was supported by higher CD31+ capillary density,
reduction of necrosis and detection of viable CS subcutaneously with
focal proliferation of mADSC and minimal (app 10%) prevalence of
apoptotic cells at 14 days post transplantation Paracrine mechanism
for CS mode of action is suggested due to the fact, that ww found
vessel growth within CS mass, which indicates possibility of
cells’ nutrition and uptake of secreted proteins from the site of
transplantation besides diffusion
Thus, our findings indicate, that CS-based delivery for treatment of
MI or PAD is a potential method in regenerative medicine in this field
and furthermore genetic modification of cells used for CS generation
may be a way to “tune up” the cells’ paracrine activity and efficacy
of their application
658 Increasing Fetal Hemoglobin Expression
With TALEN Induced Small Deletions in Peripheral
Blood Stem Cells
Christopher T Lux,1 Andrew M Scharenberg,1 Brandon K
Hadland.2
1 Seattle Children’s Research Institute, Seattle Children’s Hospital,
Seattle, WA; 2 Fred Hutchinson Cancer Research Center, Seattle,
WA.
Hemoglobinopathies are common and morbid inherited conditions
caused by mutations in hemoglobin genes that generate pathologic
globin molecules or reduced expression of wild type hemoglobin A subset of individuals with hemoglobinopathy-associated beta globin mutations have a mild clinical phenotype and are found to carry additional disease modifying mutations including small deletions
or point mutations that induce fetal hemoglobin expression This condition is referred to as hereditary persistence of fetal hemoglobin Recent progress in the arenas of gene editing and hematopoietic stem cell (HSC) expansion has raised the possibility of treating hemoglobinopathies through direct modification of genes in HSCs to increase the expression of fetal hemoglobin in the erythroid lineage
To date, most attempts to induce fetal hemoglobin have targeted the expression of transcription factors implicated in regulation of fetal hemoglobin production, such as Bcl11a, an approach which could lead to unintended and potentially deleterious effects on cellular phenotype Here, we are evaluating the application of mRNA transfection of TALENs to directly modify transcription factor binding sites within the hemoglobin locus in HSCs to achieve efficient, stable expression of fetal hemoglobin We have designed TALENs to induce small deletions in the beta hemoglobin promoter at the binding site
of KLF1, postulated to play a role in localizing the active chromatin hub to and preferentially expressing adult beta hemoglobin, as well
as other globin locus target regions implicated in the regulation of fetal hemoglobin production By combining TALEN-mediated gene modification with novel HSC expansion approaches involving the recently described small molecule UM729, we hope to realize both efficient HSC modification and expansion with a sufficient level of fetal hemoglobin production in erythrocytes to achieve a functional cure for a wide range of beta hemoglobinopathies
659 Precision Genome Engineering in Human Induced Pluripotent Stem Cells Using CRISPR/ CAS9
Jon P Connelly,1 Amber M Neilson,1 Sam T Peters,1 Rita Martinez,1 Jeffrey D Milbrandt,1 Shondra M Miller.1
1 Genetics, Washington University, St Louis, MO.
Site-specific nucleases are engineered to create a double-stranded DNA break at a defined genomic locus Subsequent repair of these DNA breaks by the cellular endogenous DNA repair pathways can then be exploited to create genetically modified cell lines Genes can
be knocked out, disease relevant mutations introduced, or proteins can be tagged for subsequent monitoring in differentiation assays and high throughput small molecule screens Human induced pluripotent stem cells (iPSCs) have the potential to differentiate into any somatic cell in the body Importantly, they can be derived from both normal individuals, as well as from patients The combination of these two technologies allows investigators to create custom modified cell lines harboring site specific genetic changes of interest relevant to their field of study Here, we present two examples of precise and efficient genome editing without the use of antibiotic selection using the CRISPR/Cas9 system in iPSCs First we demonstrate the steps involved in the creation of a knockout (SARM1) iPSC line Second,
we outline the workflow for creating a custom iPSC line that has been tagged with a fluorescent protein at the endogenous locus (CHAT)
660 Bowel/Bladder Sensation and Control
in Patients with Spinal Cord Injury Treated with Human Embryonic Stem Cell Therapy
Geeta Shroff
1 Nutech Mediworld, New Delhi, India.
Introduction: Spinal cord injury (SCI) involves a complex series of pathological events resulting in long-lasting locomotor and sensory neuron degeneration The present study evaluated the bowel/bladder sensation and control in patients with SCI after human embryonic stem cell (hESC) therapy
Trang 2Molecular Therapy Volume 23, Supplement 1, May 2015
Methods: This is a retrospective analysis (May 2005-Aug 2012)
of 226 SCI patients (mean age-28 years) treated with hESC therapy
Therapy schedule had four treatment phases (T1, T2, T3, T4) lasting
4-6 weeks separated with gap phases (4-8 months) hESCs in an
injectable form were administered at a dose of 0.25 ml (<4 million
cells) (cultured and maintained in-house in a xeno product free
patented technology) intramuscularly twice daily, 1 ml hESCs (<16
million cells) every 10 days through i.v route and 1-5 ml hESCs
every 5- 7 days by any of the supplemental routes The patients with
bowel and bladder problems were graded based on their symptoms
Results: Overall, 200 (81.5% patients had no bowel sensation of
fullness or evacuation) and 204 (77.5% had no bladder sensation of
filling or voiding) subjects had bowel and bladder sensation problems,
respectively; while, 203 (81.5% had no bowel control), and 209
(92.3% had no bladder control) subjects had bowel and bladder
control problems, respectively At the end of therapy, 7 (3.5%) patients
reached bowel sensation normalcy, 7 (3.4%) patients reached bladder
sensation normalcy, 135 (67.5%) had mild, partial or full sensation
of bowel fullness and 155 (76%) patients had mild, partial and full
sensation of bladder filling
Table 1: Bowel and Bladder sensation in SCI patients after hESCs
therapy Level Description Bowel n (%) Bladder n (%)
Admis-sion
End of treat-ment
Admis-sion
End of treat-ment
1 No sensation of full-ness and evacuation 163 (81.5) 58 (29) 158 (77.5) 42 (20.6)
2 Mild sensation of fullness but no
feel-ing of evacuation 22 (11) 80 (40) 31 (15.2)
79 (38.7)
3 Partial sensation of fullness and
4
Full sensation of
fullness and partial
sensation of
evacu-ation
7 (3.5) 19 (9.5) 6 (2.9) 27 (13.2)
5 Above description 4, or reached normalcy 0 7 (3.5) 0 7 (3.4)
Similarly, 4 (2%) reached bowel control normalcy, 4 (1.9%)
reached bladder control normalcy, 95 (46.8%) had bowel control and
113 (54%) had bladder control
Conclusion: An improvement in bowel/bladder control and
sensation was observed in SCI patients after hESC therapy
661 Assay Development and Qualification for Human Embryonic Stem Cell (hESC)-Derived Cardiomyocytes Intended for Clinical Studies
Derek Kong, Wei Dang, Aparna Krishnan, Heather Javier, Jennil Patel, David Hsu, Larry A Couture
1 Center for Applied Technology Development, Center for Biomedicine & Genetics, City of Hope, Duarte, CA.
Human embryonic stem cell (hESC)-derived cellular products have been considered as promising candidates for cell replacement therapy for multiple degenerative diseases At the Center of Biomedicine and Genetics (CBG), we have manufactured under cGMP numerous hESC-differentiated products including neuron stem cells (NSC), neuronal progenitor cells (NPC), retinal pigment epithelium (RPE) cells, dopaminergic neurons and cardiomyocytes intended for pre-clinical and clinical studies Specifically, cardiomyocytes are produced through hESC differentiation procedure utilizing adherent and notably suspension culture systems Prior to their release for clinical application, cardiomyocytes must be thoroughly tested for their identity and purity We have developed a systematic approach
to characterize hESC-derived cardiomyocytes using assays such as real-time quantitative polymerase chain reaction (RT-qPCR) and flow cytometry The development and qualification of each assay involve the selection and banking of positive and negative controls, selection and banking of critical reagents, determination of assay conditions, qualification of assay, and generation of assay qualification report and standard operating procedure (SOP) We will also examine a number of critical factors including assay specificity, sensitivity, assay controls, and establishment of assay specifications
662 Mesenchymal and Induced Pluripotent Stem Cells: General Insights and Clinical Perspectives
Helena D Zomer,1 Atanasio S Vidane,1 Natalia N Gonçalves,1 Daniele S Martins,1 Carlos E Ambrósio.1
1 Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, Pirassununga, São Paulo, Brazil.
Mesenchymal stem cells (MSC) have awakened a great deal of interest in regenerative medicine due to their plasticity, immunomodulatory and anti-inflammatory properties They are easy
in yield and can be acquired through non-invasive methods from adult tissues Besides, are non-tumorigenic and are extensively studied In the other hand, induced pluripotent stem (iPS) cells can be derived directly from adult cells through gene reprogramming The new iPS technology avoids the embryo destruction or manipulation to generate pluripotent cells, therefore, are exempt of ethical implication surrounding embryonic stem cell use The pre-differentiation of iPS cells can assure the safety of future approaches Both MSC and iPS cells can be used to autologous cell transplantations without the risk of immune rejection and represent a great opportunity to future therapies In this we discussed the therapeutic perspectives using mesenchymal and induced pluripotent stem cells and used the rabbit mouse of fat tissue stem cell and use it for IPs production Perpectives and negatives highlights of this protocols will open venue for stem cell labs to produce safe different cell types using the rabbit model