NICD and activated expression of Notch target genes (Hes, Hey). Activation of ... Human muscle-derived stem cells (hMDSCs) have been shown to promote the ...
Cell Therapies II NICD and activated expression of Notch target genes (Hes, Hey). Activation of Notch signaling upregulated expression of endothelial cell transcription factors Gata 4 and Vezf1 in CSCs but γ-secretase inhibitor prevented Notch signaling activation and endothelial cell commitment of CSCs. CONCLUSIONS: These results revealed that Notch signaling activation promote CSCs commitment toward vascular cells. These findings suggest that modulation of Notch signaling can be promising therapeutic strategy for the treatment of myocardial ischemic damage.
448. Therapeutic Angiogenesis by Subcutaneous Cell Sheet Delivery Is Superior to Cell Injection: A Study of ADSC Efficacy in a Model of Hind Limb Ischemia
Pavel I. Makarevich1, Maria A. Boldyreva2, Anastasia Yu. Efimenko1, Evgeny V. Gluhanyuk3, Konstantin V. Dergilev2, Julia O. Gallinger1, Yu-Chen Hu4, Yelena V. Parfyonova2 1 Lomonosov Moscow State University, Moscow, Russian Federation, 2Russian Cardiology Research and Production Complex, Moscow, Russian Federation, 3Federal Research and Clinical Centre of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation, 4Tsing-Hua National University, Hsinchu, Taiwan
Engineering of cell sheets (CS) is an effective approach for delivery of cells to induce angiogenesis and tissue regeneration. Basis for increased CS efficacy is better engraftment and cell survival due to absence of anoikis and intact cell-to-cell interaction in the transplant. Still question to be addressed is whether CS are superior to injection route in terms of efficacy/cell engraftment and how can we improve therapeutic output of CS delivery for stimulation of tissue repair. We conducted a comparative study of adipose-derived stromal cells (ADSC) delivery in a model of hind-limb ischemia. C57/B6 male mice with unilateral limb ischemia (n=8-10/group) were injected with 106 of passage 3 syngeneic ADSC or transplanted with equivalent amount of cells in CS shape. After that animals were monitored for limb perfusion by laser-Doppler for 2 wks and then euthanized for histology studies of vessel density and ADSC detection using a PKH26 or CMFDA fluorescent label. Obtained samples were stained for macrophage invasion, endothelial cell markers and proliferation/ apoptosis to evaluate cell fate. Our animal test data has revealed that by Day 14 delivery of ADSC by means of injection induced restoration of limb perfusion compared to negative control group (41.5±4.7% vs. 29.7±3.0% respectively; p=0.01) indicating wellknown pro-angiogenic properties of these cells. Still, subcutaneous transplantation of CS was found to be superior to injection in terms of perfusion. CS-treated animals had the highest (55.3±7.3; p=0.03 vs. injected ADSC) perfusion by the end of experiment. This data was supported by vascular density assessment, which revealed increased capillary counts in both ADSC-treated groups with significantly higher values after CS delivery compared to injection (220.9±11.4 vs. 191.3±8.8 respectively; p=0.01). Analysis of necrotic tissue span in hematoxylin/eosin-stained section found a significant decrease of necrosis in ADSC-treated animals and also found CS to have better performance in terms of tissue protection compared to injection. Furthermore, we also evaluated ADSC engraftment and found that after injection pre-labeled cells reside as scattered mass and found their number to decrease over time by Day 14. Whilst after CS transplantation the cells were compactly localized in the site of application. CS were found to be vascularized by capillary vessels and infiltrated by CD68+ macrophage indicating graft-host interaction. Interestingly, certain cells within CS were found to show signs of proliferation (Ki67+) with sporadic apoptosis (cleaved caspase-3+) with overall transplant staying intact and viable by Day 14 after delivery. Overall, our data indicates that transplantation of CS is S178
superior to injection of equivalent amount of cells. We may speculate that this is not limited to ADSC and can be utilized for novel treatment methods. In an attempt to enhance the CS efficacy we turned our attention to hybrid constructs consisting of ADSC and endothelial cells to generate pre-vascularized constructs. Our preliminary data revealed “tube-like” behavior of HUVEC seeded on top of a CS from ADSC and may be the way to overcome the diffusion distance issue and generate CS with vascularized structure, which have a closer resemblance to native tissue structures. Moreover, this unveils the possibility to generate multi-layered constructs and imitate cell-to-cell interaction for basic and applied studies.
449. IGF-1 Treatment Enhances the Myogenic Potential of Human Skeletal Muscle-Derived Stem Cells
Elizabeth Morris1, Alex Scibetta1, Aiping Lu2, Xueqin Gao2, Johnny Huard2 1 Steadman Philippon Research Institute, Vail, CO, 2University of Texas, Houston, TX
Human muscle-derived stem cells (hMDSCs) have been shown to promote the regeneration of a variety of tissues, including damaged heart, peripheral nerve, bone, articular cartilage and skeletal muscle1-3; however, without appropriate stimulation their differentiation potential remains limited.4 In this study we examined whether insulin-like growth factor 1 (IGF-1) treatment could also enhance the myogenic potential of hMDSC, since it has been shown that IGF-1 treatment increased the myogenic potential of bone marrow-derived stem cells (BMSCs).5 Methods: HMDSCs were isolated from human adult muscle biopsies via a modified preplate technique, as previously described.6 Young female hMDSCs were plated in proliferation medium at a density of 1.58 x 104 cells/cm2. After 24h in proliferation medium, the cells were cultured for an additional 7 days in myogenic medium containing various levels of IGF-1: 0ng/mL, 50 ng/mL, 100 ng/mL, or 200 ng/mL. To evaluate the extent of myogenesis, cells were fixed in MeOH and stained for fast myosin heavy chain (fMHC). Activity levels of creatine kinase, which is highly expressed in tissues like skeletal muscle that rapidly consume ATP, were measured according to the company’s protocol. Additionally, RNA was extracted from cells, and cDNA was synthesized. Semi-quantitative PCR and qRT-PCR analyses were performed for myogenin, a late myogenic differentiation marker. Results: (1) fMHC staining indicated that IGF-1 treatment increased myotube formation (Fig. 1A). However, the mortality rates of the cells in all IGF-1 treatment groups were increased compared to the non-treated group. (2) hMDSCs treated with 50 ng/ml IGF-1 had significantly higher CK activity compared to all other groups when normalized to total protein. Furthermore, cells treated with 200 ng/ml IGF-1 also had significantly more CK activity than the untreated cells. This suggests that IGF-1 may enhance CK activity at low levels. At higher IGF-1 concentrations, CK activity may be reduced due to increased cell death (Fig. 1B). (3) Semi-quantitative analysis showed that IGF-1 treatment, at all concentrations, increased myogenin expression. The 100ng/mL concentration appeared to have the greatest effect. These results were corroborated by qRT-PCR analysis, which indicated that, compared to the non treated group, the 50ng/mL, 100ng/mL, and 200ng/mL groups had a 16.1 fold, 30.1 fold, and 14.3 fold increase in myogenin expression, respectively. Conclusion: These results indicate that IGF-1 enhances the myogenic potential of hMDSCs. However, we observed more cell death in IGF-1 treatment groups, which may be a result of overgrown cells during differentiation. In the future, we will determine whether IGF-1 treated hMDSCs display a higher regenerative potential in skeletal muscle,
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Cell Therapies II after injury and disease, than non-treated hMDSCs. References 1. Sekiya et al., 2013. 2. Gao et al., 2014. 3. Distefano et al., 2013. 4. Chen et al., 2013. 5. Sacco et al., 2005. 6. Ogawa et al., 2015.
growth without media exchange. Importantly, this system is simple to use and can be placed in a regular incubator as the G-Rex does not require active agitation or perfusion. To evaluate the utility of this system for the expansion of CAR T cells, we transduced healthy donor-derived primary T cells with a CAR targeting the prostate cancer antigen - PSCA (previously generated and characterized by our group). Three days after retroviral transduction, transgenic CAR T cells (transduction efficiency of 83.6±6%) from 3 donors were transferred to G-Rex100M devices (surface area of 100cm2) in 1000ml of complete T cell media (10ml/cm2) at low (total of 25E+06 CAR T cells), intermediate (50E+06 CAR T cells) and high (100E+06 CAR T cells) cell densities (250E+03 cells/cm2, 500E+03 cells/cm2 and 1000E+03 cells/cm2, respectively). Subsequently, the cell cultures were monitored by glucose and lactic acid assessment. After 10 days of culture, the average fold-expansion was similar for all conditions (24.3±10.4, 35.5±7.8, 29.8±2.1 for low, intermediate, and high cell densities, respectively). Interestingly though, the donor-to-donor variability was decreased significantly at the higher cell density (SD of 10.4, 7.8, and 2.1 for cell densities of 250E+03 cells/cm2, 500E+03 cells/cm2 and 1000E+03 cells/cm2, respectively), highlighting the importance of identifying the optimal seeding density to support robust manufacture. Notably, no media replenishment was required and the only culture manipulation performed was the addition of IL2 (50U/ml) 3 times/week. Importantly, T cells manufactured using this optimized method expressed higher levels of central memory and activation markers (CD62L and CD25) and demonstrated superior anti-tumor activity when compared to cells maintained in conventional tissue-culture plates. To further simplify the manufacturing process, we have now developed a semi-automated, closed system (GatheRex) for cell collection, which can be paired with G-Rex and allows collection of cells in a small volume (100ml) in under five minutes.
452. RegenVOX - Translational Exploitation Strategy for Stem Cell-Based Tissue-Engineered Laryngeal Implants Undergoing Phase I/II Clinical Trial
450.
Abstract Withdrawn
451.
Robust Manufacture of CAR-T Cells
Pradip Bajgain1, John Wilson2, Dan Welch2, Helen E. Heslop1, Cliona M. Rooney1, Malcolm K. Brenner1, Ann M. Leen1, Juan F. Vera1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, Houston Methodist Hospital, Houston, TX, 2Wilson Wolf Manufacturing, New Brighton, MN Although adoptive transfer of chimeric antigen receptor (CAR)modified T cells has produced promising clinical responses, the broader application of this therapy has been hindered by prolonged and complicated cell production methods. In the current work, we have overcome this limitation through the incorporation of a gas permeable culture device (G-Rex) to support T cell expansion. This culture system consists of a suite of devices, all of which contain a gas permeable silicone membrane, which allows gas exchange to occur at the base. This configuration allows for the culture of T cells with an unconventionally large volume of media per unit of surface area (10ml of media/cm2), thereby supporting uninterrupted cell Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Emily J. Culme-Seymour1, Carla Carvalho2, Owen Bain2, Eugene Omakobia3, Sophie Wilson3, Helen Knowles4, Susan Tebbs4, Kim Champion4, Jeff Round4, Gareth Ambler5, Martin A. Birchall3, Mark W. Lowdell2, Chris Mason6 1 LRMN, London, United Kingdom, 2Centre for Cell, Gene & Tissue Therapeutics, Royal Free Hospital, London, United Kingdom, 3Ear Institute, University College London, London, United Kingdom, 4 Clinical Trials Unit, University College London, London, United Kingdom, 5Department of Statistical Science, University College London, London, United Kingdom, 6Advanced Centre for Biochemical Engineering, University College London, London, United Kingdom The larynx (‘voicebox’) regulates breathing, voice and airway protection during swallowing - all critical human activities. For patients who lose laryngeal function due to trauma or cancer, there are no satisfactory long-term solutions, hence quality of life would be dramatically improved if a living, tissue-engineered laryngeal replacement could be transplanted. Based on prior experience from ‘first-in-man’ successes, a Phase I/II clinical trial (NCT01977911) of these autologous cell-based, tissue-engineered laryngeal implants is now underway for ten UK patients with severe irreversible structural disorders of the larynx, unresponsive to conventional treatment. The trial has regulatory approval in the UK from the MHRA and the first patient has been enrolled for treatment. Safety and efficacy of the RegenVOX technology are the primary output for the Phase I/II trial. However, a considered commercialisation strategy is vital if the technology is to successfully translate to clinical application. S179
