Joanna Cwykiel

Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy

Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin–deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 106) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

A New Supermicrosurgery Training Model of Saphenous Artery and Great Saphenous Vein Anastomosis for Development of Advanced Microsurgical Skills

Background This study aimed to confirm the feasibility and reliability of saphenous artery (SA) and great saphenous vein (GSV) anastomosis as a new supermicrosurgery training model and to compare the one‐way‐up anastomosis with the currently used end‐to‐end anastomosis technique. Methods Twenty supermicrosurgical anastomoses were performed in 10 Sprague Dawley rats. The external diameters of SA and GSV were measured using Leica LAS EZ software. The right‐side SA and GSV anastomoses were performed using the standard end‐to‐end anastomosis technique. The left‐side SA and GSV anastomoses were performed using the one‐way‐up technique with 11–0 monofilament‐interrupted sutures. The duration of the surgery, patency rates, and technical challenges of the two anastomoses methods were compared. Results The mean external diameters of SA and GSV were 0.273 ± 0.03 and 0.291 ± 0.02 mm, respectively, which qualify these vessels for supermicrosurgical training. The vessels were easily accessible and both anastomosis techniques were feasible. The one‐way‐up technique was proven to be faster as compared with the end‐to‐end anastomosis technique (artery: 34 ± 2.55 vs. 40.4 ± 2.97 minutes, p = 0.02; and vein: 37 ± 4.85 vs. 44 ± 2.35 minutes, p = 0.05, respectively). Short‐term patency rates for arteries and veins were 100% for both techniques. Seven‐day anastomosis patency rates for arteries and veins were 80 and 100% for the end‐to‐end technique and 100 and 80% for the one‐way‐up technique, respectively. Conclusions We confirmed that saphenous pedicle is suitable for creating a supermicrosurgery training model for practicing the ultrafine motor skills. To the best of our knowledge, this is the first report on supermicrosurgery of SA and GSV in the rat model.

Anatomic variations of brachial and lumbosacral plexus models in different rat strains

Selection of an appropriate model for preclinical assessment of new methods of peripheral nerve injury management is crucial. This report presents anatomic variations within brachial and lumbosacral plexuses in three selected rat strains Sprague Dawley (Hsd:Sprague Dawley SD), Lewis (LEW/SsNHsd), and Athymic Nude (Hsd:RH‐Foxn1rnu) rats.

A new total hemiface allotransplantation model in rats

Vascularized composite allotransplantation (VCA), a new reconstructive option for patients suffering from extensive facial defects leads to superior functional and aesthetic outcomes compared to the standard autologous reconstruction. Among VCA recipients, each case involves different facial structures and tissues depending on the patients injury, thus drawing conclusions on the mechanism of immune interactions between the donor and recipient is challenging. This study introduces a new total hemiface VCA model, including scalp, external ear, mystacial pad, premaxilla, upper/lower lids, nose, and upper/lower lips to evaluate the effect of transplantation of multitissue VCA on the recipients immune response.

Bone Marrow-Derived Ex Vivo Created Hematopoietic Chimeric Cells to Support Engraftment and Maintain Long-Term Graft Survival in Reconstructive Transplantation

Vascularized composite allotransplantation (VCA) introduces a promising alternative approach to standard reconstructive procedures for severely disfigured patients. Currently, VCA research is focusing on the development of immunomodulatory protocols for tolerance induction. The ultimate goal is to reduce or preferably eliminate the need for toxic, lifelong immunosuppression and to prevent both acute and chronic rejection.

Cellular Therapy Models: Ex Vivo Chimera Model by Cell Fusion

Cell fusion is a ubiquitous process fundamental to physiological and pathophysiological events common to multiple cell types and species. Performed ex vivo, cell fusion is a versatile research and therapeutic tool for gene mapping, antibody production, discovering new mechanisms in biological processes, inventing alternative therapies for cell reprogramming, restoring organ function, and creating cellular therapeutics for cancer treatment. Cell fusion can be successfully applied by creating cellular therapeutic of donor – recipient chimeric cell (DRCC) in the field of solid organ and vascularized composite allotransplantation (VCA). Immunomodulatory DRCC therapy has the potential to reduce or even eliminate the need for toxic, life-long immunosuppression and to prevent both acute and chronic rejection. This innovative VCA treatment is a combination of ex vivo created chimeric cell therapy with a short-term selective protocol of monoclonal antibody and Cyclosporine A. The utilization of short-term immunosuppressive protocol will provide the opportunity for chimeric cell engraftment, proliferation, and re-education of recipient’s immune system resulting in prolongation of allograft survival. The use of chimeric cells, as a supportive treatment for VCA, would improve the conditions of severely disfigured patients by offering safe alternative approach and providing better functional and aesthetic results compared to standard reconstructive procedures. This chapter summarizes the phenomenon, current discoveries, and advancements in the field of cell fusion, as well as introduces ex vivo creation of chimeric cells and presents potential benefits of chimeric cell-based protocols. Successful application of chimeric cell protocol in VCA experimental models will advance the field of reconstructive transplantation towards clinical trials.

Development of Targeted Muscle Reinnervation Model in Hind Limb Amputated Rats

Background Targeted muscle reinnervation (TMR) is a novel approach to postamputation neuroma pain; however, this has not been explicitly studied. The purpose of this study was to develop a TMR model in hind limb amputated rats. Methods Ten hind limbs from 5 Sprague Dawley cadaver rats were used. Sciatic nerve, main branches of the sciatic nerve (common peroneal, tibial, sural), motor branches from the sciatic nerve to the biceps femoris and cauda femoris, gluteal nerve and its motor branches to the semimembranosus, and biceps femoris and femoral nerve were dissected to look for consistent nerve anatomy that can be used for TMR in the rat hind limb amputation model. Transfemoral amputation was performed and two types of coaptations were made: common peroneal nerve to motor branch to biceps femoris and tibial nerve to motor branch to semimembranosus. Results The total surgical time for the dissection, amputation, and coaptation of nerves was ˜90 minutes. A total of 100 nerves were dissected in 10 rat hind limbs. Anatomical dissections were straightforward to perform. Anatomy of the dissected nerves was consistent. Hind limb amputations were performed without damaging the target muscles and nerves. Nerve lengths were sufficient for coaptation without any tension. Conclusions To the best of our knowledge, this is the first report on TMR model in hind limb amputated rats. This model will allow for mechanical, electromyography (EMG), and histological analysis for future assessment of neuroma prevention.

2585: The effect of size discrepancy of human nerve allograft derived epineural conduit on the functional outcomes of nerve regeneration in athymic nude rat model

2585: The effect of size discrepancy of human nerve allograft derived epineural conduit on the functional outcomes of nerve regeneration in athymic nude rat model Marcin Strojny, Rafal Gendek, Joanna Cwykiel, Erzsebet Szilagyi, Malgorzata Cyran, Wojciech Malewski, Husein Karagoz, and Maria Siemionow University of Illinois at Chicago, Chicago, IL, USA Background Nerve autograft is a gold standard in peripheral nerve regeneration However, it is challenged by limited availability, donor site morbidity and scarring Nerve allografts provide an unlimited source of nerve tissue, which can be matched to the recipient’s injured nerve to support nerve recovery This study aimed to assess the effect of human Epineural Sheath Conduit (hESC) adjusted with tissue adhesive or suture on restoration of long nerve defect in an athymic nude rat model. Methods Restoration of 20 mm of nude rat sciatic nerve defect with hESC filled with saline was performed in 5 groups: Group 1: autograft controls (n D 4), Group 2: mismatched size diameter hESC (n D 2), Group 3: hESC with diameter adjusted with tissue adhesive (n D 4), Group 4: hESC with diameter adjusted with nylon 10-0 suture (n D 2), and Group 5: matched diameter hESC (n D 4) Toe-spread and pinprick analyses were performed at 1, 3, 6, 9, 12 weeks Nerve samples for toluidine blue staining and for fluorescent immunostaining for GFAP, NGF, S-100, laminin B, CD3 and CD4 were harvested at 12 weeks post-surgery Muscle denervation atrophy was assessed by Gastrocnemius Muscle Index (GMI). Results Macroscopic evaluation of nerve conduits at 12 weeks showed no tissue adhesion or local signs of inflammation and good vascularization in all groups Additionally, the shape and integrity of the conduit were preserved The best sensory and motor recovery following hESC application was observed in groups with hESC diameter adjusted with tissue adhesive (Group 3) and matched diameter hESC (Group 5), pinprick 175 and 175; toe-spread 05 and 075, respectively hESC without diameter adjustment (Group 2) and hESC adjusted with sutures (Group 4) showed the worst regeneration GMI measurements were the highest for autograft group (Group 1–035) followed by matched diameter hESC (Group 5–032) and hESC adjusted with tissue adhesive (Group 3–0277). Conclusions We confirmed the feasibility of hESC creation and diameter adjustment hESC conduit with adjusted diameter using tissue adhesive showed sensory and motor recovery comparable to hESC conduit with matched diameter to the rat sciatic nerve. Our new hESC conduit provides an alternative option to the autograft nerve gap repair. CONTACT Marcin Strojny mstrojnymd@gmail.com

2597: Assessment of engraftment and safety of a new tolerance inducing therapy of human cord blood derived ex-vivo created di-chimeric cells in NOD SCID mouse model

2597: Assessment of engraftment and safety of a new tolerance inducing therapy of human cord blood derived ex-vivo created di-chimeric cells in NOD SCID mouse model Joanna Cwykiel, MSc, Natalia Filipek, BSc, Malgorzata Cyran, Can Emre Bas, Erzsebet Szilagyi, Ewa Bryndza Tfaily, Medhat Askar, MD, PhD, and Maria Siemionow University of Illinois at Chicago, Chicago, IL, USA; Cleveland Clinic, Cleveland, OH, USA Background The aim of this study was to evaluate the phenotype, engraftment and safety of ex-vivo fused human cord blood derived di-chimeric cell therapy (DCC) in the NOD SCID mouse model. Methods A total of 36 fusions of human umbilical cord blood (UCB) mononuclear clls were performed UCB from 2 unrelated donors were separately stained with PKH26 and PKH67 dyes Fused with polyethylene glycol, double (PKH26/PKH67) stained DCC were sorted and subjected to the in vitro evaluations (15 fusions): lymphocytotoxicity (LCT) test, PCR-rSSOP, STR-PCR, viability, apoptosis, colony forming unit (CFU) assay and COMET assay DCC (3–5 £ 10 cells) from 18 fusions (n D 6/Group) were delivered: Group 1: intraosseous, Group 2: intramuscular, and Group 3: subcutaneous to the NOD SCID recipient mice Control mice in Groups 4, 5, and 6 (n D 6) received 3 £ 10UCB utilizing the same 3 delivery sites Mice were observed daily for 90 days for changes in weight, activity, posture and hair loss Moreover, mice were evaluated bi-weekly by palpation and at 90 days by magnetic resonance imaging (MRI) The presence of DCC in the blood was determined by complete blood count (CBC) and flow cytometry (FC) The migratory pathways of DCC, peripheral blood, bone marrow and lymphoid organs were assessed at 3 months after delivery using immunofluorescent staining Furthermore, H&E staining was performed to assess harvested tissues for tumor growth. Results The presence of HLA class I and II from both UCB donors was confirmed by LCT, PCR-rSSOP, and STR COMET assay showed no damage to the DNA of DCC following fusion procedure Migratory properties of DCC were confirmed at 24 hours after cell delivery Human derived cells (CD45C, CD19C, HLA class I and CD4C) were detected at a level up to 2% in the peripheral blood as tested by CBC and flow cytometry at 90 days following delivery No DCC derived tumorlike growth was observed. Conclusions Phenotype, engraftment and safety of DCC were characterized The unique concept of supportive therapy of DCC introducing cells presenting phenotype characteristics of both transplant donor and recipient is a new promising approach for tolerance induction and prolonging VCA survival. CONTACT Joanna Cwykiel, MSc jcwykiel@uic.edu

2594: In vitro and in vivo characterization of human hematopoietic chimeric cells - A novel strategy to induce tolerance in transplantation

2594: In vitro and in vivo characterization of human hematopoietic chimeric cells A novel strategy to induce tolerance in transplantation Ewa Bryndza Tfaily, PhD, Malgorzata Cyran, MD, Rafal Gendek, MD, PhD, Erzsebet Szilagyi, MD, PhD, Jason Le, MD, Anna Domaszewska-Szostek, PhD, Joanna Cwykiel, MSc, and Maria Siemionow, MD, PhD, DSc University of Illinois at Chicago, Chicago, IL, USA; Polish Academy of Science, Warszawa, Poland Background Cell-based therapies represent a new promising approach for tolerance induction in transplantation One of the methods for immune response modulation in solid organ and vascularized composite allograft (VCA) recipients is donor bone marrow (BM) transplantation We propose a new cellular therapy based on application of the ex vivo created donor-recipient chimeric cells as an alternative approach to BM-based therapies in support of solid organ and VCA transplantation The aim of this study was further characterization of human hematopoietic chimeric cells (HHCC).