Biodistribution and Safety of Human Multi-Chimeric Cells After Systemic Intraosseous and Intravenous Administration in the Experimental Mouse Model.

Cellular therapies provide promising options for inducing tolerance in transplantation of solid organs, bone marrow, and vascularized composite allografts. However, novel tolerance-inducing protocols remain limited, despite extensive research. We previously introduced and characterized a human multi-chimeric cell (HMCC) line, created through ex vivo fusion of human umbilical cord blood (UCB) cells derived from three unrelated donors. In this study, we assessed in vivo biodistribution and safety of HMCCs in the NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ NOD scid gamma (NSG) mouse model. Twenty-four NSG mice were randomly assigned to four groups (n = 6/group) and received intraosseous (IO.) or intravenous (IV.) injections of 0.6 × 106 donor UCB cells or fused HMCC: Group 1-UCB (IO.), Group 2-UCB (IV.), Group 3-HMCC (IO.), and Group 4-HMCC (IV.). Hematopoietic phenotype maintenance and presence of human leukocyte antigens (HLA), class I antigens, in the selected lymphoid and nonlymphoid organs were assessed by flow cytometry. Weekly evaluation and magnetic resonance imaging (MRI) assessed HMCC safety. Comparative analysis of delivery routes revealed significant differences in HLA class I percentages for IO.: 1.83% ± 0.79%, versus IV. delivery: 0.04% ± 0.01%, P < 0.01, and hematopoietic stem cell marker percentages of CD3 (IO.: 1.41% ± 0.04%, vs. IV.: 0.07% ± 0.01%, P < 0.05) and CD4 (IO.: 2.74% ± 0.31%, vs. IV.: 0.59% ± 0.11%, P < 0.01). Biodistribution analysis after IO. delivery confirmed HMCC presence in lymphoid organs and negligible presence in nonlymphoid organs, except for lung (IO.: 0.19% ± 0.06%, vs. IV.: 6.33% ± 0.56%, P < 0.0001). No evidence of tumorigenesis was observed by MRI at 90 days following IO. and IV. administration of HMCC. This study confirmed biodistribution and safety of HMCC therapy in the NSG mouse model, both following IO. and IV. administration. However, IO. delivery route confirmed higher efficacy of engraftment and safety profile, introducing HMCCs as a novel cell-based therapeutic approach with promising clinical applications in solid organ, bone marrow, and vascularized composite allotransplantation transplantation.

Novel Human Umbilical Di-Chimeric (HUDC) cell therapy for transplantation without life-long immunosuppression.

Background: Cell-based therapies are promising for tolerance induction in bone marrow (BM), solid organs, and vascularized composite allotransplantation (VCA). The toxicity of bone marrow transplantation (BMT) protocols precludes this approach from routine clinical applications. To address this problem, we developed a new therapy of Human Umbilical Di-Chimeric (HUDC) cells for tolerance induction in transplantation. This study established in vitro characterization of the created HUDC cells. Methods: We performed sixteen ex vivo polyethylene glycol (PEG)-mediated fusions of human umbilical cord blood (UCB) cells from two unrelated donors. Fusion feasibility was confirmed in vitro by flow cytometry (FC) and confocal microscopy (CM). The HUDC cells' genotype was assessed by lymphocytotoxicity test and short tandem repeat-polymerase chain reaction (STR-PCR) analysis, phenotype by FC, viability by LIVE/DEAD® assay, and apoptosis level by Annexin V staining. We used COMET assay to assess HUDC cells' genotoxicity after the fusion procedure. Clonogenic properties of HUDC cells were evaluated by colony forming unit (CFU) assay. Mixed lymphocyte reaction (MLR) assay assessed immunogenic and tolerogenic properties of HUDC cells. Results: We confirmed the creation of HUDC cells from two unrelated human donors of UCB cells by FC and CM. Human leukocyte antigen (HLA) class I and II typing, and STR-PCR analysis of HUDC cells confirmed the presence of alleles and loci from both unrelated UCB donors (donor chimerism: 49%±8.3%, n=4). FC confirmed the hematopoietic phenotype of HUDC cells. We confirmed high HUDC cells' viability (0.47% of dead cells) and a low apoptosis level of fused HUDC cells (15.9%) compared to positive control of PKH-stained UCB cells (20.4%) before fusion. COMET assay of HUDC cells revealed a lack of DNA damage. CFU assay confirmed clonogenic properties of HUDC cells, and MLR assay revealed a low immunogenicity of HUDC cells. Conclusions: This study confirmed creation of a novel HUDC cell line by ex vivo PEG-mediated fusion of UCB cells from two unrelated donors. The unique concept of creating a HUDC cell line, representing the genotype and phenotype of both, transplant donor and the recipient, introduces a promising approach for tolerance induction in BM, solid organs, and VCA transplantation.

Human Multi-Chimeric Cell (HMCC) Therapy as a Novel Approach for Tolerance Induction in Transplantation.

Cellular therapies are regarded as the most promising approach for inducing transplant tolerance without life-long immunosuppression in solid organ and vascularized composite allotransplantation (VCA). Currently, no therapies are achieving this goal. This study introduces a novel Human Multi-Chimeric Cell (HMCC) line created by fusion of umbilical cord blood (UCB) cells, from three unrelated donors as an alternative therapeutic approach to bone marrow transplantation and tolerance induction in solid organ and VCA transplants. We performed eighteen ex vivo polyethylene glycol mediated fusions of human UCB cells from three unrelated donors to create HMCC. Mononuclear cells labeled with PKH26, PKH67, and eFluor™ 670 fluorescent dyes were fused and sorted creating a new population of triple-labeled (PKH26/PKH67/eFluor™ 670) HMCC. The creation of HMCC from three unrelated human UCB donors was confirmed by flow cytometry and confocal microscopy. Genotyping analyses determined the tri-chimeric state of HMCC by presence of parent alleles and selected loci specific for each of three UCB donors. Phenotype characterization confirmed hematopoietic markers distribution, comparable to UCB donors. HMCC maintained viability and displayed a low apoptosis level. The COMET assay revealed absence of genotoxicity, confirming fusion safety. Colony forming units assay showed clonogenic properties of HMCC. This study confirmed the feasibility of HMCC creation from three unrelated human UCB donors and characterized tri-chimeric state, hematopoietic phenotype, viability, safety, and clonogenic properties of HMCC. The created HMCC line, representing genotype characteristics of three unrelated human UCB donors, introduces a novel therapeutic approach for bone marrow, solid organ, and VCA transplants.

Intraosseous transplant of dystrophin expressing chimeric (DEC) cells improves skeletal muscle function in mdx mouse model of Duchenne muscular dystrophy.

Introduction: We previously reported that systemic delivery of dystrophin expressing chimeric (DEC) cells of normal (wt) and dystrophin-deficient (mdx) myoblast (MB) or mesenchymal stem cell (MSC) origin restored dystrophin expression and improved cardiac function in the mdx mouse model of Duchenne muscular dystrophy (DMD). Aim: This study evaluated the effect of intraosseous delivery of murine DEC lines of MB (MB wt /MB mdx ) and MSC (MB wt /MSC mdx ) origin on function of gastrocnemius muscle (GM). Material and methods: DEC lines created by ex vivo fusion were tested in the mdx mouse model of DMD: Group 1 - vehicle (control), Group 2 - non-fused 0.25 × 106 MB wt and 0.25 × 106 MSC mdx (control), Group 3 - fused 0.5 × 106 MB wt /MB mdx DEC and Group 4 - fused 0.5 × 106 MB wt /MSCmdx DEC. In situ and in vitro muscle force tests assessed GM function at 90 days post-transplant. Results: Application of MB wt /MSC mdx and MB wt /MB mdx DEC significantly improved the fatigue ratio of GM compared to vehicle-injected controls detected by in vivo muscle force tests (0.567 ±0.116, p = 0.045 and 0.489 ±0.087, p < 0.05, respectively). MB wt /MSCmdx DEC recipients presented enhanced maximum force at tetanus (0.145 ±0.040 g/mg, p < 0.05); furthermore, recipients of MB wt /MBmdx DEC showed a significant increase in the maximum force generation rate compared to vehicle controls (4.447 ±1.090 g/s/mg, p < 0.05). The ex vivo GM force testing in MB wt /MSCmdx DEC recipients detected increased average GM force compared to vehicle and non-fused controls. Conclusions: Systemic-intraosseous administration of MB wt /MBmdx and MB wt /MSCmdx DEC therapy combining the myogenic and immunomodulatory properties of MB and MSC significantly improved skeletal muscle (GM) function of force and resistance to fatigue in an mdx mouse model of DMD.

Application of Human Epineural Conduit Supported with Human Mesenchymal Stem Cells as a Novel Therapy for Enhancement of Nerve Gap Regeneration.

Various therapeutic methods have been suggested to enhance nerve regeneration. In this study, we propose a novel approach for enhancement of nerve gap regeneration by applying human epineural conduit (hEC) supported with human mesenchymal stem cells (hMSC), as an alternative to autograft repair. Restoration of 20 mm sciatic nerve defect with hEC created from human sciatic nerve supported with hMSC was tested in 4 experimental groups (n = 6 each) in the athymic nude rat model (Crl:NIH-Foxn1rnu): 1 - No repair control, 2 - Autograft control, 3 - Matched diameter hEC filled with 1 mL saline, 4 - Matched diameter hEC supported with 3 × 106 hMSC. Assessments included: functional tests: toe-spread and pinprick, regeneration assessment by immunofluorescence staining: HLA-1, HLA-DR, NGF, GFAP, Laminin B, S-100, VEGF, vWF and PKH26 labeling; histomorphometric analysis of myelin thickness, axonal density, fiber diameter and myelinated nerve fibers percentage; Gastrocnemius Muscle Index (GMI) and muscle fiber area ratio. Best sensory and motor function recovery, as well as GMI and muscle fiber area ratio, were observed in the autograft group, and were comparable to the hEC with hMSC group (p = 0.038). Significant improvements of myelin thickness (p = 0.003), fiber diameter (p = 0.0296), and percentage of myelinated fibers (p < 0.0001) were detected in hEC group supported with hMSC compared to hEC with saline controls. At 12-weeks after nerve gap repair, hEC combined with hMSC revealed increased expression of neurotrophic and proangiogenic factors, which corresponded with improvement of function comparable with the autograft control. Application of our novel hEC supported with hMSC provides a potential alternative to the autograft nerve repair.

Human dystrophin expressing chimeric (DEC) cell therapy ameliorates cardiac, respiratory, and skeletal muscle's function in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive and lethal disease, caused by X-linked mutations of the dystrophin encoding gene. The lack of dystrophin leads to muscle weakness, degeneration, fibrosis, and progressive loss of skeletal, cardiac, and respiratory muscle function resulting in premature death due to the cardiac and respiratory failure. There is no cure for DMD and current therapies neither cure nor arrest disease progression. Thus, there is an urgent need to develop new approaches and safer therapies for DMD patients. We have previously reported functional improvements which correlated with increased dystrophin expression following transplantation of dystrophin expressing chimeric (DEC) cells of myoblast origin to the mdx mouse models of DMD. In this study, we demonstrated that systemic-intraosseous transplantation of DEC human cells derived from myoblasts of normal and DMD-affected donors, increased dystrophin expression in cardiac, respiratory, and skeletal muscles of the mdx/scid mouse model of DMD. DEC transplant correlated with preservation of ejection fraction and fractional shortening on echocardiography, improved respiratory function on plethysmography, and improved strength and function of the limb skeletal muscles. Enhanced function was associated with improved muscle histopathology, revealing reduced mdx pathology, fibrosis, decreased inflammation, and preserved muscle morphology and architecture. Our findings confirm that DECs generate a systemic protective effect in DMD-affected target organs. Therefore, DECs represents a novel therapeutic approach with the potential to preserve or enhance multiorgan function of the skeletal, cardiac, and respiratory muscles critical for the well-being of DMD patients.

Development of Donor Recipient Chimeric Cells of bone marrow origin as a novel approach for tolerance induction in transplantation.

BACKGROUND: Cell therapies and chimerism-based strategies are currently the most successful approach for tolerance induction in transplantation. This study aimed to establish and characterize novel Donor Recipient Chimeric Ccell (DRCC) therapy of bone marrow (BM) origin presenting donor-recipient phenotype to support tolerance induction. METHODS: Ex vivo fusions of fully MHC-mismatched BM cells from ACI (RT1a) and Lewis (RT1l) rats were performed using polyethylene-glycol (PEG). The creation of rat DRCC was tested by flow cytometry (FC), confocal microscopy and PCR. FC characterized DRCC's phenotype (CD3, CD4, CD8, CD45, CD90, CD11b/c, CD45RA, OX-82, or CD4/CD25) and apoptosis, while mixed lymphocyte reaction assessed DRCC's immunogenicity and colony forming unit assay tested DRCC's differentiation and proliferation. DRCC's polyploidy was evaluated using Hoechst33342 staining and COMET assay tested genotoxicity of fusion procedure. ELISA analyzed the secretion of IL-2, IL-4, IL-10, TGFß1, IFNγ and TNFα by DRCC at day 1, 5 and 14 post-fusion. The DRCC's phenotype after long-term culturing was assessed by reverse-transcription PCR. RESULTS: The chimeric state of DRCC was confirmed. Fusion did not change the expression of hematopoietic markers compared to BM controls. Although an increased number of early and late apoptotic (Annexin V+/Sytox blue- and Annexin V+/Sytox blue+, respectively) DRCC was detected at 24h post-fusion, the number significantly decreased at day 5 (38.4%±3.1% and 22.6%±2.5%, vs. 28.3%±2.5% and 13.9%±2.6%, respectively, P<0.05). DRCC presented decreased immunogenicity, increased expression of IL-10 and TGFß1 and proliferative potential comparable to BM controls. The average percentage of tetraploid DRCC was 3.1%±0.2% compared to 0.96%±0.1% in BM controls. The lack of damage to the DRCC's DNA content supported the DRCC's safety. In culture, DRCC maintained proliferation for up to 28 days while preserving hematopoietic profile. CONCLUSIONS: This study confirmed feasibility of DRCC creation via ex vivo PEG mediated fusion. The created DRCC revealed pro-tolerogenic properties indicating potential immunomodulatory effect of DRCC therapy when applied in vivo to support tolerance induction in solid organ and vascularized composite allograft transplantation.

Donor Recipient Chimeric Cells Induce Chimerism and Extend Survival of Vascularized Composite Allografts.

This study evaluated the efficacy of donor recipient chimeric cell (DRCC) therapy created by fusion of donor and recipient derived bone marrow cells (BMC) in chimerism and tolerance induction in a rat vascularized composite allograft (VCA) model. Twenty-four VCA (groin flaps) from MHC-mismatched ACI (RT1a) donors were transplanted to Lewis (RT1l) recipients. Rats were randomly divided into (n = 6/group): Group 1-untreated controls, Groups 2-7-day immunosuppression controls, Group 3-DRCC, and Group 4-DRCC with 7-day anti-αßTCR monoclonal antibody and cyclosporine A protocol. DRCC created by polyethylene glycol-mediated fusion of ACI and Lewis BMC were cultured and transplanted (2-4 × 106) to VCA recipients via intraosseous delivery route. Flow cytometry assessed peripheral blood chimerism while fluorescent microscopy and PCR tested the presence of DRCC in the recipient's blood, bone marrow (BM), and lymphoid organs at the study endpoint (VCA rejection). No complications were observed after DRCC intraosseous delivery. Group 4 presented the longest average VCA survival (79.3 ± 30.9 days) followed by Group 2 (53.3 ± 13.6 days), Group 3 (18 ± 7.5 days), and Group 1 (8.5 ± 1 days). The highest chimerism level was detected in Group 4 (57.9 ± 6.2%) at day 7 post-transplant. The chimerism declined at day 21 post-transplant and remained at 10% level during the entire follow-up period. Single dose of DRCC therapy induced long-term multilineage chimerism and extended VCA survival. DRCC introduces a novel concept of customized donor-recipient cell-based therapy supporting solid organ and VCA transplants.

Transplantation of Dystrophin Expressing Chimeric Human Cells of Myoblast/Mesenchymal Stem Cell Origin Improves Function in Duchenne Muscular Dystrophy Model.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in dystrophin gene. Currently, there is no cure for DMD. Cell therapies are challenged by limited engraftment and rejection. Thus, more effective and safer therapeutic approaches are needed for DMD. We previously reported increased dystrophin expression correlating with improved function after transplantation of dystrophin expressing chimeric (DEC) cells of myoblast origin in the mdx mouse models of DMD. This study established new DEC cell line of myoblasts and mesenchymal stem cells (MSC) origin and tested its efficacy and therapeutic potential in mdx/scid mouse model of DMD. Fifteen ex vivo cell fusions of allogenic human myoblast [normal myoblasts (MBN)] and normal human bone marrow-derived MSC (MSCN) from normal donors were performed using polyethylene glycol. Flow cytometry, confocal microscopy, polymerase chain reaction (PCR)-short tandem repeats, polymerase chain reaction-reverse sequence-specific oligonucleotide probe assessed chimeric state of fused MBN/MSCN DEC cells, whereas Comet assay assessed fusion procedure safety testing genotoxicity. Immunofluorescence and real-time PCR assessed dystrophin expression and myogenic differentiation. Mixed lymphocyte reaction (MLR) evaluated DEC's immunogenicity. To test MBN/MSCN DEC efficacy in vivo, gastrocnemius muscle of mdx/scid mice were injected with vehicle (n = 12), nonfused MBN and MSCN (n = 9, 0.25 × 106/each) or MBN/MSCN DEC (n = 9, 0.5 × 106). Animals were evaluated for 90 days using ex vivo and in vivo muscle strength tests. Histology and immunofluorescence staining assessed dystrophin expression, centrally nucleated fibers and scar tissue formation. Post-fusion, MBN/MSCN DEC chimeric state, myogenic differentiation, and dystrophin expression were confirmed. MLR reveled reduced DEC's immune response compared with controls (P < 0.05). At 90 days post-DEC transplant, increase in dystrophin expression (20.26% ± 2.5%, P < 0.05) correlated with improved muscle strength and function in mdx/scid mice. The created human MBN/MSCN DEC cell line introduces novel therapeutic approach combining myogenic and immunomodulatory properties of MB and MSC, and as such may open a universal approach for muscle regeneration in DMD.

The effect of thymus transplantation on donor-specific chimerism in the rat model of composite osseomusculocutaneous sternum, ribs, thymus, pectoralis muscles, and skin allotransplantation.

INTRODUCTION: Research on tolerance has proven that development of donor-specific chimerism (DSC) may accompany tolerance induction in vascularized composite allotransplantation (VCA). In this study, we aimed to determine the effect of thymus transplantation on the induction of DSC in rat VCA model of osseomusculocutaneous sternum (OMCS) and osseomusculocutaneous sternum and thymus (OMCST) allotransplantation. MATERIALS AND METHODS: A total of 20 Lewis-Brown Norway and Lewis rats, 5-6 weeks old, weighting between 120 and 150 g, were used in the study. OMCS (n = 5) and OMCST (n = 5) allografts were harvested from Lewis-Brown Norway donors (RT1l + n ) based on the common carotid artery and external jugular vein, and a heterotopic transplantation was performed to the inguinal region of the Lewis (RT1l ) recipients under cyclosporine A monotherapy (16 mg/kg) protocol tapered to 2 mg/kg and maintained for the duration of the study. The peripheral blood chimerism levels (T-cell, B-cell, and monocyte/granulocyte/dendritic cell-MGDC populations) were evaluated at days 7, 14, 35, 63, 100, and 150 posttransplant by flow cytometry. At Day 150, thymus, spleen, and liver samples were assessed by polymerase chain reaction (PCR) in the presence of DSC. RESULTS: Total chimerism level increased in both OMCST and OMCS groups at all time points. At 150 days posttransplant, chimerism in OMCST group was significantly higher (12.91 ± 0.16%) than that in OMCS group (8.89 ± 0.53%%, p < .01), and PCR confirmed the presence of donor-derived cells in the liver and spleen of all OMCST recipients and in one liver sample and two spleen samples in OMCS recipients without thymus transplant. CONCLUSIONS: This study confirmed the direct effects of thymus transplantation on the induction and maintenance of DSC in T-cell, B-cell, and MGDC populations. These results confirm correlation between thymus transplantation and DSC induction.

Application of epineural sheath conduit for restoration of 6-cm long nerve defects in a sheep median nerve model.

BACKGROUND: Due to limited number of studies, we tested feasibility of autologous epineural sheath conduit (ESC) in repair of 6-cm median nerve gaps in a sheep-the large animal model. MATERIALS AND METHODS: Eight ewes, 6-8 months old, 30-35 kg, were divided into three experimental groups: group 1-no defect repair (n = 4 nerves/group), group 2-autograft controls (n = 6 nerves/group), group 3-autologous ESC filled with saline (n = 6 nerves/group). ESC was constructed from a 6-cm long segment of sheep median nerve and tested for expression of laminin B, Glial fibrillary acidic protein (GFAP), S-100 and CD31 using immunofluorescent staining. At 6 months after nerve repair, nerve conduction velocity and somatosensory evoked potentials (SSEP) assessed neurosensory recovery, while histomorphometry tested nerve regeneration. RESULTS: Ex vivo characterization of ESC, before in vivo nerve gap repair, showed high laminin B expression, which supports axonal growth. At 6 months post-repair, structural integrity of ESC was preserved. ESC was well-vascularized and tissue adhesions were comparable to autograft controls. The maximal conduction velocities (29.80 ± 5.85 ms vs. 32.28 ± 6.75 ms; p = .44), action potential amplitudes (32.68 ± 17.44 mV vs. 44.14 ± 23.10 mV; p = .38) and SSEP amplitude values (6.18 ± 5.84 mV vs. 4.68 ± 2.53 mV; p = .28) were comparable between autograft and ESC groups. Presence of regenerating axons was confirmed in the distal segment of ESC at 6 months after repair. CONCLUSION: The feasibility of ESC in restoration of 6-cm long nerve defects in a sheep median nerve model was confirmed by nerve conduction assessments and correlated with axonal regeneration tested by histomorphometry. We confirmed ESC potential in support of regeneration of long nerve defects.

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.

Dystrophin Expressing Chimeric (DEC) Human Cells Provide a Potential Therapy for Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a progressive and lethal disease caused by mutations of the dystrophin gene. Currently no cure exists. Stem cell therapies targeting DMD are challenged by limited engraftment and rejection despite the use of immunosuppression. There is an urgent need to introduce new stem cell-based therapies that exhibit low allogenic profiles and improved cell engraftment. In this proof-of-concept study, we develop and test a new human stem cell-based approach to increase engraftment, limit rejection, and restore dystrophin expression in the mdx/scid mouse model of DMD. We introduce two Dystrophin Expressing Chimeric (DEC) cell lines created by ex vivo fusion of human myoblasts (MB) derived from two normal donors (MBN1/MBN2), and normal and DMD donors (MBN/MBDMD). The efficacy of fusion was confirmed by flow cytometry and confocal microscopy based on donor cell fluorescent labeling (PKH26/PKH67). In vitro, DEC displayed phenotype and genotype of donor parent cells, expressed dystrophin, and maintained proliferation and myogenic differentiation. In vivo, local delivery of both DEC lines (0.5 × 106) restored dystrophin expression (17.27%±8.05-MBN1/MBN2 and 23.79%±3.82-MBN/MBDMD) which correlated with significant improvement of muscle force, contraction and tolerance to fatigue at 90 days after DEC transplant to the gastrocnemius muscles (GM) of dystrophin-deficient mdx/scid mice. This study establishes DEC as a potential therapy for DMD and other types of muscular dystrophies.

Epineural Sheath Jacket as a New Surgical Technique for Neuroma Prevention in the Rat Sciatic Nerve Model.

BACKGROUND: Terminal neuromas resulting from severe nerve injuries and traumatic or surgical limb amputations can become a source of pain, and significantly impair patients' quality of life. Recently, the number of patients with peripheral nerve injuries increased due to modern war conflicts, natural disasters, and traffic accidents. This study investigated the efficacy of the epineural sheath jacket (ESJ) as a novel technique for neuroma prevention in the rat sciatic nerve model. METHODS: A 20-mm segment of the right sciatic nerve was excised in 18 Lewis rats, and the animals were divided into 3 experimental groups (n = 6/group): group I-control, nerve stump without protection; group II-muscle burying group, nerve stump buried in the muscle; group III-ESJ group, nerve stump protected by ESJ. The ESJ was created from the excised sciatic nerve and applied as a "cap" over the proximal nerve stump. The presence of neuropathic pain was assessed weekly by pinprick test and Tinel sign, up to 24 weeks postsurgery. At 24 weeks, assessments, such as macroscopic evaluation, retrograde neuronal labeling analysis, histomorphometry, and neural/connective tissue ratio were performed. RESULTS: Epineural sheath jacket significantly reduced neuroma formation, which was associated with decreased Tinel sign (16.7%, P < 0.05) response compared with the nerve stump control. Moreover, ESJ reduced axonal sprouting, bulb-shaped nerve ending formation and perineural adhesions, as confirmed by macroscopic evaluation. Histological evaluation confirmed that nerve stumps protected with the ESJ showed less fibrosis and presented well-organized axonal structure. Neural/connective tissue ratio and retrograde neuronal labeling analysis revealed significantly improved results in the ESJ group compared to the control nerve stump group (P = 0.032 and P = 0.042, respectively). CONCLUSIONS: The protective effect of the ESJ against neuroma formation was confirmed by behavioral and histological analyses, showing outcomes comparable to the muscle burying technique-the criterion standard of neuroma management.

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.

PURPOSE: 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. METHODS: Based on their strain eighteen rats were divided into three groups. A total of 90 brachial plexus nerves (axillary, musculocutaneous, median, ulnar, and radial nerves) and 72 lumbosacral plexus nerves (sciatic, tibial, common peroneal, and sural nerves) were analyzed for the length, diameter and correlation with the body weight. A detailed anatomic course of each nerve within the brachial and lumbosacral plexuses was outlined. RESULTS: The sural nerve was the longest nerve in all studied rat strains, whereas the sciatic nerve had the largest diameter. Comparison of all the nerves' length demonstrated that the Lewis rat sciatic and sural nerves were significantly shorter (P < 0.05). No significant differences in nerve diameters were found among the analyzed rat strain groups. Significant correlation was revealed between the length of sciatic nerve and the rats' weight, which is irrelevant to the rats' genetic background. CONCLUSIONS: This study confirmed that nerves' length within rat's brachial and lumbosacral plexus depends on the inter-individual variations within the rat strains rather than on the differences in the peripheral nerve development, which is inherent to the specific rat strain. Correlation between the nerve length and body weight, suggests that bigger rats should be considered for studies requiring access to the long nerves. © 2016 Wiley Periodicals, Inc. Microsurgery 37:327-333, 2017.

The reversed paradigm of chimerism induction: Donor conditioning with recipient-derived bone marrow cells as a novel approach for tolerance induction in vascularized composite allotransplantation.

PURPOSE: To test a new approach of donor conditioning with recipient bone marrow cells (BMC) to induce tolerance in vascularized composite allograft (VCA) transplantation. METHODS: Lewis rats' (recipients) BMC were stained with PKH-26. The ACI rats (donors) were conditioned with 80 × 106 Lewis BMC, 24 or 72 hours before VCA (groin flap) transplantation. Forty-eight VCA were performed between ACI donors and Lewis recipients. In groups I and II, donors were preconditioned (24 and 72 hours before transplantation, respectively), and recipients received 7-day anti-αß-TCR/cyclosporine-A post-transplantation. In groups III and IV, donors were preconditioned (24 and 72 hours before transplantation, respectively), and recipients received no systemic immunosuppression. In group V, recipients received 7-day anti-αß-TCR/cyclosporine-A post-transplantation. In group VI, recipients received no systemic immunosuppression. Assessment included evaluation of transplant viability and induction of donor-specific chimerism via flow cytometry, immunofluorescence, and PCR. RESULTS: Groups III, IV, and VI rejected allografts, at an average of 14 ± 5.2, 10 ± 2.7, and 8 ± 0.7 days. In groups I, II, and V, the mean survival was 80 ± 18.2 (p = 0.0002), 64 ± 27.4 (p = 0.001), and 30 ± 4.7 (p = 0.02) days. In groups I and II, donor-specific chimerism in the blood decreased from 8.8 ± 3.4% and 8.6 ± 3.4% on day 7 to 3.7 ± 1.32% (p = 0.02) and 4.7 ± 2.7% when the flaps manifested grade 3 rejection. The presence of PKH-26+ Lewis BMC was confirmed in the donor's blood, bone marrow, lymphoid organs, and liver (preconditioned at 24 and 72 hours). CONCLUSIONS: Donor preconditioning is a novel approach modifying recipient's responsiveness to donor allograft and prolonging the allograft survival under short-term immunosuppression. © 2015 Wiley Periodicals, Inc. Microsurgery 36:676-683, 2016.

A new total hemiface allotransplantation model in rats.

INTRODUCTION: 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 patient's 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 recipient's immune response. MATERIAL AND METHODS: Ten hemiface allotransplantations were performed in two groups between Lewis-Lewis (isograft) and LBN-Lewis (allograft) rats. Cyclosporine A (CsA) monotherapy was applied in the allograft group to prevent rejection. RESULTS: All flaps survived up to 100 days post-transplant. The mean warm ischemia time was 45 minutes. Histological analysis revealed normal bone, cartilage (ear and nose), conjunctiva, palpebra, and eyelashes. Flow cytometry confirmed donor-specific chimerism for T cells (CD4/RT1(n) and CD8/RT1(n)) and B cells (CD45RA/RT1(n)) in the peripheral blood of all rats in the allotransplantation group. At post-transplant day 7, chimerism levels were at 1.68% for CD4/RT1(n) , 0.46% for CD8/RT1(n) and 0.64% for CD45RA/RT1(n). However, chimerism levels for CD4/RT1(n), CD8/RT1(n), and CD45RA/RT1(n) populations decreased at long-term follow-up (at post-transplant day 100) to 0.08%, 0.04%, and 0.23%, respectively. CONCLUSION: The feasibility and long-term survival of the new hemiface VCA transplantation model was confirmed, donor-specific chimerism and post-transplant tissue changes were evaluated.

Immunomodulatory Effects of Different Cellular Therapies of Bone Marrow Origin on Chimerism Induction and Maintenance Across MHC Barriers in a Face Allotransplantation Model.

Many more patients would benefit from vascularized composite allotransplantation if less toxic and safer immunosuppressive protocols will become available. Tolerance induction protocols with donor cells co-transplantation are one of the promising pathways to reduce maintenance immunosupressive regimens. We investigated the role of donor bone marrow cells (BMC), mesenchymal stromal cells (MSC) and in vivo created chimeric cells (CC) used as supportive therapies in a fully MHC-mismatched rat face transplantation model. Twenty-four fully MHC-mismatched hemiface transplantations were performed between ACI (RT1(a)) donors and Lewis (RT1(l)) recipients under combined seven-day immunosuppressive regimen of anti-αß-T-cell receptor (TCR) monoclonal antibody and cyclosporin A. We studied four experimental groups-group 1: no cellular therapy; group 2: supportive therapy with BMC; group 3: supportive therapy with MSC; group 4: supportive therapy with CC generated in a primary chimera. We evaluated clinical and histological rejection grades, transplanted cells migration, donor-specific chimerism in the peripheral blood and bone marrow compartments, and CD4(+)/CD25(+) T-cell levels. Face allograft rejection was observed at 26.8 ± 0.6 days post-transplant (PT) in the absence of cellular therapy, at 34.5 ± 1.1 days for group 2, 29.3 ± 0.8 days for group 3, and 30.3 ± 1.38 PT for group 4. The longest survival was observed in allografts supported by co-transplantation of BMC. All support in cellular therapies delayed face allograft rejection by chimerism induction and/or immunomodulatory properties of co-transplanted cells. Survival time was comparable between groups, however, further studies, with different cell dosages, delivery routes and delivery times are required.