Skeletal muscle reprogramming enhances reinnervation after peripheral nerve injury.

Peripheral Nerve Injuries (PNI) affect more than 20 million Americans and severely impact quality of life by causing long-term disability. The onset of PNI is characterized by nerve degeneration distal to the nerve injury resulting in long periods of skeletal muscle denervation. During this period, muscle fibers atrophy and frequently become incapable of "accepting" innervation because of the slow speed of axon regeneration post injury. We hypothesize that reprogramming the skeletal muscle to an embryonic-like state may preserve its reinnervation capability following PNI. To this end, we generated a mouse model in which NANOG, a pluripotency-associated transcription factor can be expressed locally upon delivery of doxycycline (Dox) in a polymeric vehicle. NANOG expression in the muscle upregulated the percentage of Pax7+ nuclei and expression of eMYHC along with other genes that are involved in muscle development. In a sciatic nerve transection model, NANOG expression led to upregulation of key genes associated with myogenesis, neurogenesis and neuromuscular junction (NMJ) formation, and downregulation of key muscle atrophy genes. Further, NANOG mice demonstrated extensive overlap between synaptic vesicles and NMJ acetylcholine receptors (AChRs) indicating restored innervation. Indeed, NANOG mice showed greater improvement in motor function as compared to wild-type (WT) animals, as evidenced by improved toe-spread reflex, EMG responses and isometric force production. In conclusion, we demonstrate that reprogramming the muscle can be an effective strategy to improve reinnervation and functional outcomes after PNI.

Gene Editing Technologies Targeting TFAM and Its Relation to Mitochondrial Diseases.

Mitochondria are organelles present in the cytoplasm of eukaryotic cells; they play a key role in adenosine triphosphate (ATP) synthesis and oxidative phosphorylation. Mitochondria have their own DNA, mitochondrial DNA (mtDNA), keeping the function of the mitochondria. Mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters is and considered essential in mtDNA replication and transcription. More recently, TFAM has been shown to play a central role in the maintenance and regulation of mitochondrial copy number, inflammatory response, expression regulation, and mitochondrial genome activity. Gene editing tools such as the CRISPR-Cas 9 technique, TALENs, and other gene editing tools have been used to investigate the role of TFAM in mitochondrial mechanics and biogenesis as well as its correlation to mitochondrial disorders. Thus this chapter brings a summary of mitochondria function, dysfunction, the importance of TFAM in the maintenance of mitochondria, and state of the art of gene editing tools involving TFAM and mtDNA.

Morphology and morphometry of the human obturator nerve in males and females.

Peripheral nerve injury and the nerves' subsequent repair and regeneration continues to be marked clinically by poor functional recovery. The analysis of nerve morphology is an aspect which may provide an impact on successful clinical outcomes through better prediction of donor and recipient matching. In this study, we evaluated the morphological aspects of the human obturator nerve for a better understanding of its potential in nerve transplantation. Morphological characteristics of donor obturator nerves were analysed, including nerve diameter and length, fascicle count and the ratio of neural to non-neural tissue present within the cross-sectional area of the nerve's epineurium, with respect to laterality and sex. Statistical significance (p < 0.10) was determined for male obturator nerves having an average diameter of 2.67 mm compared to female obturator nerves at 1.91 mm, as well as left obturator nerves having an average of 11.21 fascicles compared to the right having an average of 10.17 fascicles. Strong positive correlations were determined between cross-sectional nerve area and limb size index, as well as between percentage of non-neural tissue and area of non-neural tissue, among males. Separately, strong correlation between percentage of non-neural tissue and area of non-neural tissue among right obturator nerves in males and females was determined . These findings indicate that there are associations and predictions that can be made about nerve morphology and that these when combined with other patient characteristics may enhance patient functional recovery following a peripheral nerve's repair.

Addition of synthetic polymer in the freezing solution of mesenchymal stem cells from equine adipose tissue as a future perspective for reducing of DMSO concentration.

The regenerative therapies with stem cells (SC) has been increased by the cryopreservation, permitting cell storage for extended periods. However, the permeating cryoprotectant agents (CPAs) such as dimethylsulfoxide (DMSO) can cause severe adverse effects. Therefore, this study evaluated equine mesenchymal stem cells derived from adipose tissue (eAT-MSCs) in fresh (Control) or after slow freezing (SF) in different freezing solutions (FS). The FS comprise DMSO and non-permeating CPAs [Trehalose (T) and the SuperCool X-1000 (X)] in association or not, totalizing seven different FS: (DMSO; T; X; DMSO+T; DMSO+X; T+X, and DMSO+T+X). Before and after cryopreservation were evaluated, viability, colony forming unit (CFU), and cellular differentiation capacity. After freezing-thawing, the viability of the eAT-MSCs reduced (P< 0.05) in all treatments compared to the control. However, the viability of frozen eAT-MSCs in DMSO (80.3 ± 0.6) was superior (P<0.05) to the other FS. Regarding CFU, no difference (P>0.05) was observed between fresh and frozen cells. After freezing-thawing, the eAT-MSCs showed osteogenic, chondrogenic, and adipogenic lineages differentiation potential. Nonetheless, despite the significative reduction in the osteogenic differentiation capacity between fresh and frozen cells, no differences (P > 0.05) were observed among FS. Furthermore, the number of chondrogenic differentiation cells frozen in DMSO+X solution reduced (P<0.05) comparing to the control, without differ (P>0.05) to the other FS. The adipogenic differentiation did not differ (P>0.05) among treatments. In conclusion, although these findings confirm the success of DMSO to cryopreserve eAT-MSCs, the Super Cool X-1000 could be a promise to reduce the DMSO concentration in a FS.

As terapias regenerativas com células-tronco (CT) têm sido incrementadas pela criopreservação, permitindo o armazenamento celular. No entanto, os agentes crioprotetores (ACPs) penetrantes, como DMSO, podem causar efeitos adversos graves. Portanto, este estudo avaliou células-tronco mesenquimais equinas derivadas de tecido adiposo (CTM-TAe) in natura (Controle) ou após congelamento lento (CL) em diferentes soluções de congelamento (SC). As SCs compreendem DMSO e ACPs não permeáveis [Trealose (T) e o SuperCool X-1000 (X)] associados ou não: (DMSO; T; X; DMSO+T; DMSO+X; T +X e DMSO+T+X). Antes e após a criopreservação foram avaliados, viabilidade, unidade formadora de colônia (UFC) e capacidade de diferenciação celular. Após o congelamento-descongelamento, a viabilidade das CTM-TAe reduziu (P< 0,05) em todos os tratamentos em relação ao controle. Entretanto, a viabilidade das CTM-TAe congeladas em DMSO (80,3 ± 0,6) foi superior (P<0,05) às demais SC. Em relação às UFC, não houve diferença (P>0,05) entre células frescas e congeladas. Após congelamento-descongelamento, as CTM-TAe apresentaram potencial de diferenciação de linhagens osteogênicas, condrogênicas e adipogênicas. No entanto, apesar da redução significativa na capacidade de diferenciação osteogênica entre células frescas e congeladas, não foram observadas diferenças (P > 0,05) entre SCs. Além disso, o número de células de diferenciação condrogênica congeladas em solução de DMSO+X reduziu (P<0,05) em relação ao controle, sem diferir (P>0,05) das demais SCs. A diferenciação adipogênica não diferiu (P>0,05) entre os tratamentos. Em conclusão, embora esses achados confirmem o sucesso do DMSO para criopreservar CTM-TAe, o Super Cool X-1000 pode ser uma promessa para reduzir a concentração de DMSO.

The effects of graft source and orientation on outcomes after ablation of a branched peripheral nerve.

Segmental peripheral nerve injuries (PNI) are the most common cause of enduring nervous system dysfunction. The peripheral nervous system (PNS) has an extensive and highly branching organization. While much is known about the factors that affect regeneration through sharp bisections and linear ablations of peripheral nerves, very little has been investigated or documented about PNIs that ablate branch points. Such injuries present additional complexity compared to linear segmental defects. This study compared outcomes following ablation of a branch point with branched grafts, specifically examining how graft source and orientation of the branched graft contributed to regeneration. The model system was Lewis rats that underwent a 2.5 cm ablation that started in the sciatic nerve trunk and included the peroneal/tibial branch point. Rats received grafts that were rat sciatic autograft, inbred sciatic allograft, and inbred femoral allograft, each of which was a branched graft of 2.5 cm. Allografts were obtained from Lewis rats, which is an inbred strain. Both branches of the sciatic grafts were mixed motor and sensory while the femoral grafts were smaller in diameter than sciatic grafts and one branch of the femoral graft is sensory and the other motor. All branched grafts were sutured into the defect in two orientations dictated by which branch in the graft was sutured to the tibial vs peroneal stumps in recipients. Outcome measures include compound muscle action potentials (CMAPs) and CatWalk gait analysis throughout the recovery period, with toluidine blue for intrinsic nerve morphometry and retrograde labeling conducted at the 36-week experimental end point. Results indicate that graft source and orientation does play a significant role earlier in the regenerative process but by 36 weeks all groups showed very similar indications of regeneration across multiple outcomes.

Pineal Gland from the Cell Culture to Animal Models: A Review.

This review demonstrates current literature on pineal gland physiology, pathology, and animal model experiments to concisely explore future needs in research development with respect to pineal gland function and neuro-regenerative properties. The pineal gland plays an integral role in sleep and recovery by promoting physiologic circadian rhythms via production and release of melatonin. Yet, the current literature shows that the pineal gland has neuroprotective effects that modulate both peripheral and central nerve injuries through several direct and indirect mechanisms, such as angiogenesis and induction of growth factors and anti-inflammatory mediators. Animal models have also shown correlations between pineal gland function and metabolic homeostasis. Studies have shown that a functional pineal gland is essential in preventing and slowing the progression of certain diseases such as diabetes, osteoporosis, vertebral osteoarthritis, and neurodegenerative processes. Lastly, the array of cell culturing methods and animal models that can be used to further develop the study of pineal gland function and nervous system injury were reviewed.

El índice de masa corporal no se asocia con indicadores metabólicos en pacientes con traumatismo ortopédico / Body mass index found not to be associated with metabolicindicators in patients with orthopedic trauma

Introduction: The relevance of the nutritional status inpatients with orthopedic trauma (OT) has been enhanced inthe literature. Objective: This study compared overweight and obese pa-tients with normal body weight patients and their relationshipwith nutritional and metabolic indicators. Methods: Patients with OT (n=108) were investigated in aprospective study for body mass index (BMI) in relation totheir calf circumference (CC), vitamin D and albumin serumlevels. The statistical analysis included the Chi-square or Fishertests and the Mann-Whitney test and Spearman’s linear cor-relation coefficient. Results: There was no association between gender, typeof fractured bone, laterality, smoking, diabetes, menopause,albumin and vitamin D and the two groups of patients classi-fied by BMI. There was an association (p <0.0001) only withCC, with lower CC values in patients with normal body weight. Conclusion: BMI was not associated with metabolic indi-cators in patients with OT.(AU)

Introducción: La relevancia del estado nutricional en pa-cientes con traumatismo ortopédico (TO) se ha reforzado enla literatura.Objetivo: Este estudio comparó pacientes con sobrepesoy obesidad con pacientes con peso corporal normal y su rela-ción con indicadores nutricionales y metabólicos. Métodos: Los pacientes con TO (n = 108) fueron investi-gados en un estudio prospectivo para el índice de masa cor-poral (IMC) en relación con su circunferencia de la pantorrilla(CC), vitamina D y niveles séricos de albúmina. El análisis es-tadístico incluyó las pruebas de Chi-cuadrado o Fisher y laprueba de Mann-Whitney y el coeficiente de correlación linealde Spearman. Resultados: No hubo asociación entre sexo, tipo de fracturaósea, lateralidad, tabaquismo, diabetes, menopausia, albúminay vitamina D y los dos grupos de pacientes clasificados porIMC. Hubo asociación (p <0,0001) solo con CC, con valores deCC más bajos en pacientes con peso corporal normal. Conclusión: El IMC no se asoció con indicadores metabó-licos en pacientes con TO. (AU)

Typical and atypical properties of peripheral nerve allografts enable novel strategies to repair segmental-loss injuries.

We review data showing that peripheral nerve injuries (PNIs) that involve the loss of a nerve segment are the most common type of traumatic injury to nervous systems. Segmental-loss PNIs have a poor prognosis compared to other injuries, especially when one or more mixed motor/sensory nerves are involved and are typically the major source of disability associated with extremities that have sustained other injuries. Relatively little progress has been made, since the treatment of segmental loss PNIs with cable autografts that are currently the gold standard for repair has slow and incomplete (often non-existent) functional recovery. Viable peripheral nerve allografts (PNAs) to repair segmental-loss PNIs have not been experimentally or clinically useful due to their immunological rejection, Wallerian degeneration (WD) of anucleate donor graft and distal host axons, and slow regeneration of host axons, leading to delayed re-innervation and producing atrophy or degeneration of distal target tissues. However, two significant advances have recently been made using viable PNAs to repair segmental-loss PNIs: (1) hydrogel release of Treg cells that reduce the immunological response and (2) PEG-fusion of donor PNAs that reduce the immune response, reduce and/or suppress much WD, immediately restore axonal conduction across the donor graft and re-innervate many target tissues, and restore much voluntary behavioral functions within weeks, sometimes to levels approaching that of uninjured nerves. We review the rather sparse cellular/biochemical data for rejection of conventional PNAs and their acceptance following Treg hydrogel and PEG-fusion of PNAs, as well as cellular and systemic data for their acceptance and remarkable behavioral recovery in the absence of tissue matching or immune suppression. We also review typical and atypical characteristics of PNAs compared with other types of tissue or organ allografts, problems and potential solutions for PNA use and storage, clinical implications and commercial availability of PNAs, and future possibilities for PNAs to repair segmental-loss PNIs.

Functional and immunological peculiarities of peripheral nerve allografts.

This review addresses the accumulating evidence that live (not decellularized) allogeneic peripheral nerves are functionally and immunologically peculiar in comparison with many other transplanted allogeneic tissues. This is relevant because live peripheral nerve allografts are very effective at promoting recovery after segmental peripheral nerve injury via axonal regeneration and axon fusion. Understanding the immunological peculiarities of peripheral nerve allografts may also be of interest to the field of transplantation in general. Three topics are addressed: The first discusses peripheral nerve injury and the potential utility of peripheral nerve allografts for bridging segmental peripheral nerve defects via axon fusion and axon regeneration. The second reviews evidence that peripheral nerve allografts elicit a more gradual and less severe host immune response allowing for prolonged survival and function of allogeneic peripheral nerve cells and structures. Lastly, potential mechanisms that may account for the immunological differences of peripheral nerve allografts are discussed.

Protocol to Study the Role of Extracellular Vesicles During Induced Stem Cell Differentiation.

Extracellular vesicles (EVs) are vesicles released by cells, which due to their cargo and cell membrane proteins induce changes in the recipient cells. These vesicles can be a novel option to induce stem cell differentiation. Here we described a method to induce mesenchymal stem cell differentiation (MSC) into neuron-like cells using small EVs from neurons. First, we will describe a method based on neurons to induce adipocyte derived stem cells differentiation, a type of MSC, by coculturing both using inserts. Secondly, we will describe a follow-up method by using only isolated neuron-derived small EVs to directly induce ADSC differentiation in neuron-like cells. Importantly, in both methods it is possible to avoid the direct cell-to-cell contact, thus allowing for the study of soluble factors role during stem cell differentiation.

HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation.

The mitochondrial transcription factor A (TFAM) is considered a key factor in mitochondrial DNA (mtDNA) copy number. Given that the regulation of active copies of mtDNA is still not fully understood, we investigated the effects of CRISPR-Cas9 gene editing of TFAM in human embryonic kidney (HEK) 293T cells on mtDNA copy number. The aim of this study was to generate a new in vitro model by CRISPR-Cas9 system by editing the TFAM locus in HEK293T cells. Among the resulting single-cell clones, seven had high mutation rates (67-96%) and showed a decrease in mtDNA copy number compared to control. Cell staining with Mitotracker Red showed a reduction in fluorescence in the edited cells compared to the non-edited cells. Our findings suggest that the mtDNA copy number is directly related to TFAM control and its disruption results in interference with mitochondrial stability and maintenance.

Pluripotent stem cells proliferation is associated with placentation in dogs.

Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs. The uteri were obtained from social castration programs for population control in Pirassununga, Sao Paulo, Brazil. It was collected 20 uteri at different stages. The samples were fixed and processed for immunohistochemical analysis of NANOG, OCT4 and SOX2 expression, knowing as pluripotent stem cells makers. Our results showed positive expression for NANOG, OCT4 and SOX2 in all stages of gestation and nonpregnant uterus; however, we highlight some quantitative different between stages. OCT4 showed more expression in non-pregnant uterus than NANOG and SOX2, and its expression increased in pregnant uterus. In pregnant uterus there was more expression of NANOG than OCT4 and SOX2. Interesting, no difference was found between these markers in the other periods. In conclusion, it was possible to identify pluripotent stem cells in all periods in dog placenta and uterus, however during the early stage of pregnancy we observed more pluripotent stem cells than in all the others periods confirming the high plasticity and regeneration capacity of the uterine tissue.

The Use of Bone Marrow-Derived Stem Cells in the Treatment of a Comminuted Tibiotarsal Fracture in a Juvenile Red-legged Seriema (Cariama cristata).

An adult red-legged seriema (Cariama cristata) presented with a comminuted fracture of the tibiotarsus and fibula. Surgery was performed, and a type II external fixator, with 2 distal and 2 proximal pins, was used to stabilize the fracture. After a 10-day stabilization period, the bird developed a second fracture on the same bone, proximal to the first fracture site. Another surgery was performed on the seriema similar to the first one. However, in this second surgical procedure a single pin, instead of 2 perpendicular pins, was placed proximally to the fracture site. After the second surgical procedure, bone marrow stem cells (BMSCs) from the seriema's left ulna were collected. Twenty-seven days after the second surgery, the BMSCs were transplanted, into the fracture sites. Twenty-four days after the stem cells were injected into the fractures (51 days after the second surgical procedure), radiographic images revealed healing bone calluses at the fracture sites. The fracture healing was relatively long for this case (a total of 75 days). The addition of bone marrow stem cell therapy to the use of external fixation may have contributed to the healing observed radiographically 24 days after administration; therefore, bone marrow stem cell therapy, in addition to traditional surgical fracture reduction and stabilization, may be a promising therapeutic approach for avian cases with similar injuries and bone anatomy. However, as this is a single case, this therapeutic modality deserves further application and study. Moreover, we suggest modifications in the bone marrow stem cell collection and therapy, which may be useful for future studies and application involving birds.

A Comparison of Automated Perfusion- and Manual Diffusion-Based Human Regulatory T Cell Expansion and Functionality Using a Soluble Activator Complex.

Absence or reduced frequency of human regulatory T cells (Tregs) can limit the control of inflammatory responses, autoimmunity, and the success of transplant engraftment. Clinical studies indicate that use of Tregs as immunotherapeutics would require billions of cells per dose. The Quantum® Cell Expansion System (Quantum system) is a hollow-fiber bioreactor that has previously been used to grow billions of functional T cells in a short timeframe, 8-9 d. Here we evaluated expansion of selected Tregs in the Quantum system using a soluble activator to compare the effects of automated perfusion with manual diffusion-based culture in flasks. Treg CD4+CD25+ cells from three healthy donors, isolated via column-free immunomagnetic negative/positive selection, were grown under static conditions and subsequently seeded into Quantum system bioreactors and into T225 control flasks in an identical culture volume of PRIME-XV XSFM medium with interleukin-2, for a 9-d expansion using a soluble anti-CD3/CD28/CD2 monoclonal antibody activator complex. Treg harvests from three parallel expansions produced a mean of 3.95 × 108 (range 1.92 × 108 to 5.58 × 108) Tregs in flasks (mean viability 71.3%) versus 7.00 × 109 (range 3.57 × 109 to 13.00 × 109) Tregs in the Quantum system (mean viability 91.8%), demonstrating a mean 17.7-fold increase in Treg yield for the Quantum system over that obtained in flasks. The two culture processes gave rise to cells with a memory Treg CD4+CD25+FoxP3+CD45RO+ phenotype of 93.7% for flasks versus 97.7% for the Quantum system. Tregs from the Quantum system demonstrated an 8-fold greater interleukin-10 stimulation index than cells from flask culture following restimulation. Quantum system-expanded Tregs proliferated, maintained their antigenic phenotype, and suppressed effector immune cells after cryopreservation. We conclude that an automated perfusion bioreactor can support the scale-up expansion of functional Tregs more efficiently than diffusion-based flask culture.

Characterization of post-edited cells modified in the TFAM gene by CRISPR/Cas9 technology in the bovine model.

Gene editing in large animal models for future applications in translational medicine and food production must be deeply investigated for an increase of knowledge. The mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters. This gene maintains mtDNA, and it is essential for the initiation of mtDNA transcription. Lately, we generated a new cell line through the disruption of the TFAM gene in bovine fibroblast cells by CRISPR/Cas 9 technology. We showed that the CRISPR/Cas9 design was efficient through the generation of heterozygous mutant clones. In this context, once this gene regulates the mtDNA replication specificity, the study aimed to determine if the post-edited cells are capable of in vitro maintenance and assess if they present changes in mtDNA copies and mitochondrial membrane potential after successive passages in culture. The post-edited cells were expanded in culture, and we performed a growth curve, doubling time, cell viability, mitochondrial DNA copy number, and mitochondrial membrane potential assays. The editing process did not make cell culture unfeasible, even though cell growth rate and viability were decreased compared to control since we observed the cells grow well when cultured in a medium supplemented with uridine and pyruvate. They also exhibited a classical fibroblastoid appearance. The RT-qPCR to determine the mtDNA copy number showed a decrease in the edited clones compared to the non-edited ones (control) in different cell passages. Cell staining with Mitotracker Green and red suggests a reduction in red fluorescence in the edited cells compared to the non-edited cells. Thus, through characterization, we demonstrated that the TFAM gene is critical to mitochondrial maintenance due to its interference in the stability of the mitochondrial DNA copy number in different cell passages and membrane potential confirming the decrease in mitochondrial activity in cells edited in heterozygosis.

Long-term neural regeneration following injury to the peroneal branch of the sciatic nerve in sheep.

Peripheral nerves (PNs) are frequently injured as a result of trauma or disease. Development of therapies to regenerate PNs requires the use of animal models, typically beginning in rodents and progressing to larger species. There are several large animal models of PN regeneration that each has their benefits and drawbacks. Sheep have been used in PN studies due to their similarities in body weight to humans and the ease and lesser expense in their care and housing relative to other species. We have investigated the use of sheep for studies of PN regeneration and have developed and tested an injury model in the peroneal branch of the sciatic nerve. Three experimental groups were tested on mature sheep: a bisection; a 5-cm reverse autograft; and sham surgery. Protocols were developed for the post-operative care for animals with this injury, and regeneration was tracked for extended time points via compound muscle action potentials (CMAPs) and endpoint assessments of nerve morphometry, muscle mass and muscle fibrosis. Results indicate the practical viability of this PN injury model and show distinctions in the degree and rate of regeneration between bisection and reverse autograft that persisted 14 months. This long-term study shows bisections lead to significantly improved CMAPS and muscle mass and lesser muscle fibrosis as compared to reverse autograft. The persistence of these discernable changes between two relatively similar experimental groups out to extended time points is an indication of the sensitivity of this nerve section and its potential applicability for comparative studies.

Analysis of N- and O-Linked Glycosylation: Differential Glycosylation after Rat Spinal Cord Injury.

Glycosylation is a fundamental cellular process that has a dramatic impact on the functionality of glycoconjugates such as proteins or lipids and mediates many different biological interactions including cell migration, cellular signaling, and synaptic interactions in the nervous system. In spinal cord injury (SCI), all of these cellular processes are altered, but the potential contributions of glycosylation changes to these alterations has not been thoroughly investigated. We studied the glycosylation of injured spinal cord tissue from rats that received a contusion SCI. The N- and O-linked glycosylation was assessed at 3 and 14 days post-injury (DPI), and compared with uninjured control and time-matched sham spinal tissue. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS/MS) were performed to analyze carbohydrate structures. Results revealed diverse and abundant glycosylation in all groups, with some carbohydrate structures differentially produced in SCI animals compared with uninjured controls and shams. One such change occurred in the abundance of the Sda structure, Neu5Ac-α-(2,3)-[GalNAc-ß-(1,4)-]Gal-ß-(1,4)-GlcNAc, which was increased in SCI samples compared with shams and non-injured controls. Immunohistochemistry (IHC) and western blot were performed on SCI and sham samples using the CT1 antibody, which recognizes the terminal trisaccharide of Sda with high specificity. Both of these metrics confirmed elevated Sda structure in SCI tissue, where IHC further showed that Sda is expressed mainly by microglia. The results of these studies suggest that SCI causes a significant alteration in N- and O-linked glycosylation.

Neurons-derived extracellular vesicles promote neural differentiation of ADSCs: a model to prevent peripheral nerve degeneration.

Potential mechanisms involved in neural differentiation of adipocyte derived stem cells (ADSCs) are still unclear. In the present study, extracellular vesicles (EVs) were tested as a potential mechanism involved in the neuronal differentiation of stem cells. In order to address this, ADSCs and neurons (BRC) were established in primary culture and co-culture at three timepoints. Furthermore, we evaluated protein and transcript levels of differentiated ADSCs from the same timepoints, to confirm phenotype change to neuronal linage. Importantly, neuron-derived EVs cargo and EVs originated from co-culture were analyzed and tested in terms of function, such as gene expression and microRNA levels related to the adult neurogenesis process. Ideal neuron-like cells were identified and, therefore, we speculated the in vivo function of these cells in acute sciatic nerve injury. Overall, our data demonstrated that ADSCs in indirect contact with neurons differentiated into neuron-like cells. Neuron-derived EVs appear to play an important role in this process carrying SNAP25, miR-132 and miR-9. Additionally, in vivo neuron-like cells helped in microenvironment modulation probably preventing peripheral nerve injury degeneration. Consequently, our findings provide new insight of future methods of ADSC induction into neuronal linage to be applied in peripheral nerve (PN) injury.

Xenotransplantation of canine spermatogonial stem cells (cSSCs) regulated by FSH promotes spermatogenesis in infertile mice.

BACKGROUND: Xenotransplantation of spermatogonial stem cells (SSCs) has become a popular topic in various research fields because manipulating these cells can provide insights into the mechanisms associated with germ cell lines and the entire spermatogenesis process; moreover, these cells can be used in several biotechnology applications. To achieve successful xenotransplantation, the in vitro microenvironment in which SSCs are cultured should be an ideal microenvironment for self-renewal and similar to the in vivo testicular microenvironment. The age of the donor, the correct spermatogenesis cycle, and the quality of the donor tissue are also important. Although cell culture-related factors, such as the in vitro supplementation of hormonal factors, are known to promote successful xenotransplantation in mice, little is known about the influence of these factors on SSCs in vitro or in vivo in other mammalian species, such as dogs (Canis lupus familiaris). In this context, the goals of this study were to test the effect of follicle-stimulating hormone (FSH) on canine spermatogonial stem cell (cSSC) cultures since this hormone is related to the glial cell-derived neurotrophic factor (GDNF) signaling pathway, which is responsible for the self-renewal and maintenance of these cells in vivo, and to investigate the microenvironment of the SSC culture after FSH supplementation. Additionally, in vivo analyses of transplanted FSH-supplemented cSSCs in the testes of infertile mice were performed to assess the capacity of cSSCs to develop, maintain, and restore spermatogenesis. METHODS: SSCs from canine prepubertal testes (aged 3 months) were cultured in vitro in the presence of FSH (10 IU L-1). GFRA1 transcript expression was detected to confirm the spermatogonia population in culture and the effect of FSH on these cells. The protein and transcript levels of late germ cell markers (GFRA1, DAZL, STRA8, PLZF, and CD49f) and a pluripotency marker (OCT4) were detected at 72 and 120 h to confirm the cSSC phenotype. In vivo experiments were performed by transplanting GFP+ cSSCs into infertile mice, and a 10-week follow-up was performed. Histological and immunofluorescence analyses were performed to confirm the repopulation capacity after cSSC xenotransplantation in the testis. RESULTS: Supplementation with FSH in cell culture increased the number of cSSCs positive for GFRA1. The cSSCs were also positive for the pluripotency and early germline marker OCT4 and the late germline markers PLZF, DAZL, C-kit, and GFRA-1. The in vivo experiments showed that the cSSCs xenotransplanted into infertile mouse testes were able to repopulate germline cells in the seminiferous tubules of mice. CONCLUSIONS: In conclusion, our results showed for the first time that the treatment of cSSC cultures with FSH can promote in vitro self-renewal, increase the population of germline cells, and possibly influence the success of spermatogenesis in infertile mice in vivo.

Localized delivery of immunosuppressive regulatory T cells to peripheral nerve allografts promotes regeneration of branched segmental defects.

Segmental injuries to peripheral nerves (PNs) too often result in lifelong disability or pain syndromes due to a lack of restorative treatment options. For injuries beyond a critical size, a bridging device must be inserted to direct regeneration. PN allografts from immunologically incompatible donors are highly effective bridging devices but are not a regular clinical option because of the expense and health risks of systemic immunosuppression (ISN). We have developed a method to deliver a single administration of ISN localized around a PN allograft that circumvents the risks of systemic ISN. Localized ISN was provided by regulatory T cells (Tregs), a potently immunosuppressive cell type, that was delivered around a PN allograft with a poly(ethylene glycol) norbornene (PEGNB) degradable hydrogel. Tregs are released from the hydrogel over 14 d, infiltrate the graft, suppress the host immune response and facilitate regeneration of the recipient rats equal to the autograft control. Furthermore, this method was effective in a segmental PN defect that included a branch point, for which there currently exist no treatment options. These results show that localized delivery of immunosuppressive cells for PN allografts is an effective new strategy for treating segmental PN defects that can also be used to regenerate complex nerve structures.