Effects of minocycline and rapamycin in gamma-irradiated human embryonic stem cells-derived cerebral organoids.

Radiation induces DNA and protein damage and free radical formation, effectively establishing cellular senescence in a variety of models. We demonstrate the effects of two known pleiotropic drugs following gamma radiation damage in neurosphere/cerebral organoid system based on human embryonic stem cells. mTORC1 repression by rapamycin prior to irradiation, or metabolic activation by minocycline after irradiation, partially rescues neuroepithelium integrity, neurite-growing capacity, ventricle formation and extracellular acidification rate as an integral measure of metabolic output. Cerebral organoid model thus provides valid and robust readouts for radiation studies in a complex 3D setting.

Autologous mesenchymal stromal cells as a therapeutic in ALS and epilepsy patients: Treatment modalities and ex vivo neural differentiation.

Stem cell therapy for incurable central nervous system disorders has long been viewed as a promising therapeutic option. In this review, we discuss the existing data and approaches on cell transplantation in the context of the neural differentiation potential of adult autologous stem cells, focusing on those of mesenchymal origin as easily accessible and well studied. Mesenchymal stromal cells (MSCs) are a heterogeneous cell population with a remarkable therapeutic plasticity, demonstrated by their ability to dampen inflammation, inhibit pathogenic immune responses and secrete neuroprotective factors. To demonstrate and discuss the broad therapeutic potential of MSCs, this review focuses on two examples of neurological conditions: amyotrophic lateral sclerosis and epilepsy. We review the lessons from animal models and clinical trials, and consider encouraging newly published clinical data on therapeutic applications of neurally induced MSCs.

Inter-microcarrier transfer and phenotypic stability of stem cell-derived Schwann cells in stirred suspension bioreactor culture.

Emerging bioreactor technologies offer an effective way for scaled-up production of large numbers of cells for cell therapy applications. One of the clinical paradigms where cell therapy can be an asset is restorative neurosciences. Nerve repair can benefit from the injections of stem cells and/or Schwann cells, acting as a source for axon myelination, myelin debris clearance, and trophic support. We have adapted microcarrier-based suspension bioreactor culture for Schwann cells (SCs) differentiated from a new stem cell source - skin-derived precursors (SKPs). SKP-derived SCs attach and grow on different types of microcarriers in both static and stirred culture, with Cytodex 3 and CultiSpher-S found most effective. Inter-microcarrier migration of SKP-SCs represents a key mechanism for rapid expansion and colonization in stirred suspension culture. We have shown that microcarrier-expanded SKP-SCs cells express Schwann cell markers p75-NTR, GFAP and S100 and retain their key ability to myelinate axons both in vitro and in vivo. Scaled-up microcarrier-based production of SKP-SCs in suspension bioreactors appears feasible for timely generation of sufficient cell numbers for nerve repair strategies.

Demyelination induces transport of ribosome-containing vesicles from glia to axons: evidence from animal models and MS patient brains.

Glial cells were previously proven capable of trafficking polyribosomes to injured axons. However, the occurrence of such transfer in the general pathological context, such as demyelination-related diseases, needs further evidence. Since this may be a yet unidentified universal contributor to axonal survival, we study putative glia-axonal ribosome transport in response to demyelination in animal models and patients in both peripheral and central nervous system. In the PNS we investigate whether demyelination in a rodent model has the potential to induce ribosome transfer. We also probe the glia-axonal ribosome supply by implantation of transgenic Schwann cells engineered to produce fluorescent ribosomes in the same demyelination model. We furthermore examine the presence of axonal ribosomes in mouse experimental autoimmune encephalomyelitis (EAE), a well-established model for multiple sclerosis (MS), and in human MS autopsy brain material. We provide evidence for increased axonal ribosome content in a pharmacologically demyelinated sciatic nerve, and demonstrate that at least part of these ribosomes originate in the transgenic Schwann cells. In the CNS one of the hallmarks of MS is demyelination, which is associated with severe disruption of oligodendrocyte-axon interaction. Here, we provide evidence that axons from spinal cords of EAE mice, and in the MS human brain contain an elevated amount of axonal ribosomes compared to controls. Our data provide evidence that increased axonal ribosome content in pathological axons is at least partly due to glia-to-axon transfer of ribosomes, and that demyelination in the PNS and in the CNS is one of the triggers capable to initiate this process.

Aligned collagen-GAG matrix as a 3D substrate for Schwann cell migration and dendrimer-based gene delivery.

The development of artificial off-the-shelf conduits that facilitate effective nerve regeneration and recovery after repair of traumatic nerve injury gaps is of fundamental importance. Collagen-glycosaminoglycan (GAG) matrix mimicking Schwann cell (SC) basal lamina has been proposed as a suitable and biologically rational substrate for nerve regeneration. In the present study, we have focused on the permissiveness of this matrix type for SC migration and repopulation, as these events play an essential role in nerve remodeling. We have also demonstrated that SCs cultured within collagen-GAG matrix are compatible with non-viral dendrimer-based gene delivery, that may allow conditioning of matrix-embedded cells for future gene therapy applications.

Early regenerative effects of NGF-transduced Schwann cells in peripheral nerve repair.

Peripheral nerve injury leads to a rapid and robust increase in the synthesis of neurotrophins which guide and support regenerating axons. To further optimize neurotrophin supply at the earliest stages of regeneration, we over-expressed NGF in Schwann cells (SCs) by transducing these cells with a lentiviral vector encoding NGF (NGF-SCs). Transplantation of NGF-SCs in a rat sciatic nerve transection/repair model led to significant increase of NGF levels 2weeks after injury and correspondingly to substantial improvement in axonal regeneration. Numbers of NF200, ChAT and CGRP-positive axon profiles, as well as the gastrocnemius muscle weights, were significantly higher in the NGF-Schwann cell group compared to the animals that received control SCs transduced with a lentiviral vector encoding GFP (GFP-SCs). Comparison with other models of NGF application signifies the important role of this neurotrophin during the early stages of regeneration, and supports the importance of developing combined gene and cell therapy for peripheral nerve repair.

Evidence for a systemic regulation of neurotrophin synthesis in response to peripheral nerve injury.

Up-regulation of neurotrophin synthesis is an important mechanism of peripheral nerve regeneration after injury. Neurotrophin expression is regulated by a complex series of events including cell interactions and multiple molecular stimuli. We have studied neurotrophin synthesis at 2 weeks time-point in a transvertebral model of unilateral or bilateral transection of sciatic nerve in rats. We have found that unilateral sciatic nerve transection results in the elevation of nerve growth factor (NGF) and NT-3, but not glial cell-line derived neurotrophic factor or brain-derived neural factor, in the uninjured nerve on the contralateral side, commonly considered as a control. Bilateral transection further increased NGF but not other neurotrophins in the nerve segment distal to the transection site, as compared to the unilateral injury. To further investigate the distinct role of NGF in regeneration and its potential for peripheral nerve repair, we transduced isogeneic Schwann cells with NGF-encoding lentivirus and transplanted the over-expressing cells into the distal segment of a transected nerve. Axonal regeneration was studied at 2 weeks time-point using pan-neuronal marker NF-200 and found to directly correlate with NGF levels in the regenerating nerve.

Dendrimer-driven neurotrophin expression differs in temporal patterns between rodent and human stem cells.

This study reports the use of a nonviral expression system based on polyamidoamine dendrimers for time-restricted neurotrophin overproduction in mesenchymal stem cells and skin precursor-derived Schwann cells. The dendrimers were used to deliver plasmids for brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) expression in both rodent and human stem cells, and the timelines of expression were studied. We have found that, despite the fact that transfection efficiencies and protein expression levels were comparable, dendrimer-driven expression in human mesenchymal stem cells was characterized by a more rapid decline compared to rodent cells. Transient expression systems can be beneficial for some neurotrophins, which were earlier reported to cause unwanted side effects in virus-based long-term expression models. Nonviral neurotrophin expression is a biologically safe and accessible alternative to increase the therapeutic potential of autologous adult stem cells and stem cell-derived functional differentiated cells.

Morphological evidence for a transport of ribosomes from Schwann cells to regenerating axons.

Recently, we showed that Schwann cells transfer ribosomes to injured axons. Here, we demonstrate that Schwann cells transfer ribosomes to regenerating axons in vivo. For this, we used lentiviral vector-mediated expression of ribosomal protein L4 and eGFP to label ribosomes in Schwann cells. Two approaches were followed. First, we transduced Schwann cells in vivo in the distal trunk of the sciatic nerve after a nerve crush. Seven days after the crush, 12% of regenerating axons contained fluorescent ribosomes. Second, we transduced Schwann cells in vitro that were subsequently injected into an acellular nerve graft that was inserted into the sciatic nerve. Fluorescent ribosomes were detected in regenerating axons up to 8 weeks after graft insertion. Together, these data indicate that regenerating axons receive ribosomes from Schwann cells and, furthermore, that Schwann cells may support local axonal protein synthesis by transferring protein synthetic machinery and mRNAs to these axons.

Clinical benefits of single vs repeated courses of mesenchymal stem cell therapy in epilepsy patients.

PURPOSE: Epilepsy is defined as "drug-resistant" when existing anti-epileptic drugs (AED) are found to have minimal to no effect on patient's condition. Therefore the search and testing of new treatment strategies is warranted. This study focuses on the effects of autologous mesenchymal stem cells (MSC) in drug-resistant epilepsy patients within a Phase I/II open-label registered clinical trial NCT02497443. MATERIALS/METHODS: A total of 67 patients was included (29 males, 38 females, mean age 33 ± 1.3 yo). The patients received either standard treatment with AEDs, or AEDs supplemented with one or two courses of therapy with autologous bone marrow-derived MSCs expanded in vitro. MSC therapy courses were 6 months apart, and each course consisted of two cell injections: an intravenous infusion of MSCs, followed within 1 week by an intrathecal injection. Primary outcome of the study was safety, secondary outcome was efficacy in terms of seizure frequency reduction and response to treatment. RESULTS: MSC injections proved safe and did not cause any severe side effects. In MSC group (n = 34), 61.7% patients responded to therapy at 6 months timepoint (p < 0.01 vs control, n = 33), and the number rose to 76.5% by 12 months timepoint. Decrease in anxiety and depression scores and paroxysmal epileptiform activity was observed in MSC group based on HADS and EEG, respectively, and MMSE score has also improved. Another observation was that concomitant administration of levetiracetam, but not other AEDs, correlated significantly with the success of MSC therapy. Second course of MSC therapy facilitated further reduction in seizure count and epileptiform EEG activity (p < 0.05 vs single course). CONCLUSIONS: Application of autologous mesenchymal stem cell-based therapy in patients with pharmacoresistant epilepsy demonstrated significant anticonvulsant potential. This effect lasted for at least 1 year, with repeated administration of MSCs conveying additional clinical benefit.

Use of polyamidoamine dendrimers to engineer BDNF-producing human mesenchymal stem cells.

We report the use of polyamidoamine (PAMAM-NH(2)) dendrimers along with other non-viral vehicles for the in vitro transfection of human bone marrow mesenchymal stem cells (hMSCs) and for engineering MSCs to secrete brain-derived neurotrophic factor (BDNF). Different generations of cationic polyamidoamine dendrimers (generations 3-6) were tested on HEK 293T cells. hMSCs were then transfected with PAMAM-NH(2) G4 dendrimers and Lipofectamine 2000, which elicited the expression of GFP reporter in around 6 and 20% of the cells, respectively. Both vehicles were then shown to elicit the expression of BDNF in MSCs from a bicistronic cassette. Non-virally induced neurotrophin expression may be a safe and easy method for adapting autologous stem cells for therapeutic treatment of diseases and neural system injuries.

Treatment of refractory epilepsy patients with autologous mesenchymal stem cells reduces seizure frequency: An open label study.

PURPOSE: Existing anti-epileptic drugs (AED) have limited efficiency in many patients, necessitating the search for alternative approaches such as stem cell therapy. We report the use of autologous patient-derived mesenchymal stem cells (MSC) as a therapeutic agent in symptomatic drug-resistant epilepsy in a Phase I open label clinical trial (registered as NCT02497443). PATIENTS AND METHODS: The patients received either standard treatment with AED (control group), or AED supplemented with single intravenous administration of undifferentiated autologous MSC (target dose of 1×106cells/kg), followed by a single intrathecal injection of neurally induced autologous MSC (target dose of 0.1×106cells/kg). RESULTS: MSC injections were well tolerated and did not cause any severe adverse effects. Seizure frequency was designated as the main outcome and evaluated at 1 year time point. 3 out of 10 patients in MSC therapy group achieved remission (no seizures for one year and more), and 5 additional patients became responders to AEDs, while only 2 out of 12 patients became responders in control group (difference significant, P=0.0135). CONCLUSIONS: MSC possess unique immunomodulatory properties and are a safe and promising candidate for cell therapy in AED resistant epilepsy patients.

Phosphorus-containing nanoparticles: biomedical patents review.

INTRODUCTION: The beginning of the nano-era started with the appearance of artificial nanosized supramolecular systems called nanomaterials and nanoparticles (NPs). AREAS COVERED: In the present review, we have analyzed the patents on phosphorus-based nanomaterials (fullerenes, quantum dots [QDs], graphene, liposomes, dendrimers, gold and silver NPs) in biology and medicine. Their impact in treatment of cancer, viral infections and cardiovascular diseases is discussed. EXPERT OPINION: Liposomes and dendrimers had the highest number of biomedical patents. The third candidates were QDs and the fourth and fifth were gold and silver NPs. Fullerenes and carbon nanotubes have the fewest applications in biology and medicine. Thus, our first conclusion was about the 'unifying nanotoxicology paradigm', that 'soft' NPs are significantly more biocompatible than 'hard' NPs. There has been a trend of these nanomaterials being applied in medicine drug and gene delivery, visualization of cells and pathologic processes, using them as antivirals and antimicrobials, contrast agents, antioxidants and photosensitizers. It was unexpected that no patents were found in which phosphorus NPs were used in 3D printing of bones and other biological tissues. The conclusion reached is that nanomaterials are promising tools in future medical applications.

Fluorescent Phosphorus Dendrimer as a Spectral Nanosensor for Macrophage Polarization and Fate Tracking in Spinal Cord Injury.

Dendrimers and dendriplexes, highly branched synthetic macromolecules, have gained popularity as new tools for a variety of nanomedicine strategies due to their unique structure and properties. We show that fluorescent phosphorus dendrimers are well retained by bone marrow-derived macrophages and exhibit robust spectral shift in its emission in response to polarization conditions. Fluorescence properties of this marker can also assist in identifying macrophage presence and phenotype status at different time points after spinal cord injury. Potential use of a single dendrimer compound as a drug/siRNA carrier and phenotype-specific cell tracer offers new avenues for enhanced cell therapies combined with monitoring of cell fate and function in spinal cord injury.

Sensory recovery after cell therapy in peripheral nerve repair: effects of naïve and skin precursor-derived Schwann cells.

OBJECT: Cell therapy is a promising candidate among biological or technological innovations sought to augment microsurgical techniques in peripheral nerve repair. This report describes long-term functional regenerative effects of cell therapy in the rat injury model with a focus on sensory recovery. METHODS: Schwann cells were derived from isogenic nerve or skin precursor cells and injected into the transected and immediately repaired sciatic nerve distal to the injury site. Sensory recovery was assessed at weeks 4, 7, and 10. Axonal regeneration was assessed at Week 11. RESULTS: By Week 10, thermal sensitivity in cell therapy groups returned to a level indistinguishable from the baseline (p > 0.05). Immunohistochemistry at 11 weeks after injury showed improved regeneration of NF+ and IB4+ axons. CONCLUSIONS: The results of this study show that cell therapy significantly improves thermal sensation and the number of regenerated sensory neurons at 11 weeks after injury. These findings contribute to the view of skin-derived stem cells as a reliable source of Schwann cells with therapeutic potential for functional recovery in damaged peripheral nerve.

How to study dendrimers and dendriplexes III. Biodistribution, pharmacokinetics and toxicity in vivo.

This paper reviews the biodistribution, toxicity and pharmacokinetics of pure dendrimers and their complexes with nucleic acids (dendriplexes) in animals, including mice, rats, rabbits, and guinea pigs. Methods and results will both be discussed. The paradigm about dendrimers' toxicity based on in vitro studies should be revised; almost all dendrimers of low and middle generations are non-toxic in vivo, despite showing some cytotoxic effects in vitro. Only the high generations of unmodified cationic dendrimers in high doses have some toxicity in vivo. Modifications of dendrimers decrease their toxicity, even when this has already been acceptable with regard to unmodified dendrimers. Several undesirable effects following administration of unmodified cationic dendrimers diminish during prolonged dosing because of the development of counteracting mechanisms. Disturbances tend to return to normal levels during the recovery period after dendrimers have ceased to be administered to animals. Neutralization of the surface charge of dendrimers in their dendriplexes leads to less toxicity in vivo. Although dendrimers and dendriplexes accumulate temporarily in liver, pancreas, heart, and kidneys, they do not do permanent damage to them, i.e. the risk of irreversible damage or malfunction of these internal organs is slight. Chemical modifications of dendrimers determine the desired location of multifunctional dendrimer-based conjugates and dendriplexes in a targeted organ. Clearance of dendrimers also strongly depends on their chemical structure. When nucleic acids are complexed with dendrimers (forming so-called dendriplexes), they are more stable, having longer circulation times than free and PEI-complexed ones. Dendrimers are highly efficient in transfection and can be targeted to any organ, e.g. brain, lung and kidneys. The vast majority of dendrimers and dendriplexes are non-immunogenic. To sum up, these promising results from in vivo studies open up the possibility of dendrimers being applied as a new generation of nano-therapeutic agents in medicine, especially in human gene therapy.

Skin derived precursor Schwann cells improve behavioral recovery for acute and delayed nerve repair.

Previous work has shown that infusion of skin-derived precursors pre-differentiated into Schwann cells (SKP-SCs) can remyelinate injured and regenerating axons, and improve indices of axonal regeneration and electrophysiological parameters in rodents. We hypothesized that SKP-SC therapy would improve behavioral outcomes following nerve injury repair and tested this in a pre-clinical trial in 90 rats. A model of sciatic nerve injury and acellular graft repair was used to compare injected SKP-SCs to nerve-derived Schwann cells or media, and each was compared to the gold standard nerve isograft repair. In a second experiment, rats underwent right tibial nerve transection and received either acute or delayed direct nerve repair, with injections of either 1) SKP-SCs distal to the repair site, 2) carrier medium alone, or 3) dead SKP-SCs, and were followed for 4, 8 or 17weeks. For delayed repairs, both transected nerve ends were capped and repaired 11weeks later, along with injections of cells or media as above, and followed for 9 additional weeks (total of 20weeks). Rats were serially tested for skilled locomotion and a slip ratio was calculated for the horizontal ladder-rung and tapered beam tasks. Immediately after nerve injury and with chronic denervation, slip ratios were dramatically elevated. In the GRAFT repair study, the SKP-SC treated rats showed statistically significant improvement in ladder rung as compared to all other groups, and exhibited the greatest similarity to the sham controls on the tapered beam by study termination. In the ACUTE repair arm, the SKP-SC group showed marked improvement in ladder rung slip ratio as early as 5weeks after surgery, which was sustained for the duration of the experiment. Groups that received media and dead SKP-SCs improved with significantly slower progression. In the DELAYED repair arm, the SKP-SC group became significantly better than other groups 7weeks after the repair, while the media and the dead SKP-SCs showed no significant improvement in slip ratios. On histomorphometrical analysis, SKP-SC group showed significantly increased mean axon counts while the percent myelin debris was significantly lower at both 4 and 8weeks, suggesting that a less inhibitory micro-environment may have contributed to accelerated axonal regeneration. For delayed repair, mean axon counts were significantly higher in the SKP-SC group. Compound action potential amplitudes and muscle weights were also improved by cell therapy. In conclusion, SKP-SC therapy improves behavioral recovery after acute, chronic and nerve graft repair beyond the current standard of microsurgical nerve repair.

Poly(amidoamine) dendrimer complexes as a platform for gene delivery.

INTRODUCTION: Gene therapy is one of the most effective ways to treat major infectious diseases, cancer and genetic disorders. It is based on several viral and non-viral systems for nucleic acid delivery. The number of clinical trials based on application of non-viral drug and gene delivery systems is rapidly increasing. AREAS COVERED: This review discusses and summarizes recent advances in poly(amidoamine) dendrimers as effective gene carriers in vitro and in vivo, and their advantages and disadvantages relative to viral vectors and other non-viral systems (liposomes, linear polymers) are considered. EXPERT OPINION: In this regard, dendrimers are non-immunogenic and have the highest efficiency of transfection among other non-viral systems, and none of the drawbacks characteristic for viral systems. The toxicity of dendrimers both in vitro and in vivo is an important question that has been addressed on many occasions. Several non-toxic and efficient multifunctional dendrimer-based conjugates for gene delivery, along with modifications to improve transfection efficiency while decreasing cytotoxicity, are discussed. Twelve paradigms that affected the development of dendrimer-based gene delivery are described. The conclusion is that dendrimers are promising candidates for gene delivery, but this is just the beginning and further studies are required before using them in human gene therapy.

The impact of motor axon misdirection and attrition on behavioral deficit following experimental nerve injuries.

Peripheral nerve transection and neuroma-in-continuity injuries are associated with permanent functional deficits, often despite successful end-organ reinnervation. Axonal misdirection with non-specific reinnervation, frustrated regeneration and axonal attrition are believed to be among the anatomical substrates that underlie the poor functional recovery associated with these devastating injuries. Yet, functional deficits associated with axonal misdirection in experimental neuroma-in-continuity injuries have not yet been studied. We hypothesized that experimental neuroma-in-continuity injuries would result in motor axon misdirection and attrition with proportional persistent functional deficits. The femoral nerve misdirection model was exploited to assess major motor pathway misdirection and axonal attrition over a spectrum of experimental nerve injuries, with neuroma-in-continuity injuries simulated by the combination of compression and traction forces in 42 male rats. Sciatic nerve injuries were employed in an additional 42 rats, to evaluate the contribution of axonal misdirection to locomotor deficits by a ladder rung task up to 12 weeks. Retrograde motor neuron labeling techniques were utilized to determine the degree of axonal misdirection and attrition. Characteristic histological neuroma-in-continuity features were demonstrated in the neuroma-in-continuity groups and poor functional recovery was seen despite successful nerve regeneration and muscle reinnervation. Good positive and negative correlations were observed respectively between axonal misdirection (p<.0001, r(2)=.67), motor neuron counts (attrition) (p<.0001, r(2)=.69) and final functional deficits. We demonstrate prominent motor axon misdirection and attrition in neuroma-in-continuity and transection injuries of mixed motor nerves that contribute to the long-term functional deficits. Although widely accepted in theory, to our knowledge, this is the first experimental evidence to convincingly demonstrate these correlations with data inclusive of the neuroma-in-continuity spectrum. This work emphasizes the need to focus on strategies that promote both robust and accurate nerve regeneration to optimize functional recovery. It also demonstrates that clinically relevant neuroma-in-continuity injuries can now also be subjected to experimental investigation.

Doxycycline-regulated GDNF expression promotes axonal regeneration and functional recovery in transected peripheral nerve.

Increased production of neurotrophic factors (NTFs) is one of the key responses seen following peripheral nerve injury, making them an attractive choice for pro-regenerative gene therapies. However, the downside of over-expression of certain NTFs, including glial cell line-derived neurotrophic factor (GDNF), was earlier found to be the trapping and misdirection of regenerating axons, the so-called 'candy-store' effect. We report a proof-of-principle study on the application of conditional GDNF expression system in injured peripheral nerve. We engineered Schwann cells (SCs) using dendrimers or lentiviral transduction with the vector providing doxycycline-regulated GDNF expression. Injection of GDNF-modified cells into the injured peripheral nerve followed by time-restricted administration of doxycycline demonstrated that GDNF expression in SCs can also be controlled locally in the peripheral nerves of the experimental animals. Cell-based GDNF therapy was shown to increase the extent of axonal regeneration, while controlled deactivation of GDNF effectively prevented trapping of regenerating axons in GDNF-enriched areas, and was associated with improved functional recovery.