Combined administration of laminin-221 and prostacyclin agonist enhances endogenous cardiac repair in an acute infarct rat heart.

Although endogenous cardiac repair by recruitment of stem cells may serve as a therapeutic approach to healing a damaged heart, how to effectively enhance the migration of stem cells to the damaged heart is unclear. Here, we examined whether the combined administration of prostacyclin agonist (ONO1301), a multiple-cytokine inducer, and stem cell niche laminin-221 (LM221), enhances regeneration through endogenous cardiac repair. We administered ONO1301- and LM221-immersed sheets, LM221-immersed sheets, ONO1301-immersed sheets, and PBS-immersed sheets (control) to an acute infarction rat model. Four weeks later, cardiac function, histology, and cytokine expression were analysed. The combined administration of LM221 and ONO1301 upregulated angiogenic and chemotactic factors in the myocardium after 4 weeks and enhanced the accumulation of ILB4 positive cells, SMA positive cells, and platelet-derived growth factor receptor alpha (PDGFRα) and CD90 double-positive cells, leading to the generation of mature microvascular networks. Interstitial fibrosis reduced and functional recovery was prominent in LM221- and ONO1301-administrated hearts as compared with those in ONO1301-administrated or control hearts. LM221 and ONO1301 combination enhanced recruitment of PDGFRα and CD90 double-positive cells, maturation of vessels, and functional recovery in rat acute myocardial infarction hearts, highlighting a new promising acellular approach for the failed heart.

The impact of initial tumor microenvironment on imaging phenotype.

Models of human cancer, to be useful, must replicate human disease with high fidelity. Our focus in this study is rat xenograft brain tumors as a model of human embedded cerebral tumors. A distinguishing signature of such tumors in humans, that of contrast-enhancement on imaging, is often not present when the human cells grow in rodents, despite the xenografts having nearly identical DNA signatures to the original tumor specimen. Although contrast enhancement was uniformly evident in all the human tumors from which the xenografts' cells were derived, we show that long-term contrast enhancement in the model tumors may be determined conditionally by the tumor microenvironment at the time of cell implantation. We demonstrate this phenomenon in one of two patient-derived orthotopic xenograft (PDOX) models using cancer stem-like cell (CSC)-enriched neurospheres from human tumor resection specimens, transplanted to groups of immune-compromised rats in the presence or absence of a collagen/fibrin scaffolding matrix, Matrigel. The rats were imaged by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and their brains were examined by histopathology. Targeted proteomics of the PDOX tumor specimens grown from CSC implanted with and without Matrigel showed that while the levels of the majority of proteins and post-translational modifications were comparable between contrast-enhancing and non-enhancing tumors, phosphorylation of Fox038 showed a differential expression. The results suggest key proteins determine contrast enhancement and suggest a path toward the development of better animal models of human glioma. Future work is needed to elucidate fully the molecular determinants of contrast-enhancement.

Investigation of drug dissolution and uptake from low-density DPI formulations in an impactor-integrated cell culture model.

Besides deposition, pulmonary bioavailability is determined by dissolution of particles in the scarce epithelial fluid and by cellular API uptake. In the present work, we have developed an experimental in vitro model, which is combining the state-of-the-art next generation impactor (NGI), used for aerodynamic performance assessment of inhalation products, with a culture of human alveolar A549 epithelial cells to study the fate of inhaled drugs following lung deposition. The goal was to investigate five previously developed nano-milled and spray-dried budesonide formulations and to examine the suitability of the in vitro test model. The NGI dissolution cups of stages 3, 4, and 5 were transformed to accommodate cell culture inserts while assuring minimal interference with the air flow. A549 cells were cultivated at the air-liquid interface on Corning® Matrigel® -coated inserts. After deposition of aerodynamically classified powders on the cell cultures, budesonide amount was determined on the cell surface, in the interior of the cell monolayer, and in the basal solution for four to eight hours. Significant differences in the total deposited drug amount and the amount remaining on the cell surface at the end of the experiment were found between different formulations and NGI stages. Roughly 50% of budesonide was taken up by the cells and converted to a large extent to its metabolic conjugate with oleic acid for all formulations and stages. Prolonged time required for complete drug dissolution and cell uptake in case of large deposited powder amounts suggested initial drug saturation of the surfactant layer of the cell surface. Discrimination between formulations with respect to time scale of dissolution and cell uptake was possible with the present test model providing useful insights into the biopharmaceutical performance of developed formulations that may be relevant for predicting local bioavailability. The absolute quantitative result of cell uptake and permeation into the systemic compartment is unreliable, though, because of partly compromised cell membrane integrity due to particle impaction and professed leakiness of A549 monolayer tight junctions, respectively.

Assembly of FN-silk with laminin-521 to integrate hPSCs into a three-dimensional culture for neural differentiation.

Three-dimensional (3D) neural tissue cultures recapitulate the basic concepts during development and disease better than what can be obtained using conventional two-dimensional cultures. Here, we use a recombinant spider silk protein functionalized with a cell binding motif from fibronectin (FN-silk) in combination with a human recombinant laminin 521 (LN-521) to create a fully defined stem cell niche in 3D. A novel method to assemble silk blended with LN-521 together with human pluripotent stem cells (hPSC) is used to create centimeter-sized foams, which upon cultivation develop into 3D cell constructs supported by a microfibrillar network. After initial cell expansion, neural differentiation was induced to form a homogenous layer of continuous neuroectodermal tissue that allows further differentiation into neuronal subtypes. The silk-supported 3D cell constructs could then be detached from the bottom of the well and cultured as floating entities, where cells appeared in distinctive radial organization resembling early neural tube. This shows that the neural progenitors retain their cellular self-organization ability in the FN-silk/LN-521-supported 3D culture. Calcium imaging demonstrated spontaneous activity, which is important for the formation of neuronal networks. Together, the results show that hPSCs integrated into FN-silk/LN-521 foam develop into neural progenitors and that these stay viable during long-term differentiations. FN-silk/LN-521 also supports morphogenesis mimicking the human brain development and can serve as base for engineering of hPSC-derived neural tissue.

Laminin-111 protein therapy enhances muscle regeneration and repair in the GRMD dog model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating X-linked disease affecting ~1 in 5000 males. DMD patients exhibit progressive muscle degeneration and weakness, leading to loss of ambulation and premature death from cardiopulmonary failure. We previously reported that mouse Laminin-111 (msLam-111) protein could reduce muscle pathology and improve muscle function in the mdx mouse model for DMD. In this study, we examined the ability of msLam-111 to prevent muscle disease progression in the golden retriever muscular dystrophy (GRMD) dog model of DMD. The msLam-111 protein was injected into the cranial tibial muscle compartment of GRMD dogs and muscle strength and pathology were assessed. The results showed that msLam-111 treatment increased muscle fiber regeneration and repair with improved muscle strength and reduced muscle fibrosis in the GRMD model. Together, these findings support the idea that Laminin-111 could serve as a novel protein therapy for the treatment of DMD.

Laminin-coated electrodes improve cochlear implant function and post-insertion neuronal survival.

The benefits of Cochlear implant (CI) technology depend among other factors on the proximity of the electrode array to the spiral ganglion neurons. Laminin, a component of the extracellular matrix, regulates Schwann cell proliferation and survival as well as reorganization of actin fibers within their cytoskeleton, which is necessary for myelination of peripheral axons. In this study we explore the effectiveness of laminin-coated electrodes in promoting neuritic outgrowth from auditory neurons towards the electrode array and the ability to reduce acoustic and electric auditory brainstem response (i.e. aABR and eABR) thresholds. In vitro: Schwann cells and neurites are attracted towards laminin-coated surfaces with longer neuritic processes in laminin-coated dishes compared to uncoated dishes. In vivo: Animals implanted with laminin-coated electrodes experience significant decreases in eABR and aABR thresholds at selected frequencies compared to the results from the uncoated electrodes group. At 1 month post implantation there were a greater number of spiral ganglion neurons and neuritic processes projecting into the scala tympani of animals implanted with laminin-coated electrodes compared to animals with uncoated electrodes. These data suggest that Schwann cells are attracted towards laminin-coated electrodes and promote neuritic outgrowth/ guidance and promote the survival of spiral ganglion neurons following electrode insertion trauma.

Angiopoietin-1 Promotes the Integrity of Neovascularization in the Subcutaneous Matrigel of Type 1 Diabetic Rats.

OBJECTIVE: This study aimed to investigate the effects of Ang-1 on neovascularization of diabetic organs by subcutaneous Matrigel angiogenesis model, established in type 1 diabetic rats. METHODS: Ang-1 adenoviral vector was constructed. The rat model was established by STZ and divided into four group. The Matrigel was inserted subcutaneously into the abdominal cavity of rats at 8 weeks, the treatment group was injected with Ang-1 adenovirus vector via tail vein, and the rats were sacrificed at 10 weeks. Neovascularization of Matrigel was observed with transmission electron microscopy. The marker of vascular endothelial cell and pericyte were detected by immunofluorescence. Immunohistochemical detection of the neovascular endothelial junction protein was performed. RT-PCR was used to determine protein expression of neovascular in Matrigel. RESULTS: Vascular cavity-like structure could be seen in subcutaneous Matrigel of diabetic rats, and the cavity was filled with a lot of red blood cells. Transmission electron microscopy showed that neovascular endothelial structure of the Matrigel was incomplete, while the Ang-1 treatment group had more vascular cavity-like structures, intact vascular endothelial structure, and reduced inflammatory cell infiltration in Matrigel. Additionally, the integrity of vascularization improved, and the marker of pericyte and the cell tight junctions protein was upregulated in Ang-1 treatment group. CONCLUSION: Hyperglycemia could induce pathological angiogenesis in subcutaneous Matrigel of diabetic rats, and Ang-1 could upregulate the expression of intercellular junction protein in subcutaneous Matrigel of diabetic rats and promote the integrity of neovascularization in the subcutaneous Matrigel of diabetic rats.

Laminin-511 Supplementation Enhances Stem Cell Localization With Suppression in the Decline of Cardiac Function in Acute Infarct Rats.

BACKGROUND: The extracellular matrix, in particular basement membrane components such as laminins (LMs), is essential for stem cell differentiation and self-renewal. LM511 and LM221 are the main extracellular matrix components of the epicardium, where stem cells were abundant. Here, we examined whether LMs affected the regeneration process by modulating stem cell activities. METHODS: In vitro, adhesive, and proliferative activities of mesenchymal stem cells (MSCs) were evaluated on LM511 and LM221. To examine the effects of LMs in vivo, we established an acute myocardial infarction model by ligation of the proximal part of the left anterior descending artery at the height of the left atrial appendage and then placed atelocollagen sheets with or without LM511 and LM221 over the anterolateral surface of the left ventricular wall. Four or 8 weeks later, cardiac function, histology, and cytokine expressions were analyzed. RESULTS: MSCs showed greater proliferation and adhesive properties on LM511 than on LM221. In vivo, at 4 weeks, isolectin B4-positive cells were significantly higher in the LM511-transplanted group than in the control group. Moreover, some isolectin B4-positive cells expressed both platelet-derived growth factor receptor α and CD90, suggesting that LM511 enhanced MSC recruitment and attachment at the implanted site. After 8 weeks, these cells were more abundant than at 4 weeks. Transplantation with LM511-conjugated sheets increased the expression of cardioprotective and angiogenic factors. CONCLUSIONS: Transplantation with LM511-conjugated sheets enhanced MSC localization to the implantation site and modulated stem cells activities, leading to angiogenesis in acute myocardial infarction rat models.

Extracellular matrix and fibroblast injection produces pterygium-like lesion in rabbits.

BACKGROUND: Translational research to develop pharmaceutical and surgical treatments for pterygium requires a reliable and easy to produce animal model. Extracellular matrix and fibroblast are important components of pterygium. The aim of this study was to analyze the effect of the subconjunctival injection of fibroblast cells (NIH3T3 cell line) and exogenous extracellular matrix in rabbits in producing a pterygium-like lesion. METHODS: Six 3-month-old white New Zealand rabbits were injected with 20,000 NIH3T3 cells and 5 µL of Matrigel in the right conjunctiva, and with only 5 µL of Matrigel in the left conjunctiva. The eyes were photographed under a magnification of 16× using a 12-megapixel digital camera attached to the microscope on day 1, 3 and 7. Conjunctival vascularization was measured by analyzing images to measure red pixel saturation. Area of corneal and conjunctival fibrovascular tissue formation on the site of injection was assessed by analyzing the images on day 3 and 7 using area measurement software. Histopathologic characteristics were determined in the rabbit tissues and compared with a human primary pterygium. RESULTS: The two treatments promoted growth of conjunctival fibrovascular tissue at day 7. The red pixel saturation and area of fibrovascular tissue developed was significantly higher in right eyes (p < 0.05). Tissues from both treatments showed neovascularization in lesser extent to that observed in human pterygium. Acanthosis, stromal inflammation, and edema were found in tissues of both treatments. No elastosis was found in either treatment. CONCLUSIONS: Matrigel alone or in combination with NIH3T3 cells injected into the rabbits' conjunctiva can promote tissue growth with characteristics of human pterygium, including neovascularization, acanthosis, stromal inflammation, and edema. The combination of Matrigel with NIH3T3 cells seems to have an additive effect on the size and redness of the pterygium-like tissue developed.

Laminin-521 Protein Therapy for Glomerular Basement Membrane and Podocyte Abnormalities in a Model of Pierson Syndrome.

Background Laminin α5ß2γ1 (LM-521) is a major component of the GBM. Mutations in LAMB2 that prevent LM-521 synthesis and/or secretion cause Pierson syndrome, a rare congenital nephrotic syndrome with diffuse mesangial sclerosis and ocular and neurologic defects. Because the GBM is uniquely accessible to plasma, which permeates endothelial cell fenestrae, we hypothesized that intravenous delivery of LM-521 could replace the missing LM-521 in the GBM of Lamb2 mutant mice and restore glomerular permselectivity.Methods We injected human LM-521 (hLM-521), a macromolecule of approximately 800 kD, into the retro-orbital sinus of Lamb2-/- pups daily. Deposition of hLM-521 into the GBM was investigated by fluorescence microscopy. We assayed the effects of hLM-521 on glomerular permselectivity by urinalysis and the effects on podocytes by desmin immunostaining and ultrastructural analysis of podocyte architecture.Results Injected hLM-521 rapidly and stably accumulated in the GBM of all glomeruli. Super-resolution imaging showed that hLM-521 accumulated in the correct orientation in the GBM, primarily on the endothelial aspect. Treatment with hLM-521 greatly reduced the expression of the podocyte injury marker desmin and attenuated the foot process effacement observed in untreated pups. Moreover, treatment with hLM-521 delayed the onset of proteinuria but did not prevent nephrotic syndrome, perhaps due to its absence from the podocyte aspect of the GBM.Conclusions These studies show that GBM composition and function can be altered in vivovia vascular delivery of even very large proteins, which may advance therapeutic options for patients with abnormal GBM composition, whether genetic or acquired.

New model of proliferative vitreoretinopathy in rabbit for drug delivery and pharmacodynamic studies.

Blinding retinal diseases become more epidemic as the population ages. These diseases, such as diabetic retinopathy and macular edema, are of chronic nature and require protracted drug presence at the disease site. A sustained intravitreal porous silicon delivery system with dexamethasone (pSiO2-COO-DEX) was evaluated in a new rabbit model of proliferative vitreoretinopathy (PVR) in a real treatment design. In contrast to the pretreatment design model, pSiO2-COO-DEX was intravitreally injected into the eyes with active inflammation. Subretinal injection of vascular endothelial growth factor (VEGF) and Matrigel induced a late-onset vitreoretinal inflammation that gradually developed into PVR. This method mimics the human disease better than PVR induced by either intravitreal cell injection or trauma. The pSiO2-COO-DEX intervened eyes had minimal PVR, while balanced saline solution or free dexamethasone intervened eyes had significantly more PVR formation. In addition, adding VEGF to the Matrigel for subretinal injection induced greater inflammation and retinal neovascularization in comparison to only Matrigel injected under the medullary ray. Clinical and pathological examinations, including fundus fluorescein angiography and optical coherence tomography, confirmed these changes. In the current study, neither subretinal injection of Matrigel or subretinal injection of VEGF and Matrigel induced choroidal neovascularization. However, the current PVR model demonstrates a chronic course with moderate severity, which may be useful for drug screening studies.

Co-delivery of a laminin-111 supplemented hyaluronic acid based hydrogel with minced muscle graft in the treatment of volumetric muscle loss injury.

Minced muscle autografting mediates de novo myofiber regeneration and promotes partial recovery of neuromuscular strength after volumetric muscle loss injury (VML). A major limitation of this approach is the availability of sufficient donor tissue for the treatment of relatively large VMLs without inducing donor site morbidity. This study evaluated a laminin-111 supplemented hyaluronic acid based hydrogel (HA+LMN) as a putative myoconductive scaffolding to be co-delivered with minced muscle grafts. In a rat tibialis anterior muscle VML model, delivery of a reduced dose of minced muscle graft (50% of VML defect) within HA+LMN resulted in a 42% improvement of peak tetanic torque production over unrepaired VML affected limbs. However, the improvement in strength was not improved compared to a 50% minced graft-only control group. Moreover, histological analysis revealed that the improvement in in vivo functional capacity mediated by minced grafts in HA+LMN was not accompanied by a particularly robust graft mediated regenerative response as determined through donor cell tracking of the GFP+ grafting material. Characterization of the spatial distribution and density of macrophage and satellite cell populations indicated that the combination therapy damps the heightened macrophage response while re-establishing satellite content 14 days after VML to a level consistent with an endogenously healing ischemia-reperfusion induced muscle injury. Moreover, regional analysis revealed that the combination therapy increased satellite cell density mostly in the remaining musculature, as opposed to the defect area. Based on the results, the following salient conclusions were drawn: 1) functional recovery mediated by the combination therapy is likely due to a superposition of de novo muscle fiber regeneration and augmented repair of muscle fibers within the remaining musculature, and 2) The capacity for VML therapies to augment regeneration and repair within the remaining musculature may have significant clinical impact and warrants further exploration.

A Three-Dimensional Culture System with Matrigel Promotes Purified Spiral Ganglion Neuron Survival and Function In Vitro.

In vitro culture of spiral ganglion neurons (SGNs) is a useful approach to investigate numerous aspects of neuronal behavior and to identify potential therapeutic targets for SGN protection and regeneration. However, the isolation of SGNs and the long-term maintenance of their structure and function in vitro remain challenging. In this study, we isolated SGNs from Bhlhb5-cre and Rosa26-tdTomato mice with fluorescence-activated cell sorting and determined the cell purity. We then encapsulated the pure SGNs in matrigel and cultured the SGNs in vitro. We found that the three-dimensional (3D)-matrigel culture environment significantly suppressed apoptosis and improved SGN survival in vitro, which enabled the long-term culture of SGNs for up to 6 months. The 3D-matrigel system also significantly promoted neurite outgrowth of the SGNs, increased the cells' polarity, promoted the area of growth cones, and significantly increased the synapse density of the SGNs. More importantly, the 3D-matrigel system helped to maintain and promote the electrophysiological properties of the SGNs. In conclusion, the 3D-matrigel culture system promoted the survival of purified SGNs in vitro and maintained their morphological structure and function and thus could be a useful tool for studying the physiology and pathophysiology of purified SGNs in long-term culture.

Extracellular matrix and fibroblast injection produces pterygium-like lesion in rabbits

BACKGROUND: Translational research to develop pharmaceutical and surgical treatments for pterygium requires a reliable and easy to produce animal model. Extracellular matrix and fibroblast are important components of pterygium. The aim of this study was to analyze the effect of the subconjunctival injection of fibroblast cells (NIH3T3 cell line) and exogenous extracellular matrix in rabbits in producing a pterygium-like lesion. METHODS: Six 3-month-old white New Zealand rabbits were injected with 20,000 NIH3T3 cells and 5 µL of Matrigel in the right conjunctiva, and with only 5 µL of Matrigel in the left conjunctiva. The eyes were photographed under a magnification of 16× using a 12-megapixel digital camera attached to the microscope on day 1,3 and 7. Conjunctival vascularization was measured by analyzing images to measure red pixel saturation. Area of corneal and conjunctival fibrovascular tissue formation on the site of injection was assessed by analyzing the images on day 3 and 7 using area measurement software. Histopathologic characteristics were determined in the rabbit tissues and compared with a human primary pterygium. RESULTS: The two treatments promoted growth of conjunctival fibrovascular tissue at day 7. The red pixel saturation and area of fibrovascular tissue developed was significantly higher in right eyes (p < 0.05). Tissues from both treatments showed neovascularization in lesser extent to that observed in human pterygium. Acanthosis, stromal inflammation, and edema were found in tissues of both treatments. No elastosis was found in either treatment. CONCLUSIONS: Matrigel alone or in combination with NIH3T3 cells injected into the rabbits' conjunctiva can promote tissue growth with characteristics of human pterygium, including neovascularization, acanthosis, stromal inflammation, and edema. The combination of Matrigel with NIH3T3 cells seems to have an additive effect on the size and redness of the pterygium-like tissue developed.

Tunable, Injectable Hydrogels Based on Peptide-Cross-Linked, Cyclized Polymer Nanoparticles for Neural Progenitor Cell Delivery.

A PEG-based cyclized vinyl polymer was synthesized via one-step RAFT polymerization and used as a precursor of injectable hydrogels. Dithiol linkers including laminin-derived peptides containing IKVAV and YIGSR sequences and DTT were used for gelation. Fast and adjustable gelation rate was achieved through nucleophile-initiated thiol-Michael reaction under physiological conditions. Low swelling ratio and moderate degradation rate of the formed hydrogels were observed. 3D encapsulation of neural progenitor cells in the synthetic hydrogel showed good cell viability over 8 days. The long-term cell survival and proliferation were promoted by the introduction of laminin-derived peptides. This hydrogel platform based on peptide-cross-linked, cyclized vinyl polymers can be used as a universal hydrogel template for 3D cell encapsulation.

Wound healing effects of collagen-laminin dermal matrix impregnated with resveratrol loaded hyaluronic acid-DPPC microparticles in diabetic rats.

An alternative formulation for the treatment of diabetic foot wounds that heal slowly is a requirement in pharmaceutical field. The aim of this study was to develop a dermal matrix consisting of skin proteins and lipids with an antioxidant that will enhance healing and balance the oxidative stress in the diabetic wound area due to the high levels of glucose. Thus a novel three dimensional collagen-laminin porous dermal matrix was developed by lyophilization. Resveratrol-loaded hyaluronic acid and dipalmitoylphosphatidylcholine microparticles were combined with this dermal matrix. Characterization, in vitro release, microbiological and in vivo studies were performed. Spherical microparticles were obtained with a high RSV encapsulation efficacy. The microparticles were well dispersed in the dermal matrix from the surface to deeper layers. Collagenase degraded dermal matrix, however the addition of RSV loaded microparticles delayed the degradation time. The release of RSV was sustained and reached 70% after 6h. Histological changes and antioxidant parameters in different treatment groups were investigated in full-thickness excision diabetic rat model. Collagen fibers were intense and improved by the presence of formulation without any signs of inflammation. The highest healing score was obtained with the dermal matrix impregnated with RSV-microparticles with an increased antioxidant activity. Collagen-laminin dermal matrix with RSV microparticles was synergistically effective due to presence of skin components in the formulation and controlled release achieved. This combination is a safe and promising option for the treatment of diabetic wounds requiring long recovery.

Reconstitution of a Patterned Neural Tube from Single Mouse Embryonic Stem Cells.

The recapitulation of tissue development and patterning in three-dimensional (3D) culture is an important dimension of stem cell research. Here, we describe a 3D culture protocol in which single mouse ES cells embedded in Matrigel under neural induction conditions clonally form a lumen containing, oval-shaped epithelial structure within 3 days. By Day 7 an apicobasally polarized neuroepithelium with uniformly dorsal cell identity forms. Treatment with retinoic acid at Day 2 results in posteriorization and self-organization of dorsal-ventral neural tube patterning. Neural tube organoid growth is also supported by pure laminin gels as well as poly(ethylene glycol) (PEG)-based artificial extracellular matrix hydrogels, which can be fine-tuned for key microenvironment characteristics. The rapid generation of a simple, patterned tissue in well-defined culture conditions makes the neural tube organoid a tractable model for studying neural stem cell self-organization.

Directed Differentiation of Dopamine-Secreting Cells from Nurr1/GPX1 Expressing Murine Embryonic Stem Cells Cultured on Matrigel-Coated PCL Scaffolds.

Parkinson's disease (PD) is a progressive neurological disorder characterized by a large number of motor and non-motor features and is known as the second most common neurodegenerative disorder after Alzheimer's disease. The hallmark pathology of PD is the damage and death of dopamine-producing neurons in the substantia-nigra of midbrain. Intrastriatal transplants of fetal mesencephalon derived DAergic neurons have provided proof-of-principle for the cell replacement strategy and have demonstrated reinnervation of the denervated striatum. However, ethical, technical, and practical limitations of deploying fetal DAergic neurons as the source for cell therapy in PD have ceased the spread of this procedure into clinical practice. Embryonic stem (ES) cells have emerged as a therapeutic alternative that can proliferate extensively and generate dopamine-producing neurons. To this extent and to surmount the obstacles related to embryonic neural cells, many investigations have focused on using pluripotent stem cells for the derivation of DAergic neurons. In the present study, a mouse embryonic stem (mES) R1 cell line was generated which could stably co-express Nurr1 (an essential transcription factor in DAergic neuron development) and GPX-1 (a neuroprotective enzyme against oxidative stress). The Nurr1/GPX-1-expressing ES cells (Nurr1/GPX-1-ES) were differentiated into DAergic-like cells via a three-dimensional culture environment consisting of Poly-ε-Caprolactone (PCL) nanofibrous scaffolds embedded by Matrigel (Mtg) in the presence of specific signaling molecules. DAergic neuron-specific genes were highly expressed in ES-derived DAergic neurons cultured and differentiated on PCL/Mtg scaffolds. Reverse-phase HPLC confirmed that the Nurr1/GPX-1-ES-cells differentiated on PCL/Mtg electrospun scaffolds could efficiently and exclusively secrete dopamine in response to stimulus. In conclusion, our results demonstrated that PCL/Matrigel nanofibrous scaffolds could efficiently support and promote the generation of functional DAergic-like cells from Nurr1/GPX-1-ES cells. The results of this study may have an impact on future tissue engineering for cell therapy of PD.

Induced Neural Differentiation of MMP-2 Cleaved (RADA)4 Drug Delivery Systems.

(RADA)4 self-assembling peptides (SAPs) are promising for neural nanoscaffolds with on-demand drug delivery capabilities due to their automated synthesis, in-situ assembly, and potential for interaction with and release of biomolecules. Neuroinflammation cued on-demand drug release, due to up-regulated proteases, may well be vital in the treatment of several neurological diseases. In these conditions, releasing neurotrophic growth factors (NTFs) could potentially lead to neuroprotection and neurogenesis. As such, (RADA)4 was made with the high and low activity matrix metalloproteinase 2 (MMP-2) cleaved sequences, GPQG+IASQ (CP1) and GPQG+PAGQ (CP2), the brain-derived NTF secretion stimulating peptide MVG (DP1) and the ciliary NTF analogue DGGL (DP2). PC-12 cell culture was performed to assess bioactive substrate cell adhesion and NTF specific neuronal differentiation. The laminin-derived IKVAV peptide, known for neural cell attachment and interaction, was tethered to (RADA)4-IKVAV and mixed in increasing increments with (RADA)4 for this purpose. With 1 nanomolar MMP-2 treatment, product formation was observed to increase over a three day period, with (RADA)4/(RADA)4-CP1/CP2 mixture, however there was little difference between groups. Smaller CP1/CP2 concentrations displayed comparable (RADA)4 nanoscale morphology to higher concentrations. Acetylcholine esterase and neural differentiation was observed over 3 days with 1 nM MMP-2 treatment according to the following makeup: 8/1/1 (RADA)4/(RADA)4-IKVAV/(RADA)4-CP1/CP2-DP1/DP2. Signalling gradually increased in all groups, and neurite outgrowth was visible after three days.

Combination of agrin and laminin increase acetylcholine receptor clustering and enhance functional neuromuscular junction formation In vitro.

Clustering of acetylcholine receptors (AChR) at the postsynaptic membrane is a crucial step in the development of neuromuscular junctions (NMJ). During development and after denervation, aneural AChR clusters form on the sarcolemma. Recent studies suggest that these receptors are critical for guiding and initiating synaptogenesis. The aim of this study is to investigate the effect of agrin and laminin-1; agents with known AChR clustering activity; on NMJ formation and muscle maturation. Primary myoblasts were differentiated in vitro on collagen, laminin or collagen and laminin-coated surfaces in the presence or absence of agrin and laminin. The pretreated cells were then subject to innervation by PC12 cells. The number of neuromuscular junctions was assessed by immunocytochemical co-localization of AChR clusters and the presynaptic marker synaptophysin. Functional neuromuscular junctions were quantitated by analysis of the level of spontaneous as well as neuromuscular blocker responsive contractile activity and muscle maturation was assessed by the degree of myotube striation. Agrin alone did not prime muscle for innervation while a combination of agrin and laminin pretreatment increased the number of neuromuscular junctions formed and enhanced acetylcholine based neurotransmission and myotube striation. This study has direct clinical relevance for treatment of denervation injuries and creating functional neuromuscular constructs for muscle tissue repair.