Search

Protein-coated polymer-based microparticles are attractive supports for cell delivery, but the interplay between microparticle properties, protein coating, and cell response is poorly understood. The interest in alternative microparticle formulations increases the need for a better understanding of how functional protein coatings form on different microparticles. In this work, microparticle formulations based on biodegradable polymers [poly (lactic-co-glycolic acid) (PLGA) and the triblock copolymer PLGA-poloxamer-PLGA] were prepared via an emulsion-based process. To explore the impact that the use of a surfactant has on the properties of the microparticles, the emulsion was stabilized by using either a surfactant, poly(vinyl alcohol), or an organic solvent, propylene glycol. Four different types of microparticles were prepared through combinations of the two types of polymers and the two types of stabilizers. The coating of microparticles with proteins/polypeptides such as fibronectin and poly-d-lysine has been demonstrated before and is an integral step for their application as microcarriers, e.g., for cell delivery; however, the impact of the microparticles' surface chemical properties on the formation (prevalence and distribution) of the mixed polypeptide coatings and the influence on subsequent cell attachment remain to be elucidated. Using a colocalization analysis approach on ToF-SIMS images of protein-coated microparticles, we show that the use of propyleneglycol over PVA as well as the substitution of PLGA by the triblock copolymer resulted in enhanced protein adsorption. Furthermore, if propyleneglycol is used, the substitution of PLGA with the triblock copolymer leads to increased stem cell adhesion. [ABSTRACT FROM AUTHOR]

Background: Tick-borne encephalitis virus (TBEV) is a member of the Flaviviridae family, Flavivirus genus, which includes several important human pathogens. It is responsible for neurological symptoms that may cause permanent disability or death, and, from a medical point of view, is the major arbovirus in Central/Northern Europe and North-Eastern Asia. TBEV tropism is critical for neuropathogenesis, yet little is known about the molecular mechanisms that govern the susceptibility of human brain cells to the virus. In this study, we sought to establish and characterize a new in vitro model of TBEV infection in the human brain and to decipher cell type-specific innate immunity and its relation to TBEV tropism and neuropathogenesis.Method: Human neuronal/glial cells were differentiated from neural progenitor cells and infected with the TBEV-Hypr strain. Kinetics of infection, cellular tropism, and cellular responses, including innate immune responses, were characterized by measuring viral genome and viral titer, performing immunofluorescence, enumerating the different cellular types, and determining their rate of infection and by performing PCR array and qRT-PCR. The specific response of neurons and astrocytes was analyzed using the same approaches after enrichment of the neuronal/glial cultures for each cellular subtype.Results: We showed that infection of human neuronal/glial cells mimicked three major hallmarks of TBEV infection in the human brain, namely, preferential neuronal tropism, neuronal death, and astrogliosis. We further showed that these cells conserved their capacity to mount an antiviral response against TBEV. TBEV-infected neuronal/glial cells, therefore, represented a highly relevant pathological model. By enriching the cultures for either neurons or astrocytes, we further demonstrated qualitative and quantitative differential innate immune responses in the two cell types that correlated with their particular susceptibility to TBEV.Conclusion: Our results thus reveal that cell type-specific innate immunity is likely to contribute to shaping TBEV tropism for human brain cells. They describe a new in vitro model for in-depth study of TBEV-induced neuropathogenesis and improve our understanding of the mechanisms by which neurotropic viruses target and damage human brain cells. [ABSTRACT FROM AUTHOR]

Regenerative medicine is a promising approach to treat neurodegenerative diseases by replacing degenerating cells like neurons or oligodendrocytes. Targeting human neural stem cells directly in the brain is a big challenge in such a strategy. The neurofilament derived NFL-TBS.40-63 peptide has recently been introduced as a novel tool to target neural stem cells. Previous studies showed that this peptide can be internalized by rat neural stem cells in vitro and in vivo, which coincided with lower proliferation and self-renewal capacity and increase of differentiation. In this study, we analyzed the uptake and potential effects of the NFL-TBS.40-63 peptide on human neural stem cells isolated from human fetuses. We showed that the peptide inhibits proliferation and the ability to produce neurospheres in vitro, which is consistent with an increase in cell adhesion and differentiation. These results confirm that the peptide could be a promising molecule to target and manipulate human neural stem cells and thus could serve as a strategic tool for regenerative medicine. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma (GB) is the most malignant brain tumor in adults. It is characterized by angiogenesis and a high proliferative and invasive capacity. Standard therapy (surgery, radiotherapy and chemotherapy with temozolomide) is of limited efficacy. Innovative anticancer drugs targeting both tumor cells and angiogenesis are urgently required, together with effective systems for their delivery to the brain. We assessed the ability of human mesenchymal stromal cells (MSCs) to uptake the multikinase inhibitor, sorafenib (SFN), and to carry this drug to a brain tumor following intranasal administration. Method: MSCs were primed with SFN and drug content and release were quantified by analytical chemistry techniques. The ability of SFN-primed MSCs to inhibit the survival of the human U87MG GB cell line and endothelial cells was assessed in in vitro assays. These cells were then administered intranasally to nude mice bearing intracerebral U87MG xenografts. Their effect on tumor growth and angiogenesis was evaluated by magnetic resonance imaging and immunofluorescence analyses, and was compared with the intranasal administration of unprimed MSCs or SFN alone. Results: MSCs took up about 9 pg SFN per cell, with no effect on viability, and were able to release 60% of the primed drug. The cytostatic activity of the released SFN was entirely conserved, resulting in a significant inhibition of U87MG and endothelial cell survival in vitro. Two intranasal administrations of SFN-primed MSCs in U87MG-bearing mice resulted in lower levels of tumor angiogenesis than the injection of unprimed MSCs or SFN alone, but had no effect on tumor volume. We also observed an increase in the proportion of small intratumoral vessels in animals treated with unprimed MSCs; this effect being abolished if the MSCs were primed with SFN. Conclusion: We show the potential of MSCs to carry SFN to brain tumors following an intranasal administration. However, the therapeutic effect is modest probably due to the pro-tumorigenic properties of MSCs, which may limit the action of the released SFN. This calls into question the suitability of MSCs for use in GB therapy and renders it necessary to find methods guaranteeing the safety of this cellular vector after drug delivery. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the suitability of the transpedicular approach (TPA) in a sheep model of IVD regenerative strategies METHODS: 24 IVD from four sheep were used. TPA and biopsies of the Nucleus pulposus (NP) were performed in 18 IVD (6 IVD control). Seven discographies were performed to assess the feasibility of injecting contrast agent. MRI, micro-CT scan, and histological analyses were performed and the accuracy of the TPA was evaluated. The effects on the vertebra and endplates were analyzed.Results: 83% of our biopsies or injections were located in the NP. Osseous fragments in IVD were observed in 50%. We observed two cases (11%) of rostral endplate fracture and five cases (27%) of breaching of the cortical pedicle and encroachment into the spinal canal. Two cases of perivertebral venous embolism and two of backflow through the canal of the TPA inside the vertebra were noted. Significant damage occurred to the bone structure of the vertebra and to the rostral endplate on which the IVD had been inserted.Conclusions: TPA induces damage to the endplates, and it may lead to neurological impairment and leakage of injected materials into the systemic circulation. These adverse effects must be fully considered before proceeding with TPA for IVD regenerative strategies. [ABSTRACT FROM AUTHOR]

Rationale: Engraftment and survival of transplanted stem or stromal cells in the microenvironment of host tissues may be improved by combining such cells with scaffolds to delay apoptosis and enhance regenerative properties. Aims: We examined whether poly(lactic-co-glycolic acid) pharmacologically active microcarriers (PAMs) releasing vascular endothelial growth factor (VEGF) enhance survival, differentiation, and angiogenesis of adipose tissue-mesenchymal stromal cells (AT-MSCs).We analysed the efficacy of transplanted AT-MSCs conjugated with PAMs in a murine model of acute myocardial infarction (AMI). Methods and results: We used fibronectin-coated (empty) PAMs or VEGF-releasing PAMs covered with murine AT-MSCs. Twelve-monthold C57 mice underwent coronary artery ligation to induce AMI, and were randomized into five treatment groups: AMI control (saline 20 µL, n = 7), AMI followed by intramyocardial injection with AT-MSCs (2.5 × 105 cells/20 µL, n = 5), or concentrated medium (CM) from AT-MSCs (20 µL, n = 8), or AT-MSCs (2.5 × 105 cells/20 µL) conjugated with empty PAMs (n = 7), or VEGF-releasing PAMs (n = 8). Sham-operated mice (n = 7) were used as controls. VEGF-releasing PAMs increased proliferation and angiogenic potential of AT-MSCs, but did not impact their osteogenic or adipogenic differentiation. AT-MSCs conjugated with VEGF-releasing PAMs inhibited apoptosis, decreased fibrosis, increased arteriogenesis and the number of cardiac-resident Ki-67 positive cells, and improved myocardial fractional shortening compared with AT-MSCs alone when transplanted into the infarcted hearts of C57 mice. With the exception of fractional shortening, all such effects of AT-MSCs conjugated with VEGF-PAMs were paralleled by the injection of CM. Conclusions: AT-MSCs conjugated with VEGF-releasing PAMs exert paracrine effects that may have therapeutic applications. [ABSTRACT FROM AUTHOR]

One of the main cause of ineffective cell therapy in repairing the damaged heart is the poor yield of grafted cells. To overcome this drawback, rats with 4-week-old myocardial infarction (MI) were injected in the border zone with human adipose-derived stem cells (ADSCs) conveyed by poly(lactic- co-glycolic acid) microcarriers (PAMs) releasing hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) (GFsPAMs). According to treatments, animals were subdivided into different groups: MI_ADSC, MI_ADSC/PAM, MI_GFsPAM, MI_ADSC/GFsPAM, and untreated MI_V. Two weeks after injection, a 31% increase in ADSC engraftment was observed in MI_ADSC/PAM compared with MI_ADSC ( p < 0.05). A further ADSC retention was obtained in MI_ADSC/GFsPAM with respect to MI_ADSC (106%, p < 0.05) and MI_ADSC/PAM (57%, p < 0.05). A 130% higher density of blood vessels of medium size was present in MI_ADSC/GFsPAM compared with MI_ADSC ( p < 0.01). MI_ADSC/GFsPAM also improved, albeit slightly, left ventricular remodeling and hemodynamics with respect to the other groups. Notably, ADSCs and/or PAMs, with or without HGF/IGF-1, trended to induce arrhythmias in electrically driven, Langendorff-perfused, hearts of all groups. Thus, PAMs releasing HGF/IGF-1 markedly increase ADSC engraftment 2 weeks after injection and stimulate healing in chronically infarcted myocardium, but attention should be paid to potentially negative electrophysiological consequences. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3012-3025, 2015. [ABSTRACT FROM AUTHOR]

For tissue-engineering studies of the infarcted heart it is essential to identify a source of cells that may provide cardiomyocyte progenitors, which is easy to amplify, accessible in adults, and allowing autologous grafts. Preclinical studies have shown that human adipose-derived stem cells (ADSCs) can differentiate into cardiomyocyte-like cells and improve heart function in myocardial infarction. We have developed pharmacologically active microcarriers (PAMs) which are biodegradable and biocompatible polymeric microspheres conveying cells on their biomimetic surface, therefore providing an adequate three-dimensional (3D) microenvironment. Moreover, they can release a growth factor in a prolonged manner. In order to implement ADSCs and PAMs for cardiac tissue engineering we first defined the biomimetic surface by studying the influence of matrix molecules laminin (LM) and fibronectin (FN), in combination with growth factors present in the cardiogenic niche, to further enhance the in vitro cardiac differentiation of ADSCs. We demonstrated that LM increased the expression of cardiac markers (Nkx2.5, GATA4, MEF2C) by ADSCs after 2 weeks in vitro. Interestingly, our results suggest that the 3D support provided by PAMs with a LM biomimetic surface (LM-PAMs) further enhanced the expression of cardiac markers and induced the expression of a more mature contractile protein, cardiac troponin I, compared with the 2D differentiating conditions after only 1 week in culture. The enrichment of the growth-factor cocktail with TGF-β1 potentiated the cardiomyogenic differentiation. These results suggest that PAMs offering a LM biomimetic surface may be efficiently used for applications combining adult stem cells in tissue-engineering strategies of the ischemic heart. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 1828-1839, 2015. [ABSTRACT FROM AUTHOR]

Resistance of transplanted mesenchymal stem cells ( MSCs) in post-ischemic heart is limited by their poor vitality. Vascular-endothelial-growth-factor-A ( VEGF-A) as such or slowly released by fibronectin-coated pharmacologically-active-microcarriers ( FN- PAM- VEGF) could differently affect survival kinases and anti-apoptotic mediator ( e.g. Bcl-2). Therefore VEGF-A or FN- PAM- VEGF could differently enhance cell proliferation, and/or resistance to hypoxia/reoxygenation (H/R) of MSCs. To test these hypotheses MSCs were incubated for 6-days with VEGF-A alone or with FN- PAM- VEGF. In addition, MSCs pre-treated for 24-hrs with VEGF-A or FN- PAM- VEGF were subsequently exposed to H/R (72-hrs 3% O2 and 3-hrs of reoxygenation). Cell-proliferation and post-hypoxic vitality were determined. Kinases were studied at 30-min., 1- and 3-days of treatment. Cell-proliferation increased about twofold ( P < 0.01) 6-days after VEGF-A treatment, but by a lesser extent (55% increase) with FN- PAM- VEGF ( P < 0.05). While MSC pre-treatment with VEGF-A confirmed cell-proliferation, pre-treatment with FN- PAM- VEGF protected MSCs against H/R. In the early phase of treatments, VEGF-A increased phospho-Akt, phospho- ERK-1/2 and phospho- PKCε compared to the untreated cells or FN- PAM- VEGF. Afterword, kinase phosphorylations were higher with VGEF, except for ERK-1/2, which was similarly increased by both treatments at 3 days. Only FN- PAM- VEGF significantly increased Bcl-2 levels. After H/R, lactate dehydrogenase release and cleaved Caspase-3 levels were mainly reduced by FN- PAM- VEGF. While VEGF-A enhances MSC proliferation in normoxia, FN- PAM- VEGF mainly hampers post-hypoxic MSC death. These different effects underscore the necessity of approaches suited to the various conditions. The use of FN- PAM- VEGF could be considered as a novel approach for enhancing MSC survival and regeneration in hostile environment of post-ischemic tissues. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2011) 119, 972-988. Abstract Cell-based therapies for global cerebral ischemia represent promising approaches for neuronal damage prevention and tissue repair promotion. We examined the potential of marrow-isolated adult multilineage-inducible (MIAMI) cells, a homogeneous subpopulation of immature human mesenchymal stromal cell, injected into the hippocampus to prevent neuronal damage induced by global ischemia using rat organotypic hippocampal slices exposed to oxygen-glucose deprivation and rats subjected to asphyxial cardiac arrest. We next examined the value of combining fibronectin-coated biomimetic microcarriers (FN-BMMs) with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) pre-treated MIAMI compared to EGF/bFGF pre-treated MIAMI cells alone, for their in vitro and in vivo neuroprotective capacity. Naïve and EGF/bFGF pre-treated MIAMI cells significantly protected the Cornu Ammonis layer 1 (CA1) against ischemic death in hippocampal slices and increased CA1 survival in rats. MIAMI cells therapeutic value was significantly increased when delivering the cells complexed with FN-BMMs, probably by increasing stem cell survival and paracrine secretion of pro-survival and/or anti-inflammatory molecules as concluded from survival, differentiation and gene expression analysis. Four days after oxygen and glucose deprivation and asphyxial cardiac arrest, few transplanted cells administered alone survived in the brain whereas stem cell survival improved when injected complexed with FN-BMMs. Interestingly, a large fraction of the transplanted cells administered alone or in complexes expressed βIII-tubulin suggesting that partial neuronal transdifferentiation may be a contributing factor to the neuroprotective mechanism of MIAMI cells. [ABSTRACT FROM AUTHOR]

We report here that curative treatment of the multiple sclerosis paradigm, chronic relapsing experimental autoimmune encephalomyelitis (EAE) of the Lewis rat, by 1,25-dihydroxyvitamin D3 (1,25-D3) leads to a rapid clinical improvement accompanied by an inhibition of CD4, MHC class II and type II nitric oxide synthase (NOS II) expression in the posterior areas of the central nervous system (CNS). In contrast, the hormone has no effect on transforming growth factor-β1 transcripts. Computer analysis of the NOS II promoter, expressed by microglia and astrocytes, reveals consensus sequence for vitamin D receptor binding, emphasizing the idea that 1,25-D3 may regulate some aspects of EAE by acting directly on CNS constituent cells. We also demonstrate that vitamin D deprivation leads to minimal effects on the kinetic profile of EAE accompanied by a moderate exacerbation of the clinical symptoms. Interestingly, curative treatment of vitamin D-deprived rats with a non-toxic-1,25-D3 analogue (MC1288) strongly inhibited EAE symptoms, thus promulgating the potential interest of such compounds in the management of multiple sclerosis. [ABSTRACT FROM AUTHOR]

The infusion of BDNF and NT-3 into Schwann cell (SC) grafts promotes regeneration of brainstem neurones into the grafts placed in adult rat spinal cord transected at T8 (Xu et al. 1995b). Here, we compared normal SCs with SCs genetically modified to secrete human BDNF, grafted as trails 5 mm long in the cord distal to a transection site and also deposited in the transection site, for their ability to stimulate supraspinal axonal regeneration beyond the injury. SCs were infected with the replication-deficient retroviral vector pL(hBDNF)RNL encoding the human preproBDNF cDNA. The amounts of BDNF secreted (as detected by ELISA) were 23 and 5 ng/24 h per 10[sup 6] cells for infected and normal SCs, respectively. Biological activity of the secreted BDNF was confirmed by retinal ganglion cell bioassay. The adult rat spinal cord was transected at T8. The use of Hoechst prelabelled SCs demonstrated that trails were maintained for a month. In controls, no SCs were grafted. One month after grafting, axons were present in SC trails. More 5-HT-positive and some DβH-positive fibres were observed in the infected vs. normal SC trails. When Fast Blue was injected 5 mm below the transection site (at the end of the trail), as many as 135 retrogradely labelled neurones could be found in the brainstem, mostly in the reticular and raphe nuclei (normal SCs, up to 22, mostly in vestibular nuclei). Numerous neurones were labelled in the ventral hypothalamus (normal SCs, 0). Also, following Fast Blue injection, a mean of 138 labelled cells was present in dorsal root ganglia (normal SCs, 46) and spinal cord (39 vs. 32) rostral to the transection. No labelled spinal neurones rostral to the transection were seen when SCs were not transplanted. Thus, the transplantation of SCs secreting increased amounts of BDNF improved the regenerative response across a transection site in the thoracic cord. Moreover, the enhanced regeneration observed with infected SCs may be specific as the ... [ABSTRACT FROM AUTHOR]

Clinical studies have demonstrated the regenerative potential of stem cells for cardiac repair over the past decades, but their widespread use is limited by the poor tissue integration and survival obtained. Natural or synthetic hydrogels or microcarriers, used as cell carriers, contribute to resolving, in part, the problems encountered by providing mechanical support for the cells allowing cell retention, survival and tissue integration. Moreover, hydrogels alone also possess mechanical protective properties for the ischemic heart. The combined effect of growth factors with cells and an appropriate scaffold allow a therapeutic effect on myocardial repair. Despite this, the effects obtained with cell therapy remain limited and seem to be equivalent to the effects obtained with extracellular vesicles, key actors in intercellular communication. Extracellular vesicles have cardioprotective effects which, when combined proangiogenic properties with antiapoptotic and anti-inflammatory actions, make it possible to act on all the damages caused by ischemia. The evolution of biomaterial engineering allows us to envisage their association with new major players in cardiac therapy, extracellular vesicles, in order to limit undesirable effects and to envisage a transfer to the clinic. This new therapeutic approach could be associated with the release of growth factors to potentialized the beneficial effect obtained. [ABSTRACT FROM AUTHOR]

For Huntington's disease (HD) cell-based therapy, the transplanted cells are required to be committed to a neuronal cell lineage, survive and maintain this phenotype to ensure their safe transplantation in the brain. We first investigated the role of RE-1 silencing transcription factor (REST) inhibition using siRNA in the GABAergic differentiation of marrow-isolated adult multilineage inducible (MIAMI) cells, a subpopulation of MSCs. We further combined these cells to laminin-coated poly(lactic-co-glycolic acid) PLGA pharmacologically active microcarriers (PAMs) delivering BDNF in a controlled fashion to stimulate the survival and maintain the differentiation of the cells. The PAMs/cells complexes were then transplanted in an ex vivo model of HD. Using Sonic Hedgehog (SHH) and siREST, we obtained GABAergic progenitors/neuronal-like cells, which were able to secrete HGF, SDF1 VEGFa and BDNF, of importance for HD. GABA-like progenitors adhered to PAMs increased their mRNA expression of NGF/VEGFa as well as their secretion of PIGF-1, which can enhance reparative angiogenesis. In our ex vivo model of HD, they were successfully transplanted while attached to PAMs and were able to survive and maintain this GABAergic neuronal phenotype. Together, our results may pave the way for future research that could improve the success of cell-based therapy for HDs. [ABSTRACT FROM AUTHOR]

We tested the hypothesis that cardiac regeneration through local delivery of adipose-derived stem cells (ASCs), activation of resident cardiac stem cells via growth factors (GFs) [hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-1):GFs] or both, are improved by pharmacologically active microcarriers (PAMs) interacting with cells/molecules conveyed on their surface. Rats with one-month old myocardial infarction were treated with ASCs, ASCs+PAMs, GF-releasing PAMs, ASCs+GF-releasing PAMs or vehicle. Two weeks later, hemodynamic function and inducibility of ventricular arrhythmias (VAs) were assessed. Eventually, the hearts were subjected to anatomical and immunohistochemical analyses. A significant ASCs engraftment and the largest improvement in cardiac mechanics occurred in ASC+GF-releasing PAM rats which by contrast were more vulnerable to VAs. Thus, PAMs may improve cell/GF-based cardiac regeneration although caution should be paid on the electrophysiological impact of their physical interaction with the myocardium. [ABSTRACT FROM AUTHOR]