Survival, differentiation, and neuroprotective mechanisms of human stem cells complexed with neurotrophin-3-releasing pharmacologically active microcarriers in an ex vivo model of parkinson’s disease

© AlphaMed Press 2015. Stem cell-based regenerative therapies hold great potential for the treatment of degenerative disorders such as Parkinson’s disease (PD). We recently reported the repair and functional recovery after treatment with human marrow-isolated adult multilineage inducible (MIAMI) cells adhered to neurotrophin-3 (NT3) releasing pharmacologically active microcarriers (PAMs) in hemiparkinsonian rats. In order to comprehend this effect, the goal of the present work was to elucidate the survival, differentiation, and neuroprotective mechanisms of MIAMI cells and human neural stem cells (NSCs), both adhering to NT3-releasing PAMs in an ex vivo organotypic model of nigrostriatal degeneration made from brain sagittal slices. It was shown that PAMs led to a marked increase in MIAMI cell survival and neuronal differentiation when releasing NT3. A significant neuroprotective effect of MIAMI cells adhering to PAMs was also demonstrated. NSCs barely had a neuroprotective effect and differentiated mostly into dopaminergic neuronal cells when adhering to PAM-NT3. Moreover, those cells were able to release dopamine in a sufficient amount to induce a return to baseline levels. Reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay analyses identified vascular endothelial growth factor (VEGF) and stanniocalcin-1 as potential mediators of the neuroprotective effect of MIAMI cells and NSCs, respectively. It was also shown that VEGF locally stimulated tissue vascularization, which might improve graft survival, without excluding a direct neuroprotective effect of VEGF on dopaminergic neurons. These results indicate a prospective interest of human NSC/PAM and MIAMI cell/PAM complexes in tissue engineering for PD.Spanish Ministry of Economy and Competitiveness (Grant SAF2010-17167), a grant from the Comunidad de Madrid (Grant S2011-BMD-2336), and Instituto de Salud Carlos III (Redes Tematicas de Investigacion Cooperativa en Salud RD12/0019/0013) to A.M.S.Peer Reviewe

The association of extracellular vesicles and microcarriers as an innovative approach in regenerative medicine.

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A Combinatorial Cell and Drug Delivery Strategy for Huntington's Disease Using Pharmacologically Active Microcarriers and RNAi Neuronally-Committed Mesenchymal Stromal Cells

International audienceFor 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

Adipose-derived stem cell adhesion on laminin-coated microcarriers improves commitment toward the cardiomyogenic lineage

International audienceFor 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.</p

A novel ex vivo Huntington’s disease model for studying GABAergic neurons and cell grafts by laser microdissection

International audienceOrganotypic brain slice cultures have been recently used to study neurodegenerative disorders such as Parkinson's disease and Huntington's disease (HD). They preserve brain three-dimensional architecture, synaptic connectivity and brain cells microenvironment. Here, we developed an innovative model of Huntington's disease from coronal rat brain slices, that include all the areas involved in the pathology. HD-like neurodegeneration was obtained in only one week, in a single step, during organotypic slice preparation, without the use of neurotoxins. HD-like histopathology was analysed and after one week, a reduction of 40% of medium spiny neurons was observed. To analyse new therapeutic approaches in this innovative HD model, we developed a novel protocol of laser microdissection to isolate and analyse by RT-qPCR, grafted cells as well as surrounding tissue of fresh organotypic slices. We determined that laser microdissection could be performed on a 400μm organoty-pic slice after alcohol dehydration protocol, allowing the analysis of mRNA expression in the rat tissue as well as in grafted cells. In conclusion, we developed a new approach for modeling Huntington's disease ex vivo, and provided a useful innovative method for screening new potential therapies for neurodegenerative diseases especially when associated with laser microdissection

Pharmacology active microcarriers delivering HGF associated with extracellular vesicles for myocardial repair

International audienceDespite the curative approaches developed against myocardial infarction, cardiac cell death causes dysfunctional heart contractions that depend on the extent of the ischemic area and the reperfusion period. Cardiac regeneration may allow neovascularization and limit the ventricular remodeling caused by the scar tissue. We have previously found that large extracellular vesicles, carrying Sonic Hedgehog (lEVs), displayed proangiogenic and antioxidant properties, and decreased myocardial infarction size when administrated by intravenous injection. We propose to associate lEVs with pharmacology active microcarriers (PAMs) to obtain a combined cardioprotective and regenerative action when administrated by intracardiac injection. PAMs made of poly-D,Llactic-coglycolic acid-poloxamer 188-poly-D,L-lactic-coglycolic acid and covered by fibronectin/poly-D-lysine provided a biodegradable and biocompatible 3D biomimetic support for the lEVs. When compared with lEVs alone, lEVs-PAMs constructs possessed an enhanced in vitro pro-angiogenic ability. PAMs were designed to continuously release encapsulated hepatocyte growth factor (PAMs HGF) and thus, locally increase the activity of the lEVs by the combined anti-fibrotic properties and regenerative properties. Intracardiac administration of either lEVs alone or lEVs-PAMs HGF improved cardiac function in a similar manner, in a rat model of ischemiareperfusion. Moreover, lEVs alone or the IEVs-PAMs HGF induced arteriogenesis, but only the latter reduced tissue fibrosis. Taken together, these results highlight a promising approach for lEVs-PAMs HGF in regenerative medicine for myocardial infarction

Early events of the inflammatory reaction induced in rat brain by lipopolysaccharide intracerebral injection: relative contribution of peripheral monocytes and activated microglia

International audienceWe have previously demonstrated that lipopolysaccharide (LPS) intracerebral injection induced only minimal inflammatory reaction in rat brain, apart from an increased number of 'brain macrophages' observed 24 h after LPS administration [Montero-Menei et al., Brain Res., 653 (1994) 101-111]. However, the nature of these 'brain macrophages' in the inflammatory response is still unclear. The present study focused on the early time-points (from 5 h to 24 h) after LPS injection or stab-lesion, and was aimed at the identification of the peripheral (monocytes) or parenchymal (microglia) origin of these 'brain macrophages'. OX42-and EDl-labeling did not clearly discriminate between monocytes/macrophages and reactive microglia, both cell types being immunoreactive. In other experiments, rats were made aplasic by irradiation prior to lesioning. These experiments clearly demonstrated that LPS induces an intense monocyte recruitment and, to a lesser extent, microglial activation since about 80% of the cells present 24 h after LPS injection consisted of recruited monocytes not observed in aplasic rats. Interestingly, our data show that LPS exerts a sequential dual action by first inhibiting the monocyte recruitment observed 5 h after stab lesion and then enhancing it at 15 h and 24 h after injection. A possible involvement of cytokines, chemokines and adhesion molecules in the mechanisms occurring in the early events of brain inflammatory reaction is discussed

In vitro and in vivo interactions between glioma and marrow isolated adult multilineage inducible (MIAMI) cells.

International audienceThe prognosis of patients with malignant glioma remains extremely poor despite surgery and improvements in radio- and chemo-therapies. We recently showed that marrow-isolated adult mutilineage inducible (MIAMI) cells, a subpopulation of human mesenchymal stromal cells (MSCs), can serve as cellular carriers of drug-loaded nanoparticles to brain tumors. However, the safety of MIAMI cells as cellular treatment vectors in glioma therapy must be evaluated, in particular their effect on glioma growth and their fate in a tumor environment. In this study, we showed that MIAMI cells were able to specifically migrate toward the orthotopic U87MG tumor model and did not influence its growth. In this model, MIAMI cells did not give rise to cells resembling endothelial cells, pericytes, cancer-associated fibroblasts (CAFs), or astrocytes. Despite these encouraging results, the effects of MIAMI cells may be glioma-dependent. MIAMI cells did not migrate toward the orthotopic Lab1 GB and they can induce the proliferation of other glioma cell lines in vitro. Furthermore, a fraction of MIAMI cells was found to be in a state of proliferation in the U87MG tumor environment. These findings indicate that the use of MIAMI cells as cellular treatment vectors for malignant tumors must be controlled. These cells may be used as “suicide vectors”: vectors for killing not only tumor cells but themselves

Combined therapy for critical limb ischaemia: Biomimetic PLGA microcarriers potentiates the pro-angiogenic effect of adipose tissue stromal vascular fraction cells

International audienceWe propose a regenerative solution in the treatment of critical limb ischaemia (CLI). Poly‐lactic/glycolic acid microcarriers were prepared and coated with laminin to be sterilized through γ‐irradiation of 25 kGy at low temperature. Stromal vascular fraction (SVF) cells were extracted through enzymatic digestion of adipose tissue. Streptozotocin‐induced diabetic mice underwent arteriotomy and received an administration of SVF cells combined or not with biomimetic microcarriers. Functional evaluation of the ischaemic limb was then reported, and tissue reperfusion was evaluated through fluorescence molecular tomography. Microcarriers were stable and functional after γ‐irradiation until at least 12 months of storage. Mice that received an injection of SVF cells in the ischaemic limb have 22% of supplementary blood supply within this limb 7 days after surgery compared with vehicle, whereas no difference was observed at Day 14. With the combined therapy, the improvement of blood flow is significantly higher compared with vehicle, of about 31% at Day 7 and of about 11% at Day 14. Injection of SVF cells induces a significant 27% decrease of necrosis compared with vehicle. This effect is more important when SVF cells were mixed with biomimetic microcarriers: −37% compared with control. Although SVF cells injection leads to a non‐significant 22% proprioception recovery, the combined therapy induces a significant recovery of about 27% compared with vehicle. We show that the combination of SVF cells from adipose tissue with laminin‐coated poly‐lactic/glycolic acid microcarriers is efficient for critical limb ischaemia therapy in a diabetic mouse model

The therapeutic potential of human multipotent mesenchymal stromal cells combined with pharmacologically active microcarriers transplanted in hemi-parkinsonian rats.: PAMs and MIAMI cells for Parkinson's disease

International audienceMultipotent mesenchymal stromal cells (MSCs) raise great interest for brain cell therapy due to their ease of isolation from bone marrow, their immunomodulatory and tissue repair capacities, their ability to differentiate into neuronal-like cells and to secrete a variety of growth factors and chemokines. In this study, we assessed the effects of a subpopulation of human MSCs, the marrow-isolated adult multilineage inducible (MIAMI) cells, combined with pharmacologically active microcarriers (PAMs) in a rat model of Parkinson's disease (PD). PAMs are biodegradable and non-cytotoxic poly(lactic-co-glycolic acid) microspheres, coated by a biomimetic surface and releasing a therapeutic protein, which acts on the cells conveyed on their surface and on their microenvironment. In this study, PAMs were coated with laminin and designed to release neurotrophin 3 (NT3), which stimulate the neuronal-like differentiation of MIAMI cells and promote neuronal survival. After adhesion of dopaminergic-induced (DI)-MIAMI cells to PAMs in vitro, the complexes were grafted in the partially dopaminergic-deafferented striatum of rats which led to a strong reduction of the amphetamine-induced rotational behavior together with the protection/repair of the nigrostriatal pathway. These effects were correlated with the increased survival of DI-MIAMI cells that secreted a wide range of growth factors and chemokines. Moreover, the observed increased expression of tyrosine hydroxylase by cells transplanted with PAMs may contribute to this functional recovery