Neuroprotective properties of marrow-isolated adult multilineage-inducible cells in rat hippocampus following global cerebral ischemia are enhanced when complexed to biomimetic microcarriers

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.

Figure 1

Representative images showing bright field images of näive MIAMI cells (A) and E/F pre-treated MIAMI cells (B), scanning electron microscopy images of FN-BMM (C), bright field (D) and scanning electron microscopy images of E/F pre-treated MIAMI/FN-BMM complexes (E) higher magnification of scanning electron microscopy of E/F pre-treated MIAMI/FN-BMM complexes (F). Images D, E and F show the 3 dimensional structures of the cells/FN-BMM complexes.

Figure 2

A) Representative images of hippocampal slice cultures. Bright-field and propidium iodide fluorescence images taken 24h after the lethal ischemic (40 min of OGD) insult of culture medium, fibroblasts, EGF/bFGF-pretreated MIAMI cells, naïve MIAMI and EGF/bFGF pre-treated MIAMI/FN-BMMs, respectively. B) Propidium fluorescence values measured in the CA1 pyramidal cells in rat organotypic slices one day after ischemia. BMMs, E/F pre-treated MIAMI cells, E/F pre-treated MIAMI cells/FN-BMMs and näive MIAMI cells were neuroprotective as compared with culture medium and human fibroblasts-injected group (p<0.01). E/F pre-treated MIAMI cells/FN-BMMs were more neuroprotective as compared to BMMs and E/F pre-treated MIAMI (p<0.05). % Cell death as defined in the methods, reflects the ratio of propidium iodide staining 24h after lethal ischemia (OGD) and propidium iodide staining 24 h after 100 μm/L NMDA treatment (total cell death).

Figure 3

Representative images of survival and neuron-like differentiation studies showing organotypic slices injected with E/F pre-treated MIAMI cells/FN-BMMs (A, B and C) or with näive MIAMI cells (D, E and F) 4 days after OGD (10×). MIAMI cells are stained with anti-human mitochondria (green) and βIII tubulin (red). C) 20× confocal images showing the colocalization of human mitochondria positive cells with βIII tubulin for E/F pre-treated MIAMI cells/FN-BMMs injected group. F) 20× confocal images showing the colocalization of human mitochondria positive cells with βIII tubulin for naive MIAMI injected group. Stem cell survival rate is clearly increased by delivering the cells complexed with FN-BMMs. A large fraction (40-60%) of the transplanted cells are positive for βIII tubulin (yellow cells). The complexes between E/F pre-treated MIAMI cells and FN-BMMs 4 days after injection are clearly visible in A and B. Cells remained adhered to particles through the implantation process. The structural support provided for the FN-BMMS to the cells is shown in A and B.

Figure 4

(A-F) Representative hematoxyline and eosin staining of the CA1 hippocampus on day 4 after ACA showing the predetermined CA1 areas where neuronal counting was performed as a measure of neuroprotection. The number of injured neurons was significantly reduced in groups MIAMI (n=3) (D) E/F pre-treated MIAMI (n=3) (E) and E/F pre-treated MIAMI/FN-BMMs (n=3) (F) compared with groups HBSS (n=4) (B) or FN-BMMs (n=4) (C) (p<0.001). (G) Treatment with naïve, E/F-treated MIAMI cells or with E/F-treated MIAMI/FN-BMM complexes significantly increased the number of normal neurons in the CA1 region compared to HBSS or FN-BMMs (p<0.001). E/F-MIAMI/FN-BMM complexes were significantly more neuroprotective than E/F-MIAMI cells injected alone (p<0.001). Results from one-way analysis of variance followed by Tukey's post hoc test. Scale bar 30 μm.

Figure 5

1) Anatomic references. The microinjection site as marked by the red dot for stem cell transplantation. The red square shows the area were images of h-mitochondria and β-III tubulin double immunofluorescent (DIF) were taken. 2) Representative fluorescence images of h-mitochondria and β-III tubulin DIF. Images are of brain sections from animals injected with HBSS (A-C), or E/F-treated MIAMI cells alone (D-F) or in complexes with FN-BMMs (G-I). Human cells were stained with anti-hu-Mito antibodies followed FITC-labeled secondary antibodies (green cells in left column). Neuronal cells in the hippocampus (red) were detected by staining with anti-β-III-tubulin antibodies (B, E and H). The brain of animals injected with HBSS show the absence of human cells, those injected with naïve (not shown) and E/F-treated MIAMI cells alone or in complexes with FN-BMMs show that a fraction of them developed features of neurons, characterized by the expression of β-III-tubulin (yellow cells in F and I) while others did not (green cells in F and I).