Mannitol-Enhanced Delivery of Stem Cells and Their Growth Factors across the Blood–Brain Barrier

Ischemic brain injury in adults and neonates is a significant clinical problem with limited therapeutic interventions. Currently, clinicians have only tPA available for stroke treatment and hypothermia for cerebral palsy. Owing to the lack of treatment options, there is a need for novel treatments such as stem cell therapy. Various stem cells including cells from embryo, fetus, perinatal, and adult tissues have proved effective in preclinical and small clinical trials. However, a limiting factor in the success of these treatments is the delivery of the cells and their by-products (neurotrophic factors) into the injured brain. We have demonstrated that mannitol, a drug with the potential to transiently open the blood–brain barrier and facilitate the entry of stem cells and trophic factors, as a solution to the delivery problem. The combination of stem cell therapy and mannitol may improve therapeutic outcomes in adult stroke and neonatal cerebral palsy

DPC co-culture protects rat neural cells against OGD.

<p>Co-culture of rat primary neural cells with DPCs afforded neuroprotection against OGD dose-dependently in both Trypan blue and MTT activity assays of cell viability (a–f). The percentages correspond to the ratio of DPCs co-cultured with rat primary neural cells as follows: 1∶1 (100%), 1∶2 (50%), 1∶4 (25%), and 1∶0 (0%). DPCs co-cultured with rat primary neural cells under non-OGD condition did not alter cell viability in both assays (g–l). Asterisk* corresponds to statistically significant difference (p<0.05 vs. 0%). a,g: 0%; b,h: 25%; c,i: 50%; d,j: 100%; e,k: Trypan blue stain quantification; f,l: MTT assay quantification.</p

DPCs display stem cell surface markers.

<p>Subcultured DPCs at day 3 in culture expressed stem cell phenotypes including Oct-4, SSEA, and CXCR4. Left panels: phenotypic marker; Middle panels: DAPI; Right panels: Merged. Scale bar  = 75 µm.</p

DPCs maintain stemness and differentiate into neural cells under appropriate conditions over multiple passages.

<p>Stem cell under proliferation medium and neural cell markers under differentiation medium were detected at passage 10. The DPCs expressed the stem cell markers, Oct-4 (A), SSEA (B), Nanog (D), and CXCR4 (E), and the neural cell markers, NeuN (C), GFAP (F), Nestin (G), MAP2 (H), and O4 (I). 1,4: phenotypic marker; 2,5: DAPI; 3,6: Merged. 1–3: 10X; 4–6: 20X.</p

DPC grafts within the peri-infarct area co-localize with Hsp27 expression.

<p>The majority of grafted DPCs were deposited within the ipsilateral peri-infarct striatal site as detected by the fluorescent dye tracker PKH26. Less than 1% of the grafts survived with no detectable differences in graft persistence whether delivered IV, IC, or IA (p>0.05). Only sparse PKH26-labeled cells were found in the contralateral intact hemisphere across all three delivery routes. Significantly increased Hsp27 expression was juxtaposed to grafted DPCs and quantitative analyses of Hsp27 expression (G) in the peri-infarct striatal site revealed robust Hsp27 expression in transplanted brains compared to controls, more pronounced in IV- and IC-delivered DPCs compared to IA-administered DPCs (*p<0.05 vs. controls). A: IV-delivered cells, ipsilateral; B: IC-delivered cells, ipsilateral; C: IA-delivered cells, ipsilateral; D: IV-delivered cells, contralateral; E: IC-delivered cells, contralateral; F: IA-delivered cells, contralateral; 1: PKH26; 2: Hsp27; 3: DAPI; 4: Merged. 1–4: 10X; 1′–4′:20×.</p

Hsp-27-mediated neuroprotection following DPC transplantation in stroke.

<p>Hsp27 appears to be targeted by DPCs for neuroprotection in stroke. Following intracerebral transplantation, DPCs survived in the stroke brain and elevate Hsp27 expression which may afford rescue of the ischemic penumbra proximal, as well as distal from the transplant site. Although the transplanted cells do not migrate far from the transplant site, the anti-oxidative stress protein Hsp27 is able to amplify the neuroprotection.</p

DPCs dose-dependently facilitate GFAP, NeuN, and MAP2 expression in primary rat cells post-OGD.

<p>Immunocytochemical analyses revealed that co-culture of DPCs modulated dose-dependent expression of GFAP, NeuN, and MAP2 in OGD-exposed primary neural cells in the following order: 100%>50%>25%>0%. Phenotypic markers labeled as A: GFAP; B: NeuN; C: MAP2; Immunofluorescence identified as 1: phenotypic marker; 2: DAPI; 3: Merged; Cell doses given as 1–3: 100%; 4–6: 50%; 7–9: 25%; 10–12: 0%. Quantitative analyses revealed dose-dependent expression of these neural markers (*p<0.05 vs. controls).</p