Traumaticbraininjury(TBI)survivorsexhibitmotorandcognitivesymptomsfromtheprimaryinjurythatcanbecomeaggravatedovertimebecauseof secondary cell death. In the present in vivo study, we examined the beneficial effects of human adipose-derived stem cells (hADSCs) in a controlled cortical impact model of mild TBI using young (6 months) and aged (20 months) F344 rats. Animals were transplanted intravenously with 4 ϫ 10 6 hADSCs (Tx), conditioned media (CM), or vehicle (unconditioned media) at 3 h after TBI. Significant amelioration of motor and cognitive functions was revealed in young, but not aged, Tx and CM groups. Fluorescent imaging in vivo and ex vivo revealed 1,1Ј dioactadecyl-3-3-3Ј,3Ј-tetramethylindotricarbocyanine iodide-labeled hADSCs in peripheral organs and brain after TBI. Spatiotemporal deposition of hADSCs differed between young and aged rats, most notably reduced migration to the aged spleen. Significant reduction in cortical damage and hippocampal cell loss was observedinbothTxandCMgroupsinyoungrats,whereaslessneuroprotectionwasdetectedintheagedratsandmainlyintheTxgroupbutnottheCM group. CM harvested from hADSCs with silencing of either NEAT1 (nuclear enriched abundant transcript 1) or MALAT1 (metastasis associated lung adenocarcinoma transcript 1), long noncoding RNAs (lncRNAs) known to play a role in gene expression, lost the efficacy in our model. Altogether, hADSCsarepromisingtherapeuticcellsforTBI,andlncRNAsinthesecretomeisanimportantmechanismofcelltherapy.Furthermore,hADSCsshowed reduced efficacy in aged rats, which may in part result from decreased homing of the cells to the spleen.

Oligodendrocytes (OLs), the predominant cell type found in cerebral white matter, are essential for structural integrity and proper neural signaling. Very little is known concerning stroke-induced OL dysfunction. Our laboratory has shown that infusion of human umbilical cord blood (HUCB) cells protects striatal white matter tracts in vivo and directly protects mature primary OL cultures from oxygen glucose deprivation (OGD). Microarray studies of RNA prepared from OL cultures subjected to OGD and treated with HUCB cells showed an increase in the expression of 33 genes associated with OL proliferation, survival, and repair functions, such as myelination. The microarray results were verified using quantitative RT-PCR for the following eight genes: U2AF homology motif kinase 1 (Uhmk1), insulin induce gene 1 (Insig1), metallothionein ( Mt3), tetraspanin 2 (Tspan2), peroxiredoxin 4 (Prdx4), stathmin-like 2 (Stmn2), myelin oligodendrocyte glycoprotein (MOG), and versican (Vcan). Immunohistochemistry showed that MOG, Prdx4, Uhmk1, Insig1 and Mt3 protein expression were upregluated in the ipsilateral white matter tracts of rats infused with HUCB cells 48 hrs after middle cerebral artery occlusion (MCAO). Furthermore, promoter region analysis of these genes revealed common transcription factor binding sites, providing insight into the shared signal transduction pathways activated by HUCB cells to enhance transcription of these genes. These results show expression of genes induced by HUCB cell therapy that could confer oligoprotection from ischemia.

Systemic administration of human umbilical cord blood (HUCB) mononuclear cells (MNC) following middle cerebral artery occlusion (MCAO) in the rat reduces infarct size and, more importantly, restores motor function. The HUCB cell preparation is composed of immature T-cells, B-cells, monocytes and stem cells. In this study we examined whether the beneficial effects of HUCB injection were attributable to one of these cell types. Male Sprague Dawley rats underwent permanent MCAO followed 48 hours later by intravenous administration of HUCB MNC preparations depleted of either CD14+ monocytes, CD133+ stem cells, CD2+ T-cells or CD19+ B cells. Motor function was measured prior to MCAO and 30 days post-stroke. When CD14+ monocytes were depleted from the HUCB MNC, activity and motor asymmetry were similar to the MCAO only treated animals. Monocyte depletion prevented HUCB cell treatment from reducing infarct size while monocyte enrichment was sufficient to reduce infarct size. Administration of monocyte-depleted HUCB cells did not suppress Iba1 labeling of microglia in the infarcted area relative to treatment with the whole HUCB preparation. These data demonstrate that the HUCB monocytes provide the majority of the efficacy in reducing infarct volume and promoting functional recovery.

The human umbilical cord blood (HUCB) mononuclear cell (MNC) fraction is a mixed population of cells that induces functional repair in rodent models of stroke when injected intravenously (i.v.). The transplanted cells are found in the infarcted hemisphere and the spleen. The goal of this project was to determine the nature of the interaction between the HUCB MNCs cells and splenic immune cells. Male Sprague Dawley rats underwent permanent middle cerebral artery occlusion (MCAO) and received i.v. injection of either vehicle (MCAO only), HUCB MNCs or MNCs depleted of CD14+ monocytes, CD133+ stem cells or CD19+ B cells 48 hours post-stroke. At 72 hours post-MCAO, the animals were euthanized and the spleens and blood MNCs harvested for flow cytometry and mitogen proliferation assays. All HUCB cell preparations decreased the percentage of T cells in the spleen and monocytes in the blood (p < 0.05). MNCs depleted of CD14+ and CD19+ decreased the percentage of macrophage (p < 0.001), while CD133 depleted MNCs increased the percentage of macrophage in spleen (p < 0.001); MNC did not alter the macrophage population from the level observed after MCAO. Only HUCB MNC significantly decreased Concanavalin A (ConA)-induced T cell stimulation (p < 0.05). These results suggest that the effects of HUCB MNC in the spleen are not due to a single HUCB population, but the interaction of all the subpopulations together.

Human umbilical cord blood (HUCB) cell therapies have shown promising results in reducing brain infarct volume and most importantly in improving neurobehavioral function in rat permanent middle cerebral artery occlusion, a model of stroke. In this study, we examined the gene expression profile in neurons subjected to oxygen-glucose deprivation (OGD) with or without HUCB treatment and identified signaling pathways (Akt/MAPK) important in eliciting HUCB-mediated neuroprotective responses. Gene chip microarray analysis was performed using RNA samples extracted from the neuronal cell cultures from four experimental groups: normoxia, normoxia + HUCB, OGD, and OGD + HUCB. Both quantitative RT-PCR and immunohistochemistry were carried out to verify the microarray results. Using the Genomatix software program, promoter regions of selected genes were compared to reveal common transcription factor-binding sites and, subsequently, signal transduction pathways. Under OGD condition, HUCB cells significantly reduced neuronal loss from 68% to 44% [one-way ANOVA, F(3, 16) = 11, p = 0.0003]. Microarray analysis identified mRNA expression of Prdx5, Vcam1, CCL20, Alcam, and Pax6 as being significantly altered by HUCB cell treatment. Inhibition of the Akt pathway significantly abolished the neuroprotective effect of HUCB cells [one-way ANOVA, F(3, 11) = 8.663, p = 0.0031]. Our observations show that HUCB neuroprotection is dependent on the activation of the Akt signaling pathway that increases transcription of the Prdx5 gene. We concluded that HUCB cell therapy would be a promising treatment for stroke and other forms of brain injury by modifying acute gene expression to promote neural cell protection.