Intranasal delivery of bone marrow derived mesenchymal stem cells, macrophages, and microglia to the brain in mouse models of Alzheimer's and Parkinson's disease

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinson’s disease (PD). The present study examined delivery of bone marrow derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD ((Thy1)-h[A30P] αS) and an APP/PS1 model of Alzheimer’s disease (AD) via intranasal application (INA). INA of microglia in naïve BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1x104) after INA of 1x106 cells, while the total amount of cells detected in peripheral organs did not exceed 3.4x103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] αS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13 month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both, MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular Amyloid beta (macrophages in APP/PS1 model) or α-Synuclein (MSCs in (Thy1)-h[A30P] αS model) immunoreactivity. Here we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs

Intranasal delivery of bone marrow derived mesenchymal stem cells, macrophages, and microglia to the brain in mouse models of Alzheimer's and Parkinson's disease

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinson’s disease (PD). The present study examined delivery of bone marrow derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD ((Thy1)-h[A30P] αS) and an APP/PS1 model of Alzheimer’s disease (AD) via intranasal application (INA). INA of microglia in naïve BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1x104) after INA of 1x106 cells, while the total amount of cells detected in peripheral organs did not exceed 3.4x103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] αS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13 month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both, MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular Amyloid beta (macrophages in APP/PS1 model) or α-Synuclein (MSCs in (Thy1)-h[A30P] αS model) immunoreactivity. Here we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs

Cell age-dependent Glu-uptake and the effects of EPO on the expression of GLAST by astroglial primary cultures (APC) under normoxic and hypoxic culture conditions.

<p>(<b>A</b>) Quantification of GLAST-positive astrocytes (GLAST+/GFAP+cells) under normoxia shows a significant (**p<0.01; ***p<0.001) increase in GLAST+/GFAP+cells upon EPO-treatment in both young and aged APC (D7 and D21); (<b>B</b>) hypoxia enhanced the effect of EPO on GLAST/GFAP+ cells. In both young and aged cells the number of GLAST/GFAP cells was increased (**p<0.01 at d7 and ***p<0.001 at d21). (<b>C</b>) The uptake of 1 mM glutamate by APC (shown in absolute values) was increased on day 14 in comparison with day 7 under normoxia (white bars, **p<0.01) and hypoxia (grey bars, ***p<0.001) and strongly decreased in 21-day old cultures (d21) exposed to hypoxia (grey bars) when compared to those from day 7 in hypoxia (grey bars d21vs. d7, p***<0.001).</p

The significance of EPOR for astroglial cell survival and utilization of glutamate by astroglial cells.

<p>(<b>A</b>) Immunostaining for EPOR (green) and β-tubulin III (red) in DIV14 APC; (<b>B</b>) Immunostaining of EPOR (green) and GFAP (red) of DIV14 APC; (<b>C</b>) Immunostaining for EPOR (green) and /ß-tubulin III (red) in DIV14 APC transfected with EPOR siRNA; (<b>D</b>) Immunostaining of and EPOR (green) and /GFAP (red) of 14-day-old APC transfected with EPOR siRNA. Cell nuclei are stained with DAPI shown in blue. Scale bar 200µm. (<b>E</b>) Caspase 3/7 activity in untreated 14-day-old APC (white bars) and those transfected with siRNA for EPOR upon normoxia (patterned white bars) (<b>F</b>) Caspase 3/7 activity in hypoxic untreated 14-day-old APC (grey bars) and those transfected with siRNA for EPOR (patterned grey bars) Treatment of original APC (EPOR+) with transfection reagent (TR) did not influence significantly the survival of APC under both normoxic (white bars) and hypoxic (grey bars) conditions. (<b>G</b>) Glu uptake in untreated (EPOR+) APC at DIV14 and those transfected with EPOR siRNA (EPOR-) under normoxia (white bars) and hypoxia (grey bars) (<b>H</b>) GS-activity in untreated (EPOR+) APC at DIV14 and those transfected with EPOR siRNA (EPOR-) under normoxia (white bars) and hypoxia (grey bars). Treatment of original APC (EPOR+) with transfection reagent (contr TR) did not influence significantly the GS-activity of APC. * p<0.05, ** p<0.01, *** p<0.001.</p

Cell age- and concentration-dependent effects of EPO on glutamine synthetase activity.

<p>(<b>A</b>) DIV7 APC under normoxia treated with (+1mM Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>B</b>) DIV7 APC under 24h normoxia /white bars) or hypoxia (grey bars) treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>C</b>) DIV14 APC under normoxia treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO; (<b>D</b>) DIV14 APC upon 24h normoxia /white bars) or hypoxia (grey bars) treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>E</b>) DIV21 APC under normoxia treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>F</b>) DIV21 APC under 24h normoxia /white bars) or hypoxia (grey bars) treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively). At all three time points in culture, Glu increased the activity of GS when compared to respective controls culture without Glu (-Glu). Treatment with EPO increased the glutamate-induced activation of GS in concentration-dependent manner when compared to control culture (cf. +Glu vs +Glu+1U E and +Glu+5U E). *, p<0.05, **, p<0.01, ***, p<0.001.</p

Expression of EPOR/GFAP in young and culture aged APC under normoxia and hypoxia.

<p>(<b>A</b>) Combined picture of double immunostaining of EPOR (green) and GFAP (red) in 7-day old APC under normoxia; (<b>B</b>) Corresponding picture of EPOR in green; (<b>C</b>) Double immunostaining of EPOR (green) and GFAP (red) of APC DIV 7 upon hypoxia; (<b>D</b>) Corresponding picture to C of EPOR (green) only; (<b>E</b>) EPOR (green) and GFAP (red) expression in APC on DIV21 under normoxia; (<b>F</b>) EPOR (green) picture corresponding to E; (<b>G</b>) EPOR(green) and GFAP(red) expression in APC at DIV21 upon hypoxia; (<b>H</b>) corresponding to G picture of EPOR (green) only in DIV21 APC under hypoxia. A-H Nuclear staining with DAPI shown in blue, scale bar 200µm.</p

Quantification of GFAP/PCNA-positive cells in APC in young and culture aged APC upon hypoxia/Glu/EPO-exposure.

<p>(<b>A</b>) GFAP/PCNA+ cell counts in APC at DIV7 exposed to Glu under normoxia (N, white bars) and hypoxia (H, grey bars) showed an increase of proliferating astrocytes upon exposure to 5U/ml EPO (5U E) only under hypoxic conditions. Hypoxia decreased the proliferation of APC (cf- N+Glu vs. H+Gu); (<b>B</b>) Quantification of GFAP/PCNA+ cells on DIV21 shows no changes in PCNA+ astrocytes exposed to EPO under normoxia (white bars), while a significant increase (**p<0.01) is detected in EPO-treated APC (H+Glu+5U E) under hypoxia (grey bars), as compared with the hypoxic control (H+Glu). The proliferation of APC in normoxic (N+Glu) and hypoxic control conditions (H+Glu) remained unchanged.</p

Cell age-dependent effects of glutamate, hypoxia on the expression of EPO, glutamine synthetase (GS), EPO release and EPO mRNA expression in rat APC.

<p>(<b>A</b>) Western blot analysis of GS and EPO in cell homogenates prepared from 7- (DIV7) and 21-day-old (DIV21) rat APC under normal and hypoxic conditions with or without exposure to Glu and EPO. (<b>B</b>) The cell-age-dependent release of EPO into the culture medium under normoxia (N, white bars) and 24h or 48h hypoxia (H, grey bars) in 7-,15- and 21-day old APC. (<b>C</b>) The effect of EPO treatment (5U/ml) on the expression of EPO mRNA in early (7-day-old) APC under normoxia (N, white bars) and hypoxia (H, grey bars). (<b>D</b>) The effect of EPO on the expression of EPO mRNA in prolonged (21-day-old) APC under normoxic (N) and hypoxic (H) culture conditions. Data are normalized to normoxic control (N) *, p<0.05, **, p<0.01, ***, p<0.001.</p