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. 2015 Sep;4(9):1011-20.
doi: 10.5966/sctm.2015-0010. Epub 2015 Jul 9.

Improving the Post-Stroke Therapeutic Potency of Mesenchymal Multipotent Stromal Cells by Cocultivation With Cortical Neurons: The Role of Crosstalk Between Cells

Valentina A Babenko  1 , Denis N Silachev  1 , Ljubava D Zorova  1 , Irina B Pevzner  1 , Anastasia A Khutornenko  1 , Egor Y Plotnikov  2 , Gennady T Sukhikh  1 , Dmitry B Zorov  2
Affiliations

Improving the Post-Stroke Therapeutic Potency of Mesenchymal Multipotent Stromal Cells by Cocultivation With Cortical Neurons: The Role of Crosstalk Between Cells

Valentina A Babenko et al. Stem Cells Transl Med. 2015 Sep.

Abstract

The goal of the present study was to maximally alleviate the negative impact of stroke by increasing the therapeutic potency of injected mesenchymal multipotent stromal cells (MMSCs). To pursue this goal, the intercellular communications of MMSCs and neuronal cells were studied in vitro. As a result of cocultivation of MMSCs and rat cortical neurons, we proved the existence of intercellular contacts providing transfer of cellular contents from one cell to another. We present evidence of intercellular exchange with fluorescent probes specifically occupied by cytosol with preferential transfer from neurons toward MMSCs. In contrast, we observed a reversed transfer of mitochondria (from MMSCs to neural cells). Intravenous injection of MMSCs in a postischemic period alleviated the pathological indexes of a stroke, expressed as a lower infarct volume in the brain and partial restoration of neurological status. Also, MMSCs after cocultivation with neurons demonstrated more profound neuroprotective effects than did unprimed MMSCs. The production of the brain-derived neurotrophic factor was slightly increased in MMSCs, and the factor itself was redistributed in these cells after cocultivation. The level of Miro1 responsible for intercellular traffic of mitochondria was increased in MMSCs after cocultivation. We conclude that the exchange by cellular compartments between neural and stem cells improves MMSCs' protective abilities for better rehabilitation after stroke. This could be used as an approach to enhance the therapeutic benefits of stem cell therapy to the damaged brain.

Significance: The idea of priming stem cells before practical use for clinical purposes was applied. Thus, cells were preconditioned by coculturing them with the targeted cells (i.e., neurons for the treatment of brain pathological features) before the transfusion of stem cells to the organism. Such priming improved the capacity of stem cells to treat stroke. Some additional minimal study will be required to develop a detailed protocol for coculturing followed by cell separation.

Keywords: Astrocytes; Intercellular communication; Ischemia; Mitochondria; Neurons; Stem cell therapy; Stroke.

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Figures

Figure 1.
Figure 1.
Phenotyping of bone marrow-derived stem cells (A) and brain cortex-derived neural cells (B, С). (A): Fluorescence-activated cell sorting analysis of typical mesenchymal multipotent stromal cell (MMSC) markers (CD44, CD90, CD105, and CD73) and blood cell markers (CD34, CD45, and CD31). (B): Phase-contrast image of neural culture at 7 days in vitro. (C): Confocal image of neural culture after immunocytochemical staining with antibodies specific to β-III-tubulin (green) and glial fibrillar acidic protein (GFAP) (red). The nuclei are stained with TO-PRO-3 (blue). A large part of the cell culture consists of neurons (β-III-tubulin-positive cells), with astrocytes (GFAP-positive cells) present in smaller quantities. Scale bars = 50 µm (B) and 20 μm (C).
Figure 2.
Figure 2.
The intercellular transfer of cytosolic probe Calcein Green between neural cells and mesenchymal multipotent stromal cells (MMSCs) or astrocytes and MMSCs was explored by monitoring the ratio of stained and unstained cells in coculture by flow cytometry. (A–C): Stained neural culture. (D, E): Stained culture of MMSCs. Two subpopulations of cells were clearly observable after 25 minutes of cocultivation (A, D), with significant coalescence or even convergence of peaks evident after 24 hours of coculture (B, E). (C): A lack of convergence of populations under cocultivating on separate slides for 24 hours was seen. (F, G): MMSCs stained with Calcein were cocultivated with astrocytes. (H–J): Astrocytes stained with Calcein were cocultivated with human MMSCs under conditions providing direct contact (H, I) or separation of cocultivated cells by a semipermeable membrane (J). The cells were analyzed on a flow cytometer after 25 minutes (F, H) and 24 hours (G, I, J) of cocultivation.
Figure 3.
Figure 3.
The transfer of cytosolic components between neural cells and MMSCs at 1.5 hours (A) and 24 hours after cocultivation (B–E). Before cocultivation, MMSCs were loaded with the fluorescent probe Calcein Red-Orange AM, and neurons were stained with Calcein Green AM. Light (C) and fluorescent (D, E) microscopy of contacting neurons (stained by Calcein Green) and MMSCs (stained by Calcein Red-Orange). After 24 hours, the fluorescence of Calcein Green was observed not only in neural cells, but also in MMSCs (D, E). Scale bars = 20 μm. Abbreviations: MMSC, mesenchymal multipotent stromal cell; N, neural cell.
Figure 4.
Figure 4.
Unidirectional transport of cytoplasm from neurons to mesenchymal multipotent stromal cells (MMSCs). (A): Calcein Green transfer from neurons to MMSCs was observed after 24 hours of cocultivation. The bright green fluorescence of Calcein Green can be observed in neurons, with weaker fluorescence in MMSCs positive for human nuclei (red fluorescence). (B): Neurons positive for β-III-tubulin (red fluorescence) did not receive Calcein Green from MMSCs loaded with the dye (green fluorescence). (C): Astrocytes positive for glial fibrillar acidic protein (red fluorescence) did not receive Calcein Green from MMSCs loaded with the dye. (D): Green fluorescent protein (GFP) transfer from neurons to MMSCs. Bright green fluorescence of GFP is observed in neurons, with weaker fluorescence in MMSCs positive for human nuclei (red fluorescence). Scale bars = 20 μm.
Figure 5.
Figure 5.
Transfer of mitochondria from MMSCs to neural cells in vitro and in vivo. MMSCs were transfected with Discosoma species red fluorescent protein fused with the mitochondrial localization signal of cytochrome c oxidase subunit VIII construct (red fluorescence in mitochondria), and neurons were transfected with green fluorescent protein (GFP) fused with the mitochondrial localization signal of cytochrome c oxidase subunit VIII (mitoGFP) construct (green fluorescence). Red mitochondria in neurons (A, arrowhead) and astrocytes (B, arrowheads) according to cell morphology are shown. (C): Miro1 levels, the protein responsible for mitochondria transfer, were increased in MMSCs after coculturing with neurons. (D): The densitometry results represent an average over three bands obtained from the three different MMSC cultures and three cocultures. Band densities for Miro1 were normalized to the density of the total actin bands. (E, F): Slices of the rat brain with noticeable injected mitoGFP-expressing MMSCs. Neuronal cells are discriminated by staining for β-III-tubulin specific for neurons (red fluorescence). Note that to observe the green fluorescent particles in neurons, the detector gain for green fluorescence was maximally increased, such that the fluorescence of MMSCs is in saturation and individual mitochondria in MMSCs cannot be resolved. Nuclei were stained with To-Pro-3 (magenta). Scale bars = 10 μm (A, B), 100 μm (E), and 20 μm (F). Abbreviations: MMSC, mesenchymal multipotent stromal cell; MMSCcocult, middle cerebral artery occlusion plus MMSCs previously cocultivated with neurons.
Figure 6.
Figure 6.
Beneficial effects of MMSC injection after experimental insult. (A): Representative T2-weighted magnetic resonance (MR) images from coronal brain sections obtained at 8 days after reperfusion. Hyperintensive regions refer to ischemic areas. (B): The volume of the ischemic lesion in the brain on day 8 after MCAO as determined from MR images. Intravenous injection with either native MMSCs or MMSCs previously cocultivated with neurons caused a significant decrease in the volume of brain damage. The injection of neurons had no effect on the size of the lesion. (C): Effect of MMSC transplantation on neurological status at different periods after insult. ∗, p < .05 compared with ischemic saline controls; #, p < .05 compared with ischemic MMSC-treated rats. Abbreviations: MCAO, middle cerebral artery occlusion; MMSC, mesenchymal multipotent stromal cell; MMSCcocult, MCAO plus MMSCs previously cocultivated with neurons.
Figure 7.
Figure 7.
Scheme of possible interaction of MMSCs with neural cells in brain according to cell-to-cell interactions in coculture. Red and blue ovals inside of cells indicate mitochondria of MMSCs and neurons, respectively. Abbreviations: BDNF, brain-derived neurotrophic factor; MMSC, mesenchymal multipotent stromal cell.
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