Co-transplantation of syngeneic mesenchymal stem cells improves survival of allogeneic glial-restricted precursors in mouse brain

doi:10.1016/j.expneurol.2015.10.008

. 2016 Jan;275 Pt 1(0 1):154-61.

doi: 10.1016/j.expneurol.2015.10.008. Epub 2015 Oct 26.

Co-transplantation of syngeneic mesenchymal stem cells improves survival of allogeneic glial-restricted precursors in mouse brain

Amit K Srivastava¹, Camille A Bulte¹, Irina Shats¹, Piotr Walczak², Jeff W M Bulte³

Affiliations

¹ Russell H. Morgan Department of Radiology and Radiological Science, Division of MR Research, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
² Russell H. Morgan Department of Radiology and Radiological Science, Division of MR Research, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Radiology, Faculty of Medical Sciences, University of Warmia and Mazury, Olsztyn, Poland.
³ Russell H. Morgan Department of Radiology and Radiological Science, Division of MR Research, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Chemical & Biomolecular Engineering, The Johns Hopkins University Whiting School of Engineering, Baltimore, MD 21218, USA; Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: jwmbulte@mri.jhu.edu.

PMID: 26515691
PMCID: PMC4688082
DOI: 10.1016/j.expneurol.2015.10.008

Co-transplantation of syngeneic mesenchymal stem cells improves survival of allogeneic glial-restricted precursors in mouse brain

Amit K Srivastava et al. Exp Neurol. 2016 Jan.

. 2016 Jan;275 Pt 1(0 1):154-61.

doi: 10.1016/j.expneurol.2015.10.008. Epub 2015 Oct 26.

Authors

Amit K Srivastava¹, Camille A Bulte¹, Irina Shats¹, Piotr Walczak², Jeff W M Bulte³

Affiliations

¹ Russell H. Morgan Department of Radiology and Radiological Science, Division of MR Research, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
² Russell H. Morgan Department of Radiology and Radiological Science, Division of MR Research, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Radiology, Faculty of Medical Sciences, University of Warmia and Mazury, Olsztyn, Poland.
³ Russell H. Morgan Department of Radiology and Radiological Science, Division of MR Research, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Chemical & Biomolecular Engineering, The Johns Hopkins University Whiting School of Engineering, Baltimore, MD 21218, USA; Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: jwmbulte@mri.jhu.edu.

PMID: 26515691
PMCID: PMC4688082
DOI: 10.1016/j.expneurol.2015.10.008

Abstract

Loss of functional cells from immunorejection during the early post-transplantation period is an important factor that reduces the efficacy of stem cell-based therapies. Recent studies have shown that transplanted mesenchymal stem cells (MSCs) can exert therapeutic effects by secreting anti-inflammatory and pro-survival trophic factors. We investigated whether co-transplantation of MSCs could improve the survival of other transplanted therapeutic cells. Allogeneic glial-restricted precursors (GRPs) were isolated from the brain of a firefly luciferase transgenic FVB mouse (at E13.5 stage) and intracerebrally transplanted, either alone, or together with syngeneic MSCs in immunocompetent BALB/c mice (n=20) or immunodeficient Rag2(-/-) mice as survival control (n=8). No immunosuppressive drug was given to any animal. Using bioluminescence imaging (BLI) as a non-invasive readout of cell survival, we found that co-transplantation of MSCs significantly improved (p<0.05) engrafted GRP survival. No significant change in signal intensities was observed in immunodeficient Rag2(-/-) mice, with transplanted cells surviving in both the GRP only and the GRP+MSC group. In contrast, on day 21 post-transplantation, we observed a 94.2% decrease in BLI signal intensity in immunocompetent mice transplanted with GRPs alone versus 68.1% in immunocompetent mice co-transplanted with MSCs and GRPs (p<0.05). Immunohistochemical analysis demonstrated a lower number of infiltrating CD45, CD11b(+) and CD8(+) cells, reduced astrogliosis, and a higher number of FoxP3(+) cells at the site of transplantation for the immunocompetent mice receiving MSCs. The present study demonstrates that co-transplantation of MSCs can be used to create a microenvironment that is more conducive to the survival of allogeneic GRPs.

Keywords: Bioluminescence imaging; Cell survival; Co-transplantation; Glial-restricted precursor; Immunomodulation; Mesenchymal stem cell.

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Figures

**Figure 1**
Characterization of bone marrow-derived-MSCs by flow cytometry analysis. Shown are histograms of analyzed markers overlaid with unstained controls. MSCs express the specific surface markers CD90 and CD105, while CD34 and CD45 expression was negative, eliminating hematopoietic and endothelial cell contamination in the cell population.

**Figure 2**
Characterization of brain-derived GRPs (E13.5) by immunofluorescence and correlation of luciferase reporter gene activity with cell number. (A) Expression of A2B5 protein (red) and Olig1 protein (green). Scale bar= 200μm. (B) BLI of GRPs *in vitro* at several cell densities. (C) Linear correlation between luciferase expression (number of cells) and BLI signal (r² =0.97).

**Figure 3**
Co-registration of CT and BL images confirm the correct placement of cells at the site of the targeted injection. Shown are (A) coronal, (B) transaxial, (C) sagittal, and (D) a 3D image of a mouse brain with engrafted cells obtained at day 1 post-transplantation.

**Figure 4**
Improved allogeneic GRP graft survival following co-transplantation of syngeneic MSCs. Immunocompetent BALB/c and immunodeficient Rag^2−/− mice were used as recipients. (A) BL images of luciferase-expressing GRPs, transplanted alone (upper panel), or together with MSCs (lower panel) at days 1 and 21. (B) Percentage loss of BLI signal compared to day 1 post-transplantation (set as 100%). (*=p<0.5, **= p<0.01, ***= p<0.001, n=10 each).

**Figure 5**
Co-transplantation of syngeneic MSCs creates a microenvironment that is more conducive to the survival of allogeneic GRPs, as assessed at 21 days post-transplantation. (A) Anti-luciferase staining (green) and anti-CD45 (red) (scale bar = 100μm), anti-GFAP (red), anti-CD11b (red), anti-CD8 (red) and anti-FoxP3 (red) (scale bar = 50μm). Needle track is indicated by arrows, c.c= corpus callosum. (B) Stereological counting of immune cells. Although a higher number of CD45⁺ cells was observed in mice transplanted with GRPs alone, the difference was not significant (p>0.05). A significant difference in CD11b⁺ cell numbers was observed between mice transplanted with GRPs alone and those transplanted together with syngeneic MSCs (***p<0.001). Stereological counting of FoxP3⁺ and CD8⁺ cells numbers revealed significant differences between animals transplanted with GRPs alone and those transplanted together with syngeneic MSCs (***p<0.001). All images were acquired using the same exposure time, with the data are expressed as mean ± S.E.M.

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References

1. Abboud SL, Bethel CR, Aron DC. Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells. J Clin Invest. 1991;88:470–475. - PMC - PubMed
1. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005;105:1815–1822. - PubMed
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[1] Abboud SL, Bethel CR, Aron DC. Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells. J Clin Invest. 1991;88:470–475. - PMC - PubMed

[2] Abboud SL, Bethel CR, Aron DC. Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells. J Clin Invest. 1991;88:470–475. - PMC - PubMed

[3] Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005;105:1815–1822. - PubMed

[4] Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005;105:1815–1822. - PubMed

[5] Apostolides C, Sanford E, Hong M, Mendez I. Glial cell line-derived neurotrophic factor improves intrastriatal graft survival of stored dopaminergic cells. Neuroscience. 1998;83:363–372. - PubMed

[6] Apostolides C, Sanford E, Hong M, Mendez I. Glial cell line-derived neurotrophic factor improves intrastriatal graft survival of stored dopaminergic cells. Neuroscience. 1998;83:363–372. - PubMed

[7] Baglio SR, Pegtel DM, Baldini N. Mesenchymal stem cell secreted vesicles provide novel opportunities in (stem) cell-free therapy. Frontiers in physiology. 2012;3:359. - PMC - PubMed

[8] Baglio SR, Pegtel DM, Baldini N. Mesenchymal stem cell secreted vesicles provide novel opportunities in (stem) cell-free therapy. Frontiers in physiology. 2012;3:359. - PMC - PubMed

[9] Bantubungi K, Blum D, Cuvelier L, Wislet-Gendebien S, Rogister B, Brouillet E, Schiffmann SN. Stem cell factor and mesenchymal and neural stem cell transplantation in a rat model of Huntington’s disease. Mol Cell Neurosci. 2008;37:454–470. - PubMed

[10] Bantubungi K, Blum D, Cuvelier L, Wislet-Gendebien S, Rogister B, Brouillet E, Schiffmann SN. Stem cell factor and mesenchymal and neural stem cell transplantation in a rat model of Huntington’s disease. Mol Cell Neurosci. 2008;37:454–470. - PubMed

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Co-transplantation of syngeneic mesenchymal stem cells improves survival of allogeneic glial-restricted precursors in mouse brain

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Co-transplantation of syngeneic mesenchymal stem cells improves survival of allogeneic glial-restricted precursors in mouse brain

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