Elsevier

Brain, Behavior, and Immunity

Volume 45, March 2015, Pages 98-108
Brain, Behavior, and Immunity

CXCR4+CD45 BMMNC subpopulation is superior to unfractionated BMMNCs for protection after ischemic stroke in mice

https://doi.org/10.1016/j.bbi.2014.12.015 Get rights and content

Highlights

  • BMMNCs reduced infarct volume and neurobehavioral deficits in tMCAO mice.

  • CXCR4+CD45 BMMNCs were more effective than unfractionated BMMNCs.

  • CXCR4+CD45 BMMNCs decreased TNF-α and increased VEGF in the infarcted brain.

  • The CXCR4+ BMMNCs exhibited robust migration into the lesion area.

  • CXCR4+CD45 BMMNCs exhibited more plasticity than did unfractionated BMMNCs.

Abstract

Cell-based therapy is considered to be a promising therapeutic strategy for stroke treatment. Although unfractionated bone marrow mononuclear cells (BMMNCs) have been tried in both preclinical and clinical trials, the effective subpopulations need to be identified. In this study, we used fluorescence-activated cell sorting to harvest the CXCR4+CD45+ and CXCR4+CD45 BMMNC subpopulations from transgenic mice that express enhanced green fluorescent protein. We then allogeneically grafted unfractionated BMMNCs or a subpopulation into mice subjected to transient middle cerebral artery occlusion (tMCAO) and compared the effects on stroke outcomes. We found that CXCR4+CD45 BMMNCs, but not CXCR4+CD45+ BMMNCs, more effectively reduced infarction volume and neurologic deficits than did unfractionated BMMNCs. Brain tissue from the ischemic hemisphere of mice treated with CXCR4+CD45 BMMNCs had higher levels of vascular endothelial growth factor and lower levels of TNF-α than did tissue from mice treated with unfractionated BMMNCs. In contrast, CXCR4+CD45+ BMMNCs showed an increase in TNF-α. Additionally, CXCR4+CD45+ and CXCR4+CD45 populations exhibited more robust migration into the lesion areas and were better able to express cell-specific markers of different linages than were the unfractionated BMMNCs. Endothelial and astrocyte cell markers did not colocalize with eGFP+ cells in the brains of tMCAO mice that received CXCR4+CD45+ BMMNCs. In vitro, the CXCR4+CD45 BMMNCs expressed significantly more Oct-4 and Nanog mRNA than did the unfractionated BMMNCs. However, we did not detect gene expression of these two pluripotent markers in CXCR4+CD45+ BMMNCs. Taken together, our study shows for the first time that the CXCR4+CD45 BMMNC subpopulation is superior to unfractionated BMMNCs in ameliorating cerebral damage in a mouse model of tMCAO and could represent a new therapeutic approach for stroke treatment.

Introduction

Cell transplantation-based regenerative therapy provides us with a promising approach for stroke treatment (Bliss et al., 2010, Burns and Steinberg, 2011, Liu et al., 2014a, Misra et al., 2012). Compared with other cell sources, bone marrow mononuclear cells (BMMNCs) have attracted the interest of many researchers because their use avoids ethical concerns, and they are easy to obtain and purify. They can be harvested allogeneically or autologously from bone marrow within hours, no cell culture procedures are needed, and they can be administered immediately into the recipient through various routes. Over the past decade, evidence from preclinical studies has shown that grafting BMMNCs after cerebral ischemia provides substantial therapeutic effects (Boltze et al., 2011, Fujita et al., 2010, Hess and Hill, 2011, Mendez-Otero et al., 2007, Prasad et al., 2012). Despite progress in this field, the detailed mechanism through which BMMNCs exert their protective effects in cerebral ischemia remains elusive.

BMMNCs harbor a heterogeneous population that contains mature and immature cells in the myeloid and lymphoid lineages, such as mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), and endothelial progenitor cells (EPCs) (Arnous et al., 2012, Civin and Gore, 1993, Crosby et al., 2000, Dominici et al., 2006, Savitz, 2013). In reality, BMMNCs harvested from bone marrow by density centrifugation contain very few stem cells (∼2% to 4% HSCs/EPCs and ∼0.01% MSCs) (Malliaras and Marban, 2011). Bone marrow-derived stromal cells, or MSCs, are currently a promising cell source in stroke therapy. MSCs are capable of self-renewal and can differentiate into various cell linages, including cartilage, bone, adipose, hepatocytes, and neurons (Duenas et al., 2014, Pittenger et al., 1999, Prockop, 1997). It has been reported that human MSCs can migrate into the rat brain and acquire a neuronal phenotype in vivo (Azizi et al., 1998). More importantly, MSCs function as a “cytokine and trophic factors factory” that supports other cell types (Caplan and Dennis, 2006). Despite the advantages of MSCs, obtaining sufficient quantities requires cell culture. Therefore, autologous MSCs cannot be obtained in the acute stage after stroke, limiting their application.

Most investigators who have studied the use of cell transplantation for cerebral ischemia have used mixed BMMNCs. However, the migration and beneficial effects of BMMNCs require the cell surface expression of CXCR4. Many studies have documented that BMMNCs expressing this marker undergo rapid mobilization during cerebral ischemia in response to the chemokine gradient formed by stromal cell-derived factor-1 (SDF-1), which is secreted in the ischemic penumbra, especially by astrocytes and endothelial cells (Hill et al., 2004, Wang et al., 2012). Compared with CXCR4 BMMNCs, CXCR4+ BMMNCs exhibit greater migratory capacity and are more effective at improving neovascularization, releasing trophic factors, and facilitating tissue repair after acute ischemia (Seeger et al., 2009). In addition, the tissue-committed stem cell (TCSC), a population of non-adherent CXCR4+ cells, express mRNA for various markers of progenitor cells and can circulate into peripheral tissues, where they contribute to regeneration after tissue damage (Kucia et al., 2005, Kucia et al., 2007, Ratajczak et al., 2004, Ratajczak et al., 2007). It has been reported that hypoxia upregulates the expression of CXCR4 in ischemic regions (Tang et al., 2009). In addition, CXCR4 knockout donor cells have significantly less survival potential than do wild-type donor cells in the recipient brain (Shichinohe et al., 2007). These findings suggest that the optimum cells for stroke therapy should be CXCR4+.

The vast majority of BMMNC populations contain committed HSCs, which maintain all blood lineages, including erythrocytes, platelets, monocytes, granulocytes, and lymphocytes (Civin and Gore, 1993). HSCs have been shown to mobilize from bone marrow to peripheral blood circulation during stroke, and the concentration of HSCs in blood correlates with neurofunctional improvements in patients after stroke (Taguchi et al., 2009). It has been reported that allogeneic grafting of HSCs reduced post-ischemic inflammation and improved outcome in a mouse stroke model (Schwarting et al., 2008). Furthermore, HSCs were shown to transdifferentiate across tissue-lineage boundaries into various terminal cell types, including non-HSC (Jang et al., 2004, Krause et al., 2001, Orlic et al., 2003), microglia, and macroglia cells (Eglitis and Mezey, 1997). However, the transdifferentiation of HSCs has been debated vigorously (Fukuda and Fujita, 2005, Murry et al., 2004, Wagers et al., 2002). Possible explanations, such as cell fusion (Terada et al., 2002, Ying et al., 2002) and epigenetic changes in recipient tissues (Hochedlinger and Jaenisch, 2003, Jaenisch, 2002), are not fully able to explain the mechanisms of HSC transdifferentiation. It has been reported that the CXCR4 receptor is widely expressed on both HSCs and TCSCs. CD45, a cell surface marker uniquely expressed on HSCs (Thomas, 1989), can be used to separate CXCR4+ BMMNCs into a CXCR4+CD45+ subpopulation enriched in HSCs and a CXCR4+CD45 subpopulation highly enriched in non-hematopoietic TCSCs (Kucia et al., 2005). To the best of our knowledge, no report has described the effects of CXCR4+CD45+ and CXCR4+CD45 BMMNCs on outcome of ischemic stroke.

In this study, we examined whether one subpopulation of BMMNCs provides better protection after ischemic stroke than unfractionated BMMNCs. We found that CXCR4+CD45 BMMNCs are superior to both CXCR4+CD45+ BMMNCs and unfractionated BMMNCs for improving stroke outcomes.

Section snippets

Transient middle cerebral artery occlusion (tMCAO) and experimental groups

All studies were carried out in accordance with the guidelines for animal research and approved by the Institutional Animal Care and Use Committee at Zhengzhou University. All efforts were made to minimize animal suffering and reduce the number of animals used. Adult male C57BL/6J mice (stock number, J000664; weight, 25–30 g; 10–12 weeks old; Animal Center of Nanjing University School of Medicine, Nanjing, China) were housed at room temperature with a 12-h light/dark cycle in a pathogen-free

Results

During this study, the mortality was 1.4% (1/72) in the sham groups, 25.0% (6/24) in the vehicle-treated tMCAO group, 21.7% (13/60) in the unfractionated BMMNC-treated tMCAO group, 23.3% (14/60) in the CXCR4+CD45+ BMMNC-treated group, and 20.0% (12/60) in the CXCR4+CD45 BMMNC-treated group. In addition, three mice died from the MCAO procedure, and two mice died from anesthesia.

Discussion

In this study, we obtained highly purified CXCR4+CD45 and CXCR4+CD45+ BMMNC subpopulations by FACS isolation and compared their effect to that of unfractionated BMMNCs in mice subjected to tMCAO. We found that the CXCR4+CD45 subpopulation is superior to unfractionated BMMNCs in ameliorating cerebral damage and neurologic deficits. Therefore, CXCR4+CD45 BMMNCs may become a promising cell source in stroke treatment. Consistent with our previous publications (Jiang et al., 2013, Wang et al.,

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by Grants from NSFC (81271284), AHA 13GRNT15730001, and NIH (K01AG031926, R01AT007317, R01NS078026). We thank Dr. Lan Huang in the Department of Biological Therapy of the First affiliated Hospital of Zhengzhou University for her kind help with FACS protocol and Claire Levine for assistance with this manuscript.

References (70)

  • X. Liu et al.

    Cell based therapies for ischemic stroke: from basic science to bedside

    Prog. Neurobiol.

    (2014)
  • J.E. Nor et al.

    Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression

    Am. J. Pathol.

    (1999)
  • H. Shichinohe et al.

    Role of SDF-1/CXCR4 system in survival and migration of bone marrow stromal cells after transplantation into mice cerebral infarct

    Brain Res.

    (2007)
  • J. Wang et al.

    Bone marrow mononuclear cell transplantation promotes therapeutic angiogenesis via upregulation of the VEGF-VEGFR2 signaling pathway in a rat model of vascular dementia

    Behav. Brain Res.

    (2014)
  • J. Wang et al.

    Bone marrow mononuclear cells exert long-term neuroprotection in a rat model of ischemic stroke by promoting arteriogenesis and angiogenesis

    Brain Behav. Immun.

    (2013)
  • S. Arnous et al.

    Bone marrow mononuclear cells and acute myocardial infarction

    Stem Cell Res. Ther.

    (2012)
  • S.A. Azizi et al.

    Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats–similarities to astrocyte grafts

    Proc. Natl. Acad. Sci. U.S.A.

    (1998)
  • J. Boltze et al.

    Histopathological investigation of different MCAO modalities and impact of autologous bone marrow mononuclear cell administration in an ovine stroke model

    Transl. Stroke Res.

    (2011)
  • M. Brenneman et al.

    Autologous bone marrow mononuclear cells enhance recovery after acute ischemic stroke in young and middle-aged rats

    J. Cereb. Blood Flow Metab.

    (2010)
  • T.C. Burns et al.

    Stem cells and stroke: opportunities, challenges and strategies

    Expert Opin. Biol. Ther.

    (2011)
  • A.I. Caplan et al.

    Mesenchymal stem cells as trophic mediators

    J. Cell. Biochem.

    (2006)
  • J. Chen et al.

    Endothelial nitric oxide synthase regulates brain-derived neurotrophic factor expression and neurogenesis after stroke in mice

    J. Neurosci.

    (2005)
  • J. Chen et al.

    Intravenous administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary zone after stroke in rats

    Circ. Res.

    (2003)
  • C.I. Civin et al.

    Antigenic analysis of hematopoiesis: a review

    J. Hematother.

    (1993)
  • J.R. Crosby et al.

    Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation

    Circ. Res.

    (2000)
  • X. Cui et al.

    The neurorestorative benefit of GW3965 treatment of stroke in mice

    Stroke

    (2013)
  • F. Duenas et al.

    Hepatogenic and neurogenic differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses

    BMC Vet. Res.

    (2014)
  • M.A. Eglitis et al.

    Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice

    Proc. Natl. Acad. Sci. U.S.A.

    (1997)
  • J.R. Faulkner et al.

    Reactive astrocytes protect tissue and preserve function after spinal cord injury

    J. Neurosci.

    (2004)
  • Y. Fujita et al.

    Early protective effect of bone marrow mononuclear cells against ischemic white matter damage through augmentation of cerebral blood flow

    Stroke

    (2010)
  • D.C. Hess et al.

    Cell therapy for ischaemic stroke

    Cell Prolif.

    (2011)
  • W.D. Hill et al.

    SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury

    J. Neuropathol. Exp. Neurol.

    (2004)
  • K. Hochedlinger et al.

    Nuclear transplantation, embryonic stem cells, and the potential for cell therapy

    N. Engl. J. Med.

    (2003)
  • R. Jaenisch

    Nuclear cloning, embryonic stem cells, and transplantation therapy

    Harvey Lect.

    (2002)
  • Y.Y. Jang et al.

    Hematopoietic stem cells convert into liver cells within days without fusion

    Nat. Cell Biol.

    (2004)
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