CXCR4+CD45− BMMNC subpopulation is superior to unfractionated BMMNCs for protection after ischemic stroke in mice
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.
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These authors contributed equally to this work.