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Bone marrow stem cells have the ability to populate the entire central nervous system into fully differentiated parenchymal microglia ¹

Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University, Québec, Canada

²Correspondence: Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University, 2705 Laurier Blvd., Québec, Canada G1V 4G2. E-mail: Serge.Rivest{at}crchul.ulaval.ca

SPECIFIC AIM

Bone marrow stem cells are capable of differentiating into multiple cell types, though it is still unclear whether they can migrate across the blood–brain barrier (BBB) in many regions of the central nervous system (CNS) and whether these cells can readily differentiate into functional parenchymal microglia. The first aim of this study was to unequivocally determine whether bone marrow-derived cells can migrate across the BBB and populate the brain parenchyma. We then assessed the differentiation fate of these cells once immigrated into the CNS.

PRINCIPAL FINDINGS

1. Bone marrow-derived stem cells can readily migrate across the BBB
To assess whether stem cells can migrate from the blood circulation to the brain parenchyma, we transplanted GFP-expressing stem cells into lethally irradiated mice. We found that donor-derived cells were present in many regions throughout the brain, from the olfactory bulb to the end of the medulla. Regions that consistently showed a great number of infiltrating GFP-positive cells included the medial part of the anterior olfactory nucleus, piriform cortex, lateral septal nucleus, hypothalamus, the amygdaloid area, the trunk region of the S1 layer of the cerebral cortex, hippocampus, substantia nigra, midbrain, medulla, cerebellum, and the area postrema. The vast majority of these cells had indeed crossed the BBB and were in the brain parenchyma since they were not associated with blood vessels. These cells were found across the whole rostro-caudal brain in neurogenic and non-neurogenic areas, which indicates that bone marrow precursors have the ability to populate the cerebral tissue in a nonspecific manner.

2. Donor-derived stem cells differentiated into microglia
One of the most striking initial observations was that nearly all of the infiltrated cells had a highly ramified morphology, closely resembling that of microglia. We thus assessed whether the donor-derived cells also expressed the microglial marker iba1. As depicted in Fig. 1 , nearly all of the GFP-positive cells coexpressed iba1, indicating these cells were fully differentiated into microglia. Immunoreactive signal levels for iba1 were very similar in donor-derived GFP and endogenous microglial cells.

View larger version (65K): [in this window] [in a new window]   Figure 1. Bone marrow-derived cells transdifferentiate into microglial cells. CD31-positive cells were stained with a rhodamine red X-conjugated secondary antibody (red blood vessels) and a primary antibody directed against iba1 was used to label microglia. They were then stained with an Alexa 633-conjugated secondary antibody (blue). Donor-derived cells (green) with a ramified morphology were always colocalized with iba1 staining (blue) and rarely colocalized with blood vessels (red). These results provide direct anatomical evidence that highly ramified cells are parenchymal microglia, which originate from circulating donor stem cells. Scale bar = 25 µm.

3. Exogenous microglia may be strong antigen-presenting cells (APCs)
Microglia and blood macrophages have similar characteristics. It is well known that blood macrophages are very efficient APCs and can express high levels of major histocompatibility complex class II (MHC-II). On the other hand, microglia are not recognized as potent APCs and have much lower MHC-II expression levels. Effective APCs express high CD11c levels whereas parenchymal microglia contain very small quantities of this protein. We assessed surface expression levels of this protein in donor-derived microglia to determine their ability to present antigens. As seen in Fig. 2 , GFP-positive microglia expressed high levels of the CD11c protein. Since resident parenchymal microglia did not express significant levels of this protein, our data suggest that donor-derived microglia could potentially be very efficient APCs.

View larger version (33K): [in this window] [in a new window]   Figure 2. Microglial cells derived from bone marrow stem cells exhibit characteristics of antigen-presenting cells (APCs). Effective APCs express high levels of the surface molecule CD11c, which was visualized by immunohistochemistry using an anti-CD11c primary antibody and a rhodamine red X-conjugated secondary antibody. Virtually all GFP-positive cells were immunoreactive to CD11c (red). Arrows indicate the highly localized cellular expression of the surface antigen CD11c. These data suggest that infiltrating microglia derived from bone marrow precursors are immunologically competent to present antigens to cells of the adaptive immunity. Scale bar: 10 µm.

CONCLUSIONS

Taken together, our data clearly show that stem cells originating from the bone marrow have the capacity to migrate across the BBB and to transdifferentiate into microglia in vivo. Thus, the generation of these cells during adulthood does not depend solely on proliferation of resident cells. Previous studies have shown that bone marrow-derived stem cells in the brain mostly differentiate into perivascular microglia and rarely cross the BBB. Our data also show that microglia of bone marrow stem cell origin express high levels of CD11c compared with residential microglia, a strong indication that these newly differentiated parenchymal microglia are likely to be effective APCs. This could have a critical effect on brain disorders that have an immune etiology, such as multiple sclerosis (MS). As depicted in Fig. 3 , macrophage-derived IL-12 stimulates the differentiation of a subset of T lymphocytes (CD4+) into T helper 1 cells (Th-1), which in turn produce interferon gamma (IFN). These activated Th-1 cells are believed to play a critical role in MS, especially during the demyelinating episodes. Our data demonstrate that immigrating microglia from blood circulation are more potent APCs than their residential counterparts. These blood-derived myeloid cells may therefore recruit and activate Th-1 cells inside the CNS and thus contribute to the development of detrimental immune processes in the cerebral environment.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic and hypothetical representation of the mechanisms involved in stem cell immigration into the CNS and their possible effects on neurodegenerative diseases. Stem cells that have developed into monocytes are attracted to endothelial cells that express the chemoattractant protein MCP-1. The monocytes then migrate across the BBB and further differentiate into fully functional microglial cells. However, these bone marrow-derived microglia express high levels of the MHC-II and CD11c proteins. Upon subsequent infection by various agents, these cells will express the antigen and produce cytokine IL-12, which will serve to induce the differentiation of T-cells into T-helper 1 cells (Th-1). Once the Th-1 cells recognize the antigen presented by the microglial cells, large amounts of TNF- and IFN will be synthesized and secreted, which may cause neurodegeneration and demyelination. See Conclusions for further details.

Monocyte chemoattractant protein 1 (MCP-1) is likely to be essential in recruiting bone marrow-derived cells, because the release of this chemokine by endothelial cells is the key mechanism for the chemoattraction of cells of monocytic lineage. MCP-1 and its receptor CCR2 have been implicated in a number of inflammatory diseases. The report that mice lacking CCR2 are resistant to experimental autoimmune encephalomyelitis (EAE), the animal model of MS, provided decisive evidence that this chemokine has a leading role in cerebral infiltrating processes. This concept is further supported by the absence of mononuclear cell inflammatory infiltrates within the cerebral tissue of CCR2-deficient mice and the fact that monocyte recruitment to the CNS is a prerequisite step for the development of inflammatory lesions in EAE. Differentiated macrophages in the brain parenchyma are believed to be directly responsible for demyelination and axonal dysfunction, as a consequence of myelin sheath destruction by these activated phagocytes. The presence of these cells in the CNS of intact animals may play a critical role in the effect the innate immune system may have on neurodegenerative disorders.

These data may also have clinical importance for cancer patients exposed to different levels of chemotherapy and radiotherapy, particularly those undergoing allogeneic hematopoietic stem cell transplantation. A similar immigration process of bone marrow precursors is expected to take place in the brain of these patients, but the physiological relevance of such a phenomenon has yet to be unraveled. Because these cells are more efficient APCs than endogenous microglia, it is tempting to propose that recruitment of bone marrow precursors may be a critical step in brain diseases with an immune etiology.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1517fje;

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