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Origin and differentiation of microglia Origin and differentiation of microglia
Ginhoux, Florent;Lim, Shawn;Hoeffel, Guillaume;Low, Donovan;Huber, Tara;
2013-01-01 00:00:00
REVIEW ARTICLE published: 17 April 2013 CELLULAR NEUROSCIENCE doi: 10.3389/fncel.2013.00045 1 2 1 1 2,3 Florent Ginhoux *, Shawn Lim , Guillaume Hoeffel , Donovan Low and Tara Huber Singapore Immunology Network, Agency for Science, Technology, and Research, Singapore Genome Institute Singapore, Agency for Science, Technology, and Research, Singapore Department of Biological Science, National University of Singapore, Singapore Edited by: Microglia are the resident macrophage population of the central nervous system (CNS). Amanda Sierra, University of the Adequate microglial function is crucial for a healthy CNS. Microglia are not only the first Basque Country EHU/UPV, Spain immune sentinels of infection, contributing to both innate and adaptive immune responses Reviewed by: locally, but are also involved in the maintenance of brain homeostasis. Emerging data are Payam Rezaie, The Open showing new and fundamental roles for microglia in the control of neuronal proliferation University, UK Miguel A. Cuadros, Universidad and differentiation, as well as in the formation of synaptic connections. While microglia de Granada Spain, Spain have been studied for decades, a long history of experimental misinterpretation meant that *Correspondence: their true origins remained debated. However, recent studies on microglial origin indicate Florent Ginhoux, Singapore that these cells in fact arise early during development from progenitors in the embryonic Immunology Network, yolk sac (YS) that seed the brain rudiment and, remarkably, appear to persist there into 8A Biomedical Grove, Immunos Building, Level 4, adulthood. Here, we review the history of microglial cells and discuss the latest advances Singapore, 138648. in our understanding of their origin, differentiation, and homeostasis, which provides new e-mail: florent_ginhoux@ insights into their roles in health and disease. immunol.a-star.edu.sg Keywords: microglia, macrophage, central nervous system, origin, yolk sac and the elimination of synaptic structures (Tremblay et al., 2010). INTRODUCTION Microglia also contribute to the remodeling of post-natal neural Microglia are the resident mononuclear phagocytes of the cen- circuits as they have been recently shown to play a role in synaptic tral nervous system (CNS), belonging to the glial system of pruning during post-natal development in mice (Paolicelli et al., non-neuronal cells that support and protect neuronal functions. 2011). Microglia are broadly distributed throughout the brain and the Thus, microglia occupy a central position in the defense and spinal cord (Lawson et al., 1990), and account for 5–20% of the maintenance of the CNS and so are attracting interest as potential total glial cell population within the CNS parenchyma (Perry, 1998). Adequate and appropriate microglial function is crucial therapeutic targets in neurological disorders and recovery from brain injury. However, in order to exploit the abilities of the for the homeostasis of the CNS in both health and disease (Perry microglial population, we must first understand their origins and et al., 2010). homeostasis before attempting to manipulate their functions. In There are two main functional aspects of microglia: immune this review we will present the latest advances in our knowledge defense and CNS maintenance. As immune cells, they act as sen- on the origin of microglia, revisit early studies in light of recent tinels, detecting the first signs of pathogenic invasion or tissue developments and highlight some of the most relevant strategies damage in this delicate immune-privileged site that is actively to generate microglia for therapeutic approaches of neurological protected by the brain blood barrier (Daneman, 2012). Under disorders. the inflammatory conditions of an active immune response how- ever, microglia must also moderate the potential damage to HISTORICAL PERSPECTIVES ON THE NATURE OF MICROGLIA the CNS and support tissue repair and remodeling. Perhaps Defining the origin of microglia has been an elusive goal for unsurprisingly, dysregulated microglial activation and microglia- induced inflammation is observed in virtually all brain patholo- generations of researchers and a longstanding issue of debate. Multiple schools of thought have emerged. The first descrip- gies; emerging evidence suggests that microglia exert direct effects on neurons, contributing to disease progression (Perry et al., tion of the cells came from the work of Franz Nissl in the late 2010; Kettenmann et al., 2011; Kingwell, 2012). nineteenth century, who described rod cells (“Stabchenzellen”) as In recent years there has been an increasing appreciation of reactive glial elements with migratory, phagocytic and prolifera- the importance of microglia for normal CNS function. In addi- tive potential. In the late nineteenth century, W. Ford Robertson introduced the term “mesoglia” to describe mesoderm-derived tion to their immune functions, emerging data are showing new and fundamental roles for microglia in the control of neu- phagocytic elements in the nervous system that had origins dis- tinct from those of neurons and neuroglia. Neuroglia were first ronal proliferation and differentiation as well as in the formation of synaptic connections (Graeber, 2010; Hughes, 2012). In the described by Virchow, in 1856, who named them “nevernkitt” meaning nerve-glue, later translated as “neuroglia,” though in fact steady state, microglial cells constantly survey their local microen- vironment, extending their motile processes to make transient they corresponded to the macroglial population, which comprises astrocytes and oligodendrocytes (Rio-Hortega, 1939). While this contact with neuronal synapses, contributing to the modification Frontiers in Cellular Neuroscience www.frontiersin.org April 2013 | Volume 7 | Article 45 | 1 Ginhoux et al. Origin and differentiation of microglia idea had merit, in fact, Robertson’s mesoglia similarly turned Lassmann and co-workers were the first to demonstrate that res- out to correspond mainly to oligodendrocytes. Santiago Ramon ident microglia in rats are a very stable cell pool, in contrast to y Cajal later renamed the same cells the “third element of the meningeal and perivascular macrophages, which in adult animals nervous system” to further differentiate them from neurons and are only exceptionally replaced by circulating blood cells, even neuroglia, and stated that they were of probable mesodermal ori- after recovery from severe brain inflammation (Lassmann et al., gin. This “third element” concept was refined further in 1919 1993). Such observations were later confirmed in mice by Priller, by del Rio-Hortega, a student of Ramon y Cajal, who made where the majority (85–95%) of microglial cells remained of host the distinction between various cell types within the cells of the origin up to 15 weeks after bone marrow transplantation (Priller “third element” based on morphological and functional differ- et al., 2001). The importance of the observation that newborn ences. Del Rio-Hortega introduced the term “microglial cell” to microglia are not replaced by donor bone marrow-derived cells describe the non-neuronal, non-astrocytic third element as dis- (De Groot et al., 1992) will be discussed later, as it has the sig- tinct from neurectodermal oligodendroglia or oligodendrocytes nificant implication that the adult microglial population can be (Rio-Hortega, 1939)(Forhistoric review see Rezaie and Male, maintained solely by local radio-resistant precursors which are 2002). present in the brain prior to birth. Although both W. Ford Robertson and Santiago Ramon y Other hypotheses on the origin of microglia included their Cajal suspected a mesodermal origin of what were to become derivation from the pericytes associated with blood vessels (Mori known as “microglial cells” (their mesoglia/third element of the and Leblond, 1969; Baron and Gallego, 1972)orfromthe nervous system), it was commonly held at the time that all glial subependyma adjacent to the lateral ventricles (Lewis, 1968). cells were of neuro-ectodermal origin. Further dissecting the At the same time, a second school of thought was devel- heterogeneity of the mesoglia, del Rio-Hortega was the first to oping which paralleled del Rio-Hortega’s original hypothesis. introduce the term “microglia” to discriminate true mesodermal His conviction of the mesodermal origin of microglia was sup- elements from oligodendrocytes, which were previously consid- ported both by studies coupling light/electron microscopy and ered a component of the mesoglia. Del Rio-Hortega exploited immunohistochemistry, which recognized typical morphologi- silver staining techniques to describe the two types of cells as cal features of macrophages in the various stages of microglial differing in origin, distribution, form, and function: the major development (Murabe and Sano, 1982), and by the demonstra- population, called oligodendroglia, that lacked phagocytic activ- tion that microglial cells reacted positively to antisera recognizing ity, and the minor population of ramified resting cells. This minor monocyte/macrophage antigens (Hume et al., 1983; Murabe and population was then clearly defined as the “third element of the Sano, 1983). Despite the emerging evidence of the relation- CNS” with a mesodermal origin, containing phagocytic corpus- ship of microglia to macrophages, other reports led to variable cles and with migratory and phagocytic activity (Rio-Hortega, interpretations due to a lack of homology between monocytes 1932). and mature microglia in the expression of certain antigens, Despite del Rio-Hortega’s seminal work, his theories were complicating the issue (Oehmichen et al., 1979; Wood et al., largely overlooked and have only recently come back to the fore- 1979). Nevertheless, the data showing phenotypic homologies front of scientific thinking (Rezaie and Male, 2002). At the time, between monocytes/macrophages and microglia were eventu- there was much support for the belief that microglia shared a ally validated by immunohistochemical studies that reported the neuro-ectodermal origin with the other glial cells. Several stud- specific expression of macrophage markers, including F4/80, Fc ies supported this belief well into the twentieth century, including receptor and CD11b in mouse microglia (Perry et al., 1985), as reports from Fujita who proposed a common, matrix-derived well as FcGRI, and CD11b in their human counterparts (Akiyama progenitor for microglia, astrocytes, and oligodendrocytes (Fujita and McGeer, 1990). Finally, a pivotal genetic study revealed that and Kitamura, 1975). The work of Kitamura was similarly inter- mice lacking PU.1, a crucial transcription factor for myeloid cells, preted to indicate that microglia, as well as astrocytes, originated were also devoid of microglia (McKercher et al., 1996; Beers from neuro-ectodermal-derived glioblasts (Kitamura et al., 1984). et al., 2006). This unequivocally established the myeloid nature As late as the 1990’s, new studies continued to emerge that of microglia and simultaneously suggested that these cells might seemed to show a common origin of astrocytes and microglia; be ontogenetically related to macrophages. Hao reported that cultures of either murine embryonic neuro- epithelial cells or astrocytes could differentiate in vitro to give THE ORIGIN(S) OF MURINE MICROGLIA microglial-like cells, an idea which was supported by Fedoroff’s Although there is a consensus about the myeloid origin of work showing that clonal cultures of disaggregated neopallial cells microglia, much controversy remains regarding the precise nature from newborn mice gave rise to mixed microglial-astroglial cells of microglial progenitors. Initial studies described the pres- (Hao et al., 1991; Fedoroff et al., 1997). In addition, data show- ence of microglial cells during early development, suggesting ing that donor bone marrow cells failed to contribute to the adult that microglia arise from embryonic progenitors. While del microglial population in either newborn (De Groot et al., 1992) Rio-Hortega proposed that microglia originate from meningeal or adult rodents (Matsumoto and Fujiwara, 1987) was interpreted macrophages penetrating the brain during embryonic develop- to mean that the majority of microglial cells were of local neuro- ment, many authors including del Rio-Hortega himself, claimed ectodermal origin. However, this interpretation was soon updated that brain parenchymal microglia could also be derived from in response to the finding that microglia (in contrast to other blood monocytes. Monocytes are indeed recruited to the neonatal blood leucocyte populations) are highly radio-resistant. In 1993, and adult brain, in the latter case most often under inflammatory Frontiers in Cellular Neuroscience www.frontiersin.org April 2013 | Volume 7 | Article 45 | 2 Ginhoux et al. Origin and differentiation of microglia conditions, where they can differentiate into microglia-like cells. Altogether, these seminal studies strongly suggested that This knowledge long supported the prevailing viewpoint that microglia derive from embryonic hematopoietic precursors that circulating blood monocytes represent microglial progenitors, seed the CNS prior to birth and, more importantly, before the replacing those seeding the brain during embryonic develop- onset of bone marrow hematopoiesis. However, the exact tissue ment. In fact, until recently, the most consensual hypothe- origin and developmental cell lineage of precursors that migrate sis was that embryonic and peri-natal hematopoietic waves of to the CNS to give rise to the “first” endogenous wave of microglia microglial recruitment and differentiation occurred in the CNS remained unknown and a topic of debate until recently. (Chan et al., 2007). However, we now know that the situation is somewhat different. Here, we will describe and discuss the MOUSE AND HUMAN EMBRYONIC HEMATOPOIESIS recent advances in understanding of the origin of microglia, and A major challenge in defining the embryonic microglial precursor will also revisit the data from earlier studies in light of these was the complication of the dual source of blood cell formation developments. during embryogenesis. Two major hematopoietic sites contribute to this process: the extra-embryonic yolk sac (YS) and the fetal EARLY DEVELOPMENT liver (Tavian and Peault, 2005; Orkin and Zon, 2008). In mice, In addition to having first described microglia, del Rio-Hortega primitive hematopoiesis initiates in the YS around E7.0, shortly also proposed that they might initially arise in the early stages of after the onset of gastrulation, leading mainly to the produc- development from mesodermal cells of the pia mater, the inner- tion of erythrocytes and macrophages (Moore and Metcalf, 1970; most layer of the meninges (the membranes surrounding the Palis et al., 1999; Bertrand et al., 2005). Primitive macrophages brain and spinal cord). From his work on embryonic brains, he first appear in the blood islands of the mouse YS on the ninth reported the “migration of embryonic corpuscles from the pia day of gestation and their pattern of differentiation is unique into the nerve centers” with morphological similarity to lym- in the sense that they do not go through a monocytic inter- phocytes (Rio-Hortega, 1939). However, del Rio-Hortega also mediate stage, as seen in adult macrophages (Takahashi et al., proposed that “microglia may eventually arise from other related 1989). YS-derived primitive macrophages will spread into the elements, chiefly the blood mononuclears” based on the similari- embryo proper through the blood after the circulatory system ties in morphology and phagocytic activities of the microglia and has been fully established (from E8.5 to E10) (McGrath et al., monocytes (Rio-Hortega, 1939), thereby founding the “origin of 2003) and migrate to various tissues, including the brain. Once microglia” controversy. in the tissues, they differentiate into so-called “fetal macrophage The immunohistochemical study conducted by Perry et al., populations” even before the onset of monocyte production by more precisely described this phenomenon using macrophage the fetal liver (Naito et al., 1990). These fetal macrophages have markers such as F4/80. They concluded that as early as embry- high proliferative potential, not only in the YS where they are onic day 16 (E16) of development, macrophage-like cells that produced but also in the tissues that they colonize (Takahashi had extravasated into the brain parenchyma were localized in et al., 1989; Sorokin et al., 1992; Naito et al., 1996; Lichanska “hot spots,” from where they subsequently invaded the brain and Hume, 2000). After E8.5, with the determination of the and differentiated through a series of transitional forms to intra-embryonic mesoderm toward the hematopoietic lineage, finally become ramified microglia (Perry et al., 1985). Other a new wave of hematopoietic progenitors is generated within studies later detected dispersed F4/80 expressing macrophages the embryo proper, first in the para-aortic splanchnopleura distributed within loose connective tissue surrounding the neuro- (P-Sp) region and then in the aorta, gonads, and mesonephros ectoderm in E12 rat embryos (Morris et al., 1991). Also in (AGM) region (Godin et al., 1993; Medvinsky et al., 1993). The the rat, amoeboid microglial cells expressing monocytic mark- hematopoietic stem cells generated within the AGM will lead to ers are present as early as E12 in the neuro-epithelium (Wang the establishment of definitive hematopoiesis (Orkin and Zon, et al., 1996). Interestingly, such embryonic cells were proposed 2008). Around E10.5, YS- and AGM-derived hematopoietic pro- to be microglial progenitors not only due to the similarities in genitors colonize the fetal liver (Kumaravelu et al., 2002), which phenotype and morphology, but because of their potent prolif- serves as the major hematopoietic organ after E11.5, generating all erative response to mitogenic stimulation in vitro (Alliot et al., hematopoietic lineages, including monocytes (Naito et al., 1990). 1991). A recent study highlighted further differences between primi- Similarly, in human fetuses, microglia-like cells with a range tive and definitive hematopoiesis, showing that the latter relies of morphologies can be detected from as early as 3 weeks of esti- on the transcription factor Myb, while YS-derived macrophages mated gestational age (EGA) (Hutchins et al., 1990). However, it are Myb-independent. This further underlines the fact that YS- appears that maturation of the microglial compartment is ongo- derived macrophages constitute an independent lineage, distinct ing throughout the majority of gestation: colonization of the from the progeny of definitive hematopoietic stem cells (Schulz spinal cord begins at around 9 weeks, the major influx and distri- et al., 2012). bution of microglia commences at about 16 weeks, and ramified Human hematopoiesis also begins in the YS around day microglial forms take up to 22 weeks to become widely distributed 19 of the EGA, and YS-derived stem cells are similarly lim- within the intermediate zone (Rezaie and Male, 1999; Rezaie et al., ited/committed to myelo-erythroid development. Hematopoiesis 2005). It is only close to term, at 35 weeks, that well-differentiated then moves transitorily to the fetal liver around 4–5 weeks EGA, microglial populations can be detected (Esiri et al., 1991)(for before being definitively established in the BM approximately at review Rezaie, 2003 and Verney et al., 2010). 10.5 weeks EGA (Tavian and Peault, 2005). Frontiers in Cellular Neuroscience www.frontiersin.org April 2013 | Volume 7 | Article 45 | 3 Ginhoux et al. Origin and differentiation of microglia THE YOLK SAC HYPOTHESIS OF MICROGLIAL ORIGIN Interestingly, the YS derivation of microglia appears to be con- served across diverse species as shown in the zebrafish (Herbomel Ashwell was the first to report the presence of round and amoe- boid microglial cells in the fetal mouse cerebellum (Ashwell, et al., 2001), and in avians (Cuadros and Navascues, 1998). However, what differs in mice is the requirement for a func- 1990) and then in rat forebrain (Ashwell and Waite, 1991)asearly as E11.0. Sorokin soon after detected macrophage-like cells and tional blood circulation for the spreading of YS macrophages in the embryo proper. In zebrafish, this colonization appears inde- their precursors in blood vessels and the embryonic mesenchyme in rat embryos from 10.5, and noted that the developing brain was pendent of the blood circulation as YS macrophages first directly the first organ to be colonized (Sorokin et al., 1992). Interestingly, invade the whole cephalic mesenchyme, and from there invade epithelial tissues including the brain, while other macrophages cells with the capacity to differentiate into microglia-like cells in vitro (expressing Mac-1, Mac-3, F4/80 and Fc antigens, with a enter the blood circulation (Herbomel et al., 1999). Similarly, using chick-quail blood chimeras, Kurz showed that in avians, macrophage-like morphology and ultrastructure) can be detected in the developing neuro-epithelium at days E8.5/E9.0, suggesting YS macrophages do not penetrate through the wall of embry- onic CNS vessels, but rather from the pial surface (Kurz et al., that in mice, the earliest developmental stage at which seeding of cells with myeloid features occurs in the brain is at E8.5/E9.0 2001). In contrast, in mouse embryos, there is a clear require- −/− ment for the circulatory system as E9.5–E10.5 Ncx-1 embryos, (Alliot et al., 1991). Later reports confirmed the presence at sim- ilar stages of amoeboid cells expressing macrophage (Alliot et al., which lack a heartbeat and therefore have no functional blood circulation due to a defect in the sodium calcium exchanger 1 1999; Ginhoux et al., 2010) and microglial markers (Chan et al., 2007; Mizutani et al., 2012) in both the cephalic mesenchyme and (Koushik et al., 2001), also lack microglial progenitors (Ginhoux the neuro-epithelium, in accordance with the idea that the YS et al., 2010), as well as other fetal macrophages. However, this −/− defect does not affect YS hematopoiesis as Ncx-1 embryos contributes to microglial genesis. However, theevidencefor a YS origin ofsuch microglial have similar numbers of YS macrophages as their control, normal phenotype littermates (Ginhoux et al., 2010). Whether murine YS progenitors was, at first, mixed. Initially, data from one of the aforementioned in vitro studies were interpreted to support the macrophages enter via the blood circulation directly in the brain parenchyma or first enter in the cephalic mesenchyme and then hypothesis that these macrophage-like cells that will give rise to microglia originated from the neuro-ectoderm (Hao et al., migrate to the neuro-epithelium (Chan et al., 2007)remains to be clearly defined. 1991). Takashi and Naito drew a different conclusion after they described the first emergence of immature macrophages within The overall conclusion of these studies in rodents, humans, and other species is that microglia derive from the YS blood islands of embryonic YS at fetal day 9 in both mouse (Takahashi et al., 1989)and rat (Takahashi and Naito, 1993). macrophages that seed the brain rudiment during early fetal development (Figure 1). However, these reports could not Following the establishment of fetal blood circulation, these exclude the possibility that others progenitors could supersede cells colonize the embryonic tissues, including the brain rudi- ment. By [3H]-thymidine autoradiography, YS macrophages were the YS contribution. In fact, some data that will be discussed below, continued to emerge that suggested a requirement for the shown to possess high proliferative potential, which suggested that these fetal macrophages were in fact primitive macrophages contribution of blood-borne cells to both generate the post-natal microglial compartment, and to maintain it into adulthood. from the YS (Takahashi and Naito, 1993). Alliot also clearly and convincingly proposed that such cells were true microglial THE EARLY POST-NATAL CONTRIBUTION OF MONOCYTES progenitors of YS origin, as, at that stage, the YS is the only TO THE MICROGLIAL POPULATION hematopoietic site in the embryo. This group then conclusively Shortly after birth in rodents, the microglial population expands documented the presence of potential microglial progenitors in the YS and then the brain rudiment, with their numbers increas- dramatically (Alliot et al., 1999; Tambuyzer et al., 2009), leading to the suggestion that the proliferation of embryonic microglial ing dramatically from E9.0/E9.5 until around 2 weeks after birth cells alone could not account for the steep rise in numbers and (Alliot et al., 1999). A similar pattern of events is likely observed in humans, where, that there must be a fresh influx of cells from another compart- ment. As initially suggested by del Rio-Hortega, blood monocytes from 4.5 weeks gestation, amoeboid microglial cells (charac- terized by the expression of Iba1, CD68, CD45, and MHC-II) were believed to invade the CNS in the perinatal period and give rise to microglia, replacing the embryonic microglial cells. enter the cerebral wall from the ventricular lumen and the lep- tomeninges (Rezaie et al., 2005; Monier et al., 2007). In the YS There was support for this belief from several studies, notably an early report where round, amoeboid, phagocytic cells were and mesenchyme at 4–6 weeks after fertilization, two populations of cells with a dendritic morphology could be distinguished: a seen in rat corpus callosum during the first few days of life and then disappeared coincident with the appearance of rami- majority that expressed monocyte/macrophage-associated mark- ers but no detectable HLA-DR antigen, while the minority con- fied microglia. These cells were typical macrophages, but some displayed features of monocytes, while others appeared to be stitutively expressed MHC class II (HLA-DR and -DP) but no transitional between the two types. The authors of this study con- monocyte/macrophage-associated markers (Janossy et al., 1986). The emergence of this heterogeneity preceded the formation cluded that circulating monocytes enter the developing brain to assume the form of ameboid microglia that subsequently evolved of both thymus and bone marrow, suggesting the independent development of these macrophage populations (for review Verney to become ramified microglia (Ling, 1976). Subsequent studies gave neonatal rats an intra-peritoneal pulse of [3H]-thymidine to et al., 2010). Frontiers in Cellular Neuroscience www.frontiersin.org April 2013 | Volume 7 | Article 45 | 4 Ginhoux et al. Origin and differentiation of microglia FIGURE 1 | Brain development and microglial homeostasis. Primitive gestation and post-natal development as well as in the injured adult brain macrophages exit the yolk sac blood islands at the onset of circulation in reaction to inflammation. Nevertheless, during certain inflammatory and colonize the neuroepithelium from E9.5 to give rise to microglia. The conditions found for example after bone marrow transplantation, the blood brain barrier starts to form from E13.5 and may isolate the recruitment of monocytes or other bone marrow-derived progenitors can developing brain from the contribution of fetal liver hematopoiesis. supplement the microglial population to some extent. However, we do Embryonic microglia expand and colonize the whole CNS until adulthood. not understand yet whether these cells persist and become integrated in Importantly, in steady state conditions, embryonically-derived microglia the microglial network, or are a temporary addition to the endogenous will maintain themselves until adulthood, via local proliferation during late population. allow tracking of labeled blood cells by autoradiography. Labeled Nevertheless, later studies employing the PU.1 knockout (KO) immature amoeboid cells were detected in the corpus callosum mouse model, that lacks embryonic microglia, demonstrated few hours after administration, while the majority of newly- the capacity of bone marrow-derived cells to contribute to the ramified microglia were labeled one week later. These observa- post-natal microglial population. In this study, neonates received tions implied that labeled microglial cells must therefore have wild-type bone marrow transplants within 24 h of birth, which come from the transformation of immature amoeboid cells that resulted in de novo generation of the full microglial compartment acquired the tracer earlier (Imamoto and Leblond, 1978). This (Beers et al., 2006). Therefore, it must be concluded that, at least possibility was further tested by injecting a suspension of car- under exceptional circumstances such as in the PU.1 KO mouse bon particles into the circulation of rats of various ages to enable where endogenous embryonic microglia are completely absent, tracing of carbon-labeled monocytes, or by direct adoptive trans- some bone marrow-derived cells have the capacity to infiltrate fer of carbon-labeled monocytes. Later on, carbon particles were the CNS and assume the morphology and phagocytic capacity of sequentially found in amoeboid cells of the corpus callosum and microglia. then on ramified microglial cells, suggesting again that blood A CONTRIBUTION OF MONOCYTES TO THE ADULT MICROGLIAL monocytes, after ingesting carbon particles in the circulation or after transfer, migrated to the corpus callosum and differenti- POPULATION IN THE STEADY STATE? ated into microglial cells via an amoeboid stage (Ling, 1979; Ling Following the observations that monocytes might be able to con- et al., 1980; Leong and Ling, 1992). However, while such data sug- tribute to the microglial population immediately after birth, it gest that circulating blood monocytes can enter the CNS right became implicitly accepted that they could also do so in adults. after birth, perhaps in a specific site, it is important to note that The idea that monocytes, or any bone marrow-derived cells, such studies were rather qualitative and did not clearly address might then be able to be engineered and used as a delivery system the exact relative contributions of post-natal monocytes versus into the CNS for therapies, the “Trojan Horse” theory, motivated embryonic progenitors to adult microglial homeostasis. In fact, investigators to discover the underlying mechanisms. The main the authors had themselves clearly recognized that such events hypothesis became that embryonic microglia disappear and are were infrequent (Ling et al., 1980). replaced by post-natal bone marrow-derived cells. Frontiers in Cellular Neuroscience www.frontiersin.org April 2013 | Volume 7 | Article 45 | 5 Ginhoux et al. Origin and differentiation of microglia That is not to say that there were not data supporting source for maintaining the microglial population, although this the hypothesis of a role of monocytes in maintaining the might change during CNS inflammation or disease (Vallieres and adult microglial population: a seminal study employing [3H]- Sawchenko, 2003; Ladeby et al., 2005; Mildner et al., 2007). In thymidine incorporation and autoradiography in normal adult fact, in response to CNS inflammation and damage, an increase mice concluded that cells can be recruited from the circulat- in microglial number is often observed, a phenomenon called ing monocyte pool through an intact blood-brain barrier (BBB) reactive microgliosis, which has become a hallmark of many and rapidly differentiate into resident microglia (Lawson et al., CNS pathologies. However, it remained to be elucidated whether 1992). However, the authors also noted that the resident microglia such increases in number rely on local expansion of mature were proliferating, which suggested that the microglial popula- microglia or are achieved by recruitment of blood precursors such tion might maintain itself through either mechanism, or perhaps as monocytes. both. Other data supporting the idea that adult bone marrow- Two recent studies clarified the relative contribution of blood derived cells can give rise to microglia included the observation monocytes to microglia in experimental models of CNS patholo- that following total bone marrow transplantation, some donor gies. Both revealed that the irradiation regimen used to prepare hematopoietic cells differentiated into microglia within the brains recipient animals for bone marrow transplants is necessary for of adult mice (Eglitis and Mezey, 1997; Mezey et al., 2000; Simard the recruitment and differentiation of monocytes into microglia and Rivest, 2004). (Ajami et al., 2007; Mildner et al., 2007). Mildner showed that However, these results were in slight disagreement with a pre- recipientmicein which theCNS was shielded toprotect from the vious, and importantly, more quantitative, study which showed irradiation and its associated inflammation, which induces the that the majority of microglial cells remained of host origin up release of pro-inflammatory cytokines and chemokines, did not to 15 weeks after bone marrow transplantation in mice (Priller experience a significant invasion of bone marrow-derived cells et al., 2001). Similar results were also initially reported in rats as into the brain, in contrast to the unshielded mice (Mildner et al., several investigators concluded that there was little or no con- 2007). Beyond the irradiation issue, these data also suggest that tribution of bone marrow-derived cells to the adult microglial microglial engraftment from the blood requires pre-conditioning pool (Matsumoto and Fujiwara, 1987; Lassmann et al., 1993). In of the CNS that likely disrupts the BBB. Additional clarity came addition, itwas shownthatwhile microglia are notbonemarrow- from experiments in parabiotic mice, which have undergone derived in adults, the closely-associated meningeal and perivascu- surgery to physically link their circulatory systems, providing a lar macrophages are, perhaps going some way to explaining the more physiological means to study the turnover of hematopoi- confusion. Schelper and Adrian bluntly concluded that “mono- etic cells for prolonged periods without the need for irradiation cytes become macrophages; they do not become microglia,” in (Ajami et al., 2007). Ajami used such mice to show that in con- this case, following CNS lesions (Schelper and Adrian, 1986), trast to what was observed in irradiated and transplanted mice, while Hickey and Kimura showed that the stable pool of resi- there was no microglial progenitor recruitment from the circu- dent microglia is only exceptionally supplemented by hematopoi- lation in either denervation or CNS neurodegenerative disease, etic cells, even after recovery from severe brain inflammation despite the fact that the mixing of leucocyte populations can reach (Hickey and Kimura, 1988). Similarly, Vallieres reported that up to 50% in the blood of both parabionts. In agreement with many of these cells were in fact perivascular macrophages and their findings, we found no contribution of bone marrow-derived that newly-formed parenchymal microglia were found in signifi- cells to CNS microglia up to 12 months after parabiosis (Ginhoux cant numbers only in the cerebellum and at injury sites (Vallieres et al., 2010). Such data suggest that maintenance and local expan- and Sawchenko, 2003). Importantly, in humans, taking advan- sion of microglia are solely dependent on the self-renewal of tage of sex-mismatched donor bone marrow transplant (male CNS-resident cells in these models. into female) where Y-chromosome specific in situ hybridization Interestingly, with this parabiotic model, in the context of can be performed to follow the origin of cells, similar results irradiation of one parabiont, no further contribution from the were obtained. The only donor male cells detected corresponded other parabiont was detected in contradiction with the results of to mononuclear leucocytes within the vessel lumen and infiltrat- Mildner. However, Ajami further clarified that although irradi- ing the perivascular space and parenchyma, and perivascular cells ation is required for donor cells to engraft, it is not sufficient; (Unger et al., 1993). In fact, the observation that bone marrow- another important, but overlooked, requirement is the artificial derived microglia were only found in notable amounts under introduction of a critical number of bone marrow cells into the certain conditions highlighted some significant shortfalls of the blood circulation (where they are not normally found) in con- earlier studies: while it was successfully shown that monocytes junction with the inflammation of BBB caused by irradiation, a could differentiate into cells that resembled microglia, few had situation found only upon lethal total bone marrow transplan- quantified the effect or attempted to define the phenomenon tation (Diserbo et al., 2002; Li et al., 2004; Linard et al., 2004). in space and time, or to monitor the persistence of the bone More recently, the same group used a similar approach combining marrow-derived microglia. parabiosis and myelo-ablation to show that recruited monocytes do not persist and therefore do not contribute to the resident A CONTRIBUTION OF MONOCYTES TO THE ADULT MICROGLIAL microglial pool. However, recruited monocytes contribute to POPULATION DURING INFLAMMATION? the severity of disease in multiple sclerosis and the experimen- What became clear was that although the monocyte-to-microglia tal autoimmune encephalitis mouse model (Ajami et al., 2011). path may exist in adult brain, it is unlikely to be a significant Similarly, in transgenic mouse models of Alzheimer’s disease, Frontiers in Cellular Neuroscience www.frontiersin.org April 2013 | Volume 7 | Article 45 | 6 Ginhoux et al. Origin and differentiation of microglia irradiation was shown to condition the brain for engraftment to the steady-state microglial population in adults: are the embry- of myeloid cells, a phenomenon that does not occur normally onic microglia responsible for maintaining the adult pool or do during disease progression (Mildner et al., 2011). Interestingly, embryonic and adult microglia in fact have different origins? One in this study, perivascular macrophages, rather than microglia of the studies already discussed, from De Groot, had implied and monocytes, modulated β-amyloid deposition in the brains that embryonic microglia were the sole contributors to the adult of AD transgenic mice by clearing Aβ in a CCR2-dependent microglial pool. This study observed that donor bone marrow fashion (Mildner et al., 2011). This highlights the distinct and cells failed to contribute to the adult microglial population in non-redundant roles of microglia, monocyte, and perivascular a model of newborn transplantation, and concluded therefore macrophages in acute injury and autoimmune inflammation that the adult microglial population was totally independent (Jung and Schwartz, 2012). Finally, Capotondo recently clarified of post-natal bone marrow-derived circulating precursors from that the conditioning regimen also contributes to the ablation birth onward (De Groot et al., 1992). Recently, we revisited their of endogenous microglia, thereby allowing the local proliferation experiments with a more quantitative aim and found that while of invading blood cells (Capotondo et al., 2012). In conclusion, most circulating leucocytes were of donor origin, the majority parabiotic mice provided, for the first time, unequivocal evidence of microglia remained of host origin for more than 3 months that the microglial population during the steady state is able after transplantation, confirming that post-natal hematopoietic to maintain itself throughout adult life by local renewal, inde- precursors, including monocytes, likely do not contribute to the pendent of circulating precursors in steady state. Conversely, in adult microglial population (Ginhoux et al., 2010). transplant models, which are perhaps not so much reflections of We also employed a more advanced technique of YS progen- normal physiology, a fraction of microglia can arise from adult itor fate mapping to definitively answer this question. Our fate bone marrow. mapping mouse model expresses a fluorescent protein (eYFP) As discussed before, we know that adult bone marrow cells can exclusively in YS progenitors and their progeny, which include YS also enter into the CNS and differentiate into microglia in excep- macrophages. Briefly, this mouse model expresses a tamoxifen- tional circumstances: when the endogenous microglial niche is activated MER-Cre-MER recombinase gene under the control of completely vacant, such as in the PU.1 KO (Beers et al., 2006), one of the endogenous promoters of the runt-related transcrip- or experimentally depleted, for example using Gancyclovir in a tion factor 1 (Runx1) locus (Samokhvalov et al., 2007). When mouse model expressing the thymidine kinase under the CD11b crossed with a Cre-reporter mouse strain, recombination can be promoter (Varvel et al., 2012). Importantly, microglial repopula- induced in embryos by a single injection of 4-Hydroxytamoxifen tion in the latter study did not require any conditioning regimen (4 OHT) into pregnant females. Active recombination in these such as irradiation, as the microglial pool reconstituted itself after knock-in mice occurs in a short time frame that does not cessation of the Gancyclovir treatment. However, the effect of exceed 24 h post-injection and leads to irreversible expression of Gancyclovir on the permeability of the BBB was not evaluated eYFP in Runx1 cells and their progeny (Samokhvalov et al., in this model and we do not know if the bone marrow progeni- 2007). tors require additional “help” in order to cross the BBB, perhaps Although both YS and fetal liver hematopoietic progeni- through the presence of as yet undefined inflammatory media- tors express Runx1, YS progenitors are the only cells present tors. In addition, in their experimental setting, the authors could at E7.5 and so injection of tamoxifen at E7.5 will therefore not formally track the origin of the cells that repopulate the allow the specific and irreversible tagging of YS progenitors and microglia and therefore were unable to exclude local repopula- their progeny but not of fetal liver-derived progeny. In con- tion from non-depleted microglial cells. Nevertheless, the fact trast, injection of tamoxifen at later time points (from E8.5) that adult bone marrow cells can give rise to microglia in the will favor the tagging of AGM-derived hematopoietic progeni- context of hematopoietic cell transplantation with a conditioning tors and not the YS progenitors (North et al., 1999; Samokhvalov regimen open the door for invaluable therapeutic strategies for et al., 2007). We can use this model to accurately ask about the correction of CNS conditions in which defects of microglia are the origins of different cell types; for example, in the case of implicated. For example, bone marrow transplantation of mouse microglia, if they are predominantly derived from YS tagged models for metachromatic leukodystrophy (Biffi et al., 2004), progenitors, they should express eYFP in the adult CNS when the obsessive compulsive disorder trichotillomania (Chen et al., 4 OHT is injected at E7.5 and not at E8.5. In contrast, circu- 2010), and Rett syndrome (Derecki et al., 2012) has been shown lating leukocytes, including monocytes that are known to derive to ameliorate disease symptoms. from AGM hematopoietic progenitors, will present the opposite In conclusion, in these transplant models, which are per- profile, expressing eYFP when 4 OHT is injected at E8.5 instead haps not so much reflections of normal physiology, a fraction of of E7.5. In addition, if the microglial population does predom- microglia are of bone marrow origin. However, during the steady inantly derive from YS progenitors without a significant contri- state, monocytes or other bone marrow-derived cells do not enter bution from fetal liver- or bone marrow-derived hematopoiesis, the CNS and do not significantly contribute to the microglial they should be tagged at a higher level than circulating leuko- population. cytes, which derive predominantly from mature hematopoiesis. To test this hypothesis, we injected the mice with tamoxifen at closely spaced time points of gestation and compared the EVIDENCE FOR THE PERSISTENCE OF THE EMBRYONIC WAVE OF MICROGLIA number of eYFP-tagged microglia and circulating monocytes in What remained unclear in the field, however, was the relative con- the mice as adults. Strikingly, the relative number of tagged tribution of embryonic and post-natal hematopoietic progenitors microglia in mice injected at E7.25 was much greater than of Frontiers in Cellular Neuroscience www.frontiersin.org April 2013 | Volume 7 | Article 45 | 7 Ginhoux et al. Origin and differentiation of microglia blood monocytes or other circulating leukocytes (Ginhoux et al., of which are time-consuming and generally yield few cells. While 2010). of limited use in rodents, this approach is entirely unfeasible for In contrast, the relative number of tagged microglia in mice obtaining human microglia. injected from E8.0 onwards decreased dramatically to reach Although there has been some success producing microglia- undetectable levels as soon as E8.5, while the relative number like cells from bone marrow stem cells and circulating mono- of eYFP leukocytes, including monocytes, increased progres- cytes, they are perhaps poor models of “true” microglia as these sively in adult blood. This opposing pattern of recombination in cell populations do not share the embryonic origin of the vast microglia compared to circulating leukocytes strongly supports majority of microglia in the homeostatic brain. Furthermore, the idea that the major contribution to microglial numbers comes their relatively advanced states of differentiation also make them from YS progenitors, and formally excludes the contribution of unsuitable for asking questions about the intrinsic and extrin- definitive hematopoiesis. Altogether these results establish that sic regulators of microglial development and specification. In microglia originate from E7.25 Runx1 YS-derived hematopoietic contrast, pluripotent stem cells are already widely used in inves- progenitors, with little, if any, contribution from hematopoietic tigations of embryonic development, have undergone directed progenitors arising later in embryonic development. Recent stud- differentiation into astrocytes and neuronal subtypes of the CNS, ies confirmed our findings (Schulz et al., 2012; Kierdorf et al., and even been used for cellular transplantation therapies for neu- 2013). In particular, the latest study from Kierdorf refined the rodegenerative diseases (Park et al., 2008a; Kiskinis and Eggan, characterization of the YS precursors that give rise to microglia 2010; Wu and Hochedlinger, 2011; Ben-David et al., 2012). and identified them as early E8 primitive c-kit erythromyeloid The exciting discovery that terminally-differentiated somatic cells + − YS precursors which develop into CD45 c-kitloCX3CR1 cells can be reprogrammed into an embryonic-like “induced pluripo- before their maturation and migration into the developing brain tent stem cell” (iPSC) has also opened up new possibilities + + as CD45 c-kit-CX3CR1 cells (Kierdorf et al., 2013). for disease modeling and developing patient-specific therapies Altogether, these studies conclusively demonstrated that prim- (Takahashi and Yamanaka, 2006; Okita et al., 2007; Takahashi itive macrophages are the embryonic source of the steady-state et al., 2007; Wernig et al., 2007; Yu et al., 2007; Park et al., adult microglial population, which was particularly interesting 2008b). as it implied that microglia not only have a unique func- Both embryonic stem cells (ESCs) and iPSCs have vir- tional specialization within the CNS, but also a unique ori- tually unlimited expansion potential, can be cultured under gin, arising from YS progenitors that maintain themselves by defined conditions to ensure reproducible and scalable differ- proliferating in situ throughout adulthood (Figure 1). Beyond entiation protocols, and are amenable to genetic manipula- the case of microglia, it also provided startling evidence for a tion to create tools for deeper functional studies. Large-scale broader conclusion, that primitive macrophages are the ulti- in vitro generation of microglia would also allow us to per- mate source of a functional immune compartment that persists form high-throughput genetic screens aiming to uncover key throughout adulthood. However, the case of the microglia seems transcription factors responsible for specifying the microglial to be unique, as other fetal macrophage populations in the phenotype. Even more attractively, we could potentially dif- embryo will mostly be replaced by fetal liver-derived monocytes ferentiate iPSCs from human patients to re-create a “disease that seed the tissues later and differentiate into macrophages, in a dish,” forming a crucial bridge between animal models as we have recently shown in the case of Langerhans cells (which are often deficient) and pathological disease states in (Hoeffel et al., 2012). Lack of differentiation of fetal liver- humans. Thus far, efforts to recapitulate neurological disease derived monocytes into microglial progenitors could result from features in vitro from human iPSCs have mainly focused on their lack of intrinsic differentiation potential or lack of access the afflicted neurons (Dimos et al., 2008; Park et al., 2008b; to the developing brain. Corroborating the latter hypothesis, Lee et al., 2009; Soldner et al., 2009; Marchetto et al., 2010). the BBB in rodents is starting to be established at approx- The development of more complex disease models incorpo- imately E13.5, at the time of fetal liver monocyte release rating multiple cell types, particularly microglia, remains a into the blood circulation (Daneman et al., 2010), but after challenge. YS-derived macrophages start to invade the neuro-epithelium In contrast to the numerous protocols for efficient gener- from E9.5 (Ginhoux and Merad, 2010), possibly restricting ation of astrocytes and neurons from pluripotent stem cells the access of fetal liver-derived cells to the embryonic brain (Lee et al., 2000; Zhang et al., 2001; Hu et al., 2010), there is (Figure 1). little literature reporting methods for obtaining phenotypically- correct microglia from the same cell sources. An early study MOVING TOWARD HARNESSING MICROGLIA TO IMPROVE on the differentiation of mouse ESCs (mESCs) into CNS HUMAN HEALTH cells in retinoic acid-induced embryoid bodies (EBs) showed It is well established that microglia are intimately involved in the that incidental cells expressing microglial markers were gen- pathology of neurological disease. However, efforts to elucidate erated, in addition to neurons and astrocytes (Angelov et al., the specific roles of microglia, their activation phenotypes and 1998). This preliminary success was the motivation for sub- how they can be harnessed to ameliorate disease, are hampered by sequent attempts to generate microglia from mESCs via neu- the lack of access to sufficient numbers of cells for comprehensive ronal differentiation strategies (Tsuchiya et al., 2005; Napoli in vitro studies. Isolation of primary rodent microglia is gener- et al., 2009). In brief, mESCs were induced to differentiate as ally achieved either by cell sorting or stepwise cell culture, both EBs following withdrawal of leukemia inhibitory factor (LIF), Frontiers in Cellular Neuroscience www.frontiersin.org April 2013 | Volume 7 | Article 45 | 8 Ginhoux et al. Origin and differentiation of microglia and then progenitors were expanded and further differenti- recapitulate YS hematopoiesis in vitro (Figure 2). In the mouse, ated with neuronal-supportive media and cytokine stimulation. hemangioblast precursors migrate from the posterior primi- low + After 21–50 days CD45 /CD11b putative microglia-like cells tive streak into the YS proper, where they form the blood were observed in these cultures; they expressed surface mark- islands and surrounding endothelial cells (Huber et al., 2004). ers consistent with primary microglia, responded to classical YS hematopoiesis yields primitive erythroblasts as early as E7.0, immune activators such as lipopolysaccharide and interferon-γ, followed by definitive erythroblasts and macrophage progeni- and appeared to survive implantation into the mouse brain. tors between E8.5–9.0 (Palis et al., 1999). However, it has been However, the low yields and prolonged culture period required, known for some time that these developmental stages can be together with our current understanding of the ontogeny of closely mirrored in mESC differentiation in vitro, specifically microglia, suggest that these microglia-like cells perhaps are in terms of the kinetics of hematopoietic gene expression, as arising as a side population in the neuro-ectodermal differ- well as the order in which hematopoietic progenitors appear. In entiation process. On the other hand, we should not dis- two modalities of differentiation, either in a co-culture with a count the possibility that neural cells present in these cultures hematopoietic-supportive stromal cell layer such as OP9 cells, might have provided a signaling milieu supportive of genuine or as EBs, mESCs sequentially generate in vitro equivalents of microglial maturation. For example, brain- and bone marrow- the primitive streak, hemangioblast, and YS hematopoietic pro- derived Mac-1 progenitors co-cultured on a supportive layer genitors (Risau et al., 1988; Wiles and Keller, 1991; Nakano of astroglial cells proliferated and matured into microglial-like et al., 1996; Ogawa et al., 1999; Kennedy and Keller, 2003; cells (Alliot et al., 1991). Sievers and colleagues also showed Hirai et al., 2005). These processes in mESCs are controlled that co-culture with astrocytes induced blood monocytes and by the same molecular regulators as those operative during spleen macrophages to adopt a ramified morphology akin to early hematopoiesis in vivo. For example, mouse embryos with microglia (Sievers et al., 1994a,b). In lightof our currentunder- targeted gene disruption of the hematopoietic master regula- standing that adult microglia originate as primitive macrophages tor SCL/Tal-1 have no YS hematopoiesis and fail to develop from the embryonic YS, a more effective strategy for approach- beyond E9.5 (Robb et al., 1995; Shivdasani et al., 1995); likewise ing pluripotent stem cell differentiation may be to attempt to abrogation of SCL expression in both mouse and human ESCs FIGURE 2 | Strategy for directed differentiation of microglial onset of circulation, primitive macrophages exit the yolk sac and seed precursors from pluripotent stem cells. Microglial differentiation the developing brain, forming microglia. Likewise, pluripotent stem cells + + in vitro can be achieved by recapitulating the steps of yolk sac can be differentiated into Bry Flk cells with hemangioblast properties; hematopoiesis. Hemangioblast cells arise in the posterior primitive these cells are then further differentiated into both primitive and streak and migrate into the yolk sac, giving rise to the blood islands. definitive eryothroblasts and primitive macrophages. In vitro-derived Primitive erythroblasts are observed from E7.0–7.5, followed by primitive macrophages may be the functional equivalent to primitive definitive erythroblasts and primitive macrophages at E8.5. At the microglia in the embryo. Frontiers in Cellular Neuroscience www.frontiersin.org April 2013 | Volume 7 | Article 45 | 9 Ginhoux et al. Origin and differentiation of microglia completely represses early hematopoietic specification (D’souza of primitive macrophages and their YS derivation throughout et al., 2005; Real et al., 2012). Similarly, PU.1 KO ESCs could not evolution and across diverse species suggests that microglia be differentiated into macrophages (Henkel et al., 1996; Anderson play an important physiological role in the development of the et al., 1998), consistent with the finding that PU.1 KO embryos CNS. Furthermore, microglial cells are present in all stages of lack both macrophages and microglia (McKercher et al., 1996). brain development, including the early prenatal stages of neu- However, useful the mouse developmental model may be, the ronal circuit building as well as the post-natal stage of synapse paucity of equivalent human in vivo models makes it imperative elimination. to develop accurate in vitro alternatives using human pluripotent An earlier report had highlighted that, in contrast to their stem cells. Classical lineage tracing studies of blood development broad distribution in the adult brain, embryonic microglia have a in human embryos showed that many essential features of YS strikingly uneven distribution during embryogenesis (Perry et al., hematopoiesis are conserved from mice to humans (Migliaccio 1985). Microglial cells accumulate in hot spots that were initially et al., 1986; Huyhn et al., 1995; Palis and Yoder, 2001). As was the proposed to be most likely related to the clearance of apoptotic case in the mouse system, human pluripotent stem cells could also bodies and the remodeling of brain tissues. In light of the recent be differentiated into cells representative of the early hematopoi- work which has shown that microglia contribute to the control etic developmental stages, albeit with extended kinetics (Wang of synaptic connections, it will be interesting to verify whether et al., 2004; Zambidis et al., 2005; Kennedy et al., 2007), sug- such hotspots co-localize with areas crucial for neuronal devel- gesting that hematopoietic cell emergence in the YS might be opment. This will indicate that microglia play an important role closely modeled by human ESC differentiation (Zambidis et al., in development of neuronal circuits of the brain and proposes 2005). more questions to be answered regarding the integrated develop- Given their direct ontogenetic relationship, we postulate ment of the neural and immune systems. Such questions also have that techniques for directing primitive macrophage fate spec- tremendous implications beyond the simple biology of microglia. ification from pluripotent stem cells will also yield microglial Do defects affecting microglial development have a long-term precursors. Our proposed approach is to further refine pro- impact on the functional vulnerability of CNS? And do defects in tocols for recapitulating YS hematopoiesis, with the aim of microglial function perhaps contribute to synaptic abnormalities increasing the yield of primitive macrophages and levels of seen in some neurodevelopmental disorders? In support of such reproducibility. Serum-based protocols introduce intrinsic vari- hypotheses, prenatal inflammation, which triggers the activation ability, so we will turn to a defined serum-free and feeder cell- of microglia, is thought to be a risk factor for the development free procedure, using only specific combinations of cytokines of neuropsychiatric disorders such as schizophrenia and autism to replicate the developmental signals during embryogenesis. spectrum disorders in the unborn child (Patterson, 2009, 2011). The key challenge in this approach will be to screen appro- Importantly, embryonic microglia will maintain themselves priate combinations of factors as well as the time window until adulthood via local proliferation during late gestation and for treatment. Candidate primitive macrophage/microglial cells post-natal development as well as in the injured adult brain can then be isolated based on surface marker expression, and in reaction to inflammation. They are unlikely to be replaced assayed for microglial-appropriate phenotypes such as response by blood-derived monocytes or any bone marrow-derived cells. to immune stimulation, morphological analysis and phago- Nevertheless, during certain inflammatory conditions found for cytic ability (Giulian and Baker, 1986; Sedgwick et al., 1991). example after bone marrow transplantation or in chronic neu- Eventually, the ability to engraft within the brain, with classic rodegenerative diseases such as Multiple Sclerosis and Alzheimer’s resting microglial morphology will be the true test of successful disease (Simard and Rivest, 2006; Jung and Schwartz, 2012), the differentiation. recruitment of monocytes or other bone marrow-derived progen- itors can supplement the microglial population to some extent, CONCLUSION but we do not understand whether these cells persist and become The “origin of murine microglia controversy” is now resolved integrated, or are a temporary addition to the endogenous pop- in steady state conditions and in a few mouse models of CNS ulation. The interactive dynamic between embryonic and adult pathologies. We have learned that microglia arise from YS microglial populations requires further study: We need now to macrophages that seed the brain rudiment from the cephalic understand to what extent the endogenous microglia is replaced, mesenchyme very early during development, as predicted ear- where and how it is done, and if engrafted cells have a selec- lier by the founder of the microglia field, Pio del Rio-Hortega. tive advantage over the endogenous microglia, how well they Importantly, embryonically-derived microglia will maintain “compete” against the endogenous embryonic microglial popu- themselves until adulthood. While much progress has been made lation and how long they will persist. Finally, we do not know yet in terms of our understanding of both the origin and importance precisely if these bone marrow-derived microglia can fulfill the of microglia, many questions remain unanswered. functional roles of the endogenous population. As discussed before, this knowledge may have implications ACKNOWLEDGMENTS for the use of embryonically-derived microglial progenitors in the treatment of brain inflammatory diseases. Moreover, these This work was supported by the Singapore Immunology Network results have fundamental implications for the understanding of core grant. We thank Dr. L. Robinson for critical review and microglial function in CNS development. First, the conservation editing of the manuscript. Frontiers in Cellular Neuroscience www.frontiersin.org April 2013 | Volume 7 | Article 45 | 10 Ginhoux et al. Origin and differentiation of microglia Bertrand, J. Y., Jalil, A., Klaine, M., can be differentiated into motor zebrafish embryo. 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Conflict of Interest Statement: The are credited and subject to any copy- Wernig, M., Meissner, A., Foreman, Yu, J., Vodyanik, M. A., Smuga-Otto, authors declare that the research right notices concerning any third-party R., Brambrink, T., Ku, M., K., Antosiewicz-Bourget, J., Frane, J. was conducted in the absence of any graphics etc. Frontiers in Cellular Neuroscience www.frontiersin.org April 2013| Volume7| Article45 | 14
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