Basic NeuroscienceA novel in vitro human microglia model: Characterization of human monocyte-derived microglia
Graphical abstract
Highlights
► New culture protocol for the generation of human monocyte-derived microglia in vitro. ► Morphological, phenotypic and functional characterization of human monocyte-derived microglia. ► Expression of surface markers by human microglia. ► Chemokine receptors expression pattern in human microglia. ► Comparison of human microglia with monocytes and dendritic cells.
Introduction
Microglia are resident innate immune cells of the central nervous system (CNS) and play a crucial role in maintaining the healthy physiological homeostasis. Microglia also contribute substantially to inflammation in response to injury, toxins, pathogens and degenerative processes (Streit, 2001, Streit, 1996, Howell et al., 2010, Lee et al., 2010, Politis et al., 2011). Microglia are characterized by a quick response to various stimuli, resulting in activation with rapid changes in morphology, phenotype and function. Morphological changes include shortening of cell processes and cellular hypertrophy. To date, there is no specific microglia marker clearly separating this cell type from other cells of the monocyte/macrophage lineage. Some studies suggested that primary microglia could be distinguished from other tissue macrophages according to expression levels of markers such as CD11b and CD45 (Aloisi et al., 2000, Ford et al., 1995). Under healthy condition, resting microglia with typical ramified morphology show low expression levels of these common myeloid lineage markers (Nimmerjahn et al., 2005). However, microglia have been shown to share expression of surface markers common to other immune cells of the macrophage family such as CD45, CD14, MHC-class II, CD68, immunoglobulin Fc receptors and β2 integrins (Lambertsen et al., 2009, Wirenfeldt et al., 2005). Expression levels of these surface markers may depend on the inflammatory status of the CNS and on microglia activation. Similarly, functional inflammatory changes are dominated by secretion of pro-inflammatory cytokines, chemokines, neurotrophic factors and up-regulation of corresponding receptors, as well as production of nitric oxide and reactive oxygen intermediates (Aloisi, 2001, Tambuyzer et al., 2009).
Microglia have recently been shown in a mouse model to originate from a myeloid yolk sac population during embryogenesis (Ginhoux et al., 2010). In humans, the microglia origin is still not known. Furthermore, little is known about the turnover rate of microglia in the healthy CNS. It has been postulated that proliferation of the resident microglia population is rather low and there may be immigration of bone marrow derived precursor cells into the CNS. Invasion of bone-marrow derived microglia have been shown to migrate into the CNS using chimeric irradiated mice (Simard et al., 2006, Zhang et al., 2007). However, the proportion of bone-marrow progenitor cells in replenishment of microglia is controversial.
A variety of cytokines have been shown to contribute to microglia development and differentiation. Colony stimulating factor-1/M-CSF and its receptor CSF-1R play an important role in the development of macrophage populations (Chitu and Stanley, 2006). Recently, Ginhoux et al. (2010) reported the constant absence of microglia throughout life in CSF-1R deficient mice, indicating that M-CSF is essential for microglia development and differentiation. In addition, granulocyte-macrophage colony stimulating factor (GM-CSF) also contributes to microglia development and differentiation (Aloisi et al., 2000, Esen and Kielian, 2007). Both, M-CSF and GM-CSF have crucial effects on proliferation and survival of primary human fetal and adult microglia in culture with GM-CSF having a greater impact on proliferation (Esen and Kielian, 2007, Lee et al., 1994). M-CSF plays a crucial role in the final maturation stage of microglia. In op/op mice, a model of human osteopetrosis with a functional mutation the M-CSF gene, the number of microglia are decreased and the cells are smaller and have impaired activation ability in response to injury (Kalla et al., 2001, Węgiel et al., 1998). Importantly, GM-CSF or interleukin-3 does not substitute for M-CSF (Blevins and Fedoroff, 1995).
Nerve growth factor (NGF), a member of neurotrophins, has also been shown to act on the proliferation and survival of microglia (Zhang et al., 2003). NGF binds and acts through the P75 receptor, which has been shown to be expressed in microglia in multiple sclerosis lesions (Valdo et al., 2002). It has also been shown that microglia express the nerve growth factor receptor TrkA (Tonchev, 2011). NGF induces migration of the microglia cell through activation of the TrkA (TrkA activation) (De Simone et al., 2007). NGF is expressed by activated microglia, astrocytes and hippocampal neurons (Friedman, 2000, Saez et al., 2006, Tonchev, 2011). However, inflammation certainly induces neurotrophin secretion by human microglia (Heese et al., 1998, Nakajima et al., 2001).
Chemokines have also been shown to play an important role in microglia biology. Monocyte chemoattractant protein-1 (MCP-1), or CCL2, is one of the prominent chemokines in the regulation of the microglia migration to the site of inflammation in experimental models (Leonard et al., 1991, Zhang et al., 2007). CCL2 acts through its specific receptor CCR2 which is expressed by microglia and other cells of monocytic lineage, such as macrophages and dendritic cells (Rebenko-Moll et al., 2006). CCL2 acts also through CCR4, but little information is available about CCR4 expression in microglia (Craig and Loberg, 2006, Zhang et al., 2006). Studies on normal rat CNS showed that CCL2 is constitutively expressed by astrocytes and neurons and it can be found in various brain regions, including the cerebral cortex, the hippocampus and the hypothalamus (Banisadr et al., 2005). CCL2 is also secreted by astrocytes and neurons under inflammatory condition (Banisadr et al., 2005, Farina et al., 2007, Tanuma et al., 2006). It has been shown that CCL2 is secreted by both undamaged and damaged spinal sensory neurons in rat, resulting in activation of spinal microglia and initiating neuropathic-like pain (Thacker et al., 2009). Up-regulation of CCL2 expression during Alzheimer's disease and multiple sclerosis correlates with activation of microglia and may contribute to pathogenesis of neurodegenerative diseases (Conductier et al., 2010, Simpson et al., 2000a). In that respect, CCL2 contributes to recruitment of mononuclear cells into the inflamed CNS, followed by activation of the microglia. Zhang et al. (2007) showed that CCL2 can induce spinal microglia activation in mice. They also reported in chimeric mice that CCL2 recruits bone marrow-derived macrophages, which proliferate and differentiate into microglia in the spinal cord and induce inflammation and microgliosis after nerve injury. In experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis, CCR2 deficient mice do not develop the mononuclear cell infiltration and inflammation. Indeed, no chemokines increase was detected in the CNS in that model (Izikson et al., 2000). On the other hand, CCR2 deficiency in a mouse model of Alzheimer's disease impairs significantly microglia accumulation at sites of plaque formation, resulting in a decrease of β-amyloid (Aβ) clearance and in accelerating early disease progression (El Khoury et al., 2007).
In vitro culture of mouse and human microglia has often been used as a model by various researchers. Thereby, astrocyte-conditioned medium (ACM) has been used to keep microglia in culture with a ramified resting morphology. ACM contains M-CSF, GM-CSF and transforming growth factor β (TGF β), all known to be secreted by astrocytes. Microglia lose their ramified morphology in the presence of antibodies against the mentioned cytokines, indicating that M-CSF and GM-CSF are essential for microglia culturing (Schilling et al., 2001). GM-CSF, even at low concentration in serum free medium, keeps microglia at a resting state with morphological ramifications (Fujita et al., 1996). A recent study reported the proliferative effect of CCL2 on neonatal and embryonic primary rat microglia in culture without inducing additional changes in morphology, phenotype and cytokine expression (Hinze and Stolzing, 2011).
Most interestingly, murine bone marrow-derived precursor cells can be differentiated towards microglia-like cells when cultured in the presence of astrocytes or in mixed glial cultures. Thereby, M-CSF alone is not sufficient for microglia differentiation, which indicates the importance of additional astrocyte-derived factors (Noto et al., 2010). Differentiation of murine bone marrow stem cells toward microglia-like cells has recently also been successful, using ACM supplemented with GM-CSF (Hinze and Stolzing, 2011). Culturing fetal and adult human microglia in the presence of GM-CSF induces functional maturation towards an antigen presenting cell type, especially in adult microglia. However, GM-CSF does not induce maturation of microglia to acquire the complete phenotype and function of mature dendritic cells (Lambert et al., 2008, Re et al., 2002).
In vitro studies on microglia used mainly primary microglia culture from embryonic or neonatal murine brains (Bassett et al., 2012, Hinojosa et al., 2011). In human, brain-derived microglia is difficult to obtain for ethical reasons. In addition, only low numbers of cells are collected. Also, fetal microglia seems to be quite different from adult microglia. Only few human microglia cell lines have been generated, including HMO6 cells (Nagai et al., 2005) and HMC3 cells (Janabi et al., 1995). These cell lines cannot be considered as an optimal model for microglia cells due the significant modification in morphology and function as result of genetic manipulation and long-term culture. Therefore, a more convenient and more appropriate model for in vitro microglia human studies is still missing.
Most importantly, Leone et al. (2006) showed that human blood-derived monocyte, cultured with astrocytes-conditioned medium (ACM), acquired the ramified morphology of microglia and expressed substance P, calcium binding protein Iba1 and dimly MHCII, three typical characteristics of microglia. There are also reports showing successful differentiation of rat blood monocytes towards microglia using ACM (Schmidtmayer et al., 1994, Sievers et al., 1994). Recently, Hinze and Stolzing (2011) showed the differentiation of murine bone marrow stem cells towards microglia-like cells in the presence of ACM with similar phenotypic and functional properties to primary brain-derived microglia cultures. In the light of these data, we developed a new in vitro human microglia model using human blood peripheral mononuclear cells, a serum-free culture condition and a panel of factors, including M-CSF, GM-CSF, NGF and CCL2. This new human microglia model has then been assessed for morphology, phenotype, and function.
Section snippets
Cell Isolation
Human blood mononuclear cells (PBMC) were isolated from buffy coats (50 buffy coats were used for this study) of healthy donors (Australian Red Cross Blood Service (ARCBS), Perth, WA, Australia; ethics approval was granted by UWA and ARCBS) using Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) as published before (Meagher et al., 2005). To obtain monocytes (adherent PBMC), the isolated blood cells were cultured in T25 tissue culture flasks (Sarstedt, Numbrecht, Germany) (2 × 106 to 5 × 106
Morphological and phenotypic changes of monocyte-derived microglia in culture
Adherent PBMC, representing mainly the monocytic population of blood leukocytes, were cultured for up to 2 weeks in the presence M-CSF, GM-CSF, NGF-β and CCL2, in RPMI 1640 without addition of serum to create optimally standardized conditions. The cells were characterized for morphological changes over the period of 2 weeks in culture (Fig. 1). The well established HMC3 human microglia cells line was used as comparison. After 5 days in culture, the small (10–15 μm diameter) round-shaped and
Discussion
Microglia play crucial roles in maintaining homeostasis and contributing to neuroinflammation in response to any disturbances, such as injuries, toxins and pathogen (Streit, 2001, Streit, 1996). To date, in vitro studies on microglia have used mainly primary cells derived from embryonic or neonatal murine and human brains (Bassett et al., 2012, Hinojosa et al., 2011). This study provides the first simple, standardized and reliable protocol to generate human microglia-like cells from blood
Conclusion
We developed a new in vitro protocol for the generation of human microglia from blood monocytes using a serum-free culture condition and a novel mixture of 4 recombinant human cytokines, i.e. M-CSF, GM-CSF, NGF and CCL2. Detailed characterization of M-MG revealed a cell population representing resting microglia with their specific morphological, phenotypic and functional properties. The described protocol is easy to handle, well standardized and very reproducible, as it uses only well defined
Conflict of interest statement
None of the authors have any conflict of interest to disclose.
Acknowledgements
Microscopes were provided by Cell Central, School of Anatomy, Physiology and Human Biology. In that respect, we thank Guy Ben-Ary and Steve Parkinson for their excellent support. The authors acknowledge the facilities (Confocal microscopy and flow cytometry), scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, a facility funded by the University, State
References (77)
- et al.
Structure and function of the blood–brain barrier
Neurobiol Dis
(2010) - et al.
Effects of methylmercury on the secretion of pro-inflammatory cytokines from primary microglial cells and astrocytes
NeuroToxicology
(2012) - et al.
Colony-stimulating factor-1 in immunity and inflammation
Curr Opin Immunol
(2006) - et al.
Induction of macrophage-derived chemokine/CCL22 expression in experimental autoimmune encephalomyelitis and cultured microglia: implications for disease regulation
J Neuroimmunol
(2002) - et al.
The role of monocyte chemoattractant protein MCP1/CCL2 in neuroinflammatory diseases
J Neuroimmunol
(2010) - et al.
NGF promotes microglial migration through the activation of its high affinity receptor: modulation by TGF-[beta]
J Neuroimmunol
(2007) - et al.
Astrocytes are active players in cerebral innate immunity
Trends Immunol
(2007) - et al.
Regulation of chemokine receptor expression in human microglia and astrocytes
J Neuroimmunol
(2003) - et al.
Blood monocytes consist of two principal subsets with distinct migratory properties
Immunity
(2003) - et al.
Establishment of human microglial cell lines after transfection of primary cultures of embryonic microglial cells with the SV40 large T antigen
Neurosci Lett
(1995)
CX3CR1 deficiency alters microglial activation and reduces beta-amyloid deposition in two Alzheimer's disease mouse models
Am J Pathol
Chemokines, mononuclear cells and the nervous system: heaven (or hell) is in the details
Curr Opin Immunol
Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease
Neuron
Expression of the [beta]-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue
J Neuroimmunol
Expression of the β-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue
J Neuroimmunol
CCL5 induces a pro-inflammatory profile in microglia in vitro
Cell Immunol
Microglia and macrophages in the developing CNS
Neurotoxicology
CCL2 is a key mediator of microglia activation in neuropathic pain states
Eur J Pain (London, England)
Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster
Trends Immunol
Reduced number and altered morphology of microglial cells in colony stimulating factor-1-deficient osteopetrotic op/op mice
Brain Res
Neurotrophins regulate proliferation and survival of two microglial cell lines in vitro
Exp Neurol
Immune function of microglia
Glia
Functional maturation of adult mouse resting microglia into an APC is promoted by granulocyte-macrophage colony-stimulating factor and interaction with Th1 cells
J Immunol
Characterization of chemokines and their receptors in the central nervous system: physiopathological implications
J Neurochem
Highly regionalized neuronal expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) in rat brain: evidence for its colocalization with neurotransmitters and neuropeptides
J Comp Neurol
Microglia in colony-stimulating factor 1-deficient op/op mice
J Neurosci Res
Titanium uptake, induction of RANK-L expression, and enhanced proliferation of human T-lymphocytes
J Orthop Res
CCL2 (Monocyte Chemoattractant Protein-1) in cancer bone metastases
Cancer Metastasis Rev
Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease
Nat Med
Lipopolysaccharide differentially regulates microglial trk receptor and neurotrophin expression
J Neurosci Res
Temporal expression and cellular origin of CC chemokine receptors CCR1, CCR2 and CCR5 in the central nervous system: insight into mechanisms of MOG-induced EAE
J Neuroinflamm
Effects of low dose GM-CSF on microglial inflammatory profiles to diverse pathogen-associated molecular patterns (PAMPs)
J Neuroinflamm
Human dendritic cells phagocytose and process Borrelia burgdorferi
J Immunol
Brain dendritic cells and macrophages/microglia in central nervous system inflammation
J Immunol
Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared
J Immunol
Neurotrophins induce death of hippocampal neurons via the p75 receptor
J Neurosci
Effects of GM-CSF and ordinary supplements on the ramification of microglia in culture: a morphometrical study
Glia
Fate mapping analysis reveals that adult microglia derive from primitive macrophages
Science
Cited by (64)
Human monocyte-derived microglia-like cell models: A review of the benefits, limitations and recommendations
2023, Brain, Behavior, and ImmunityCitation Excerpt :Whereas untreated monocytes typically show a round morphology, MDMi cells derived from both fresh and frozen PBMCs consistently grow branches from Day 5. By Days 10–14, cells typically appear as ramified microglia with a small soma and multiple processes (Banerjee et al., 2021; Bertin et al., 2012; Bortolotti et al., 2019; Etemad et al., 2012; Leone et al., 2006; Ohgidani et al., 2014; Ormel et al., 2020; Rai et al., 2020; Rawat and Spector, 2017; Sellgren et al., 2019; Sellgren et al., 2017). This is distinguishable from macrophages, which display a ‘fried-egg’ shape with an enlarged soma (Etemad et al., 2012).
Patient-Derived In Vitro Models of Microglial Function and Synaptic Engulfment in Schizophrenia
2022, Biological PsychiatryMicroglial innate memory and epigenetic reprogramming in neurological disorders
2021, Progress in NeurobiologyCitation Excerpt :Our lack of knowledge about microglial IIM compared with other myeloid cells, like peripheral macrophages, may be due to the difficulty of translating in vivo microglial physiology to in vitro experiments. However, recent strategies for microglial culturing, such as using those derived from human pluripotent stem cells (Abud et al., 2017; Douvaras et al., 2017; Haenseler et al., 2017; Muffat et al., 2016; Pandya et al., 2017), or the more unconventional monocyte-derived microglia (Etemad et al., 2012; Leone et al., 2006; Ohgidani et al., 2014; Rawat and Spector, 2017; Ryan et al., 2017), are promising and may encourage progress in IIM research. Furthermore, greater attention must be paid not only to post-translational histone modifications, which have been briefly addressed in microglial IIM, but also to DNA methylation, which is important for microglial functioning and is a mediator of immune memory in other innate immune cells.
Astrocytes and microglia in neurodegenerative diseases: Lessons from human in vitro models
2021, Progress in NeurobiologyA characterization of the molecular phenotype and inflammatory response of schizophrenia patient-derived microglia-like cells
2020, Brain, Behavior, and Immunity