Expression profiling of human glial precursors
© Campanelli et al; licensee BioMed Central Ltd. 2008
Received: 08 April 2008
Accepted: 23 October 2008
Published: 23 October 2008
We have generated gene expression databases for human glial precursors, neuronal precursors, astrocyte precursors and neural stem cells and focused on comparing the profile of glial precursors with that of other populations.
A total of 14 samples were analyzed. Each population, previously distinguished from each other by immunocytochemical analysis of cell surface markers, expressed genes related to their key differentiation pathways. For the glial precursor cell population, we identified 458 genes that were uniquely expressed. Expression of a subset of these individual genes was validated by RT-PCR. We also report genes encoding cell surface markers that may be useful for identification and purification of human glial precursor populations.
We provide gene expression profile for human glial precursors. Our data suggest several signaling pathways that are important for proliferation and differentiation of human glial precursors. Such information may be utilized to further purify glial precursor populations, optimize media formulation, or study the effects of glial differentiation.
Glial differentiation is a specific developmental process that has been extensively characterized in both rodents and human . Embryonic stem cells generate a neuroectoderm that undergoes rostrocaudal and dorsoventral patterning and significant expansion to make a large number of neural stem cells (NSCs). NSCs first generate neuronal precursors and subsequently differentiate into glial precursors that further mature into two major types of glia: oligodendrocytes and astrocytes. During this process, multiple growth factors, transcription factors and molecules of different signal transduction pathways are involved, including fibroblast growth factors (FGFs), epidermal growth factors (EGFs), transforming growth factors beta (TGFβ) family members, Notch-Hes, inhibitor of DNA-binding (ID) family and Wnt pathways (reviewed in [2, 3]). Identification of these molecules (markers) is important for the isolation, purification, and characterization of human glial precursors, which may find extensive applications in transplantation studies and regenerative medicine.
Studies of glial development and differentiation in rodents have identified antibodies that recognize markers for isolating human glial cells [4–7]. For instance, A2B5, which reacts with ganglioside epitope GT3 , characterizes a glial precursor population [6, 7]. Upon further differentiation, A2B5+ cells give rise to oligodendrocyte precursors that express PDGFRα, Sox10, and NG2 [2, 3]. Multiple lineage pathways have been suggested for astrocyte development , including maturation of radial glia [10, 11], a neuron-astrocyte precursor and an oligodendrocyte-astrocyte precursor [7, 12–15]. Our laboratory has used antibodies to an extracellular matrix transmembrane protein CD44 to isolate astrocyte precursors from rat, mouse and human neural tissue. These CD44+ cells only gave rise to astrocytes in vitro and in vivo and did not differentiate into neuronal or oligodendrocytic lineages, even in conditions where glial progenitors or stem cells readily differentiated into such phenotypes . Growth factors that are important for glial differentiation include bone morphogenetic proteins (BMP) 2 and 4, leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF, [16–18]).
Although there are significant similarities in the differentiation of glia in chick, rodent and human systems, there are nevertheless differences as well. For example, A2B5, which characteristically labels glial precursors in rodent cells, has been reported to occasionally recognize cells of the neuronal lineage derived from human embryonic stem cells (hESCs) . This is not surprising since even between rat and mouse spinal cord, the A2B5 staining pattern is slightly different (Liu and Rao, unpublished). Likewise the pattern of CD44 expression in mouse and rat is distinct [19, 20] and the growth factor requirements of early precursors appears to differ as well .
We have previously created large scale gene expression profiles of hESCs, NSCs and multipotent neural precursor cells and identified a list of markers, which can be used to generate antibodies, design PCR primers, and characterize developmental stages for human neural cells [4, 22]. In this manuscript, we extended these studies to compare human glial progenitors to NSCs and more differentiated progeny. We confirmed the utility of some of the glial precursor markers that were defined in rodent, and proposed novel markers and signaling pathways that may be important for proliferation and differentiation of human glial precursor cells.
Preparation of fetal brain derived cell populations
Tissue from fetal cadavers of gestational age 20 to 23 weeks was procured by Procurement Specialists employed by Advanced Bioscience Resources (ABR) following Donor ID and Informed Consent SOPs, Donor Medical Record Review procedures. All protocols and procedures were reviewed by the Western Institutional Review Board and deemed that any further IRB oversight was unnecessary. Each population of cells included in the present study was derived from a different biological donor.
Human fetal tissue derived glial restricted precursor cell populations (NAE and AE, as described above) were harvested and grown on poly-L-ornithine and laminin-coated dishes for six days in vitro before RNA was extracted for analysis. Cells were grown in DMEM/F12 (with L-glutamine and 15 mM Hepes, no phenol red, Invitrogen), 0.01% human serum albumin, 1× N1 supplement (Sigma), Pen/Strep Fungizone, basic fibroblast growth factor (20 ng/ml), platelet derived growth factor AA (10 ng/ml), and Neurotrophin-3 (5 ng/ml). Human astrocyte precursor cells (hAPCs, CD44+) were derived from human neural precursor cells (ScienCell Research Laboratories) and cultured as described . Culture of other cell populations was described in Shin et al . Unpurified cells (SM) and NCAM+ cells (NE) were not grown in culture and RNA was extracted shortly after cell purification.
NAE, NE, AE and SM cells were seeded on chamber slides (LabTek) and grown overnight before they were fixed with 2% paraformaldehyde. For cell surface antigens, cells were blocked with detergent free blocking buffer (10% normal goat serum and 5% bovine serum albumin in PBS) and for internal antigens Triton X-100 and 0.05% Tween-20 were added to the blocking buffer. The following antibodies were used: A2B5 (1:1000), PSA-NCAM (1:5000), PDGFRα (BD Pharmingen, 1:200), NG2 (Millipore, 1:200), GFAP (DAKO, 1:4000), Tuj1 (Sigma, 1:4000), Nestin (BD Bioscience, 1:200) and NeuN (Sigma, 1:200). Appropriate fluorescent labeled secondary antibodies (Invitrogen) were used and nuclei were revealed by counter staining with DAPI.
Illumina bead array, statistical analysis and RT-PCR verification
Primers for RT-PCR
Human glial precursors and neuronal precursors can be isolated from fetal tissue
Samples included in this study
Unsorted fetal brain cells
A2B5+, PSA-NCAM- cells
A2B5+, PSA-NCAM- cells
A2B5+, PSA-NCAM- cells
A2B5+, PSA-NCAM- cells
Neurosphere from whole brain
Neurosphere from frontal brain
Human astrocyte line
Human Schwann line
Signal abundance of all samples
Signal ≥ 100
hAPC (replicate 1)
hAPC (replicate 2)
Fold change in the expression of selected known glial markers in glial precursors NAE versus neuronal precursors NE and unsorted population SM
Expression of neuronal specific genes is downregulated in glial precursors NAE versus neuronal precursors NE and unsorted population SM
Glial precursors show distinct gene expression profiles
Novel cell surface markers can be identified for glial precursors
In search of novel cell surface markers from A2B5+/NCAM- magnetically sorted glial precursor NAE cells, we compared NAE with unsorted fetal brain cells (SM), and with NCAM+ sorted neuronal precursor NE cells. We have found that the expression of several known glial specific cell surface markers, such as NG2 (chondroitin sulfate proteoglycan 4, melanoma-associated, CSPG4), was upregulated in NAE. BCAN, the gene encoding the proteoglycan molecule Brevican,, was found to be enriched in NAE as its expression was nearly 15-fold higher in NAE than in unsorted brain cells SM and 2–4 fold higher than in NCAM+ sorted NE and A2B5+ single sorted AE cells (the signal intensity was 6873.3 in NAE, 466.2 in SM, 2930.8 in NE and 1561.9 in AE). Another group of cell surface molecules that were differentially expressed by NAE were members of the integrin family, particularly integrin beta 5 (ITGB5) and integrin beta 8 (ITGB8). Both were expressed higher (15- and 6-fold, respectively) in glial precursors than in unpurified cells (the signal intensity of ITGB5 was 4712.6 in NAE and 301.7 in SM; the signal intensity of ITGB8 was 29.7 in NAE and 4.9 in SM).
Transcription factors and molecules of major signal transduction pathways show distinct profile in A2B5+/NCAM- glial precursors
Screening of transcription factors, cell surface markers, receptors, and extracellular matrix molecules showed distinct profile for glial precursor population NAE. Sixty-one transcription factors were expressed at 2-fold or higher in glial precursors NAE, which included known transcription factors that drove glial specification such as Olig2, ID3 and Nkx2.2. GJA1 (Connexin 43), a known marker for glial precursors, was detected 4-fold higher in glial precursors (signal intensity 375.3) than in neuronal precursors (signal intensity 96.2).
Important pathways in NAE
Two secreted frizzled-related proteins, SFRP1 and SFRP2, which are important antagonists of the Wnt pathway, were expressed three to five fold higher in the glial precursor NAE. On the contrary, a Wnt inducer, IL11, was also upregulated in NAE (signal intensity 62.8) and not detected in NSCs (WBFB, signal intensity <0). These data indicated that at least Wnt molecules were differentially regulated in NAE and direct evidence is needed to establish the role of SFRPs and other Wnt molecules in glial differentiation [30–32].
FGFs are important in both proliferation and differentiation of NSCs. Here we have found that FGF9 and FGF12 were highly expressed (3~14-fold) in glial precursors NAE compared to NSCs WBFB (signal intensity was 58.2 and 116.3 in NAE, and 16.9 and 8 in WBFB). Further examination of these two growth factors for their role in directing NSCs or glial precursors toward mature glia is warranted.
Steroids have been shown to affect and regulate glial gene expression . We have found that corticotropin releasing hormone (CRH or CRF) was highly expressed in NAE (signal intensity 1989.8) but was 48-fold lower in WBFB (signal intensity 41.2). Additionally, expression of nuclear receptor family members such as NR0B1, was upregulated in NAE as compared to WBFB as well.
Several cell cycle regulators and key genes of the MAPK pathway, CDKN2B (cyclin-dependent kinase inhibitor 2B, or p15) which inhibits CDK4, and CCNA1 (Cyclin A1), were found to be expressed at 7–10 fold higher in NAE than in WBFB.
The TGFβ pathway is a key pathway involved in glial differentiation [16, 34] and our findings were consistent with the published data. Expression of several key components of the TGFβ pathway, for example, nuclear receptor subfamily 0, group B, member 1 (NR0B1), bone morphogenetic protein 6 (BMP6), transforming growth factor beta-induced (TGFBI), and BMP and activin membrane-bound inhibitor homolog (BAMBI), was detected more than 10-fold higher in glial precursor NAE than in NSCs WBFB.
Cyclic AMP also plays an important role in neural and glial differentiation . Several cAMP related genes, including calbindin 1 (CALB1), proenkephalin (PENK), neuropeptide Y (NPY) and secretogranin II (chromogranin C) (SCG2) were highly expressed in NAE but were expressed much lower (7~20-fold) or were absent in the NSC population WBFB.
In this manuscript, we generated global gene expression profiles for human glial precursors, astrocyte precursors, neuronal precursors and NSCs isolated from fetal tissues using Illumina bead array. We focused on detailed marker and pathway analysis for glial precursors by comparing their profile to other populations.
Differential expression of markers on the plasma membrane of glial precursors was used to isolate these cells from a general population of fetal brain cells. The general cellular population was depleted for those expressing NCAM followed by enrichment of those cells that were recognized by the A2B5 antibody. The resultant population (A2B5+ and NCAM-, NAE) has been shown to have characteristics of glial precursors and give rise to only oligodendrocytes and astrocytes upon further differentiation in vitro and in vivo (Figure 2 and data not shown).
Generally accepted glial precursor markers such as Olig1, Olig2, Nkx2.2, ID2, ID4, and glutamate transporter genes were expressed higher in the glial precursor populations (Table 4, see Additional file 1). Expression data collected for glial precursors, astrocyte precursors, neuronal precursors, and NSCs showed that glial precursors NAE possessed a distinct gene expression profile, suggesting several core signaling pathways that might be important in glial differentiation. For instance, major components of the TGFβ pathway, BMP6, BMP7, TGFBI, and TGFBII, were upregulated in glial precursors when compared to NSCs or neuronal precursors. These data are consistent with previous observations that BMPs can induce glial differentiation [16–18].
We also found several novel pathways or molecules that might be involved in glial differentiation. For instance, the SLIT and NTRK-like family proteins (Slitrk). Slitrk's are integral membrane proteins, with two leucine-rich repeat (LRR) domains similar to Slit family and C-term domains similar to Trk proteins and might possess the Trk kinase function. Genes of Slitrk family are found to be predominantly expressed in the brain and glial tumors including astrocytoma, oligodendroglioma and glioblastoma . Consistent with these reports, three Slitrk proteins Slitrk1, Slitrk4 and Slitrk5, as well as Slit2 were differentially highly expressed in all glial precursor populations described in this work. These combined indicate that the Slit pathways are important to glial differentiation. Since Slitrk's are integral transmembrane proteins, these molecules may also serve as new cell surface markers for isolating glial precursors.
Similarly, Ephrins, proteins involved in axon guidance, were found to be differentially expressed in our glial precursor populations as well. For example, EPHA2, EPHA3, EPHA5, EPHB6 were expressed 2~4-fold higher in glial precursors than in NSCs or neuronal populations. In line with our data, the EphB2 receptor has recently been reported to be released at the neuron-glia interaction from neurons and endogenous EphB2 was expressed by glia simultaneously . In addition, the Ephrin family receptor tyrosine kinases were shown to be upregulated in response to CNS injury as part of the function of glia . The above mentioned pathways have previously been implicated in neuronal pathfinding and synaptogenesis. Their expression in glial lineages may reflect the growing body of evidence pointing to the importance of glia in neuronal development events [39, 40].
Differential expression of genes in the Pax-Six-Eya-Dach (paired box-sine oculis homeobox-eyes absent-dachshund) gene regulatory network was also observed. Dach was expressed 700 fold higher in glial precursors than NSCs (Figure 4 and see Additional file 1), and Eya2 and Six5 were expressed 2~4-fold higher in glial precursors than in neuronal precursors (see Additional file 1). The Pax-Six-Eya-Dach network has been shown to be important for inner ear and other sensory organ development [41–44], it may be worthwhile to initiate studies that will elucidate this pathway's role in glial biology.
Several extracellular matrix metalloproteinases showed differential expression in glial populations and other cell types. For instance, MMP7 was expressed 40- to 200-fold higher in glial cells than in NSCs (Figure 4 and see Additional file 1). This is not totally unexpected as MMP7 has been reported to be involved in the canonical Wnt pathway as well as during FGF2-mediated response . Expression of another metalloproteinase related molecule, tissue inhibitor of metalloproteinase 3 (TIMP3), was elevated in glial precursors as well (the signal intensity in NAE was 496.7). These data indicate that the metalloproteinase family may actively participate in glial differentiation.
Several frizzled-related protein molecules of the Wnt pathway, SFRP1, SFRP2 FRPB, IL11, and FRAT1, were highly expressed in glial precursors when compared to NSCs. However, similar expression was detected for these Wnt molecules in neuronal precursors, supporting the conclusion that these proteins are involved in general neural cell differentiation instead of glial specification.
Another gene that was brought to our attention was PENK (proenkephalin). It was expressed higher in glial precursors NAE and HOPC (signal intensities 400–3000) but was not detected in NSCs (signal intensity <0, Additional file 1). However, when we examined the expression levels of MAPK1 and MAPK3 (p38), major components of the MAPK-ERK pathway, which regulates PENK expression, no significant changes were found. Possible reasons might be that phosphorylation status or other posttranslational modifications are more important in determining the pathway activity than mRNA levels. To confirm this, proteomic analysis needs to be done as we and others have performed for several cell types including hESCs . This is worth mentioning as protein expression and function are regulated at multiple levels while the present work focuses on the mRNA levels only.
Genes relevant to the signal transduction pathways Akt, CNTF, SDF-1/CXCR4, PDGF, glial cell line-derived nerve growth factor (GDNF) and receptor tyrosine kinase (RTK) were found to be differentially expressed as well. As expected, members of the PDGF pathway were expressed higher in glial precursors. For the EGF receptor family, NRG1 and EGFR, ERBB3, and the downstream effector SOS were also observed to be differentially upregulated in glial populations as compared to NSCs and neuronal precursors.
Recently, several zinc finger proteins have been implicated in various processes of neural development and glial differentiation [47, 48]. Here we have found that zinc finger proteins Znf135, Znf136, Znf161, Znf24, Znf91, Znf435, Znf154, Znf232, and Znf219, were expressed as much as 14-fold or higher in glial precursors than in astrocyte precursors hAPC. Znf494, a zinc finger protein of unknown function  was expressed 415~663-fold higher in glial precursors than in NSCs. Conversely, another set of zinc finger proteins, Znf145, Znf537 Znf345, Znf370, Znf51 (Bcl6), Znf268, Znf375, and Znf373, were highly expressed in hAPC. The zinc fingers identified in this work may warrant further study.
Analyses of these expression data have led to the identification of novel cell surface markers which may be used for identification or isolation of glial precursors. Our results indicate that in addition to the well accepted surface marker NG2, Brevican and Integrin beta 5 and beta 8 are potential cell surface molecules for glial precursors. Brevican is a neural-specific chondroitin sulfate proteoglycan and is expressed in mature astrocytes and downregulated in adult rat spinal cords [50, 51]. β integrins have been reported to be involved in spinal cord injury and regeneration process. Both Brevican and integrins can be regulated by the TGFβ pathway [50, 52]. Our results show that Brevican expression is significantly increased in glial precursors. In line with this, expression of ADAMTS4, a gene that encodes a disintegrin and metalloproteinase that selectively cleaves Brevican/Lectican, also is elevated in the glial precursor population. A detailed time course study of its expression in glial development in the human fetal brain is underway to validate its utility as a marker for isolating glial precursors.
We have compared gene expression profile of glial precursors to neuronal precursors, astrocyte precursors and NSCs. Such information may be utilized to further purify glial precursor populations, optimize media formulation, or study the effects of glial cell differentiation.
staring material (unsorted fetal brain cells)
NCAM flow through: A2B5 Eluate (A2B5+: PSA-NCAM- cells)
A2B5 Eluate (A2B5+ cells)
NCAM Eluate (PSA-NCAM+ cells)
human oligodendrocyte precursors (A2B5+: PSA-NCAM- cells)
Neurosphere from whole brain
Neurosphere from frontal brain
human astrocyte precursor cells (CD44+ cells)
human embryonic stem cells
mouse embryonic stem cells
neural stem cells
fibroblast growth factor
epidermal growth factor
transforming growth factors beta
bone morphogenetic proteins
leukemia inhibitory factor
ciliary neurotropic factor
SLIT and NTRK-like family proteins
platelet derived growth factor
glial cell line-derived nerve growth factor
receptor tyrosine kinase
magnetic cell sorting
fluorescence activate cell sorting
This work was supported by Invitrogen Corporation and Q Therapeutics Inc. The authors would like to thank Dr. Steve Goldman, Dr. Ahmet Hoke, and Cognate Inc. for providing samples. We thank all members of our laboratories for constant stimulating discussions. MSR acknowledges the contributions of Dr. S. Rao that made undertaking this project possible.
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