Expression profiling of human glial precursors

BMC Developmental Biology BioMed Central Research article Open Access Expression profiling of human glial precursors James T Campanelli1, Robert W Sandrock1, Will Wheatley1, Haipeng Xue2, Jianhua Zheng2, Feng Liang2, Jonathan D Chesnut2, Ming Zhan3, Mahendra S Rao2 and Ying Liu*2 Address: 1Q Therapeutics, Inc. 615 Arapeen Dr., Ste. 102, Salt Lake City, UT 84108, USA, 2Invitrogen Corporation, 5781 Van Allen Way, Carlsbad, California 92008, USA and 3Bioinformatics Unit, Branch of Research Resources, National Institute on Aging, NIH, Baltimore, MD 21224, USA Email: James T Campanelli - ; Robert W Sandrock - ; Will Wheatley - ; Haipeng Xue - ; Jianhua Zheng - ; Feng Liang - ; Jonathan D Chesnut - ; Ming Zhan - ; Mahendra S Rao - ; Ying Liu* - * Corresponding author Published: 23 October 2008 BMC Developmental Biology 2008, 8:102 doi:10.1186/1471-213X-8-102 Received: 8 April 2008 Accepted: 23 October 2008 This article is available from: http://www.biomedcentral.com/1471-213X/8/102 © 2008 Campanelli et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: 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. Results: 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. Conclusion: 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. Background Glial differentiation is a specific developmental process that has been extensively characterized in both rodents and human [1]. 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 characterizaPage 1 of 12 (page number not for citation purposes) BMC Developmental Biology 2008, 8:102 tion 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 [8], 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 [9], including maturation of radial glia [10,11], a neuronastrocyte 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 [13]. 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) [5]. 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 [21]. http://www.biomedcentral.com/1471-213X/8/102 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. The isolation of the cells used in the present study is described as follows (Figure 1). Fetal forebrain was dissociated into a single cell suspension using enzymatic and mechanical methods according to published procedures and these cells are named as Starting Material: SM [23]. Purified precursors were obtained by single or double immunomagnetic cell sorting (magnetic cell sorting, MACS) using Miltenyi superparamagnetic bead technology according to the manufacturer's instructions. Briefly, the single cell suspension was incubated in anti-PSA-NCAM antibody (NCAM; 1:5000, Millipore, MAB5324) followed by incubation with bead conjugated anti-IgM antibody (Mitenyi). The NCAM positive cells were purified from the mixture first by passage through a magnetic field, these cells were collected and termed NE for NCAM Eluate. Unretained cells (NCAM negative, i.e., NCAM flow through) were collected afterwards and incubated with A2B5 antibody (1:1000, R&D Systems, MAB1416) followed by incubation with bead conjugated anti-IgM antibody before they were passaged through a second magnetic field. Then the retain (...truncated)