Isolation and characterization of murine neural stem/progenitor cells based on Prominin-1 expression
Introduction
Neural stem cells (NSCs) are a unique population of CNS cells characterized by self-renewal and multilineage differentiation properties (Muller et al., 2006). NSCs reside in the germinal layers of the developing brain, initially in the early neuroepithelium, later in the ventricular (VZ) and subventricular zone (SVZ) during embryogenesis (Gotz and Huttner, 2005), and finally in some areas of adult brain such as the SVZ and the dentate gyrus of the hippocampus (Alvarez-Buylla et al., 2001).
Basic knowledge of NSC properties is of considerable relevance both for the understanding of CNS development and for cell-mediated therapies for neurodegenerative disorders, as NSCs represent a potential source of transplantable cells (Martino and Pluchino, 2006).
The neurosphere-forming assay has become a standard method to prospectively isolate neural stem and progenitor cells. It has been proposed that the ability to self-renew over an extended period of time is required to fulfil the criteria of a putative stem cell (Reynolds and Rietze, 2005). In fact, progenitors cells have a proliferative capacity that allows them to generate secondary or tertiary spheres upon passage, while NSCs presented self-renewal ability over longer period of time (more than five passages) (Reynolds and Rietze, 2005). Furthermore, neurospheres are a complex mixed population of NSCs, progenitors and differentiated cells (Reynolds and Weiss, 1992, Davis and Temple, 1994). The development of new strategies capable of identifying and isolating defined NSC populations in vivo and in vitro are, therefore, of particular importance. At present, different antigens have been associated to the NSC phenotype such as nestin (Lendahl et al., 1990), Musashi (Sakakibara et al., 2002), SOX2 (D'Amour and Gage, 2003) and SOX1 (Barraud et al., 2005). However, the intracellular distribution of these proteins limits their use as selective antigens. A strategy to circumvent this limitation is the use of a gene reporter such as green fluorescence protein (GFP) under the control of the promoters of these specific NSC genes. However, the need for cell transfection procedures often with viral vectors hampered this method (D'Amour and Gage, 2003, Kawaguchi et al., 2001, Keyoung et al., 2001).
Recent studies have aimed at selecting NSCs for a particular pattern of cell distribution in the FACS analysis based on cell size combined with a number of negative selection criteria (Rietze et al., 2001), such as the exclusion of the DNA binding dye Hoechst 33342 (side population) (Kim and Morshead, 2003) or the high aldehyde dehydrogenase (ALDH) activity combined with low side scatter (Corti et al., 2006). A consolidated cell purification strategy highly validated for purification of hematopoietic stem cells (HSCs) is the use of cell surface antigens suitable for MACS and FACS (Yin et al., 1997). In fact, the isolation of NSCs using the expression of the surface antigen Lewis X (Capela and Temple, 2002) in the adult mouse forebrain and Prominin (CD133) in human neural stem/progenitor cells (NSPCs) (Uchida et al., 2000) and cerebellum (Lee et al., 2005) has been already previously described.
Prominin is a five-transmembrane protein that was originally identified in mouse neuroepithelial stem cells (Weigmann et al., 1997). Remarkably, in neuroepithelial cells prominin immunoreactivity was associated mostly, if not exclusively, with microvilli and plasma membrane protrusions, and it was not detected in the planar areas of the apical plasma membrane. Because of this preferential localization, it has been proposed to name this antigen “prominin” (from the Latin word “prominere,” to stand out, to be prominent) (Weigmann et al., 1997). In humans, this protein was originally identified as an antigenic marker expressed on hematopoietic stem cells and referred to as CD133 antigen (Miraglia et al., 1997) and it has been used to enrich human HSCs and NSPCs (Uchida et al., 2000, Corbeil et al., 2000). Although this antigen displayed an identical membrane topology to mouse prominin (Weigmann et al., 1997, Miraglia et al., 1997), there was uncertainty as to their relationship (Corbeil et al., 1998, Miraglia et al., 1998) because of the low level of amino acid identity (Miraglia et al., 1997). The demonstration that both mouse prominin and the human CD133 antigen show similar cellular distribution and subcellular localization supports the conclusion that CD133 is the orthologue of murine prominin (Corbeil et al., 2000). For these reasons, the CD133 antigen was proposed to be called “prominin (mouse)-like 1” (PROML1) (Corbeil et al., 2000). The similar morphological pattern of CD133 distribution in human and mouse hematopoietic stem cells and neuroepithelial tissues has been observed, however few studies have been conducted to demonstrate that human and mouse CD133-positive cells present similar characteristics from a functional point of view. It has been recently described that murine postnatal NSPCs from cerebellum can be prospectively isolated based on their expression of the prominin-1 and the absence of markers of neuronal and glial lineages (lin−). Prominin+ lin− cells can form neurospheres and differentiate in vitro into the three neuroectodermal lineages astrocytes, oligodendrocytes and neurons. Furthermore, when transplanted in vivo in the cerebellum they can generate neurons and glial cells (Lee et al., 2005). Despite these data that support the utility of CD133 for perspective isolation of NSPCs from cerebellar tissues, the role of CD133 for the isolation of NSPCs in forebrain murine areas has never, to our knowledge, been investigated.
In this study, we therefore evaluated whether CD133 may represent a marker for the isolation of murine stem cells in developing and adult murine brain. We showed that CD133 antigen characterizes a cell population capable of generating neurospheres and differentiating into the three neuroectodermal lineages. Moreover, transplantation of the CD133 cell fraction in mouse brain evidences an efficient cell engraftment in the host tissue and differentiation into neurons. In conclusion, the CD133 antigen appears to be a useful marker for the isolation of brain-derived murine NSPCs.
Section snippets
Cell harvesting
Cells were isolated from the forebrain germinal zones of murine brain at embryonic day 12.5 (E12.5) and from adult forebrain subventricular dissection as described previously (Tropepe et al., 1999).
For the in vitro and in vivo experiments, we derived NSPCs either from C57BL/6 mice or from transgenic mice expressing an “enhanced” GFP cDNA under the control of a chicken β-actin promoter in all the tissues (TgN(ACTbEGFP)1Osb mice) (Okabe et al., 1997) or from B6.Cg-TgN(Thy1–YFP)16Jrs mice
Isolation of Prominin-1-positive cells
To determine whether Prominin-1 selection may be useful for NSPCs isolation, murine brain at embryonic day 12.5 (E12.5) and adult dissociated brain were stained with Prominin-1 and then analyzed by flow cytometry (Figs. 1A–F). Prominin-1 was expressed on 16.5 ± 4.6% of cells from E12.5 brain and on 3.2 ± 1.4% of adult brain-derived cells. Histochemical analysis of E12.5 brain with CD133 antibody showed a positive staining of the apical ventricular side of neuroepithelial cells (Fig. 1G). In the
Discussion
Prominin-1 (also named CD133) is a membrane glycoprotein with five transmembrane domains specifically associated with plasma membrane protrusions whose function is unknown at present (Jaszai et al., in press). Prominin-1 has been indicated as a marker for several stem cell populations including human HSCs (Yin et al., 1997) and NSPCs (Uchida et al., 2000). Indeed, human NSPCs can be directly isolated by using an anti-CD133 antibody (Uchida et al., 2000). Human CD133-positive cells form
Acknowledgments
This work was supported by research grants from the Italian Ministry of Health, Ricerca Finalizzata 2004 “Studio di protocolli di terapia cellulo-mediata nelle patologie neurodegenerative e nelle distrofie muscolari”, MIUR (Ministero Istruzione Università di Ricerca Scientifica) Italian Ministery PRIN 2007 “Molecular pathogenesis of the motoneuron disorders as a tool for the identification of novel biomolecular and cellular therapeutic targets”, FIRST 2006, Telethon grant: GGP06043 “Development
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