WO2004101772A2

WO2004101772A2 - Neural cells

Info

Publication number: WO2004101772A2
Application number: PCT/EP2004/005233
Authority: WO
Inventors: Andreas Bosio; Harold Cremer; Sandra Pennartz
Original assignee: Memorec Biotec Gmbh
Priority date: 2003-05-14
Filing date: 2004-05-14
Publication date: 2004-11-25
Also published as: WO2004101772A3

Abstract

Cell population comprising at least 5% neural stem cells, said stem cells being characterized by an expression of ASCT2.

Description

Neural cells

Field of the invention

The present invention relates to neural stem cells.

Background of the invention

Multipotent neural stem cells (NCS) represent the starting point for the generation of the myriad of diverse neuronal and glial cells, which make up the vertebrate nervous system (Williams et al., 1991) (Davis and Temple, 1994) (Weiss et al., 1996). In recent years, several research groups showed that such self- renewing multipotent precursors can be found not only in the developing organ- ism, but also in the adult brain (Gage, 2000).

Besides the developmental significance of NCS in neurogenesis their therapeutic potential is of great interest. Certain factors can induce their differentiation into diverse neural cell types, which could subsequently be transplanted to contribute to the treatment of Alzheimer's, Parkinson's disease and stroke, for example. Advantages versus other cell types like embryonic stem (ES) cells are

i) that NSC can be derived from the very patient and thus do not provoke rejection by the immune system after grafting, and ii) that their isolation is ethically unobjectionable.

NSC are multipotent; they are at least able to differentiate to astrocytes, oli- godentritic cells and neurons.

The precise localization of the stem cells' niche in the central nervous system is still unclear: Johansson et al. described ependymal cells along the lumen of the adult ventricular zone with features characteristic of multipotent stem cells (Johansson et al,, 1999), while Doetsch et al. identified a subpopulation of astro- cytes in the subventricular zone (SVZ, also known as subependymal zone) as NSC (Doetsch et al., 1999). The latter thesis was corroborated by further publications (Capela and Temple, 2002; Imura et al., 2003; Laywell et al., 2000). The identification and characterization of neural differentiation steps and the factors involved in controlling them is a prerequisite for understanding the NCS' role in neurogenesis and for their therapeutic usage. However, the isolation of adult NCS is at present not possible due to the poor characterization of this cell type and the lack of suitable markers (Morshead and van der Kooy, 2004). Neu- rospheres, which are clonal aggregates derived from NSC and enriched in NSC, contain diverse differentiating cells and thus would not fulfill the requirement of homogeneity (Gritti et al., 1996). A homogeneous cell population is essential for the generation of a reliable gene expression profile.

Summary of the invention

It is one of the object of the present invention to provide a cell population comprising neural stem cells.

It is another object of the present invention to provide a method for isolating such a cell population.

A further object is to provide a medicament comprising the cell population of the present invention.

The strategy for the identification of potential NCS markers is based on the hypothesis that NCS can be considered as a multipotent differentiation intermediate between pluripotent ES cells and lineage-restricted, polysialylated NCAM ex- pressing (PSA⁺) neuronal precursors. These two cell populations represent the developmental stages immediately up-stream and down-stream of NCS, respectively. There are already indications that ES cells and NSC have genes in common. Ramalho-Santos et al. demonstrated a "global overlap between genes expressed in ESC and NSC" (Ramalho-Santos et al., 2002).

PSA⁺ neuronal precursor cells were isolated from the adult mouse brain by fluorescent activated cell sorting (FACS) for transcriptome analysis (Pennartz et al., 2004). Bruce-4 ES cells were chosen for the second SAGE library because they are derived from the inbred mouse strain C57BL/6J (Kontgen, 1993) that was used for PSA⁺ cell isolation. Bruce-4 ES cells have been used in many gene tar- geting experiments showing excellent germ line transmission, which proves their pluripotent state. Prior to the RNA isolation of ES cells, the ratio of ES cells to contaminating fibroblasts was reduced from 60: 1 after initial plating to 80: 1 (1.25%).

SAGE (Serial Analysis of Gene Expression) (Velculescu et al., 1995) was used to perform a genome-wide expression screen in murine ES cells and PSA⁺ precursors. A SAGE library for adult total brain (ATB) was prepared. The selection criterion for potential NSC markers was the following expression pattern: high expression in ES cells, low or no expression in PSA⁺ precursors and ATB. The candidate genes were further analyzed using in situ hybridizations to determine if their cellular localization is compatible with the localization of neurogenic areas, the SVZ and the hippocampus.

The selection strategy for potential NSC markers is shown in figure 1. ES cells, NSC, PSA⁺ cells and ATB are shown in a hierarchical order in terms of developmental potential starting with the greatest one on the left. All cell types are ac- cessible and can be isolated homogeneously, except for the NSC because of a lack of marker genes. Given that there is an overlap in the genetic programs of ES cells and NSC, a group of genes that are highly expressed in ES cells and downregulated or absent in PSA⁺ cells and ATB should contain a subset of genes which are also expressed by the vanishingly small number of stem cells in the brain.

Isolation of adult murine PSA⁺ neuronal precursor cells

In the adult mammalian forebrain, large numbers of PSA⁺ neuronal precursors are permanently generated in the SVZ lining the lateral ventricle. From here, they migrate along a well-defined pathway, the rostral migratory stream (RMS), into the olfactory bulb (OB), (Altman, 1969; Lois and Alvarez-Buylla, 1994; Luskin, 1993). Expression of PSA-NCAM is found exclusively on these migrating precursors in the adult mouse brain. Therefore, a specific antibody, the α-PSA mAb menB (1: 100) (Rougon et al., 1986), allowed the pure isolation of this cell population by FACS (Figure 2). SAGE analysis for PSA⁺ cells and ES cells

SAGE is based on the generation of short DNA. segments -15 bp SAGE tags- at defined positions close to 3' termini, allowing the assignment of these tags to the corresponding individual transcripts (Blackshaw et al., 2001; Velculescu et al., 1995).

SAGE was performed for PSA⁺ cells as well as for C57BL/6J derived ES cells (Bruce-4) (Kontgen et al., 1993) (Tab. 1) (Pennartz et al., 2004). The mapping of SAGE tags to UniGene and Swissprot is consistent with that observed in other SAGE databases (Gunnersen et al., 2002) (Figure 3). Since SAGE analysis dis- plays the expression level of genes by absolute tag numbers, different SAGE projects can be directly compared. The other reference SAGE library contained tags from adult total brain (ATB), i.e. mature neuronal and glial cells. These identified genes have unambiguous identification numbers provided for example under the name "SAGEmap" from the National Centre for Biotechnology Informa- tion (NCBI) accessable under www.ncbi.nlm.nin.gov/sage.

Table 1

SAGE tag numbers and distribution of tag frequencies for PSA⁺ cells and ES cells

Selection of potential NSC marker genes

Provided that the genetic programs of ES cells and NSC overlap, some of the genes that are highly expressed in ES cells and downregulated or absent in PSA⁺ cells and ATB should also be expressed by stem cells in the brain. The selection of these genes from the three SAGE libraries was based on significantly differential upregulation (p<0.05) and 10-fold expression in ES cells versus PSA⁺ cells and ATB (Tab. 2). Genes that code for cell surface proteins represent the most promising potential NSC markers since antibodies generated against them would allow an isolation of the cells using MACS or FACS.

Table 2

SAGE tag Tag number Tae number Tag number UniGene Annotation Memorec ES cells PSA+ cells ATB number number

GTGGACTCAAT 108.7 0.0 0.7 Mm.l756δl Ifitml, interferon induced transmembrane OP219H04 protein 1 (before RIKEN 1110036C17)

AGAGGTTGCCC 34.3 0.0 0.7 Mm.27134 RKEN 2610033C09 OP237G07

TACAGATCACG 34.4 0.9 0.0 Mm.4770 Frizzled-7 OP015E02

AGTCTCGAGGG (2; ) 37.2 2.8 2.1 Mm.1056 ASCT2, Slcla₅ OP011E02

CCCCACCCCAC 22.8 0.0 2.2 Mm.l₅3963 KIAA0152 (before CD8 antigen) OP279A06

CGTATCCTGTC 14.3 0.0 0.0 Mm.90003 Gap junction protein beta3, Connexin31 OP11D09

AATCACTGTGT 14.3 0.0 0.7 Mm.33240 Eva, epithelial V-li e antigen OP237H04

Table 2: Genes coding for cell surface proteins which represent potential NSC markers. The selection of these genes was based on significantly differential upregulation (p<0.05) and 10-fold expression in ES cells versus PSA⁺ cells and ATB according to the SAGE data. In cases, where several tags belong to one transcript, the main tag is shown with the number of different tags indicated in parentheses. Total tag numbers are normalized to 100,000.

Investigation of NSC candidates

Criteria for potential NSC marker genes

Criteria were defined for the investigation of the potential NSC markers: • Assumed expression pattern: high in ES cells, absent/low in PSA+ cells and adult brain

• Preferably cell surface protein for later isolation

• Expression in undifferentiated embryoid bodies (Ebs) • Expression in neurospheres

• Expression in adult mouse brain in single cells in the SVZ (striatal side)

• Decreasing expression from birth to adulthood

Besides showing the described expression pattern and being transmembrane proteins, they should be expressed in neurospheres since these are inhomogene- ous cell aggregates containing about 3-4% NSC. Embryoid bodies (EBs) were used as control for in situ hybridizations in neurospheres. EBs are morphologically similar to neurospheres, and since they are derived from ES cells they should express the candidate gene.

In the adult mouse brain, the frequency of NSC is extremely low. According to rough estimates, 0.3% of the cells in the small neurogenic area, the SVZ, represent self-renewing, multipotent NSC. Therefore, NSC marker genes should label only single cells in the SVZ of adult mice. Since adult NSC can be viewed as remnants from brain development, a decrease in the number of brain cells expressing a potential marker gene would be expected from the time around birth to adulthood (Pevny and Rao, 2003).

Expression in embryoid bodies and neurospheres

In situ hybridizations were used to analyze the expression of the potential NSC markers (Tab. 2) in EBs and secondary neurospheres. Antisense and sense (negative control) digoxigenin-labeled RNA probes were generated by in vitro transcription for each candidate. EBs were grown in culture for 24 h. Neurospheres were produced from dissected brain tissue containing the SVZ of P3 or P4 mice. Primary neurospheres were grown for 5-7 days. To ensure that the neurospheres had the capability of self-renewal, they were dissociated to produce secondary neurospheres. EBs and secondary neurospheres were fixed, frozen and cryostat sections were produced. In situ hybridization of the antisense probe in both, EBs (Figure 4) and neurospheres (Figure 5), resulted in a relatively stronger staining than the corresponding sense probe only in the case of RIKEN 2610033C09 and ASCT2. Under more stringent hybridization conditions, the ASCT2 antisense probe produced a weaker overall staining in neurospheres while only few cells retained a strong signal (Figure 6). The other candidates were excluded from further investigations because the sense probe produced an equal or stronger staining than the antisense probe. Frz-7 was analyzed by immunofluorescence with an anti-Frz-7 rabbit pAb (Orbigen, 1 :66). Neurospheres and EBs were weakly stained (data not shown). The SAGE tag "CCCCACCCCAC" had been assigned to CD8 antigen due to a falsely annotated EMBL sequence in the corresponding UniGene cluster. Recently, the correct UniGene cluster Mm.153963 KIAA0152 was discovered. This is a further potential marker, which could be used instead of ASCT2 to identify NSC.

Expression analysis of potential NSC markers in mouse brain

The expression of RIKEN 2610033C09, ASCT2 and Frz-7 was examined in adult mouse brain. For RIKEN 2610033C09, staining was observed not only in the SVZ but also in the entire brain for different hybridization conditions (data not shown). For Frz-7, neither immunofluorescence nor the Vectastain ABC System produced labeling in mouse brain cryosections (data not shown).

By contrast, in situ hybridization for ASCT2 mouse brain generated a striking expression pattern (Figure 7). In P3 and P14 brain strong labeling is observed in the few cell layers adjacent to the lateral ventricle. While in P3 brain the staining extends around the entire ventricle, in P14 brain it is confined to the striatal side, the SVZ and the beginning RMS. In P3 brain almost all the cells are labeled with different intensity, whereas in the older brain relatively less cells are labeled creating a mottled pattern. Similar results like for P14 were seen in P32 mice (data not shown).

These experiments demonstrate expression of ASCT2 in the SVZ, the neurogenic area of the forebrain, where expression of a potential NSC marker gene is to be expected. The tendency of a decreasing expression in terms of cell number from birth to adulthood was confirmed. Figures 8 and 9 provide a comparison of ASCT2 antisense and sense probe, respectively. The sense probe produces only background staining and therefore confirms the specificity of the antisense staining. The sense probe does not label P3 brain either (data not shown).

Higher magnifications of the P14 brain show that only single cells and small groups of cells are labeled in the SVZ and the beginning RMS (Figure 8). The ASCT2 staining appears to be restricted to the cells in the SVZ excluding the ependyma, the single cell layer bordering the lateral ventricle. This finding is in agreement with the fact that the population of ependymal cells is unlikey to con- tain the NSC (Capela and Temple, 2002).

In summary, the present invention has identified a gene, ASCT2, which was selected by high expression in ES cells, low expression in PSA⁺ neuronal precursors and ATB, which is expressed by EBs and neurospheres and which is expressed in the neurogenic area of the mouse forebrain in an appropriate age- dependent manner. Thus, ASCT2 is a NSC marker.

Function and localization of ASCT2

ASCT2 (also known as ATB⁰, SLC1A2, SLC1A7) is a neutral amino acid transporter, which was cloned from mouse testis (Utsunomiya-Tate et al., 1996). This transporter is part of the ASC system that is responsible for Na⁺-dependent transport of L-alanine, L-serine, and L-cysteine (Palacin et al., 1998). ASCT2 participates in the glutamate-glutamine cycle which transfers glutamine between astrocytes and neurons (Broer and Brookes, 2001) and was found to mediate the L-isomer-selective transport of L-aspartic acid at the brain-blood barrier (Tetsuka et al., 2003).

Northern blots failed to detect ASCT2 in mouse brain tissue (Utsunomiya-Tate et al., 1996), but the protein was discovered in embryonic brain and upregulated in astrocytes cultures (Broer et al., 1999). Broer et al. conclude that ASCT2 might play a role during brain development and its expression might be associated with proliferative astrocytes. (Broer and Brookes, 2001). Studies in rabbits showed expression for ASCT2 in the intestine and highest expression in the kidney. More precisely, the protein is confined to the brush- border membrane of the proximal tubular cell and the enterocyte (Avissar et al., 2001).

Immunohistochemical analysis in mouse showed that the ASCT2 protein is localized on the abluminal side of brain capillaries in the cortex constituting the brain- blood barrier (Tetsuka et al., 2003). Here, GFAP staining did not colocalize with ASCT2 staining, but enclosed it.

A polyclonal antibody made in rabbit against the human ASCT2 protein is already available (Cat. num. AB5468, Chemicon).

Based on these findings, the subject matter of the present invention is a cell population comprising at least 5% neural stem cells, said stem cells being characterized by an expression of genes identified by a SAGE tag mentioned in Table 2, especially by an expression of ASCT2 or KIAA0152. Preferably, the cell popula- tion comprises at least 10% of these neural stem cells, preferably at least 25%, more preferably more than 50%. Higher purities are preferred even up to 90 or 95% purity.

In one embodiment, the cells are derived from rodents, e.g. mouse or rat. These cells are specially useful for further research.

In a further embodiment, the cells are derived from human. These cells are specially useful for diagnostic and development of medicaments.

Neural stem cells are obtainable by isolating them for example from brain. While embryonic stem cells are, by definition, localized in the inner cell mass of the embryo, the neural stem cells are present in the adult organism and localized in the nervous system. While ES cells and populations of differentiated cells derived from them were shown to form teratomas when introduced into the organism, neurospheres have not been observed to form teratomas and therefore NSC are unlikely to do so.

A further part of the invention is a method for isolating the cell population of the present invention comprising the following steps: a) taking a sample from nervous tissue, preferably brain b) isolating neural stem cells expressing ASCT2 or a) differentiating embryonic stem cells to neural stem cells, b) isolating neural stem cells expressing ASCT2 or a) trans-differentiating of adult none-neural stem cells to neural stem cells, b) isolating neural stem cells expressing ASCT2 or a) differentiating of adult neural precursor cells to neural stem cells, b) isolating neural stem cells expressing ASCT2 or a) differentiating of immortalised cells of neural stem cells, b) isolating neural stem cells expressing ASCT2 or a) producing neurospheres in culture, b) isolating neural stem cells expressing ASCT2.

Alternatively, KIAA0152 could be used as a marker.

A further embodiment of the present invention is a medicament comprising the cell population of the present invention. Such a medicament can be used to treat neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, injury of the brain, stroke, birth defects of the brain.

A further embodiment is a monoclonal antibody directed against ASCT2 or KIAA0152.This antibody can be a murine antibody directed against the murine protein. Further embodiment is a monoclonal antibody directed against human ASCT2 or KIAA0152. Brief description of the drawings

Figure 1 shows the selection strategy for potential NSC cells

Figure 2 shows the isolation of PSA⁺ neuronal precursors from the adult forebrain. a-PSA mAb specifically recognizes PSA-NCAM on the surface of the imma- ture neuronal precursors in the RMS (a). Use of this antibody permitted the isolation of a pure population of PSA⁺ cells by FACS. Rl marks the population of living cells (b) from which PSA⁺ cells are selected in Ml by subtracting the autofluores- cence of the cells and the non-specific labeling of the secondary a-IgM antibody (c). Total yields of PSA⁺ cells and RNA from several FACS experiments (d). Scale bar: 25 μm.

Figure 3 shows the mapping of (a) PSA⁺ cell SAGE tags and (b) ES cell SAGE tags to UniGene clusters and SwissProt.

Figure 4 shows in situ hybridizations with antisense and sense probes for potential NSC markers in undifferentiated embryoid bodies. Only for RIKEN 2610033C09 (oP237G07), ASCT2 (OP011E02) did the antisense probe produce a stronger staining than the corresponding sense probe. Hybridization conditions: Ifitml (OP219H04): 45°C, 1000 ng/ml; RIKEN 2610033C09 (oP237G07), ASCT2 (oP011E02), Gap junction protein beta3, Connexin31 (oPHD09), Eva, epithelial V-like antigen (oP237H04): 50°C, 800 ng/ml. Scale bar 40 μm.

Figure 5 shows in situ hybridizations with antisense and sense probes for potential NSC markers in secondary neurospheres. The hybridization of the antisense probe resulted in a stronger staining than the corresponding sense probe only in the case of RIKEN 2610033C09 (oP237G07) and ASCT2 (oP011E02). Hybridization conditions: Ifitml (oP219H04): 45°C, 1000 ng/ml; RIKEN 2610033C09 (oP237G07), ASCT2 (oP011E02), Gap junction protein beta3, Connexin31 (OP11D09), Eva, epithelial V-like antigen (oP237H04): 50°C, 800 ng/ml. Scale bar 40 μm.

Figure 6 shows in situ hybridization with the antisense probe for ASCT2, in secondary neurospheres under more stringent conditions (50°C, 600ng/ml) shows that occasionally few cells stand out from the others. Scale bar 25 μm. Figure 7 shows in situ hybridization for ASCT2 in the mouse forebrain. The ASCT2 antisense probe produces a specific staining in the majority of the cells in the first cell layers surrounding the lateral ventricle of P3 mice (a). In P14 mice, relatively less cells are labeled creating a spot-like staining pattern restricted to the SVZ and the beginning RMS (b). LV: lateral ventricle, RMS: rostral migratory stream, SP: septum, ST: striatum. Scale bar 50 μm.

Figure 8 shows in situ hybridization with the ASCT2 antisense probe at the lateral ventricle in P14 mouse forebrain. In situ hybridization for ASCT2 labels single cells and small groups of cells within the SVZ and the RMS while no staining is observed on the septal side of the lateral ventricle. LV: lateral ventricle, RMS: rostral migratory stream, ST: striatum. Scale bar 25 μm.

Figure 9 shows in situ hybridization with the ASCT2 sense probe (negative control) at the lateral ventricle in P14 mouse forebrain. The sense probe produces only background staining and confirms the specificity of the antisense staining. LV: lateral ventricle, RMS: rostral migratory stream, ST: striatum. Scale bar 25 μm.

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Claims

1. Cell population comprising at least 5% neural stem cells, said stem cells being characterized by an expression of ASCT2.

2. Cell population according to claim 1, wherein at least 25% of the cells are neural stem cells.

3. Cell population according to anyone of claims 1 or 2, wherein it is a murine, human or rat cell population.

4. Cell population according to anyone of claims 1 to 3, wherein the neural stem cells are obtainable by isolation from brain tissue.

5. A method for isolating the cell population according to any of claims 1 to 4, with the following steps:

a) taking a sample from nervous tissue, b) isolating neural stem cells expressing ASCT2 or a) differentiating embryonic stem cells to neural stem cells, b) isolating . neural stem cells expressing ASCT2 or a) trans-differentiating of adult non-neural stem cells to neural stem cells, b) isolating neural stem cells expressing ASCT2 or a) de-differentiating of adult neural precursor cells to neural stem cells, b) isolating neural stem cells expressing ASCT2 or a) differentiating of immortalised cells of neural stem cells, b) isolating neural stem cells expressing ASCT2 or a) producing neurospheres in culture, b) isolating neural stem cells expressing ASCT2.

6. Medicament comprising a cell population according to anyone of claims 1 to 4.

7. Use of a cell population according to anyone of claims 1 to 4 for the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, injury of brain or stroke, birth defects of the brain.

8. A monoclonal antibody directed against ASCT2.

9. Use of ASCT2 as a marker in diagnostics and research.

10. Cell population comprising at least 5% neural stem cells, said stem cells being characterized by an expression of KIAA0152.