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发表于 2009-3-5 00:54 |只看该作者 |倒序浏览 |打印

作者：Martina Maisela, Alexander Herrb, Javorina Milosevicc, Andreas Hermanna, Hans-Jrg Habischd, Sigrid Schwarzc, Matthias Kirsche, Gregor Antoniadisf, Rolf Brennerg, Susanne Hallmeyer-Elgnera, Holger Lerched, Johannes Schwarzc, Alexander Storcha 8 u, N2 G- u1 Y- o2 \' z  H5 R



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      【摘要】8 I; k$ {3 v6 e/ g  g* k
   Global gene expression profiling was performed using RNA from adult human hippocampus-derived neuroprogenitor cells (NPCs) and multipotent frontal cortical fetal NPCs compared with adult human mesenchymal stem cells (hMSCs) as a multipotent adult stem cell control, and adult human hippocampal tissue, to define a gene expression pattern that is specific for human NPCs. The results were compared with data from various databases. Hierarchical cluster analysis of all neuroectodermal cell/tissue types revealed a strong relationship of adult hippocampal NPCs with various white matter tissues, whereas fetal NPCs strongly correlate with fetal brain tissue. However, adult and fetal NPCs share the expression of a variety of genes known to be related to signal transduction, cell metabolism and neuroectodermal tissue. In contrast, adult NPCs and hMSCs overlap in the expression of genes mainly involved in extracellular matrix biology. We present for the first time a detailed transcriptome analysis of human adult NPCs suggesting a relationship between hippocampal NPCs and white matter-derived precursor cells. We further provide a framework for standardized comparative gene expression analysis of human brain-derived NPCs with other stem cell populations or differentiated tissues.
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Disclosure of potential conflicts of interest is found at the end of this article.
      【关键词】 Gene expression profile Neural stem cells Neuroprogenitors Mesenchymal stem cells Gene chips2 L( `8 G5 J8 b8 m" |
   INTRODUCTION

Neural progenitor cells (NPCs) are multipotent stem cells that are capable of self-renewal and generate all three cell types of the central nervous system (neurons, oligodendrocytes, and astrocytes). NPCs hold the promise of repairing and finally curing degenerative disorders, such as Parkinson's disease or amyotrophic lateral sclerosis, stroke, or head trauma . These human NPCs are typically expanded as neurosphere cultures and differentiate exclusively in neural cell types using specific culture conditions. Understanding the genes that govern the special properties of NPCs has implications for both basic stem cell biology as well as therapeutic applications. Although there is a great interest and potential of NPCs in cell replacement therapy of neurological diseases, there is lack of data about genetic programs for human NPCs, particularly with respect to adult NPCs (aNPCs).
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Wright and coworkers .

In this report, we focus on the transcriptome analysis of human adult and fetal NPCs propagated in neurospheres to provide (a) knowledge of the molecular mechanisms of governing self-renewal and multipotent differentiation capacity restricted to the neural cell fate and (b) a framework for standardized comparative gene expression analysis of human NPCs with other stem cell populations or differentiated tissues. We therefore applied the Affymetrix (Santa Clara, CA, http://www.affymetrix.com/index.affx) gene chip technology to RNA derived from human hippocampal aNPCs, human fNPCs from frontal cortex, multipotent adult human mesenchymal stem cells (hMSCs) as a multipotent adult stem cell control and adult (differentiated) human hippocampal tissue. In addition, we correlated the expression profiles of adult and fetal neural tissues available in public databases. This approach has allowed us to define a large number of genes that delineate the phenotypical differences of adult and fetal NPCs.* }2 D0 Q# Q& N% x9 @# F% k

MATERIALS AND METHODS1 @2 e  m; C1 M; T1 @+ o& K5 R7 M

Adult Human Hippocampal NPC Isolation

Adult NPCs were isolated from human hippocampal tissue obtained from routine epilepsy surgery procedures (selective amygdala-hippocampectomy or anterior temporal lobectomy performed in patients with pharmacoresistant temporal lobe epilepsy. The procedure was in accordance with the Declaration of Helsinki and approved by the local Institutional Review Board (IRB)s. Dissected tissue was stored in ice-cold Hanks' balanced salt solution supplemented with 11 mM glucose and 1% penicillin/streptomycin (both from Sigma, St. Louis, http://www.sigmaaldrich.com) until further preparation. NPCs were expanded only if high-resolution magnetic resonance imaging and neuropathological investigations showed no evidence for tumor formation or infection. Tissue samples were manually minced and digested using 0.1% trypsin (Sigma) for 30 min at room temperature (RT), incubated in DNase (40 mg/ml; both from Sigma) for 10 min at 37¡ãC and homogenized to a quasi single cell suspension via gentle triturating. The cells were added to 25-cm2 flasks (2¨C3 x 106 viable cells per flask) in Knock-Out Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Tulsa, OK, http://www.invitrogen.com), supplemented with 10% serum replacement (Invitrogen), 0.5 mM glutamine, 1% penicillin/streptomycin, and 20 ng/ml epidermal growth factor (EGF), and fibroblast growth factor 2 (FGF-2) (Sigma) at 5% CO2, 92% N2, and 3% O2 using an incubator equipped with an O2-sensitive electrode system (Heraeus, Hanau, Germany, http://www.heraeus-tenevo.com/en/index.html). After 10¨C20 days, neurosphere formation could be observed, and these spheres were then expanded for additional 5 weeks (in total 7 weeks, five passages). The medium was changed once a week, and the growth factors were added twice a week. The characterization of neurospheres revealed that the aNPCs expressed all major properties of neural progenitor cells: They were CD15low/¨C, CD34¨C, CD45¨C, CD133¨C, and Nestin . They self-renewed, expressed telomerase, and differentiated into astroglia, oligodendroglia, and functional neurons . For RNA analysis, adult NPCs from the hippocampus of one patient were used after an expansion period of 7 weeks (five passages; 7¨C10 population doublings). The patient was a 17-year old white girl who had experienced a severe meningoencephalitis at the age of 6 months and had suffered from pharmacoresistant, left-sided temporal lobe epilepsy with epigastric auras and complex focal seizures since the age of 4 years. Carbamazepine, sultiam, vigabatrin, oxcarbazepine, and levetiracetam were applied without sufficient success. Before the epilepsy surgery, she was treated with a combination of oxcarbazepine (1050 mg per day) and levetiracetam (2750 mg per day) and still had daily complex focal seizures. On magnetic resonance imaging, she had left-sided widespread temporal lobe atrophy and hippocampal sclerosis. Levetiracetam was reduced and completely discontinued 7 days before surgery, and the patient was put on 1,500 mg oxcarbazepine daily as monotherapy 2 days before surgery.: c/ l, M$ ]9 N# t* {' d& r4 v

Fetal Human Frontal (Cortical) Neural Progenitor Cell Isolation: u  m# Y! E  R  W2 g0 {
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Human fetal brain tissue samples (15 weeks after fertilization) were harvested and supplied by Advanced Bioscience Resources Inc. (Alameda, CA) according to NIH and local IRB guidelines. If there was no evidence of a pathologic condition in the fetal tissue and informed consent was obtained, parts of the brain were transferred to our laboratory. The quality of the tissue was confirmed by initiating primary cultures and immunohistochemical characterization. NPCs were isolated from fetal frontal cortex and induced to proliferate as free-floating neurospheres. For that purpose, the tissue samples were mechanically dissociated with a scalpel, sequentially incubated in 0.1% trypsin and 40 mg/ml DNase, and expanded using EGF and FGF-2 as mitogens under reduced atmospheric oxygen (3%) as described previously . Fetal neurosphere formation was observed after 10¨C20 days. Neurospheres could be propagated up to 12 weeks. RNA from two different tissue samples was isolated after 4 weeks of propagation (4 and 5 passages, respectively; 20¨C28 populations doublings).

Adult Human Mesenchymal Stem Cell Isolation

Adult human bone marrow samples were harvested from routine surgical procedures (pelvic osteotomies; three samples; age, 14¨C40 years) after informed consent and in accordance with the terms of the ethics committees of the University of Ulm or the Technical University of Dresden. hMSC were isolated and cultured as described previously . In brief, hMSCs were cultured in a basal medium consisting of DMEM with 10% fetal calf serum and 1% penicillin/streptomycin (all from Invitrogen) at 37¡ãC, 5% CO2 in 95% humidity. Cells were passaged once a week. The hMSC phenotype was proven by immunocytochemistry (all cells expressed high levels of fibronectin), fluorescence-activated cell sorting analysis (cells were CD9 , CD90 , CD105 , and CD166 , as well as CD14¨C, CD34¨C, and CD45¨C) and by testing the potential to differentiate into osteoblasts, chondrocytes, and adipocytes. hMSCs from three different donors (32, 14, and 40 years of age) were expanded for 2¨C10 weeks (2¨C4 passages; 5¨C30 population doublings) and used for RNA isolation as described below.

Adult Human Hippocampal Tissue Isolation

Adult hippocampal tissue was obtained by surgical resection from one patient with epilepsy (male, 35 years of age) after informed consent of the patient. There was no evidence for tumor formation or infection. The tissue was stored immediately in RNA later (QIAGEN, Hilden, Germany, http://www.qiagen.com) until RNA isolation.6 a2 V6 A/ d! x% {9 ]

RNA Isolation and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction

Total cellular RNA was extracted from hippocampal tissue and cultured neurospheres using the RNeasy Total RNA Purification Kit (Qiagen) followed by treatment with RNase-free DNase (QIAGEN). Quantitative real-time one step RT-PCR was carried out using the LightCycler System (Roche, Mannheim, Germany) as described previously . Relative expression was measured in relation to the expression of a housekeeping gene (hydroxymethylbilane synthase). Primer sequences (100¨C200 bp) used for the RT-PCR are given in additional data accompanying this report (supplemental online Table 1).

Single-Channel Oligonucleotide Microarray Analysis0 I4 q# {. r: |1 t  h- Q

The experimental protocol is indicated in Figure 1. Total RNA was reverse-transcribed, T7-amplified, fragmented, and labeled according to standard procedures recommended by Affymetrix (Santa Clara, CA). We hybridized the labeled cRNA to Affymetrix U133A chips containing 22,215 probe sets representing at least 12,905 individual genes. Washing and scanning was performed following the manufacturer's (Affymetrix) standard protocol. Chip intensity data was retrieved in cel-files. Additional expression data sets in form of cel-files were obtained from the web (http://www.ncbi.nlm.nih.gov/geo/) or kindly provided per DVD (http://wombat.gnf.org/suppl.html#reqdata_geneatlas). Datasets included the NCI60-dataset (http://wombat.gnf.org/samples/NCI60_sample_info.htm), the GNF-SymAtlas . Gene categorization was based on literature reviews.) J+ l0 n4 j- @: z# o

Figure 1. Transcriptional profiling of human NPCs. Human fetal frontal (cortical) NPCs and adult hippocampal NPCs, as well as adult human MSCs, serving as a multipotent adult stem cell control and tissue from adult human hippocampus serving as a differentiated tissue control, were isolated, and their cRNAs were hybridized to Affymetrix U133A-DNA chips. The arrays were analyzed by use of the RMA-software, followed by public database searches and functional annotation. At the bottom, we indicated genes that were described previously to be specifically expressed in each of the two stem cell-types and tissue, as verified by reverse transcription-polymerase chain reaction. Abbreviations: MSC, mesenchymal stem cell; NPC, neural progenitor cell.  L/ \! P1 t* c

RESULTS7 I' t! f5 @. y) ^- O- j

Global Transcriptome Complexity of Human Neural and Mesenchymal Progenitors
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Our dataset consisted of 37 individual chips from three different sources. Eight tissues were represented in replicas processed from different labs. All eight pairs were clustered together, thus demonstrating the performance of our fitting procedure (Fig. 2A). Furthermore, multiple microarray analyses of one respective progenitor cell type from different tissue samples/patients (n = 2¨C3) showed a 93%¨C94% overlap of specifically overexpressed genes.

Figure 2. Overlaps between genes enriched in human NPCs compared to various neural tissue and cell types. (A): Hierarchical clustering of 31 neural tissues. Expression data (i.e., the 1,000 most variant genes) were clustered using Pearson's correlation as similarity measure and average linkage as agglomeration rule. External sources of chip data are denoted in brackets: gnf from SymAtlas . Own experimental datasets are marked with colors. All biological replicates are clustered together illustrating the performance of the chosen fitting procedure. Our hippocampus chip forms a branch together with hippocampus (tp) and the two amygdala chips (gnf, tp). The fetal NPCs fall in one cluster along with fetal brain tissues (gnf, tp). The adult NPCs cluster together with corpus callosum (gnf) and spinal cord (gnf, tp). (B, C): Venn diagrams of the number of genes enriched in each human NPC population and adult hippocampus (B) or human mesenchymal stem cells (C) and their overlaps. Abbreviations: hMSC, human mesenchymal stem cell; NPC, neural progenitor cell.

We then profiled the gene expression of adult hippocampus initially to evaluate the comparability between our chip and the data obtained elsewhere (Fig. 2A). After completing the fitting procedure a correlation coefficient (R2) of 0.9935 was reached between our hippocampus data and the hippocampus data retrieved from literature , providing strong evidence that the experimental protocol used is valid. The two NPC subtypes do not fall into the same cluster and showed rather limited overlap in gene expression. Fetal NPCs cluster together with their tissue of origin, fetal brain. In contrast, adult NPCs cluster together with corpus callosum and spinal cord, mainly consisting of white matter tissue and thus do not resemble their respective tissue of origin, the hippocampus.
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To further capture an overall estimate of the similarities and differences between the analyzed tissues, we compared the transcriptome of aNPCs, fNPCs, hMSCs, and adult hippocampal tissue on a global scale in which cell types were compared in a pairwise manner (refer to supplemental online Fig. 1). The transcriptomes of all these cell/tissue types were significantly different, with lower correlation coefficients of 0.612¨C0.664. These data were confirmed by analyzing the expression of enriched genes in all cell types showing only a small overlap of gene expression in all analyzed cell/tissue samples (see Fig. 2B for Venn diagrams).0 H- S1 X$ X# n) t6 I! u
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Figure 1 and Table 1 show that most previously known stem cell markers were enriched in their respective progenitor cell population providing additional validation of the protocol. The human brain-derived aNPCs showed high expression of the neural stem cell marker NES (nestin), as well as of OLIG2, MSl1, NKX2-2, and CD44. NES and MSI1 were already reported in aNPCs by Hermann et al .

Table 1. Genes expressed in aNPCs, fNPCs, human hippocampus, or hMSCs

Table 1. (Continued): E( S+ K7 L' B: w* M

The fNPCs showed a high expression, for example, of NES, PROM1 (prominin1, CD133), Olig2, MSI1, as well as SOX genes such as SOX2, SOX3, SOX4, SOX9, and SOX11. In contrast, hMSCs expressed various extracellular matrix proteins, such as various collagen isoform and other genes related to connective tissue and its metabolism (LOXL1, LOXL2, LUM). Fully differentiated neural tissue like the hippocampus expressed neuronal and glial genes like NTRK2, NTRK3, MBP, and GFAP. The presence or absence of gene expression of the most prominent genes (CCR7, EDNRB, GPR17, LAMA4a, NES, P75 , PDGFR and TNC) was confirmed by real-time RT-PCR in all cell types (data not shown).( c- R2 |4 G+ r3 \
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Transcriptome Analysis Clusters aNPCs with White Matter Tissues

Hierarchical clustering of aNPCs and tissues containing mainly white matter, such as spinal cord and corpus callosum, is characterized by a strong expression of MHCII genes and myelin-components such as peripheral myelin protein two (PMP2), myelin-associated glycoprotein (MAG), or the myelin-oligodendrocyte glycoprotein (MOG). The overexpression of CD44 and GDF1/LASS1 might point to a restricted stem cell potential. We found that a number of genes specific for neurons were transcribed only on reduced levels: the 25-kDa synaptosome-associated protein (SNAP25), neurogranin (NRGN), and others. When looking for genes specifically expressed in aNPCs, a large number of such genes can be retrieved. We found the mRNA for the low-affinity nerve growth factor receptor precursor (p75, NGFR) and for Nestin (NES) in a very high abundance. In addition, the dual specific phosphatase 10 (DUSP10), the CC-motif containing chemokine receptor seven (CCR7), the transcription factor four (TCF4), the G protein-coupled receptor 17 (GPR17), and the chondroitin sulfate proteoglycan four (CSPG4) were specifically highly expressed in the aNPCs (Table 1). The gene CSPG4 encodes for the epitope NG2, which is reported to be specifically expressed on oligodendrocyte precursor cells. Interestingly, only three genes were exclusively expressed in aNPCs, namely NGFR, GPR17, and CCR7.

No Adult NPC Markers in Total Adult Hippocampus
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Unlike fetal NPCs, adult NPCs showed little overlap with their tissue of origin (Figs. 2A, 2B). There was no elevated expression of specific markers for adult NPCs in hippocampus. Instead, markers typically expressed in olfactory bulb and spinal cord tissue were detected ) or the myelin associated glycoprotein (MAG). This observation might be explained by the very low frequency of NPCs in the adult brain.& R; G6 G" i( r

Fetal Neural Precursors Maintain an Expression Pattern of Undifferentiated Cells* o& J# H; d# R3 D

The expression pattern of fNPCs, clearly distinguishable from those of cells from the peripheral nervous system (Fig. 2A), was also different from adult central nervous system (CNS) tissue, but in closest relation with fetal brain. Both tissues lacked the mRNA for myelin components of mature oligodendrocytes: myelin basic protein (MBP), proteolipid protein one (PLP1), myelin-associated oligodendrocyte basic protein (MOBP), and others. The neurofilament heavy chain (NEFH) or the GABA-receptor, 1 (GABRA1) were not expressed. Instead, a number of SOX- and homeobox genes were highly expressed compared with adult brain: SOX4, SOX11, SOX12, and the LIM homeobox gene two (LHX2), or the distal-less homeobox gene 2 (DLX2). In addition, we could detect an up-regulation of prominin one (PROM1 or CD133) in both tissues. Important genes uniquely overexpressed in fNPCs are predominantly associated with cell proliferation-like topoisomerase II (TOP2A), histone acetyltransferase one (HAT1, , the endothelin receptor type B (EDNRB), the myeloid ecotropic viral integration site 1 homolog (MEIS1), and the tissue factor pathway inhibitor (TFPI).
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Adult NPCs Share Only Few Similarities with Fetal NPCs. }! J; o, z) I4 o+ b0 o
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Despite the global differences between transcriptomes of aNPCs and fNPCs, a few similarities were observed (Fig. 2B, Table 2). Most of these similarities could be attributed to genes associated with cell cycle progression, such as a disintegrin and metalloproteinase domain nine (ADAM9), HAT1-, protein kinase-, DNA-activated, catalytic polypeptide (PRKDC), or RNA binding motif protein 3 (RBM3). Both cell types overexpressed other genes commonly associated with a neural stem/progenitor cell fate: Jagged 1 (JAG1) ) was also overexpressed in both adult and fetal NPCs. The categorization of the genes by gene ontology terms were similar in both NPC populations (refer to supplemental online Fig. 2), whereas the genes themselves overlapped only occasionally (refer to supplemental Fig. 2; supplemental Tables 2 and 3 for genes showing greater than 10-fold differences between aNPCs and fNPCs).* |& l$ q0 n, R4 [- U+ K8 R9 S  Y

Table 2. The 38 genes enriched in both adult and fetal NPCs

Comparison of Adult NPCs with hMSCs
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We also included hMSCs in our expression analysis, primarily as a multipotent adult stem cell control and to have a predefined outgroup for rooting the hierarchical dendrogram (Fig. 2A). We found that hMSCs overexpress a number of genes commonly found in connective tissues (decorin , and various collagen proteins), similar to peripheral nervous system (PNS) tissues. These genes are likely responsible for the observed association of hMSCs with tissues of the PNS upon cluster analysis, being completely separated from all neural tissues including adult and fetal NPCs (data not shown). However, there are various genes expressed by both aNPCs and hMSCs, mainly extracellular matrix proteins (COL1A1, COL1A2, COL3A1, LOXL2) and genes such as insulin-like-growth-factor 3 and 5 (IGFBP3 and IGFBP5) (refer to Table 3). Three of the four genes expressed in aNPCs, fNPCs, and hMSCs were also extracellular matrix components, namely laminin-4 (LAMA4), tenascin C (TNC), and integrin-7 (ITGA7). The other gene was pleiotrophin (PTN) reported to be involved in various developmental processes.
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Table 3. The 28 genes enriched in both adult NPCs and adult hMSCs

In contrast, there was no relevant overlap of gene expression with respect to cell cycle, proliferation, stem cell markers or genes known to maintain an undifferentiated cell fate (refer to supplemental Fig. 3 and supplemental Tables 4 and 5 for genes showing greater than 10-fold differences between aNPCs and hMSCs).9 c% H* ^. \1 P4 K
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DISCUSSION3 |3 o* d4 D$ ]+ D; j* k8 M9 B+ ]

For the first time, we describe the global gene expression profiles of human adult NPCs isolated from the human hippocampus of adult patients with temporal lobe epilepsy and fetal NPCs isolated from frontal cortex. It is noteworthy that we used diseased hippocampal tissue to isolate aNPCs that are most likely containing mesiotemporal sclerosis, which is reported to include more neural progenitor cells in vivo . Both cell types were propagated as neurosphere cultures in vitro for at least 4¨C7 weeks (7¨C20 cell doublings) under similar conditions using EGF and FGF-2 as mitogens. Gene expression profiles show that both NPC subtypes express core neural stem cell makers but neither share global expression profile with each other nor with multipotent hMSCs or adult brain (hippocampal) tissue. In contrast, aNPCs share expression profiles with white matter brain tissue, whereas fNPCs show a close relation with fetal brain. Together, our data strongly suggest that adult and fetal human NPCs use divergent paths to maintain the neuroprogenitor cell state.
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Technical Issues with Expression Profiling

Gene chip technology is a powerful tool to generate global gene expression profiles of cells and tissues. It is exceptionally valuable when looking for novel genes associated with a defined biological process. However, despite largely standardized protocols and technical equipment (in this case the Affymetrix technology), the deduced results display a varying degree of specificity. The analysis algorithm (i.e., MAS5) specified by Affymetrix is very basic and delivers rather noisy data, which makes it especially challenging to interpret low-intensity signals. The inherent difficulties have led to a number of third-party software approaches to improve the situation . Low-level signals close to the background levels remain difficult to interpret. "No signal" could have a biological meaning that the gene is not expressed¡ªor worse, the probeset is not properly designed to detect a high signal. The latter is often caused by underestimation of the length of 3'-UTR of genes placing the probe set very far from the poly(A)-tail and thus exceeding the average length of the reverse-transcribed sequences. Therefore, an "absence" call should only be issued when there is positive knowledge that the probe set allows for detection at all. We estimate that the U133A-chip contains up to 3,000 (out of 22,215) "dead probe sets" unable to deliver a signal. This assumption is based on the observed lack of an appropriate signal in all the analyzed 385 chips, which covered a wide range of normal and cancerous tissues, stem cells, and cell lines. Multiple gene array analyses of a respective progenitor cell type derived from different tissue donors revealed a >90% overlap of gene expression demonstrating high similarity of transcriptomes within one progenitor cell type independent of the donor. However, (a) to make this necessary positive control available and (b) to provide an internal reference, the individual chips required extensive scaling and normalization finally leading to a technical variance being smaller than the biological variance. We achieved this goal by showing that hierarchical clustering groups biological replicas regardless of the lab conducting the experiments (Fig. 2A).

Comparison with Prior Knowledge

Two studies investigated the gene expression profile of human fetal NPCs , we could detect expression of SOX2 and SOX13 in our fetal NPC cultures and extend the list of stem cell biology-related genes, including MELK (maternal embryonic leucine zipper kinase), EMX2 (empty spiracles homolog 2), and others (Table 1).
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Gene Expression in Human Adult and Fetal NPCs
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In general, adult and fetal NPCs display a level of overlap comparable with any other pair of the analyzed neural tissues retrieved from databases. The expression profile of our population of adult NPCs rather resembled the expression patterns obtained from corpus callosum and spinal cord, but this similarity was not based on the shared expression of markers for adult progenitor cells (e.g., NGFR, GPR17) and¡ªbased on expression analysis¡ªthere is no clear sign for a noteworthy subpopulation of NPCs in spinal cord or corpus callosum. The mentioned NGFR gene was expressed in high abundance in our adult hippocampus-derived neurospheres. NGFR plays a central role for the decision between survival and death in neural cells. The confusingly complex pattern of signal transduction involving a wide range of coreceptors and various ligands was so far mainly of interest for the delineation of the molecular pathways governing the fate of neurons. However, for the oligodendroglial lineage a clear assignment as pro-mitotic or pro-apoptotic signal remains to be resolved. There are reports on NGFR expression in postmitotic oligodendrocytes in vivo and in vitro, often associated with apoptotic activity , all of which are involved in stem cell function and/or brain development.8 I, o: p3 b7 I# H  z! i3 k

Our fetal NPC population was grouped together with whole fetal brain by hierarchical clustering likely indicating that the fetal brain at week 12 contains a large proportion of uncommitted stem cells. By looking for genes that are specifically over-expressed in fetal neurospheres but not in whole fetal brain, we could retrieve a large list containing genes associated with mitosis (CDC2, MCM2, PCNA, or CDKN3) or being critical for maintaining an undifferentiated state, such as SOX2, SOX3, SOX4, and SOX9. This category also includes the transcription factor eight (TCF8, also named Zfhep/deltaEF1), which was found to be expressed in early neural progenitors in the rodent brain . Our MEIS1 expression pattern fits that observation and suggests a SOX2-like role for the gene in human neural development., ?0 J% Z; q* H" n# q! A- Y1 U
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CONCLUSIONS/ Y% G. Z+ V9 l" A' r. W- d+ g# ?
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In conclusion, we have used Affymetrix gene chip technology to obtain a genomic expression profile of human adult and fetal NPCs, which were compared with profiles generated from mesenchymal stem cells and from adult hippocampus. The profiles indicate that both NPCs share the expression of a variety of genes known to be related to signal transduction, cell metabolism, and neuroectodermal tissue determination. These comparisons led to the identification of novel genes involved in stem cell and neural development. In contrast, adult NPCs and hMSCs both express genes mainly involved in extracellular matrix. Global comparison of transcriptomes using hierarchical cluster analysis of various neuroectodermal cell/tissue types revealed a strong relationship of aNPCs with various white matter tissues, whereas fetal NPCs were strongly correlated with fetal brain tissue. Thus, our analysis suggests a relationship between adult hippocampal NPCs and white matter-derived precursor cells. The capability of NPCs to survive, proliferate, and integrate into the damaged brain underscores the great therapeutical potential of these cells. This report is a further step toward the full characterization of these cells, which is a vital prerequisite to clinical transplantation. We further provide a framework for standardized comparative gene expression analysis of human adult brain-derived NPCs with other stem cell populations or differentiated tissues.+ I0 }( f/ d& C( T- ?! f! d

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST( X: ^* z+ C9 F2 R8 Y7 R+ N9 P
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS) [# M$ e  E: G5 T% m* T" S5 D
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We thank Sylvia Kanzler, Thomas Lenk, and Nancy Meyer for excellent technical assistance. We thank Stefan Liebau for his help with quantitative RT-PCR. This work was supported in part by the Interdisziplinäres Zentrum f¨¹r Klinische Forschung (IZKF) Ulm (Project D6) (to A.S.), the BMBF (Verbundvorhaben "Tissue Engineering"; AZ 0312126) (to A.S. and J.S.), and the Landesstiftung Baden-W¨¹rttemberg (Förderprogramm "Adulte Stammzellen"; AZ 37610) (to A.S.). M.M. and A.H. contributed equally to this work.) q7 `+ {& y+ T2 B: y
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