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Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0003-3811-5439
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2005 (English)In: BMC neuroscience (Online), ISSN 1471-2202, Vol. 6, 55- p.Article in journal (Refereed) Published
Abstract [en]

Background: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro.

Results: We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP.

Conclusion: Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed.

Place, publisher, year, edition, pages
2005. Vol. 6, 55- p.
Keyword [en]
CENTRAL-NERVOUS-SYSTEM, ADULT-MOUSE BRAIN, PARKINSONS-DISEASE, MICROARRAY DATA, DENTATE GYRUS, DIFFERENTIATION, TRANSPLANTATION, AMPLIFICATION, PROGENITORS, NEURONS
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-6168DOI: 10.1186/1471-2202-6-55ISI: 000231880900001Scopus ID: 2-s2.0-26444590557OAI: oai:DiVA.org:kth-6168DiVA: diva2:10800
Note
QC 20100914Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2010-10-06Bibliographically approved
In thesis
1. Mining the transcriptome - methods and applications
Open this publication in new window or tab >>Mining the transcriptome - methods and applications
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Regulation of gene expression occupies a central role in the control of the flow of genetic information from genes to proteins. Regulatory events on multiple levels ensure that the majority of the genes are expressed under controlled circumstances to yield temporally controlled, cell and tissue-specific expression patterns. The combined set of expressed RNA transcripts constitutes the transcriptome of a cell, and can be analysed on a large-scale using both sequencing and microarray-based methods.

The objective of this work has been to develop tools for analysis of the transcriptomes (methods), and to gain new insights into several aspects of the stem cell transcriptome (applications). During recent years expectations of stem cells as a resource for treatment of various disorders have emerged. The successful use of endogenously stimulated or ex vivo expanded stem cells in the clinic requires an understanding of mechanisms controlling their proliferation and self-renewal.

This thesis describes the development of tools that facilitate analysis of minute amounts of stem cells, including RNA amplification methods and generation of a cDNA array enriched for genes expressed in neural stem cells. The results demonstrate that the proposed amplification method faithfully preserves the transcript expression pattern. An analysis of the feasibility of a neurosphere assay (in vitro model system for study of neural stem cells) clearly shows that the culturing induces changes that need to be taken into account in design of future comparative studies. An expressed sequence tag analysis of neural stem cells and their in vivo microenvironment is also presented, providing an unbiased large-scale screening of the neural stem cell transcriptome. In addition, molecular mechanisms underlying the control of stem cell self-renewal are investigated. One study identifies the proto-oncogene Trp53 (p53) as a negative regulator of neural stem cell self-renewal, while a second study identifies genes involved in the maintenance of the hematopoietic stem cell phenotype.

To facilitate future analysis of neural stem cells, all microarray data generated is publicly available through the ArrayExpress microarray data repository, and the expressed sequence tag data is available through the GenBank.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 62 p.
Series
Theses in philosophy from the Royal Institute of Technology, ISSN 1650-8831
Keyword
transcriptome, gene expression profiling, EST, microarray, RNA amplification, stem cells, neurosphere
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4115 (URN)91-7178-436-5 (ISBN)
Public defence
2006-10-13, FR4, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100927Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2010-09-27Bibliographically approved
2. Transcript profiling of small tissue samples using microarray technology
Open this publication in new window or tab >>Transcript profiling of small tissue samples using microarray technology
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Through a number of biological, technological and computational achievements during the 20th century and the devoted work of hundreds of researchers the sequence of the human and other genomes are now available in public databases. The current challenge is to begin to understand the information encoded by the DNA sequence, to elucidate the functions of the proteins and RNA molecules encoded by the genes as well as how they are regulated. For this purpose new technologies within the area of functional genomics are being developed. Among those are powerful tools for gene expression analysis, such as microarrays, providing means to investigate when and where certain genes are used.

This thesis describes a method that was developed to enable gene expression analysis, on the transcriptome level, in small tissue samples. It relies on PCR amplification of the 3’-ends of cDNA (denoted 3’-end signature tags). PCR is a powerful technology for amplification of nucleic acids, but has not been used much for transcript profiling since it is generally considered to introduce biases, distorting the original relative transcript levels. The described method addresses this issue by generating uniformly sized representatives of the transcripts/cDNAs prior to amplification. This is achieved through sonication which, unlike restriction enzymes, does not require a specific recognition sequence and fragments each transcript randomly. The method was evaluated using cDNA microarrays, Affymetrix™ oligonucleotide arrays and real-time quantitative PCR. It was shown to perform well, yielding transcript profiles that correlate well to the original, unamplified material, as well as being highly reproducible.

The developed method was applied to stem cell biology. The variability in gene expression between different populations of cultured neural stem cells (neurospheres) was investigated. It was shown that neurospheres isolated from different animals or passaged to different degrees show large fluctuations in gene expression, while neurospheres isolated and cultured under identical conditions are more similar and suitable for gene expression analysis. A second study showed that withdrawing epidermal growth factor (EGF) from the culture medium when treating the cells with an agent of interest has profound effects on gene expression, something which should be taken into consideration in future neurosphere studies.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. 89 p.
Keyword
Biology, Gene expression analysis, transcriptomics, microarray analysis, 3’-tag signature amplification, neural stem cells, Biologi
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-158 (URN)91-7283-989-9 (ISBN)
Public defence
2005-04-08, Kollegiesalen, KTH, Valhallavägen 79, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20101006Available from: 2005-04-04 Created: 2005-04-04 Last updated: 2010-10-06Bibliographically approved

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