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Gene expression profile of neuronal progenitor cells derived from hESCs: activation of chromosome 11p15.5 and comparison to human dopaminergic neurons.

Freed WJ, Chen J, Bäckman CM, Schwartz CM, Vazin T, Cai J, Spivak CE, Lupica CR, Rao MS, Zeng X - PLoS ONE (2008)

Bottom Line: Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated.This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons.IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurobiology Research Branch, Intramural Research Program (IRP), National Institute on Drug Abuse, National Institutes of Health (NIH), Baltimore, Maryland, USA. wfreed@mail.nih.gov

ABSTRACT

Background: We initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription.

Methodology: Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined.

Principal findings: A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture.

Conclusions: The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons.

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Chromosomal distribution of genes in NCAM+ sorted cells mapped to chromosomes.(A) Plot of percentages of total detected genes expressed on each chromosome. (B) Representation of the locations of the highly-expressed genes in 11p15.5, as described in the text. (C) Expression levels (tpm) for genes in the 11p15.5 region in NCAM+ neuronal precursor cells as compared to undifferentiated hESCs.
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pone-0001422-g003: Chromosomal distribution of genes in NCAM+ sorted cells mapped to chromosomes.(A) Plot of percentages of total detected genes expressed on each chromosome. (B) Representation of the locations of the highly-expressed genes in 11p15.5, as described in the text. (C) Expression levels (tpm) for genes in the 11p15.5 region in NCAM+ neuronal precursor cells as compared to undifferentiated hESCs.

Mentions: When the 232 uniquely or highly expressed in NCAM+ cells were sorted for chromosomal locations, they were generally distributed across all chromosomes, with relatively higher numbers of genes on chromosomes 1, 5, 10, 11, 14, 17, and 19 (Fig. 3A). Examination of cytogenetic map locations (Table S4) revealed a number of localized clusters of genes that were highly expressed in NCAM+ cells; most notably, five genes located on chromosome 11p15.5, at or near the H19-IGF2 imprinting center were highly expressed. These were H19, IGF2, CDKN1C, TSSC4, and HBG2. Representations of locations and expression levels of genes in this chromosomal region, from 1.9 to 5.5 MB from the telomere, in NCAM+ cells as compared to undifferentiated hESCs are shown in Figure 3B and 3C. It is clear that several genes in this region are highly expressed in NCAM+ sorted cells, but not in undifferentiated hESCs (Fig. 3C). Among the genes in this region are H19 and IGF2, the only two genes with expression above 50,000 tpm. Some of the same genes are expressed in embryoid bodies and oligodendrocyte precursors as well, although not at the high levels seen for NCAM+ sorted cells (data not shown). There was also a prominent cluster of highly expressed genes at 14q24.3 (see Table S4).


Gene expression profile of neuronal progenitor cells derived from hESCs: activation of chromosome 11p15.5 and comparison to human dopaminergic neurons.

Freed WJ, Chen J, Bäckman CM, Schwartz CM, Vazin T, Cai J, Spivak CE, Lupica CR, Rao MS, Zeng X - PLoS ONE (2008)

Chromosomal distribution of genes in NCAM+ sorted cells mapped to chromosomes.(A) Plot of percentages of total detected genes expressed on each chromosome. (B) Representation of the locations of the highly-expressed genes in 11p15.5, as described in the text. (C) Expression levels (tpm) for genes in the 11p15.5 region in NCAM+ neuronal precursor cells as compared to undifferentiated hESCs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2170519&req=5

pone-0001422-g003: Chromosomal distribution of genes in NCAM+ sorted cells mapped to chromosomes.(A) Plot of percentages of total detected genes expressed on each chromosome. (B) Representation of the locations of the highly-expressed genes in 11p15.5, as described in the text. (C) Expression levels (tpm) for genes in the 11p15.5 region in NCAM+ neuronal precursor cells as compared to undifferentiated hESCs.
Mentions: When the 232 uniquely or highly expressed in NCAM+ cells were sorted for chromosomal locations, they were generally distributed across all chromosomes, with relatively higher numbers of genes on chromosomes 1, 5, 10, 11, 14, 17, and 19 (Fig. 3A). Examination of cytogenetic map locations (Table S4) revealed a number of localized clusters of genes that were highly expressed in NCAM+ cells; most notably, five genes located on chromosome 11p15.5, at or near the H19-IGF2 imprinting center were highly expressed. These were H19, IGF2, CDKN1C, TSSC4, and HBG2. Representations of locations and expression levels of genes in this chromosomal region, from 1.9 to 5.5 MB from the telomere, in NCAM+ cells as compared to undifferentiated hESCs are shown in Figure 3B and 3C. It is clear that several genes in this region are highly expressed in NCAM+ sorted cells, but not in undifferentiated hESCs (Fig. 3C). Among the genes in this region are H19 and IGF2, the only two genes with expression above 50,000 tpm. Some of the same genes are expressed in embryoid bodies and oligodendrocyte precursors as well, although not at the high levels seen for NCAM+ sorted cells (data not shown). There was also a prominent cluster of highly expressed genes at 14q24.3 (see Table S4).

Bottom Line: Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated.This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons.IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurobiology Research Branch, Intramural Research Program (IRP), National Institute on Drug Abuse, National Institutes of Health (NIH), Baltimore, Maryland, USA. wfreed@mail.nih.gov

ABSTRACT

Background: We initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription.

Methodology: Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined.

Principal findings: A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture.

Conclusions: The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons.

Show MeSH