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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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Expression of H19, CDKNIC and 1GF1 in mature human dopaminergic neurons.(A) Amplification plot for tyrosine hydroxylase copy RNA from neuromelanin-positive cells collected by laser capture microdissection in 4 control and 4 Parkinson disease cases (samples 1–8 in Fig. 2). For all samples analyzed, a sequence specific for tyrosine hydroxylase (Ct 22-32) was amplified, indicating the selection of dopaminergic cells. (B) Images of ethidium bromide gels showing the PCR products amplified from reversed-transcribed RNA extracted from the substantia nigra. Lines 1–8 correspond to amplified copy RNA from neuromelanin (+) neurons collected by laser capture microdissection from Parkinson's disease patients (1–4) and age-matched controls (5–8). Line 9 corresponds to total RNA isolated from the substantia nigra of an age-matched control. DNA size markers (1 kb plus ladder, Life tech) are shown on left and right.
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pone-0001422-g005: Expression of H19, CDKNIC and 1GF1 in mature human dopaminergic neurons.(A) Amplification plot for tyrosine hydroxylase copy RNA from neuromelanin-positive cells collected by laser capture microdissection in 4 control and 4 Parkinson disease cases (samples 1–8 in Fig. 2). For all samples analyzed, a sequence specific for tyrosine hydroxylase (Ct 22-32) was amplified, indicating the selection of dopaminergic cells. (B) Images of ethidium bromide gels showing the PCR products amplified from reversed-transcribed RNA extracted from the substantia nigra. Lines 1–8 correspond to amplified copy RNA from neuromelanin (+) neurons collected by laser capture microdissection from Parkinson's disease patients (1–4) and age-matched controls (5–8). Line 9 corresponds to total RNA isolated from the substantia nigra of an age-matched control. DNA size markers (1 kb plus ladder, Life tech) are shown on left and right.

Mentions: We also examined expression of H19, IGF2, and CDKN1C in laser-captured dopamine neurons, identified on the basis of neuromelanin presence, from a series of human postmortem RNA samples from human cases of Parkinson's disease and controls (see Table 1). TH was amplified from each of the samples, verifying that the collected neurons were dopaminergic (Fig. 5A). H19 was found to be expressed in the midbrain, but was not expressed in dopamine neurons per se. CDKN1C was expressed in all samples of laser-captured dopamine neurons (Fig. 5B). These samples were probed for two IGF2 transcripts, and one was positive (NM_001007139), while BC000531 was negative (Fig. 5B). Therefore, certain genes within the H19 cluster, including TH, CDKN1C, and one IGF2 transcript are expressed in mature human dopamine neurons in vivo.


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)

Expression of H19, CDKNIC and 1GF1 in mature human dopaminergic neurons.(A) Amplification plot for tyrosine hydroxylase copy RNA from neuromelanin-positive cells collected by laser capture microdissection in 4 control and 4 Parkinson disease cases (samples 1–8 in Fig. 2). For all samples analyzed, a sequence specific for tyrosine hydroxylase (Ct 22-32) was amplified, indicating the selection of dopaminergic cells. (B) Images of ethidium bromide gels showing the PCR products amplified from reversed-transcribed RNA extracted from the substantia nigra. Lines 1–8 correspond to amplified copy RNA from neuromelanin (+) neurons collected by laser capture microdissection from Parkinson's disease patients (1–4) and age-matched controls (5–8). Line 9 corresponds to total RNA isolated from the substantia nigra of an age-matched control. DNA size markers (1 kb plus ladder, Life tech) are shown on left and right.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2170519&req=5

pone-0001422-g005: Expression of H19, CDKNIC and 1GF1 in mature human dopaminergic neurons.(A) Amplification plot for tyrosine hydroxylase copy RNA from neuromelanin-positive cells collected by laser capture microdissection in 4 control and 4 Parkinson disease cases (samples 1–8 in Fig. 2). For all samples analyzed, a sequence specific for tyrosine hydroxylase (Ct 22-32) was amplified, indicating the selection of dopaminergic cells. (B) Images of ethidium bromide gels showing the PCR products amplified from reversed-transcribed RNA extracted from the substantia nigra. Lines 1–8 correspond to amplified copy RNA from neuromelanin (+) neurons collected by laser capture microdissection from Parkinson's disease patients (1–4) and age-matched controls (5–8). Line 9 corresponds to total RNA isolated from the substantia nigra of an age-matched control. DNA size markers (1 kb plus ladder, Life tech) are shown on left and right.
Mentions: We also examined expression of H19, IGF2, and CDKN1C in laser-captured dopamine neurons, identified on the basis of neuromelanin presence, from a series of human postmortem RNA samples from human cases of Parkinson's disease and controls (see Table 1). TH was amplified from each of the samples, verifying that the collected neurons were dopaminergic (Fig. 5A). H19 was found to be expressed in the midbrain, but was not expressed in dopamine neurons per se. CDKN1C was expressed in all samples of laser-captured dopamine neurons (Fig. 5B). These samples were probed for two IGF2 transcripts, and one was positive (NM_001007139), while BC000531 was negative (Fig. 5B). Therefore, certain genes within the H19 cluster, including TH, CDKN1C, and one IGF2 transcript are expressed in mature human dopamine neurons in vivo.

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
Related in: MedlinePlus