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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 neuronal markers in hESCs during dopaminergic differentiation and in FACS-isolated NCAM+ cells.(A) A culture of hESCs differentiated for 16 days on PA6 cells immunostained for β-III-tubulin. (B) Immunostaining for TH after 21 days. Bar = 50 µm. (C) Quantification of hESC colonies positive for PSA-NCAM, during differentiation of hESCs co-cultured with PA6 cells. (D) Expression of PSA-NCAM in day 14 hESCs by immunocytochemistry. Bar = 50 µm. (E) Morphology of FACS-isolated hESC by PSA-NCAM after 14 days of subsequent differentiation. (F) Expression of markers for undifferentiated hESC and neuronal markers in FACS-isolated NCAM+ cells by RT-PCR.
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pone-0001422-g001: Expression of neuronal markers in hESCs during dopaminergic differentiation and in FACS-isolated NCAM+ cells.(A) A culture of hESCs differentiated for 16 days on PA6 cells immunostained for β-III-tubulin. (B) Immunostaining for TH after 21 days. Bar = 50 µm. (C) Quantification of hESC colonies positive for PSA-NCAM, during differentiation of hESCs co-cultured with PA6 cells. (D) Expression of PSA-NCAM in day 14 hESCs by immunocytochemistry. Bar = 50 µm. (E) Morphology of FACS-isolated hESC by PSA-NCAM after 14 days of subsequent differentiation. (F) Expression of markers for undifferentiated hESC and neuronal markers in FACS-isolated NCAM+ cells by RT-PCR.

Mentions: On day 0, undifferentiated BG03 cells were transferred to co-culture with mouse stromal PA6 cells. TH-positive cells were first detected in small numbers at days 10–12. Numerous β-III tubulin (Tuj1)+ cells and TH+ cells were identified in the cultures by days 16 and 21, respectively (Fig. 1A–B).


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 neuronal markers in hESCs during dopaminergic differentiation and in FACS-isolated NCAM+ cells.(A) A culture of hESCs differentiated for 16 days on PA6 cells immunostained for β-III-tubulin. (B) Immunostaining for TH after 21 days. Bar = 50 µm. (C) Quantification of hESC colonies positive for PSA-NCAM, during differentiation of hESCs co-cultured with PA6 cells. (D) Expression of PSA-NCAM in day 14 hESCs by immunocytochemistry. Bar = 50 µm. (E) Morphology of FACS-isolated hESC by PSA-NCAM after 14 days of subsequent differentiation. (F) Expression of markers for undifferentiated hESC and neuronal markers in FACS-isolated NCAM+ cells by RT-PCR.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001422-g001: Expression of neuronal markers in hESCs during dopaminergic differentiation and in FACS-isolated NCAM+ cells.(A) A culture of hESCs differentiated for 16 days on PA6 cells immunostained for β-III-tubulin. (B) Immunostaining for TH after 21 days. Bar = 50 µm. (C) Quantification of hESC colonies positive for PSA-NCAM, during differentiation of hESCs co-cultured with PA6 cells. (D) Expression of PSA-NCAM in day 14 hESCs by immunocytochemistry. Bar = 50 µm. (E) Morphology of FACS-isolated hESC by PSA-NCAM after 14 days of subsequent differentiation. (F) Expression of markers for undifferentiated hESC and neuronal markers in FACS-isolated NCAM+ cells by RT-PCR.
Mentions: On day 0, undifferentiated BG03 cells were transferred to co-culture with mouse stromal PA6 cells. TH-positive cells were first detected in small numbers at days 10–12. Numerous β-III tubulin (Tuj1)+ cells and TH+ cells were identified in the cultures by days 16 and 21, respectively (Fig. 1A–B).

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