Limits...
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.

Show MeSH
Verification of genes uniquely expressed in NCAM+ cells by RT-PCR and qPCR.Eighteen genes that were highly expressed in NCAM+ cells were selected for verification by RT-PCR. (A) Expression of EMP3, SLC7A7, Pitx1, Msx1, Pitx2, SDF2L1, NPY and NINJ1 was detected in NCAM+ cells by RT-PCR. Of these, two (SDF2L1 and NINJ1) were also expressed in undifferentiated cells, and the other 6 genes (EMP3, SLC7A7, Pitx1, Pitx2, Msx1 and NPY) were expressed only in NCAM+ cells but were absent or expressed at very low levels in undifferentiated hESCs. (B) qPCR results for Pitx1 and Sdf2l1 expression in NCAM+ cells and undifferentiated hESCs. (C) Expression of seven unknown genes lacking known protein products in undifferentiated hESCs and in NCAM+ sorted cells. Hs.534052, Hs.473109, Hs.479491, Hs.19193 and Hs.551588 (H19) were detected only in NCAM+ sorted cells. Hs.446315 was detected in undifferentiated hESC, but at a lower level than for NCAM+ sorted cells. No difference in expression was detected for Hs.109798 or Hs.211282. (D) RT-PCR examination of IGF2 and TSSC4 expression in undifferentiated hESCs and NCAM+ sorted cells IGF2 and TSSC4 were also examined in 12-day differentiated, unsorted cultures (Diff D12). IGF2 was not detected in the undifferentiated hESCs, whereas no difference was seen for TSSC4.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2170519&req=5

pone-0001422-g004: Verification of genes uniquely expressed in NCAM+ cells by RT-PCR and qPCR.Eighteen genes that were highly expressed in NCAM+ cells were selected for verification by RT-PCR. (A) Expression of EMP3, SLC7A7, Pitx1, Msx1, Pitx2, SDF2L1, NPY and NINJ1 was detected in NCAM+ cells by RT-PCR. Of these, two (SDF2L1 and NINJ1) were also expressed in undifferentiated cells, and the other 6 genes (EMP3, SLC7A7, Pitx1, Pitx2, Msx1 and NPY) were expressed only in NCAM+ cells but were absent or expressed at very low levels in undifferentiated hESCs. (B) qPCR results for Pitx1 and Sdf2l1 expression in NCAM+ cells and undifferentiated hESCs. (C) Expression of seven unknown genes lacking known protein products in undifferentiated hESCs and in NCAM+ sorted cells. Hs.534052, Hs.473109, Hs.479491, Hs.19193 and Hs.551588 (H19) were detected only in NCAM+ sorted cells. Hs.446315 was detected in undifferentiated hESC, but at a lower level than for NCAM+ sorted cells. No difference in expression was detected for Hs.109798 or Hs.211282. (D) RT-PCR examination of IGF2 and TSSC4 expression in undifferentiated hESCs and NCAM+ sorted cells IGF2 and TSSC4 were also examined in 12-day differentiated, unsorted cultures (Diff D12). IGF2 was not detected in the undifferentiated hESCs, whereas no difference was seen for TSSC4.

Mentions: To verify the differential expression in NCAM+ cells detected by MPSS, we selected 18 genes (EMP3, SLC7A7, Pitx1, Msx1, Pitx2, SDF2L1, NPY and NINJ1, Hs.551588 (H19), Hs.19193, Hs.473109, Hs.109798, Hs.534052, Hs.446315, Hs.211282 and Hs.479491, TSSC4, and IGF2) for RT-PCR analysis (Fig. 4).


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)

Verification of genes uniquely expressed in NCAM+ cells by RT-PCR and qPCR.Eighteen genes that were highly expressed in NCAM+ cells were selected for verification by RT-PCR. (A) Expression of EMP3, SLC7A7, Pitx1, Msx1, Pitx2, SDF2L1, NPY and NINJ1 was detected in NCAM+ cells by RT-PCR. Of these, two (SDF2L1 and NINJ1) were also expressed in undifferentiated cells, and the other 6 genes (EMP3, SLC7A7, Pitx1, Pitx2, Msx1 and NPY) were expressed only in NCAM+ cells but were absent or expressed at very low levels in undifferentiated hESCs. (B) qPCR results for Pitx1 and Sdf2l1 expression in NCAM+ cells and undifferentiated hESCs. (C) Expression of seven unknown genes lacking known protein products in undifferentiated hESCs and in NCAM+ sorted cells. Hs.534052, Hs.473109, Hs.479491, Hs.19193 and Hs.551588 (H19) were detected only in NCAM+ sorted cells. Hs.446315 was detected in undifferentiated hESC, but at a lower level than for NCAM+ sorted cells. No difference in expression was detected for Hs.109798 or Hs.211282. (D) RT-PCR examination of IGF2 and TSSC4 expression in undifferentiated hESCs and NCAM+ sorted cells IGF2 and TSSC4 were also examined in 12-day differentiated, unsorted cultures (Diff D12). IGF2 was not detected in the undifferentiated hESCs, whereas no difference was seen for TSSC4.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001422-g004: Verification of genes uniquely expressed in NCAM+ cells by RT-PCR and qPCR.Eighteen genes that were highly expressed in NCAM+ cells were selected for verification by RT-PCR. (A) Expression of EMP3, SLC7A7, Pitx1, Msx1, Pitx2, SDF2L1, NPY and NINJ1 was detected in NCAM+ cells by RT-PCR. Of these, two (SDF2L1 and NINJ1) were also expressed in undifferentiated cells, and the other 6 genes (EMP3, SLC7A7, Pitx1, Pitx2, Msx1 and NPY) were expressed only in NCAM+ cells but were absent or expressed at very low levels in undifferentiated hESCs. (B) qPCR results for Pitx1 and Sdf2l1 expression in NCAM+ cells and undifferentiated hESCs. (C) Expression of seven unknown genes lacking known protein products in undifferentiated hESCs and in NCAM+ sorted cells. Hs.534052, Hs.473109, Hs.479491, Hs.19193 and Hs.551588 (H19) were detected only in NCAM+ sorted cells. Hs.446315 was detected in undifferentiated hESC, but at a lower level than for NCAM+ sorted cells. No difference in expression was detected for Hs.109798 or Hs.211282. (D) RT-PCR examination of IGF2 and TSSC4 expression in undifferentiated hESCs and NCAM+ sorted cells IGF2 and TSSC4 were also examined in 12-day differentiated, unsorted cultures (Diff D12). IGF2 was not detected in the undifferentiated hESCs, whereas no difference was seen for TSSC4.
Mentions: To verify the differential expression in NCAM+ cells detected by MPSS, we selected 18 genes (EMP3, SLC7A7, Pitx1, Msx1, Pitx2, SDF2L1, NPY and NINJ1, Hs.551588 (H19), Hs.19193, Hs.473109, Hs.109798, Hs.534052, Hs.446315, Hs.211282 and Hs.479491, TSSC4, and IGF2) for RT-PCR analysis (Fig. 4).

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