Abstract
Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson disease models, but can generate teratomas. Purification of dopamine neurons derived from embryonic stem cells by fluorescence-activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive enhanced green fluorescent protein (eGFP) expression in mES cells. First, we evaluated 2.5-kilobase (kb) and 9-kb TH promoter fragments and showed that clones generated using the 9-kb fragment produced significantly more eGFP+/TH+ neurons. We selected the 9-kb TH clone with the highest eGFP/TH overlap for further differentiation, FACS, and transplantation experiments. Grafts contained large numbers of eGFP+ dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP+ cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings, we examined the expression of stem cell markers in our eGFP+ population. We found that a majority of eGFP+ cells were stage-specific embryonic antigen-positive (SSEA-1+) and that the genetically engineered clones contained more SSEA-1+ cells after differentiation than the original D3 mES cells. By negative selection of SSEA-1, we could isolate a neuronal eGFP+ population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell-derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression.
| Original language | English |
|---|---|
| Pages (from-to) | 1126-1135 |
| Number of pages | 10 |
| Journal | Stem Cells |
| Volume | 25 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - 2007 May 1 |
| Externally published | Yes |
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Keywords
- CD-15
- Fluorescence-activated cell sorting
- Genetic engineering
- Parkinson disease
- Stage-specific embryonic antigen 1
ASJC Scopus subject areas
- Cell Biology
Cite this
Selection of embryonic stem cell-derived enhanced green fluorescent protein-positive dopamine neurons using the tyrosine hydroxylase promoter is confounded by reporter gene expression in immature cell populations. / Hedlund, Eva; Pruszak, Jan; Ferree, Andrew; Viñuela, Angel; Hong, Sunghoi; Isacson, Ole; Kim, Kwang Soo.
In: Stem Cells, Vol. 25, No. 5, 01.05.2007, p. 1126-1135.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Selection of embryonic stem cell-derived enhanced green fluorescent protein-positive dopamine neurons using the tyrosine hydroxylase promoter is confounded by reporter gene expression in immature cell populations
AU - Hedlund, Eva
AU - Pruszak, Jan
AU - Ferree, Andrew
AU - Viñuela, Angel
AU - Hong, Sunghoi
AU - Isacson, Ole
AU - Kim, Kwang Soo
PY - 2007/5/1
Y1 - 2007/5/1
N2 - Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson disease models, but can generate teratomas. Purification of dopamine neurons derived from embryonic stem cells by fluorescence-activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive enhanced green fluorescent protein (eGFP) expression in mES cells. First, we evaluated 2.5-kilobase (kb) and 9-kb TH promoter fragments and showed that clones generated using the 9-kb fragment produced significantly more eGFP+/TH+ neurons. We selected the 9-kb TH clone with the highest eGFP/TH overlap for further differentiation, FACS, and transplantation experiments. Grafts contained large numbers of eGFP+ dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP+ cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings, we examined the expression of stem cell markers in our eGFP+ population. We found that a majority of eGFP+ cells were stage-specific embryonic antigen-positive (SSEA-1+) and that the genetically engineered clones contained more SSEA-1+ cells after differentiation than the original D3 mES cells. By negative selection of SSEA-1, we could isolate a neuronal eGFP+ population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell-derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression.
AB - Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson disease models, but can generate teratomas. Purification of dopamine neurons derived from embryonic stem cells by fluorescence-activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive enhanced green fluorescent protein (eGFP) expression in mES cells. First, we evaluated 2.5-kilobase (kb) and 9-kb TH promoter fragments and showed that clones generated using the 9-kb fragment produced significantly more eGFP+/TH+ neurons. We selected the 9-kb TH clone with the highest eGFP/TH overlap for further differentiation, FACS, and transplantation experiments. Grafts contained large numbers of eGFP+ dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP+ cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings, we examined the expression of stem cell markers in our eGFP+ population. We found that a majority of eGFP+ cells were stage-specific embryonic antigen-positive (SSEA-1+) and that the genetically engineered clones contained more SSEA-1+ cells after differentiation than the original D3 mES cells. By negative selection of SSEA-1, we could isolate a neuronal eGFP+ population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell-derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression.
KW - CD-15
KW - Fluorescence-activated cell sorting
KW - Genetic engineering
KW - Parkinson disease
KW - Stage-specific embryonic antigen 1
UR - http://www.scopus.com/inward/record.url?scp=34247590317&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34247590317&partnerID=8YFLogxK
U2 - 10.1634/stemcells.2006-0540
DO - 10.1634/stemcells.2006-0540
M3 - Article
C2 - 17234989
AN - SCOPUS:34247590317
VL - 25
SP - 1126
EP - 1135
JO - Stem Cells
JF - Stem Cells
SN - 1066-5099
IS - 5
ER -