Aim
To find a safe source for dopaminergic neurons, we generated neural progenitor cell lines from human embryonic stem cells.
Conclusion
Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.
Methods
The human embryonic stem (hES) cell line H9 was used to generate human neural progenitor (HNP) cell lines. The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers, including beta-III tubulin (TUJ1) and tyrosine hydroxylase (TH). To assess the risk of teratoma or other tumor formation, HNP cell lines and mouse neuronal progenitor (MNP) cell lines were injected subcutaneously into immunodeficient SCID/beige mice.
Results
We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells. These cell lines, which can be stored in liquid nitrogen for several years, have the potential to differentiate in vitro into dopaminergic neurons. Following day 30 of differentiation culture, the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH, indicating differentiation into dopaminergic neurons. In contrast to H9 ES cells, the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection. Similarly, no tumors developed after injection of MNP cells. Notably, mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90% of the recipients.