Genetic selection of sox1GFP-expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation.
Because of their ability to proliferate and to differentiate into diverse cell types, embryonic stem (ES) cells are a potential source of cells for transplantation therapy of various diseases, including Parkinson's disease. A critical issue for this potential therapy is the elimination of undifferentiated cells that, even in low numbers, could result in teratoma formation in the host brain. We hypothesize that an efficient solution would consist of purifying the desired cell types, such as neural precursors, prior to transplantation. To test this hypothesis, we differentiated sox1-green fluorescent protein (GFP) knock-in ES cells in vitro, purified neural precursor cells by fluorescence-activated cell sorting (FACS), and characterized the purified cells in vitro as well as in vivo. Immunocytofluorescence and RT-PCR analyses showed that this genetic purification procedure efficiently removed undifferentiated pluripotent stem cells. Furthermore, when differentiated into mature neurons in vitro, the purified GFP+ cell population generated enriched neuronal populations, whereas the GFP- population generated much fewer neurons. When treated with dopaminergic inducing signals such as sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF8), FACS-purified neural precursor cells responded to these molecules and generated dopaminergic neurons as well as other neural subtypes. When transplanted, the GFP+ cell population generated well contained grafts containing dopaminergic neurons, whereas the GFP- population generated significantly larger grafts (about 20-fold) and frequent tumor-related deaths in the transplanted animals. Taken together, our results demonstrate that genetic purification of neural precursor cells using FACS isolation can effectively remove unwanted proliferating cell types and avoid tumor formation after transplantation.
Chung
S
S
Udall Parkinson's Disease Research Center of Excellence, McLean Hospital, Belmont, MA 02178, USA.
Shin
B-S
BS
Hedlund
E
E
Pruszak
J
J
Ferree
A
A
Kang
Un Jung
UJ
Isacson
Ole
O
Kim
Kwang-Soo
KS
D000818:Animals; D001932:Brain Neoplasms / Q000209:etiology / Q000503:physiopathology / Q000517:prevention & control*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics*; D004298:Dopamine / Q000378:metabolism; D051524:Fibroblast Growth Factor 8 / Q000378:metabolism / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics; D053823:Hedgehog Proteins; D006609:High Mobility Group Proteins / Q000235:genetics*; D007381:Intermediate Filament Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011183:Postoperative Complications / Q000209:etiology / Q000503:physiopathology / Q000517:prevention & control; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors; D033581:Stem Cell Transplantation / Q000009:adverse effects / Q000379:methods*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013724:Teratoma / Q000209:etiology / Q000503:physiopathology / Q000517:prevention & control*; D015534:Trans-Activators / Q000378:metabolism / Q000494:pharmacology
D004268:DNA-Binding Proteins; C496457:Fgf8 protein, mouse; D053823:Hedgehog Proteins; D006609:High Mobility Group Proteins; D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D011993:Recombinant Fusion Proteins; D055748:SOXB1 Transcription Factors; C465570:Shh protein, mouse; C098693:Sox1 protein, mouse; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D051524:Fibroblast Growth Factor 8; D004298:Dopamine