Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension.
Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x(L) and oxygen tension on dopaminergic differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-x(L) over-expressing subline (hVMbcl-x(L)) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10 days at 20% oxygen, hVMbcl-x(L) cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 +/- 0.8% to 17.2 +/- 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q-PCR-analysis revealed expression of several dopaminergic markers besides of TH just as dopamine was detected in the culture medium by HPLC analysis. Although Bcl-x(L)-over-expression reduced cell death in the cultures, it did not alter the relative content of GABAergic, neurons, while the content of astroglial cells was reduced in hVMbcl-x(L) cell cultures compared with control. We conclude that Bcl-x(L) and lowered oxygen tension act in concert to enhance dopaminergic differentiation and survival of human neural stem cells.
Krabbe
Christina
C
Department of Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Odense C, Denmark.
Courtois
Elise
E
Jensen
Pia
P
Jørgensen
Jesper R
JR
Zimmer
Jens
J
Martínez-Serrano
Alberto
A
Meyer
Morten
M
D051033:Apoptosis Inducing Factor / Q000378:metabolism; D001973:Bromodeoxyuridine / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005576:Colforsin / Q000494:pharmacology; D003995:Dibutyryl Cyclic GMP / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000502:physiology*; D005333:Fetus; D015870:Gene Expression / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics; D015707:HN Protein / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008636:Mesencephalon / Q000166:cytology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009474:Neurons / Q000502:physiology*; D010100:Oxygen / Q000494:pharmacology*; D010703:Phorbol Esters / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics / Q000502:physiology*
C494829:AIFM1 protein, human; D051033:Apoptosis Inducing Factor; D015707:HN Protein; D036341:Intercellular Signaling Peptides and Proteins; D007476:Ionophores; C489788:MAP2 protein, human; D008869:Microtubule-Associated Proteins; D010703:Phorbol Esters; D051020:bcl-X Protein; D049452:Green Fluorescent Proteins; D005576:Colforsin; D003995:Dibutyryl Cyclic GMP; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C032974:12-O-retinoylphorbol-13-acetate; D007770:L-Lactate Dehydrogenase; D014446:Tyrosine 3-Monooxygenase; D001973:Bromodeoxyuridine; D010100:Oxygen; D004298:Dopamine