Culture of embryonic-like stem cells from human umbilical cord blood and onward differentiation to neural cells in vitro.
This 3-week protocol produces embryonic-like stem cells from human umbilical cord blood (CBEs) for neural differentiation using a three-step system (cell isolation/expansion/differentiation). The CBE isolation produces a highly purified fraction (CD45-, CD33-, CD7-, CD235a-) of small pluripotent stem cells (2-3 microm in diameter) coexpressing embryonic stem cell markers including Oct4 and Sox2. Initial CBE expansion is performed in high density (5-10 millions per ml) in the presence of extracellular matrix proteins and epidermal growth factor. Subsequent neural differentiation of CBEs requires sequential introduction of morphogenes, retinoic acid, brain-derived neurotrophic factor and cyclic AMP. Described methods emphasize defined media and reagents at all stages of the experiment comparable to protocols described for culturing human embryonic stem cells and cells from other somatic stem cell sources. Neural progenitor and cells generated from CBEs may be used for in vitro drug testing and cell-based assays and potentially for clinical transplantation.
Newcastle Centre for Cord Blood, North East England Stem Cell Institute and Institute of Human Genetics, Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK. c.mcguckin@novussanguis.org
Colin
C
McGuckin
Marcin
M
Jurga
Hamad
H
Ali
Marko
M
Strbad
Nicolas
N
Forraz
D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002469:Cell Separation; D036101:Cord Blood Stem Cell Transplantation; D016895:Culture Media, Serum-Free; D053595:Embryonic Stem Cells / Q000166:cytology*; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D007231:Infant, Newborn; D009474:Neurons / Q000166:cytology*; D039904:Pluripotent Stem Cells / Q000166:cytology
D016895:Culture Media, Serum-Free