Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells.
Human embryonic stem cells (hESCs) have been directed to differentiate into neuronal cells using many cell-culture techniques. Central nervous system cells with clinical importance have been produced from hESCs. To date, however, there have been no definitive reports of generation of peripheral neurons from hESCs. We used a modification of the method of Sasai and colleagues for mouse and primate embryonic stem cells to elicit neuronal differentiation from hESCs. When hESCs are cocultured with the mouse stromal line PA6 for 3 weeks, neurons are induced that coexpress (a) peripherin and Brn3a, and (b) peripherin and tyrosine hydroxylase, combinations characteristic of peripheral sensory and sympathetic neurons, respectively. In vivo, peripheral sensory and sympathetic neurons develop from the neural crest (NC). Analysis of expression of mRNAs identified in other species as NC markers reveals that the PA6 cells induce NC-like cells before neuronal differentiation takes place. Several NC markers, including SNAIL, dHAND, and Sox9, are increased at 1 week of coculture relative to naive cells. Furthermore, the expression of several NC marker genes known to be downregulated upon in vivo differentiation of NC derivatives, was observed to be present at lower levels at 3 weeks of PA6-hESC coculture than at 1 week. Our report is the first on the expression of molecular markers of NC-like cells in primates, in general, and in humans, specifically. Our results suggest that this system can be used for studying molecular and cellular events in the almost inaccessible human NC, as well as for producing normal human peripheral neurons for developing therapies for diseases such as familial dysautonomia.
Pomp
Oz
O
Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
Brokhman
Irina
I
Ben-Dor
Israel
I
Reubinoff
Benjamin
B
Goldstein
Ronald S
RS
D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D046508:Culture Techniques; D015536:Down-Regulation; D004622:Embryo, Mammalian / Q000166:cytology*; D006609:High Mobility Group Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D009432:Neural Crest / Q000166:cytology*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor; D000071250:Snail Family Transcription Factors; D013234:Stem Cells / Q000166:cytology*; D013564:Sympathetic Nervous System / Q000502:physiology; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D029961:Zebrafish Proteins
D051792:Basic Helix-Loop-Helix Proteins; D006609:High Mobility Group Proteins; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D029961:Zebrafish Proteins; C498765:HAND2 protein, human; C498766:Hand2 protein, mouse; C527932:SOX9 protein, human; C527933:Sox9 protein, mouse; C410915:hand2 protein, zebrafish