Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation.
Embryonic stem (ES) cells differentiate into neuroectodermal progenitors when cultured as floating aggregates in serum-free conditions. Here, we show that strict removal of exogenous patterning factors during early differentiation steps induces efficient generation of rostral hypothalamic-like progenitors (Rax(+)/Six3(+)/Vax1(+)) in mouse ES cell-derived neuroectodermal cells. The use of growth factor-free chemically defined medium is critical and even the presence of exogenous insulin, which is commonly used in cell culture, strongly inhibits the differentiation via the Akt-dependent pathway. The ES cell-derived Rax(+) progenitors generate Otp(+)/Brn2(+) neuronal precursors (characteristic of rostral-dorsal hypothalamic neurons) and subsequently magnocellular vasopressinergic neurons that efficiently release the hormone upon stimulation. Differentiation markers of rostral-ventral hypothalamic precursors and neurons are induced from ES cell-derived Rax(+) progenitors by treatment with Shh. Thus, in the absence of exogenous growth factors in medium, the ES cell-derived neuroectodermal cells spontaneously differentiate into rostral (particularly rostral-dorsal) hypothalamic-like progenitors, which generate characteristic hypothalamic neuroendocrine neurons in a stepwise fashion, as observed in vivo. These findings indicate that, instead of the addition of inductive signals, minimization of exogenous patterning signaling plays a key role in rostral hypothalamic specification of neural progenitors derived from pluripotent cells.
Wataya
Takafumi
T
Organogenesis and Neurogenesis Group and Division of Human Stem Cell Technology, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
Ando
Satoshi
S
Muguruma
Keiko
K
Ikeda
Hanako
H
Watanabe
Kiichi
K
Eiraku
Mototsugu
M
Kawada
Masako
M
Takahashi
Jun
J
Hashimoto
Nobuo
N
Sasai
Yoshiki
Y
D000818:Animals; D015415:Biomarkers; D002454:Cell Differentiation; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D053595:Embryonic Stem Cells / Q000166:cytology*; D005136:Eye Proteins / Q000378:metabolism; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000378:metabolism; D007031:Hypothalamus / Q000166:cytology* / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism
D015415:Biomarkers; D017077:Culture Media, Conditioned; D005136:Eye Proteins; D018398:Homeodomain Proteins; D007328:Insulin; D036341:Intercellular Signaling Peptides and Proteins; C105441:Rax protein, mouse; D014157:Transcription Factors; D051057:Proto-Oncogene Proteins c-akt