Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation

Abstract

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.chemically defined ͉ hypothalamus ͉ patterning ͉ vasopressin D ifferentiation culture of mouse ES (mES) cells is a versatile in vitro tool for understanding molecular and cellular controls in early mammalian neurogenesis (1-5). We previously established a mES cell culture system with reduced exogenous signals, namely, serum-free culture of embryoid body-like aggregates (SFEB culture) (4). In this method, ES cells are dissociated (to minimize possible effects of culture substrate matrix), reaggregated (over one day), and cultured as floating aggregates in serum-free medium containing knockout serum replacement (KSR) (6), but with no major exogenous inductive factors, such as Fgf, BMP, Wnt, or Nodal. SFEB-cultured mES cells spontaneously differentiate into neural progenitors that acquire a rostral forebrain fate and efficiently generate telencephalic progenitors positive for Bf1 (FoxG1; (7); see Fig. 1A). Efficient forebrain differentiation is also seen in SFEB-cultured human ES cells (8).In contrast to telencephalic development, relatively little has been known about regulatory signals for early diencephalic development, including the initial specification of the mammalian hypothalamic anlage (9, 10). In the present study, using a similar SFEB culture approach, we wished to steer ES cell differentiation into hypothalamic tissues, which arises from the rostral forebrain region adjacent to the embryonic telencephalon in vivo. However, we noticed at the beginning of this study that hypothalamic mark...

Defining the Integration Capacity of Embryonic Stem Cell-Derived Photoreceptor Precursors

“…1E). This expression profile is consistent with previous observations of neural differentiation using this protocol and the derivation of early retinal progenitors after 7-10 days of culture [8,9,14].…”

“…We sought to further investigate this method of retinal differentiation, with the specific purpose of generating photoreceptor precursors for transplantation. Using the same EB5 mouse ESC line, which contains the eGFP reporter inserted into the first exon of the Rax gene (kind gift of Y. Sasai) [14], we examined the stage-specific differentiation of early retinal progenitors. In chimeric embryos, the expression of Rax.GFP is restricted to the diencephalon, in particular to the eye field and subsequent optic vesicle ( Fig.…”

“…An EB5 mouse ESC line, Rax.GFP (kind gift of Y. Sasai) [14], was maintained and differentiated as previously described [8,9]. Details of the adapted protocol and the generation of the Rax.GFP/Nrl.RFP reporter line are described in Supporting Information Methods.…”

Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue

“…68) under 5% CO 2 at 37°C. The differentiation medium is gfCDM 69 supplemented with 10% KSR, while gfCDM contains 45% Iscove's modified Dulbecco's medium (IMDM, Gibco), 45% Hams F12 (F12, Gibco), Glutamax, 1% chemically defined lipid concentrate (Gibco), monothioglycerol (450 mM, Sigma), 100 U ml À 1 penicillin and 100 mg ml À 1 streptomycin (Gibco). Defining the day on which the SFEBq culture was started as day 0, recombinant human BMP4 (R&D) was added to the culture to the final 1.5 nM (55 ng/ml) on day 6, and its concentration was diluted into half by half medium change every 3rd day.…”

Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells

“…1C; supplementary material Table S1). As these functions are fundamental, the dysfunction of human hypothalamic neurons has been linked to common diseases, such as obesity, hypertension, mood disorders and sleep disorders, that place a substantial burden on Western healthcare systems (MeyerLindenberg et al, 2011;Spiegelman and Flier, 2001;Swaab, 1999Swaab, , 2006Wataya et al, 2008) (supplementary material Table S1). …”