Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules - PubMed (original) (raw)

Comparative Study

Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules

Xue-Jun Li et al. Stem Cells. 2008 Apr.

Abstract

Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these naïve pluripotent cells in regenerative medicine. Determination of the nontarget differentiated populations, which is lacking in the field, is also crucial. Here, we show an efficient differentiation of motor neurons ( approximately 50%) by a simple sequential application of retinoid acid and sonic hedgehog (SHH) in a chemically defined suspension culture. We also discovered that purmorphamine, a small molecule that activates the SHH pathway, could replace SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+, Irx3+, and Pax7-), with the exception of motor neurons (HB9+) and their progenitors (Olig2+). Thus, the directed neural differentiation system with small molecules, even without further purification, will facilitate basic and translational studies using human motoneurons at a minimal cost.

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Conflict of interest statement

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

Figures

Figure 1

Figure 1. Near complete specification of ventral spinal progenitors from human ESCs in suspension culture

(A): Schematic procedure for ventral spinal progenitor differentiation.(B): Primitive NE (d10), after treatment with RA for 1 week, were isolated and cultured in suspension without (control; upper row) or with (lower row) RA for another week (total 24 d). RA induced the expressionof Hoxb4 but inhibited Otx2 expression.Very few cells expressed Phox2b in the RA-treated cultures. (C): Posteriorized neuralprogenitors (d17) were cultured in the absence(upper row) or presence (lower row)of SHH, and expression of transcriptional factors along the dorsal-ventral axis was examined at d28. In the absence of SHH, a small population of cells expressed Nkx2.2 and Olig2, whereas more cells were positive for Irx3, among which some also expressed Pax7. When SHH (100 ng/ml) was added (lower row), a large portion of cells expressedOlig2 or Nkx2.2, whereas few cells were positive for Irx3 and no cells werepositively stained for Pax7 (second row).Blue indicates Hoechst-stained nuclei. Scalebars = 50 µm. Abbreviations: d, days; E, embryonic day; NE, neuroepithelia; RA, retinoidacid; SHH, sonic hedgehog.

Figure 2

Figure 2. Highly efficient generation of motoneurons in the continual presence of sonic hedgehog

(A): Olig2+ motoneuron progenitors peaked at approximately 4 weeks after differentiation, when HB9+ postmitotic motoneurons began to appear. Subsequently, the population of HB9+ motoneurons increased and peaked at 5 weeks. (B): A confocal image showing the separation of most Olig2- and HB9-positive cells at 5 weeks after differentiation. (C): Diagram showing the change of population of Olig2+ and HB9+ cells at 4–5 weeks after differentiation. Data are presented as mean ± SEM; n = 15–17. Blue indicates Hoechst-stained nuclei. Scale bars = 50 µm. Abbreviation: w, weeks.

Figure 3

Figure 3. SHH promotes proliferation of Olig2+ progenitors

(A): Olig2-enriched clusters were dissociated and plated on polyornithine/laminin-coated coverslips in the neural medium supplemented with B27 in the absence or presence of SHH (100 ng/ml) for 24 hours. More Olig2+ and Ki67+/ Olig2+ cells were seen with SHH than without SHH. TUNEL staining showed no difference between the SHH and non-SHH groups. Blue indicates Hoechst-stained nuclei. Scale bars = 50 µm. (B): Quantitative analyses indicated that there were more Olig2+, Ki67+/Olig2+ cells in the SHH-treated cultures than in the control cultures without SHH, whereas the numbers of Ki67+ and TUNEL+ cells in the total differentiated cells were similar between the SHH and non-SHH-treated groups. Data are presented as mean ± SEM; n = 7–8.*, analysis of variance test between SHH and non-SHH-treated groups, p < .05. Abbreviations: SHH, sonic hedgehog; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Figure 4

Figure 4. Efficient generation of spinal progenitors and motor neurons by purmorphamine

(A): Caudalized neuroepithelia (NE) (day 17) were treated with RA and different concentrations of purmorphamine. At 3.5 weeks after human ESC differentiation, Olig2 was induced by purmorphamine in a dose-dependent manner. (B): Cell populations were quantified by fluorescence-activated cell sorting, as exemplified by Olig2-expressing cells in the purmorphamine (1 µM) group. (C): Diagram showing time-dependent change of population of Olig2+ and HB9+ cells after differentiation. Data are presented as mean ± SEM; n = 5–7. (D): At 4.5 weeks, the expression of Olig2 and HB9 increased to more than 40%. (E): After another week of differentiation in adherent cultures, most HB9+ motoneurons also expressed ChAT. (F): Synapsin-positive neurites colocalized with α-BTX-stained acetylcholine receptors on the surface of the myotube (arrow) after 2 weeks of coculture of motor neurons and C2C12 myoblasts, as shown on a 0.5-µm confocal section. (G): Reverse transcription-polymerase chain reaction analyses indicated expression of transcriptional factors by caudalized NE that were cultured with RA (0.1 µM), purmorphamine, SHH, or purmorphamine plus RA for 1 week (day 24). Blue indicates Hoechst-stained nuclei. Scale bars = 50 µm (A, D, E) and 30 µm (F). Abbreviations: ChAT, choline acetyltransferase; Pur, purmorphamine; RA, retinoid acid; SHH, sonic hedgehog; w, weeks.

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