Differentiation of human pluripotent stem cells into Medial Ganglionic Eminence vs. Caudal Ganglionic Eminence cells.

paper Cited Public

Authors: Ahn, Sandra; Kim, Tae-Gon; Kim, Kwang-Soo; Chung, Sangmi
Year: 2016
Journal: Methods (San Diego, Calif.)
PMID: 26364591
DOI: 10.1016/j.ymeth.2015.09.009
PMCID: PMC4786472

Fig. 1

(a–d) Bright-field images taken during in vitro differentiation of H9 cells. (e and f) H9-derived MGE cells, assayed after 25 days of differentiation, highly expressed independent ventral telencephalic marker Olig2 and telencephalic marker FoxG1 (adapted from [46]). Scale bar: 100 µm.

Fig. 2

FGF8 and FGF19 regulate rostral/caudal identity of ventral telencephalic cells (adapted from [46]). FGF8 treatment induced MGE phenotype while FGF19 induced CGE phenotype, as shown by immunocytochemistry and cell counting analysis after 3 weeks of differentiation of H9 cells (Mean ± SEM; n = 3, P < 0.05, two tailed t-test). The image in the insets is Hoechst-labeled nuclei of the same microscope field. Scale bar: 100 µm.

Fig. 3

(a) Overview of the optimized MGE derivation protocol. (b) Overview of the optimized CGE derivation protocol.

Fig. 4

FACS analysis of fixed cells (adapted from [46]). (a) FACS analysis of MGE cells generated from H9 cells after Nkx2.1 staining at day 21 of differentiation. (b–d) Combined and temporal treatment with IWP-2, SAG and FGF8 resulted in robust induction of MGE cells from H9 and H7 hESCs as well as iPSCs, assayed after 25 days of differentiation. The image in the insets is Hoechst-labeled nuclei of the same microscope field. Scale bar: 100 µm.

Fig. 5

Gene expression analysis during MGE derivation of H9 cells (adapted from [46]), assayed by real-time PCR (Mean ± SEM; n = 3) on days 0, 3, 7, 14 and 21 of differentiation. The expression level of Nanog, Oct4, Nkx2.1, Gsx2, Dlx2 and Lhx6 was analyzed at each time point after differentiation. The expression level of each gene was normalized to that of GAPDH.

Fig. 6

Human MGE cells generated GABAergic interneurons (adapted from [46] and [29]). (a–l) Immunocytochemistry and cell counting analysis after 6 weeks of differentiation (Mean ± SEM; n = 4, P < 0.05, two tailed t-test), showing the expression of β-tubulin, GABA, Lhx6, GAD, Sox6, Calbindin (Calb), Parvalbumin (PV) and Somatostatin (SST). Immunostaining was performed on H9-derived cells except a and c, in which H7-derived cells were analyzed. The image in the insets is Hoechst-labeled nuclei of the same microscope field. (m–s) Immunohistochemical analysis of transplanted H7-derived MGE cells 4 months after transplantation, showing the expression of SST, PV, Calretinin (Calr), Neuropeptide Y (NPY), Calb among transplanted human nuclei (hNuc)+ cells. Scale bar: 100 µm.

Fig. 7

Human CGE cells generated VIP+ or Calretinin+ neurons (adapted from [46]). Immunocytochemistry after 60 days of differentiation of H9 cells. Scale bar: 100 µm.

#	Section	Preview
0	1. Introduction	During early neurodevelopment, cortical interneuron progenitors arise from the ventral telencephalic…
1	1. Introduction	Dysfunction of interneurons has been implicated in various brain diseases, such as epilepsy,…
2	1. Introduction	In this study, we provide a detailed description of the method for the stepwise and combinatorial…
3	2. Materials — 2.1. Reagents — 2.1.1. Cells	Human embryonic stem cells (ESCs): H9 cells, WA09 (WiCell, Madison, WI, passage 45–55)Human ESCs:…
4	2. Materials — 2.1. Reagents — 2.1.2. Growth media and supplements	DMEM (Life Technologies, Carlsbad, CA, cat. no. 11965–118)Essential 8 medium kit (E8 medium; Life…
5	2. Materials — 2.1. Reagents — 2.1.3. Enzymes and other reagents	Matrigel, hESC-qualified matrix (BD Biosciences, San Jose, CA, cat. no. 354277)DPBS, without calcium…
6	2. Materials — 2.1. Reagents — 2.1.3. Enzymes and other reagents	donkey serum (NDS; Sigma-Aldrich, St. Louis, MO, cat. no. D9663)Triton X-100 (Sigma-Aldrich, St.…
7	2. Materials — 2.1. Reagents — 2.1.4. Antibodies	Table 1.
8	2. Materials — 2.1. Reagents — 2.1.5. Primers	Table 2.
9	2. Materials — 2.2. Equipment and software	Laminar flow hoodCO2 incubator (Sanyo, Osaka, Japan)Refrigerator, 4 °C (Sanyo, Osaka,…
10	2. Materials — 2.2. Equipment and software	(Chemglass life Sciences, Vineland, NJ, cat. no. CLS-1760-012)Standard 24-well tissue culture plates…
11	3. Methods — 3.1. PSC culture — 3.1.1. Preparation of Matrigel-coated plates	Matrigel was used as a substrate to support the expansion of hPSCs. At least 1 day before the…
12	3. Methods — 3.1. PSC culture — 3.1.2. Passaging and maintenance of PSCs	Human PSCs were passaged using 0.5 mM EDTA every 4–5 days when confluence reached ~80%. To prepare…
13	3. Methods — 3.1. PSC culture — 3.1.2. Passaging and maintenance of PSCs	Prior to passaging, the cells were inspected under an inverted microscope. In the rare case that any…
14	3. Methods — 3.1. PSC culture — 3.1.2. Passaging and maintenance of PSCs	pipette to lift the colonies off the plate and collect at the bottom of the plate, which was tilted…
15	3. Methods — 3.2. Differentiation into ventral telencephalic lineage — 3.2.1. Neural induction of floating spheres	For differentiation, PSCs were passaged and grown as floating aggregates (Fig. 1b) in low adherent…
16	3. Methods — 3.2. Differentiation into ventral telencephalic lineage — 3.2.2. Induction of ventral telencephalic lineage	For ventral telencephalic lineage induction, cells were treated with IWP-2 (5 µM) from day 0 to day…
17	3. Methods — 3.3. Further specification of MGEvs. CGE phenotype — 3.3.1. Induction of MGE vs. CGE phenotype	For induction of MGE phenotype, differentiating spheres were initially treated with FGF8 (100 ng/ml)…
18	3. Methods — 3.3. Further specification of MGEvs. CGE phenotype — 3.3.2. Maturation of interneurons	After 3 weeks of differentiation, cells were trypsinized, and droplets of 100,000 cells in 5 µl…
19	3. Methods — 3.4. FACS analysis of fixed cells	To quantitatively analyze the phenotype of generated cells, we performed FACS analysis of stained…

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