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. 2013 May 1;8(5):e61956.
doi: 10.1371/journal.pone.0061956. Print 2013.

Use of "MGE Enhancers" for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons

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Free PMC article

Use of "MGE Enhancers" for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons

Ying-Jiun J Chen et al. PLoS One. .
Free PMC article

Abstract

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6(+) cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6(+) cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP(+) cells, while enhancer 1056 is active in Olig2(+) cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments.

Conflict of interest statement

Competing Interests: JLRR, AAB, ARK, SCB, and CRN have affiliations with Neurona, Inc. However, this does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Comparison of various conditions for mouse ES cells differentiation using Lhx6-GFP+ cell percentage as a criteria for optimization.
J14-derived ES cell line J6M1 (J14 carrying lentiviral enhancer 692-mCherry) were tested for differentiation using four conditions listed. (A): In condition 1 and 2 (shown in blue and green), cells were differentiated in GMEM+10% KSR media while in condition 3 and 4 (shown in red and purple), cells were differentiated in Neurobasal media supplemented with B27 without retinoic acid (NB/B27), a commonly used media for neural progenitor differentiation . Either 100 or 200 ng/ml Dkk-1 was added on day 0 of differentiation (D0), (B): Among all four conditions, KSR-containing media surpassed NB/B27 media in the generation of Lhx6-GFP+ cells. Addition of 2× more Dkk-1 on D0 did not improve the efficiency of Lhx6-GFP+ cells with KSR-containing media. (C): FACS analyses of Lhx6-GFP+ cells with Condition 1. The X-axis showed green fluorescent gating with the log scale.
Figure 2
Figure 2. MGE differentiation protocol from mouse ES cells (ES-MGE) and characterization of MGE-like differentiated J14 (Lhx6-GFP) cells.
(A): Schema outlining the ES-MGE differentiation protocol. The black horizontal line: time line of days after initiation of differentiation. Days when a treatment was introduced are indicated (see Materials and Methods for details). From day 0 (D0) to day 6 (D6), cells were cultured with GMEM and 10% KSR (shown in purple) in a lipidure-coated 96-well plate (shown in cyan). Dkk-1 (100 ng/ml) was added on D0 and SAG (6 nM) was added on D3 shown in red. On D6, cell aggregates were collected and transferred to a bacterial grade sterile petri dish in DMEM/F-12 supplemented with N2. Additional SAG (6 nM) was added to the medium on D6. Starting on D9 (and the following days), aggregates were collected either for immunofluorescent staining, FACS analysis, or FACS purification followed by gene expression microarray analysis, or transplantation. (B–E″): Nkx2-1 expression is shown in red; Lhx6-GFP expression is shown in green; DAPI stains the nucleus blue. B-B″: D10; C-C″: D12; D-D″: D14; E-E″: D16. White arrows indicate cells co-expressing Nkx2-1 and Lhx6-GFP. (F): Dlx2 (red) and Lhx6-GFP (green) expression on D12. White arrows indicate co-localization of Dlx2 and Lhx6-GFP. (G): Foxg1 (red) and Lhx6-GFP (green) expression on D12. White arrows indicate co-localization of Foxg1 and Lhx6-GFP. (H): Islet1 (red) and Lhx6-GFP (green) expression on D12. (I): There were only a few Mki67+ (red) cells that expressed Lhx6-GFP (green) on D11. (J): No Tbr1+ (red) cells were detected on D12. (K): Olig2+ (red) cells and Lhx6-GFP+ (green) cells were mutually exclusive on D12. Scale bar for all panels: 100 µm.
Figure 3
Figure 3. Supervised clustering showing all differentially expressed (DE) genes.
Microarray comparison of RNA expression from primary E12.5 MGE Lhx6-GFP+ cells, ES-Lhx6-GFP+ and ES-Lhx6-GFP cells. Heatmap includes 1821 probes that exhibit a fold change (FC) of greater than 4 in any one of the possible 3 pairwise comparisons. Heatmap colors correspond to the signal intensity relative to the global average for that probe. Color spectrum ranges from red (5) to black (0) to green (-4): red blocks represent sample-specific expression that is elevated relative to the average across all samples; green blocks represent genes whose transcripts are relatively less abundant. Two areas (A and B, bracketed on the right side) in the supervised heatmap contain many of the genes that regulate and/or mark developing cortical interneurons (see Tables S2 and S3 in File S2).
Figure 4
Figure 4. Expression of MGE enhancers in embryonic forebrains, and lentiviral constructs used to transduce them into primary MGE cells and ES cells.
(A–D): MGE enhancers driving β-galactosidase expression (X-Gal staining) of E11.5 telencephalic sections from transient transgenic mice. Coronal sections are shown from rostral to caudal (left to right). Each transgene is composed of one enhancer element 422 (A), 692 (B), 1056 (C), or 1538 (D), followed by an hsp68 minimal promoter that drives expression of LacZ (β-galactosidase). (E): Lentiviral constructs harboring each enhancer reporter cassette for making stable mouse embryonic stem cell clones. Each construct is flanked by a lentiviral 5′LTR and a 3′LTR, and contains two separated gene expression cassettes: the first is the enhancer/promotor driving a mCherry reporter gene; the second is Rex-1 promoter driving the Blasticidin resistant gene (BlaR) . Three lentiviral constructs differed in the first cassette were tested: one without minimal promoter, one with the heat shock protein 68 (hsp68) minimal promoter, and the last one with the β-globin (βg) minimal promoter. The enhancers tested in this study were: mouse DlxI12b enhancer (a shorter version of enhancer 422), three novel human enhancers (692, 1056, and 1538), and a mouse Lhx6 proximal enhancer/promoter DNA element .
Figure 5
Figure 5. Characterization of DlxI12b-βglobin-mCherry in E14 & J14 ES cells differentiated with ES-MGE protocol.
Marker expression analyses were done with immunofluorescence of sections from aggregates of differentiated ES cells (ES EBs). (A–C): mCherry expression (red) driven by the DlxI12b-βglobin enhancer/promoter; Lhx6-GFP expression (green) in panels A-A″ (D11 EBs), B-B″ (D13), C-C″ (D15). (D): DlxI12b-βg-mCherry (red) and Nkx2-1 (green) expression on D13 of differentiation. (E): DlxI12b-βg-mCherry (red) and Dlx2 (green) expression on D13. (F): DlxI12b-βg-mCherry (red) and Foxg1 (green) expression on D11. (G): DlxI12b-βg-mCherry (red) and Islet1 (green) expression on D13. (H): DlxI12b-βg-mCherry (red) and Olig2 (green) expression on day 12. (I): Most of the DlxI12b-βg-mCherry+ (red) cells also express Calbindin (green). Scale bar, 100 µm. White arrows indicate markers co-labeling. (J) FACS analyses of Lhx6-GFP+ cells (on x-axis) and DlxI12b-βg-mCherry+ cells (y-axis) from day 0, day 9, day 11, day 13 and day 15 of ES-MGE differentiation.
Figure 6
Figure 6. Enhancer 692-βg-mCherry was active in 70% of Lhx6 GFP+ cells.
(A–D″): mCherry expression (red) driven by 692-βg and Lhx6-GFP (green) expression on D9 (A-A″), D11 (B-B″), D13(C-C″), and D15 (D-D″) ES EB aggregates. On D13 and D15, about 70% of the 692-mCherry+ cells were labeled with Lhx6-GFP (white arrows). (E): 692-βg-mCherry (red) and Nkx2-1 (green) expression on D15. (F): 692-βg-mCherry+ (red) cells are postmitotic, as they don't express Mki67 (green) on D15 (and other earlier time points). (G): E14 cells line carrying 692-mCherry was examined with Sox6 expression. All of the 692-mCherry+ (red) cells express Sox6 (green). White arrows indicate markers co-labeling. Scale bar, 100 µm. (H) FACS analyses of Lhx6-GFP+ cells (on x-axis) and 692-mCherry+ cells (y-axis) from day 0, day 9, day 11, day 13 and day 16 of ES-MGE differentiation. Though 692-mCherry+ (and 692-βg-mCherry+) cells was detected with immunostaining and showed extensive co-localization with Lhx6-GFP signals, their endogenous intensity was too low to be detected by FACS (no staining was done with FACS analyses).
Figure 7
Figure 7. Characterization of 1056-βg-mCherry in J14 ES cells differentiated with ES-MGE protocol.
Enhancer 1056-βg-mCherry+ cells are Olig2+ and don't express markers of MGE-derived neurons. (A–E″): mCherry expression (red) driven by the 1056-βg and Olig2 (green) expression are shown in panels A-A″ (D9 aggregates), B-B″ (D11), C-C″ (D13), D-D″ (D15) and E-E″ (D17). Almost all of the 1056-βg-mCherry+ cells express Olig2 (white arrows) on all the time points examined. Only a few 1056-βg-mCherry+ cells are Olig2 (white arrowheads in B-B″ and C-C″). (F): 1056-βg-mCherry (red) and Lhx6-GFP (green) expression on D11. (G):, 1056-βg-mCherry (red) and Nkx2-1 (green) expression on D11. Some of the Nkx2-1+ cells are also 1056-βg-mCherry+. (H): A few 1056-βg-mCherry (red)+ cells are still mitotically active, as indicated by Mki67+ (green) staining on D11. (I): 1056-βg-mCherry (red) and Calbindin (green) expression on D11. (J): 1056-βg-mCherry (red) and Islet1 (green) expression on D11. White arrows indicate co-labeling of respective markers shown. Scale bar for all panels, 100 µm. (K) FACS analyses of Lhx6-GFP+ cells (on x-axis) and 1056-βg-mCherry+ cells (y-axis) from day 0, day 9, day 11, day 13, day 15 and day 17 of ES-MGE differentiation. There was no detectable GFP/mCherry double positive cell.
Figure 8
Figure 8. Enhancer 1538-βg-mCherry+ labeled 40% of Lhx6-GFP+ cells.
(A–D″): mCherry expression (red) driven by 1538-βg and Lhx6-GFP (green) expression in panels A-A″ (D10 aggregates), B-B″ (D12), C-C″ (D14), and D-D″ (D16). On D14, 40% of Lhx6-GFP+ cells are 1538-mCherry+ and more than 90% of the 1538-βg-mCherry+ cells were also labeled with Lhx6-GFP (white arrows). (E): 1538-βg-mCherry (red) and Nkx2-1 (green) expression on D14. (F): Most of the 1538-βg-mCherry (red)+ cells were postmitotic, as they don't express Mki67 (green) on D14 (and other earlier time points). There were a few exceptions (white arrows). Scale bar, 100 µm. (G) FACS analyses of Lhx6-GFP+ cells (on x-axis) and 1538-βg-mCherry+ cells (y-axis) from day 0, day 8, day 10, day 12, day 14 and day 16 of ES-MGE differentiation. Similar to the enhancer 692, the mCherry expression activity of enhancer 1538 appeared too low to be detected by FACS (no staining was done with FACS analyses).

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