Analysis of SOX2-expressing cell populations derived from human pluripotent stem cells

Abstract

SOX2 is involved in several cell and developmental processes, including maintenance of embryonic stem cells, differentiation of neural progenitor cells, and patterning of gut endoderm. To study its role in a human system, we generated a human embryonic stem cell (hESC) line harboring a reporter gene encoding GFP in the SOX2 locus. This SOX2 reporter line faithfully recapitulates expression of the SOX2 gene in undifferentiated human pluripotent stem cells (hPSCs), neural progenitor cells (NPCs), and anterior foregut endoderm (AFE). In undifferentiated hESCs, GFP expression corresponds to those cells with highest levels of expression of genes associated with the pluripotent state. In NPCs, expression of GFP can be employed to isolate cells expressing markers associated with NPC multipotency. In AFE, we used transcriptome-wide expression analysis to identify cell surface markers with elevated expression in this population, thereby facilitating isolation and purification of this hPSC-derived cell population.

Figure 1

Generation and Characterization of SOX2-GFP Clones (A) Schematic of SOX2-GFP-targeting strategy. The top diagram represents the rAAV targeting vector used for targeting of the SOX2 locus. The middle diagram depicts the genomic locus of SOX2, a single exon gene, and the bottom diagram illustrates the properly targeted SOX2 locus. The genetic elements are not displayed to scale. (B) Southern blot using probe-1 (see diagram in [A])-confirmed targeting of the GFP gene to the endogenous SOX2 locus in hSOX2-23 (23). The bands specific to the targeted allele are not observed in nontargeted wild-type cells (H9). Blots hybridized with probe 2 as well as uncropped blots can be found in Figure S1. (C) Using fluorescence-based cell sorting, undifferentiated hSOX2-23 hESCs were separated on the basis of GFP expression. Wild-type (WT) nonfluorescing H9 hESCs were used as a control to set gates for cell sorting. NFC, nonfluorescent channel. (D) Gene expression analysis by quantitative RT-PCR (qRT-PCR) reveals that pluripotency markers SOX2, OCT4, and NANOG were enriched in the GFP⁺ population. Data represent the mean ± SEM from three independent sorting experiments. Populations were compared using Student’s t test. The asterisk denotes p < 0.05. (E) Representative images of GFP, α-NANOG, and α-OCT4 IF of GFP⁺ and GFP⁻ cells (scale bar represents 200 μm). See also Figures S1 and S2.

Figure 2

Differentiation of SOX2-GFP hESC to Neurectoderm Lineages (A) Outline of protocol for differentiation of hESCs to NPCs. The soluble factors, substrate, and culture media at each stage are indicated. KO, knockout; KSR, KnockOut serum replacement. (B) Gene expression analysis for the neurectoderm marker PAX6 during hESC differentiation to rosettes and NPCs (n = 3 independent experiments; error bars represent ± SEM; ^∗∗p < 0.01). (C) Flow cytometry analysis of TRA1-81 and GFP during NPC differentiation. Isotype controls used are listed in Table S4. (D) IF analysis of GFP and SOX2 showed colocalization in NPCs (scale bar represents 100 μm). (E) Flow cytometry analysis of SOX1 expression in SOX2-GFP NPCs. SOX1 shows high coexpression with GFP. Isotype controls used are listed in Table S4. (F) IF of SOX2-GFP hESCs differentiated to neural rosettes (scale bar represents 500 μm). (G) SOX2-GFP neural rosette cells were sorted on the basis of GFP expression. WT H9 rosettes were used as a control to set gates for cell sorting. (H) Gene expression analysis of sorted GFP⁺ and GFP⁻ cells showed high expression of NPC markers SOX2, SOX1, NESTIN, and PAX6 in GFP⁺ cells. Data represent the mean ± SEM from three independent sorting experiments. Populations were compared using Student’s t test. The asterisk denotes p < 0.05 and double asterisks denote p < 0.01. (I) Flow cytometry analysis of SOX1 expression in FACS-purified GFP⁺ cells. Replated GFP⁺ cells maintained high expression of GFP and SOX1.

Figure 3

Differentiation of hESCs to Anterior Foregut and Lung Endoderm (A) Outline of protocol for differentiation of hESCs to anterior foregut and lung progenitor cells. The soluble factors and culture media at each stage are shown. (B) Gene expression analysis of markers of undifferentiated hESCs (NANOG, SOX2), definitive endoderm (DE; SOX17), anterior foregut endoderm (AFE; SOX2, FOXA2, TBX1), and lung progenitor cell (LPC; NKX2.1, SOX9; n = 3 independent experiments; error bars represent ± S.E.M; ^∗p < 0.05; ^∗∗p < 0.01). (C) IF for NKX2.1 on day 13 LPC cultures (mean ± SD; scale bar represents 200 μm). See also Figure S3.

Figure 4

Characterization of SOX2-GFP Reporter hESCs Differentiation to AFE (A) Flow cytometry analysis of SOX2-GFP dynamics during hESC differentiation to AFE and LPC. (B) IF analysis of day 8 SOX2-GFP AFE cultures (scale bar represents 200 μm). (C) Gene expression analysis showed that AFE markers (SOX2, TBX1, PAX9, HOXA1, HOXA2) were highly enriched in GFP⁺ cells. The expression levels of markers of the posterior foregut endoderm (PFE; HNF1B, HNF4A, GATA6, CDX2, PDX1) were higher in GFP⁻ cells (n = 3 independent experiments; error bars represent ± SEM). (D) IF analysis of GFP⁺, GFP⁻, or unsorted control cells that were purified using fluorescence-based cell sorting at day 8 of differentiation, replated, and differentiated to LPCs. Expression of GFP and the LPC marker NKX2.1 was enriched in in-vitro-differentiated GFP⁺ cells versus GFP⁻ or unsorted control cells (mean ± SD; scale bar represents 200 μm).

Figure 5

Genome-wide Expression Analysis of SOX2-GFP AFE (A) Day 8 AFE SOX2-GFP cells were separated by fluorescence-based cell sorting on the basis of GFP expression. (B) Scatter plot of log₁₀ RPKM in GFP⁺ and GFP⁻ day 8 AFE cells. Genes with a statistically significant difference are shown in red. (C) Selection of differentially expressed genes highlighting differences in gene expression patterns related to patterning and differentiation of the foregut endoderm. See also Table S1.

Figure 6

Cell Surface Markers Expressed in hESC-Derived AFE (A) HESC-differentiated AFE cells were sorted based on levels of CD56 and CD271 expression. Double-positive CD56⁺CD271⁺ and double-negative CD56⁻CD271⁻ cells were replated and further differentiated in vitro to LPCs. (B) Flow cytometry analysis demonstrated that CD56 and CD271 expression correlates with GFP expression in day 8 AFE SOX2-GFP cells. (C) HESC-differentiated AFE cells were sorted on the basis of CD56 and CD271. (D) Flow cytometry analysis shows that GFP expression is highest in double-positive CD56⁺CD271⁺ compared to single-positive CD56⁺CD271⁻ or CD56⁻CD271⁺ cells or double-negative CD56⁻CD271⁻ cells. (E) Gene expression analysis reveals that the expression of the AFE markers SOX2, TBX1, and PAX9 were highly enriched in the CD56⁺CD271⁺ cells. As expected, expression of CD56 and CD271 was enriched in CD56⁺CD271⁺ cells. Conversely, expression of the PFE markers GATA6, HNF1B, HNF4A, CDX2, and PDX1 were enriched in CD56⁻CD271⁻ cells. (F) Expression of LPC markers NKX2.1 and SOX9 was enriched in in-vitro-differentiated CD56⁺CD271⁺ cells. Data represent the mean ± SEM from three independent sorting experiments. Populations were compared using Student’s t test. The number sign denotes p > 0.05, asterisk denotes p < 0.05, and double asterisks denote p < 0.01. (G) IF analysis of CD56⁺CD271⁺ and CD56⁻CD271⁻ cells that were purified by fluorescence-based cell sorting at day 8, replated, and differentiated to LPCs. Expression of the LPC marker NKX2.1 was enriched in in-vitro-differentiated CD56⁺CD271⁺ cells versus CD56⁻CD271⁻ cells (scale bar represents 200 μm). See also Figures S4 and Table S2.

Figure 7

CD56 and CD271 Do Not Mark a SOX2⁺ hESC or Neural Population Gene expression analysis of GFP⁺ and GFP⁻ undifferentiated hESCs (A) and neural rosette cells (B) shows that expression of CD56 and CD271 is not enriched in GFP⁺ or GFP⁻ cell populations (n = 3 independent experiments; error bars represent ± SEM, #p > 0.05). Flow cytometry analysis demonstrates that CD56 and CD271 do not correlate with GFP expression in undifferentiated hESCs (C) and neural rosettes (D). Double-positive CD56⁺CD271⁺ AFE cells are not enriched for hESC- (E) or neural- (F) related markers (n = 3 independent experiments; error bars represent ± SEM; ^∗∗p < 0.01. DP, double-positive CD56⁺CD271⁺ AFE; DN, double-negative CD56⁻CD271⁻ AFE; NS, no statistically significant difference).