Constitutive Differences in Gene Expression Predict In Vitro Differentiation Response of NT2/D1 and 27X-1 Cells
ATRA and BMP-2, respectively, mediate neural [2, 3] and epithelial [4, 5] differentiation in NT2/D1 cells, whereas both morphogens promote PE differentiation in 27X-1 cells [6, –8]. To understand whether constitutive differences in patterns of gene expression between NT2/D1 and 27X-1 cells in part account for the observed differences in morphogen-mediated differential lineage induction in these cells, we determined gene expression profiles of both cell lines in triplicate and compared the relative levels of gene expression by the SAM program. SAM analysis identified 283 and 759 transcripts (Q value cutoff = 0), respectively, that were expressed at ≥2-fold levels in NT2/D1 and 27X-1 cells (supplemental online Tables 3 and 4).
Functional annotation of the 50 most differentially expressed transcripts in NT2/D1 cells compared with 27X-1 cells (Table 1) revealed an abundance of transcripts associated with neurectodermal development. These included genes that function in transcription regulation and signaling (ID2, PBX1, OTX2, LDB2, SFRP1, SMCY, and DUSP23), neurite outgrowth (NEFH, MLLT11 [AF1Q], and SP8), axonal guidance (FEZ1 and NAV1), and neural development (SGNE1, NMA2, OPN3, GNG4, C5ORF13, BEX1, and C7ORF16). A list of remaining transcripts are provided as supplemental information (supplemental online Table 3), which includes several additional neurogenic transcription factors (PAX8, SOX13, GLI3, HKR1, and BHLHB3) and those involved in neural development (NELL2, ARHGEF9, PROM1, EPHA1, BEX2, BEXL1, FGF19, NBEA, ADCY1, SYT6, SYN2, and DCMKL1).
Table Table 1.. Fifty most highly overexpressed transcripts in NT2/D1 cells relative to 27X-1 cells
On the other hand, functional annotation of the 50 most differentially overexpressed transcripts in 27X-1 cells compared with NT2/D1 (Table 2) identified a number of transcripts associated with non-neural developmental pathways such as mesodermal (MEST, FER1L3, CDH11, CD44, COL1A1, COL5A1, and ANXA3) and epithelial (CDH1, SFN, and KRT19) lineages. The remaining transcripts are presented as supplemental data (supplemental online Table 4), which include multiple additional genes involved in epithelial (EPPK1, DSP, PKP3, VCL, AHNAK, and CTNNA1) and mesendodermal (EOMES, DSC96, TAGLN, MYL9, TEAD4, GATA3, MTMR1, CNN1, MYADM, PDLIM1, PDLIM2, and CALD1) development.
Table Table 2.. Fifty most highly overexpressed transcripts in 27X-1 cells relative to NT2/D1
Furthermore, NT2/D1 and 27X-1 cells also differed in the expression of a number of known stem cell markers. Whereas NT2/D1 cells expressed higher levels of SOX2, ECAT8, LEFTY2, and PROM1, 27X-1 cells expressed higher levels of DPPA3 (STELLA), ZFP42, DSC96, and EOMES. SOX2, LEFTY2, and ZFP42 are considered core markers associated with stemness or pluripotentiality in human ES cells , whereas PROM1 (CD133) is a known stem cell marker expressed in neural, hematopoietic, and embryonic stem cells . DSC96 and EOMES expressed at higher levels in 27X-1 cells are characteristic of mesenchymal lineages . The two cell types also differed in the expression of transcripts associated with Nodal and Activin signaling, which are known to play a significant role in early embryonic development . Whereas 27X-1 cells expressed higher levels of both NODAL and INHBA (Activin A), NT2/D1 cells expressed a known nodal signaling inhibitor, LEFTY2. Thus, by virtue of constitutive gene expression, NT2/D1 and 27X-1 cells seem predisposed to respond with neural/epithelial and extra-embryonal lineage fates upon treatment with the respective morphogens.
To exclude the possibility that the observed differences in gene expression patterns between the two cell types are due to a contaminating fraction of spontaneously differentiated cells, we first performed flow-activated cell sorting (FACS) analysis of NT2/D1 and 27X-1 cells for four of cell surface stemness markers such as TRA-1-61, TRA-1-80, SSEA3, and SSEA4. The large majority (∼93%–99%) of both cell types expressed all four markers. We next collected the SSEA3-positive populations by FACS, isolated the RNA, and performed semiquantitative RT-PCR analyses for six representative transcripts (GLI3, PROM1, NEFH, CD44, MEST, and ALCAM) that were indicated by microarray analyses as differentially expressed between the total populations of NT2/D1 and 27X-1 cells. The “pluripotentiality/stemness-associated” transcript POU5F1 was included as control, which was expressed at comparable levels between the two cell types based on our array data. The RT-PCR analyses confirmed higher levels of expression of GLI3, NEFH, and PROM1 in NT2/D1 cells. In contrast, MEST, ALCAM, and CD44 were expressed at higher levels in 27X-1 cells. These differences were observed for both the total cell populations and the SSEA3-positive population (supplemental online Fig. 1). The stemness marker POU5F1 was expressed at comparable levels between the two cell types in both total and SSEA3-positive fractions, consistent with the microarray data. Our data thus show that differences in gene expression between NT2/D1 and 27X-1 cells observed by microarray analysis reflect gene expression patterns in the undifferentiated cells and are not due to spontaneous differentiation. Our gene expression data were further corroborated by immunocytochemical data confirming stemness (POU5F1) and proliferation-associated (Ki67) marker expression in nearly all the cells in both cell types (supplemental online Fig. 2). As a representative marker differentially expressed in microarray and RT-PCR analyses, we performed immunocytochemistry for PROM1. Our data revealed higher expression of PROM1 in NT2/D1 cells compared with 27X-1 cells.
ATRA and BMP-2 Induce Distinct Sets of Transcription Factors and Phenotypic Markers in NT2/D1 and 27X-1 Cells
To understand whether ATRA and BMP-2 induce distinct sets of transcription factors and lineage-specific markers in NT2/D1 and 27X-1 cells to modulate respective embryonal versus extra-embryonal lineages in the two cell types, we performed time course gene expression profiling of 27X-1 cells in response to both morphogens and compared them with NT2/D1 expression profiles in response to ATRA  and in response BMP-2 . Upon exposure of 27X-1 cells to ATRA and BMP-2, 3,496 transcripts (4,405 probe sets) and 3,371 transcripts (4,269 probe sets), respectively, exhibited a ≥2-fold change in expression in triplicate at least at one time point during each of the course. Notably, 1,671 transcripts (2,089 probe sets) were altered in both time courses, consistent with previous observations that both morphogens induce differentiation of 27X-1 cells towards a PE lineage [6, –8]. Early transcriptional targets were distinguished from those regulated subsequently by K-means temporal clustering, altered during the first 48 hours, and those altered during the entire time course. This analysis identified nine and 12 clusters comprising transcripts regulated during the first 48 hours of ATRA and BMP-2 treatment, respectively (supplemental online Fig. 3a and Fig. 3b), and 14 clusters each from the total time course for either morphogen (supplemental online Fig. 3c and Fig. 3d).
As expected, a number of known direct targets of ATRA (e.g., CYP26A1, RARA, RARB, and HOX family members) and BMP-2 (e.g., MSX1, MSX2, and TFAP2A) were induced in the early response clusters in the respective time course programs (Fig. 1A, 1B; supplemental online Fig. 3a [clusters 1 to 3]; supplemental online Fig. 3b [clusters I–III]). However, consistent with the reported role of BMP signaling in ATRA-mediated extra-embryonic endoderm differentiation , we noted induction of multiple BMP-responsive genes at later times, mediated by ATRA, which coincided with the induction of BMP7 (e.g., TFAP2A, GATA2, GATA3, ID2, and ID3, Fig. 1B). Furthermore, in agreement with the onset of a differentiation response, a number of key transcripts associated with stemness/pluripotentiality (e.g., NANOG, POU5F1, and UTF1; Fig. 1C) and cell proliferation (e.g., CCNA2, CCNE2, and CDC2; Fig. 1D) were downregulated, whereas multiple cell-cycle inhibitors such as CDKN1A, CDKN2A, and CDKN2B were induced by both morphogens (Fig. 1E).
Figure Figure 1.. Heat maps of the representative gene expression alterations noted during the time course of ATRA and BMP-2-mediated parietal endoderm differentiation in 27X-1 cells. (A): ATRA-responsive transcripts, (B) BMP-2-responsive transcripts, (C) stemness/pluripotentiality-associated transcripts, (D) cell proliferation-associated transcripts, (E) cell-cycle inhibitors, and (F) transcripts associated with parietal endoderm differentiation. Color scale displayed at the bottom shows the average log fold change of the expression data in triplicate. Abbreviations: ATRA, all-trans retinoic acid; BMP-2, bone morphogenetic protein-2.
Download figure to PowerPoint
Accompanying these changes, we also noticed a robust induction of several transcripts associated with PE differentiation by both morphogens (Fig. 1F). These transcripts belonged to multiple functional categories such as transcription factors and signaling molecules (GATA6, SOX7, CITED2, EPAS1, and DAB2), structural components associated with extracellular matrix (COL4A5, COL4A6, LAMA1, LAMA5, LAMB1, LAMC1, LAMC2, NID, FN1, and HSPG2), post-translational modification and protein processing associated with extracellular matrix (P4HA2, PLOD2, KDELR3, and NAGLU), and molecules secreted by parietal endoderm (PLAT, LCN7, and MDK) (Fig. 1F) [23, , , –27]. In contrast to PE lineage markers, several characteristic transcripts related to VE derivation (e.g., AFP, APOA1, APOB, TTR, HNF4A, HNF3B, and FOXA2) remained unaltered in both morphogen programs, in agreement with previous observations . Phase-contrast images of both the untreated control and the morphogen-induced differentiated 27X-1 cells are provided as supplemental information (supplemental online Fig. 4). We performed semiquantitative RT-PCR and immunocytochemical analyses to validate the array expression data. Whereas RT-PCR confirmed the induction of RARB, ID2, GATA6 (by ATRA), and TFAP2A (by BMP-2) during each of the time course programs, (supplemental online Fig. 5), immunocytochemical analyses detected the loss of POU5F1 expression, accompanied by the induction of several phenotypic markers (keratin-7, keratin-19, keratin-8 [Endo A], and Nidogen) modulated by both morphogens (supplemental online Fig. 6). Single channel images depicted significant POU5F1 levels in nearly all the untreated cells. (supplemental online Fig. 7).
We next compared the gene expression changes associated with ATRA and BMP-2-mediated ExE differentiation in 27X-1 cells with those of neural and epithelial cell fates in NT2/D1 cells, mediated by the same morphogens [2, 4]. Using the same criteria as those used in analyzing changes in 27X-1, ATRA and BMP-2 were found to modulate 915 and 1,197 transcripts, respectively, in NT2/D1 cells during the course of neural  and epithelial  differentiation.
ATRA induced a number of transcripts associated with neural development in NT2/D1 but not in 27X-1 cells. These transcripts belonged to diverse functional categories such as transcription factors (PAX6, HOXB3, HOXC4, HOXC5, HOXD4, ARNT2, ASCL1 [MASH1], BTEB1, ZIC1, BHLHB2 [DEC1], BACH1, NHLH2, EGR3, NR2F1, SMARCD3, and ZFHX1B), developmental growth factors/receptors (PDGFRA, NRG1, EPHA2, FGF9, DLK1, WNT13, NTRK2, NELL1, TMEFF1, FYN, and BDNF), factors associated with neurite outgrowth/axonal guidance (DCX, NFM, AMPH, RIT1, WASF1, WASF3, PFN2, NRP2, ENPP2, SLIT2, CRMP1, and TMSNB), and miscellaneous other genes involved in neural development (PCP4, BAI1, PENK, NCALD, SH3GL3, and CBLN).
Likewise, BMP-2 induced a number of transcripts characteristic of epithelial (CDH1, TJP3, CTNNA2, and CLDN3) and smooth muscle differentiation (ACTA2, SMTN, TPM2, CRIP1, CRIP2, SSPN, CNN1, SGCG, FLNC, MYH11, MYH6, SILV, TNNI1, TNNT2, DMN, TLN2, MYL4, MYL1, TMOD, TTN, and CALD1) in NT2/D1 but not in 27X-1 cells. Conversely, both ATRA and BMP-2 induced key transcripts associated with ExE lineage such as GATA6 and SOX7 only in 27X-1 but not in NT2/D1. Thus, the specific response of these two EC cell lines appears to be linked to the level of pluripotency defined by their constitutive gene expression pattern.
Comparison of Gene Expression Between PE Differentiation In Vitro and Yolk Sac Tumors In Vivo Identifies Markers Associated with VE Development
Differentiation of EC tumors towards a yolk sac phenotype involves both PE and VE lineages , unlike the in vitro differentiation of 27X-1 cells that involves only the PE lineage [6, –8]. We reasoned that by comparing the gene expression patterns between PE differentiation in vitro and yolk sac tumor development in vivo, the pathways associated with development of the VE and PE components of yolk sac tumors could be distinguished.
To this end, we first identified differentially expressed transcripts between 10 pure yolk sac and 15 pure EC tumors using SAM analysis . A total of 1,570 transcripts were differentially expressed (Q value cutoff = 0.08) in yolk sac tumors in comparison with EC tumors. Of these 1,570 transcripts, 492 were found to alter during PE differentiation of 27X-1 cells and thus represented the PE component of in vivo yolk sac tumor differentiation (supplemental online Table 5A). This left 1,078 transcripts, of which 481 were expressed at twofold or higher in yolk sac tumors compared with EC tumors (supplemental online Table 5B). Functional annotation of the 50 most abundantly expressed transcripts among these (sixfold and above, Table 3) indicated that a number of well-characterized (AFP, TTR, BMP-2, HNF4A, HNF3B, APOA1, APOB, and SOX17) and putative (C5, OTX2, CER1, HP, TF, APOC3, FOXA2, and VIL1) transcripts associated with VE lineage were induced during yolk sac tumor differentiation [6, 26, 28, –30]. None of these transcripts were induced during PE differentiation of 27X-1 cells. We also noted that several MAGE (MAGEA3, MAGEA6, MAGEA12, and MAGEA2) and RAGE family members were expressed sixfold or higher in yolk sac tumors (Table 3), suggesting that these transcripts may be associated with VE differentiation.
Table Table 3.. Fifty most highly overexpressed transcripts in pure yolk sac (YS) tumors relative to pure embryonal carcinoma (EC) tumors after a subtraction of all-trans retinoic acid and bone morphogenetic protein-2-mediated time course data associated with parietal endoderm lineage establishment in 27X-1 cells