|Same Network Different Outcomes - Distinct Lineage Specification Roles for NANOG, OCT4, and SOX2 in hESCs|
NANOG, OCT4 and SOX2 are often given the grand title of master regulators of pluripotency, being tightly associated with human embryonic stem cell (hESC) identity and acting as some of the key reprogramming factors used in common protocols for the production of induced pluripotent stem cells (iPSCs). Studies in mouse ESCs (mESCs) have shown that the loss of each of these genes generally leads to differentiation towards specific lineages; however in hESCs the findings have been more confusing, suggesting that an all-encompassing mechanistic overview of NANOG, OCT4 and SOX2 function in humans is required. This has now been completed by the researchers from the group of Natalia Ivanova at the Yale Stem Cell Center, Yale University, USA, and by using specific knockdowns (KD) and overexpressions (OE) they identify general and cell-line specific requirements for NANOG, OCT4, and SOX2 in hESCs which suggests that, rather than being pan-repressors, each factor represses a specific cell fate (Wang et al).
H1, H7 and H9 hESCs were chosen with regards to their relative dissimilarity to each other, based on previous research (Bock et al), to ensure that any findings where not due to cell-specific effects. Lentiviral vectors expressing both shRNA (short hairpin RNA) and Hygromycin-mCherry fusion protein were utilised, and a rapid decrease in mCherry+ cells was noted when NANOG and OCT4 were downregulated. However, SOX2 downregulation only led to a decrease in the amount of mCherry positive cells in H1 cells, suggesting its dispensability to the self-renewal of some hESCs. Over 2-4 days, NANOG-KD cells downregulated TRA-181 and OCT4, but maintained SOX2, indicative of a neuroectodermal (NE) fate, while SOX2-KD cells maintained TRA-181 and OCT4 expression. However, OCT4-KD was cell specific, as while SSEA-4, TRA-181 and NANOG decreased with time in all cell lines, only H9 cells retained SOX2, again suggestive of a NE fate. Specific differentiation patterns determined by micro-array analysis found that while SOX2-KD cells remained similar to control hESCs, significant changes were observed for OCT4- and NANOG-KD and, interestingly, that differentiated OCT4-KD hESCs and NANOG-KD hESCs microarray patterns were significantly different suggesting that the cells react in different ways during differentiation.
Detailed analysis found that OCT4 was required for the repression of differentiation towards extraembryonic (exEC) and epiblast-derived lineages in hESCs, as OCT4-KD in H1 and H7 cells upregulated exEC markers (CDX2, HAND1, GATA3, TFAP2A, and KRT7), markers expressed in both exEC and embryonic ectoderm (eEC) (DLX3, DLX5, and MSX1/2) and primitive endoderm (pEN) markers (SOX7, GATA6, and GATA4), while markers of primitive streak (PS) and NE were not induced. However, OCT4-KD H9 cells (which retained SOX2) induced different markers; NE markers (PAX6 and ZIC1) and a neural rosette marker (PLZF), suggesting that the exact differentiation route may be specified by additional hESC-line-specific components. Comparison of OCT4-KD H1 and H9 cell microarray data found that BMP4, previously known to induce exEC markers in hESCs (Xu et al) and its downstream targets—ID1, ID2, and SMAD6, were upregulated upon OCT4-KD in H1 cells only. Indeed, OCT4-KD H1 cells treated with BMP4 inhibitor NOGGIN pushed differentiation toward a NE fate at high concentrations, while addition of BMP4 to OCT4-KD H9 cells blocked NE differentiation and induced markers observed in the OCT4-KD H1 and H7 cells.
Gene expression changes in OCT4-KD H1 cells and BMP4-treated H1 cells compared to their respective controls were very similar and the genes involved were mainly associated with differentiation and development, containing markers of the extraembryonic and PS lineages (CDX2, HAND1, TFAP2A, and GATA3). Changes unique to the OCT4-KD H1 cells included genes associated with cell proliferation, metabolism and exEC markers (DLX3 and KRT7), while changes unique to BMP4 included genes associated with development, differentiation and mesendodermal (ME)-specifying genes (T, MIXL1, and EOMES). Immunofluorescence studies additionally demonstrated that while H1 cells treated with BMP4 for 2 days were positive for T, CDX2 and OCT4, by day 4 the cells had downregulated OCT4 and T and upregulated HAND1, a marker of exEC and cardiac/lateral ME. In the OCT4-KD H1 cells, CDX2+HAND1+ cells (exEC) were abundant alongside KRT7+ cells that resembled multinucleated syncytiotrophoblast, but markers of trophoblast stem cells (TSCs) (EOMES AND ELF5) where not induced while CDX2+SOX7+ (pEN marker) cells and GATA6+TFAP2A+ (exEC marker) cells were also present. Overall, combinations of OCT4 and BMP gave four cellular outcomes; 1) high OCT4 and no BMP4 allows self renewal, 2) high OCT4 and high BMP4 allows ME differentiation, 3) low OCT4 and no BMP4 allows NE differentiation and finally 4) low OCT4 and high BMP4 allows for exEC and pEN differentiation.
SOX2-KD hESCs showed no apparent changes to SSEA4, NANOG or OCT4 expression suggesting that the pluripotency network was intact in these cells. However, ME-specifying genes (T and EOMES) and a dEN marker (FOXA2) were detected suggesting a bias towards the PS fate, while addition of ACTIVIN A, which promotes the differentiation of PS cells towards a dEN fate, led to SOX17+ cell cluster formation in both control and SOX2-KD cells. Interestingly, SOX2-KD cells could also form SOX17+ cell clusters in the absence of ACTIVIN A. ME-differentiation, as tested by cardiomyocyte differentiation and expression of a marker gene (cTNT), found that SOX-KD allowed for the formation of multiple cTNT+ cell clusters, with none observed in the control, while haematopoietic differentiation assays demonstrated that the marker genes CD34 and CD31 were markedly increased in SOX2-KD cultures compared to control. eEC differentiation assays found that while NE differentiation was altered (SOX2-KD cells did not form PAX6+ cells), they could form TP63+ epidermal progenitors; which combined with previous data suggests that while SOX2-KD hESCs do remain pluripotent, their differentiation capacity is increased. SOX3 has the ability to compensate for SOX2, and it was demonstrated that SOX3 was found to be induced in SOX2-KD cells and loss of both SOX2 and SOX3 together led to hESC differentiation towards a dEN fate (GATA6, GATA4, FOXA2, and SOX17 induction) and a ME fate (T, MIXL1, and EOMES induced) alongside FOXA2 and CDX2 expression suggesting the emergence of distinct PS populations.
NANOG-KD in hESCs has been associated with both an exEC and pEN differentiation fate or NE differentiation and in this study two major populations were found; OCT4-SOX2+ NANOG-KD hESCs which could differentiate to EN and a significant OCT4interSOX2+ population which could represent another lineage. mRNA analysis of NANOG-KD hESCs found that eEC markers were induced, alongside NE markers (PAX6, ZIC1 and PLZF) and neural crest markers (GBX2 and PAX3), and the treatment of NANOG-KD H1 cells with various factors induced different fates; NOGGIN enhanced NE differentiation, bFGF enhanced NC differentiation and BMP4 induced exEC and pEN differentiation. Differentiation studies also demonstrated that NANOG-KD H1 cells had no defect in dEN or haematopoietic differentiation, suggesting overall that NANOG specifically represses NE and NC cell fates.
Overexpression (OE) studies were next analysed, as previous data in mESCs had suggested that pluripotency factors can function as lineage specifying factors (Loh and Lim) and therefore overexpression may force or alter differentiation. OCT4- and SOX2-OE H1 cells did not express differential levels of lineage markers, but NANOG-OE H1 cells induced dEN markers (EOMES, FOXA2, and SOX17) although only very few FOXA2+SOX17+ cells were identified by immunofluorescence. Further genome-wide expression analysis found that in fact only a few sets of genes were perturbed upon overexpression. However, subsequent differentiation of these cells towards NE and PS lineages found that SOX2-OE cells had less SOX17+ dEN progenitors and enhanced NE differentiation, OCT4-OE cells had enhanced dEN and suppressed NE differentiation, and NANOG-OE cells led to a block in NE differentiation.
Comparison of the KO data with a previously reported data set on promoter occupancies of OCT4, NANOG and SOX2 in hESCs (Boyer et al) allowed correlations with differentially expressed genes in KD hESCs and genes bound by each factor. Over 80% of OCT4-, NANOG- and SOX2-bound genes showed gene expression changes in their matched KD hESC partners, and it was noted that direct targets represented only a minor fraction of all differentially expressed genes (5% for OCT4). Direct targets were genes associated with transcriptional control and regulation of differentiation, while the indirect targets were enriched for a broader range of cellular functions. A small set of genes (111) required all three factors for expression, while larger sets of genes are co-regulated by NANOG and OCT4 (252 genes) or OCT4 and SOX2 (158), and these genes tended to be involved in differentiation and development. However, OCT4 regulated genes were uniquely associated with vascular development, regulation of the cell cycle, and cell adhesion, while NANOG regulated genes were associated with neurogenesis and brain development and SOX2 regulated genes with tissue morphogenesis.
This work represents an impressive new level of understanding of the pluripotent state, the factors which govern this and how they ultimately function to maintain pluripotency. Over-arching points which we can draw from this data include; 1) the usage of pluripotency factors in hESCs differs from mESCs, 2) the usage of pluripotency factors varies among hESC lines, 3) that NANOG, OCT4, and SOX2 function as differentiation repressors, and 4) that each factor mediates the repression of specific fates.
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