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Heightened potency of human pluripotent stem PNAS PLUS lines created by transient BMP4 exposure

Ying Yanga,1, Katsuyuki Adachib,1, Megan A. Sheridanc, Andrei P. Alexenkoa, Danny J. Schustb, Laura C. Schulzb, Toshihiko Ezashia, and R. Michael Robertsa,c,2

aDivision of Animal Sciences, Bond Life Sciences Center and cDepartment of Biochemistry, University of Missouri, Columbia, MO 65211; and bDepartment of Obstetrics, Gynecology, and Women’s Health, University of Missouri, Columbia, MO 65212

Contributed by R. Michael Roberts, March 10, 2015 (sent for review October 30, 2014; reviewed by James Cross and Michael J. Soares) Human pluripotent stem cells (PSCs) show -type pluripo- traembryonic cell types (1). However, there are several reports tency that is maintained with ACTIVIN/FGF2 signaling. Here, we indicating that mouse ESCs, given a suitable stimulus, can dif- report the acquisition of a unique phenotype by both ferentiate to (9–11). human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) hESCs and iPSCs will also differentiate to the extraembryonic in response to transient (24–36 h) exposure to bone morphogenetic trophoblast lineage either when they are allowed to form em- protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and bryoid bodies (12, 13) or when BMP4, or certain of its homologs, FGF2 (PD173074), followed by trypsin dissociation and recovery of including BMP2, BMP5, BMP7, BMP10, and BMP13, are pre- colonies capable of growing on a gelatin substratum in standard sent in the medium and FGF2 is absent (13–24). The BMP- medium for human PSCs at low but not high FGF2 concentrations. driven process is accelerated and directionality is enhanced if the The self-renewing cell lines stain weakly for CDX2 and strongly for signaling pathways that maintain the pluripotent state are inhibited NANOG, can be propagated clonally on either Matrigel or gelatin, (25). Trophoblast markers become up-regulated as differentiation + and are morphologically distinct from human PSC progenitors on proceeds, and an invasive HLA-G population and a syncytial cell either substratum but still meet standard in vitro criteria for pluri- population expressing CGA, CGB, ERVW1, and other signature potency. They form well-differentiated in immune-compro- gradually emerge (17, 18, 25–27). The colonies of human cells mised mice that secrete human chorionic gonadotropin (hCG) into the BIOLOGY release human chorionic gonadotropin (hCG), placental growth

host mouse and include small areas of trophoblast-like cells. The DEVELOPMENTAL factor (PGF), placental lactogen (CSH1), and progesterone into the cells have a distinct transcriptome profile from the human PSCs from medium (25). All of this trophoblast differentiation can occur in which they were derived (including higher expression of NANOG, LEFTY1,andLEFTY2). In nonconditioned medium lacking FGF2, the either a complex medium conditioned by mouse embryonic fibro- colonies spontaneously differentiated along multiple lineages, in- blasts (MEFs) or in a chemically defined medium (25). On the cluding trophoblast. They responded to PD173074 in the absence other hand, if FGF2 is not removed from the culture medium of both FGF2 and BMP4 by conversion to trophoblast, and especially before addition of BMP4, a more complex differentiation , whereas an A83-01/PD173074 combination fa- pattern materializes, with both and , as vored increased expression of HLA-G, a marker of extravillous well as extraembryonic tissues, emerging in amounts that appear trophoblast. Together, these data suggest that the cell lines ex- to depend on the relative concentrations of BMP4, ACTIVIN, hibit totipotent potential and that BMP4 can prime human PSCs to and FGF2 (5). Consequently, it might be inferred that both mouse a self-renewing alternative state permissive for trophoblast devel- opment. The results may have implications for regulation of line- Significance age decisions in the early . Human ES cells (ESCs) and induced pluripotent stem cells (iPSCs) biological sciences | | pluripotent stem cells | can differentiate along all the major cell lineages of the embryo totipotent | trophoblast proper, but there is evidence that they can also give rise to ex- traembryonic placental trophoblast. This observation is contro- ouse ES cells, the “naive” type, are obtained from outgrowths versial because human ESCs (hESCs) are considered to arise from Mof the /early epiblast of (1) and are a part of the embryo that does not contribute to trophoblast. dependent on the growth factor leukemia inhibitory factor (LIF) Here, we describe stable, self-renewing stem cell lines derived for maintenance of pluripotency. They may be passaged by dis- from hESCs and iPSCs by brief exposure to bone morphogenetic persal to single cells with trypsin and can be maintained on a gelatin protein 4 (BMP4) that appear poised to differentiate readily substratum. Human ES cells (hESCs) and induced pluripotent stem along all the main developmental cell lineages, including pla- cells (iPSCs) form flattened colonies that resemble, and are con- cental trophoblast. BMP4 signaling may thus play a role in the sidered functionally comparable to, pluripotent cells derived from early embryo by establishing a cell state permissive for tro- the mouse epiblast, or “primed”-type stem cells (1–5). hESCs and phoblast development. iPSCs are maintained with activators of two signaling pathways: the BMP receptor type-1A (BMPR1A) (ALK3) pathway via ACTIVIN Author contributions: Y.Y., K.A., T.E., and R.M.R. designed research; Y.Y., K.A., M.A.S., and A.P.A. performed research; Y.Y., K.A., A.P.A., D.J.S., L.C.S., T.E., and R.M.R. analyzed and the mitogen-activated protein kinase kinase/ERK signaling data; and Y.Y., K.A., T.E., and R.M.R. wrote the paper. – pathway via FGF2 (6 8). They do not readily survive single-cell Reviewers: J.C., University of Calgary Faculty of Medicine, Calgary, Canada; and M.J.S., dispersion by proteinases; rather, they must be passaged by either University of Kansas Medical Center. mechanical breakage of colonies after dispase treatment or gentle The authors declare no conflict of interest. dissociation with chelating agents in presence of Rho-associated Freely available online through the PNAS open access option. kinase (ROCK) inhibitors to small clusters of cells. Moreover, they Data deposition: The data reported in this paper have been deposited in the Ex- can be grown on a feeder layer or substratum coated with Matrigel pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE62065). (Corning), but not gelatin. Both mouse and hESCs and iPSCs are 1Y.Y. and K.A. contributed equally to this work. considered pluripotent and capable of differentiating to the 2To whom correspondence should be addressed. Email: [email protected]. three embryonic lineages but are generally not regarded as to- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tipotent, because they are not considered to contribute to ex- 1073/pnas.1504778112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1504778112 PNAS Early Edition | 1of10 Downloaded by guest on October 1, 2021 ESCs and hESCs, when placed in an appropriate environment, PSC lines, each is provided with the same cell line designation can expand their developmental potential and differentiate followed by the subscript BP (BAP-primed). Hence, the de- to trophoblast. rivative cell lines from H1 cells are named H1BP cells. That these The ability of BMP4 to initiate trophoblast differentiation H1BP colonies are distinct from the precursor H1 cells is evident from hESCs has been puzzling. In naive-type ESCs, BMP4 from their comparative morphologies. For example, the H1BP augments pluripotency rather than destabilizing it (28, 29), al- cells appeared to be flatter and to have a larger surface area, though mouse epiblast-derived ESCs in absence of ACTIVIN reflecting a greater cytoplasm-to-nuclear ratio, than the parental signaling also respond to BMP4 by expressing markers charac- H1 cells (Fig. 3 A and E). They also displayed more distinct cell teristic of primitive endoderm and trophoblast (2). On the other margins (Fig. S1A). These morphological differences were also hand, embryologists have expressed doubt as to whether “epi- observed during culture on Matrigel (Fig. 3E), and so were not a blast”-type stem cells could give rise to trophoblast, a lineage consequence of a particular substratum. However, when H1 and that segregates from the inner cell mass before the epiblast has H1BP cells were evaluated for relative size after dispersion to formed (30, 31). single cells, their diameters did not differ [6.33 ± 0.17 μm for H1 Here, we present evidence that BMP4 acts to enhance the cells vs. 6.17 ± 0.17 μm for H1BP cells (n = 3, with each exper- potency of hESCs and iPSCs, making them readily capable of iment performed on >5 × 105 cells)], suggesting that the differ- forming both embryonic and trophoblast lineages. Previously, ences evident during culture were a reflection of their respective we reported that only a transient, 24-h exposure to BMP4 was interactions with the substratum. When H1BP colonies were necessary for hESC colonies to commit to trophoblast. There- dispersed to single cells by TrypLE and plated on a gelatin after, the ACTIVIN signaling inhibitor A83-01 and FGF2 sig- substratum, 73 ± 5% (n = 3) cells attached to the substratum naling inhibitor PD173074, in the complete absence of BMP4, within 24 h and formed well-developed colonies within 3 d were sufficient to promote a full display of markers for differ- (Table S1). By contrast, parental H1 cells did not survive complete entiated trophoblast sublineages (25). The goal of the current dispersion to single cells by TrypLE and could not be propagated study was to maintain stable cell lines in the state reached just on a gelatin substratum (Fig. S2 and Table S1). H1 cells passaged after brief exposure to BMP4, A83-01, and PD173074 (BAP). in the standard manner as small clumps (∼100 μm in diameter) We had initially predicted that these cells would be present early by dispase treatment, followed by mechanical dissociation with a in the trophoblast lineage, possibly even in trophoblast stem cutting tool, also failed to grow on gelatin (Fig. S2C). cells. Instead, these lines represent a unique type of human plu- The protocol described above works successfully for H1 cells, ripotent stem cell (PSC). H9 cells, and iPSCs (Fig. 2), and it has been performed re- Results peatedly with H1 cells (Fig. 2). For each of the cell lines, colony self-renewal can be extended over multiple passages with no Isolation of BAP-Converted ESC and iPSC Lines. H1 (WA01) cells, measurable change in population doubling times. However, ex- H9 (WA09) cells, and iPSC cells were routinely cultured on a tended passage (>25 passages) of H1 and H9 lines has led to Matrigel substratum on mTeSR1 medium (STEMCELL Tech- BP BP variable degrees of chromosomal instability (Fig. S3). For ex- nologies) (32). To initiate differentiation toward trophoblast, ample H1 cells have been noted with trisomy 12 and other colonies were subcultured; on the following day, the culture me- BP signs of chromosomal instability in late-passage cells when trypsin dium was changed from the defined mTeSR1 medium to the MEF- had been used for regular cell passage. H9 cells also show conditioned DMEM/F12/KOSR medium (MEF-CM), which con- BP tains lower concentrations of recombinant FGF2 (4 ng/mL) than karyotype abnormalities (e.g., translocations involving chromo- mTeSR1 medium and no supplementary TGF-β (Fig. 1A). After a somes 8 and 18) as passage numbers are extended (Fig. S3). further 24 h, the medium was changed again to one lacking FGF2 Such aberrations associated with trypsin passage have been but containing BMP4 (10 ng/mL), the ALK4/5/7 inhibitor A83-01 commonly noted in hESCs (33). A more gentle dispersion to (1 μM), and the FGF2-signaling inhibitor PD173074 (0.1 μM) single cells with nonenzymatic reagents, rather than the con- (BAP treatment) in nonconditioned DMEM/F12/KOSR medium tinued use of trypsin, has so far provided chromosomal stability (hESCM). Control cultures continued to be grown in the same over 16 passages (Fig. S3). basal medium in the presence of FGF2 and in the absence of BMP4 Colonies can also be derived if the initiating BAP conditions and inhibitors. At this stage, the BAP cultures remained negative are extended to 36 h, but not to 48 h or beyond (Fig. 2 and Fig. for KRT7, but many cells, particularly on the periphery of the S1B). The emergence of colonies is also sensitive to the con- colonies, stained weakly for CDX2 (Fig. 1B), although they centration of FGF2 in the culture medium. No lines have been + + converted completely to a KRT7 /CDX2 state if kept in this successfully derived when FGF2 exceeded 10 ng/mL (Fig. 2, – medium for a further day (25). conditions 3 5 and 10). Similarly, the H1BP cells could neither be After the initial 24-h exposure to BAP conditions, the culture generated nor maintained in mTeSR1 medium, which contains medium was changed to MEF-CM containing FGF2 (4 ng/mL) high concentrations of FGF2 (100 ng/mL) (Fig. 2 condition 6). (Fig. 1A). After 24 h on this medium, all cells stained positively H1BP colonies can be generated, and subsequently maintained, for CDX2 but KRT7 staining was weak and less uniform, with on conditioned medium without supplemental FGF2 (Fig. 2, many areas of the colonies remaining negative for this antigen conditions 2 and 11); however, as shown later (Fig. S1C), such + − (Fig. 1C, Upper). After 3 d, defined patches of CDX2 /KRT7 colonies stain less intensely for NANOG and POU5F1 than the cells were evident and appeared to be organizing into colonies ones grown with added FGF2. These results are consistent with (Fig. 1C, Lower). These regions of clustered cells that stained for the conclusion that the H1BP cells require only low concentra- + CDX2 were encompassed by KRT7 cells. At day 4, soon after tions of FGF2, which can be supplied minimally in the MEF-CM these disorganized colonies became evident, the cultures were (34). Importantly, self-renewing H1BP colonies could not be dispersed into single cells with TrypLE and passaged at a 1:2 generated in nonconditioned ESC medium, even in presence of ratioonto0.1%gelatin-coatedculturewellsinthesameMEF-CM FGF2 (Fig. 2, condition 10), suggesting that in addition to FGF2, with 10 ng/mL FGF2. some other factor released by MEFs and present in conditioned In most experiments, well-formed colonies began to emerge medium is required for growth. within 3–8 d (Fig. 1D), and where colonies had not emerged so Finally, no colonies could be generated under conditions used early, they did so after the next passage. Thereafter, cells from to derive and maintain trophoblast stem cells from mouse con- colonies can be passaged approximately every 3 d at a ratio of 1:3 ceptuses (35, 36) (Fig. 2, conditions 8 and 9). Colonies did form or 1:4. To distinguish these new cell lines from their progenitor when FGF4 was used in association with MEF-CM, but there

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Fig. 1. Procedure for deriving PSCBP colonies. (A) Human ESC lines (H1 and H9) and a well-characterized human iPSC line were progressively cultured on mTeSR1 medium (green line) and conditioned medium containing FGF2 (4 ng/mL) (CM + FGF2) for 24 h (yellow line), and then treated with BAP (BMP4, 10 ng/mL; A83-01, 1 μM; PD173074, 0.1 μM; red line) for 24 h (25). To prevent further progression along the trophoblast lineage, the medium was changed to standard ESCM lacking BAP (blue line) and containing FGF2 (10 ng/mL). After a further 3 d (day 4), the colonies were dispersed to single cells with TrypLE and cultured on the same FGF2-containing medium on a gelatin substratum. (B) Images of H1 colonies treated with BAP for 24 h (red-line phase). + Cells had an epithelium-like morphology in a phase-contrast image, and some, near the periphery of colonies, were CDX2 . KRT7 immunofluorescence was very faint. Nuclear stain with DAPI was captured at the same site. (C) Images of H1 colonies in standard ESCM with FGF2 after the transient BAP treatment (blue-line phase). (Upper) H1 colonies 24 h after removal of BAP (day 2) when most cells were CDX2+. Variable KRT7-expressing cells within the colonies were also detectable. (Lower) H1 colonies 3 d after removal of BAP (day 4). Cells in the colony were consistent with cells seemingly organized into + − patches of strongly CDX2 but KRT7 cells and cells with down-regulated CDX2 and highly up-regulated KRT7. (D) Colonies emerged among a back- ground of scattered surrounding cells between days 3 and 8 after passage with TrypLE (days 7–12). (Scale bars: phase-contrast images, 500 μm; immu- nofluorescence images, 200 μm.)

was no indication that this colony formation occurred more ef- plated, the background of epithelial cells was markedly reduced ficiently than when the FGF4 was omitted. (Fig. S1F). The second approach, shown here for H1BP cells but also successful with other lines, was to passage the cells repeatedly Phenotype of the BAP-Primed ESC and iPSC Lines. The PSCBP col- at a lower split ratio (1:6–1:8). Over extended passage, the cultures + onies were distinct in several features from the initiating H1 progressively lost the KRT7 components and became composed cells, H9 cells, and iPSCs (Fig. 3A and Table S1). They were solely of colonies similar to those colonies shown in Fig. S1F. weakly positive for CDX2, negative for KRT7, but strongly posi- + Thus, the KRT7 supplemental cells were not required to main- tive for POU5F1 and NANOG (Fig. 3B and Fig. S1 D and E). By contrast, the scattered surrounding cells still present in the cul- tain H1BP self-renewal. The increased expression of POU5F1 and NANOG transcripts, tures at early passage did not form colonies and were negative for POU5F1 and NANOG, but positive for KRT7, indicating they inferred from immunohistochemistry (Fig. 3B and Table S2), was had probably converted to trophoblast. Two approaches were used confirmed by real-time quantitative PCR (qPCR) (Fig. 3C and to minimize the contribution of these trophoblast cells as culture Table S3). Expression of POU5F1 and NANOG was higher in continued. The first approach was to dissociate the colonies to H1BP cells than in H1 cells. CDX2, GATA3, and TFAP2A tran- single cells, such that no clumps were present, and then to allow scripts were also significantly up-regulated, but levels of all three the cell suspension to settle. Under these conditions, the larger were low, requiring more than 28 amplification cycles when + KRT7 cells sank more quickly, causing the upper layer to become the internal control, GAPDH, was detected after only about enriched with cells from the colonies. When the latter were re- 17 cycles.

Yang et al. PNAS Early Edition | 3of10 Downloaded by guest on October 1, 2021 Fig. 2. Summary of conditions used to derive and maintain of PSCBP. Trypsin is TrypLE (recombinant trypsin).

Flow cytometry demonstrated that dissociated colonies of both Microarray Analysis. RNA was isolated from control H1 cells, from H1BP cells (passaged by single-cell dissociation) and H1 cells H1 cells exposed to BAP conditions for 24 and 48 h, from H1BP (passaged by mechanical dispersion but dissociated by TrypLE colonies picked individually at two different passage numbers (p7 before fixing and staining for flow cytometry) were highly uni- and p18), and from H1BP cells that had been allowed to differentiate form in terms of expression of POU5F1 and were >99% nega- spontaneously in the absence of FGF2 (Fig. 4A). The latter cells are tive for KRT7 staining (Fig. 3 F and G and Table S4). H1 discussed in greater detail in the next section. Unsupervised hierar- BP chical clustering and principal component analysis of the data (Fig. 4 colonies also provided a highly homogeneous population of cells A and B) showed that the H1 cells had distinct transcriptional positive for NANOG, whereas H1 cells were more heteroge- BP profiles from the progenitor H1 cells and H1 cells treated with BAP neous for NANOG staining and included some cells that stained for 24 h. H1BP cells at different passage numbers had also only weakly, if at all, for this . These exper- diverged somewhat in terms of their profiles. iments have been repeated on at least three different occasions Analysis by the pluripotency test (PluriTest) (Fig. 4C), a bio- with similar outcomes. In the case of H1BP cells, the flow informatics tool that provides an assessment of whether or not cytometry was performed with different clonal populations of human cells are pluripotent by their expression of a relatively large cells. Each was highly homogeneous in terms of POU5F1 and number of gene markers consistently associated with hESCs and NANOG staining. iPSCs (37), indicated that the H1BP cells had transcriptional Western blot analysis performed on colony lysates verified that profiles consistent with pluripotency and pluri-raw scores com- H1BP cells expressed POU5F1, NANOG, , CDX2, and parable to the pluripotency and pluri-raw scores of H1 cells. The GATA3 (Fig. 3D and Table S5). The up-regulation of NANOG, novelty scores, which essentially measure deviation from the CDX2, and GATA3 in H1BP cells relative to H1 cells was clearly expected gene expression pattern of an idealized human pluripo- evident in these Western blotting experiments. However, these tent cell line, were also low. By contrast, the cultures of H1 cells that had been treated with BAP for 24 h and from which the H1BP data were not as clear-cut for H9BP cells and iPSCBP.Withthe H9 cells, there appeared to be increased expression of POU5F1, cells ultimately emerged had already diverged from their parental BP H1 cells and lost pluripotency. This drift from pluripotency was NANOG, and SOX2 relative to the parental H9 cells (Fig. S1D, further accentuated after 48 h of BAP treatment. Left), but expression of CDX2 and GATA3 was much lower than A total of 110 genes were up-regulated greater than twofold noted in the H1BP cell line. In iPSCBP/iPSC comparisons, ex- in the two H1BP cell samples relative to H1 cells (Fig. S4 and pression of POU5F1, NANOG, and SOX2 was similar, whereas Dataset S1). Among the most strongly up-regulated genes were CDX2 expression was increased in iPSCBP (Fig. S1D, Right). NODAL, CER1 (which encodes , a BMP antagonist), Despite these differences, both of these cell lines successfully F2RL1, LEFTY1, LEFTY2, GDF3, GAL, and SCGB3A2 (which survived complete proteolytic dispersion to single cells, could encodes secretoglobin, a surfactant protein). In addition to these be maintained on a gelatin substratum (Fig. 2, conditions 16 and genes, there was significant up-regulation of at least two other 17), and displayed no sign of differentiation. potential trophoblast stem cell markers, namely, EOMES and

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Fig. 3. Characterization of PSCBP colonies. (A) Typical colony morphologies of H1 cells, H9 cells, and iPSCs (Upper) and of H1BP cells, H9BP cells, and iPSCBP (Lower). (Scale bar: 500 μm.) (B)H1BP colonies immunostained for CDX2, KRT7, NANOG, and POU5F1. (Scale bar: 100 μm.) (C) Real-time PCR assessments (n = 3; i.e., three RNA preparations from three independent experiments) of relative concentrations of transcripts for POU5F1, NANOG, CDX2, GATA3, and TFAP2A

in H1BP cells relative to H1 cells (with GAPDH as an endogenous standard). To provide comparisons, the mean concentration of each transcript in H1 cells has been assigned a value of 1 (*P < 0.05; **P < 0.01; mean ± SD). (D) Western blotting of SDS gels used for analysis of proteins present in 30 μg of H1 and H1BP cell extracts. The arrow indicates the anticipated mobility of GATA3 and T (Brachyury) based on their migration rate (Mr). All analyses were performed on the same 10% polyacrylamide gel. TUBA, α-tubulin. (E) Morphology of H1 cells cultured on Matrigel in mTeSR1 medium or CM supplemented with 10 ng/mL FGF2

and H1BP cells cultured on Matrigel in CM supplemented with 10 ng/mL FGF2. (Scale bar: 100 μm.) Flow cytometry histograms for POU5F1, NANOG, and KRT7 expression in H1BP (F) and H1 (G) cells are shown. For the negative control (NC), cells were exposed to IgG and a second antibody without prior exposure to primary antibodies.

TFAP2C, whose increased expression in the H1BP colonies was detail was insufficient to determine whether syncytial structures confirmed by qPCR (Fig. 4D). were present (Fig. 5B). A few regions within the tumors showed positive HLA-G and KRT7 staining (Fig. 5B). The presence of Formation. H1 cells, H1BP cells,H9cells,H9BP cells, HLA-G and CGB transcripts in the teratomas was confirmed by iPSCs, and iPSCBP all gave rise to teratomas in immunodeficient RT-PCR (Fig. 5C). In addition to these potential indicators of mice. In each case, the tumors included tissue representative of trophoblast, mice carrying tumors derived from H1BP or H9BP (neural epithelium and melanin-producing cells), endo- cells, but not mice with H1 cell, H9 cell, or iPSCBP tumors, had derm (gut-like epithelium accompanied by secretory glands), and serum concentrations of hCG significantly higher than control mesoderm (muscle and cartilage tissues, shown for H1BP and mice and mice harboring H1 and H9 teratomas (Fig. 5D). To- H9BP cells in Fig. 5A). Immunostaining of sections for KRT7, gether, these data indicated that all of the cell lines were plu- which is a broadly used marker for trophoblast, and for the tran- ripotent and that the H1BP and H9BP teratomas contained small scription factor GATA3 revealed a few small regions of positive amounts of tissue that was possibly trophoblast. tissue adjacent to open, vacuole-like structures resembling lacunae of the early human (Fig. 5B). Such regions were also In Vitro Differentiation. Although the PSCBP lines could self-renew, positive for CGA, and hence are potential trophoblast, but cellular removal of FGF2 and replacement of the MEF-CM with

Yang et al. PNAS Early Edition | 5of10 Downloaded by guest on October 1, 2021 Fig. 4. Comparison of the transcriptomes of H1 colonies and H1BP colonies at two different passage numbers (p7 and p18) in H1 colonies that were exposed to BAP conditions for 24 and 48 h and H1BP colonies that had been allowed to differentiate spontaneously [10 d on defined hESCM medium (i.e., nonconditioned ESC medium minus FGF2 and BMP4, differentiated H1BP cells). Unsupervised hierarchical clustering (A) and principal component analysis of microarray data (B), outcomes of analysis by the PluriTest (www.pluritest.org) based on overall gene expression (C), and confirmation of the >twofold up-regulation of two genes

(EOMES and TFAP2C) in H1BP vs. H1 cells by real-time PCR (D). Conditions were as in Fig. 3C (**P < 0.01). PC, principal component.

nonconditioned ESC medium lacking FGF2 (hESCM) led to only a modest increase relative to controls, PD173074 had a initiation of differentiation within the colonies (Fig. 6A and major effect. These effects of PD173074 were much greater on Fig. S5A). Within 10 d of culture in hESCM, many colonies the H1BP cells than on the H1 cells. Not only were daily amounts possessed regions of cells that were positive for NESTIN, T of hCG and progesterone enhanced by PD173074 but the onset (BRACHYURY), and SOX17 (Fig. 6A). The colonies also of detectable production was always earlier than in the controls. + release α-fetoprotein into the medium, consistent with a Quantification of a CGB syncytial area on day 10 of treatment partial endodermal phenotype (Fig. S5B). Additionally, there was consistent with the ELISA results (Fig. 6I). Double-staining were regions that were positive for CGA and GATA2 (Fig. the colonies for desmoplakin (DSP), a component of functional 6B), and hence likely to be trophoblast. There was also con- desmosomes and CGB, indicated that the hCG production was siderable heterogeneity in colony morphologies and in the largely limited to cellular regions where the cytoplasm appeared distribution of markers among the colonies within a particular continuous and contained several nuclei (Fig. 6H), whereas the culture well. The differentiated colonies produced hCG, pro- cells that were negative for CGB contained a single nucleus and gesterone, and PGF, although in relatively small amounts rela- were enclosed by a DSP-staining outer surface. tive to when differentiation was driven by PD173074 (0.1 μM), The combination of PD173074 and A83-01 led to a lower A83-01 (1 μM), or both inhibitors together (Fig. 6E and Table production of hCG and progesterone than with PD173074 alone, S6). Thus, all three main germ layers, as well as trophoblast, although amounts under both treatments remained higher than appeared to be represented among these differentiating colonies. with A83-01 alone. That the H1BP cells were responding differ- The expression of additional markers for trophoblast (transcripts ently to PD173074 than to PD173074 and A83-01 in combination for CGB, PGF, and HLA-G) and their increased expression is also illustrated in Fig. 6D. Here, it is clear that CGB and in differentiated H1BP cells relative to undifferentiated cells HLA-G expression had been boosted by PD173074, whereas the (maintained on MEF-CM plus 10 ng/mL FGF2) was confirmed combination of both drugs led to a much augmented production by qPCR (Fig. 6C). of HLA-G relative to CGB. These data for CGB are consistent Differentiation to trophoblast was increased in the presence of with the qPCR outcomes (Fig. 6C), where PD173074 alone in- PD173074 or A83-01 and PD173074 (Fig. 6 C–F), as reflected in creased CGB transcript levels ∼10-fold in H1BP cells relative to the release of hCG and progesterone by the cultured cells H1 cells exposed to the same conditions. qPCR data for HLA-G assessed at 24-h intervals from day 6, when the production of were more complex but confirmed the enhanced expression in these hormones was initiated, until day 10 of treatment. Under response to PD173074 and A83-01 in combination than with all three inhibitor treatment conditions, the H1BP cells produced PD173074 alone. Experiments to follow PGF expression (Fig. 6G) significantly greater amounts of the two hormones than when the confirmed that the control cultures maintained in hESCM pro- inhibitors were omitted, although both hCG and progesterone duced low amounts (∼100 pg/mL) of hormone and that production were measurable in the hESCM controls, albeit in small amounts was enhanced ∼100-fold with PD173074 and, to a lesser extent, in (Fig. 6E and Table S6). However, although A83-01 promoted response to PD173074 and A83-01 together. Comparable results

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7 Fig. 5. In vivo differentiation of PSCBP.(A) Histological analysis of teratomas generated from H1BP (Upper) and H9BP (Lower) cells after injecting 10 cells into the dorsal flanks of nonobese diabetic SCID-γ mice for 6.0 and 6.9 wk, respectively. The tissue sections were stained by H&E and indicate the presence of representative ectoderm-derived [Left, neural epithelium; Lower Center, melanin-producing cells (white arrow)], endoderm-derived (Right, gut-like epi- thelium accompanied with secretory glands), and mesoderm-derived (Center, cartilage tissues; Lower Right, muscle) tissues. (Scale bar: 100 μm.) (B) Immu- + + + + + + nohistological images show areas of presumptive trophoblast staining for GATA3 and KRT7 , CGA and KRT7 , and HLA-G and KRT7 cells from H1BP teratomas. (Scale bar: 100 μm.) (C) Real-time PCR assessments (n = 3; i.e., three PCR reactions from the same RNA preparation from each teratoma) of relative

concentrations of transcripts for HLA-G and CGB in an H1BP teratoma relative to an H1 teratoma (with GAPDH as an endogenous standard). To provide comparisons, the mean concentration of each transcript in H1 cells has been assigned a value of 1 (**P < 0.01; mean ± SD). (D) Comparisons of hCG con-

centrations (mIU/mL) in sera of control mice (first column) and mice bearing teratomas from H1, H1BP, H9, and H9BP cells (second–fifth columns). Values (mean ± SD) for mice carrying teratomas from H1BP and H9BP cells are significantly higher than in controls (*P < 0.05; **P < 0.01), where hCG concentrations for control mice and mice with H1 and H9 teratomas were close to the detection limit of the ELISA.

have been obtained with the other cell lines. Fig. S5D, for example, differentiated cell populations that expressed a suite of trophoblast indicates that H9BP cells also have contrasting responses to markers (Fig. 6). However, it soon became obvious that rather PD173074 and to PD173074 and A83-01 together in terms of hCG than being , the cells were quite closely related to, but production. H9 cells, although responding to PD173074 with a nevertheless distinct from, their pluripotent progenitors (Fig. 3). In later onset of hCG production than H9BP cells, did produce large the discussion that follows, we first consider the identities of these quantities of the hormone, particularly by day 10 of culture cells. Second, we ask whether or not these cells are totipotent. (compare Fig. 6E with Fig. S5D). Differences with regard to re- Third, we propose that ESCBP and iPSCBP can potentially provide sponses to inhibitors between H1 and H9 ESCs have been noted improved models for following syncytiotrophoblast and extra- previously (25). villous trophoblast differentiation by varying the concentration of PD173074 and A83-01 in the culture medium. Finally, we read- Discussion dress the ongoing controversy relating to how hESCs and iPSCs Our original goal was to isolate trophoblast stem cells from might harbor trophoblast potential and discuss the role played by hESCs by brief exposure to BAP. Together, this regimen of BMP4 in this process. + factors efficiently drives hESCs to a uniformly KRT7 state in 48 h (25). The hypothesis was that after about 24 h of treatment, Are ESCBP/iPSCBP (PSCBP) Distinct from Their Pluripotent Progenitors? + and around the time they became CDX2 , the cells would be There seems little doubt that H1BP cells and their H9 and iPSC passing through a transient trophoblast stem cell stage before homologs are pluripotent as gauged by the usual criterion of being differentiating into more advanced trophoblast lineages. Confi- able to differentiate along the three main lineages (Figs. dence that this hypothesis was correct was raised when it proved 5 and 6). The undifferentiated BP cells also express the usual possible to derive colonies from H1 and H9 cells exposed to this transcription factors associated with the pluripotent state and differentiation protocol under culture conditions that would not “passed” the PluriTest (37) (Fig. 4). Nevertheless, the BP cell lines support the growth and passage of the initiating ESCs themselves differ in several key respects (Table S1) from the cells from which (Fig. 3 and Fig. S2). The cells comprising these colonies also they were derived. In particular, they are morphologically distinct possessed a number of features that initially led us to believe that from the initiating ESCs whether they are cultured on Matrigel or they might indeed be trophoblast stem cells. For example, under gelatin (Fig. 3 and Fig. S1). They can be propagated clonally on appropriate culture conditions, they were self-renewing and gelatin from single cells after trypsin treatment and have tran- weakly positive for CDX2, and could be readily converted to scriptome profiles that distinguish them from their ESC/iPSC

Yang et al. PNAS Early Edition | 7of10 Downloaded by guest on October 1, 2021 Fig. 6. In vitro differentiation of H1BP cells. (A) Differentiation of H1BP cells to colonies containing presumptive ectoderm (NESTIN), mesoderm (T, Brachyury), and endoderm (SOX17) by culturing in basal, chemically-defined hESCM without any growth factors for 10 d. (Scale bar: 100 μm.) (B) Some colonies cultured as in A also contained areas of presumptive trophoblast, which were positive for both CGA (green) and GATA2 (red). (C) Comparison of relative expressions of

CGB, PGF, and HLA-G in H1 and H1BP colonies that had been cultured under five different culture conditions [1, controls in MEF feeder cell-conditioned medium plus FGF2; 2, hESCM with no additions (hESCM); 3, hESCM plus A83-01 (hESCM + A); 4, hESCM plus PD173074 (hESCM + P); 5, hESCM plus A83-01 and PD173074

(hESCM + AP)] for 10 d. The values are the mean ± SD for three separate experiments. Comparisons between H1 and H1BP cells under each culture condition were evaluated by the Student’s t test (*P < 0.05; **P < 0.01). Values across treatments were assessed by ANOVA (different letters indicate values differed from

each other by at least P < 0.05). (D) Representative Western blot comparing relative concentrations of HLA-G and CGB in H1 and H1BP cells cultured in either hESCM with no additions or in the same medium supplemented with A, P, or A plus P. The data were all from the same blot of a 10% SDS-polyacrylamide blot

(30 μg of protein per lane). The loading control is TUBA. Comparison between H1 (blue bars) and H1BP (red bars) cells in production of three placental hormones [hCG (E), progesterone (F), and PGF (G)] over time of culture in four different media (hESCM, hESCM + A, hESCM + P, and hESCM + AP as defined above) for up to 10 d. The values are the mean ± SD for three separate experiments. The medium was replaced daily, such that values represent daily production and release of the hormones to the medium. Analyses of PGF were limited to days 6, 8, and 10 and to just three of the media for cost considerations. The values for hCG production are also illustrated in Table S6 to demonstrate that measurable amounts of hormone were secreted in the hESCM by both cell types. (H)Inthe presence of PD173074 (hESCM + P), large areas of were detected by double staining for DSP (green) and CGB (red), indicating that the + syncytial areas had a continuous cytoplasm and contained multiple nuclei. (I) Quantification of syncytial areas (immunostaining for CGB ) from H1 and H1BP cells in four different media (hESCM, hESCM + A, hESCM + P, and hESCM + AP as defined above) for 10 d. CGB+ areas from nine frames of each experiment were automatically counted by imageJ software (NIH) and averaged.

progenitors (Fig. 4 and Fig. S4). The relevance of elevated, was the coexpression of CDX2 with NANOG, because the two somewhat varied expression of several genes encoding transcrip- transcription factors have mutually antagonistic effects in mouse tion factors that may direct trophoblast formation (38–40) ESCs and preimplantation (46). (e.g., GATA3, TFAP2A, TFAP2C, CDX2, EOMES) is unclear but One possible origin of the PSCBP is that they were selected might reflect the fact that such cells are hovering on the point of from a small residual population able to survive in a pluripotent differentiating along that lineage (i.e., transcriptionally primed) state despite exposure (24 h or 36 h) to BAP conditions. Con- as soon as the pluripotent gene networks are down-regulated. ceivably, such cells existed in a “sporadic superstate” (47) and Although the observation that CDX2 was coexpressed with were already present as a minority population of NANOG- POU5F1 in the undifferentiated H1BP cells might be considered overexpressing cells within the parental ESC and iPSC colonies. surprising, CDX2 expression has been consistently observed in rat Heterogeneity exists in ESC colonies (48–50) and is evident for ESCs (41–43) and associated with POU5F1 expression in human NANOG staining of H1 cells (Fig. 3G). Such heterogeneity is not (44) and bovine (45) embryonic trophectoderm. More unexpected apparent for H1BP cells, which are highly homogeneous for

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1504778112 Yang et al. Downloaded by guest on October 1, 2021 NANOG and POU5F1 expression. Moreover, lines resembling understanding the pathobiology of a disease like pre-eclampsia, PNAS PLUS H1BP and H9BP cell lines cannot be derived directly from pa- where placental development appears to be abnormal (58). rental H1 and H9 cell colonies by simply dispersing the latter to single cells and capturing survivors on a gelatin substratum (Fig. Role for BMP4 in the Generation of Trophoblast from epi-ESCs. We S2). Furthermore, our stock populations of ESCs and iPSCs are propose that short-term exposure to BMP4 in presence of the routinely maintained on mTeSR1 medium, which does not sup- two inhibitors causes the PSC to undergo a transition to a “BMP- ” port H1BP cell self-renewal and would presumably have purged primed state, such that the cells remain self-renewing and such cells from the population. Accordingly, we believe that such continue to display a pluripotent signature but are poised to cells arise transiently in response to brief BAP treatment. differentiate to trophoblast much more efficiently than if they had not been BMP4-exposed. A somewhat similar role has been Are PSCBP Totipotent? ESCs and iPSCs are considered pluripotent envisioned for BMP4 in conserving a stable “ground state” for in that they differentiate to all three embryonic germ layers but naive-type stem cells from the mouse (28, 29), where in associ- are not generally believed to be capable of giving rise to extra- ation with the growth factor LIF, BMP4 helps preserve the po- embryonic tissue, including trophoblast (1). Although the H1BP cell tential of the ESCs for multilineage differentiation, lines differentiated spontaneously along the three main germ cell formation, and clonal propagation. lineages in what appears to be a stochastic manner when FGF2 and As discussed earlier, the idea that BMP4 can drive hESCs to MEF-CM were withdrawn and replaced with a defined medium trophoblast has been controversial (31), despite evidence that that lacked any growth factors, they also readily formed trophoblast both mouse ESCs and hESCs have the potential to differentiate (Fig. 6B). Additionally, H1BP and H9BP cells differentiated pre- along extraembryonic lineages under appropriate culture con- dominantly, if not completely, to trophoblast when PD173074 ditions (9–24). Moreover, recent evidence suggests that BMP alone or in combination with A83-01 was added to the defined signaling has a role in ensuring the correct development of the ESCM (Fig. 6 C–E). Interestingly, cell lines with the capacity to trophoblast lineage in preimplantation mouse embryos (59, 60) contribute to extraembryonic as well as embryonic tissues have and that, by the 16-cell stage, only the outer cells are fully been described recently for the mouse (49, 51). We suggest that equipped to respond to signals initiated by BMP4 and related both the aforesaid mouse lines and the PSCBP lines described here ligands, such as BMP7 (59). Conceivably, human PSCBP lines are have achieved a state that is permissive to form trophoblast in analogous to these outer cells. Their phenotype has been molded

addition to differentiated derivatives of the three main germ layers. by brief BMP exposure, and they remain pluripotent yet can BIOLOGY A number of genes associated with pluripotency are up-regulated

transform to trophoblast as soon as the pluripotent network DEVELOPMENTAL in PSCBP, especially LEFTY1, LEFTY2,andNANOG, and could begins to be down-regulated. prove to be useful markers of this BMP-primed state. Consistent with this idea were the observations on the H1BP Materials and Methods and H9BP teratomas, which were composed largely of an array of Animal Care. All animal experiments were approved by the University of fetal tissue types (Fig. 5A) but also included small regions that Missouri Institutional Animal Care and Use Committee under Protocol 7170. were trophoblast-like in that they were positive for KRT7 and GATA3 and for KRT7 and CGA, as well as being located ad- Pluripotent Stem Cell Culture. Human H1 (WA01) and H9 (WA09) ESCs were jacent to vacuolar-like spaces that might be analogous to the from the WiCell Research Institute, whereas the iPSC line was derived from lacunae present in the immature human placenta (52) (Fig. 5B). human umbilical cord reprogrammed with five factors and TP53 Moreover, the mice carrying tumors from the H1 and H9 shRNA by using episomal plasmid transfection (61) as described by Lee et al. BP BP (62). For routine maintenance, all cell lines were cultured in six-well culture cells had significantly elevated concentrations of hCG in their plates (Nunc) coated with Matrigel (BD Bioscience) in the defined mTeSR1 (Fig. 5D). Care must be taken in evaluating the teratoma medium, containing FGF2 at 100 ng/mL and 0.6 ng of TGF-β at 0.6 ng/mL data, however. Many human tumors express CGB (53–55), and (STEMCELL Technologies) (63), The medium in all wells was changed daily. some secrete placental CG and CSH1 (56), whereas others, in- Cells were passaged at a 1:6 ratio every 5–6 d by using dispase (1 mg/mL; cluding aggressive breast , contain areas that are both STEMCELL Technologies) for 7 min at 37 °C, followed by breakage into small + + GATA3 and KRT7 (57). Because the areas of teratomas ex- clumps with the Stempro EZpassage (InVitrogen) cutting tool. pressing trophoblast markers were sporadic and not well charac- terized histologically, the proof that they represent fetal placenta Establishing Self-Renewing PSCBP Colonies. To establish the BP self-renewing remains weak. In addition, it is clear from the relative amounts of colonies (Fig. 1A), H1 cell, H9 cell, hESC, and iPSC colonies were subcultured ∼ × 4 different tissues within the tumors that introduction of the stem to provide a transfer of 2.4 10 cells per square centimeter. On the fol- cell under the skin of the mice favored differentiation along the lowing day, the culture medium was changed from the defined mTeSR1 medium (STEMCELL Technologies) to the standard medium for hESCs (64, three main germ-line images and not to trophoblast. 65), which had been conditioned by a monolayer of γ-irradiated MEF feeder cells (21, 66) containing FGF2 (4 ng/mL). After a further 24 h, the medium PSCBP as an Improved Model for Trophoblast Differentiation. The was changed to one lacking FGF2 but containing BMP4 (10 ng/mL; R&D outcomes with different inhibitor combinations on the ESCBP Systems), the ALK4/5/7 inhibitor A83-01 (1 μM; Tocris Bioscience), and the were not identical. With the FGF2-signaling inhibitor (PD173074) FGF2-signaling inhibitor PD173074 (0.1 μM; Sigma–Aldrich) in hESC basal alone, markers of syncytiotrophoblast predominated, which rep- medium not conditioned with MEF feeder cells. Control cultures continued resents an outcome quite similar to the outcome observed by to be grown in the presence of FGF2 and in the absence of BAP. Following Sudheer et al. (20), who noted that another FGF inhibitor, namely this exposure to BMP4 and inhibitors, the culture medium was changed to SU5402, in the presence of 10 ng/mL BMP4 drove H1 and H9 cells MEF-CM with FGF2 (10 ng/mL). Culture medium was replenished daily for an – largely to syncytiotrophoblast. Intriguingly, in our experiments, the additional 2 4 d. On either day 5 or 6, cells were dispersed with TrypLE Express (Gibco) for 3–4 min or with Gentle Cell Dissociation Reagent presence of the second inhibitor, A83-01, which targets the ALK4, (STEMCELL Technologies) for 6–7 min at 37 °C and transferred to 0.1% ALK-5, and ALK-7 receptors, and hence ACTIVIN/NODAL/ + gelatin-coated culture dishes. TGF-β signaling, led to a smaller area of CGB cells within colo- nies than PD173074 (Fig. 6I), reduced the amount of placental ACKNOWLEDGMENTS. We thank Dr. Aihua Dai, Dr. Alexander Jurkevich, hormones released into the medium, and accentuated expression and Ms. Yuchen Tian for experimental contributions and Mr. Dennis Reith of HLA-G, suggesting that the colonies contained a higher pro- for his editorial assistance. We also thank Drs. Yun Lian and Jinchun Zhou (University of Texas Southwestern Microarray Core Laboratory) for performing portion of . Confirming these hypotheses the microarray analysis and for their extensive discussion of the microarray will require additional experiments, but the ability to bias tropho- data, and the reviewers for their helpful insights. This work was supported by blast differentiation toward either might be of value in NIH Grants R01HD067759 and R01HD077108.

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