Heightened potency of human pluripotent stem cell 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 epiblast-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 stem cell phenotype by both ferentiate to trophoblast (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 teratomas in immune-compro- genes gradually emerge (17, 18, 25–27). The colonies of human cells mised mice that secrete human chorionic gonadotropin (hCG) into the release human chorionic gonadotropin (hCG), placental growth host mouse and include small areas of trophoblast-like cells. The 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 syncytiotrophoblast, whereas an A83-01/PD173074 combination fa- pattern materializes, with both mesoderm and endoderm, 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 embryo. Human ES cells (ESCs) and induced pluripotent stem cells (iPSCs) biological sciences | developmental biology | 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 inner cell mass/early epiblast of blastocysts (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 Gene 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. 2 considered pluripotent and capable of differentiating to the To whom correspondence should be addressed. Email: [email protected]. BIOLOGY three embryonic lineages but are generally not regarded as to- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. DEVELOPMENTAL tipotent, because they are not considered to contribute to ex- 1073/pnas.1504778112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1504778112 PNAS | Published online April 13, 2015 | E2337–E2346 Downloaded by guest on September 24, 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.