. 2015 Dec 1;142(23):4010-25.

doi: 10.1242/dev.122846. Epub 2015 Oct 19.

Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification (V体育ios版)

Tamara Zdravkovic¹, Kristopher L Nazor², Nicholas Larocque¹, Matthew Gormley¹, Matthew Donne³, Nathan Hunkapillar¹, Gnanaratnam Giritharan⁴, Harold S Bernstein⁵, Grace Wei⁶, Matthias Hebrok⁷, Xianmin Zeng⁸, Olga Genbacev¹, Aras Mattis⁹, Michael T McMaster¹⁰, Ana Krtolica⁴, Diana Valbuena¹¹, Carlos Simón¹¹, Louise C Laurent¹², Jeanne F Loring², Susan J Fisher¹³

Affiliations

¹ Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Division of Maternal Fetal Medicine, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Human Embryonic Stem Cell Program, University of California San Francisco, San Francisco, CA 94143, USA.
² Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA.
³ Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Division of Maternal Fetal Medicine, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Anatomy, University of California San Francisco, San Francisco, CA 94143, USA.
⁴ StemLifeLine, San Carlos, CA 94070, USA.
⁵ The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143, USA.
⁶ The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Diabetes Center, Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA.
⁷ Diabetes Center, Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA.
⁸ Buck Institute for Research on Aging, Novato, CA 94945, USA.
⁹ The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA.
¹⁰ The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Human Embryonic Stem Cell Program, University of California San Francisco, San Francisco, CA 94143, USA Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, CA 94143, USA.
¹¹ Fundación Instituto Valenciano de Infertilidad (IVI), Parc Científic Universitat de València, 46980, Valencia, Spain.
¹² Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA Department of Reproductive Medicine, University of California San Diego, La Jolla, CA 92093, USA.
¹³ Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Division of Maternal Fetal Medicine, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Human Embryonic Stem Cell Program, University of California San Francisco, San Francisco, CA 94143, USA Department of Anatomy, University of California San Francisco, San Francisco, CA 94143, USA sfisher@qiuluzeuv.cn.

PMID: 26483210
PMCID: PMC4712832
DOI: 10.1242/dev.122846

Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification

Tamara Zdravkovic et al. Development. 2015.

. 2015 Dec 1;142(23):4010-25.

doi: 10.1242/dev.122846. Epub 2015 Oct 19.

Authors

Affiliations

¹ Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Division of Maternal Fetal Medicine, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Human Embryonic Stem Cell Program, University of California San Francisco, San Francisco, CA 94143, USA.
² Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA.
³ Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Division of Maternal Fetal Medicine, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Department of Anatomy, University of California San Francisco, San Francisco, CA 94143, USA.
⁴ StemLifeLine, San Carlos, CA 94070, USA.
⁵ The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143, USA.
⁶ The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Diabetes Center, Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA.
⁷ Diabetes Center, Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA.
⁸ Buck Institute for Research on Aging, Novato, CA 94945, USA.
⁹ The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA.
¹⁰ The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Human Embryonic Stem Cell Program, University of California San Francisco, San Francisco, CA 94143, USA Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, CA 94143, USA.
¹¹ Fundación Instituto Valenciano de Infertilidad (IVI), Parc Científic Universitat de València, 46980, Valencia, Spain.
¹² Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA Department of Reproductive Medicine, University of California San Diego, La Jolla, CA 92093, USA.
¹³ Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA Division of Maternal Fetal Medicine, University of California San Francisco, San Francisco, CA 94143, USA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA The Eli & Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA Human Embryonic Stem Cell Program, University of California San Francisco, San Francisco, CA 94143, USA Department of Anatomy, University of California San Francisco, San Francisco, CA 94143, USA sfisher@qiuluzeuv.cn.

PMID: 26483210
PMCID: PMC4712832
DOI: 10.1242/dev.122846 (VSports注册入口)

Abstract

Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active β-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos VSports手机版. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines. .

Keywords: Blastomere; Epigenome; Fate specification; Human embryo; Human embryonic stem cell; Human trophoblast stem cell; Transcriptome. V体育安卓版.

PubMed Disclaimer

Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

**Fig. 1.**
**Directed differentiation of the UCSFB lines into neuronal precursors, cardiomyocytes, endocrine precursors and pancreatic endoderm.** (A) Neuronal precursors derived from UCSFB5-7 expressed SOX1 and nestin (left panels) and β-III-tubulin (middle panels). Two of the lines tested (UCSFB5 and USCSFB6) formed dopaminergic neurons as assessed by immunostaining for tyrosine hydroxylase (TH, right panels). Scale bars: 100 µm. (B, graph) In duplicate, rotary orbital suspension was used to form 300 EBs of uniform size from single cell suspensions of UCSFB6 and WA09 hESCs. Over 20 days, the percentage of adherent EBs demonstrating spontaneous contractile activity in differentiation medium was scored. UCSFB6 formed cardiomyocytes with the same efficiency as conventional hESCs by day 20 (14.0±1.4% versus 12.9±1.0%; P>0.1, Student's t-test). (B, table) Distribution of cardiomyocyte subtypes differentiated from UCSFB6 compared with WA09 hESCs. qRT-PCR showed that EBs formed embryonic atrial, right and left ventricular, and specialized conduction (nodal) cells with efficiencies similar to WA09 cells (N=30; P=0.70 [χ2 1.41, DF 3]). (C, upper panels) UCSFB5-7 formed endocrine precursors and pancreatic endoderm. Immunostaining of UCSFB lines cultured in conditions that promoted endoderm differentiation showed that they expressed SOX17 and PDX1, markers of endocrine precursors. (C, lower panels) When transplanted under the kidney capsule of SCID-beige mice for 100 days, UCSFB5-7 immunostained for Pdx1 and insulin, markers of pancreatic endoderm. Scale bars: 100 µm.

**Fig. 2.**
**The UCSFB lines and conventional hESCs had distinct transcriptomes.** (A) A 3D principal component analysis and an unbiased nearest neighbor clustering plot of global transcriptional profiling data (shown in B) showed that the UCSFB lines were distinct from 228 samples of 72 conventional hESCs according to: (left) 100% of the detected autosomal transcripts; (middle) the most variable transcripts at a 50% level; and (right) the most variable transcripts at a 20% level. (B) Heatmap of the gene expression data (P<0.01) shown in A. Colors at the top indicate lines as follows: yellow, UCSFB lines; blue, conventional lines; light blue, conventional hESCs that were distinguished from UCSFB by a single bifurcation in the array tree dendrogram (detailed in Fig. S2). (C) Gene enrichments, as determined by GREAT, showed that mRNAs involved in trophoblast and cholesterol biosynthetic pathways were upregulated in the UCSFB lines. Conversely, mRNAs that regulate various aspects of embryonic development were upregulated in the conventional lines. Bars depicting the fold enrichment for each gene set were annotated with the −log10 P-values. (D) mRNA expression levels for a gene set in a Naïve Bayes classifier that distinguished UCSFB lines from conventional hESCs. They included regulators of fundamental developmental processes such as *DISP1* and *NOTCH1.* TS, Theiler stage.

**Fig. 3.**
**The UCSFB lines were hypomethylated compared with conventional hESCs in genomic regions that control trophoblast differentiation and basic developmental processes.** (A) A 3D principal component analysis and an unbiased nearest neighbor clustering plot of the methylation data (shown in B) differentiated the UCSFB (yellow) and conventional hESC (blue) lines. (B) Heatmap showing differentially methylated cytosines (P<0.01, beta-value≥0.1) of UCSFB5-7, which were derived from a single embryo, compared with 46 samples from 21 conventional hESC lines. (C) Functional enrichments for differentially methylated cytosines between UCSFB lines and conventional hESCs as determined by GREAT. Bars depicting the fold enrichment for each gene set were annotated with the −log10 P-values. The hypomethylated regions, which dominated, encoded genes involved in trophoblast/placental differentiation and key aspects of developmental processes such as cytoplasmic organization, cell adhesion and blastoderm segmentation. The hypermethylated regions were involved in PDCD1 signaling and ZAP-70 functions at the immunological synapse. (D-G) Box and whisker plots of genes driving enrichments for spongiotrophoblast differentiation, cytoplasmic organization, blastoderm segmentation/embryo axis formation and PD-1 signaling, respectively. Cytosine genomic coordinates are plotted on the x-axis. The associated transcripts are listed in the bottom right-hand corners of the panels.

**Fig. 4.**
**Sequence determinants of methylation in conventional and blastomere-derived hESCs.** (A) The significance of differential methylation increased as a function of CpG density (left). CpH methylation was significantly lower in conventional hESCs versus lines that were derived in physiological hypoxia and was restricted to regions of low CpG density (right). (B, left) For the UCSFB lines, the hypomethylated CpGs were enriched within a 1 MB window, centered on the transcription start site (TSS)±∼500 kb, in regions of high CpG density (fourth quartile for all 450 K probes) (left graphs). In regions of very low CpG density (first quartile), CpH methylation (right graphs) was spread across the same 1 MB window. In regions of higher CpG density (second quartile), this modification became restricted to the TSS and flanking regions.

**Fig. 5.**
**Gene expression patterns diverged among the UCSFB lines.** (A) The UCSFB lines were derived from embryos that were donated by one couple. The ‘pinwheel’ diagrams depict the single blastomere derivation scheme. In some cases, multiple hESC lines were established from the same embryo (red and blue cells). (B) An ANOVA analysis (P<0.01, fold change ≥1.5) comparing the transcriptomes of the UCSFB lines identified 3620 mRNAs as differentially expressed. The heatmap was created by plotting the results from weighted gene correlation network analysis according to rank. Four covariant gene expression modules emerged. As shown in Fig. S4, they included transcripts encoding genes involved in: (module 1) the cell cycle and cholesterol biosynthesis; (module 2) MYC targets and cell type-specific pathways; (module 3) hypoxia responses, glucose metabolism and catabolic responses; (module 4) extra-embryonic development and migration. (C) A heatmap of expression data for the same genes from 72 conventional lines revealed hESCs with portions of these modules, but not the entire program. B, blastomere; E, embryo.

**Fig. 6.**
**Immunoanalyses of cleavage-stage human embryos revealed differential nuclear staining for EOMES, T, GDF15 and active β-catenin among blastomeres.** Microarray analyses showed that mRNAs encoding *EOMES*, T and *GDF15* were differentially expressed among the UCSFB lines (Fig. S5), suggesting possible differences in blastomere expression of these transcription factors. Additionally, the WNT pathway appeared to be activated in only a portion of the lines (Fig. S4), predicting possible asymmetric distribution of active β-catenin (ABC). An immunolocalization approach was taken to localize these molecules using the antibodies listed in Table S1. The binding of primary antibodies was detected by using species-specific secondary antibodies and nuclei were stained with DAPI. Six embryos were examined for expression of each antigen with the same result. (A,D,G,J) The confocal images of entire embryos were merged into a single micrograph. To image the interior of the embryo shown in G, the surface micrographs at opposite poles were omitted from the merged image. (B,E,H,K) The nuclear staining patterns were extracted from the merged images. (C,F,I,L) Immunostaining was quantified using Volocity software. The dotted lines show division of the nuclei for the purpose of quantification. (A-C) EOMES cytoplasmic and nuclear immunoreactivity was variable (high to low) among the blastomeres. (D-F) Immunostaining for T primarily localized to a single nucleus. Immunoreactivity (presumably nonspecific) was also detected in association with the zona pellucida (ZP). (G-I) Immunolocalization of GDF15 revealed stronger staining in some nuclei and weaker antibody reactivity in others. This growth factor was also detected in the cytoplasm, particularly near the embryo surface, and in association with the ZP. (J-L) As expected, anti-ABC localized to the plasma membrane, but also strongly stained a subset of the blastomere nuclei. px, pixels. Scale bars: 50 µm.

**Fig. 7.**
**UCSFB6 EBs spontaneously formed CDX2-positive cells, which gave rise to human trophoblast stem cells.** (A) By day 3, adherent EBs produced large outgrowths in which downregulation of POU5F1 (OCT4) was associated with upregulation of CDX2 expression in a nuclear pattern. (B) Cells at the periphery of outgrowths that no longer expressed CDX2 stained with anti-KRT7, a trophoblast antigen. (C) In some areas of the outgrowths, phase contrast microscopy revealed cells that appeared to have fused (arrow), consistent with a syncytiotrophoblast identity. (D) Other areas of the outgrowths were composed of mononuclear cells with membrane projections indicative of migration, a property of invasive cytotrophoblasts. (E) Areas corresponding to the CDX2-positive outgrowths (shown in A) were manually dissected and propagated under conditions that enabled derivation of human trophoblast progenitors from the chorion (see Materials and Methods). A phase contrast image of the cultures at p11 showed a mononuclear population. (F) Plating of the cells at either p11 or p14 showed that they grew at a consistent rate. (G-I) In an undifferentiated state, they exhibited nuclear staining for transcription factors that drive a trophoblast fate: TEAD4, CDX2 and GEMININ. (J-M) They also immunostained, in a nuclear pattern, for GATA3, ELF5, EOMES and GCM1, transcription factors that are required at later stages of trophoblast differentiation. (N-Q) The cells expressed other stem cell and trophoblast markers including HMGA2, LIFR, GDF15 and LGR5. (R) They also displayed nuclear expression of the active form of β-catenin, which is required for the generation of implantation competent trophoblasts. Scale bars: 100 µm in A,B; 50 µm in C-E,G-R. TSCs, trophoblast stem cells.

**Fig. 8.**
**UCSFB6-derived human trophoblast stem cells formed the mature cell types that carry out the specialized functions of the human placenta.** (A) Upon differentiation, the TSCs formed mononuclear invasive cytotrophoblasts. When they were plated on a thin coating of Matrigel, phase contrast microscopy showed numerous lamellipodia, a feature of migrating cells. (B,C) As the cells moved, they continued to express cytokeratin (KRT7) and upregulated integrin α1, which is required for this process (Zhou et al., 1993). (D) Plating the differentiating TSCs on a Matrigel plug on top of a Transwell filter promoted their aggregation, which was visualized by phase contract microscopy. This behavior mirrors that of primary cytotrophoblasts when they are plated under the same conditions. (E) The aggregates gave rise to invasive cytotrophoblasts that penetrated the Matrigel and migrated through the filter pores to the underside, where numerous KRT7-positive processes were visible (arrows). (F) The invasive cytotrophoblasts immunostained for HLA-G. Among all normal human cells, expression of this MHC class Ib molecule is limited to this trophoblast subpopulation. (G) They also exhibited the integrin α6→α1 transition that accompanies uterine invasion (Zhou et al., 1993). Arrows indicate direction of CTB migration/invasion. (H,I) The cytotrophoblasts executed the unusual epithelial-to-endothelial transition that accompanies invasion (Zhou et al., 1997), exemplified by the upregulated expression of VE cadherin and VCAM1. (J) Upon differentiation, the TSCs also formed multinuclear syncytiotrophoblasts. (K) Fusion was confirmed by immunolocalization of Ezrin, which showed that the nuclei were not separated by plasma membranes. (L,M) DAPI staining was used to estimate the ploidy of the nuclei in KRT7-expressing cells, which showed that a substantial number were hyperdiploid, another unusual feature of human trophoblasts (Weier et al., 2005). (N,O) The TSC progeny also immunostained for hCG and CSH1 (hPL). Scale bars: 50 µm.

See this image and copyright information in PMC

"V体育ios版" References

1. Altmäe S., Reimand J., Hovatta O., Zhang P., Kere J., Laisk T., Saare M., Peters M., Vilo J., Stavreus-Evers A. et al. (2012). Research resource: interactome of human embryo implantation: identification of gene expression pathways, regulation, and integrated regulatory networks. Mol. Endocrinol. 26, 203-217. 10.1210/me.2011-1196 - "VSports手机版" DOI - PMC - PubMed
1. Amita M., Adachi K., Alexenko A. P., Sinha S., Schust D. J., Schulz L. C., Roberts R. M. and Ezashi T. (2013). Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4. Proc. Natl. Acad. Sci. USA 110, E1212-E1221. 10.1073/pnas.1303094110 - DOI - PMC - PubMed
1. Barbash-Hazan S., Frumkin T., Malcov M., Yaron Y., Cohen T., Azem F., Amit A. and Ben-Yosef D. (2009). Preimplantation aneuploid embryos undergo self-correction in correlation with their developmental potential. Fertil. Steril. 92, 890-896. 10.1016/j.fertnstert.2008.07.1761 - DOI - PubMed
1. Berg D. K., Smith C. S., Pearton D. J., Wells D. N., Broadhurst R., Donnison M. and Pfeffer P. L. (2011). Trophectoderm lineage determination in cattle. Dev. Cell 20, 244-255. 10.1016/j.devcel.2011.01.003 - DOI - PubMed
1. Bernardo A. S., Faial T., Gardner L., Niakan K. K., Ortmann D., Senner C. E., Callery E. M., Trotter M. W., Hemberger M., Smith J. C. et al. (2011). BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell 9, 144-155. 10.1016/j.stem.2011.06.015 - "VSports app下载" DOI - PMC - PubMed

Publication types

Actions (VSports注册入口)

MeSH terms

Actions
Actions (VSports在线直播)
"VSports在线直播" Actions
"VSports最新版本" Actions
VSports注册入口 - Actions
Actions
V体育平台登录 - Actions
Actions
"VSports app下载" Actions
Actions
Actions
Actions
Actions
Actions
Actions
"VSports注册入口" Actions
Actions
Actions
Actions
"VSports" Actions
Actions
Actions

"V体育官网入口" Substances

Actions
V体育官网 - Actions
Actions
Actions

Associated data

Actions
- Search in PubMed
- Search in GEO

Grants and funding

LinkOut - more resources

V体育平台登录 - Full Text Sources
Other Literature Sources
- scite Smart Citations
Molecular Biology Databases
- "V体育ios版" NIAID Data Ecosystem - Find datasets on Infectious and Immune-mediated Diseases
Research Materials
- Cellosaurus - a cell line knowledge resource
- NCI CPTC Antibody Characterization Program
Miscellaneous
- NCI CPTAC Assay Portal (VSports在线直播)

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification (V体育ios版)

Affiliations

Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

"V体育ios版" References

Publication types

MeSH terms

"V体育官网入口" Substances

Associated data

Grants and funding

LinkOut - more resources

V体育平台登录 - Full Text Sources

Other Literature Sources

Molecular Biology Databases

Research Materials

Miscellaneous