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Derivation and differentiation of haploid human embryonic stem cells

Abstract

Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species1,2,3,4,5,6, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.

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Figure 1: Derivation of haploid human ES cells.
Figure 2: Haploid human ES cells display pluripotent stem cell characteristics and enable loss-of-function screening.
Figure 3: Molecular and cellular comparisons of haploid and diploid ES cells.
Figure 4: Differentiation of haploid human cells.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

All high-throughput data have been deposited at the Gene Expression Omnibus (GEO) under accession number GSE71458.

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Acknowledgements

We thank all members of the Benvenisty and Egli laboratories for input and support. We thank Y. Avior and W. Breuer for their assistance with experimental procedures. I.S. is supported by the Adams Fellowships Program for Doctoral Students, G.C. is supported by the A*STAR International Fellowship, U.W. is a Clore Fellow, D.E. is a NYSCF-Robertson Investigator, and N.B. is the Herbert Cohn Chair in Cancer Research. This work was partially supported by The Rosetrees Trust and by The Azrieli Foundation (to N.B.), by the Russell Berrie Foundation Program in Cellular Therapies of Diabetes, by the New York State Stem Cell Science (NYSTEM) IIRP Award number C026184, and by the New York Stem Cell Foundation (to D.E.).

Author information

Affiliations

Authors

Contributions

I.S., D.E. and N.B. designed the study and wrote the manuscript with input from all authors. I.S. isolated and characterized haploid human ES cell lines, performed differentiation experiments and analysed the data. G.C. developed and performed the centromere quantification analysis and carried out neuronal differentiation. T.G.-L. assisted in tissue culture and performed karyotype analyses and tissue sectioning. I.S., M.P., U.W. and O.Y. were involved in the genetic screening. M.P. and U.W. assisted with teratoma assays. L.S. assisted with pancreatic differentiation. M.V.S. was involved in all aspects of oocyte donation and research. D.E. derived human pES cell lines from haploid oocytes. D.E. and N.B. supervised the study.

Corresponding authors

Correspondence to Dieter Egli or Nissim Benvenisty.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Derivation of haploid human ES cell lines h-pES10 and h-pES12.

a, Left panel, diploidization rate model for a haploid egg with a theoretical diploidization probability of 10%, overlaid with an exponential decay fit (red curve, see Supplementary Notes). Approximated cell cycle numbers for different ES cell line derivation stages are indicated. Right panel, diploidization dynamics of h-pES10 over seven passages by flow cytometry, overlaid with an exponential fit to the data (red curve). Bars indicate the mean across biological replicates. b, Establishment of a haploid-enriched human ES cell line from pES12 cells after repeated sorting and enrichment of 1c cells using Hoechst 33342 staining. Top to bottom, DNA content profiles of unsorted diploid cells, partially purified haploid cells at the third sort, and mostly-purified haploid cells at the fifth sort. c, chromosomal copies. c, Karyotypes and haploid metaphase percentage over the course of enrichment and passaging. d, e, DNA FISH (d) and centromere protein immunofluorescence staining (e) in haploid-enriched pES12 cells (upper panels) and unsorted diploid pES10 cells (lower panels). Magnified insets show representative haploid and diploid nuclei with single or double hybridization signals (d) and 23 or 46 centromeres (e), respectively. Scale bars, 10 μm. f, SNP array-based CNV analysis comparing haploid pES10 and pES12 cells with their unsorted diploid counterparts (logarithmic scale).

Extended Data Figure 2 Determination of ploidy at single-cell level by quantification of centromere foci.

a, The counted number of centromeres correlates with ploidy. 1n, haploid-enriched pES10 cells grown for 4 passages after the fourth sort (n = 33; 76% haploids by this assay); 2n, unsorted diploid pES10 cells (n = 34); 3n, soPS2 cells23 (n = 27); 4n, Hybrid1 cells41 (n = 27). Black horizontal lines indicate mean ± s.e.m. and dashed lines mark expected chromosome numbers. b, Quantification of haploid and diploid cells by DNA FISH in the haploid-enriched (n = 152; 73% haploids by this assay) and diploid (n = 135) cells in a. c, DNA content profile of the haploid-enriched cells in a (73% haploids by this assay). c, chromosomal copies. d, e, Co-staining of centromeres and either phospho-histone 3 (pH3, Ser10) (d) or 5-ethynyl-2′-deoxyuridine (EdU) (e) for distinguishing between different stages of interphase in haploid pES12 cells. In blue, DNA staining. Scale bar, 5 μm. f, Quantification of centromere counts in the different cell cycle stages shown in d and e. n indicated in parenthesis. Black horizontal lines indicate mean ± s.e.m. See Supplementary Notes for details.

Extended Data Figure 3 Pluripotent stem cell markers in haploid human ES cells.

Co-staining of pluripotency markers NANOG, OCT4, SOX2, SSEA4 and TRA-1-60 (red), centromeres (green) and DNA (blue) in h-pES10 and h-pES12 at colony resolution (upper panels; scale bars, 50 μm) and single-cell resolution (lower panels; scale bars, 10 μm). Magnified insets show representative haploid cells with 23 centromeres.

Extended Data Figure 4 Analysis of parental imprinting and gene trap mutagenesis in haploid human parthenogenetic ES cells.

a, b, Clustering analysis of diploid in vitro fertilization (IVF) ES cells and G1-sorted haploid and diploid parthenogenetic ES (pES) cells by expression levels of imprinted genes (n = 75, see Supplementary Table 4) (a) and DNA methylation levels at imprinted differentially methylated regions (iDMRs, n = 35)29 (b). The symbols (1) and (2) indicate biological replicates. c, Relative mean expression levels ± s.e.m. of representative paternally expressed imprinted genes across seven chromosomes in the samples shown in a (RPKM ratios). d, Mean DNA methylation levels ± s.e.m. at representative paternally methylated and maternally methylated iDMRs (typically intermediately methylated in bi-parental control cells, and respectively hypomethylated and hypermethylated in parthenogenetic cells) in the samples shown in b. β values range from complete hypomethylation (0) to complete hypermethylation (1). e, Schematic outline of the piggyBac gene trap system. The gene trap vector26 is flanked by piggyBac inverted terminal repeats (ITRs) and FRT flox sites, and carries a 5′ splice acceptor (SA), an internal ribosome entry site (IRES) element followed by a promoterless puromycin resistance gene (PuroΔtk) and a 3′ poly(A) signal (pA). In the presence of the PiggyBac transposase (encoded on a separate plasmid27, not shown), the gene trap vector undergoes random transposition into the genome. Insertion into a transcriptionally active gene results in truncation of the endogenous transcript and introduction of resistance to puromycin.

Extended Data Figure 5 Comparative analyses of isogenic haploid and diploid human ES cells.

a, Sorting purity of haploid and diploid ES cells in G1. b, Log-scaled volcano plots of relative differential gene expression between haploid and diploid human ES cells, divided into panels by all genes (top), autosomal genes (middle) and X chromosomal genes (bottom). Q, false discovery rate (FDR). Significantly downregulated and upregulated genes (greater than twofold change, Q < 0.05) in haploid cells are marked in red and blue, respectively, and their totals are indicated to the right. Note that XIST is the most downregulated transcript in haploid cells. c, Smoothed distributions of the 1n/2n gene expression ratios for all expressed genes, all expressed autosomal genes and all expressed X chromosomal genes (expression threshold, mean RPKM >0.1). d, Genome-wide moving median plot of the gene expression ratio between haploid and diploid pES10 cells in G1 by expression microarray analysis (window size = 100 genes). e, f, Model for genome-wide autosomal gene expression level reduction in haploid human ES cell as inferred by differential X chromosome inactivation status. e, DNA content, RNA expression levels relative to total RNA and presumed equality of absolute X chromosomal gene dosage in haploid (Xa) and diploid (XaXi) human ES cells, enable the estimation of total RNA levels per haploid cell. Xa and Xi denote active (blue) and inactive (red) X chromosomes, respectively. A, autosomes; X, X chromosome; R, total RNA. f, Schematic genome-wide representation of relative and absolute RNA levels in the cells shown in e. g, Average diameter and calculated surface area and volume of G1-sorted haploid and diploid ES cells. Error bars represent s.d. *P < 0.01 (two-tailed unpaired Student’s t-test). h, i, Functional annotation enrichment analysis for relatively downregulated genes and differentially methylated regions (DMRs) (h), as well as relatively upregulated genes (i) in haploid ES cells compared with diploid ES cells.

Extended Data Figure 6 EB differentiation of haploid human ES cells.

a, Representative image of plated cells dissociated from h-pES12-derived 21-day EBs. Karyotype is shown in Fig. 4b. Scale bar, 100 μm. b, DNA content profiles of dissociated EBs derived from haploid-enriched and diploid pES12 cells. c: chromosomal copies. c, Expression levels (RPKM) of tissue- and pluripotency-specific genes in undifferentiated (ES) and differentiated (EB) G1-sorted haploid (1n) pES10 cells.

Extended Data Figure 7 Directed differentiation of haploid human ES cells.

a, b, Flow cytometry analysis with co-staining of DNA and NCAM1 in h-pES10 cells following neural differentiation. a, Gating for NCAM1-positive cells (right) based on a negative secondary antibody stained control sample (left). b, DNA content profiles of the entire cell population (left) and NCAM1-positive cells (right). c, chromosomal copies. c, d, Expression levels of neural- and pluripotency-specific genes in haploid G1-sorted ES cells and NPCs. e, XIST expression levels in haploid and diploid pES10-derived EBs and NPCs. f, TUJ1 staining in h-pES12-derived neurons. Scale bar, 100 μm. g, DNA FISH in the neurons shown in f. Magnified insets show representative haploid and diploid nuclei with single and double hybridization signals, respectively. Scale bar, 10 μm. h, TNNT2 staining in G1-sorted haploid pES12-derived cardiomyocytes. Scale bar, 10 μm. i, j, Flow cytometry analysis with co-staining of DNA and PDX1 in h-pES10 cells following pancreatic differentiation. i, Gating for PDX1-positive cells (right) based on a negative secondary antibody stained control sample (left). j, DNA content profile of the entire cell population (related to Fig. 4k). c, chromosomal copies.

Extended Data Figure 8 In vivo differentiation of haploid human ES cells.

a, Haematoxylin and eosin histological sections of teratomas derived from h-pES10 and h-pES12. Scale bar, 50 μm. b, TUJ1 (ectoderm), α-SMA (mesoderm), AFP (endoderm) and OCT4 (pluripotency) staining in an h-pES10-derived teratoma. DNA staining is shown in blue. Note the absence of nuclear OCT4 staining. Scale bars, 100 μm.

Extended Data Table 1 Identification of haploid cells in early-passage human parthenogenetic ES cell lines by metaphase spread analysis
Extended Data Table 2 Isolation of haploid cells from early-passage human parthenogenetic ES cell lines by sub-2c-cell sorting

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-4, a Supplementary Discussion, Supplementary Notes and additional references. (PDF 290 kb)

Spontaneous beating of h-pES12-derived cardiomyocytes

This file shows spontaneous beating of h-pES12-derived cardiomyocytes. (MOV 9789 kb)

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Sagi, I., Chia, G., Golan-Lev, T. et al. Derivation and differentiation of haploid human embryonic stem cells. Nature 532, 107–111 (2016). https://doi.org/10.1038/nature17408

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