Supplementary information, Figure S1 Derivation of human diploid ESCs from parthenogenetic blastocysts generated by chemical activation.

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1 Supplementary Information Supplementary information, Figure S1 Derivation of human diploid ESCs from parthenogenetic blastocysts generated by chemical activation. (A) No 1c peak in the initial cell sorting of ESCs derived from parthenogenetic blastocysts that were generated by chemical activation. Shown are representative images of initial cell sorting of two cell lines (hcpes1 and hcpes2). (B) No haploid cells after two times of FACS enrichment of sub-2c cells for expansion. A DAPI filter was used to detect signal of Hoechst-stained DNA. Diploid ESCs from H9 cell line were used as control. 1

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3 Supplementary information, Figure S2 Derivation of human haploid ESCs from parthenogenetic blastocysts generated by removal of male pronucleus from zygotes. (A) Generation and development of human parthenogenetic haploid embryos. Shown are representative images of different stages of human haploid embryos. (B) Outgrowth (passage 0) and ESC colony (passage 4) of hpges2 cells. Scale bars, 100 μm. (C) Establishment of hpges2 cell line after two times of FACS enrichment of haploid cells. A DAPI filter was used to detect signal of Hoechst-stained DNA. High ratio of haploid cells could be detected at passage 19 after two round of FACSenrichment. Diploid ESCs from H9 cell line were used as control. (D) Karyotype of hpges1 (passage 17) and hpges2 (passage 17) showing the normal haploid set of 23 chromosomes (22+X). 3

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5 Supplementary information, Figure S3 Pluripotency of human haploid ESCs. (A) Expression of human ESC markers in hpges2 cells, including NANOG, OCT4, SSEA4, SOX2, TRA-1-60 and TRA Shown are the representative immunostaining images of human haploid ESC colonies. Scale bars, 200 μm. (B) Alkaline phosphatase staining of hpges1 and hpges2 cells. Scale bars, 50 μm. (C) RT-PCR analysis of the expression of pluripotent markers in hpges1, hpges2 and control cells. (D) Scatter plots of log2-transformed average gene expression profiles showing high correlation in gene expression between human haploid ESCs and diploid ESCs. (E) Expression of pluripotent genes and fibroblast-specific genes based on RNA-seq analysis. (F) EBs from hpges1 and hpges2 cells. Scale bars, 100 μm. (G) Histochemical analysis of teratomas from hpges1 and hpges2. Representative images indicated the existence of tissues from all three embryonic germ layers, such as neural epithelial (ectoderm), cartilage (mesoderm) and intestinal epithelium (endoderm). Scale bar, 100 μm. 5

6 Supplementary information, Figure S4 Haploidy in differentiated cells. (A) FACS enrichment of haploid cells from EBs. Undifferentiated haploid ESCs used as control. (B) Immunostaining analysis of FACS-enriched haploid cells from EBs. Expression of markers of various cells from different germ layers in haploid cells enriched from EBs, including TUJ1 (ectoderm), α-sma (mesoderm) and AFP (endoderm). Scale bar, 50 μm. 6

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8 Supplementary information, Figure S5 Human haploid ESCs stably sustain maternally imprinting state. (A) Expression of typical imprinted genes in human haploid ESCs and diploid ESCs based on RNA-seq analysis. (B) Methylation state of maternally imprinted gene (SNRPN) and paternally imprinted genes (H19 and MEG3) in hpges1 and hpges2 cells at different passages. Both cell lines stably maintain maternal imprints during cell passage. (C) DNA methylation levels at DMRs of representative paternally methylated and maternally imprinted genes based on RRBS results. (D) Clustering analysis of haploid ESCs and diploid control cells based on DNA methylation levels at DMRs of all imprinted genes. 8

9 Supplementary information, Table S1 Summary of generation of human haploid ESCs Strategy Embryos Human Blastocysts Human ES Cell Lines Haploid ES Cell Lines Pronuclear Removal Parthenogenetic Activation Total

10 Supplementary information, Table S2 STR analysis of human haploid ESCs STR hpges1 7904S 30778B hpges2 Male Female Male Female D8S D21S D7S CSF1PO D3S THO D13S D16S vwa TPOX D18S D5S FGA D2S D19S Amelogenin X Y X X X X Y X X X 10

11 Materials and Methods Source of human oocytes and embryos Our research was approved by the Institutional Review Board of Reproductive Medicine of Shandong University, and informed consent was obtained from all the participants. Preparation of human parthenogenetic haploid embryos Immature MI oocytes were collected from patients for IVF or ICSI treatment. Oocyte donors gave informed consent. MI oocytes were cultured in G1 medium in vitro for the first polar body extrusion by observation every 2hr. 3hr after polar body extrusion, ICSI was performed and embryos were cultured in timelapse incubator (vitrolife embryoscope) at 37 in 5% CO2, 5% O2, and 90% N2. Approximately 7-8h after fertilization, zygote was positioned using a holding pipette to a position that the male pronucleus can be clearly observed. The male pronucleus that usually locates in the distal position of second polar body could be removed using 5μm injection pipet. The constructed embryos were cultured in G1 medium and 74h after fertilization were transferred into G2 medium for further culturing. The embryos were allowed to develop into blastocysts that were used for ESC derivation. Human parthenogenetic haploid ESC line derivation and maintenance The trophectodermal cells of parthenogenetic blastocysts were ablated by laser system. Each isolated inner cell mass was transferred into one well of 4-well plate seeding with mitotically inactivated mouse embryonic fibroblasts in human ESC derivation medium, composed of Knock-out DMEM (Gibco), 20% KO-SR replacement, 0.1mM nonessential amino acids (Invitrogen), 2mM L-glutamine, 0.1mM β-mercaptoethanol, 50U/ml penicillin, 50 µg/ml streptomycin, 20 ng/ml bfgf (Gibco), 2 μm Thiazovivin (Selleck) and 10 μm Y (Selleck). Three days after plating, medium was changed every 1-2 days, and inner cell mass cells were allowed to grow for days in culture until the appearance of outgrowth. The outgrowth was manual picked and transferred into a new plate with a fresh feeder layer in fresh medium for ESC line derivation. Established ESCs were expanded in ESC culture medium, which is similar to ESCderivation medium, with modification of supplementation with 10 ng/ml bfgf and no addition of 2 mm Thiazovivin and 10 μm Y ROCK inhibitor. The culture medium was changed daily, and ESC colonies were split every 3-5 days manually or disaggregated by Accutase with addition of 10 μm Y ROCK inhibitor for one day. Cells were maintained in an incubator at 37 C and 5% CO2 for routine cell culture. FACS-enrichment of haploid cells Haploid ESC lines were established by sorting the 1c-cell population in early passages. To sort haploid cells, ESCs were washed with PBS, dissociated by Accutase into single cells, and then incubated with 15 μg/ml Hoechst in human ESC medium in a 37 C water bath for 15min. Following centrifugation, cells were rinsed with PBS once for removal of the remaining Hoechst 33342, and suspended in ESC medium 11

12 with 10 μm ROCK inhibitor Y The cell suspension was then filtered through a 40μm cell strainer. Haploid cells were sorted using the 355 nm laser in either BD FACSAria II or BD Influx (BD Biosciences). Normal diploid ESC sample was prepared as a negative control for each sorting. For maintenance of haploidy, cells with 1c DNA content (1c peak) were harvested and plated in new fresh feeder layer with fresh ESC medium containing 10μM ROCK inhibitor Y h later, ESCs were cultured in normal human ESC medium. It usually takes 5-8 days for colony formation and cell passage. Karyotype analysis of human haploid ESCs ESCs were split with Accutase in appropriate density. Approximately 20hr later, ESCs were incubated with 400 ng/ml demecolcine (Sigma) for 1 h. After being tripinsized, the ESCs were resuspended in M KCl at 37 C for 30 min. Hypotonic solution-treated cells were fixed in methanol:acetic acid (3:1 in volume) for 30 min and dropped onto precleaned slides. Chromosome spreads were Giemsabanded and photographed. Genome integrity analysis Copy number variation (CNV) analysis was carried out on DNA samples of hphes1, hphes2 and diploid ESCs from the H9 cell line using Affymetrix CytoHD. DNA samples were extracted using DNeasy Blood & Tissue Kit (QIAGEN) following the manufacturer s instructions. The DNA was purified by using 1% agarose gel electrophoresis, and quality of DNA was checked using NanoDrop (ThermoFisher). Raw data passed quality control were further analyzed by Affymetrix Chromosome Analysis Suite (Affymetrix) Genomic DNA isolation, digestion, sample labeling, array hybridization and data acquisition were performed at the Shanghai Biochip Company following the manufacturer s instructions. Immunofluorescence staining For immunofluorescence staining, cells on glass coverslips were washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized using 0.2% Triton X-100 in PBS for 15 min, and blocked in 1% BAS in PBS. Cells were incubated with primary antibodies against NANOG, SOX2, OCT4, SSEA4, TRA-1-60 and TRA-1-81 overnight at 4. As for secondary antibodies, the cells were treated with a fluorescently coupled secondary antibody and then incubated for 1 h at room temperature. DAPI was used for DNA staining, and all antibodies were diluted in blocking solution. Images were taken using fluorescence microscope. Reverse transcription PCR Total RNA was isolated from the cells using Trizol reagent (Invitrogen). RNA amounts were quantified using NanoDrop. One microgram of total RNA was reverse transcribed using a First Strand cdna Synthesis kit (TOYOBO). Perform Reverse transcription PCR with cdna as template. All of the gene expression levels were normalized to the internal standard gene, GADPH. RNA sequencing and transcriptome analysis Total RNA was extracted from two haploid ESC lines (hpges-1 and hpges-2, G1/G0 phase), two recently diploidized ESCs from both lines (G1/G0 phase), normal 12

13 diploid ESCs from the H9 cell line (G1/G0 phase) and human fibroblasts using TRIZOL Reagent (Life Technologies) following the manufacturer s instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 (Agilent Technologies). Qualified total RNA was further purified by RNAClean XP Kit (Beckman Coulter) and RNase-Free DNase Set (QIAGEN). RNA-seq libraries were prepared using TruSeq RNA Sample Pre Kit v2 Set A/ B (Illumina). RNA-Seq was performed using Illumina HiSeq 2500, reads were mapped to the human reference genome hg38 version using Tophat (version:2.0.9). Normalized gene expression levels were determined in units of FPKM (Fragments Per Kilobase per Million mapped reads). An unsupervised hierarchical clustering for all genes or imprinting genes was individually performed in cluster 3.0. RNA amplification, labeling, array hybridization and data acquisition were performed at the Shanghai Biochip Company following the manufacturer s instructions. The relatedness of transcription profiles was determined by calculating the Pearson s correlation coefficient (r). Embryoid body differentiation Human haploid ESCs were digested into small clumps using 1 mg/ml collagenase IV (Gibco), and suspension culture was performed with these small clumps in lowattachment plates for 7 days. Then, formed EBs were trypsinized with TrypLE Express (Gibco) and replated into plates with matrigel-coated glass slides for additional 4 days for immunofluorescence staining. The culture medium was composed of Knock-out DMEM (GIBCO) with 15% FBS (Hyclone), 2 mm L- glutamine (Invitrogen), 0.1mM nonessential amino acids, 0.1 mm β-mercaptoethanol, 50 U/ml penicillin and 50 µg/ml streptomycin. Teratoma formation assay For teratoma formation, we harvested approximately cells of each human haploid ES cell line in 140µl F12/DMEM and 60ul matrigel (BD Bioscience), followed by subcutaneous injection into NOD SCID mice. After 8-12 weeks, teratomas were harvested and dissected. Sections were stained with hematoxylin and eosin. Bisulphite sequencing Total DNA was isolated using the Blood &Tissue DNA Isolation Kit (TIANGEN). Bisulphite conversion was performed by EZ DNA methylation Gold kit (ZYMO Research). The PCR products were cloned into pmd19-t vectors (Takara) and individual clones were sequenced. RRBS and DNA methylation analysis Total DNA was extracted from two haploid ESC lines (hpges-1 and hpges-2, G1/G0 phase), two recently diploidized ESCs from both lines (G1/G0 phase), normal diploid ESCs from the H9 cell line (G1/G0 phase) and human fibroblasts using DNeasy Blood & Tissue Kit (QIAGEN). The DNA was purified by using 1% agarose 13

14 gel electrophoresis, and quality of DNA was checked using NanoDrop (ThermoFisher). DNA methylation analysis was performed on genomic DNA from the samples using Infinium HumanMethylation450 BeachChips (Illumina). Genomic DNA isolation, RRBS library generation and sequencing were performed at the Shanghai Biochip Company following the manufacturer s instructions. The raw data were processed and normalized by using subset-quantile within array normalization (SWAN) and adjusted using the R package minif, and then filtered the methylation difference of CpG sites and regions between samples with R package IMA. β values range from 0 to 1, indicating completely unmethylated and completely methylated CpG sites, respectively. The formula of β as followed: max (y (!,!"#$%), 0) beta! = max y!,!"#$%, 0 + max y (!,!"#$%&'), Accession numbers All CNV, DNA methylation and RNA-seq data sets are available through GEO under the accession numbers GEO: GSE80208, GEO: GSE80226, and GEO: GSE Also you can use the SuperSeries GSE81025 that links all these three data sets together. 14