SUPPLEMENTARY APPENDIX Down Syndrome-Related Transient Abnormal Myelopoiesis Is Attributed to a Specific Erythro-Megakaryocytic Subpopulation with GATA1 Mutation

SUPPLEMENTARY APPENDIX Down syndrome-related transient abnormal myelopoiesis is attributed to a specific erythro-megakaryocytic subpopulation with GATA1 mutation

Yoko Nishinaka-Arai, 1,2* Akira Niwa, 1* Shiori Matsuo, 1 Yasuhiro Kazuki, 3,4 Yuwna Yakura, 3 Takehiko Hiroma, 5 Tsutomu Toki, 6 Tet - sushi Sakuma, 7 Takashi Yamamoto, 7 Etsuro Ito, 6 Mitsuo Oshimura, 3 Tatsutoshi Nakahata 8 and Megumu K. Saito 1 1Department of Clinical Application, Center for iPS cell Research and Application, Kyoto University, Kyoto; 2Department of Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto; 3Chromosome Engineering Research Center, Tottori University, Tottori; 4Division of Genome and Cellular Functions, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, Tottori; 5Perinatal Medical Center, Nagano Children’s Hospital, Nagano; 6Department of Pediatrics, Hirosaki University Graduate School of Medicine, Hirosaki; 7Divi - sion of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima and 8Drug Discovery Technology Development Office, Center for iPS cell Research and Application, Kyoto University, Kyoto, Japan *YNA and AN contributed equally as co-first authors Correspondence: MEGUMU K. SAITO - [email protected] AKIRA NIWA - [email protected] doi:10.3324/haematol.2019.242693 Supplemental Materials and methods

Establishment of TAM-iPS

To establish a PSC-based in vitro model representative of the hematological abnormalities observed in TAM,

CD41+ blast cells were sorted using a FACSAriainstrument (BD Biosciences, San Jose, CA, USA). Episomal plasmid vectors carrying reprogramming factors were introduced into blasts using the Amaxa Human CD34 cell

Nucleofector Kit (Lonza, Germany). After electroporation, cells were plated on BD Falcon tissue culture plates

(#3516, CORNING, New York, USA) coated with 0.25 µg/cm2 LM511-E8, which is a recombinant short fragment of laminin 511 (#892011, Nippi, Osaka, Japan), in StemSpan-ACF medium (STEMCELL Technologies, Vancouver,

BC, Canada) containing 100 ng/mL SCF (#255-SC-010, R&D), 100 ng/mL TPO (#288-TP-005, R&D), 100 ng/mL

Flt-3 Ligand (#308-FK-005, R&D), 50 ng/mL IL-6 (#206-IL-010, R&D), and 10 ng/mL IL-3 (#203-IL-010, R&D).

The medium was changed to StemFit AK02N medium (Ajinomoto, Tokyo, Japan) gradually.

Maintenance of human PSCs

PSCs were maintained in StemFit AK02N medium on BD Falcon tissue culture plates coated with 0.25 µg/cm2

LM511-E8. At each passage, the cells were treated with 0.5 x TrypLE select (#12563011, Thermo, Waltham, MA,

USA) for 3 min at room temperature, harvested in AK02N medium with 10 µM Rho-Associated Protein Kinase

Inhibitor Y-27632 (#08945-71, Nacalai Tesque, Kyoto, Japan), and re-plated on plates coated with LM511-E8 at a density of 265 cells/cm2.

Transcription activator-like effector nucleases (TALEN) genome editing

To correct the mutation of GATA1 gene, we used TALEN genome editing. We set the TALEN targeting site at intron 2 of GATA1 gene and constructed TALEN plasmids using the Platinum Gate TALEN Kit (Addgene, Kit

#1000000043) with a CAG promoter-driven destination vector. The right and left target sequences used are as follows: right, 5′-TCTGCACTTAACTAGGGA-3′; left, 5′-TGGAAGCTTCTCAAATGG-3. For TALEN genome editing, PSCs were dissociated with TrypLE Select, and 1x106 cells were transfected with 2 µg of each TALEN 3 plasmid and 6 µg of the targeting vector plasmid in an N1 cuvette using the NEPA21 Super Electroporator

(NEPAGENE) following the manufacturer’s instruction. Then the PSCs were replated onto BD Falcon tissue culture plates coated with LM511-E8 with AK02N medium and 10 µM Y-27632. Cells were selected with 100 mg/mL

G418 (Wako) 24 h after the transfection, and surviving clones were isolated 13 days after the drug selection.

Selected clones were dissociated with TrypLE Select, and 1x106 cells were transfected with Cre expression plasmid using FuGene HD Transfection Reagent (Promega) following the manufacturer’s instruction. Cells were selected with 500 ng/mL puromycin (InvivoGen) 2 days after the transfection. Four days after the transfection, the surviving cells were dissociated and expanded.

Monolayer hematopoietic differentiation system

First, the PSCs were plated onto LM511-E8-coated plates at a density of 2.7 to 3.6 cells/cm2 and maintained as described in the previous subsection. When the undifferentiated colonies reached 750 to 1000 µm in diameter, the culture media was replaced with Essential-8 medium (#A1517001, Thermo) containing 80 ng/mL BMP4 (#314-

BP-010, R&D), 80 ng/mL VEGF 165 (#293-VE-010, R&D), and 2 µM GSK-3 inhibitor CHIR99021 (#038-23101,

Wako, Osaka, Japan). The media was changed every two days as follows. Day 2: Essential-6 medium (#A1516401,

Thermo) containing 80 ng/mL VEGF, 25 ng/mL bFGF (#064-05381, Wako), 2 µM ALK5 inhibitor SB431542

(#031-24291, Wako), and 50 ng/mL SCF; Day 4: Stemline-II medium (#S0192, Sigma-Aldrich, St. Louis, MO,

USA) containing 80 ng/mL VEGF, 50 ng/mL each of SCF, IL-6, IL-3, and Flt-3 Ligand, and 5 ng/mL TPO; and

Day 6: Stemline-II containing 50 ng/mL each of SCF, IL-6, IL-3, and Flt-3 Ligand, 5 ng/mL TPO, and 10 U/mL

EPO (#329871, Merk, Darmstadt, Germany). On day 9, the cells were harvested by flushing out colonies with PBS and filtering them through a 40 µm cell strainer. The collected cells were adjusted to 1x104 cells/cm2 and transferred to lineage-specific suspension culture, which consisted of Stemline-II medium with 50 ng/mL each of SCF and IL-

6, and either 10 U/mL EPO (for erythroid differentiation) or 20 ng/mL TPO (for megakaryocytic differentiation).

On day 13, half the culture volume of fresh media containing 150 ng/mL SCF and either 30 U/mL EPO (for erythroid lineage) or 60 ng/mL TPO (for megakaryocytic lineage) was added to the culture.

Progenitor assay

4 For the progenitor assay, each HPC subpopulation sorted on day 9 was adjusted to 5x104 cells/mL and maintained under the appropriate lineage-specific suspension culture condition. After one week, the cells were counted and subjected to cell surface marker analysis.

RNA extraction and real-time quantitative RT-PCR

RNA samples were initially prepared using silica gel membrane-based spin-columns (RNeasy Kit; Qiagen,

Valencia, CA, USA) following the manufacturer’s instructions. Extracted RNAs were subjected to reverse transcription followed by cDNA amplification following a previously reported method with minimal modification

1, 2. Predesigned assays were used for the targeted detection of hematopoietic differentiation-related genes

(PrimeTime predesigned qPCR assays; Integrated DNA Technologies, Coralville, IA, USA; see Supplemental Table

4), and quantitative RT-PCR experiments were performed with the StepOnePlus™ Real-Time PCR System

(Applied Biosystems, Foster City, CA, USA) using FastStart Universal Probe Master (#04913949001, Roche, Basel,

Switzerland) following the manufacturer’s instructions. Quantitative assessment of the gene expressions was performed using the standard ΔCT method. A combination of genes including GUSB, HPRT1, PGK1, ACTB,

GAPDH, TBP, B2M, and PPIA served as multiple references.

Analysis of real-time PCR data

For principle component analysis (PCA), data sets were filtered by the criterion requiring positive values in at least one sample and subjected to variance-stabilizing transformation and interquartile range filtering, followed by upper-quartile normalization. Next, individual analyses were performed in R using the prcomp function after removing unwanted variations between clones by applying the open-source package “RUV-seq” 3, and the results were visualized by the “Scatterplot3D” package obtained from the Bioconductor Project. Gene sets specifically relevant to the phenotypes of interest were extracted according to the correlations of the factor loadings between each gene and phenotype in a factorical space given in the PCAs, in which each phenotype was assigned as categorical data 4. GENEMANIA 5 and ReactomeFI 6 followed by ClueGO 7 plugin in Cytoscape (http://www.cytoscape.org) were used to visualize the network.

Ethics

Peripheral blood blast cells used for the iPSC generation were obtained from a Down syndrome patient (ID:

CiRA12345 at Kyoto University and 778 at Hirosaki University) with written informed consent from the patients guardians in accordance with the Declaration of Helsinki. The use of human ESCs was approved by the Ministry of

Education Culture, Sports, Science and Technology of Japan (MEXT). This study was approved by the Ethics

Committee and the recombinant DNA Experiments Safely Committee of Kyoto University. All methods were performed in accordance with the relevant guidelines and regulations.

May-Giemsa staining and microscopic observation

Phase contrast images of the culture were obtained by using BIOREVO BZ-9000 (Keyence) through a CFI Plan

Fluor DL 4x objective lens (Nikon). For the microscopic observations of blood cells, floating cells were centrifuged onto PLATINUMPRO glass slides (Matsunami glass, Osaka, Japan) by using a Shandon Cytospin 4 Cytocentrifuge

(Thermo, Pittsburgh, PA) and analyzed by BIOREVO BZ-9000 after May-Grunwald and Giemsa staining (Merck

Millipore). A PlanApo 40x/0.95 objective (Nikon) and the BZ-II Viewer software program (Keyence) were used for the image acquisition.

Chromosome analysis

PSCs were cultured on 6-cm dishes until confluent and fixed with KaryoMAX (Thermo). Q-banding analyses were performed at Chromocenter (Kobe, Japan).

Sanger sequencing

Genomic DNA was extracted from the cells by using the PureLink Genomic DNA Kit (Invitrogen, Carlsdad, CA,

USA) and subjected to PCR with the primers 5 -TTCTGAGAAAGTTAGAGGGCAAGATACAACAG-3

(forward) and 5-GTACAGGGGTTCCTATCTGGTATAAGTTTCTT-3 (reverse). After treatment with USB®

ExoSAP-IT (Affymetrix, SC, CA), the amplicons were applied for Sanger sequencing, which was performed with the BigDye Terminator® v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA). The products were

6 purified using the BigDye XTerminator® Purification Kit (ThermoFisher) and read by a 3500xL Sequencer

(Applied Biosystems). Data were analyzed using Codon Code Aligner software (CodonCode Corporation) and

4Peaks (A. Griekspoor and Tom Groothuis).

Flow cytometry and antibodies

Flow cytometric analyses and cell sorting were performed using a FACS AriaII and FlowJo software (Flowjo LLC,

Ashland, OR, USA). The following antibodies were used: BV421-conjugated anti-human CD235a (#562938) and

BV605-conjugated anti-human CD34 (#343530), FITC-conjugated anti-human CD45 (#304006, BD HorizonTM,

San Diego, CA, USA), and PE-conjugated anti-human CD42b (#303906), APC-conjugated anti-human CD71

(#334108), Alexa Flour® 647-conjugated anti-human CD309 (#338909), PE-Cy7-conjugated anti-human CD43

(#343208), APC-Cy7-conjugated anti-human CD41 (#303716), and PerCP-Cy5.5-conjugated anti-human CD11b

(#301328), which were all purchased from BioLegend (San Diego, CA, USA).

Statistics analyses

Experiments were repeated at least 3 times independently. Values are described as the mean ± standard deviation

(SD). Data were compared with Students t test and the Pearson correlation using GraphPad Prism (La Jolla, CA,

USA). The alpha level of all tests or the p value was set to 0.05.

7 Supplemental references

1. Kurimoto K, Yabuta Y, Ohinata Y, et al. An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis. Nucleic acids research. 2006;34(5):e42.

2. Ohta R, Niwa A, Taniguchi Y, et al. Laminin-guided highly efficient endothelial commitment from human pluripotent stem cells. Scientific reports. 2016;6:35680.

3. Risso D, Ngai J, Speed TP, et al. Normalization of RNA-seq data using factor analysis of control genes or samples. Nature biotechnology. 2014;32(9):896-902.

4. Kolenikov S. AG. The Use of Discrete Data in PCA: Theory, Simulations, and Applications to Socioeconomic Indices. Meas Eval wp. 2005:04-85 SRC - BaiduScholar.

5. Montojo J, Zuberi K, Rodriguez H, et al. GeneMANIA Cytoscape plugin: fast gene function predictions on the desktop. Bioinformatics (Oxford, England). 2010;26(22):2927-2928.

6. Wu G, Haw R. Functional Interaction Network Construction and Analysis for Disease Discovery. Methods in molecular biology (Clifton, NJ). 2017;1558:235-253.

7. Bindea G, Mlecnik B, Hackl H, et al. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics (Oxford, England). 2009;25(8):1091-1093.

8 Supplemental Figure 1

A B patient-iPSCs (TAM-G1s) corrected-iPSCs (TAM-WT) patient-iPSCs (TAM-G1s) corrected-iPSCs (TAM-WT) 47, XX, +21 47, XX, +21

100µm 100µm

★ GATA1 short C Targeting allele Ex 1 Ex 2 Ex 3 Ex 4 Ex 5 Ex 6 (G1s) NF CF

5’ arm pA PGK Neo pA 3’ arm Targeting vector LoxP LoxP

Corrected allele Ex 1 Ex 2 Ex 3 Ex 4 Ex 5 Ex 6 GATA1 full AD NF CF (WT)

D GATA1 full (WT) GATA1 short (G1s)

st nd patient-iPSCs 1 ATG 2 ATG ➡ 10bp dup. insert Ex 1 Ex 2 Ex 3 (TAM-iPS-G1s)

corrected-iPSCs (TAM-iPS-WT)

Supplemental Figure 1. Establishment of isogenic iPSC panels from DS-TAM patient primary blood cells

(A) Morphology (phase contrast imaging) and (B) chromosomal analysis (Q-banding) of established iPSCs before

(left panel) and after (right panel) gene correction. Scale bars, 100 µm. (C) Schema of the gene correction of GATA1 mutation using TALEN-mediated gene editing. (D) Representative chromatograph of Sanger sequencing of GATA1 gene from iPSCs before (upper panel) and after (lower panel) the gene correction.

3 Supplemental Figure 2 (%) A B 120 WT Ery Mk Mye Erythrocytes HPCs Megakaryocytes 100 day9 day13 day16 80 ** *** SCF : 50 ng/mL SCF : 50 ng/mL IL-6 : 50 ng/mL EPO : 10 U/mL EPO : 10 U/mL TPO : 20 ng/mL 60 TPO : 20 ng/mL Heparin : 50 U/mL (day 16) (day 40 n.s. Stemline-Ⅱ * 20 *** *** Frequency of lineage cells Frequency 0 WT G1s WT G1s Ts21-ES TAM-iPS C D E WT G1s (%) HPCs Erythrocytes 100 *** 72.7 0.14 ***

day9 day13 day16 ) + 80

SCF : 50 ng/mL SCF : 50 ng/mL CD235a IL-6 : 50 ng/mL EPO : 10 U/mL EPO : 10 U/mL 60 CD71 -

Stemline-Ⅱ 40

Erythroid cells 20 (CD235a

CD71 0 Ts21-ES TAM-iPS

F G (CD235a-CD41+ >) H (%) HPCs Megakaryocytes WT G1s 150 day9 day13 day16 62.8 0 SCF : 50 ng/mL SCF : 50 ng/mL CD42b 120 IL-6 : 50 ng/mL TPO : 20 ng/mL cells TPO : 20 ng/mL Heparin : 50 U/mL + ** *** 37.2 100 90 Stemline-Ⅱ CD41

+ 60 ** *** 30 G1s

CD41 in CD42b 0 WT G1s WT Propotion of megakaryoblastsPropotion Ts21-ES TAM-iPS Mega (CD42bbrightCD41+) Megablast (CD42bdimCD41+)

Supplemental Figure 2. Difference in hematopoietic specification between WT and G1s Ts21-PSCs

(A) Schematic process for erythroid-megakaryocytic differentiation from hematopoietic progenitor cells. (B)

Percentages of erythroid-, megakaryocytic-, and myeloid-lineage commitment according to the differentiation protocol shown in (A). P values compare Ery (erythrocytes), Mk (megakaryocytes), and Mye (myeloids) between

WT and G1s Ts21-ESCs: < 0.0001, 0.0131, and 0.0016, respectively; and between WT and G1s TAM-iPSCs: <

0.0001, 0.3858, and < 0.0001, respectively. These differences in hematopoiesis were not recovered even under conditions specifically designed to induce erythroid (C-E) or megakaryocytic lineages (F-H). (C) Protocol for erythroid-specific differentiation. (D) Representative FACS panel and (E) percentages of erythroid cells. Dead cells

(DAPI positive) were excluded from the analysis. P values compare Ery in erythroid lineage-inducing condition between WT and G1s Ts21-ESCs, < 0.0001, and between WT and G1s TAM-iPSCs, < 0.0001. (F) Protocol for megakaryocyte-specific differentiation. (G) Representative FACS panel and (H) percentage of megakaryocytic cells.

While megakaryocytic cells from the WT lines contained both CD42bdim (immature) and CD42bbright (more mature) populations, the G1s lines showed only CD42bdim cells, which corresponds to the phenotype seen in the primary blood cells of patients with TAM. Dead cells (DAPI positive) were excluded from the analysis. P values compare

Mega (megakaryocytes) and Megablast (megakaryoblasts) in megakaryocytic lineage-inducing condition between

WT and G1s Ts21-ESCs, 0.0029, and between WT and G1s TAM-iPSCs, 0.0008. Student’s t-test. Data are shown as the mean + standard deviation (SD). *P < .05, **P < .01, ***P < .001; n.s, not significant. (n = 4-7).

4 Supplemental Figure 3

A 170731_Day000609_Kh_nzVS2_FUQB RUVr PCA Contribution day 9 P-erymk 41 (-) C day 9 CD235a+CD41+ day 0 1.0 WT day 0 G1s WT day 6 CD34+CD43+ G1s day 6 WT + + day 9 P-erymk 41 (+) CD34 CD43 G1s

10% WT day 9 P-erymk 41 (-) day 9 G1s P-erymk 41 (+) WT 15% day 9 P-mye 0.8 G1s day 9 P-mye WT day9 CD235a+CD41+

33% 0.6

tion 170731_Day000609_Kh_nzVS2 Boxplot C r D

170731_Day000609_Kh_nzVS2_FUQRUVr PCA Contribution

22 alue)

v 1.0 Propo

11 Log(1+ 0.4 0.8

0 Log (1+Value) 0

0.6

170731_Day000609_Kh_nzVS2_FUQ Boxplot tion r

D00_Kh1WD00_Kh1W D00_Kh1SD00_Kh1S D06_Kh1WD06_Kh1W D06_Kh1SD06_Kh1S D09_34p41p235an71p_Kh1W D09_34p41p235an71p_Kh1SD09_34p41p235an71p_Kh1S D09_34p41n235an11bn43p_Kh1WD09_34p41n235an11bn43p_Kh1SD09_34p41n235an11bn43p_Kh1S D09_41n235an11bp_Kh1W D09_41n235an11bp_Kh1SD09_41n235an11bp_Kh1S D09_41p235ap_Kh1W

Sample Propo 0.4

0.2

alue) 0.2 v

0.0 11 Log(1+ PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PC11 PC12

Log (1+Value) Principal component

00 WT G1s WTG1s WT WT G1s WT G1s WT G1s

WT G1s WT G1s WT SampleG1s WT G1s WT G1s WT 0.0 P-mye CD43 CD41 SSEA4 + + + Day 0 CD34 P-erymk 41(-) P-erymk 41 (+) CD235a Day 9 P-mye TRA-1-60 Day 6 CD34+CD43 Day 9 P-erymk 41(-) Day 9 P-erymk 41(+) Day 9 CD235a+CD41+ day 0 day 6 day 9 day E F ) )

iPS (day 0) CD235a-CD34+ (day 9) P-erymk 41(+) (day 9) P-erymk 41(-) (day 9) / ACTB TAM-WT-iPS 1 / ACTB TAM-WT-iPS Relative expression level (ERG Relative expression level (RUNX

Supplemental Figure 3. Traced differentiation expression profiles using hematopoiesis-focused PCR array

(A-B) Integrative analysis of the expression profiles using hematopoiesis-focused real-time PCR arrays outlined the transition of gene expression profiles during the differentiation of WT- and G1s-clones and depicted by principal component analysis (A) and a heatmap (B). Data from WT and G1s Ts21-ESCs are shown. (C) Box plots of relative log expressions for unnormalized (upper) and normalized (lower) values. The bottom and top of the boxes indicate the first and third quartiles, respectively; thick lines inside the boxes indicate the medians. (D) Chart of the variance proportion (white) and cumulative variance proportion (gray). (E-F) Relative expression levels of representative hematopoietic genes RUNX1 (E) and ERG (F). These data were analyzed using DDCt methods normalized by the

ACTB expression level in each sample and gene expression level in WT TAM-iPSCs (day 0).

5 Supplemental Figure 4 A B (%) (%) 20 20 n.s. n.s. 15 15 n.s. n.s. cells (day 4) (day cells cells (day 6) (day cells + 10 + 10 CD34 CD43 + 5 + 5 WT KDR 0 CD34 0 Ts21-ES TAM-iPS Ts21-ES TAM-iPS

Supplemental Figure 4. Mesodermal and early hematopoietic differentiation efficacy

(A-B) Percentages of (A) KDR+CD34+ mesodermal (Day 4) and (B) CD34+CD43+ hematopoietic (Day 6) progenitors.

6 Supplemental Figure 5

1.0 A B 33 C

22 0.8

11 0.6 tion r

Log (1+Value) 00

Propo 0.4 Proportion PC2 (24%)

0.2 33

22 0.0 PC1 PC2 PC3 PC4 PC5 PC6

11 PC1 (30%)

00 WT_P-erymk 41(+) Log (1+Value) G1s_P-erymk 41(+) WT G1s WT G1s WT G1s WT_P-erymk 41(-) G1s_P-erymk 41(-) P-erymk 41(+) P-erymk 41(-) P-mye WT_P-mye G1s_P-mye D E F 1.0 33

22 0.8

0.6

0 Log (1+Value) 0 PC3 (27%)

0.4 Proportion

33 0.2

0.0 PC2 (28%) PC2 PC1 PC2 PC3 PC4

PC1 (47%) 0 0 Log (1+Value)

WT_ P-erymk 41(+) WT G1s WT G1s G1s_P-erymk 41(+) WT_P-erymk 41(-) P-erymk 41(+) P-erymk 41(-) G1s_P-erymk 41(-)

P-erymk 41(-) cluster A

TNF P-erymk 41(-) cluster E TGFB1 ID2

FOS ITGA5 P-erymk 41(+) cluster D FZD5 G ITGB1 H JUNB RHEB NRP2 GATA2 VEGFA STK25 CCNG1

MSC P-erymk 41(+) cluster B SPP1 RAF1 ANG PGF THBS2 IDH2 CREBL2 Group P-value ENG DERL1 Lineage specification PDHA2 FOSL2 SLC4A1 APC BIRC5 WNT5B BCR PIM2 ODC1 ELK3 TOMM40L Cluster GO ID GO term Corrected with PDHB FBXW5 RBPMS TUBB2A TFRC THBS3 SOD1 CD164 APOBEC2 CEBPA FZD1 PCNA ARRB2 BCL2L1 PHF21A BRIP1 PF4CACNA1C P-erymk 41(-) cluster D Pro Ery Mk Mye Bonferroni step down TRAP1 MAPK8 FOXO3 CDK7 PRDX2 CCR1 EIF4E1B RRM2 CCL23 EPOR SRXN1 EYA4 MEIS2 SOD2 CAST WBP4 GADD45A HBZ TAB2 ASXL1 CCL14 P-erymk 41(+) GO: 0010935 regulation of macrophage cytokine production 0.02807078 PRDX6 SUCLG1 SUCLA2RRM1 SMURF1 CAT CDX1 HLX DCAF11 PDK4 SMAD4 CSNK2A1 SDHB HMOX2 CEP57 CCL15 FZD6 MAP2K1 ENO2 ANAPC4 PDP1 MAPK3 GO: 0002719 negative regulation of cytokine production involved in immune response 0.030283099 DENND1B FMO1 PRELID1 A XRCC5 WNT5A ADD1 LAMTOR5 WNT6 ANK1 RAB24 GLRX PTPN11 TAL1 HOXC6 PPP2R4 PDHA1 KLF1 PRPS2 MSRA GSR EIF4E2 MDH2 DLAT KCNJ13 TCF12 GO: 0002756 MyD88-independent toll-like receptor signaling pathway 0.006458183 PRDX4GAPDH DLD CSNK1A1 TMOD1 TBX5 UBC EPO DVL1 GABPB1 TPI1 UBE2B GLA TRIM10 SKI NQO1 VRK1 LDHA ALDOB PDK3 ALAS2 P-erymk 41(+) GO: 0002755 MyD88-dependent toll-like receptor signaling pathway 0.007385797 SHFM1 GABPA RNH1 MAPK9SDHA SPTB BIK CTCF POLD3 CCS SCAF4 LDHB TGFBR1 MAP3K7 NTF4 SIM2 PRPS1 PFKM TXN GO: 0070423 nucleotide-binding oligomerization domain containing signaling pathway 0.007385797 PRDX1 PNLIPRP1 MYB TP53 SREBF1 B GLRX2 RPA1 CHD2 LEF1 RAD54B SIRT1 DPPA4 FEN1 NBN VEGFB PPIA TXNRD1 NUFIP2 RAG2 PFKL SOCS2 RCOR1 POLD4 FKBP8 CD47 MET PGK1 GO: 0002223 stimulatory C-type lectin receptor signaling pathway 0.020412957 VIP ASH2L EIF4E PFKP TP53BP1 TGFB2 ID3 MAP4K5 BCL2 CDK6 RPA3 SELP ATP2B4 EGFR BCL6 ITGA6 NDE1 EIF4EBP1 STAT1 MPV17L PELI2 LIG1 KDM1A MBNL1 PITX2 ● GO: 0060218 hematopoietic stem cell differentiation 0.0024106535 LTF ATF4 SPARC PTK2B MDC1 MSI2 ITGAV ATF1 H2AFX VIMP TGFB3 TOMM40 ECHDC2 SLC2A3 CD9 BUB1 RPA2 PIK3R3 RAP1B DLL1 POLD2 CBL RAD51C DDX11 STAT3 WNT2 LOX TGFBR2 GTSE1 PRDM1 MSX1 LIFR P-erymk 41(+) GO: 0030888 regulation of B cell proliferation 0.01963206 PIK3CG SIN3A POLD1 BMP7 EPAS1 ITGA7 PDLIM1 FHL1 P-erymk 41(-) SIRPG SRC IGF1R GPX1 SOD3 TGFBR3 TGFBI XRCC3 PTK2 SOCS1 LAMP3 ISL1 FN1 RHOBFLT1 CDKN2D GATA6 ITGA3 cluster C HOXC4 ITGB3 CAV1 GO: 0060334 regulation of interferon-gamma-mediated signaling pathway 0.005517673 CSF1R PTCH1 TNC C PTPRC SBNO2 AMHR2 VEGFC A2M APOBEC3G LAMB1 BMP2 BDNF THY1 CSF1 JUN NFIB HIF1A NTRK2 SOCS3 JAK2 CALD1 STC2 IL10RA KIT CD14 SLC18A2 IGF2 TIMP2 CTHRC1 GO: 1903708 positive regulation of hematopoiesis 0.070336045 CD38 TLR4 THBS1 IL10 ETS2 LSP1 PDGFRA MEIS1 SIRPA KDR SMAD7 OSMR GP1BA VPS18 IL6ST GPX3 TF SELPLG SOX5 F2R IL1RL1 TWIST1 PLEKHG6 ● GO: 0030218 erythrocyte differentiation 9.76476E-06 SERTAD1 OSM SDPR CHST15 NRCAM RGN FOXA1 GRID2 LIF ACVRL1 LHX6 EN1 P-erymk 41(-) cluster B DMD HOXC12 VASN P-erymk 41(+) ● GO: 0043249 erythrocyte maturation 0.000428154 KLHL13 ERG VTN P-erymk 41(+) cluster C AGP2 D ● GO: 0061515 myeloid cell development 0.001676536 ADAMTSL1 P-erymk 41(+) cluster A ● GO: 0030219 megakaryocyte differentiation 0.016272944 ● GO: 0045639 positive regulation of myeloid cell differentiation 0.017309068 I

Group P-value Lineage specification Cluster GO ID GO term Corrected with Pro Ery Mk Mye Bonferroni step down ● GO: 0002573 Myeloid leukocyte differentiation 3.36959E-05 GO: 1903708 positive regulation of hematopoiesis 0.001718853 P-erymk 41(-) ◯ GO: 0060319 Primitive erythrocyte differentiation 0.001822897 A GO: 0060754 Positive regulation of mast cell chemotaxis 0.00377464 GO: 0030225 Macrophage differentiation 0.008782239 GO: 0030316 Osteoclast differentiation 0.009006904 ● GO: 1902105 Regulation of leukocyte differentiation 0.010426746 ● GO: 0045637 Regulation of myeloid cell differentiation 0.029841227 P-erymk 41(-) GO: 0046632 Alpha-beta T cell differentiation 0.011070477 C GO: 0042093 T-helper cell differentiation 0.024089469 ● GO: 1902036 Regulation of hematopoietic stem cell differentiation 0.02759787

Supplemental Figure 5. GATA1 mutation impacts P-erymk41(+) (CD34+CD43+CD235-CD11b-CD71+CD41+) subpopulations

Post-hoc comparison analyses on the expression profiles identified P-erymk41(+) as a responsible population. (A-

C) Principal component analysis (PCA) showing differences in the gene expression profiles of P-erymk41(+), P- erymk41(-), and P-mye subpopulations. (A) PCA highlighted differences between CD11b+ and both CD11b- populations by mapping them far from each other in the PC1 direction. (B) Box plots of relative log expressions for unnormalized (upper) and normalized (lower) values. The bottom and top of the boxes indicate the first and third quartiles, respectively; thick lines inside the boxes indicate the median. (C) Chart of the variance proportion (white) and cumulative variance proportion (gray). (D) PCA showing gene expression differences in a post-hoc comparison between P-erymk41(+) and P-erymk41(-). (E) Box plots of relative log expressions for unnormalized (upper) and normalized (lower) values. The bottom and top of the boxes indicate the first and third quartiles, respectively; thick lines inside the boxes indicate the median. (F) Chart of the variance proportion (white) and cumulative variance proportion (gray). (G-I) Clustering algorithm for automated functional annotations on a weighted composite functional interaction network based on 261 genes and extracted by the correlation index > 0.58 in a factorical space given by PCA revealed that the CD11b-CD71+CD41+ cells were significantly enriched for pathways related to multi- lineage differentiation, including erythroid, megakaryocytic, and myeloid differentiation, whereas CD11b-

CD71+CD41- cells were enriched in pathways associated with more limited differentiation (P values corrected with

Bonferroni step down < 0.03). (G) An expanded map showing the clusters of genes extracted by positive correlations

(correlation index > 0.58 in 3D factorial space in the PCA shown in (D)) with P-erymk41+ (blue) and P-erymk (-)

(magenta). Closed circles depict genes originally included in the PCR array. Open circles depict genes replenished by GENEMANIA. Each gene is listed in Supplementary Table 2. (H-I) The intracellular pathways specifically enriched in (H) P-erymk41(+) and (I) P-erymk41(-). GO terms with p <0.05 in Bonferroni step down analysis are listed. Closed circles in (H) and (I) show the lineage specifications indicated by the GO terms. The open circle in

(I) depicts the indicated relationship with primitive erythrocyte differentiation. There are no significant GO terms for gene sets categorized to P-erymk41(-) clusters B, D or E were enriched.

7 Supplemental Figure 6

A 180416R_Day09_KhPt_Pair(CD41p)_nzVS Boxplot B 180416R_Day09_KhPt_Pair(CD41p)_nzVS_FUQ Boxplot 170731_Day000609_Kh_nzVS2_FUQRUVr PCA Contribution

33 33

22 22 alue) alue) v v 1.0 11 11 Log(1+ Log(1+ Log (1+Value) Log (1+Value)

00 00

KhWT_D09_41pKhWT_D09_41p PtWT_D09_41pPtWT_D09_41p KhMT_D09_41pKhMT_D09_41p PtMT_D09_41pPtMT_D09_41p KhWT_D09_41pKhWT_D09_41p PtWT_D09_41pPtWT_D09_41p KhMT_D09_41pKhMT_D09_41p PtMT_D09_41pPtMT_D09_41p Ts21-ES TAM-iPS Ts21-ES TAM-iPS Ts21-ES TAM-iPS Ts21-ES TAM-iPS 0.8 Sample Sample WT G1s WT G1s Day 9 P-erymk 41(+) Day 9 P-erymk 41(-)

0.6

tion 180416R_Day09_KhPt_Pair(CD41p)_nzVS_FUQ PCA Contribution Cr

1.0 Propo 0.8 0.4

0.6 tion r Proportion

Propo 0.4 0.2

0.2

0.0 PC1 PC2 PC3 PC4 0.0 Principal component

Supplemental Figure 6. Quality check data from principal component analysis of gene expression profiles of P-erymk41(+) derived from WT- and G1s-clones on day 9.

(A-B) Box plots of relative log expressions for unnormalized (A) and normalized (B) values. The bottom and top of the boxes indicate the first and third quartiles, respectively; thick lines inside the boxes indicate the median. (C)

Chart of the variance proportion (white) and cumulative variance proportion (gray).

8 Supplemental Figure 7 A B cells cells 4 4 * ** **

** from 1x10 from 1x10 * * Number of lineage cells on day 16 Number of lineage cells on day 16 ** * *

C Myeloid-specific condition D Erythro-megakaryocytic-specific condition

400000 ** 200000 n.s. n.s.

300000 150000

*** cells 4 cells

4 n.s. ** *** n.s. 200000 100000 from 1x10

from 1x10 100000 50000 Number of total cells on day 16 Number of total cells on day 16 0 0 WT G1s WT G1s WT G1s WT G1s WT G1s WT G1s WT G1s WT G1s Ts21-ES TAM-iPS Ts21-ES TAM-iPS Ts21-ES TAM-iPS Ts21-ES TAM-iPS

P-erymk 41(+) P-erymk 41(-) P-erymk 41(+) P-erymk 41(-)

Supplemental Figure 7. Results of the progenitor assay under erythroid-megakaryocytic lineage-specific differentiation condition.

(A-B) Number of lineage cells derived from 1 x 104 sorted (A) Ts21-ES and (B) TAM-iPS of each P-erymk subpopulation under erythro-megakaryocytic lineage-specific differentiation condition. Student’s t-test. Data are presented as the mean + SD. *P < .05, **P < .01, ***P < .001; no mark, not significant. (A) P values compare Ery (erythrocytes), Meg (megakaryocytes), Megablast (magakaryoblasts), and Mye (myeloids) between Ts21-ES-WT and -G1s P-erymk 41(+): 0.0802, 0.0193, 0.0012 and 0.0022; and P-erymk 41 (-):

0.0014, 0.3376, 0.0928 and 0.0034, respectively. (n = 4-6) (B) P values compare Ery, Meg, Megablast, and

Mye between TAM-iPS-WT and -G1s P-erymk 41(+): 0.0343, 0.0269, 0.0737 and 0.1912; and P-erymk 41

(-): 0.0245, 0.2098, 0.6173 and 0.0490, respectively. (n = 3-5) (C-D) Total cell number on day 16 derived from

1 x 104 sorted Ts21-ES and TAM-iPS of each P-erymk subpopulation (C) under myeloid lineage-specific differentiation condition and (D) under erythro-megakaryocytic lineage-specific differentiation condition.

Multiple comparisons using the Holm-Sidak method. Data are presented as the mean + SD. *P < .05, **P

< .01, ***P < .001; n.s., not significant. (C) P values compare Ts21-ES, TAM-iPS between WT and G1s P- erymk 41(+): <0.001 and 0.004811; and P-erymk 41 (-): 0.002213 and <0.0001, respectively. (n = 3-7) (D) P values compare Ts21-ES, TAM-iPS between WT and G1s P-erymk 41(+): 0.612962 and 0.141503; and P- erymk 41 (-): 0.950503 and 0.633504, respectively. (n = 3-6)

9 1K9D IHLJIG 1K9M 6 IHLJIG 1K9M 0 IHLJIG 1K9D 16 IHLJIG 1K9M 19 IHLJIG 1K9DF 7 IHLJIG 1K9D 959 IHLJIG 1K9M 9 IHLJIG 1K9 0EHE 1K9 5 0EHE 1K9 . 0EHE 1K9D 8 0EHE 1K9DF 9 0EHE 1K9 0EHE 1K9D 5 0EHE 1K9 -65 0EHE 1K9D -- 0EHE 1K9DF -- 0EHE 1K9DF -6 0EHE 1K9D 0 0EHE 1K9D 0 0EHE 1K9 2- 0EHE 1K9DF 5-8 0EHE 1K9D 5-8 0EHE 1K9DF 61 0EHE 1K9 79 0EHE 1K9 79 0EHE 1K9D 70 0EHE 1K9D 709 0EHE 1K9D 74 0EHE 1K9 74 0EHE 1K9 757 0EHE 1K9 9 0EHE 1K9D 9 0EHE 1K9D 9 0EHE 1K9D 98. 0EHE 1K9 98. 0EHE 1K9F 98. 0EHE 1K9 98. 0EHE 1K9 98. 0EHE 1K9 98.- 0EHE 1K9 98. 0EHE 1K9 98.0 0EHE 1K9 98.1 0EHE 1K9 98. 0EHE 1K9 988 0EHE 1K9 9A 0EHE 1K9D 9 0EHE 1K9DF 0EHE 1K9 15 0EHE 1K9 A5 0EHE 1K9DF 0EHE 1K9D 6 0EHE 1K9 7 0EHE 1K9D 8A 0EHE 1K9D 9 0EHE 1K9 C27 0EHE 1K9 07 0EHE 1K9 5 0EHE 1K9DF 5 0EHE 1K9DF 55 0EHE 1K9DF 5 0EHE 1K9D 0EHE 1K9D -1 0EHE 1K9DF 2 0EHE 1K9D 56 0EHE 1K9D 62 0EHE 1K9D 69 0EHE 1K9DF 69 0EHE 1K9D 69 0EHE 1K9D 69 0EHE 1K9 7 0EHE 1K9DF 0EHE 1K9DF 0EHE 1K9 . 0EHE 1K9 29 0EHE 1K9 7 0EHE 1K9D 7 0EHE 1K9DF 5- 0EHE 1K9D 56 0EHE 1K9D 97 0EHE 1K9DF 0EHE 1K9D 0EHE 1K9DF 0EHE 1K9 0EHE 1K9D 5 0EHE 1K9 - 0EHE 1K9F 5 0EHE 1K9D 7 0EHE 1K9D 7 0EHE 1K9 7 0EHE 1K9D 7- 0EHE 1K9 7- 0EHE 1K9D 7. 0EHE 1K9D 7 0EHE 1K9D 70 0EHE 1K9D 71 0EHE 1K9D 7 0EHE 1K9DFK 7 0EHE 1K9F 0EHE 1K9DF - 0EHE 1K9F - 0EHE 1K9D -- 0EHE 1K9D - 0EHE 1K9D - 0EHE 1K9DF - 0EHE 1K9 - 0EHE 1K9D -- 0EHE 1K9 - 0EHE 1K9 - 0EHE 1K9 - 0EHE 1K9D - 0EHE 1K9 - 0EHE 1K9D - 0EHE 1K9 - 0EHE 1K9 - 0EHE 1K9 - 0EHE 1K9 - 0EHE 1K9 -.9 0EHE 1K9 - 0EHE 1K9 -1 0EHE 1K9F -4 0EHE 1K9 -4 0EHE 1K9 -4 0EHE 1K9F -4 0EHE 1K9 -4 0EHE 1K9 -4 0EHE 1K9F -47 0EHE 1K9 -47 0EHE 1K9 -47 0EHE 1K9 -47- 0EHE 1K9 -47 0EHE 1K9F .9 0EHE 1K9F .9 0EHE 1K9 .9. 0EHE 1K9 .90 0EHE 1K9 .5 0EHE 1K9 .9 0EHE 1K9F 11- 0EHE 1K9 1- 0EHE 1K9 1- 0EHE 1K9FK 169 0EHE 1K9 169 0EHE 1K9 1 0EHE 1K9 21 0EHE 1K9 4 0EHE 1K9 4 0EHE 1K9 5 0EHE 1K9 6 0EHE 1K9 729 0EHE 1K9F . 0EHE 1K9 . 0EHE 1K9 .9 0EHE 1K9 .5 0EHE 1K9 .6 0EHE 1K9 0EHE 1K9 0EHE 1K9 0EHE 1K9F 0EHE 1K9 0EHE 1K9 74 0EHE 1K9F 74 0EHE 1K9 74 0EHE 1K9F 74 0EHE 1K9 0EHE 1K9 1 0EHE 1K9 77 0EHE 1K9 77 0EHE 1K9 5 0EHE 1K9 5 0EHE 1K9 5 0EHE 1K9F A5 0EHE 1K9F A 0EHE 1K9D A 0EHE 1K9 B 0EHE 1K9 -6 0EHE 1K9 - 0EHE 1K9F - 0EHE 1K9 -7 0EHE 1K9 -- 0EHE 1K9 --A 0EHE 1K9 -.77- 0EHE 1K9 -1 0EHE 1K9 -5 0EHE 1K9F -5- 0EHE 1K9 -55 0EHE 1K9 -55 0EHE 1K9F -6- 0EHE 1K9 -76 0EHE 1K9 -7 0EHE 1K9 -99 0EHE 1K9 .1- 0EHE 1K9 . 0EHE 1K9 .057 0EHE 1K9 .0 0EHE 1K9 .2.9 0EHE 1K9 .2 0EHE 1K9F .57. 0EHE 1K9 .55 0EHE 1K9 .55 0EHE 1K9 .54 0EHE 1K9 .6. 0EHE 1K9 .7 0EHE 1K9 .70 0EHE 1K9 .78 0EHE 1K9 .78 0EHE 1K9 .78 0EHE 1K9 .78A 0EHE 1K9 .9 0EHE 1K9 .9 0EHE 1K9 .9 0EHE 1K9F .98 0EHE 1K9 .98 0EHE 1K9 . 0EHE 1K9 .0 0EHE 1K9 . 0EHE 1K9 . 0EHE 1K9 . 0EHE 1K9 .B 0EHE 1K9D 0EHE 1K9F A 0EHE 1K9 A 0EHE 1K9 .7 0EHE 1K9 . 0EHE 1K9 0 0EHE 1K9 1 0EHE 1K9 15 0EHE 1K9 52 0EHE 1K9 57 0EHE 1K9 5 0EHE 1K9 5 0EHE 1K9 550 0EHE 1K9 68 0EHE 1K9 7 0EHE 1K9 85 0EHE 1K9 8A 0EHE 1K9 8A8 0EHE 1K9F 8A8 0EHE 1K9 8A8 0EHE 1K9 8A9 0EHE 1K9F C- 0EHE 1K9F C- 0EHE 1K9 C- 0EHE 1K9F C- 0EHE 1K9 C- 0EHE 1K9F C- 0EHE 1K9 09- 0EHE 1K9 09 0EHE 1K9 0-- 0EHE 1K9FK 0-- 0EHE 1K9FK 0--0 0EHE 1K9D 09-1 0EHE 1K9 0 0EHE 1K9 0 0EHE 1K9 0 0EHE 1K9 0 0EHE 1K9 05 0EHE 1K9 056 0EHE 1K9 02 0EHE 1K9 02 0EHE 1K9FK 05 0EHE 1K9 05A 0EHE 1K9 05A 0EHE 1K9F 09 0EHE 1K9 092 0EHE 1K9F 09A 0EHE 1K9F 09A 0EHE 1K9FK 09A 0EHE 1K9 02- 0EHE 1K9 027 0EHE 1K9 04 0EHE 1K9 0 0EHE 1K9 01 0EHE 1K9 0. 0EHE 1K9F 0B9 0EHE 1K9F 1A 0EHE 1K9FK 1C 0EHE 1K9 1- 0EHE 1K9 1- 0EHE 1K9 1- 0EHE 1K9 1- 0EHE 1K9 1-0 0EHE 1K9 11.A 0EHE 1K9 12 0EHE 1K9 1294 0EHE 1K9 14 0EHE 1K9 14 0EHE 1K9 15A 0EHE 1K9 168A 0EHE 1K9 18A 0EHE 1K9F 18A 0EHE 1K9 18A 0EHE 1K9F 18A 0EHE 1K9 18A 0EHE 1K9F 18A 0EHE 1K9 18A 0EHE 1K9 18A 0EHE 1K9 18A 0EHE 1K9 18A 0EHE 1K9 18A 0EHE 1K9F 18A 0EHE 1K9 18A 0EHE 1K9M 19 0EHE 1K9F 26 0EHE 1K9F 2- 0EHE 1K9 2- 0EHE 1K9F 2- 0EHE 1K9 2-1 0EHE 1K9 20 0EHE 1K9 20 0EHE 1K9 20 0EHE 1K9 24C 0EHE 1K9 24C 0EHE 1K9 25 0EHE 1K9F 25 0EHE 1K9 25 0EHE 1K9F 25 0EHE 1K9 25 0EHE 1K9 255 0EHE 1K9 25 0EHE 1K9 25 0EHE 1K9 25 0EHE 1K9F 25 0EHE 1K9 25 0EHE 1K9F 25 0EHE 1K9 25 0EHE 1K9F 25 0EHE 1K9 25 0EHE 1K9 25 0EHE 1K9 25 0EHE 1K9 271 0EHE 1K9 271 0EHE 1K9 29. 0EHE 1K9 2A 0EHE 1K9 25 0EHE 1K9 20 0EHE 1K9 20 0EHE 1K9 20 0EHE 1K9 20 0EHE 1K9 20 0EHE 1K9 20 0EHE 1K9 20 0EHE 1K9 20. 0EHE 1K9 206 0EHE 1K9 20 0EHE 1K9 20 0EHE 1K9F 20 0EHE 1K9D 30 0EHE 1K9 30 0EHE 1K9 34 0EHE 1K9F 37 0EHE 1K9F 37 0EHE 1K9 473 0EHE 1K9 4-6 0EHE 1K9F 4-6 0EHE 1K9 4- 0EHE 1K9 42 0EHE 1K9 4250 0EHE 1K9 45 0EHE 1K9 45 0EHE 1K9D 45 0EHE 1K9 4515 0EHE 1K9 497 0EHE 1K9 497 0EHE 1K9 56 0EHE 1K9F 569 0EHE 1K9 569 0EHE 1K9 569 0EHE 1K9 568 0EHE 1K9 5-1 0EHE 1K9 5-1 0EHE 1K9 5. 0EHE 1K9 51A 0EHE 1K9F 52 0EHE 1K9 52 0EHE 1K9 520 0EHE 1K9 520 0EHE 1K9 5264 0EHE 1K9 527 0EHE 1K9 568 0EHE 1K9 58A 0EHE 1K9 569 0EHE 1K9 59 0EHE 1K9 5 0EHE 1K9 55 0EHE 1K9FK 5B5 0EHE 1K9 5B 0EHE 1K9F 65 0EHE 1K9 665 0EHE 1K9 694 0EHE 1K9 694 0EHE 1K9 694 0EHE 1K9 694 0EHE 1K9 694 0EHE 1K9 694 0EHE 1K9 694 0EHE 1K9 694 0EHE 1K9 694 0EHE 1K9 694 0EHE 1K9 694 0EHE 1K9 65 0EHE 1K9 675 0EHE 1K9 69 0EHE 1K9 65 0EHE 1K9 66 0EHE 1K9 66 0EHE 1K9 66 0EHE 1K9 66 0EHE 1K9 6- 0EHE 1K9 6-1 0EHE 1K9 6.2 0EHE 1K9 6.2 0EHE 1K9 60 0EHE 1K9 62 0EHE 1K9 62A5 0EHE 1K9F 669 0EHE 1K9 67 0EHE 1K9 695 0EHE 1K9 698 0EHE 1K9 695 0EHE 1K9 6. 0EHE 1K9 62 0EHE 1K9 6 0EHE 1K9 6A 0EHE 1K9F 6 0EHE 1K9 6B 0EHE 1K9 6B1 0EHE 1K9 6B 0EHE 1K9 77 0EHE 1K9 76 0EHE 1K9 7-7 0EHE 1K9 78 0EHE 1K9 7-. 0EHE 1K9 7-0 0EHE 1K9 7- 0EHE 1K9 7- 0EHE 1K9 7- 0EHE 1K9 7.4 0EHE 1K9 7 0EHE 1K9 72 0EHE 1K9 71.3 0EHE 1K9 7151 0EHE 1K9 74A 0EHE 1K9 754 0EHE 1K9 78 0EHE 1K9 78 0EHE 1K9 781 0EHE 1K9 781 0EHE 1K9 781 0EHE 1K9 78A 0EHE 1K9 78A 0EHE 1K9 78A 0EHE 1K9 78A 0EHE 1K9 796 0EHE 1K9 78 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 79 0EHE 1K9 7 0EHE 1K9 729 0EHE 1K9 76 0EHE 1K9 76 0EHE 1K9 80-1 0EHE 1K9F 896 0EHE 1K9 8 0EHE 1K9F 86 0EHE 1K9 86 0EHE 1K9 9A 0EHE 1K9 9A 0EHE 1K9 9A 0EHE 1K9F 99 0EHE 1K9 9 0EHE 1K9 97 0EHE 1K9 9-0 0EHE 1K9 9-1 0EHE 1K9F 9-1 0EHE 1K9 9-4 0EHE 1K9 9-4 0EHE 1K9 9-4 0EHE 1K9 9-4 0EHE 1K9 9-526 0EHE 1K9 9-9 0EHE 1K9 9.6 0EHE 1K9 9.52 0EHE 1K9F 9 0EHE 1K9 945 0EHE 1K9 946 0EHE 1K9 949 0EHE 1K9 904 0EHE 1K9 905 0EHE 1K9 918A 0EHE 1K9 9240 0EHE 1K9 924 0EHE 1K9 925 0EHE 1K9 926 0EHE 1K9 926 0EHE 1K9F 92A 0EHE 1K9 95 0EHE 1K9 95.410 0EHE 1K9 969 0EHE 1K9FK 9749 0EHE 1K9 975299 0EHE 1K9F 976 0EHE 1K9 985- 0EHE 1K9F 985- 0EHE 1K9 985- 0EHE 1K9F 985- 0EHE 1K9 9855 0EHE 1K9 990 0EHE 1K9 990 0EHE 1K9 999 0EHE 1K9DF 9-6 0EHE 1K9 9-A 0EHE 1K9F 9-A 0EHE 1K9 9-A 0EHE 1K9 9-A 0EHE 1K9 9.52- 0EHE 1K9 94C 0EHE 1K9 94- 0EHE 1K9F 979 0EHE 1K9 99 0EHE 1K9 99 0EHE 1K9 91 0EHE 1K9 997 0EHE 1K9 997 0EHE 1K9 99 0EHE 1K9 95 0EHE 1K9F 0EHE 1K9 0EHE 1K9 0EHE 1K9 - 0EHE 1K9 - 0EHE 1K9 - 0EHE 1K9 - 0EHE 1K9 - 0EHE 1K9 - 0EHE 1K9 -5 0EHE 1K9 9 0EHE 1K9 0EHE 1K9 8A 0EHE 1K9 93 0EHE 1K9 96 0EHE 1K9 07 0EHE 1K9 0 0EHE 1K9DF 18 0EHE 1K9 526 0EHE 1K9 84 0EHE 1K9 8 0EHE 1K9 8 0EHE 1K9 9 0EHE 1K9 9 0EHE 1K9 9 0EHE 1K9F 9 0EHE 1K9 9. 0EHE 1K9 7A 0EHE 1K9D 7A 0EHE 1K9F 9 0EHE 1K9 78 0EHE 1K9 0EHE 1K9 . 0EHE 1K9 -1 0EHE 1K9 -9 0EHE 1K9 .5. 0EHE 1K9 .59 0EHE 1K9 .- 0EHE 1K9 A7 0EHE 1K9 029 0EHE 1K9F 1 0EHE 1K9 1- 0EHE 1K9 16 0EHE 1K9 18A 0EHE 1K9 29 0EHE 1K9 2 0EHE 1K9 2A 0EHE 1K9 42 0EHE 1K9 49 0EHE 1K9 56 0EHE 1K9 5 0EHE 1K9 5 0EHE 1K9 5 0EHE 1K9 5 0EHE 1K9 5 0EHE 1K9 5 0EHE 1K9 5 0EHE 1K9 5 0EHE 1K9 6- 0EHE 1K9 6- 0EHE 1K9 6- 0EHE 1K9 6- 0EHE 1K9 6- 0EHE 1K9F 6- 0EHE 1K9 6- 0EHE 1K9 6- 0EHE 1K9 6 0EHE 1K9 6 0EHE 1K9 6 0EHE 1K9F 8 0EHE 1K9F 8 0EHE 1K9 8 0EHE 1K9 8 0EHE 1K9 8- 0EHE 1K9 8- 0EHE 1K9 8- 0EHE 1K9F 8A 0EHE 1K9 8A 0EHE 1K9 9 0EHE 1K9 92 0EHE 1K9 97 0EHE 1K9 99 0EHE 1K9 9B 0EHE 1K9 9 0EHE 1K9 9 0EHE 1K9 0EHE 1K9 - 0EHE 1K9F 0EHE 1K9 A7 0EHE 1K9 9 0EHE 1K9 05 0EHE 1K9 0EHE 1K9 0EHE 1K9 0EHE 1K9 0EHE 1K9 0EHE 1K9 0EHE 1K9 0EHE 1K9 0EHE 1K9 26 0EHE 1K9 4 0EHE 1K9 5 0EHE 1K9 68 0EHE 1K9 - 0EHE 1K9 0EHE 1K9 0EHE 1K9 0EHE 1K9 5 0EHE 1K9 A 0EHE 1K9 0EHE 1K9 5 0EHE 1K9 .4 0EHE 1K9 . 0EHE 1K9 . 0EHE 1K9D 0EHE 1K9 0 0EHE 1K9 0 0EHE 1K9 0 0EHE 1K9 0 0EHE 1K9 02 0EHE 1K9 0 0EHE 1K9 0 0EHE 1K9 1 0EHE 1K9 1 0EHE 1K9 1 0EHE 1K9 198 0EHE 1K9 1B 0EHE 1K9 2. 0EHE 1K9 269 0EHE 1K9 269 0EHE 1K9 269 0EHE 1K9 57 0EHE 1K9F 5 0EHE 1K9F 5 0EHE 1K9F 5A 0EHE 1K9 6.6 0EHE 1K9 68- 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9F 8 0EHE 1K9 99 0EHE 1K9F 92 0EHE 1K9F 96 0EHE 1K9 9 0EHE 1K9 26 0EHE 1K9 - 0EHE 1K9D 98 0EHE 1K9 0EHE 1K9F 0EHE 1K9 2 0EHE 1K9 A7 0EHE 1K9 A7- 0EHE 1K9 A7- 0EHE 1K9 . 0EHE 1K9 7 0EHE 1K9 0EHE 1K9 6 0EHE 1K9 .0 0EHE 1K9 26 0EHE 1K9 269 0EHE 1K9 29 0EHE 1K9F 77 0EHE 1K9 77 0EHE 1K9FK 9 0EHE 1K9 4 0EHE 1K9F 7 0EHE 1K9 0EHE 1K9 9 0EHE 1K9 - 0EHE 1K9 15 0EHE 1K9F 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 7 0EHE 1K9 A 0EHE 1K9 A 0EHE 1K9 A 0EHE 1K9 A 0EHE 1K9 A 0EHE 1K9 B. 0EHE 1K9F C1 0EHE 1K9 C96 0EHE 1K9 C6B7- 0EHE 1K9 C 0EHE

Supplemental Table 1. Gene lists included in the multiplex RT-PCR analysis.

ACTB, B2M, GUSB, HMBS, HPRT1, RNA18S5, RPLP0 and TBP were used as the multiple control genes.

Supplemental Table 2. Values of gene expressions normalized by the multiple control genes.

11 && ,8 HMKLGJ, 2GIG8,94, ,A7 HMKLGJ, ,JJFN ,,87 HMKLGJ, ,JJFN ,2 HMKLGJ, ,JJFN ,83 HMKLGJ, ,JJFN ,- HMKLGJ, ,JJFN -91 HMKLGJ, 2GIG8,94, -8 HMKLGJ, 2GIG8,94, ,7 HMKLGJ, ,JJFN ,A HMKLGJ, ,JJFN + HMKLGJ, ,JJFN 3 HMKLGJ, ,JJFN 3 HMKLGJ, ,JJFN 1 HMKLGJ, ,JJFN 3 HMKLGJ, ,JJFN 77 HMKLGJ, ,JJFN 8 HMKLGJ, ,JJFN 09 HMKLGJ, ,JJFN 0, HMKLGJ, ,JJFN 02 HMKLGJ, ,JJFN 1 HMKLGJ, 2GIG8,94, 137 HMKLGJ, ,JJFN 17 HMKLGJ, ,JJFN 19 HMKLGJ, ,JJFN 1C, HMKLGJ, ,JJFN 2,, HMKLGJ, ,JJFN 2C HMKLGJ, ,JJFN 2C HMKLGJ, ,JJFN 341, HMKLGJ, ,JJFN 4 HMKLGJ, 2GIG8,94, 421 HMKLGJ, ,JJFN 477 HMKLGJ, ,JJFN 47 HMKLGJ, ,JJFN && 42, HMKLGJ, ,JJFN 42, HMKLGJ, ,JJFN 42, HMKLGJ, ,JJFN 42,A HMKLGJ, 2GIG8,94, 42- HMKLGJ, ,JJFN 6 HMKLGJ, ,JJFN 6737 HMKLGJ, ,JJFN 7,8- HMKLGJ, 2GIG8,94, 73C HMKLGJ, ,JJFN 741 HMKLGJ, ,JJFN 7C HMKLGJ, ,JJFN 804 HMKLGJ, ,JJFN 8C HMKLGJ, ,JJFN 914- HMKLGJ, ,JJFN 9,8 HMKLGJ, 2GIG8,94, 91 HMKLGJ, ,JJFN 96 HMKLGJ, 2GIG8,94, 8 HMKLGJ, ,JJFN 21, HMKLGJ, ,JJFN 748 HMKLGJ, ,JJFN 4C HMKLGJ, ,JJFN 3 HMKLGJ, ,JJFN 29 HMKLGJ, ,JJFN 3- HMKLGJ, ,JJFN HMKLGJ, ,JJFN 07 HMKLGJ, ,JJFN 7, HMKLGJ, ,JJFN 8, HMKLGJ, 2GIG8,94, HMKLGJ, ,JJFN C HMKLGJ, ,JJFN , HMKLGJ, ,JJFN 1 HMKLGJ, 2GIG8,94, 21- HMKLGJ, ,JJFN && 21- HMKLGJ, ,JJFN 21-4 HMKLGJ, ,JJFN 21- HMKLGJ, ,JJFN 21- HMKLGJ, 2GIG8,94, 3- HMKLGJ, ,JJFN 3D HMKLGJ, ,JJFN 48 HMKLGJ, ,JJFN 7 HMKLGJ, ,JJFN 9 HMKLGJ, ,JJFN B4 HMKLGJ, ,JJFN A,9 HMKLGJ, ,JJFN A021 HMKLGJ, 2GIG8,94, A HMKLGJ, ,JJFN A9 HMKLGJ, ,JJFN B9 HMKLGJ, ,JJFN ,9, HMKLGJ- ,JJFN ,37 HMKLGJ- ,JJFN ,C7 HMKLGJ- ,JJFN ,1 HMKLGJ- ,JJFN -4 HMKLGJ- ,JJFN -4 HMKLGJ- ,JJFN HMKLGJ- ,JJFN 6 HMKLGJ- ,JJFN 0 HMKLGJ- ,JJFN 96, HMKLGJ- ,JJFN 96, HMKLGJ- ,JJFN 1 HMKLGJ- ,JJFN 099- HMKLGJ- ,JJFN 7, HMKLGJ- 2GIG8,94, 7 HMKLGJ- ,JJFN 09 HMKLGJ- ,JJFN 2,3 HMKLGJ- ,JJFN 27, HMKLGJ- ,JJFN && 27C HMKLGJ- ,JJFN 27C HMKLGJ- ,JJFN 2 HMKLGJ- ,JJFN 38C HMKLGJ- ,JJFN 7,8 HMKLGJ- ,JJFN 73, HMKLGJ- 2GIG8,94, 73- HMKLGJ- ,JJFN 8,6 HMKLGJ- ,JJFN 8,6 HMKLGJ- ,JJFN 8,6+ HMKLGJ- ,JJFN 8-97 HMKLGJ- ,JJFN 83 HMKLGJ- ,JJFN 8A7 HMKLGJ- ,JJFN 84 HMKLGJ- ,JJFN 9-9 HMKLGJ- ,JJFN 9 HMKLGJ- ,JJFN 914 HMKLGJ- ,JJFN HMKLGJ- 2GIG8,94, 9, HMKLGJ- ,JJFN 3, HMKLGJ- 2GIG8,94, 3, HMKLGJ- ,JJFN 3- HMKLGJ- 2GIG8,94, 6 HMKLGJ- ,JJFN HMKLGJ- ,JJFN 26 HMKLGJ- ,JJFN C HMKLGJ- 2GIG8,94, C HMKLGJ- ,JJFN C HMKLGJ- ,JJFN C HMKLGJ- ,JJFN HMKLGJ- ,JJFN HMKLGJ- ,JJFN ,- HMKLGJ- ,JJFN , HMKLGJ- ,JJFN && 8 HMKLGJ- 2GIG8,94, 8 HMKLGJ- 2GIG8,94, ,1 HMKLGJ- ,JJFN 3, HMKLGJ- ,JJFN 3- HMKLGJ- 2GIG8,94, 318 HMKLGJ- ,JJFN 81 HMKLGJ- ,JJFN C9 HMKLGJ- ,JJFN 7, HMKLGJ- ,JJFN 72 HMKLGJ- 2GIG8,94, ,- HMKLGJ- ,JJFN 21- HMKLGJ- ,JJFN , HMKLGJ- ,JJFN C9 HMKLGJ- 2GIG8,94, C9 HMKLGJ- ,JJFN - HMKLGJ- 2GIG8,94, -0- HMKLGJ- ,JJFN A48 HMKLGJ- ,JJFN A6 HMKLGJ- ,JJFN B- HMKLGJ- ,JJFN C HMKLGJ- ,JJFN ,-02 HMKLGJ ,JJFN -7 HMKLGJ ,JJFN -8 HMKLGJ ,JJFN -7 HMKLGJ ,JJFN HMKLGJ ,JJFN HMKLGJ ,JJFN HMKLGJ ,JJFN 1 HMKLGJ 2GIG8,94, 03 HMKLGJ ,JJFN 021 HMKLGJ 2GIG8,94, 0 HMKLGJ ,JJFN 2-, HMKLGJ ,JJFN && 24 HMKLGJ ,JJFN 3C HMKLGJ ,JJFN 3C HMKLGJ ,JJFN 421 HMKLGJ 2GIG8,94, 47 HMKLGJ ,JJFN 47, HMKLGJ 2GIG8,94, 47 HMKLGJ ,JJFN 5,6 HMKLGJ ,JJFN 59 HMKLGJ 2GIG8,94, 64 HMKLGJ 2GIG8,94, 7,8 HMKLGJ ,JJFN 741 HMKLGJ ,JJFN 7 HMKLGJ ,JJFN 71 HMKLGJ ,JJFN 80 HMKLGJ 2GIG8,94, 8 HMKLGJ 2GIG8,94, 462 HMKLGJ ,JJFN 46 HMKLGJ ,JJFN 8 HMKLGJ ,JJFN 6 HMKLGJ 2GIG8,94, 6- HMKLGJ 2GIG8,94, HMKLGJ ,JJFN -9 HMKLGJ ,JJFN 0772 HMKLGJ 2GIG8,94, 0, HMKLGJ ,JJFN 4, HMKLGJ ,JJFN 42 HMKLGJ 2GIG8,94, 7, HMKLGJ ,JJFN HMKLGJ ,JJFN HMKLGJ ,JJFN HMKLGJ ,JJFN HMKLGJ ,JJFN , HMKLGJ ,JJFN && , HMKLGJ ,JJFN A4 HMKLGJ ,JJFN ,7, HMKLGJ 2GIG8,94, ,7- HMKLGJ ,JJFN ,96 HMKLGJ ,JJFN , HMKLGJ ,JJFN ,-0 HMKLGJ ,JJFN ,- HMKLGJ ,JJFN -77 HMKLGJ ,JJFN - HMKLGJ ,JJFN ,9, HMKLGJ ,JJFN , HMKLGJ ,JJFN , HMKLGJ 2GIG8,94, ,1 HMKLGJ ,JJFN A7 HMKLGJ 2GIG8,94, 0 HMKLGJ 2GIG8,94, 0 HMKLGJ ,JJFN 18 HMKLGJ ,JJFN 1C HMKLGJ 2GIG8,94, 1E HMKLGJ ,JJFN 1E HMKLGJ ,JJFN 2,, HMKLGJ ,JJFN 3-E HMKLGJ ,JJFN 43 HMKLGJ ,JJFN 695 HMKLGJ ,JJFN 671 HMKLGJ ,JJFN 8,6 HMKLGJ 2GIG8,94, 8,6 HMKLGJ ,JJFN 8, HMKLGJ ,JJFN 6 HMKLGJ ,JJFN 1 HMKLGJ ,JJFN 974 HMKLGJ ,JJFN 9 HMKLGJ ,JJFN && ,1 HMKLGJ 2GIG8,94, 4 HMKLGJ ,JJFN 7, HMKLGJ ,JJFN HMKLGJ ,JJFN - HMKLGJ ,JJFN 6 HMKLGJ ,JJFN ,7 HMKLGJ ,JJFN 1 HMKLGJ ,JJFN 8 HMKLGJ ,JJFN 48 HMKLGJ ,JJFN --, HMKLGJ ,JJFN B9- HMKLGJ ,JJFN B9 HMKLGJ ,JJFN 7 HMKLGJ, 2GIG8,94, 7 HMKLGJ, 2GIG8,94, 7 HMKLGJ, 2GIG8,94, HMKLGJ, ,JJFN 0-, HMKLGJ, ,JJFN 076 HMKLGJ, ,JJFN 092 HMKLGJ, ,JJFN 0D, HMKLGJ, ,JJFN 1-CB HMKLGJ, ,JJFN 1 HMKLGJ, 2GIG8,94, 17 HMKLGJ, ,JJFN 2,, HMKLGJ, ,JJFN 37C HMKLGJ, ,JJFN 4 HMKLGJ, ,JJFN 42, HMKLGJ, ,JJFN 42- HMKLGJ, 2GIG8,94, 59- HMKLGJ, ,JJFN 804 HMKLGJ, ,JJFN 8 HMKLGJ, 2GIG8,94, 9 HMKLGJ, 2GIG8,94, && 21 HMKLGJ, 2GIG8,94, -8 HMKLGJ, ,JJFN 30- HMKLGJ, 2GIG8,94, HMKLGJ, 2GIG8,94, 21- HMKLGJ, 2GIG8,94, 3- HMKLGJ, ,JJFN 3- HMKLGJ, ,JJFN 91 HMKLGJ, 2GIG8,94, A021, HMKLGJ, ,JJFN -- HMKLGJ- 2GIG8,94, 6 HMKLGJ- 2GIG8,94, 69 HMKLGJ- ,JJFN C HMKLGJ- ,JJFN 109 HMKLGJ- 2GIG8,94, 20 HMKLGJ- ,JJFN 3,1C HMKLGJ- 2GIG8,94, 68, HMKLGJ- ,JJFN 742 HMKLGJ- ,JJFN 8 HMKLGJ- ,JJFN 90 HMKLGJ- ,JJFN 70632 HMKLGJ- ,JJFN 7 HMKLGJ- 2GIG8,94, 7 HMKLGJ- ,JJFN 7 HMKLGJ- 2GIG8,94, 7 HMKLGJ- 2GIG8,94, 4, HMKLGJ- 2GIG8,94, , HMKLGJ- 2GIG8,94, ,- HMKLGJ- ,JJFN , HMKLGJ- 2GIG8,94, , HMKLGJ- ,JJFN HMKLGJ- ,JJFN 88 HMKLGJ- 2GIG8,94, HMKLGJ- 2GIG8,94, && - HMKLGJ- 2GIG8,94, C HMKLGJ- ,JJFN , HMKLGJ 2GIG8,94, ,1 HMKLGJ 2GIG8,94, -7 HMKLGJ ,JJFN -46 HMKLGJ 2GIG8,94, , HMKLGJ ,JJFN 0410 HMKLGJ 2GIG8,94, 2,-, HMKLGJ ,JJFN 2,-- HMKLGJ 2GIG8,94, 3C HMKLGJ ,JJFN 701 HMKLGJ ,JJFN 8,6 HMKLGJ ,JJFN 8D- HMKLGJ ,JJFN 074 HMKLGJ ,JJFN ,2 HMKLGJ 2GIG8,94, HMKLGJ 2GIG8,94, 48 HMKLGJ 2GIG8,94, 49, HMKLGJ 2GIG8,94, 64 HMKLGJ ,JJFN 8, HMKLGJ 2GIG8,94, 1 HMKLGJ 2GIG8,94, B9, HMKLGJ 2GIG8,94, HMKLGJ 2GIG8,94, C HMKLGJ 2GIG8,94, 0410- HMKLGJ 2GIG8,94, 0410 HMKLGJ 2GIG8,94, 0410- HMKLGJ ,JJFN 16- HMKLGJ 2GIG8,94, 167 HMKLGJ 2GIG8,94, 168 HMKLGJ ,JJFN 16 HMKLGJ ,JJFN HMKLGJ ,JJFN && 074 HMKLGJ ,JJFN ,- HMKLGJ ,JJFN 93 HMKLGJ 2GIG8,94, 0-1 HMKLGJ 2GIG8,94, -C HMKLGJ ,JJFN 4 HMKLGJ ,JJFN A021- HMKLGJ 2GIG8,94, ,92 HMKLGJ0 ,JJFN 92 HMKLGJ0 2GIG8,94, 0-7 HMKLGJ0 ,JJFN 07 HMKLGJ0 2GIG8,94, 1E HMKLGJ0 ,JJFN 31, HMKLGJ0 2GIG8,94, 48 HMKLGJ0 ,JJFN HMKLGJ0 ,JJFN 887 HMKLGJ0 2GIG8,94,

Supplemental Table 3. Gene sets specifically relevant to P-erymk41(+) and P-erymk41(-) cells.

“Category & Cluster” shows the group to which genes were categorized by PCA and the following ReactomeFI analysis. “Origin” column indicates 261 genes included in the original gene lists (Supplemental Table 1) and 94 genes complemented by the GeneMANIA network algorithm.

12 && ,5 DIGHCF FFBJ 96 DIGHCF FFBJ 4, DIGHCF FFBJ , DIGHCF CEC561 9 DIGHCF CEC561 ,, DIGHCF CEC561 ,66, DIGHCF CEC561 ,09 DIGHCF CEC561 04 DIGHCF CEC561 01 DIGHCF CEC561 03 DIGHCF CEC561 15 DIGHCF FFBJ 1, DIGHCF CEC561 1, DIGHCF FFBJ 1 DIGHCF CEC561 1 DIGHCF FFBJ 1 DIGHCF FFBJ 1 DIGHCF FFBJ 14 DIGHCF FFBJ 1 DIGHCF FFBJ 1 DIGHCF FFBJ 1 DIGHCF CEC561 670 DIGHCF CEC561 5 DIGHCF FFBJ 9 DIGHCF FFBJ DIGHCF CEC561 1 DIGHCF CEC561 6 DIGHCF CEC561 DIGHCF CEC561 DIGHCF FFBJ 4 DIGHCF FFBJ DIGHCF CEC561 9 DIGHCF CEC561 7 DIGHCF FFBJ DIGHCF CEC561 41 DIGHCF FFBJ 0, DIGHCF CEC561 0, DIGHCF FFBJ 53 DIGHCF CEC561 51 DIGHCF FFBJ 92 DIGHCF CEC561 96 DIGHCF CEC561 96 DIGHCF FFBJ 96 DIGHCF CEC561 5, DIGHCF FFBJ 76 DIGHCF CEC561 4 DIGHCF CEC561 ,9, DIGHCF FFBJ ,, DIGHCF CEC561 0 DIGHCF FFBJ 4, DIGHCF FFBJ 5 DIGHCF CEC561 7 DIGHCF FFBJ 79 DIGHCF CEC561 DIGHCF FFBJ 09 DIGHCF CEC561 34 DIGHCF CEC561 ,0 DIGHCF CEC561 ,3 DIGHCF FFBJ DIGHCF FFBJ DIGHCF CEC561 DIGHCF CEC561 DIGHCF FFBJ 66 DIGHCF CEC561 A DIGHCF FFBJ 6 DIGHCF, CEC561 9 DIGHCF, CEC561 91 DIGHCF, CEC561 DIGHCF, CEC561 3 DIGHCF, CEC561 63 DIGHCF, CEC561 63 DIGHCF, FFBJ 63 DIGHCF, FFBJ ,, DIGHCF, FFBJ 749 DIGHCF, FFBJ 749 DIGHCF, FFBJ 749 DIGHCF, CEC561 7492 DIGHCF, FFBJ 93 DIGHCF, CEC561 916 DIGHCF, FFBJ DIGHCF, CEC561 6 DIGHCF, FFBJ 4 5DIGHCF CEC561 99 5DIGHCF FFBJ 6 5DIGHCF CEC561 53 5DIGHCF CEC561 , 5DIGHCF CEC561 , 5DIGHCF CEC561 0, 5DIGHCF FFBJ 5DIGHCF FFBJ 9 5DIGHCF FFBJ 0 5DIGHCF FFBJ ,65 5DIGHCF FFBJ ,700 5DIGHCF CEC561 ,73 5DIGHCF CEC561 9 5DIGHCF FFBJ 9 5DIGHCF FFBJ 5DIGHCF CEC561 07 5DIGHCF FFBJ 07 5DIGHCF FFBJ 07 5DIGHCF FFBJ 1 5DIGHCF FFBJ 53 5DIGHCF FFBJ 54 5DIGHCF FFBJ ,3 5DIGHCF FFBJ 15 5DIGHCF CEC561 5 5DIGHCF CEC561 45 5DIGHCF FFBJ 5DIGHCF CEC561 5DIGHCF FFBJ 0 5DIGHCF FFBJ 07 5DIGHCF CEC561 915 5DIGHCF FFBJ 5DIGHCF FFBJ 4 5DIGHCF FFBJ 9, 5DIGHCF CEC561 ,39 5DIGHCF FFBJ ,39 5DIGHCF CEC561 ,36 5DIGHCF CEC561 63 5DIGHCF FFBJ 7 5DIGHCF FFBJ 1, 5DIGHCF FFBJ 26 5DIGHCF FFBJ 3,5 5DIGHCF CEC561 54 5DIGHCF CEC561 5 5DIGHCF CEC561 5 5DIGHCF CEC561 5 5DIGHCF FFBJ 5 5DIGHCF FFBJ 6,9 5DIGHCF CEC561 6 5DIGHCF FFBJ 5DIGHCF FFBJ 1 5DIGHCF FFBJ 16 5DIGHCF CEC561 15 5DIGHCF CEC561 16 5DIGHCF FFBJ 5, 5DIGHCF CEC561 5DIGHCF FFBJ 5DIGHCF CEC561 9 5DIGHCF CEC561 0 5DIGHCF CEC561 04 5DIGHCF FFBJ 6 5DIGHCF CEC561 6 5DIGHCF CEC561 6 5DIGHCF CEC561 60 5DIGHCF FFBJ , 5DIGHCF FFBJ , 5DIGHCF CEC561 ,3 5DIGHCF FFBJ ,3 5DIGHCF CEC561 ,3 5DIGHCF CEC561 ,36 5DIGHCF FFBJ 4 5DIGHCF CEC561 ,4 5DIGHCF FFBJ 1 5DIGHCF CEC561 9 5DIGHCF FFBJ 4,0 5DIGHCF CEC561 4,0 5DIGHCF FFBJ 56 5DIGHCF FFBJ 60 5DIGHCF CEC561 65 5DIGHCF CEC561 3 5DIGHCF CEC561 3 5DIGHCF FFBJ ,3 5DIGHCF, CEC561 ,, 5DIGHCF, CEC561 ,0 5DIGHCF, FFBJ 0 5DIGHCF, CEC561 53 5DIGHCF, CEC561 5, 5DIGHCF, CEC561 65 5DIGHCF, CEC561 35 5DIGHCF, CEC561 74, 5DIGHCF, CEC561 74, 5DIGHCF, FFBJ 9, 5DIGHCF, FFBJ 9, 5DIGHCF, CEC561 9 5DIGHCF, FFBJ 9 5DIGHCF, FFBJ 9 5DIGHCF, FFBJ 5DIGHCF, FFBJ 5DIGHCF, FFBJ 9 5DIGHCF, FFBJ 5DIGHCF FFBJ ,6 5DIGHCF FFBJ ,9 5DIGHCF FFBJ 63 5DIGHCF FFBJ

Supplemental Table 4. Gene sets specifically relevant to P-erymk41(-) from WT- and G1s-clones.

“Category & Cluster” shows the group to which genes were categorized by PCA and the following ReactomeFI analysis. “Origin” column indicates 103 genes included in the original gene lists (Supplemental Table 1) and 91 genes complemented by the GeneMANIA network algorithm.

Acknowledgements

We are grateful to Ms. Setsuko Inoue and Mr. Hirotsuna Watanabe for their technical assistance. We would also like to thank Dr. Mitsujiro Osawa, Dr. Ritsuko Shimizu, and Dr. Tutomu Toki for providing helpful scientific comments, Ms. Harumi Watanabe for providing administrative assistance, and Dr. Peter Karagiannis for proofreading the paper. This work was supported by grants from the Ministry of Health, Labour and

Welfare of Japan, Ministry of Education, Culture, Sports, Science and Technology of Japan, Japan Society for the Promotion of Science (JSPS) Research Fellowships for Young Scientists [Y.N.A], KAKENHI Grant

Number 19K17358 [Y.N.A.], KAKENHI Grant Number 25221308 [M.O.] and KAKENHI Grant Number

16K10026 [A.N.], Regional Innovation Support Program from the Ministry of Education, Culture, Sports,

Science and Technology of Japan [M.O. and Y.K.], the Leading Project of the Ministry of Education, Culture,

Sports, Science and Technology [T.N.], the Funding Program for World-Leading Innovative Research and

Development on Science and Technology of the Japan Society for the Promotion of Science [T.N. and M.K.S.],

CREST [T.N.], the Core Center for iPS Cell Research of Research Center Network for Realization of

Regenerative Medicine from the Japan Agency for Medical Research and Development (AMED) [T.N. and

M.K.S.], the Program for Intractable Diseases Research utilizing Disease-specific iPS cells of AMED

(17935423) [Y.N.A., T.N. and M.K.S.], and Practical Research Project for Rare/Intractable Diseases of AMED

(17930095) [M.K.S.].