www.sciencetranslationalmedicine.org/cgi/content/full/6/264/264ra163/DC1

Supplementary Materials for

Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa

Vittorio Sebastiano, Hanson Hui Zhen, Bahareh Haddad, Elizaveta Bashkirova,

Sandra P. Melo, Pei Wang, Thomas L. Leung, Zurab Siprashvili, Andrea Tichy, Jiang Li, Mohammed Ameen, John Hawkins, Susie Lee, Lingjie Li, Aaron Schwertschkow, Gerhard Bauer, Leszek Lisowski, Mark A. Kay, Seung K. Kim, Alfred T. Lane,

Marius Wernig,* Anthony E. Oro*

*Corresponding author. E-mail: wernig@stanford.edu (M.W.); oro@stanford.edu (A.E.O.)

Published 26 November 2014, Sci. Transl. Med. 6, 264ra163 (2014) DOI: 10.1126/scitranslmed.3009540

The PDF file includes:

Materials and Methods Fig. S1. Immunohistochemical characterization of patient-specific iPS clones. Fig. S2. GMP production of patient-specific iPS clones. Fig. S3. Genetic and karyotypic characterization of patient-specific iPS clones after loop-out. Fig. S4. CRISPR versus AAV-DJ targeting efficiency at the LAMA3 locus in LAMA3-deficient primary keratinocytes. Fig. S5. Optimization and validation of keratinocyte differentiation protocol. Fig. S6. Histology of corrected iPSC xenograft. Table S1. Persistent variant genes during corrected iPS cell generation. Table S2. List of genes, categorized by GO term, differentially expressed between iPS-KC and NHK. References (36–39)

Materials and Methods

GMP lentiviral vector manufacturing

Lentiviral vector manufacturing was carried out in the UC Davis GMP facility applying

Standard Operating Procedures (SOPs) and Quality Control (QC). Certificates of Analysis

(COAs) were obtained for all reagents, including fetal bovine serum (FBS) suitable for GMP

manufacturing. COAs were also generated after QC tests on the manufactured vector were

completed. Briefly, certified, GMP grade 293T cells (National Gene Vector Biorepository,

NGVB, Indiana University) were plated at a density of approximately 8 million cells per

225cm2 flask 24 hours prior to transient transfection with 3 certified plasmids carrying the

gag/pol genes, VSV-G envelope and the genes of interest (reprogramming genes, provided by

Stanford University). Plasmids were also manufactured in house and quality controlled for

concentration, identity and purity. 293T cell growth medium was formulated using DMEM

(Gibco - Life Technologies), 10% Fetal Bovine Serum (Hyclone) (DMEM/FBS).

Transfection mix consisted of TransIT transfection reagent (Mirus) and 3mL DMEM without

FBS. In a separate tube, 25μg vector reprogramming plasmid, 25μg of gag/pol plasmid, and

5μg of envelope plasmid were combined. After 20 minutes, plasmid DNA was added to the

transfection solution and incubated at room temperature for 30 minutes. The TransIT/DNA

mix was then added drop wise to the 293 T cell cultures and gently rocked for even

distribution. Twenty-five hours later the DNA containing medium was removed from the cells

and replaced with 30 mL of UltraCulture serum free media (Lonza) per flask and incubated

for 48 hours at 37°C. Supernatant was then collected, clarified from cell debris by

centrifugation, and benzonase treated for 2 hours at room temperature. The supernatant was

then added to Centricon plus-70 filtration units (Millipore) and centrifuged for 25-35 minutes;

the retentate, concentrated vector, was collected, filtered through a 0.45μm filter, aliquoted

into cryovials and placed into a -80°C freezer for long term storage. Several aliquots were

tested for transducing titer, replication competent lentivirus, sterility, endotoxin and

mycoplasma. Transducing titer was ~108/ml as tittered on 293T cells.

iPS derivation under GMP culture conditions

All steps were conducted in the UC Davis GMP facility applying SOPs and QC. Patient

fibroblasts were pre-cultured using DMEM/FBS. From healthy cultures, for transduction, 105

fibroblasts per well were plated into 6 well plates and allowed to adhere overnight. Lentiviral

vector aliquots were then thawed and kept at 4°C. Transduction solution (500μL) was

prepared for each well of a 6-well plate. The amount of vector was adjusted to multiplicity of

infection (MOI) of approximately 50 in DMEM/FBS. Clinical grade protamine sulfate (PPC

Fresenius) was then added to the transduction solution at a concentration of 0.02 mg/ml.

Supernatant from each culture well was removed and 0.5ml of transduction solution was

added to each well. The cell cultures were incubated at 37°C for 2 hours. Following the

incubation period, the plates were removed from the incubator and 1ml of DMEM/FBS was

added to each well. The plates were then returned to the incubator and left for 24 hours, after

which the cells were trypsinized using TrypLE (Life Technologies) and re-plated on a layer of

qualified irradiated human foreskin fibroblasts (GobalStem) in iPSC medium. iPSC medium

consisted of Knockout DMEM/F12, 20% Knockout Serum Replacement (KSR), 20ng/ml

basic fibroblast growth factor (bFGF), 2mM of L-glutamine, 1% Minimum Essential Medium

– Non Essential Amino Acids (MEM-NEAA), and 0.1mM of β-mercaptoethanol (all reagents

from Life Technologies).

iPSC colonies could be observed approximately one week after transduction. iPSC

colonies were inspected visually on a daily basis and atypical colonies, identified

morphologically, were removed. Colonies showing signs of differentiation were marked using

a marking objective on the microscope. To remove colonies, in a biosafety cabinet inside the

UC Davis GMP facility, a 1000μL pipette tip was used to scratch the center of each marked

area, then a pipette was used to rinse the surface of each well. After removing the undesirable

colonies and re-feeding the wells with growth medium, the plates were returned to the

incubator. After only morphologically correct colonies could be identified, such colonies

were expanded, stained for pluripotency markers and karyotyped.

Conventional gene targeting

iPSCs (1x107) were electroporated with 20 µg of Targeting Vector plasmid using a Bio-Rad

electroporator. Cells were treated with 10 μM ROCK inhibitor Y27632 (Sigma) 1 hour prior

to and 24 hours following electroporation. Cells were let to grow until small colonies began to

emerge and then selected in media containing 100 ng/ml G148 and gancyclovir for at least 15

consecutive days. Resistant colonies were manually picked and clonally expanded in

mTeSR1. Cell pellets from each clone was then analysed by PCR and subsequently by

Southern blotting to verify correct targeting of the COL7A1 locus.

AAV-mediated gene targeting

Plasmid and DNA analysis. Targeting vectors were constructed using classical

molecular cloning techniques. The arms for each targeting vector were PCR amplified from

genomic DNA and ligated to the respective selection cassettes. For the AAV-targeting vector,

the constructed DNA piece shown in Fig. 2 was inserted between inverted terminal repeat

(ITR) sequences cloned from AAV-2 isolate. The packaging plasmids used for viral

preparation have been described before (34). Plasmid and genomic DNA were isolated using

an Endofree maxiprep kit (Qiagen) and phenol-chloroform extraction, respectively.

AAV viral preparation. All AAV vectors used in the study were produced by

Ca3(PO4)2 triple transfection and purified by CsCl gradient centrifugation as previously

reported (34). Viral DNA was extracted by the sodium iodide method, using a DNA Extractor

kit (Wako Chemicals USA) and titrated by a quantitative dot blot assay.

AAV transduction of iPSCs. iPSCs were transduced by seeding 0.25×106 cells each to

multiple wells of a 6-well matrigel coated plate in NutriStem XF/FF Culture media. After cell

attachment, cells were transduced with AAV viral preparations at MOI of 2000. After two

days of transduction, cells were passaged using Accutase onto regular 10-cm matrigel coated

culture dishes. Following 10 days of selection with G418 (50 µg/ml), colonies were cloned

and expanded for downstream analysis.

Southern blotting

Genomic DNA was separated on a 0.8% agarose gel following restriction digest, transferred

to a nylon membrane (Amersham), and hybridized with 32P-labeled probes made by random

priming (Agilent Prime-It II kit). The restriction enzymes and probes for the different

southern blots are indicated in the figures.

Immunocytochemistry

Cells were fixed in 4% PFA for 10 minutes at room temperature and either incubated in

blocking solution (2% FBS in PBS) or permeabilized in 0.2% Triton X-100 followed by

incubation in blocking solution for detection of cell surface markers and transcription factors,

respectively. The primary antibodies used in the study are the following: anti-OCT4 (mouse,

Santa Cruz; 1:500); anti h-Nanog (Cosmo Bio); anti SSEA-3 (Developmental Studies

Hybridoma Bank); anti TRA-1-60 and anti (Millipore); anti CD31 (R&D Systems); anti-

desmin (Thermo Scientific). All primary antibodies were diluted 1:200 in blocking solution

and incubated overnight at room temperature. Secondary antibodies (Alexa-488 and Alexa

594, Invitrogen) were diluted 1:1000 and incubated for two hours at room temperature. For

differentiated cultures, cells or frozen sections were fixed in 4% paraformaldehyde (15min,

room temperature) before permeabilization and blocking in phosphate buffer solution (PBS)

supplemented with 0.1% Triton X-100, 0.05% Tween 20 and 10% horse serum. Primary

antibodies were incubated overnight at 4°C in blocking buffer. Antibodies comprised rabbit

anti-keratin 14 (1:2000, Covance), mouse anti-keratin 18 (1:800 Abcam), rabbit anti-keratin

10 (1:500, Covance), mouse anti-p63 (1:50, Santa Cruz Biotech), mouse anti-Oct4 (1:400,

Santa Cruz Biotech), mouse human specific N terminal anti-collagen VII LH7.2 (1:250

Millipore), mouse anti-collagen VII C-terminal LH24 (gift of Markinovich lab) and mouse

anti-laminin 332 (1:100 gift of Marinkovich lab). Primaries were visualized with appropriate

secondary antibody and co-stained with DAPI (4,6L-diamidino-2-phenylindole).

Western blotting

Culture media (D-KSFM) were collected from growing iPS-Ks (collagen VII mutation

corrected and non-corrected) and NHK and concentrated by using centrifugal filter unit

(Millipore). Protein concentration was determined and equal amount of protein samples were

run on 3-8% gradient gel (Life Technology). Rabbit anti-collagen VII antibody (1:500

Millipore) was used in immunoblotting.

Microarray

To determine the similarity between our iPS-Ks and normal keratinocytes, we performed a

microarray experiment using a Human-CT12 Expression Bead Chip (Illumina). We extracted

RNA in two biological replicates from the indicated samples. We normalized the data by

performing quantile normalization and log 2-transformation. We then performed LIMMA

analysis, adjusting the p-value to 0.01 and implementing a 2-fold cutoff, to obtain

significantly differentially expressed genes. We generated a heatmap using hierarchical

clustering and used DAVID for GO-term enrichment analysis.

FACS Analysis

Cells were trypsinized and fixed in BD Cytofix/Cytoperm(BD Bioscience, 20 min, room

temperature), permeabilized and blocked with 5% goat serum+5% HI AB serum (30 min,

4°C). Fluorochrome-labeled antibodies were added (30 min, room temperature) and cells

were analyzed on a FACScalibur system by use of CellQuest software (BD Biosciences).

10,000 events were analyzed for each experiment. Three independent experiments were done

for each cell type.

Genome Sequencing

Whole genome sequencing data was performed by Complete Genomics, Inc., including

sequencing, mapping and variants calling. We then uploaded all the SNVs (excluding indels)

variants to Ingenuity Variant Analysis (IVA) tool and removed those with low confidence

score (call quality >40, read depth >10). Variants were further filtered if they were common

variants (present in dbSNP, >1% frequency in 1000 genomes; Complete Genomics genomes

or the NHLBI ESP exomes) and did not result in a predicted change to the protein coding

sequence (variants resulting in frameshift or missense). For each set of variants, functional

profiling using Gene Ontology (GO) enrichment was performed by DAVID (36)

(http://david.abcc.ncifcrf.gov/).

For targeted resequencing, custom Nimblegen EZ capture resequencing beads were developed

to the top 13 SCC associated genes (Fig. 3D) and the manufacturer’s instructions were

followed to bind and precipitate selected DNAs and submit bar-coded paired end sequencing

(average 700X coverage). Sequencing reads were aligned to the human reference genome

sequence (hg19) using Burrows-Wheeler Aligner (37) (BWA) with default parameters.

Variants were called by Samtools mpileup (38). A minimum alternative allele frequency of

5% at a position with a read depth >100 was required to make calls. Identified variants were

annotated using ANNOVAR (39) to exclude variants reported in dbSNP and to identify

variants that had nonsynonymous consequences or affected splice sites.

SUPPLEMENTARY FIGURES

Figure S1. Immunohistochemical characterization of patient-specific iPS clones. Characterization of iPS cells derived from the keratinocytes of patient AO3 (clones K3-1 and K3-4) revealing their bona fide undifferentiated and pluripotent state. Expression of a set of markers (OCT4, NANOG, TRA-1-60, and SSEA3) was identified by immunofluorescence. Normal karyotype was confirmed by G-banding. Pluripotency was assessed by teratoma formation and differentiation into cells derivatives of ectoderm, mesoderm, and endoderm.

Figure S2. GMP production of patient-specific iPS clones. (A) GMP manufacturing of the lentiviral vector followed a well established standard operating procedure. All reagents were received with certificates of analysis (COAs) to demonstrate identity, sterility, and purity of the reagents, and their suitability for use in a GMP manufacturing procedure. All steps were controlled, reviewed and signed off by Quality Control (QC) and Quality Assurance (QA) personnel. Certificates of analysis were generated for all steps indicated. All manufacturing occurred in a GMP facility in a Class 10,000 clean room. Storage of the lentiviral vector was carried out in a validated, continuously monitored freezer inside the GMP facility. (B) Example of characterization of one GMP iPS lines obtained from RDEB patients. Cells were undifferentiated as revealed by the expression of SOX2 and showed a normal diploid karyotype as reveled by the G-banding on metaphase chromosome spreads.

Figure S3. Genetic and karyotypic characterization of patient-specific iPS clones after loop-out. (A) PCR detecting the presence or the absence of the STEMCCA cassette after transient expression od CRE recombinase in original iPS clones. STEMCCA cassette was detected in the original iPS clone F12 but not detected in parental somatic cells and looped-out subclones. (B) Southern blot analysis confirming successful looping-out of STEMCCA cassette in iPS clones after transient expression of CRE recombinase.

Figure S4. CRISPR versus AAV-DJ targeting efficiency at the LAMA3 locus in LAMA3-deficient primary keratinocytes. The LAMA3 locus was corrected using independent or combination of either an AAV-DJ viral preparation containing a 4-kb portion of the wild-type LAMA3 gene or transfected with Cas9, guide RNA and a donor vector equivalent to the one used for AAV-DJ. Because LAMA3 mutant keratinocytes are adhesion deficient, cells were selected based on their ability to attach to regular cell culture dishes. Data are average (standard mean) number of colonies obtained after 10 days of adhesion selection (n = 3 biological replicates). Error bars represent standard deviation.

Figure S5. Optimization and validation of keratinocyte differentiation protocol. (A) Human ESC line H9 ability to differentiate into keratinocytes when cultured on CF1 mouse embryonic fibroblast feeders, feeder-free culture conditions (mTeSR-1 maintenance media and Matrigel coating), or feeder-free followed by feeders (5 passages). n = 10 cultures. (B) Bright field image showing quality of differentiation when iPSCs (line K3-1) were formed into embryoid bodies in AggreWell 400 plates for three days prior to differentiation, in comparison to differentiation as a monolayer. Images were taken at day 59, and are representative of n = 4. (C) Immunofluorescence analysis of undifferentiated H9 and H9 after 7 days of differentiation with RA and BMP4 in a monolayer with K18 and K14. A keratinocyte lineage-committed cell is shown by white arrowhead. Arrow indicates a mature keratinocyte (K14+/K18-). Scale bar, 37.5 μM. (D) Line-to-line variability in the differentiation of patient-derived COL7A1-corrected iPSC lines towards keratinocytes. Quality assessment based on morphology and the ability of iPSC-derived keratinocytes to form stratified epithelium (several layers expressing keratin 10 and involucrin) in in vitro skin reconstitution assays (n ≥ 8).

Figure S6. Histology of corrected iPSC xenograft. H&E staining of a 2-week c-iPS-KC1 epithelial graft on NSG mice. Note stratum granulosum and corneum consistent with stratified epidermis. 10X magnification.

SUPPLEMENTARY TABLES

Table S1. Persistent variant genes during corrected iPS cell generation. Variants analysis from whole-genome sequencing revealed a set of genes present throughout donor, o-iPS and c-iPS cell lines in the three production sets. Listed are the gene identification information and the variant found.

Table S2. List of genes, categorized by GO term, differentially expressed between iPSC-

KC and NHK.

GO ID Term P-Value log10(P-

value) Genes

GO:0007049

Cell cycle

1.03×10-42 42.0 ADCY3, KIFC1, PRC1, KNTC1, TTK, PKMYT1, AURKA, AURKB, PTTG1, CDCA8, CDKN2B, OIP5, INCENP,

CDCA2, LOC727803, CCNA2, CDCA5, ASPM, CDCA3, PDPN, SGOL2, LIG1, RBL1, POLE, ESPL1, TACC3, DDIT3, NCAPD3, NCAPD2, UHRF1, MAD2L1, TIMELESS, SPAG5,

ZWINT, C14ORF106, DSCC1, CCDC99, BLM, NEK2, CHEK1, ANLN, CDC34, SPC24, SPC25, TUBB, NCAPG2,

MNS1, FBXO5, CLASP2, LFNG, HELLS, ERCC6L, CCPG1, MKI67, CKAP5, PCNT, SUV39H1, NDC80, TPD52L1, CDC20, RAD54L, BRCA1, CDKN1C, CDKN1A, PLK1,

POLD1, UBA3, CHAF1A, CHAF1B, KIF23, KIF22, E2F2, GTSE1, CDT1, CCNE2, FAM83D, KIF2C, MCM7, FANCI,

PSMD1, PSMD2, RANBP1, KLK10, C11ORF82, SIK1, CDC7, ARHGEF2, KIF11, CCNF, TPX2, NUSAP1, MCM2,

PBK, UBE2C, MCM3, CDK2, MCM6, ERN1, BUB1B, HAUS8, KPNA2, PPP1R15A, LIN9, FOXM1, POLA1, CEP55, CYP27B1, HSPA2, CENPA, NCAPG, HJURP,

BUB1, ZWILCH, THBS1, TRIP13, TXNIP, EXO1, MSH6, GMNN, NASP, DLGAP5, PSRC1, KIF18A, CENPF, CENPE, BIRC5, ILF3, RACGAP1, CDC25C, CDKN3, CENPJ, SMC2,

SC65, GSG2, CDC25A, AVPI1, SMC4, NAE1, CCNB1, CCNB2, DUSP1, PTP4A1, CKS2, KIF20B, CHTF18,

C13ORF34 GO:002240

3 Cell cycle phase

1.32×10-39 38.9 ADCY3, KIFC1, PRC1, KNTC1, PKMYT1, TTK, AURKA, PTTG1, AURKB, CDCA8, CDKN2B, OIP5, INCENP,

CDCA2, LOC727803, CDCA5, CCNA2, ASPM, CDCA3, SGOL2, POLE, ESPL1, TACC3, NCAPD3, NCAPD2, MAD2L1, TIMELESS, SPAG5, ZWINT, C14ORF106,

DSCC1, CCDC99, BLM, NEK2, ANLN, CHEK1, CDC34, SPC24, SPC25, TUBB, NCAPG2, FBXO5, MNS1, CLASP2, LFNG, HELLS, ERCC6L, MKI67, CKAP5, PCNT, CDC20, NDC80, TPD52L1, RAD54L, CDKN1C, CDKN1A, PLK1,

POLD1, KIF23, KIF22, GTSE1, FAM83D, KIF2C, RANBP1, CDC7, ARHGEF2, KIF11, CCNF, TPX2, NUSAP1, PBK,

UBE2C, CDK2, BUB1B, HAUS8, KPNA2, POLA1, CEP55, HSPA2, NCAPG, BUB1, ZWILCH, TRIP13, EXO1, MSH6, DLGAP5, KIF18A, CENPF, BIRC5, ILF3, CENPE, CDKN3, CDC25C, SMC2, CDC25A, SC65, SMC4, CCNB1, CCNB2,

KIF20B, CKS2, C13ORF34

GO:0000279

M phase 1.40×10-38 37.9 ADCY3, KIF23, KIFC1, KIF22, PRC1, KNTC1, PKMYT1, TTK, AURKA, AURKB, PTTG1, FAM83D, KIF2C, CDCA8,

OIP5, INCENP, CDCA2, LOC727803, RANBP1, CDCA5, CCNA2, ASPM, CDCA3, ARHGEF2, KIF11, SGOL2, CCNF, TPX2, NUSAP1, ESPL1, PBK, TACC3, UBE2C, NCAPD3, CDK2, NCAPD2, MAD2L1, TIMELESS, SPAG5, ZWINT, C14ORF106, BUB1B, HAUS8, KPNA2, DSCC1, CCDC99,

NEK2, ANLN, CHEK1, CEP55, SPC24, SPC25, TUBB, HSPA2, NCAPG, NCAPG2, BUB1, FBXO5, MNS1,

CLASP2, ZWILCH, LFNG, HELLS, ERCC6L, TRIP13, EXO1, MSH6, MKI67, CKAP5, DLGAP5, PCNT, KIF18A, CENPF, CDC20, CENPE, NDC80, ILF3, BIRC5, CDC25C, SMC2, RAD54L, SC65, CDC25A, SMC4, CCNB1, CCNB2,

PLK1, KIF20B, CKS2, C13ORF34 GO:002240

2 Cell cycle

process

1.06×10-35 35.0 ADCY3, KIFC1, PRC1, KNTC1, PKMYT1, TTK, AURKA, AURKB, PTTG1, CDCA8, CDKN2B, OIP5, INCENP,

CDCA2, LOC727803, CCNA2, CDCA5, ASPM, CDCA3, SGOL2, POLE, ESPL1, TACC3, NCAPD3, DDIT3, NCAPD2, MAD2L1, TIMELESS, SPAG5, ZWINT,

C14ORF106, DSCC1, CCDC99, BLM, NEK2, CHEK1, ANLN, CDC34, SPC24, SPC25, TUBB, NCAPG2, MNS1,

FBXO5, CLASP2, LFNG, HELLS, ERCC6L, MKI67, CKAP5, PCNT, NDC80, TPD52L1, CDC20, RAD54L,

BRCA1, CDKN1C, CDKN1A, PLK1, POLD1, KIF23, KIF22, GTSE1, FAM83D, KIF2C, PSMD1, PSMD2, RANBP1, C11ORF82, CDC7, ARHGEF2, KIF11, CCNF, TPX2,

NUSAP1, PBK, UBE2C, CDK2, ERN1, BUB1B, HAUS8, KPNA2, PPP1R15A, POLA1, CEP55, CYP27B1, HSPA2,

CENPA, NCAPG, BUB1, THBS1, ZWILCH, TRIP13, EXO1, MSH6, DLGAP5, KIF18A, CENPF, BIRC5, ILF3, CENPE, RACGAP1, CDC25C, CDKN3, SMC2, CENPJ, CDC25A,

SC65, SMC4, CCNB1, CCNB2, KIF20B, CKS2, C13ORF34 GO:000027

8 Mitotic

cell cycle 2.74×10-34 33.6 KIF23, KIFC1, KIF22, PRC1, KNTC1, PKMYT1, TTK,

AURKA, AURKB, PTTG1, GTSE1, FAM83D, KIF2C, CDCA8, CDKN2B, OIP5, INCENP, PSMD1, CDCA2,

PSMD2, CDCA5, CCNA2, ASPM, CDCA3, CDC7, ARHGEF2, KIF11, POLE, CCNF, TPX2, NUSAP1, ESPL1,

PBK, UBE2C, NCAPD3, CDK2, NCAPD2, MAD2L1, TIMELESS, SPAG5, ZWINT, C14ORF106, BUB1B, HAUS8,

KPNA2, DSCC1, CCDC99, BLM, NEK2, POLA1, ANLN, CHEK1, CDC34, CEP55, SPC24, SPC25, TUBB, CENPA, NCAPG, NCAPG2, BUB1, FBXO5, CLASP2, ZWILCH, HELLS, ERCC6L, CKAP5, DLGAP5, KIF18A, CENPF, CDC20, CENPE, TPD52L1, BIRC5, NDC80, CDKN3, CDC25C, SMC2, CDC25A, SMC4, CDKN1C, CCNB1,

CDKN1A, CCNB2, PLK1, POLD1, UBA3, KIF20B,

C13ORF34

GO:0007067

Mitosis 3.47×10-34 33.5 KIF23, KIF22, KIFC1, KNTC1, PKMYT1, AURKA, AURKB, PTTG1, FAM83D, KIF2C, CDCA8, OIP5, INCENP,

CDCA2, CDCA5, CCNA2, ASPM, CDCA3, ARHGEF2, KIF11, CCNF, TPX2, NUSAP1, ESPL1, PBK, UBE2C,

NCAPD3, CDK2, NCAPD2, MAD2L1, TIMELESS, SPAG5, ZWINT, C14ORF106, BUB1B, HAUS8, DSCC1, CCDC99,

NEK2, ANLN, CEP55, SPC24, SPC25, TUBB, NCAPG, NCAPG2, BUB1, FBXO5, CLASP2, ZWILCH, HELLS, ERCC6L, CKAP5, DLGAP5, KIF18A, CENPF, CDC20,

BIRC5, NDC80, CENPE, CDC25C, SMC2, CDC25A, SMC4, CCNB1, CCNB2, PLK1, KIF20B, C13ORF34

GO:0000280

Nuclear division

3.47×10-34 33.5 KIF23, KIF22, KIFC1, KNTC1, PKMYT1, AURKA, AURKB, PTTG1, FAM83D, KIF2C, CDCA8, OIP5, INCENP,

CDCA2, CDCA5, CCNA2, ASPM, CDCA3, ARHGEF2, KIF11, CCNF, TPX2, NUSAP1, ESPL1, PBK, UBE2C,

NCAPD3, CDK2, NCAPD2, MAD2L1, TIMELESS, SPAG5, ZWINT, C14ORF106, BUB1B, HAUS8, DSCC1, CCDC99,

NEK2, ANLN, CEP55, SPC24, SPC25, TUBB, NCAPG, NCAPG2, BUB1, FBXO5, CLASP2, ZWILCH, HELLS, ERCC6L, CKAP5, DLGAP5, KIF18A, CENPF, CDC20,

BIRC5, NDC80, CENPE, CDC25C, SMC2, CDC25A, SMC4, CCNB1, CCNB2, PLK1, KIF20B, C13ORF34

GO:0048285

Organelle fission

6.78×10-34 33.2 KIF23, KIF22, KIFC1, KNTC1, PKMYT1, AURKA, AURKB, PTTG1, FAM83D, KIF2C, CDCA8, OIP5, INCENP,

CDCA2, CDCA5, CCNA2, ASPM, CDCA3, ARHGEF2, KIF11, CCNF, TPX2, NUSAP1, ESPL1, PBK, UBE2C,

NCAPD3, CDK2, NCAPD2, MAD2L1, TIMELESS, SPAG5, ZWINT, C14ORF106, BUB1B, HAUS8, DSCC1, CCDC99,

NEK2, ANLN, CEP55, SPC24, SPC25, TUBB, NCAPG, NCAPG2, BUB1, FBXO5, CLASP2, ZWILCH, HELLS, ERCC6L, CKAP5, DLGAP5, KIF18A, CENPF, CDC20,

BIRC5, NDC80, CENPE, CDC25C, SMC2, CDC25A, SMC4, CCNB1, CCNB2, PLK1, BAX, KIF20B, C13ORF34

GO:0000087

M phase of mitotic cell cycle

1.22×10-33 32.9 KIF23, KIF22, KIFC1, KNTC1, PKMYT1, AURKA, AURKB, PTTG1, FAM83D, KIF2C, CDCA8, OIP5, INCENP,

CDCA2, CDCA5, CCNA2, ASPM, CDCA3, ARHGEF2, KIF11, CCNF, TPX2, NUSAP1, ESPL1, PBK, UBE2C,

NCAPD3, CDK2, NCAPD2, MAD2L1, TIMELESS, SPAG5, ZWINT, C14ORF106, BUB1B, HAUS8, DSCC1, CCDC99,

NEK2, ANLN, CEP55, SPC24, SPC25, TUBB, NCAPG, NCAPG2, BUB1, FBXO5, CLASP2, ZWILCH, HELLS, ERCC6L, CKAP5, DLGAP5, KIF18A, CENPF, CDC20,

BIRC5, NDC80, CENPE, CDC25C, SMC2, CDC25A, SMC4, CCNB1, CCNB2, PLK1, KIF20B, C13ORF34

GO:0051301

Cell division

9.25×10-26 25.0 KIF23, KIFC1, PRC1, KNTC1, AURKB, PTTG1, CCNE2, FAM83D, CDCA8, OIP5, INCENP, CDCA2, CDCA5, CCNA2, ASPM, CDCA3, CDC7, ARHGEF2, KIF11,

PIK3CB, SGOL2, LIG1, CCNF, NUSAP1, ESPL1, UBE2C, NCAPD3, MCM5, CDK2, NCAPD2, MAD2L1, TIMELESS,

SPAG5, ZWINT, C14ORF106, BUB1B, HAUS8, NEK2, ANLN, CEP55, ZBTB16, SPC24, SPC25, NCAPG, NCAPG2,

BUB1, FBXO5, CLASP2, ZWILCH, HELLS, ERCC6L, CKAP5, CENPF, CDC20, BIRC5, NDC80, CENPE,

RACGAP1, CDC25C, CENPJ, SMC2, CDC25A, SMC4, CCNB1, CCNB2, PLK1, KIF20B, CKS2, C13ORF34