immunology.sciencemag.org/cgi/content/full/3/30/eaau4292/DC1

Supplementary Materials for

Direct reprogramming of fibroblasts into antigen-presenting dendritic cells

Fábio F. Rosa, Cristiana F. Pires, Ilia Kurochkin, Alexandra G. Ferreira, Andreia M. Gomes, Luís G. Palma, Kritika Shaiv,

Laura Solanas, Cláudia Azenha, Dmitri Papatsenko, Oliver Schulz, Caetano Reis e Sousa, Carlos-Filipe Pereira*

*Corresponding author. Email: filipe.pereira@med.lu.se

Published 7 December 2018, Sci. Immunol. 3, eaau4292 (2018)

DOI: 10.1126/sciimmunol.aau4292

The PDF file includes:

Methods Fig. S1. Candidate transcription factors to instruct DC cell fate. Fig. S2. Clec9a-based reporter to identify transcription factors to induce DC fate. Fig. S3. Screening for transcription factors to activate Clec9a-tdT. Fig. S4. DC morphology and dendrites are established in mouse fibroblasts. Fig. S5. Spi1, Irf8, and Batf3 are enriched in cDC1 cells. Fig. S6. Global single-cell gene expression analysis during iDC reprogramming. Fig. S7. Stepwise transitions during iDC reprogramming. Fig. S8. iDC reprogramming does not induce alterations associated with tumorigenesis. Fig. S9. Analysis of iDC maturation states. Fig. S10. Induced DCs are functional APCs. Fig. S11. Polycistronic vector induces cDC1-like transcriptional program. Fig. S12. PIB induce human iDC reprogramming. Fig. S13. FACS gating strategies. Legends for tables S1 to S5 Legends for movies S1 and S2 References (53–55)

Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/3/30/eaau4292/DC1)

Table S1 (Microsoft Excel format). Analysis of candidate transcription factors. Table S2 (Microsoft Excel format). Single-cell mRNA-seq data analysis. Table S3 (Microsoft Excel format). Pseudotime single-cell mRNA-seq data analysis. Table S4 (Microsoft Excel format). Population mRNA-seq data analysis. Table S5 (Microsoft Excel format). Raw data.

Movie S1 (.avi format). Time lapse showing Clec9a-tdT+ cell emergence. Movie S2 (.mp4 format). Time lapse displaying dead cell incorporation.

Supplementary Methods

Scanning electron microscopy (SEM)

PU.1, IRF8, and BATF3 (PIB)-transduced Clec9a-tdTomato (Clec9a-tdT) mouse

embryonic fibroblasts (MEFs) and PIB polycistronic (PIBpoly)-transduced human embryonic

fibroblasts (HEFs) were sorted (tdT+ and CD45+ HLA-DR+, respectively) at day 7, plated in

gelatin-coated coverslips and analyzed at day 9 along with M2rtTA-transduced MEFs and HEFs.

Samples were washed in 0.1M Sorensen´s phosphate buffer and fixed with 0.1M Sorensen´s

phosphate buffer pH 7.4, 1.5% formaldehyde and 2% glutaraldehyde at room temperature for 30

min. After fixation, samples were washed in 0.1M Sorensen´s buffer. Samples were then

dehydrated in a graded series of ethanol (50%, 70%, 80%, 90% and twice in 100%), critical point

dried and mounted on 12.5 mm aluminum stubs. Samples were then sputtered with 10nm Au/Pd

(80/20) in a Quorum Q150T ES turbo pumped sputter coater and examined in a Jeol JSM-7800F

FEG-SEM.

Population mRNA-seq library preparation and sequencing

Total RNA was extracted with TRIzol (Ambion). cDNA was generated with the SMART-

Seq v4 Ultra low input RNA kit (Takara) and amplified with 8 cycles. cDNA was analyzed using

the Agilent High sensitivity DNA kit on an Agilent Bioanalyzer 2100 (Agilent). Library

preparation was performed using the Nextera XT DNA library preparation kit (Illumina)

according to the manufacturer’s protocol. Following tagmentation of cDNA, forward and reverse

indexes were added by 12 cycles of PCR. Normalization was performed by normalizing beads

and libraries were mixed in equal volumes and sequenced on an Illumina NextSeq 500 (75-bp

paired-end).

Population mRNA-seq data processing and analysis

Paired-end reads were mapped to mouse genome (Ensembl, release 93) using STAR v2.5.3a

(53) with default settings except sjdbOverhang 74 --quantMode GeneCounts. The resulting gene

counts were further processed with R package DESeq2 (54) and normalized using RLE method.

Additional variables, obtained by sva package (55), that capture unwanted variation in the data

were introduced into design matrix for normalization. DESeq2 package were used for

performing differential expression analysis based on Wald test.

DC-specific gene list was defined as differential expression behavior between cDC1 and

cDC2, cDC1 and pDC, cDC2 and pDC (adjusted P-value < 0.1 and log2foldChange > 0.5) and

excluding genes that were over expressed in MEF compared to cDC1, cDC2 and pDC

(log2foldChange > 0.5). In order to find the relationship between all samples we calculated

Pearson correlation and clustered the samples using method Ward.d2. The resulting clustering

was reordered according to first principal component and visualized using pheatmap package.

Also, principal component analysis was performed to explore relationships among day 5, 7, 8

and 9 iDC samples.

Gene List Enrichment Analysis

GO (biological processes, cellular components), KEGG pathways and WikiPathways’

enrichment analysis was performed using Enrichr (http://amp.pharm.mssm.edu/Enrichr/) and

DAVID (david.ncifcrf.gov/). MicroRNA target interaction analysis was performed using

miRTarBase 2017, Enrichr website (http://amp.pharm.mssm.edu/Enrichr/). Mouse loss-of-

function phenotypes analysis was performed using Network2canvas

(http://www.maayanlab.net/N2C/#.WmRvOjLc8yk). Outputs are listed in table S2, S3, and S4.

Gene set enrichment analysis (GSEA) between biological sample groups was performed against

C7: immunologic signatures (4872 gene sets), from MSigDB, and NetPath Databases. GSEA

was performed using census counts file using default parameters (p=1). GSEA reports are

included in table S2 and S3.

Fig. S1. Candidate transcription factors to instruct DC cell fate. (A) Gene ontology

biological processes (left) and mouse loss-of-function mutant phenotypes (right) enrichment

analysis was performed for the candidate 18 transcription factors using Enrichr and

Network2canvas, respectively. Lists show the most enriched terms (top 19) and left columns

show respective P values. Top enriched biological processes enriched are leucocyte

differentiation (P = 2.51E-12), leucocyte activation (P = 1.02E-11), DC differentiation (P =

9.58E-12) and DC activation (P = 6.31E-11), whilst mutant phenotypes include abnormal

adaptive immunity (P = 1.19E-04) and abnormal antigen presentation (P = 1.25E-03). (B) Heat

map showing gene expression of the 18 candidate factors across multiple mouse tissues

(GeneAtlas MOE430). The majority of the 18 factors are specifically enriched in DCs (black

box) but not in other tissues. (C) Heat maps showing increased gene expression of the 18 factors

in mouse DCs when compared with macrophages (Mϕ) derived from bone marrow cultures (left,

GSE62361). Heat maps displaying gene expression of the 18 transcription factors in common

dendritic cell precursors (CDP), pre-conventional DCs (pre-cDC1 and pre-cDC2) and

conventional DCs (cDC1 and cDC2) (right, GSE66565). Gene expression data were analyzed by

Cluster 3.0 and displayed by Treeview. Red indicates increased expression, whereas blue

indicates decreased expression over the mean.

Fig. S2. Clec9a-based reporter to identify transcription factors to induce DC fate. (A)

Expression profile of Clec9a in DCs and several hematopoietic cell lineages obtained from data

available in the Immunological Genome Project (www.immgen.org). (B) Gene expression of

Clec9a in monocyte DC progenitors (MDP) and DC-committed precursors (common dendritic

cell precursor, CDP; and pre-conventional DC, pre-cDC) (GSE60781). (C) Gene expression of

Clec9a in DC precursors (CDP, pre-DC1 and pre-DC2) and mature cells (cDC1 and cDC2)

(GSE60782). (D) Expression of tdTomato in 98.6% of cDC1 cells (MHC-II+ CD11c+ CD8α+)

and 76.7% of cDC2 cells (MHC-II+ CD11c+ CD11b+) isolated from spleens of double transgenic

Clec9a-tdTomato animals. CD8+ T cells (MHC-II- CD11c- CD8α+) and myeloid non-DC

population (CD11b+ CD11c-) were included as controls. (E) Expression of tdTomato on 88.4%

of plasmacytoid DCs (pDCs) (CD11clow Bst2+ Siglec-H+ B220+) isolated from spleens of double

transgenic Clec9a-tdTomato animals. The percentages of tdTomato+ cells within control B cell

(CD11c- B220+) populations are shown. (F) Gating strategy to remove residual CD45+ and

tdTomato+ cells from Clec9a-tdTomato reporter mouse embryonic fibroblast (MEF) cultures.

Double negative MEFs, approximately 97% of the population, were purified by FACS (left).

Purity of the sorted population corresponds to > 99% of CD45- tdTomato- cells (right).

Fig. S3. Screening for transcription factors to activate Clec9a-tdT. (A) Clec9a-tdTomato

mouse embryonic fibroblasts (MEFs) were transduced with M2rtTA (as control), all 18

candidate transcription factors and pools of 3 to 4 transcription factors and analyzed by

fluorescent microscopy and flow cytometry 5 days after addition of doxycycline. Scale bar, 200

(B) Quantification of tdTomato+ (tdT+) cells after transduction with M2rtTA, all 18

transcription factors or smaller pools at day 8 (n = 2 to 7, mean ± SD). (C) Surface expression of

CD45 was assessed by flow cytometry in MEFs transduced with PU.1 + C/EBPα at day 10. (D)

Quantification of tdT+ cells after removal of individual transcription factors from the pool of 4

transcription factors or their individual expression at day 8 (n = 3 to 4, mean ± SD) (E)

Quantification of tdT+ cells in MEFs transduced with PU.1, IRF8 and BATF3 (PIB) and cultured

in different conditions (different media compositions: DMEM or RPMI 1640-based complete

growth media with or without supplementation with 2-mercaptoethanol (2-ME), 4mM L-

glutamine (2xGlut), GM-CSF, IL-4, Flt3l or stimulation with LPS) at day 10 (n = 2, line

indicates mean). (F) Absolute numbers of tdT+ cells in MEFs transduced with PIB in gelatin-

coated dishes (black bar) and cocultured with OP9 and OP9-DL1 cells for 10 days (n = 2, line

indicates mean). OP9 and OP9-DL1 cultures were included as controls. (G) Absolute number of

tdT+ cells (sorted live single cells, tdT+ at day 9) cultured in the presence or absence of Flt3l for

3, 6 and 9 days (n =2, line indicates mean). (H) Quantification of tdT+ cells after transduction

with PU.1, IRF8, and BATF3 (PIB), PIB plus TCF4 or upon their individual removal at day 8 (n

= 2, line indicates mean). (I) Flow cytometry histograms showing side scatter (SSC) in PIB-

transduced (gated in tdT+ or total population) and M2rtTA-transduced cells as a control. Non-

significant, ns; **P < 0.01; ***P < 0.001; ****P < 0.0001; one-way ANOVA with Bonferroni’s

test.

Fig. S4. DC morphology and dendrites are established in mouse fibroblasts. (A) TdTomato+

cell morphology at day 5 after transduction with PU.1, IRF8 and BATF3 (PIB). Scale bars, 50

µm. (B) Scanning electron microscopy analysis of M2rtTA-transduced MEFs. High

magnification image highlighting fibroblast cell surface is shown in the right panel. Scale bars,

20 µm. (C) Scanning electron microscopy analysis of a tdTomato+ cell transduced with PIB and

analyzed at day 9. High magnification images highlighting complex cell membrane and

protrusions are shown in the right panel (i, ii, iii). Scale bars, 10 µm.

Fig. S5. Spi1, Irf8, and Batf3 are enriched in cDC1 cells. (A) Representative plots of flow

cytometry analysis of CD8α, CD4, CD11b, B220, F4/80, CD64 and SIRPα expression in PU.1,

IRF8 and BATF3 (PIB)-transduced mouse embryonic fibroblasts (MEFs) 7 days after addition of

doxycycline. MEFs and tdTomato- (tdT-) population are shown as controls. (B) Analysis of PIB

factors expression in mouse cells/tissues and characterization of PIB-transduced cells surface

phenotype. The combination of Spi1, Irf8 and Batf3 is mostly enriched in mouse conventional

DCs type 1 (cDC1s) among 96 tissues and cell-types. Gene expression data from 96 mouse

tissues and cell types was downloaded from BioGPS database (GeneAtlas MOE430),

transformed to log-space and normalized to bring the expression values to 0-1 range for each

gene across samples. The resulting data was then searched for samples with the highest averaged

expression for Spi1 + Irf8 + Batf3 and used to classify the tissues where combinations of

transcription factors were most enriched (best fit = 1). (C) Gene expression of Spi1, Irf8 and

Batf3 in single DC precursor cells (monocyte DC progenitor, MDP; common DC precursor,

CDP; and pre-conventional DC, pre-cDC; GSE60781). Gene expression level is shown in reads

per kilobase of exon model per million mapped reads (RPKM) values. (D) Gene expression of

Spi1, Irf8 and Batf3 in DC precursors (CDP, pre-DC1 and pre-DC2) and mature cells (cDC1 and

cDC2) (GSE60782). (E) Heat map showing Clec9a, Spi1, Irf8 and Batf3 expression in

mononuclear phagocyte subsets including DCs and monocytes available in the Immunological

Genome Project (www.immgen.org). Gene expression data were analyzed by Cluster 3.0 and

displayed by Treeview. Red indicates increased expression, whereas blue indicates decreased

expression over the mean. (F) Flow cytometry analysis of MHC-II and CD40 expression of GM-

CSF bone marrow-derived DCs (BM-DCs) (gated in CD11c+ viable cells) with or without LPS

stimulation overnight. Upon LPS stimulation, expression of CD86 in MHC-II+ CD40+ and

MHC-II- CD40+ populations is shown (right).

Fig. S6. Global single-cell gene expression analysis during iDC reprogramming. (A)

Principal Component Analysis (PCA) of genome-wide transcriptomes showing clustering of the

profiled 163 single cells. Each dot represents an individual cell (27 mouse embryonic fibroblasts,

MEF; 16 day 3 iDC; 30 day 7 iDC; 29 day 9 iDC and 61 splenic conventional DC type 1 cells,

cDC1 (CD11c+ MHC-II+ CD8+)). (B) Complete­linkage hierarchical clustering of the 163

transcriptomes. Complementary to dimension reduction techniques, we have performed single-

cell consensus clustering. The quality and numbers of clusters was estimated based on silhouette

plot. The clustering dendrogram of the consensus matrix clustering for the optimal number of

clusters was then plotted. (C) Gene ontology mouse loss-of-function mutant phenotypes (top),

KEGG pathways (middle) and microRNA target (bottom) enrichment analysis was performed on

the 4 gene clusters (relative to Fig. 3C, 6525 most variable genes). Lists show the most enriched

terms and left columns show respective P values by fold change in relation to the top enriched

term (complete lists are included in table S2). Relevant terms to DC biology are highlighted in

red. (D) Expression levels of the chemokine receptors Ccr1, Ccr5 and Ccr7 involved in DC

migration in iDCs at days 3, 7 and 9 after addition of doxycycline. MEFs and cDC1 cells were

included as controls. (E) Cumulative median expression levels of cDC1 and cDC2 gene

signatures in Flt3l and Flt3l/Notch BM-derived DCs (GSE110577).

Fig. S7. Stepwise transitions during iDC reprogramming. (A) Gene set enrichment analysis

(GSEA) for stepwise iDC reprogramming was performed using gene sets from NetPath-

annotated signaling pathways. Mouse embryonic fibroblast (MEF) to d3 refers to the pathways

up-regulated at day 3 versus MEFs, d3 to d7 refers to the pathways up-regulated at day 7 versus

day 3, and d7 to d9 refers to the up-regulated pathways at day 9 versus day 7. Datasets were

ordered according to the normalized enrichment score (NES). False Discovery Rate (FDR) q

values are represented on left columns. The right panel shows the enrichment plots for the IL-4

(day 3) and Oncostatin M (day 9) gene sets. (B) Transcription factor covariance networks during

iDC reprogramming for each step-wise transition. A pairwise matrix using Pearson correlation

was computed for mouse transcription factors annotated on the DBD transcription factor

database. Transcription factors which have logFC = 0.5 for each transition were identified. Next,

we selected transcription factors with a Pearson correlation > 0.35 with at least five other

transcription factors (table S2). In order to determine the probability of transcription factors

passing this threshold by chance, we performed 100 random permutations of the expression

matrix of all transcription factors across cells on the 3 transitions. All randomized data frames

resulted in 0 transcription factors. igraph R package was used to generate a network graph.

Nodes (transcription factors) with more than five edges are shown, with each edge reflecting a

correlation > 0.35 between connected transcription factors. Less than 15% of transcription

factors are overlapped between all cell transitions. At day 3, PU.1, IRF8, and BATF3

(highlighted in red) interact with 36, 37 and 42 transcription factors, respectively. At day 9,

PU.1, IRF8, and BATF3 interact with 16, 13 and 12 transcription factors, respectively. (C) Heat

maps showing expression of transcription factors enriched for each stepwise transition during

iDC reprogramming in DC precursors (common DC precursor, CDP; and pre-conventional DC

type 1, pre-cDC) and mature cells (conventional DC type 1, cDC1) (GSE60782). Gene

expression data were analyzed by Cluster 3.0 and displayed by Treeview. Red indicates

increased expression, whereas blue indicates decreased expression over the mean. (D) PU.1,

IRF8, and BATF3-transduced MEFs at day 3, 5, 7, 10 and 25 after addition of doxycycline were

assayed for hematopoietic colony formation (n = 3, mean ± SD) in 1% methylcellulose media

(Methocult M3434, Stem Cell Technologies). Sorted splenic cDC1 cells (live single cells, MHC-

II+ CD11c+ CD8α+), splenocytes (spleen) and bone marrow cells (BM) were included as

controls. Hematopoietic colonies were counted after 7 days of culture and numbers (Colony

Forming Units; CFUs) confirmed after 10 days of culture.

Fig. S8. iDC reprogramming does not induce alterations associated with tumorigenesis. (A)

Karyotype analysis of PU.1, IRF8, and BATF3 (PIB)-transduced mouse embryonic fibroblasts

(MEFs) at day 9 after addition of doxycycline. G-band karyotype analysis (Karyologic) was

performed in PIB-transduced MEFs replated at day 7 at a density of 500000 cells per T75 flask.

(B and C) Expression levels of (B) oncogenes Myc, Kras and Mdm2 and (C) cell-cycle arrest

genes Cdkn1a, Trp63 and Tsc1 in iDCs at day 3, 7 and 9 obtained by single-cell mRNA-seq.

MEF and splenic conventional DC type 1 (cDC1; live single cells, MHC-II+ CD11c+ CD8α+)

cells were included as controls. Expression levels are shown as census counts median values ±

95% confidence interval.

Fig. S9. Analysis of iDC maturation states. (A) Pseudotime ordering of transcriptomes of

single cells using TSCAN. Mouse embryonic fibroblast (MEF, blue), iDCs at day 3 (d3, green),

day 7 (d7, orange), day 9 (d9, red) and splenic conventional DC type 1 (cDC1; grey, live single

cells, MHC-II+ CD11c+ CD8α+) are shown. Each dot represents an individual cell. (B) Five

kinetic clusters of branch-dependent genes identified by branched expression analysis modeling.

(C) Top 5 gene ontology biological processes and mouse loss-of-function mutant phenotypes

enrichment analysis for Cluster I to V. (D) Gene set enrichment analysis for day 9 iDCs and

cDC1 showing enrichment for antigen processing and presentation and dendritic cell maturation

MSigDB gene sets (left). Normalized enrichment score (NES) and false discovery rate (FDR) q

values are represented on the enrichment plots. Violin plots (right) show expression distribution

of the day 9 iDC enriched genes Tapbp, Lgmn, Lgal9 and Ptpn1.

Fig. S10. Induced DCs are functional APCs. (A) Violin plots showing expression distribution

of genes regulating toll-like receptor (TLR) signaling (Tlr4, Ticam2, Traf6 and Ikkε). (B)

Cytokine secretion of iDC cultures after stimulation with LPS or polyI:C (PIC) overnight at day

10. (C) Violin plots for genes involved receptor-mediated endocytosis (Fcgr2b, Tfr2 and Mrc1)

and macropinocytosis of cells (Axl, Lrp1 and Scarf1). (D) PU.1, IRF8, BATF3 (PIB)-transduced

Clec9a-tdTomato mouse embryonic fibroblasts (MEFs) at day 7 were incubated with 2.5%

yellow-green fluorescent-coupled solid latex beads (carboxylate-modified polystyrene, Sigma) at

1:1000 ratio. 16 hours later, cells were extensively washed in PBS with 5% FBS to remove

floating beads and fixed with 4% paraformaldehyde (PFA) for 10 min. After washing, DAPI (1

μg/mL, Sigma) was used for nuclear staining and analyzed by fluorescent microscopy. (E) FACS

sorted tdTomato- (tdT-) and tdTomato+ (tdT+) cells were incubated with AlexaFluor647-labeled

Ovalbumin (OVA-Alexa647, Life Technologies) for 20 min at 37°C AT day 11. Controls were

kept on ice (4ºC). After washing with PBS with 5% FBS, cells were analyzed by flow cytometry.

(F) iDCs at day 11 were incubated overnight with dead cells labeled with DAPI and analyzed by

fluorescent microscopy. (G) Representative plots of proliferation (CFSElow) of CD4+ T cells after

7 days of coculture with iDCs and splenic conventional DCs (cDC; CD11c+ MHC-II+ cells) in

the presence or absence of LPS. (H) Violin plots showing expression distribution of genes

involved in antigen cross-presentation (Cybb, Atg7, Tap1 and Tap2). (I) β-lactamase’s export to

cytosol of iDCs at day 16 measured as CCF4 cleavage by flow cytometry. The percentage of live

single tdT+ cells with a high blue-to-green (V450/V500) fluorescence ratio was used as a

measure of the efficiency of antigen export into the cytosol. (J) Representative plots of

proliferation (celltrace violet, CTVlow) and CD44 expression of CD8α+ T cells after 3 days of

coculture with tdT- cells (sorted at day 8) and Flt3l/GM-CSF BM-derived DCs (BM-DCs) with

or without previous 10-hour pulse incubation with OVA protein in the presence of PIC. (K)

Representative plots of CTVlow CD8α+ T cells after 3 days of coculture with MEFs, tdT-, tdT+,

splenic cDC1 (CD11c+ MHC-II+ CD8α+) cells and BM-DCs after pulse incubation with OVA

protein in the presence or absence of PIC.

Fig. S11. Polycistronic vector induces cDC1-like transcriptional program. Mouse embryonic

fibroblasts (MEFs) transduced with the polycistronic vector encoding PU.1, IRF8, and BATF3

(PIBpoly) were FACS-sorted (tdTomato+ MHC-II+ CD45+) and profiled using population mRNA-

seq at day 5, 7, 8 and 9. MEF, splenic conventional DC type 1 (cDC1; live single cells, CD11c+

MHC-II+ CD8α+), cDC2 (CD11c+ MHC-II+ CD11b+) and plasmacytoid DC (pDC; CD11clow

Bst2+ MHC-II+ Siglec-H+ B220+) cells were used as controls (fig. S13). (A) Heat map of

differentially expressed genes between sDC1, sDC2 and pDC cells. (B) Venn diagrams showing

genes up-regulated in day 9 iDCs shared with sDC1, sDC2 and pDC cells. (C) Pearson

correlation matrix showing expression of the differentially expressed genes between sDC1, sDC2

and pDC cells for all samples. (D) Expression levels of Xcr1, Clec9a, Rbpj, Ifi205, Ptprc, Naaa, Sirpα, Esam, Cd4 and Siglech. (E) Gene analysis of expression of sorted iDCs at day 5 (d5), 7

(d7), 8 (d8) and 9 (d9). (F) Gene ontology biological processes (left) and cellular components

(right) enrichment analysis for each step-wise iDC reprogramming transition. MEF to d5 refers

to the genes up-regulated at day 5 versus MEFs, d5 to d7 refers to the genes up-regulated at day

7 versus day 5, d7 to d8 refers to the up-regulated genes at day 8 versus day 7 and d8 to d9 refers

to the up-regulated genes at day 9 versus day 8.

Fig. S12. PIB induce human iDC reprogramming. (A) Violin plots showing expression

distribution of CLEC9A, SPI1, IRF8 and BATF3 in human blood dendritic cells (DC1 to DC6)

and monocytes (Mono1 to Mono4) populations as defined by Villani et al. (GSE94820). (B)

Kinetics of CD45 expression in human embryonic fibroblasts (HEFs) transduced with

polycistronic vector encoding PU.1, IRF8, and BATF3 (PIBpoly). M2rtTA-transduced HEFs were

included as control. (C) PIBpoly-transduced HEFs were cultured with or without supplementation

with GM-CSF or Flt3l at day 2 after addition of doxycycline. CD45 and HLA-DR expression

was assessed at day 9 (n = 2, line indicates the mean). Non-stimulated M2rtTA-transduced HEFs

were included as control. (D) PIBpoly-transduced HEFs at day 9 were incubated overnight with

FITC-labeled beads and analyzed by fluorescent microscopy. CellVue far red (red) labels cell

membranes and DAPI (blue) nuclei.

Fig. S13. FACS gating strategies. (A) Splenic conventional DC type 1 (cDC1; CD11c+ MHC-

II+ CD8α+) were sorted for single cell mRNA-seq. (B) PU.1, IRF8, BATF3-transduced mouse

embryonic fibroblasts (MEFs) were sorted for single-cell mRNA-seq based on single expression

of tdTomato (day 3 and day 7) and double positive cells for tdTomato and MHC-II (day 9). (C)

MEFs transduced with polycistronic vector encoding PU.1, IRF8, and BATF3 were sorted 5, 7, 8

and 9 days after the addition of doxycycline based on expression of MHC-II, tdTomato and

CD45 markers for population mRNA-seq. (D) Splenic populations of cDC1 and cDC2

populations defined as CD11c+ MHC-II+ CD8α+ (cDC1, red) and CD11c+ MHC-II+ CD11b+

(cDC2, blue), respectively, were sorted for population mRNA-seq. (E) Splenic plasmacytoid DC

population defined as CD11clow Bst2+ MHC-II+ Siglec-H+ B220+ was sorted for population

mRNA-seq.

Table S1. (separate file) Analysis of candidate transcription factors. Relative to list of 18

candidate DC-inducing transcription factors; GPSmatch output files; accession numbers for

datasets used in this study; GPSgenes output files and list of antibodies used in this study.

Table S2. (separate file) Single-cell mRNA-seq data analysis. Relative to single-cell mRNA-

seq data analysis from Fig. 3, fig. S6 and fig. S7. Differential expression analysis; gene ontology

and pathway enrichment analysis for gene clusters; gene set enrichment analysis of NetPath-

annotated signaling pathways; and transcription factor covariance network analysis during iDC

reprogramming.

Table S3. (separate file) Pseudotime single-cell mRNA-seq data analysis. Relative to single-

cell mRNA-seq data analysis from Fig. 4 and fig. S9: differential expression analysis between

cell states; gene ontology and pathway enrichment analysis for differentially expressed genes

between cell states; gene ontology enrichment analysis for gene clusters; and gene set

enrichment analysis of Immunologic signatures.

Table S4. (separate file) Population mRNA-seq data analysis. Relative to population mRNA-

seq data analysis from fig. S11: differential expression analysis for iDCs collected at different

time points, splenic conventional DC type 1 (cDC1), cDC2 and plasmacytoid DC (pDC) and

mouse embryonic fibroblast (MEF); gene signatures for cDC1, cDC2 and pDC; gene overlap

between cDC1, cDC2 and pDC and iDCs; and gene ontology enrichment for transitions between

iDC and MEF.

Table S5. (separate file) Raw data.

Movie S1. (separate file) Time lapse showing Clec9a-tdT+ cell emergence. Clec9a-tdTomato

reporter activation occurs approximately 30 hours after PU.1, IRF8, and BATF3 induction.

Specified tdTomato+ cells show morphology changes, develop cytoplasmic projections and

display migratory behavior. Pictures were taken every hour over a period of 6 days after addition

of doxycycline.

Movie S2. (separate file) Time lapse displaying dead cell incorporation. Clec9a-TdTomato+

cells (red) at day 10 after addition of doxycycline were incubated with DAPI-labeled dead cells

(blue). TdTomato+ cells actively phagocyte dead cells, accumulating several cells in the

cytoplasm (arrow). On the left side, one tdTomato+ cell develops cytoplasmic protrusions and

engulfs a dead cell (arrowhead). Pictures were taken every hour over a period of 35 hours.