MEF, HEK293T, Platinum-E, and 4T1 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). Raji and Reh cells were cultured in RPMI Medium 1640 basic (Gibco) supplemented with 10% FBS and 1% PS. MCC intermediate cells and iPSCs were cultured in the iPSC medium [Knockout DMEM, plus 20% FBS, 1% PS, mouse LIF (1000 U/ml), non-essential amino acids (NEAA, 100×), L-glutamine (100×), and β-mercaptoethanol (100×)]. iMac cells were cultured in macrophage differentiation medium [RPMI Medium 1640 basic (Gibco) supplemented with 10% FBS, 1% PS, and 100 ng/ml M-CSF (PeproTech)].

Generation of virus

The retroviral constructs pMXs-GFP, pMXs-Klf4, pMXs-Sox2, pMXs-Oct4, and pMXs-c-Myc were purchased from Addgene (Cambridge, MA). To produce retrovirus, the indicated plasmid was transfected into Platinum-E retroviral packaging cells using Hieff Trans® Liposomal Transfection Reagent (Yeasen) as previously described [13]. To produce lentivirus, the indicated plasmid with pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) was transfected into HEK293T cells. The virus supernatant was collected at 48 h and 72 h after transfection.

Reprogramming fibroblasts into macrophages

MEFs were seeded on a 6-well plate and transfected with the virus in the medium supplemented with 8 µg/ml polybrene. After transfection for 48 h, the culture medium was changed into iPSC medium. MEFs with overexpression of c-Myc were cultured in the iPSC medium for 7 days. Then the cells in suspension (named P0 MCC intermediates) were collected and resuspended in iPSC medium to get MCC intermediates that were passaged every 3 days. For macrophage differentiation, the iPSC medium was removed and replaced with macrophage differentiation medium on day 10. On day 15, cells (named induced macrophages, iMac) were harvested and analyzed.

Compared to the iPSC medium, erythroid culture medium (EC-M) and granulocyte culture medium (GC-M) were used. EC-M: IMDM (50%) and Ham’s F12 (50%) plus ITS-X (100x), chemically defined lipid concentrate (100x), L-glutamine (100x), ascorbic acid (0.05 mg/ml), BSA (5 mg/ml), 1-thioglycerol (200 µM), SCF (100 ng/ml), IL-3 (10 ng/ml), erythropoietin (2 U/ml), IGF-1 (40 ng/ml), dexamethasone (1 µM), and holo transferrin (100 µg/ml). GC-M: IMDM (50%) and Ham’s F12 (50%), plus ITS-X (100x), chemically defined lipid concentrate (100x), L-glutamine (100x), ascorbic acid (0.05 mg/ml), BSA (5 mg/ml), 1-thioglycerol (200 µM), thrombopoietin (100 ng/ml), SCF (100 ng/ml), Flt3 ligand (100 ng/ml), granulocyte-colony stimulating factor (G-CSF) (100 ng/ml), and IL-3 (10 ng/ml).

Macrophage polarization and cytokine analysis

Mouse bone marrow cells were collected, lysed using red blood lysis buffer, and prepared into a single-cell suspension. Then, cells were cultured in RPIM1640, 10% FBS, 1% PS, and 20 ng/ml M-CSF to differentiate into macrophages. For polarization, iMac and bone marrow-derived macrophages were induced with LPS (100 ng/ml) and IFN-γ (20 ng/ml), or IL-4 (20 ng/ml) for 24–48 h. Cell supernatants were collected to detect cytokine levels by Luminex-MultiDTM, and cells were digested and collected for flow cytometry.

Flow cytometry

Cells were collected and separated into single cells using 100 μm cell strainers. Cells were collected, separated into single cells, and resuspended in 100 µl flow cytometry staining buffer. For cell surface markers, cells were incubated with antibodies at 4℃ for 30 min. For intracellular staining, cells were fixed and permeabilized with Intracellular Fixation and Permeabilization Buffer Set (eBioscience), then incubated with antibodies for 30 min. Samples were detected by BD Canto Plus or sorted by Beckman Coulter MoFlo XDP. FlowJo v 10.7.2 was used for data analysis. The following antibodies were used: anti-mouse CD16/32 (clone S17011E, Biolegend), APC-Cy7 anti-mouse CD45 (clone 30-F11, Biolegend), PE anti-mouse Sca-1 (clone E13-161.7, Biolegend), APC-eFluor™ 780 anti-mouse c-Kit (clone 2B8, eBioscience), PE-Cy7 anti-mouse/human B220 (clone RA3-6B2, Biolegend), PE anti-mouse Lineage Cocktail (Biolegend), PE-Cy7 anti-mouse CD3 (clone 17A2, eBioscience), APC anti-mouse CD11b (clone M1/70, eBioscience), Super Bright™ 436 anti-mouse F4/80 (clone BM8, Invitrogen), FITC anti-mouse CD11c (clone N418, eBioscience), Pacific Blue anti-mouse CD69 (clone H1.2F3, eBioscience), PE anti-mouse CD49b (clone DX5, eBioscience), PE anti-mouse CD206 (clone MR6F3, Invitrogen), Alexa Fluor™ 488 anti-mouse iNOS (clone CXNFT, eBioscience), PE-Cy7 anti-human/mouse Arginase1 (clone A1exF5, eBioscience), FITC anti-mouse CD107a (clone 1D4B, Biolegend), PE anti-human CD7 (clone CD7-6B7, Biolegend), APC-Cy7 anti-human CD45 (clone 2D1, Biolegend).

Cell transplantation

For cell transplantation, 6-8-week-old female C57BL/6J mice [Shanghai Laboratory Animal Center (SLAC), n = 5 per group] were used. MEFs were co-transfected with pMXs-GFP and pMXs-c-Myc to get MCC-GFP. In the first transplantation, a total of 1 × 106 MCC intermediate cells (labeled with GFP) or fetal liver mononuclear cells were first injected into lethally irradiated recipient mice via the tail vein. The peripheral blood of the mice was collected and analyzed by flow cytometry at indicated time points. Three months after the first transplantation, cells from different organs of one batch of mice were isolated and analyzed by flow cytometry. In the second transplantation, 1 × 107 bone marrow cells from recipient mice of the first transplantation were injected into lethally irradiated recipient mice via the tail vein. One month later, the peripheral blood of the mice was collected, and bone marrow was analyzed for lineage constitution.

Tumor xenograft animal models

In Reh leukemia animal model, 6-8-week-old female severe combined immunodeficiency (SCID) (SLAC, n = 5 per group) mice were used. SCID mice were irradiated with 2 Gy before transplantation. A total of 2 × 105 GFP-labeled Reh (Reh-GFP) cells with different quantities of MCC intermediates were co-injected into animals via the tail vein. On day 28 after injection, mice were euthanized when leukemia symptoms developed. The proportion of leukemia cells in BM and spleen was measured by flow cytometry.

In 4T1 breast cancer xenograft model, 6-8-week-old female BALB/c mice (SLAC, n = 5 per group) were used. A total of 2 × 105 Luciferase-labeled 4T1 (4T1-Luc) cells were injected into mice via the tail vein. On day 5, mice were randomized and injected with 1 × 106 iMac (from mice of the same strain) or vehicle (PBS) via the tail vein. Bioluminescence intensity (BLI) followed tumor growth using the IVIS Lumina imaging station (Caliper Life Sciences). Mouse lung tissues were analyzed by hematoxylin and eosin staining, immunohistochemistry and flow cytometry.

Patient-derived tumor xenograft (PDX) model

In the PDX model, 6-week-old male NCG mice (Gem Pharmatech Co., Ltd., Nanjing, China, n = 5 per group) were injected with 1 × 106 PDX cells by tail vein. On day 14 and 21, 2 × 106 iMac cells or vehicle (PBS) were injected into animals by tail vein. On day 35 after injection, mice were euthanized when leukemia symptoms developed. The proportion of leukemia cells in BM and spleen was measured by flow cytometry.

Western blotting assay

Cells were collected and lysed in RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM Na3VO4, 5 mM NaF, 1% Triton X-100) with a Protease inhibitor cocktail (Invitrogen) and eluted by SDS loading buffer. The proteins were resolved in SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. Blots were incubated with primary antibodies overnight at 4℃ followed by HRP-conjugated species-specific antibodies. The membrane was visualized and analyzed by an imaging system (Bio-Rad Laboratories) after incubation with appropriate primary and secondary antibodies.

Wright-Giemsa stain

Cells were collected, then centrifuged and immobilized on the slides. Slides were stained with a Wright-Giemsa Stain kit (BaSO, Zhuhai) as previously described [19]. Images were acquired with an upright light microscope.

Hematopoietic colony-forming cell assay

Hematopoietic colony-forming cell (CFC) assay was performed in 35-mm low adherent plastic dishes using MethoCult GF M3434 (StemCell Technologies) according to the manufacturer’s instructions. Briefly, cells were pre-passed through a 100 µM cell strainer, and 3000 single cells were plated in a well. Colony-forming units (CFU) were identified after 14 days of incubation according to their colony morphology as granulocyte (CFU-G), macrophage (CFU-M), granulocyte-macrophage (CFU-GM), granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), and erythroid (CFU-E).

shRNA knockdown

The sequences of all small hairpin RNA (shRNA) oligonucleotides were synthesized and cloned into an FH1TUTG vector. The murine MafB shRNA sequence (5’-CTACAAGGTCAAGTGCGAGAA-3’) was from Sigma. Virus packaging was performed as previously described. MEFs were infected with pMXs-c-Myc and FH1TUTG-shMafB and induced with doxycycline hyclate (2 µg/ml).

Hematoxylin and eosin (H&E) staining

Frozen sections were fixed with acetone, treated with hematoxylin and 1% ethanol hydrochloride, and then washed. The sections were dehydrated and sealed after being treated with Eosin Y solution.

Immunohistochemistry

Tissue sections were dewaxed with xylene and ethanol. Endogenous peroxidase was inactivated with 3% H2O2. For antigen retrieval, tissue sections were treated with citrate buffer. Tissue sections were blocked for 1 h at room temperature. After that, tissue sections were incubated overnight at 4℃ with primary antibody. Tissue sections were incubated with HRP-conjugated secondary antibody and DAB staining solution.

Single-cell RNA sequencing and analysis

MCC intermediates and iMac cells were collected for single-cell RNA sequencing. DNBelab C4 System or Illumina NovaSeq 6000 was utilized for scRNA-seq library preparation according to the manufacturer’s protocol. Raw sequencing data were aligned and quantified using the CellRanger. The output was converted to a Seurat object using the R Seurat package for downstream analysis. To remove low-quality cells, we used the following criteria: less than 1000 unique genes expressed, or more than 20% of reads mapping to mitochondria. The gene expression matrix of the remaining cells was normalized through the function of ScaleData. Cell clustering was performed using the FindClusters, and the clusters were annotated by the expression of canonical marker genes. FindAllMarkers was used to define marker genes for each cluster. Functional enrichment analysis was done by clusterProfiler. Monecle2 was used to infer the potential developmental trajectory. Enriched motif for each cell type was done using pySCENIC. Pathway activation score was calculated by GSVA on average expression on each subtype.

Bulk RNA sequencing and analysis

Total RNA (iMac, MCC, MEF, iPSC, BM, and PB CD45+ cells) was extracted using TRIzol (Thermo Fisher Scientific) and sequenced by Mingma Technologies Co., Ltd. at Shanghai according to the manufacturer’s instructions. Libraries were sequenced on Illumina NovaSeq 6000 or Illumina HiSeqX Ten platform. Raw reads were mapped to the mouse genome (mm10) and GENECODE vM25 using STAR. Quantification for each expressed gene was performed using featureCounts from Rsubread. Differentially expressed genes were calculated by DESeq2 with cutoffs as follows: Padj < 0.05 and absolute log2 fold change > 1. Functional annotations were done using enrichKEGG from clusterProfiler package.

Quantitative RT-PCR (qRT-PCR)

Cells were collected, and RNA was extracted with TRIzol. The eluted RNA was reverse-transcribed using Hifair II 1st Strand cDNA Synthesis SuperMix for qPCR (11123ES10, YEASEN) according to the manufacturer’s instructions. Quantitative RT-PCR was performed with qPCR SYBR Green Master Mix (11201ES03, YEASEN), and results were analyzed using BioRad CFX Maestro Software. Primers were as follows: 5’-CCAAGTGCTGCCGTCATTTTC-3’ and 5’-GGCTCGCAGGGATGATTTCAA-3’ for Cxcl10, 5’-GAGCCTGGCTAGGAAGGTG-3’ and 5’-TGCTGAAACCAATAAGGAACCTG-3’ for Fpr2, 5’-ATGCCAATCACTCGAATGCG-3’ and 5’-TTGTATCGGCCTGTGTGAATG-3’ for Irf1.

Chromatin immunoprecipitations (ChIP)-qPCR

For each ChIP assay, 1 × 106 cells were harvested and followed the manufacturer’s protocol of Simple ChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads, Cell Signaling Technology, 9005S). ChIP DNA was purified and subsequently quantified by qPCR. Primers: 5’-ggggccacaagagtcctcttca-3’ and 5’-gcgcttcccgagtacgcg-3’.

Promoter reporter and dual luciferase assays

The MafB promoter was amplified by PCR and then inserted into a pGL3.0-Basic vector. pGL3.0 basic-MafB promoter was co-transfected with or without c-Myc WT or mutants according to the manufacturer’s protocol. For normalization of transfection efficiency, the pRL-TK (Renilla luciferase) reporter plasmid was added to each transfection. Luciferase activity was quantified with a Dual-Specific Luciferase Assay Kit (E1910; Promega).

Immunofluorescence

Cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Then, cells were penetrated with 0.3% Triton X-100 in PBS for 20 min and blocked with 10% goat serum for 60 min. For the immunofluorescence assay, cells were incubated with primary antibody overnight at 4℃ followed by secondary antibody for 1 h at room temperature. Nuclei were counterstained with DAPI. Images were captured by Leica SP8 confocal microscope.

Fluorescent beads phagocytosis assay

Fluroresbrite Polychromatic Red 1.0-µm Latex beads (Polyscience Inc., 18660) were added to the cell culture medium and incubated with cells for 24 h. Then, cells were washed several times and analyzed by Leica SP8 confocal microscope or flow cytometry.

Tumor cell phagocytosis assay

Raji and Reh cell lines were washed with PBS, resuspended in 1 ml CFSE staining solution, and incubated for 20 min at 37℃. To remove free dye remaining in the solution, 5 ml complete culture medium was added to the cells and incubated for 5 min. Cells were centrifuged and resuspended in culture medium. MEFs were pre-stained with DiD dye, then iMac and MEF cells were co-incubated with tumor cells at a ratio of 1:4 at 37℃ for 2 h. For flow cytometry, cells in iMac group were harvested and stained with CD11b antibody, and the percentage of phagocytosis was the ratio of CFSE-positive cells in the CD11b+ population. In MEF group, cells were harvested, and the percentage of phagocytosis was the ratio of CFSE-positive cells in the DiD+ population.

Statistical analysis

All grouped data are presented as mean ± SEM. All in vitro experiments were repeated at least three times. Two-tailed unpaired Student’s t test was used to analyze the data from two groups, and one-way ANOVA was used to analyze multiple groups. For the survival analysis, Kaplan-Meier survival curves were analyzed by using log-rank test comparing the different mouse groups. Data were considered statistically significant when P values were less than or equal to 0.05 in this study (*P < 0.05, ** P < 0.01, *** P < 0.001). Statistical analyses were performed with GraphPad Prism 8.2.1.