Physiologic medium renders human iPSC-derived macrophages permissive for M. tuberculosis by rewiring organelle function and metabolism

ABSTRACT In vitro studies are crucial for our understanding of the human macrophage immune functions. However, traditional in vitro culture media poorly reflect the metabolic composition of blood, potentially affecting the outcomes of these studies. Here, we analyzed the impact of a physiological medium on human induced pluripotent stem cell (iPSC)-derived macrophages (iPSDM) function. Macrophages cultured in a human plasma-like medium (HPLM) were more permissive to Mycobacterium tuberculosis (Mtb) replication and showed decreased lipid metabolism with increased metabolic polarization. Functionally, we discovered that HPLM-differentiated macrophages showed different metabolic organelle content and activity. Specifically, HPLM-differentiated macrophages displayed reduced lipid droplet and peroxisome content, increased lysosomal proteolytic activity, and increased mitochondrial activity and dynamics. Inhibiting or inducing lipid droplet formation revealed that lipid droplet content is a key factor influencing macrophage permissiveness to Mtb. These findings underscore the importance of using physiologically relevant media in vitro for accurately studying human macrophage function. IMPORTANCE This work compellingly demonstrates that the choice of culture medium significantly influences M. tuberculosis replication outcomes, thus emphasizing the importance of employing physiologically relevant media for accurate in vitro host-pathogen interaction studies. We anticipate that our work will set a precedent for future research with clinical relevance, particularly in evaluating antibiotic efficacy and resistance in cellulo.

components that have been identified in plasma through mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy analysis (3,5,6).Typically, these basal media are enriched with fetal bovine serum (FBS), which introduces a complex and often overlooked mixture of polar metabolites and lipids, alongside essential growth factors and hormones necessary for cell growth, which may differ depending on the lot and origin of the serum (7,8).
Over recent years, there have been numerous initiatives aimed at modifying cell culture media compositions to more closely mimic physiological conditions.These initiatives have led to the development of two media that mirror the metabolic composition of human plasma, specifically, human plasma-like medium (HPLM) and Plasmax (3,5,8,9).These media contain more than 60 polar metabolites such as amino acids, nucleic acids, sugars, and small organic acids at concentrations found in human plasma.To minimize the impact of serum on plasma metabolite composition, the developers of Plasmax medium lowered its serum concentration to 2.5%, whereas the developers of the HPLM medium opted to substitute the typical serum with a dialyzed version devoid of polar metabolites (3,5).
Adding further complexity, human macrophage differentiation in vitro, either from human blood-derived monocytes or iPSC-derived monocytes, typically involves the cytokines macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF).M-CSF is continuously produced in most tissues and regulates the population of macrophages in numerous organs.In contrast, GM-CSF is primarily produced in the lungs and typically has low baseline levels in the circulation, which tend to increase in response to immune or inflammatory events (10).M-CSF operates through the c-FMS receptor (CD115), a single-pass transmembrane protein tyrosine kinase that belongs to the PDGF family.Conversely, GM-CSF utilizes CD116, an α/β heterodimeric receptor, the β-chain of which is shared with interleukin-3 (IL-3) and IL-5 (10,11).Although previous studies have compared the gene expression profiles of human macrophages differentiated with M-CSF and GM-CSF (12), the rationale behind selecting a specific differentiation protocol and the impact of these differentiation programs on macrophage organelle activity and composition remain poorly investiga ted.
In this study, we address these critical gaps by integrating RNA sequencing, extracellular flux analyses, and high-content single-cell imaging to comprehensively evaluate the effects of culture media composition on human macrophage organelle activity and host cell response to the intracellular pathogen Mycobacterium tuberculo sis (Mtb).We discovered that macrophages in physiological media displayed distinct proteolytic and lipid droplet content, mitochondrial activity, and metabolic properties with an impact on the ability of these cells to control Mtb.We anticipate that this study will not only contribute to establishing more standardized iPSC-derived cells culture practices but also illuminating the mechanisms by which modulating organelle activity influences macrophage effector functions.

HPLM-differentiated macrophages are more permissive for Mtb replication
Open-source medium (OXM) has been established to culture both induced pluripotent stem cells (iPSC) and induced pluripotent stem cell-derived macrophages (iPSDM).The OXM is a differentiation medium based on Advanced DMEM/F-12 (aDMEM/F-12) that has been developed as an alternative to commercial, serum-free media of undis closed composition, such as X-VIVO 15 (1,4).Although the use of OXM offers practi cal advantages and provides a more standardized method for iPSDM production, its chemical composition still differs significantly from human plasma.For instance, the glucose concentration of OXM is 16.7 mM, approximately 3 to 4 times the concentration found in human plasma (3)(4)(5).To overcome this limitation and establish a differentiation method using physiologic medium, we compared and functionally characterized iPSDM, differentiated using M-CSF or GM-CSF in either X-VIVO15, OXM, or Human Plasma-Like Medium (HPLM) (3) while maintaining the iPSC factories for monocyte production using OXM as previously described (4) (Fig. 1A).We first determined by flow cytom etry different monocyte and macrophage surface markers to evaluate macrophage phenotypic differences among the different media.We found that the surface marker abundance of CD16, CD14, CD119, CD206, CD163, CD169, and CD86 was similar in macrophages cultured in X-VIVO15, OXM, or HPLM consistent with previous reports (1, 2) (see Fig. S1A and B at https://doi.org/10.25418/crick.26048884).
Given that one of the most relevant functions of macrophages is phagocytosis and bacterial killing (13), we studied the functional outcomes of macrophage differentiation under physiologic and non-physiologic media in infection.We investigated the ability of macrophage populations to restrict the replication of the human pathogen Mtb using single-cell high-content imaging.We observed that Mtb replication in HPLM-differentiated macrophages was greater than with OXM and X-VIVO15 (Fig. 1B through D).In addition, GM-CSF-differentiated macrophages presented less Mtb growth in comparison to M-CSF differentiation across all the media tested (Fig. 1E through G).Overall, our results highlight important consequences for the host cell response to Mtb infection when macrophages are differentiated in HPLM and identify that macrophages growing in a physiologic medium represent a more permissive environment for Mtb replication.

X-VIVO15-and OXM-differentiated macrophages show a lipid metabolismassociated transcriptional signature
To further investigate the phenotypic distinctions between HPLM-, OXM-, and X-VIVO15differentiated macrophages with either M-CSF or GM-CSF, we employed bulk RNA sequencing (RNA-seq) analysis.Utilizing gene set enrichment analysis (GSEA) with REACTOME, we identified over 100 distinct pathways selectively enriched among the various media tested (Fig. 2; see Fig. S2 and Table S1 at https://doi.org/10.25418/crick.26048884).We found that GM-CSF differentiation induced enrichment in sev eral IFN-associated pathways, including interferon, interferon gamma, and interferon alpha beta signaling, as well as interleukin (IL) pathways such as IL-10 and IL-13, compared with M-CSF-differentiated iPSDM (see Fig. S2 and S3 at https://doi.org/10.25418/crick.26048884).Although these alterations constituted a common transcrip tional signature across the three different media tested, both OXM and X-VIVO15 differentiations triggered an enrichment in lipid metabolism-associated programs, such as sphingolipid, phospholipid, cholesterol, and fatty acid metabolism pathways, compared with HPLM-differentiated iPSDM (Fig. 2A through D; see Table S1 at https:// doi.org/10.25418/crick.26048884).Notably, this enhanced lipid metabolism-associated transcriptional signature was prevalent for both M-CSF and GM-CSF differentiation programs and more pronounced in X-VIVO15-differentiated HPLM.Consistent with these findings, a previous study demonstrated enrichment in lipid metabolic pathways in X-VIVO15-differentiated iPSDM compared with OXM media (4).However, we observed an unappreciated increase in lipid metabolism that also applies to macrophages differentiated in OXM when compared with HPLM-differentiated iPSDM (Fig. 2A through D; see Tables S1 and S2 at https://doi.org/10.25418/crick.26048884).To further explore the effect of cell culture media on macrophage transcriptional programs, we analyzed the number of genes up-and downregulated when comparing GM-CSF with M-CSFdifferentiated macrophages, independent of the media used (Fig. 2E; see Table S3 at https://doi.org/10.25418/crick.26048884).Intriguingly, only 18.1% (810 transcripts) of the differentially expressed genes (DEGs) were commonly upregulated.These genes were mainly associated with interferon signaling pathways.Additionally, a smaller fraction of genes (10.6%) was commonly downregulated, with transcripts associated with rRNA processing and Rho pathways, among others (Fig. 2E; see Table S3).Overall, these results highlight the profound impact of the cell culture media on macrophage transcript signatures.

Physiologic medium-differentiated macrophages display increased meta bolic polarization
Because our gene expression analysis suggested important changes in the metabolism of HPLM-differentiated macrophages, we next investigated the homeostatic metabolism and the response to mitochondrial activity uncouplers of iPSDM cultured in physiologic and non-physiologic media by extracellular flux analysis ( 14) (Fig. 3A).Although we observed that HPLM-differentiated macrophages in M-CSF showed the lowest basal oxygen consumption rate (OCR; indicative of OXPHOS), we found that GM-CSF triggered a macrophage metabolic polarization toward an energetic phenotype, characterized by an increase in OCR and extracellular acidification rate (ECAR; indicative of glycolysis) in all the media evaluated (Fig. 3B through D).These results align with previous studies that demonstrated the polarization of GM-CSF-differentiated macrophages toward a glycolytic profile (15,16).Moreover, HPLM-differentiated macrophages displayed the greatest increase in OCR and ECAR when compared GM-CSF with M-CSF differentiation (Fig. 3B through D).In line with these results, the response to mitochondrial stressors evaluated through ATP-linked respiration and maximal respiration parameters was also greater for GM-CSF-differentiated macrophages cultured in HPLM, in comparison with OXM and X-VIVO 15 media (Fig. 3A, E, and F).This observation underscores the differential sensitivity of macrophages to their metabolic environment, suggesting that GM-CSF-differentiated macrophages may exhibit a heightened responsiveness to media composition in terms of metabolic activity.Overall, our results argue that medium resembling human plasma composition results in a more extensive metabolic plasticity (17) for human macrophages.

Physiologic medium-differentiated macrophages show low lipid droplet content and increased lysosomal proteolytic activity
Macrophage metabolism is recognized as one of the main factors that regulate immune function, and it has been primarily studied in vitro.Given that our RNA-seq analysis revealed a selective enrichment in lipid metabolism-associated pathways for macrophages differentiated in X-VIVO 15 and OXM in comparison to HPLM-differentiated iPSDM, we sought to determine how these different transcriptional programs impact iPSDM organelle homeostasis.We first evaluated the macrophage lipid droplet (LD) content by using high-content single-cell imaging and fluorinated boron-dipyr romethene (BODIPY) staining (18).Strikingly, we observed that X-VIVO15-differentiated macrophages showed the highest amount of LD content per cell (evaluated as mean BODIPY intensity and mean droplet area per cell), followed by OXM-differentiated macrophages (Fig. 4A through D; see Fig. S4A and B at https://doi.org/10.25418/crick.26048884).In contrast, HPLM-differentiated macrophages showed very low levels of LD.We also observed a decrease in the LD content in GM-CSF-differentiated macro phages when compared with M-CSF-differentiated macrophages (Fig. 4E).Endolysoso mal degradation is central to lipid homeostasis via clearance of extracellular lipoproteins and autophagy (19,20).Thus, we investigated if the observed differences in LD content were related to differing lysosomal and proteolytic activities.By using a pan-cathepsin activity-based probe (21), we found that HPLM-differentiated macrophages showed a 2-fold increase in proteolytic activity in comparison with OXM-or XVIVO15-differentiated macrophages (Fig. 4F through I).These results were consistent with a marked increase in lysosomal content (evaluated as lysosomal area per cell) observed in HPLM-differentiated macrophages (see Fig. S4C and D at https://doi.org/10.25418/crick.26048884).In addition, we observed that GM-CSF differentiation triggered an increase in lysosomal proteolytic activity in comparison with M-CSF differentiation for OXM-differentiated macrophages or XVIVO15-differentiated macrophages (Fig. 4H through J).Overall, these results show an inverse correlation between lipid droplet content and proteolytic activity for HPLM-differentiated iPSDM, suggesting that media composition is the main factor regulating macrophage organelle content, activity, and function.

Physiologic medium-differentiated macrophages display increased mito chondrial activity
Lipids constitute a primary mitochondrial source of energy; however, there is evidence suggesting that they can also act as uncouplers and inhibitors of OXPHOS (22).A singlecell mitochondrial segmentation analysis to quantify the mitochondrial accumulation of the membrane-sensitive dye tetramethylrhodamine methyl ester (TMRM) (14) showed that HPLM-differentiated macrophages had increased mitochondrial activity (Fig. 5A through D).Moreover, we observed that GM-CSF differentiation increased the mitochon drial activity of iPSDM cultured in X-VIVO 15, OXM, and HPLM (Fig. 5E).Although we did not observe changes in the mitochondrial mass (quantified as total mitochondrial area per cell) (Fig. 5F and G), we found that HPLM-differentiated macrophages displayed a more elongated mitochondrial network (Fig. 5H and I).Altogether, these results confirm an increased metabolic reprogramming ability observed in HPLM-differentiated

Physiologic medium-differentiated macrophages display increased mito chondrial dynamics and different peroxisome contents
We next evaluated whether the changes in mitochondrial activity observed in HPLM-dif ferentiated macrophages impact mitochondrial dynamics.A live-cell super-resolution and 3D z-stack analysis of the mitochondrial network (23) show that although the mean length and number of mitochondrial branches did not change over time, macrophages cultured in HPLM consistently exhibited a greater number of mitochondrial branches compared with X-VIVO 15-differentiated macrophages.This suggests that HPLM induces a more dynamic mitochondrial network in macrophages (Fig. 6A through D).Consistent with our high-content imaging analysis, the total mitochondrial volume was also greater in HPLM-differentiated macrophages (Fig. 6E).Together with LD and mitochondria, peroxisomes are central modulators of lipid metabolism that actively participate in metabolic processes such as fatty acid beta-oxidation and ether phospholipid synthesis (24).We quantified the peroxisome numbers per cell using single-cell high-content imaging (25) and found that X-VIVO 15-differentiated macrophages with either M-CSF or GM-CSF showed an increased number of peroxisomes per cell compared with iPSDM cultured in OXM or HPLM (Fig. 6F and G).Altogether, these data show that media composition is a main factor regulating macrophage metabolism-related organelle content, particularly lysosomes, LD, mitochondria, and peroxisomes with changes in metabolism.

Lipid droplet content defines M. tuberculosis replication outcomes in human macrophages
To validate whether the enrichment in LD content and lipid metabolism-associated pathways that we observed using non-physiological cell culture media has an impact on the macrophage host response, we pharmacologically modulated the LD content.Using the diacylglycerol acyltransferase 1 (DGAT1) inhibitor pradigastat (PDG) (18), we almost completely blocked LD production in X-VIVO 15-differentiated macrophages (Fig. 7A and  B).In contrast, HPLM-differentiated macrophages, characterized by a small abundance of LD, significantly enhanced their LD abundance in the presence of triacylglycerolenriched very low-density lipoproteins (VLDL) (26, 27) (Fig. 7A through C).Importantly, PDG-treated showed an increase in Mtb replication after 72 h of infection compared with X-VIVO 15-differentiated macrophages.On the other hand, HPLM-differentiated macrophages in the presence of VLDL particles presented a more restrictive environment for Mtb (Fig. 7E and F).Altogether, our data highlight the phenotypic impact of the cell culture media that closely reflects the metabolic profile of human plasma on organelle function and provides evidence that LD content is an intracellular determinant for Mtb replication and host control of infection.

DISCUSSION
Conventional cell culture media, formulated decades ago, inadequately replicate the metabolic composition of human blood (7).Despite the advancing understanding of environmental influences on metabolism, these traditional media continue to serve as the standard for in vitro studies across diverse biological research disciplines (7).The emergence of physiologic media, along with other initiatives aimed at enhancing the modeling capabilities of cell culture (28), harbors significant promise for advancing our understanding of fundamental cellular processes (5,(29)(30)(31).This is particularly relevant in the context of the establishment of defined and robust culture conditions for iPSC and iPSC-differentiated cell types.
Here, we investigated how media composition influences human macrophage organelle content and activity in the context of host-pathogen responses to M. tuberculosis infection.We found profound changes at the transcriptional, metabolic,  and organelle levels in macrophages cultured in traditional media compared with those cultured in media that closely reflect metabolite availability in human blood.iPSDM cultured in OXM or X-VIVO15 were characterized by enrichment in lipid metabo lism transcriptional programs, reduced metabolic plasticity, and diminished lysosomal proteolytic activity and mitochondrial dynamics.The role of host lipids, and particularly LD, during mycobacterial infections has received significant attention in the context of tuberculosis (TB) pathogenesis, often yielding contrasting results that vary based on the cell type, infection conditions, and species studied (18,26,(32)(33)(34).However, whether these differences might be influenced by or caused by the cell culture media employed has not been characterized.Our results suggest that the cell culture media employed could be a critical factor to consider when interpreting results in the context of LD in macrophages.In agreement with previous evidence showing a protective role for LD in the host defense to Mtb (18,33) and other mycobacteria (26), we identified in iPSDM that increased LD content induced by OXM or X-VIVO15 culture media created a restrictive environment for Mtb replication in comparison to HPLM-differentiated iPSDM.
Traditionally, macrophage phenotypes and subtype classification have mainly been defined by distinct cell surface markers (35).However, our data challenge this paradigm, showing that macrophages with similar surface marker profiles can exhibit substantial variations in organelle content and metabolic activity.These differences significantly impact their effector functions, as demonstrated in their response to Mtb.Consequently, a more comprehensive analysis, including the quantification of organelles and metabolic characterization, would enhance our understanding of the full spectrum of macrophage states, from homeostasis to activation, revealing the nuanced differences that contribute to their functional heterogeneity.
Increasing evidence demonstrates that understanding organellar homeostasis is central to efforts aiming to identify new innate immune pathways (36).Our data, revealing the contrasting organelle and metabolic functions of human iPSDM cultured in different media, underscores the importance of adopting physiologic media as a standardized practice for in vitro functional studies.There is also a significant impact of medium composition on gene essentiality in human cells (29).Considering these findings, we anticipate that our study will pave the way for future investigations aimed at unraveling molecular mechanisms that are only discernible when employing a human plasma-like medium.

Cells iPSC and iPSDM culture
KOLF2 human iPSCs were sourced from Public Health England Culture Collections (catalog numbers 77650059 and 77650100, respectively) and maintained in Vitronectin XF (StemCell Technologies) coated plates with E8 medium (ThermoFisher Scientific).Cells were authenticated by short tandem repeats (STR) profiling upon receipt and were checked monthly for Mycoplasma contamination by PCR.Cells were passaged 1:6 once at 70% confluency using Versene (Gibco).Monocyte factories were set up following a previously reported protocol (van Wilgenburg et al., (1).Briefly, a single cell suspension of iPSCs was produced with TryplE (Gibco) at 37°C for 5 min and resuspended in E8 plus 10 µM Y-27632 (Stem Cell Technologies) and seeded into AggreWell 800 plates (StemCell Technologies) with 4 × 10 6 cells/well and centrifuged at 100 g for 3 min.The forming embryonic bodies (EBs) were fed daily with two 50% medium changes with E8 supple mented with 50 ng/mL hBMP4 (Peprotech), 50 ng/mL hVEGF (Peprotech), and 20 ng/mL hSCF (Peprotech) for 3 days.On day 4, the EBs were harvested by flushing out of the well with gentle pipetting and filtered through an inverted 40 µm cell strainer.EBs were seeded at 100-150 EBs per T175 or 250-300 per T225 flask in factory medium consisting of OXM (4) supplemented with Glutamax (Gibco), 50 µM β-mercaptoethanol (Gibco), 100 ng/mL hM-CSF (Peprotech), and 25 ng/mL hIL-3 (Peprotech).These monocyte factories were fed weekly with factory medium for 5 weeks until plentiful monocytes were observed in the supernatant.Up to 50% of the supernatant was harvested weekly and factories were fed with 10-20 mL factory medium.The supernatant was centrifuged at 300 g for 5 min, and cells were resuspended in X-VIVO15 (2) (Lonza), OXM (4), or HPLM (A4899101) (3) supplemented with 100 ng/mL hM-CSF and plated at 4 × 10 6 cells per 10 cm petri dish to differentiate over 7 days.HPLM medium was also supplemented with 10% dialyzed FBS (26400044, Thermo Fischer) as previously described (3).On day 4, a 50% medium change was performed.To detach cells, iPSDM plates were washed once with PBS and then incubated with EDTA for 15 min at 37°C and 5% CO 2 before diluting 1:3 with PBS and gently scraping.Macrophages were centrifuged at 300 g and plated for experiments containing the respective media.

Mtb infection
Mtb experiments were performed under BSL-3 conditions.Mtb H37Rv WT and Mtb H37Rv ΔRD1 were kindly provided by Prof. Douglas Young (The Francis Crick Institute, UK) and Dr Suzie Hingley Wilson (University of Surrey, UK).Fluorescent Mtb strains were generated as previously reported (37).E2Crimson Mtb was generated by transformation with pTEC19 (Addgene 30178, deposited by Prof. Lalita Ramakrishnan).Strains were verified by sequencing and tested for PDIM positivity by thin-layer chromatography of lipid extracts from Mtb cultures.Mtb strains were cultured in Middlebrook 7H9 supplemented with 0.2% glycerol, 0.05% Tween-80, and 10% albumin dextrose catalase (ADC).Mid-logarithmic-phase bacterial cultures (OD 600 0.5-1.0)were centrifuged at 2,000 × g for 5 min and washed twice in PBS.Pellets were then shaken vigorously for 1 min with 2.5-3.5 mm glass beads (VWR, 332124 G), and bacteria were resuspended in 10 mL macrophage culture medium before being centrifuged at 300 × g for 5 min to remove large clumps.The top 7 mL of bacterial suspension was taken; the OD 600 was recorded, and the suspension was diluted appropriately for infection.To maintain consistency with the high-content organelle activity evaluations, HPLM medium was used to prepare the bacterial solution and was also used for the 2-hour uptake time point for all conditions.After 2 hours of uptake, extracellular bacteria were removed with two washes in PBS, and macrophages were incubated at 37°C and 5% CO 2 in the corresponding media.At the required time post-infection, cells were collected or fixed in 4% paraformaldehyde (PFA).A target multiplicity of infection (MOI) of 1 was used for all the experiments, assuming OD 600 of 1 is 1 × 10 8 bacteria mL −1 .
After finishing the assay, cells were fixed with PFA 4% for 15 min.After that, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and imaged using an EVOS microscope (Thermo Fischer).The Analyze Particles command from ImageJ was used for nuclei quantification, and when required, cell number normalization was performed.WAVE software, version 2.6.1 (Agilent Technologies), was used for data analysis.

Imaging
High-content live-cell imaging iPSDM (30,000) were seeded into a 96-well flat-bottom PhenoPlate (PerkinElmer).After overnight incubation, iPSDM were imaged after incubation with the indicated probes or infected with M. tuberculosis as described above.The plate was sealed with parafilm and placed in a pre-heated (37°C) Opera Phenix microscope (PerkinElmer) with 5% CO 2 .Water-immersion lenses (40× or 60×) were used, and capture settings were as follows: BODIPY 493/503 (D3922, Thermo Fischer) was excited with the 488 nm laser at 5% power with 100 ms exposure, Image-iT TMRM (I34361, Thermo Fischer) was excited with the 561 nm laser at 10% power with 100 ms exposure, and iABP probe and Mtb E2crimson were excited with the 640 nm laser at 10% power with 100 ms exposure.DAPI was excited with the 405 nm laser at 20% power with 100 ms exposure.At least 20 fields per well were imaged in all the experiments.Images were acquired at 1,020 × 1,020 pixels using Harmony 4.9 high-content imaging and analysis software (PerkinElmer).

Organelle staining in live cells
After the indicated treatments, cells were washed once with PBS and incubated at 37°C and 5% CO 2 with Image-iT TMRM Reagent (1:1,000 solution, 20 min); BODIPY 493/503 (1 mg/L solution, 30 min); iABP probe (1 mm solution, 30 min) for the evaluation of mitochondrial membrane potential, LD content and lysosomal proteolytic activity, respectively.To avoid differences in probe uptake dependent on the media, the probes were always resuspended in HPLM, and all the measurements were carried out in the same media.Nuclear staining was done using 300 nM DAPI (Life Technologies, D3571), or NucBlue Live ReadyProbes Reagent (Hoechst 33342) and incubated simultaneously with the mitochondrial probe.After that, cells were gently washed with PBS and replaced with fresh HPLM media before imaging acquisition.Fluorescence intensities were quantified after single-mitochondrion and single-cell segmentation as described later (imaging analysis).

PEX14 staining
After the indicated treatments, iPSDM were washed once with PBS and fixed with 4% methanol-free PFA in PBS for 15 min.After three washes with PBS, cells were permea bilized using a 0.5% Triton X-100 (Sigma)/PBS solution for 10 min.Cells were then immunostained using anti-PEX14 antibody (1:400 dilution) (ab183885, Abcam).After 1 h, cells were washed twice with PBS and incubated with a 1:700 solution of anti-rabbit Alexa Fluor 546 antibody (A-11035, Thermo Fischer).Antibodies were diluted in PBS containing 5% FBS and incubated for 1 h at room temperature.iPSDM were then washed with PBS, and DAPI was added for nuclear detection and cell segmentation (imaging analysis).

Super-resolution live-cell imaging
iPSDM incubated with Image-iT TMRM were imaged on a VT-iSIM super-resolution imaging system (Visitech International), using an Olympus IX83 microscope, 100 ×/1.5 Apochromat objective (Olympus), ASI motorized stage with piezo Z, and 2 × Prime BSI Express scientific CMOS cameras (Teledyne Photometrics).Cells were always in the stage incubator at 37°C and 5% CO 2 .mCherry imaging was done using the 560 nm laser excitation and ET600/50 m emission filters (Chroma).Z-stacks (100 nm z-step) were acquired at the intervals indicated in the figure legends.The microscope was controlled with CellSens software (Olympus).

FIG 1
FIG 1 iPSDM cultured in the physiological medium are more permissive to M. tuberculosis replication.(A) Schematic illustrating the experimental conditions evaluated.(B, C) Quantification of Mtb replication (bacteria area per cell) in iPSDM differentiated with M-CSF or GM-CSF and cultured with the indicated media, n = 3, two-way analysis of variance (ANOVA).(D) Representative images of Mtb-infected iPSDM after 72 h (MOI = 1) and cultured in different media.Scale bar = 20 mm.(E-G) Quantification of Mtb replication (bacteria area per cell) in iPSDM differentiated with M-CSF or GM-CSF and cultured with X-VIVO15, OXM, or HPLM, n = 3, two-way ANOVA, Sidak's multiple comparison test.

FIG 2
FIG 2 iPSDM cultured in a physiological medium shows a decrease in lipid metabolism-associated transcriptional signature.Top 25 pathways significantly enriched in GSEA (P adj < 0.05) ranked by NES (normalized enrichment score).(A, B) Plots show the GSEA results of iPSDM differentiated with M-CSF and cultured with X-VIVO15 or OXM in comparison with HPLM.(C, D) Plots show the GSEA results of iPSDM differentiated with GM-CSF and cultured with X-VIVO15 or OXM in (Continued on next page)

FIG 2 (
FIG 2 (Continued) comparison with HPLM.n = 3 technical replicates.Note that only the top 25 regulated pathways are plotted; for a comprehensive list of all significantly regulated pathways, please refer to Table S1 at https://doi.org/10.25418/crick.26048884.(E) Venn diagrams show upregulated (left) or downregulated (right) DEGs (filtered by fold change > 1.2 and adjusted P-value < 0.05 when comparing GM-CSF with M-CSF) in each culture medium.

FIG 4
FIG 4 iPSDM cultured in physiological medium changes iPSDM protease activity and lipid droplet content.(A, B) BODIPY intensity density plot (left) and representative images (right) of iPSDM differentiated with M-CSF or GM-CSF and cultured with the indicated media in the presence of BODIPY.(C, D) Bar plots show BODIPY intensity quantification of iPSDM differentiated with M-CSF and GM-CSF and cultured with the indicated media.P values were calculated using ANOVA, Tukey post-hoc test.(E) BODIPY intensity quantification showing the effect of M-CSF and GM-CSF differentiation program on iPSDM cultured with the indicated media.P values were calculated using a two-way ANOVA, Sidak's multiple comparison test.(F, G) iABP (lysosomal protease activity-based probe) intensity density plot (left) and representative images (right) of iPSDM differentiated with M-CSF or GM-CSF and cultured with the indicated media in the (Continued on next page)

FIG 4 (
FIG 4 (Continued) presence of iABP probe.(H, I) Bar plots show iABP intensity quantification of iPSDM differentiated with M-CSF and GM-CSF and cultured with the indicated media.P values were calculated using ANOVA, Tukey post-hoc test.(J) iABP intensity quantification showing the effect of M-CSF and GM-CSF differentiation program on iPSDM cultured with the indicated media.P values were calculated using a two-way ANOVA, Sidak's multiple comparison test.n = 3 independent experiments, scale bar: 10 mm.

FIG 5
FIG 5 Cell culture media modulates mitochondrial activity.(A, B) iTMRM (mitochondrial activity probe) intensity density plot (left) and representative images (right) of iPSDM differentiated with M-CSF or GM-CSF and cultured with the indicated media in the presence of iTMRM.(C, D) plots show iTMRM intensity quantification of iPSDM differentiated with M-CSF and GM-CSF and cultured with the indicated media.P values were calculated using ANOVA, Tukey post-hoc test.(E) iTMRM intensity quantification showing the effect of M-CSF and GM-CSF differentiation program on iPSDM cultured with the indicated media.P values were calculated using a two-way ANOVA, Sidak's multiple comparison test.(F, G) Density plots showing the mitochondrial area values (normalized to cell area) of iPSDM differentiated with M-CSF and GM-CSF and cultured with the indicated media.(H, I) Density plots showing the mean mitochondrial length values of iPSDM differentiated with M-CSF and GM-CSF and cultured with the indicated media.n = 3 independent experiments, scale bar: 10 mm.

FIG 6
FIG 6 Cell culture media impacts on mitochondrial dynamics.(A) Live-cell super-resolution imaging evaluating mitochondrial dynamics in iPSDM differentiated with M-CSF and cultured using X-VIVO15 (top panel) or HPLM (bottom panel).Scale bar: 10 mm and 2 mm (zoom-in).(B) Schematic showing the 4D analysis sequence.(C-E) Dot plots show the quantification of mitochondrial mean branch length, number of branches, and volume over 20 time points (30 s timeframe) from one out of three independent experiments using iPSDM as described in as in panel A. (F, G) Histogram showing the quantification of PEX14-positive peroxisome spots per cell and representative images of iPSDM differentiated with M-CSF or GM-CSF, cultured in the indicated media and stained for PEX-14.n = 3 independent experiments.Scale bar: 20 mm

FIG 7
FIG 7 Culture media-induced lipid droplet content generates iPSDM more permissive or restrictive to M. tuberculosis replication.(A) Representative images of BODIPY-stained M-CSF differentiated iPSDM cultured in XVIVO-15 in the presence or absence of pradigastat (PDG).(B) Bar plots show BODIPY intensity quantification of iPSDM cultured in X-VIVO15 in the presence or absence of PDG.(C) Representative images of BODIPY-stained M-CSF differentiated iPSDM cultured in HPLM in the presence or absence of VLDL particles.(D) Bar plots show BODIPY intensity quantification of iPSDM cultured in HPLM in the presence or absence of VLDL particles.n = 3 independent experiments, t-test.(E) Scheme illustrating the experimental strategy to evaluate the impact of LD on Mtb replication.(F) Quantification of Mtb replication (bacteria area per cell) in iPSDM cultured in X-VIVO15 in the presence or absence of PDG.(G) Quantification of Mtb replication (bacteria area per cell) in iPSDM cultured in HPLM in the presence or absence of VLDL particles.n = 3 independent experiments, two-way ANOVA, Sidak's multiple comparison test.