We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in greater release of Ca2+ from the endoplasmic reticulum stores, which triggers sustained Ca2+ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility.The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer’s disease (AD). Because abnormalities in Ca Ca2+ depletion from the ER in turn activates ER STIM1 proteins to translocate proximally to puncta where closely apposed plasma membrane (PM) Orai1 channels are activated. This mechanism underlies store-operated Ca2+ entry (SOCE) in many cell types  transcriptional states . P2Y12 ay by ADP . P2Y12 rl injury , NLRP3 il injury , neuronal injury , and blol injury . While pl injury , mechani2+ signaling (2+ responses in mouse models of Alzheimer’s disease (AD) by mechanisms that are not fully understood , and amyloid-β peptides. Upon ligand binding, TREM2 signals through its adaptor protein DAP12 to activate a host of downstream pathways . Loss ofel of AD , althoug2+ signaling has not yet been extensively profiled in these models. In this study, we compared purinergic Ca2+ signaling and motility characteristics in wild type (WT) and TREM2 knockout (KO) human iPSC-microglia, and examined the mechanisms that underlie enhanced purinergic Ca2+ signaling in microglia lacking TREM2. We find that motility is differentially tuned by Ca2+ in TREM2 KO cells with consequences for chemotaxis.We previously developed methods to generate human induced pluripotent stem cell-derived microglia (iPSC-microglia) , which c2+ signaling, we compared cytosolic Ca2+ responses to the purinergic agonist ADP in isogenic, CRISPR-modified wild type (WT) and TREM2 KO human iPSC-microglia. ADP stimulation induced a biphasic Ca2+ response – a rapid initial peak followed by a secondary phase of sustained Ca2+ elevation lasting several minutes, in line with previous observations in mouse microglia  in both WT and TREM2 KO microglia  inhibitor, thapsigargin (TG), in both WT and TREM2 KO microglia  isoforms in WT and TREM2 KO cells  in WT and TREM2 KO cells  and P2Y13 (MRS 2211) receptor antagonists, confirming the role of these two purinergic receptors in ADP enhancement of microglial motility . Furthermore, even before process extension was activated with ADP, cells treated with P2Y antagonists showed significantly fewer and shorter processes, suggesting that baseline purinergic signaling may regulate resting microglial process dynamics. Altogether, these results demonstrate that activation of purinergic signaling through P2Y12 and P2Y13 receptors is required for ADP-driven microglial process extension and motility.In addition, some microglia responded to ADP by extending processes and altering their morphology rather than increasing motility . Microgl2+ influx, we analyzed microglial motility in response to ADP stimulation in the absence of extracellular Ca2+  vs. time and compared cell track overlays (flower plots), which showed that ADP enhances motility in TREM2 KO cells to a greater extent than in WT microglia . Baselinlar Ca2+ . MSD andKO cells . In the WT cells . We conc,2+-free medium did not induce process extension in WT cells, and only a modest increase in TREM2 KO cells  Ca2+ response is completely abrogated in the presence of P2Y12 and P2Y13 receptor inhibitors; (2) RNA-sequencing data shows significant increase in expression of P2Y12 and P2Y13 receptor transcripts but minimal fold change in other regulators of Ca2+ signaling ; and (3) labeling of surface P2Y12 receptors shows greater PM expression in the TREM2 KOs. Furthermore, functional assays rule out any role for Ca2+ clearance mechanisms or any difference in maximal IP3 and SOCE activity as a cause of increased sustained Ca2+ signal in TREM2 KO cells. Mechanistically, this increase in Ca2+ signals is driven by enhanced IP3-mediated ER store release coupled to SOCE. Indeed, based on the dose–response curves for peak ADP-Ca2+ responses in Ca2+-free buffer, TREM2 KO cells have an EC50 at least 10-fold lower than WT cells. As a functional consequence, TREM2 KO microglia exhibit a defect in turning behavior and show greater displacement over time despite moving with similar speeds as the WT cells. The increased frequency in turning in WT microglia (relative to TREM KO cells) reflects greater canceling of the velocity vectors, which take the direction of motility into account. This restricts cell motility to more confined regions, potentially allowing for more frequent path correction. It is important to note that these motility differences with ADP are observed after acute treatment and in the absence of any gradient.We provide several lines of evidence to show that hyper-responsiveness to purinergic ADP signaling in TREM2 KO microglia is driven primarily by increased purinergic P2Y2+ signaling in TREM2 KO cells may result in Ca2+ signaling domains that are no longer restricted to the cell region near to the highest ADP concentrations and disrupt the polarity of key signaling molecules that drive directed cell motility.Interestingly, deletion of TREM2 had no significant impact on scratch wound closure rates, over a time scale of 24 hr in the presence of a constant concentration of ADP . However2+ signals shape specificity of downstream cellular responses. Our experiments with ADP in Ca2+-free medium revealed that a transient Ca2+ signal is insufficient to induce microglial motility in either WT or TREM2 KO cells. Previous studies have shown that mouse microglia with genetic deletion of STIM1 or Orai1 also show defects in cell migration to ATP  and -independent (DAG) arms of the GPCR signaling pathway may be useful to control microglial activation and motility. However, our results suggest that altering downstream Ca2+ flux may be sufficient, and thus, CRAC (Orai1) channel blockers that would specifically inhibit the sustained Ca2+ signals without affecting the initial Ca2+ transient or the activation of DAG may provide a more targeted approach.The studies presented here provide evidence that reducing purinergic receptor activity may be clinically applicable in Alzheimer’s patients with TREM2 loss-of-function mutations . Pharmac2+ in human iPSC-microglia is critical. Beyond TREM2, we have found that protective variants in MS4A6A and PLCG2 gene expression also decrease P2Y12 and P2Y13 receptor expression (unpublished data), suggesting that this mechanism of microglial activation could be common across several microglial AD risk loci.Currently, TREM2 activating antibodies are being examined in early stage clinical trials for AD , making 2+ influx, which increases cell displacement by decreasing cell turning. Despite this, TREM2 KO microglia show a defect in chemotaxis that is likely due to their inability to sense ADP gradients and make appropriate course corrections. Decreasing purinergic signaling in TREM2 KO microglia rescues directional chemotactic migration. We suggest that purinergic modulation or direct modulation of Ca2+ signaling could provide novel therapeutic strategies in many AD patient populations, not solely those with reduced TREM2 function.In summary, deletion of TREM2 renders iPSC-microglia highly sensitive to ADP, leading to prolonged Cahere and here.Human iPSC lines were generated by the University of California, Irvine Alzheimer’s Disease Research Center (UCI ADRC) Induced Pluripotent Stem Cell Core from subject fibroblasts under approved Institutional Review Boards (IRB) and human Stem Cell Research Oversight (hSCRO) committee protocols. Informed consent was received from all participants who donated fibroblasts. Reprogramming was performed with nonintegrating Sendai virus in order to avoid integration effects. To validate the karyotype and identity of iPSC lines, cells were examined via Microarray-based Comparative Genomic Hybridization . Sterility and confirmation of mycoplasma negativity was examined every 10 passages and proceeding experimentation via MycoAlert (Lonza). Pluripotency was verified by Pluritest Array Analysis and trilineage in vitro differentiation. Additional GFP- and RFP-αtubulin-expressing iPSC lines (AICS-0036 and AICS-0031-035) were purchased from Coriell and originally generated by Dr. Bruce Conklin. Each Coriell line is provided with a corresponding certificate of analysis that verifies the correct reporter sequence insertion site, lack of plasmid integration, growth rate, expression of pluripotency markers, normal karyotype, sterility including mycoplasma negative, and identity of line via short tandem repeat (STR). See cgctgaccacgactacccac) targeting the second exon of ORAI1. The resulting ORAI1 clones were then validated to exhibit a normal for karyotype, identity, pluripotency, and sterility via Microarray-based Comparative Genomic Hybridization , tri-lineage differentiation, and MycoAlert mycoplasma testing.Genome editing to delete TREM2 was performed as in iPSC-microglia were generated as described in 2+ imaging, microglia process, and cell motility analysis are indicated in the respective sections.Unless otherwise stated, cells were imaged on an Olympus FV3000 confocal laser scanning inverted microscope equipped with high-speed resonance scanner, IX3-ZDC2 Z-drift compensator, 40× silicone oil objective (NA 1.25), and a Tokai-HIT stage top incubation chamber (STXG) to maintain cells at 37°C. To visualize Salsa6f, 488 nm and 561 nm diode lasers were used for sequential excitation of GCaMP6f  and TdTomato , respectively. Fluo-4 and Fura-red were both excited using a 488 nm diode laser . Two high-sensitivity cooled GaAsP PMTs were used for detection in the green and red channels, respectively. GFP was excited using the same settings as GCaMP6f. Other image acquisition parameters unique to Ca2+ imaging was done using Fluo-4 AM and Fura-red AM dyes as described previously  1 or 2 mM Ca2+ Ringer’s solution comprising 155 mM NaCl, 4.5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH adjusted to 7.4 with NaOH); (2) Ca2+-free Ringer’s solution containing 155 mM NaCl, 4.5 mM KCl, 1.5 mM MgCl2, 10 mM glucose, 1 mM EGTA, 10 mM HEPES, pH 7.4. Live-cell imaging was performed as described earlier. Cells were treated with ADP as indicated in the ‘Results’ section.iPSC-microglia were plated on fibronectin-coated (5 μg/mL) glass-bottom 35 mm dishes  overnight at 60% confluence. Ratiometric Caeviously . BrieflyTime-lapse images were acquired in a single Z-plane at 512 × 512 pixels (X = 318.2 μm and Y = 318.2 μm) and at 2–3 s time intervals using Olympus FV3000 software. Images were time averaged over three frames to generate a rolling average and saved as .OIR files.https://imagej.net/Fiji), converted to TIFF files (16-bit), and background-subtracted. Single-cell analysis was performed by drawing ROIs around individual cells in the field, and average pixel intensities in the green and red channels were calculated for each ROI at each time point. GCaMP6f/ TdTomato (G/R Ratio) and Fluo-4/Fura-red ratio was then obtained to further generate traces showing single-cell and average changes in cytosolic Ca2+ over time. Single-cell ratio values were used to calculate peak Ca2+ signal and responses at specific time points after agonist application as previously reported /Δt(s–1) over a 10 s time frame of maximum initial rise after Ca2+ addback. Area under the curve (AUC) was calculated using the AUC function in GraphPad Prism.Time-lapse videos were exported to Fiji- and 30 min after application of ADP. Images were acquired as a Z-stack using the Galvo scanner at Nyquist sampling. Adjacent fields of view were combined using the Stitching function of the Olympus FV3000 Software and saved as .OIR files.https://imagej.net/OlympusImageJPlugin). Maximum intensity projection (MIP) image from each Z-stack was used for further processing and analysis. MIP images were converted to 8-bit grayscale images, to which a threshold was applied to obtain 8-bit binary images. The same threshold was used for all sets of images, both before and after ADP application. Noise reduction was performed on the binary images using the Process -> Noise -> Unspeckle function. Outlier pixels were eliminated using Process -> Noise -> Outliers function. The binary images were then skeletonized using the Skeletonize2D/3D Plugin for ImageJ (https://imagej.net/plugins/skeletonize3d). Sparingly, manual segmentation was used to separate a single skeleton that was part of two cells touching each other. The Analyze Skeleton Plugin  was then applied to the skeletonized images to obtain parameters related to process length and number of branches for each cell in the imaging field. Processes were considered to be skeletons > 8 μm. The data was summarized as average process length and number of branches, before and after ADP application for a specific imaging field, normalized to the number of cells in the field that allowed for pairwise comparison. Additionally, single-cell data across all experiments were also compared in some instances.The basic workflow for microglia process analysis was adapted from 3, a poorly metabolized IP3 analog, was performed as previously described  plus 0.1% Pluronic F-127 in Microglia Basal Medium. Cells were washed and incubated in the dark for further 30 min in a HEPES-buffered salt solution (HBSS) whose composition was (in mM) 135 NaCl, 5.4 KCl, 1.0 MgCl2, 10 HEPES, 10 glucose, 2.0 CaCl2, and pH 7.4. Intracellular Ca2+ ([Ca2+]i) changes were imaged by employing a Nikon Eclipse Ti microscope system (Nikon) equipped with a 40× oil immersion objective  and an Orca Flash 4.0LT CMOS camera (Hamamatsu). Cal520 or Cal590 were excited by a 488 or a 560 nm laser light source (Vortran Laser Technologies), respectively. i-IP3 uncaging was achieved by uniformly exposing the imaged cells to a single flash of ultraviolet (UV) light (350–400 nm) from a xenon arc lamp. UV flash duration, and thus the amount of released i-IP3 was set by an electronically controlled shutter.Whole-field uncaging of i-IPescribed  with minhttps://flika-org.github.io/; 0, where F0 is the basal fluorescence intensity and ∆F the relative fluorescence change (Fx – F0). Data are reported as superplots (t-test.Image acquisition was performed by using Nikon NIS (Nikon) software. After conversion to stack TIFF files, image sequences were analyzed with Flika, a custom-written Python-based imaging analysis software . Plates were washed 3× before addition of secondary antibodies  and Hoechst (Thermo Fisher Scientific). Images were captured on an Olympus FV3000RS confocal microscope with identical laser and detection settings. Images were analyzed with Imaris 9.7.0 software. We note that our attempt to verify Orai1 expression at the protein level was unsuccessful as the antibody used  did not stain WT microglia in either immunostaining or western blot experiments.g for 5 min at 4°C. The cell pellet was subsequently resuspended in FACS buffer (1× PBS + 0.5% FBS). Fc receptors were blocked with a blocking buffer (BioLegend TruStain FcX in 1× PBS + 10% FCS). Cells were then incubated with Brilliant Violet 421-labeled anti-human P2Y12 receptor antibody  or with IgG2a isotype control antibody  for 30 min at 4°C. Cells were washed, pelleted, and then resuspended in FACS buffer. Clone S16001E binds to the extracellular domain of the P2Y12 and permits labeling of PM P2Y12 receptors. Data were acquired using Novocyte Quanteon flow cytometer (Agilent) and analyzed using FlowJo analysis software .iPSC-derived microglia were seeded on fibronectin-coated 12-well plates at 200,000 cells/well. Cells were harvested and centrifuged in FACS tubes at 300 × Nondirectional motility was analyzed using Essen Incucyte WoundMaker. iPSC-microglia were plated on fibronectin (STEMCELL Technologies) at 90% confluence. Scratches were repeated 4× to remove all cells from the wound area. Scratch wound confluency was imaged every hour until scratch wound was closed (15 hr). Confluence of cells within the original wound ROI was calculated using IncuCyte 2020C software.2+ fluorescence and motility per cell. In both conditions, tracks were defined by Brownian motion with the maximum distance jump of 4 µm and 10 frame disturbance with no gap filling. Tracks shorter than 3 min in length were eliminated from analysis. After automated track formation, tracks underwent manual quality control to eliminate extraneous tracks, merge falsely distinct tracks, and add missed tracks. After export, data was plotted in Prism 9.1.0 or analyzed in Excel using DiPer Macros for Plot_At_Origin (translation of each trajectory to the origin) and MSD(t) = 4D(t-P(1-e^(-t/P))), where D is the diffusion coefficient, t is time, and P represents directional persistence time   . From Im+ clones. Genomic DNA was extracted from positive clones using Extracta DNA prep for PCR (Quantabio) and amplified using Taq PCR Master Mix (Thermo Fisher Scientific) to confirm diallelic integration of the Salsa6f cassette. A clone confirmed with diallelic Salsa6f integration in the AAVS1 SHL was then retargeted as previously described . Salsa6f-AAVS1 SHL plasmid template  and RNP complex formed by incubating Alt-R S.p. HiFi Cas9 Nuclease V3  was fused with crRNA:tracrRNA (IDTDNA) duplex for 15 min at 23°C. This complex was combined with the cellular suspension and nucleofected using the Amaxa Nucleofector program B-016. To recover, cells were plated in TeSR-E8 (STEMCELL Technologies) media with 0.25 μM thiazovivin (STEMCELL Technologies) and CloneR (STEMCELL Technologies) overnight. The following day, cells were mechanically replated to 96-well plates in TeSR-E8 media with 0.25 μM thiazovivin and CloneR supplement for clonal isolation and expansion. Plates were screened visually with a fluorescence microscope to identify TdTomatoescribed  to knockStaphylococcus aureus (Thermo Fisher Scientific), or 2  μg/mL fluorescent beta-amyloid (AnaSpec). Image masks for fluorescence area and phase were generated using IncuCyte 2020C software.Phagocytosis of transgenic iPSC-microglia was validated using IncuCyte S3 Live-Cell Analysis System (Sartorius) as in 12 receptors. To characterize motility, we monitored the number of recruited microglia in the central chamber for 4 days under the fully automated Nikon TiE microscope .iPSC-microglia were loaded into the angular chamber (2–5K cells/device) to test activation and chemotaxis toward the central chamber containing either ADP (100 ng/mL or 234 nM) or vehicle. When noted, PSB 0739 (10 μM) was added to both the central and angular chamber to inhibit P2Y2+ over time are shown as mean ± standard error of the mean (SEM). Accompanying bar graphs with bars depicting mean ± SEM provide a summary of relevant parameters  as indicated. Details of the number of replicates and the specific statistical test used are provided in the individual figure legends.GraphPad Prism (versions 6.01 and 8.2.0) was used to perform statistical tests and generate p-values. We used standard designation of p-values throughout the figures . Traces depicting average changes in cytosolic Ca Overall, this is a significant advance in the field of microglial regulation by calcium signaling. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Mohamed Trebak (Reviewer #2).Thank you for submitting your article "TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. Reviewers had concerns on two broad aspects of the paper: the calcium signaling studies, and the results relating to cell motility. In addition, other concerns were raised which are described in the individual reviewer comments.Essential revisions:2+ signaling proteins  that could affect the results. Westerns should be performed for STIM1 and STIM2, Orai1 and IP3R 1/2/3, for which there are reliable Abs. Although the IF as performed for Orai1 is acceptable, the authors do not show positive and negative controls and a thorough validation of this Orai1Ab in IF. This is strongly recommended given the poor quality of Orai1 Abs in general. Likewise, staining for P2YR12 appears internal and the Ab should be validated. A WB on P2YR12 and P2RY13 in addition would help.1) A more rigorous examination of protein expression should be performed with antibody validation to determine if there are compensatory changes at the protein level of other relevant Ca2) Important: It is clear that the TREM2 KO cells have significantly lower STIM1 expression, which would be expected to impact SOCE under conditions of physiological agonists stimulation . The conclusion based on maximal activation with thapsigargin does not address this issue. What is the impact of reduced STIM1 expression on the Ca signals and motility?2+ off/on protocol with ADP  as done for thapsigargin should be performed. In Figure 2B, ADP was applied in the absence of external Ca2+, but sadly those recordings did not subsequently replenish Ca2+ to gauge Ca2+ entry. Low concentrations of Gd3+  reduce the initial Ca2+ release peak, suggesting that there is a contribution of Ca2+ entry to this initial response. Also, it would have been helpful to include in Figure 1 Suppl1 the control (non KO cells) in these experiments for reference.3) A Ca4) The conclusion that SOCE is mediated by Orai1 should be validated with a genetic approach as 2-APB and La3+ are non-specific.5) Conversely, it is not clear if store content is depleted to a greater extent following ADP receptor stimulation in the KO cells. Assess ER calcium content in response to ADP in WT and KO cells or with an ionomycin pulse.6) Figure 3: The WT data in this figure don't include KO cells. How do the TREM2 KOs compare to WT in the scratch wound closure assay? The KO cells seem to be tested, but only in the absence of ADP . Is there a difference in wound closure after ADP treatment? This point should be evaluated to assess the effects of altered ADP Ca signaling on non-directional cell movement in a physiologically relevant paradigm.7) Terminology: The term "Ca clamp" in the way it has been used here could be misconstrued by readers. Cellular Ca is not clamped. It varies within each cell depending on the activity of the pumps and other clearance mechanisms in the cell. The motility measurements are simply done after extracellular Ca is restored to the cells after thapsigargin treatment, at time points where the cytosolic Ca is lower in 0.2 mM Ca compared to 2 mM Ca. This phrasing (Ca clamp) needs to be changed to eliminate confusion that cellular calcium is "clamped" analogous to voltage-clamp.8) Figure 6D TREM2 KOs (but not WT) cells show differences in motility between 0.2 mM and 2 mM extracellular Ca even after thapsigargin treatment with no ADP exposure. This is strong indication that motility differences in TREM2 KO cells are due to more than just alterations in purinergic signaling. . What is the role of these intrinsic changes in cell motility in response to cellular Ca elevations in the presence and absence of nucleotides?9) Figure 6D: The data for WT cells should be directly compared to the KOs at each extracellular Ca concentration to dissect out differences in KO cells at each Ca condition.10) The data in Figure 4K should be reanalyzed to compare differences in the absolute levels of branch complexity (# of branches and process length) in the two groups.11) The apparent disconnect between the larger mean square distance travelled in the absence of nucleotides in the KO with no change in the mean speed needs to be clarified. Is this due to cancelling of speed vectors with opposite directions?Reviewer #1 (Recommendations for the authors):1) Figure 3: The WT data in this figure don't include KO cells. How do the KOs compare to the WT in the scratch wound closure assay? The KO cells seem to be tested, but only in the absence of ADP . Is there a difference in wound closure after ADP treatment? This point should be evaluated to assess the effects of altered ADP Ca signaling on non-directional cell movement in a physiologically relevant paradigm.2) Figure 4: This reviewer is not convinced that reduced turning accounts for the similar speed in the KO despite the larger mean square distance travelled. If turning is reduced and the mean square distance is doubled in the KO, then effectively, the average distance travelled over time (speed) should be greater in the KO than in the WT. The logic of this inference and how it explains the data (similar average speed but much greater root mean square displacement in the KO) needs to be better clarified. Since the mean square distance is so much larger, it suggests that the speed vectors with opposite directions are cancelling out in the KO cells to yield the same average velocity .3) Figure 4K: It seems that the baseline branch complexity is low to start with in the KOs, hence the fold change from baseline seems larger. From the images shown, there appears to be no difference following ADP treatment between the two groups. This data should be reanalyzed to compare differences in the absolute levels of branch complexity (# of branches and process length) in the two groups.4) The use of the term Ca clamp is misleading in the way it has been used here. Cellular Ca is, in fact, not clamped, but changes within each cell depending on the activity of pumps and other calcium clearance mechanisms. The motility measurements are simply done after extracellular Ca is restored to cells after thapsigargin treatment, at time points where the cytosolic Ca is lower in 0.2 mM Cacompared to 2 mM Ca. This phrasing (Ca clamp) needs to be changed to eliminate confusion that cellular Ca levels are "clamped" to defined values analogous to voltage clamp.5) Figure 6D: It is noteworthy that TREM2 KOs, but not WT cells, show differences in motility between 0.2 mM and 2 mM extracellular Ca even after thapsigargin treatment with no ADP exposure. This is strong suggestion that motility differences in TREM2 KO cells are due to more than just alterations in purinergic signaling. In fact, this inference is consistent by the lack of additional effects of adding ADP in Figure 6 Supplement 1. These intrinsic changes in cell motility in response to cellular Ca elevations need further clarification.6) Figure 6D: Related to this figure, the data for WT cells should be directly compared to the KOs at each extracellular Ca concentration to dissect out differences in KO cells at each Ca condition.Other:– Stim1 is expressed at different levels between the WT and TREM2 KO. It has a significant p-value . This decrease could affect multiple aspects of microglia including cell motility in the presence of physiologically relevant agonists. This needs to be addressed.– A technical point related to Figure 4 is that figures 4C and 4G should be labelled "velocity" rather than speed, as it seems the only way in which the mean squared distance can be twice as large in the KO but not the velocity if the speed vectors with opposite directions are cancelling out in the KO.– The dose of ADP used in Figure 7 is extremely high. 100ng/mL . By contrast, Figure 4 used 2.5uM. The 100 fold higher concentration in the chemotaxis assay needs to be justified. Further, it should be determined whether the ADP used diffuses into both chambers might be expected. This super high dose of ADP may have non-physiological effects that could explain the seemingly differing phenotypes in Figures 4 and 7.– Figure 5 is a great set of experiments to show that the Salsa6f does not affect end points of the study, but may be better as a supplementary figure rather than a main figure. If the paper is about the TREM2 KO versus WT then the main figures should stick with looking at differences between the two with maybe a sub figure to show validation.Reviewer #2 (Recommendations for the authors):2+ signaling proteins . Preferably Westerns should be performed for STIM1 and STIM2, Orai1 and IP3R 1/2/3, for which there are reliable Abs. Although IF as performed for Orai1 is acceptable, the authors do not show positive and negative controls and a thorough validation of this Orai1Ab in IF.– A more rigorous approach could be used to document the presence or absence of compensatory changes at the protein protein level of other Ca– Along the same lines as comment above, Staining for P2YR12 appears internal. Has the Ab been validated? Can authors perform a WB on P2YR12 and P2RY13 in addition?– Did the authors check on whether the expression of SECRA is altered at the protein level? Did the authors perform CRAC channel recordings in Control and TREM2 KO cells?2+ off/on protocol with ADP  as they have done with thapsigargin. In Figure 2B, ADP was applied in the absence of external Ca2+, but sadly those recordings did not subsequently replenish Ca2+ to gauge Ca2+ entry. One can rationalize the result of 2-APB , a non-specific channel blocker that also inhibits IP3Rs. However, the fact that low concentrations of Gd3+  reduce the initial Ca2+ release peak, suggests that there is a contribution of Ca2+ entry to this initial response. Also, it would have been helpful to include in Figure 1 Suppl1 the control (non KO cells) in these experiments for reference.– It is quite surprising that the authors did not opt to use the CaAdditional thoughts to the attention of the authors:2+ might lead to a "spill" outside the restricted Ca2+ nanodomain, thus disrupting polarity. Do the authors have any evidence of altered location of leading edge vs trailing edge proteins or focal adhesion proteins in TREM2 KO cells?– The authors have speculated on the fact that increased cytosolic Ca– Have the authors performed RNAseq on WT control and TREM2 KO cells? If so, does pathway analysis shows changes in the Cell motility genes between these two groups? This might offer some interesting clues to pursue in future studies.Reviewer #3 (Recommendations for the authors):2+ responses to ADP stimulation in TREM2KO cells, and that this leads to a defect in turning behaviour and hence no directed migration to a chemotactic signal.This study by Jairaman et al. describes how iPSC-derived microglia exhibit exaggerated cytosolic Ca2+ responses to ADP and then extend their findings to cell movement and directed migration. Given the substantial body of evidence linking microglia to the pathogenesis of Alzheimer's disease, and the role for TREM2, the work by Jairaman et al. is of translational significance. As an aside, the introduction of the calcium-sensitive reporter Salsa6F is a welcome new tool in the arsenal for recording cytosolic calcium. Overall, this is an elegant, novel and important study. Nevertheless, I have a few comments/suggestions.Overall, the experiments are well conducted, carefully controlled and the findings are new and exciting. The authors nicely dissect out the underlying molecular basis for the exaggerated Ca2+ plateau to ADP in TREM2KO cells is due to enhanced Ca2+ release from the stores ie greater store depletion. Evidence is presented that maximal SOCE is not compromised in TREM2KO cells, that store content under resting conditions is unaffected, InsP3R activities are similar. But the authors do not demonstrate that store content falls more following ADP receptor stimulation in the KO cells. This could be shown by directly measuring ER calcium to ADP in WT and KO cells or by applying ADP in Ca-free solution and then assessing store content with an ionomycin pulse. The ionomycin response should be smaller in the TREM2KO cells, after ADP exposure.1. The authors argue that the increased Ca2+ release from the ER. However, the increase in P2Y12 protein levels is modest at best, and the increase for P2y13 is less than 2-fold. The authors' point would be strengthened by showing InsP3 levels are increased in the KO cells compared with WT ones, for the same dose of ADP. Single cell InsP3 probes are available. Alternatively, a population measurement could be carried out.2. The authors suggest that an increase in P2Y12 and P2Y13 receptor expression in the KO cells accounts for the increased Ca2+-permeable channels could contribute to the Ca2+ plateau. The authors should therefore knock down Orai1 or at least use more selective inhibitors such as Synta66 or the GSK compound.3. The evidence for an involvement of SOCE is based on two rather non-specific inhibitors (2-APB and Gd3+). P2YRs, like other GPCRs, activate TRPC proteins and these Ca4. The authors should include some controls to show the P2Y antibodies are indeed specific. For example, they could use a cell line that lacks P2Y12/13. In Figure 2I, J, what happens to the ADP response when both P2Y inhibitors are present at the same time? It would be important to show that the combination of P2Y12 and P2Y13 suppress ADP responses fully.5. The authors should add a justification for the concentrations of agonists they have used . A dose-response curve is included for ADP but a comment on the doses selected would be helpful.2+ influx in TREM2 KO cells leads to microglia motility and process extension to a greater extent than WT cells. This is attributed to the increased SOCE. If so, then one might expect raising external Ca2+ in WT cells to have the same effect. Is this the case?6. It is nicely shown that Ca2+ signal leads to a chemotaxis defect? The authors' data would suggest that chemotaxis might have a bell-shaped dependence on cytosolic Ca2+, with too little or too much impeding migration towards a cue.7. Is anything known mechanistically how the exaggerated Ca2+ rise or via a global cytosolic Ca elevation?8. Do the authors know whether the effects of SOCE are mediated through a local Ca9. Perhaps I missed something but Figure 1 S2(D) seems to show a significant decrease in STIM1 levels in the KO cells.10. The authors present SOCE as the peak signal remaining after 5 minutes. It is not clear whether the value has been subtracted from the pre-stimulation levels. In some graphs, the signal after 5 minutes looks the same as the resting level, but the bar charts show higher values. Reviewers had concerns on two broad aspects of the paper: the calcium signaling studies, and the results relating to cell motility. In addition, other concerns were raised which are described in the individual reviewer comments.Essential revisions:2+ signaling proteins  that could affect the results. Westerns should be performed for STIM1 and STIM2, Orai1 and IP3R 1/2/3, for which there are reliable Abs. Although the IF as performed for Orai1 is acceptable, the authors do not show positive and negative controls and a thorough validation of this Orai1Ab in IF. This is strongly recommended given the poor quality of Orai1 Abs in general. Likewise, staining for P2YR12 appears internal and the Ab should be validated. A WB on P2YR12 and P2RY13 in addition would help.1) A more rigorous examination of protein expression should be performed with antibody validation to determine if there are compensatory changes at the protein level of other relevant Ca2+ imaging readouts in combination with pharmacological and genetic tools to assess P2Y receptor sensitivity to agonists and antagonists and downstream signaling events that include IP3R activation, ER store-release, and SOCE in WT and TREM2 KO microglia. Given our focus on functional data that are concordant with RNA expression results and point toward the differential expression of P2Y receptors, we did not see a strong rationale for further dissecting the expression of various STIM, Orai, IP3R or pump isoforms at the protein level. New data in the paper strengthen this conclusion. These include the ionomycin and TG-pulse experiments  together with the ADP-Ca2+ addback experiments , showing that ADP depletes ER Ca2+ stores to a greater extent in TREM2 KO cells. The new data reinforce the conclusion that increased expression of P2Y receptors in TREM2 KO microglia drives downstream calcium signaling events.Our approach in this study relies on functional Ca12 and P2Y13 receptor antibodies as described below.We concur with the reviewer that appropriate antibody controls are needed to support data related to protein expression, and we attempted to address this issue for the Orai1 antibody and P2Y2+ measurements in the newly generated Orai1 KO iPSC-microglia cell-line, and with a more specific pharmacological inhibitor of Orai channels (Synta66) to unambiguously establish the role of Orai1 in mediating SOCE and in maintaining sustained Ca2+ signals by ADP in microglia .In response to reviewer comments, we generated Orai1 CRISPR-knockout iPSC-microglia and further tested the Orai1 Ab  for immunostaining in WT and Orai1 KO iPSC-microglial cell line; antibody staining was found to be nonspecific. We therefore agree with the reviewer that, because of poor quality, Orai1 antibody staining does not provide solid and quantifiable evidence for Orai1 expression or function. Accordingly, we have removed Orai1 immunofluorescence staining data  from the revised manuscript. Instead, we performed Ca12 and P2Y13 receptors to validate the P2Y12 and P2Y13 receptor antibodies  for immunostaining. We agree with the reviewer that there is significant non-membrane staining of the cells with the P2Y12 receptor antibody making visualization of P2Y12 receptors on the membrane problematic. Upon further investigation, the P2Y13 receptor antibody was also found to be non-specific, in line with recent reports on the widespread lack of efficacy and specificity of available antibodies to label P2Y13 receptors in ex vivo and in vitro settings including the one used in our study . We have therefore removed P2Y12 and P2Y13 immunofluorescence data from the paper. Instead, we now include flow cytometry data using a different antibody that targets the extracellular domain of the P2Y12 receptor (clone 16001E) to show that these receptors are expressed to a greater level on the plasma membrane (PM) of TREM2 KO cells . The appropriate isotype control is included in this assay.Concerning P2Y expression, we performed control experiments using iPSC microglia treated with siRNA against P2Y2) Important: It is clear that the TREM2 KO cells have significantly lower STIM1 expression, which would be expected to impact SOCE under conditions of physiological agonists stimulation . The conclusion based on maximal activation with thapsigargin does not address this issue. What is the impact of reduced STIM1 expression on the Ca signals and motility?We thank the reviewer for bringing attention to this issue. The volcano plot comparing the transcriptomic expression of STIM1 in WT and TREM2 KO cells shows that STIM1 mRNA expression is below the threshold for what would be considered significant in an RNA seq experiment. In fact, this is the case for all the STIM and Orai isoforms. For RNAsequencing experiments, tens of thousands of genes are tested against the null hypothesis which has led to the use of false discovery rates (FDR) rather than traditional p-values (PMID: 12883005). However, statistical significance based on FDR alone is not enough to determine a meaningful result in RNA-sequencing experiments. Due to highly accurate sequencing, samples may show low variability leading to highly “significant” FDRs with a fold change less than 1. In many cases, this small change in expression would not yield biological differences. For these reasons, we consider only genes that reach both the FDR threshold and the fold change threshold. The data shown here were originally published in (PMID: 33097708) with cutoffs of FDR < 0.05 and -1 < FC < 1.12 and P2Y13 receptor transcripts for comparison . STIM1 mRNA expression is modestly reduced in TREM2 KO cells which is consistent with the modestly reduced maximum functional SOCE response (measured after store-depletion with TG). To address the issue about SOCE under conditions of physiological agonist stimulation, we also measured SOCE in response to store-depletion with ADP; new Figure 3D shows that ADP produces greater store-release and therefore engages SOCE to a greater extent in TREM2 KO cells. Based on this, we conclude that the modestly reduced STIM1 expression in TREM2 KO microglia does not play a significant role in determining the differences in ADP-mediated Ca2+ signals between WT and TREM2 KO cells.Unfortunately, the bar-graph in old Figure 1—figure supplement 2D may have given the false impression that differences in STIM1 expression are quite large. We have now remade this bar-graph (Y-axis range from 0 – 1.2) and have also included relative expression of P2Y2+ off/on protocol with ADP  as done for thapsigargin should be performed. In Figure 2B, ADP was applied in the absence of external Ca2+, but sadly those recordings did not subsequently replenish Ca2+ to gauge Ca2+ entry. Low concentrations of Gd3+  reduce the initial Ca2+ release peak, suggesting that there is a contribution of Ca2+ entry to this initial response. Also, it would have been helpful to include in Figure 1 Suppl1 the control (non KO cells) in these experiments for reference.3) A CaWe thank the reviewer for three excellent suggestions .2+ off/on protocol in the presence or absence of Synta66, a more specific inhibitor of Orai channels than Gd3+ or 2-APB. Synta66 significantly inhibited SOCE triggered by TG and ADP in WT and TREM2 KO microglia . Additionally, we compared ADP-induced SOCE in WT and TREM2 KO microglia . The implications of this experiment have been discussed above in Essential revision comment 1 and 2.3a (i) To demonstrate SOCE in response to ADP, we now include a Ca2+ influx contributes to the initial Ca2+ peak in two ways. First, we compared the height of the initial Ca2+ peak after application of ADP in 1mM Ca2+ and Ca2+ free buffer ; there was no significant difference between the two conditions in either WT or TREM2 KO microglia suggesting that the initial Ca2+ peak is driven primarily by store-release. We further tested this by acute addition of Gd3+ or 2-APB with ADP without pre-incubation . In this instance, we did not find an inhibition of initial Ca2+ peak. We speculate that the pre-incubation of cells with Gd3+ before addition of ADP in old Figure 1—figure supplement 1 might have non-specifically caused reduction of the initial Ca2+ peak.3a (ii) We re-examined the issue of whether Ca3+ and 2-APB on Ca2+ signaling in microglia, we did not repeat the Gd3+ and 2-APB experiments in WT microglia. Instead, we include new data as outlined above in Figure 3 and Figure 3—figure supplement 1 showing involvement of Orai channels using Synta66 and using the Orai1 KO line .3a (iii) Given the complex effects of Gd4) The conclusion that SOCE is mediated by Orai1 should be validated with a genetic approach as 2-APB and La3+ are non-specific.Thank you for the suggestion. As described above, we now include data using a more specific inhibitor (Synta66) and a newly generated Orai1 KO microglial cell line .5) Conversely, it is not clear if store content is depleted to a greater extent following ADP receptor stimulation in the KO cells. Assess ER calcium content in response to ADP in WT and KO cells or with an ionomycin pulse.2+ free buffer. The Ca2+ release peak in response to ADP was higher in the TREM2 KO cells as expected, and the subsequent ionomycin peak was significantly reduced. These results  indicate greater ER Ca2+ store-release by ADP in TREM2 KO cells. As for the suggestion to directly monitor ER calcium content, transfection of iPSC-microglia shifts them from a resting to a highly activated state in which they downregulate their P2Y receptors, as reported . Thus, transfection of genetically-encoded probes to measure ER Ca2+ or to measure IP3 levels is problematic.We thank the reviewer for this suggestion. We examined the extent of ER store-depletion in response to ADP by sequentially pulsing the cells first with ADP and then with ionomycin in Ca6) Figure 3: The WT data in this figure don't include KO cells. How do the TREM2 KOs compare to WT in the scratch wound closure assay? The KO cells seem to be tested, but only in the absence of ADP . Is there a difference in wound closure after ADP treatment? This point should be evaluated to assess the effects of altered ADP Ca signaling on non-directional cell movement in a physiologically relevant paradigm.As suggested by the reviewer, we performed the scratch wound assay in the presence of ADP . Interestingly, while ADP speeds up closure of scratch wounds, we found no differences in the wound closure rates between WT and TREM2 KO microglia. The differences in purinergic signaling are more decisive in shaping chemotaxis to ADP. The effects of TREM2 deletion, and the subsequent effects of increased purinergic signaling on microglial motility appear to depend on the specific physiological context, and this will require further investigation in a follow up study.7) Terminology: The term "Ca clamp" in the way it has been used here could be misconstrued by readers. Cellular Ca is not clamped. It varies within each cell depending on the activity of the pumps and other clearance mechanisms in the cell. The motility measurements are simply done after extracellular Ca is restored to the cells after thapsigargin treatment, at time points where the cytosolic Ca is lower in 0.2 mM Ca compared to 2 mM Ca. This phrasing (Ca clamp) needs to be changed to eliminate confusion that cellular calcium is "clamped" analogous to voltage-clamp.2+ clamp” from the manuscript. Instead, we refer to this protocol as a method to investigate the effects of Ca2+ elevation that bypasses purinergic receptor activation on microglial motility.We agree with this comment, and have accordingly removed the term “Ca8) Figure 6D TREM2 KOs (but not WT) cells show differences in motility between 0.2 mM and 2 mM extracellular Ca even after thapsigargin treatment with no ADP exposure. This is strong indication that motility differences in TREM2 KO cells are due to more than just alterations in purinergic signaling. . What is the role of these intrinsic changes in cell motility in response to cellular Ca elevations in the presence and absence of nucleotides?2+-dependent and -independent  pathways. Experiments in Figure 6 (using a protocol to maintain different levels of cytosolic Ca2+ over time using thapsigargin) were done with the goal of isolating the effects of sustained cytosolic Ca2+ levels on motility. The key observation is that motility in TREM2 KO microglia responds to changes in cytoplasmic Ca2+ levels to a greater extent than in WT cells, suggesting that Ca2+ tunes motility differently in WT and TREM2 KO cells. Addition of ADP had no further effect, as the reviewer correctly observes . We speculate in the Results section (lines 291-292) that the rise in Ca2+ in this assay may override the complex effects of ADP on motility; this may reflect intrinsic differences in Ca2+-dependent regulation of motility between WT and TREM2 KO cells. We note that the baseline motility characteristics are similar between WT and TREM2 KO microglia .We agree with the comment. The question raised has great potential for further work that is beyond the scope of this study. Purinergic stimulation produces a complex downstream response that includes activation of both Ca9) Figure 6D: The data for WT cells should be directly compared to the KOs at each extracellular Ca concentration to dissect out differences in KO cells at each Ca condition.2+ levels. Additionally, the histogram in Figure 6G compares the percent of cells with instantaneous speeds > 10 µm/min as a function of cytosolic Ca2+ in WT and TREM2 KO cells. This comparison reveals a unimodal relationship in TREM2 KO cells with low cell speeds at high and low cytosolic Ca2+ levels and higher speeds at intermediate Ca2+ levels.Yes, this is shown in Figure 6F: instantaneous speeds of WT and TREM2 KO microglia over a range of different cytosolic Ca10) The data in Figure 4K should be reanalyzed to compare differences in the absolute levels of branch complexity (# of branches and process length) in the two groups.), “Comparison of the absolute number of branches and process length after ADP treatment, as well as the relative fold-increase in these parameters from baseline indicated that process extension is not affected in TREM2 KO microglia”. We note that the absolute number of branches and the process length  was calculated and is shown in the left panels of Figure 5L and M. These results are also shown as paired plots comparing the absolute increase in the average number of branches and length of the processes after ADP treatment in the WT and KO groups per imaging field .We fully agree with the reviewer that the lower baseline branch complexity in the KOs explains the greater fold change in the KOs after ADP application. We also agree that the absolute number and length of branches  after ADP treatment is similar between WT and KOs. Based on these comments, we made the following change in the Results section (lines 267-27011) The apparent disconnect between the larger mean square distance travelled in the absence of nucleotides in the KO with no change in the mean speed needs to be clarified. Is this due to cancelling of speed vectors with opposite directions?We thank the reviewer for bringing attention to this. The MSD vs time plots  show that TREM2 KO cells travel farther away from the origin than WT cells after ADP treatment. This could either be because (1) KO cells move faster than WT cells or (2) because they change direction less frequently (or travel in straighter paths) than WT cells while moving at similar speed. Comparing mean track speeds and track straightness  affirms the latter possibility. The mean track speed  is the mean of all instantaneous speeds for a given track. It is calculated without any regard to the direction of cell motility, and therefore does not have negative values. We agree with the reviewer that the velocity vectors (which take into account the direction of cell movement) cancel each other out to a greater degree in the WT cells, which is reflected in lower values of track straightness. Our data suggest that WT cells remain confined to a smaller region of random walk because they turn more frequently. We have observed analogous distinctions in motility patterns between regulatory T (Treg) cells and inflammatory Th17 cells in the spinal cord of mice, with Treg cells executing a back-n-forth motion with similar mean speeds as Th17 cells, and being confined to smaller regions of the cord while Th17 cells traverse larger regions because they change directions less frequently (PMID:32732436). We now include a sentence in the Discussion section (lines 349-350) to indicate that velocity vectors cancel each other out to a greater extent in WT cells. For clarity, we added a new paragraph in the methods section (lines 922-937) detailing how the motility parameters were calculated.Reviewer #1 (Recommendations for the authors):1) Figure 3: The WT data in this figure don't include KO cells. How do the KOs compare to the WT in the scratch wound closure assay? The KO cells seem to be tested, but only in the absence of ADP . Is there a difference in wound closure after ADP treatment? This point should be evaluated to assess the effects of altered ADP Ca signaling on non-directional cell movement in a physiologically relevant paradigm.We thank the reviewer for this suggestion. We have addressed this under Essential revision comment no. 5.2) Figure 4: This reviewer is not convinced that reduced turning accounts for the similar speed in the KO despite the larger mean square distance travelled. If turning is reduced and the mean square distance is doubled in the KO, then effectively, the average distance travelled over time (speed) should be greater in the KO than in the WT. The logic of this inference and how it explains the data (similar average speed but much greater root mean square displacement in the KO) needs to be better clarified. Since the mean square distance is so much larger, it suggests that the speed vectors with opposite directions are cancelling out in the KO cells to yield the same average velocity .We thank the reviewer for bringing attention to this issue. We have addressed this under Essential revision comment no. 10.3) Figure 4K: It seems that the baseline branch complexity is low to start with in the KOs, hence the fold change from baseline seems larger. From the images shown, there appears to be no difference following ADP treatment between the two groups. This data should be reanalyzed to compare differences in the absolute levels of branch complexity (# of branches and process length) in the two groups.We thank the reviewer for bringing attention to this issue. We have addressed this under Essential revision comment no. 9.4) The use of the term Ca clamp is misleading in the way it has been used here. Cellular Ca is, in fact, not clamped, but changes within each cell depending on the activity of pumps and other calcium clearance mechanisms. The motility measurements are simply done after extracellular Ca is restored to cells after thapsigargin treatment, at time points where the cytosolic Ca is lower in 0.2 mM Cacompared to 2 mM Ca. This phrasing (Ca clamp) needs to be changed to eliminate confusion that cellular Ca levels are "clamped" to defined values analogous to voltage clamp.2+ clamp” from the manuscript .We agree with this comment, and have accordingly removed the term “Ca5) Figure 6D: It is noteworthy that TREM2 KOs, but not WT cells, show differences in motility between 0.2 mM and 2 mM extracellular Ca even after thapsigargin treatment with no ADP exposure. This is strong suggestion that motility differences in TREM2 KO cells are due to more than just alterations in purinergic signaling. In fact, this inference is consistent by the lack of additional effects of adding ADP in Figure 6 Supplement 1. These intrinsic changes in cell motility in response to cellular Ca elevations need further clarification.We have addressed this under Essential revision comment no. 7.6) Figure 6D: Related to this figure, the data for WT cells should be directly compared to the KOs at each extracellular Ca concentration to dissect out differences in KO cells at each Ca condition.We have addressed this under Essential revision comment no. 8.Other:– Stim1 is expressed at different levels between the WT and TREM2 KO. It has a significant p-value . This decrease could affect multiple aspects of microglia including cell motility in the presence of physiologically relevant agonists. This needs to be addressed.We thank the reviewer for this comment. This point was made by other reviewers as well and needs better clarification. We have addressed this under Essential revision comment no. 2.– A technical point related to Figure 4 is that figures 4C and 4G should be labelled "velocity" rather than speed, as it seems the only way in which the mean squared distance can be twice as large in the KO but not the velocity if the speed vectors with opposite directions are cancelling out in the KO.These graphs  plot the mean track speed, which represents the mean of all instantaneous speeds over the total time of tracking. The instantaneous speed is calculated at each time point for a given track as the scalar equivalent to object velocity, without taking into account directionality . We agree that MSD is lower in WTs; this is because WT cells turn more frequently (reflected as lower track straightness compared with TREM2 KOs), leading to greater cancellation of velocity vectors. For clarification, we now include definition of the different motility parameters in lines 922-937 of the methods section.– The dose of ADP used in Figure 7 is extremely high. 100ng/mL . By contrast, Figure 4 used 2.5uM. The 100 fold higher concentration in the chemotaxis assay needs to be justified. Further, it should be determined whether the ADP used diffuses into both chambers might be expected. This super high dose of ADP may have non-physiological effects that could explain the seemingly differing phenotypes in Figures 4 and 7.The reviewer’s calculation of concentration is incorrect. 100 ng/ml of ADP is equivalent to a concentration of 234 nM, not 234 µM. In the methods section on the chemotaxis assay (line 962), we now also report the concentration of ADP in nM. This dose of ADP is not super high.– Figure 5 is a great set of experiments to show that the Salsa6f does not affect end points of the study, but may be better as a supplementary figure rather than a main figure. If the paper is about the TREM2 KO versus WT then the main figures should stick with looking at differences between the two with maybe a sub figure to show validation.We thank the reviewer for this suggestion and are happy to comply. Because we ended up using the Salsa6f expressing lines in several additional experiments to confirm key findings in the study, the Salsa6f validation data are now presented in Figure 1 figure supplement 1.Reviewer #2 (Recommendations for the authors):2+ signaling proteins . Preferably Westerns should be performed for STIM1 and STIM2, Orai1 and IP3R 1/2/3, for which there are reliable Abs. Although IF as performed for Orai1 is acceptable, the authors do not show positive and negative controls and a thorough validation of this Orai1Ab in IF.– A more rigorous approach could be used to document the presence or absence of compensatory changes at the protein protein level of other CaWe thank the reviewer for these comments and suggestions. We have addressed this under Essential revision cmment no. 1.– Along the same lines as comment above, Staining for P2YR12 appears internal. Has the Ab been validated? Can authors perform a WB on P2YR12 and P2RY13 in addition?We have addressed this under Essential revision comment no. 1.– Did the authors check on whether the expression of SECRA is altered at the protein level? Did the authors perform CRAC channel recordings in Control and TREM2 KO cells?2+ signaling molecules  are now shown in Figure 2—figure supplement 1D and E. We note that among these, only P2Y12 and P2Y13 receptor transcripts showed significant fold change in expression between WT and TREM2 KO cells, which prompted us to focus our efforts in that direction. Additionally, we found that Ca2+ clearance rate after SOCE is similar in WT and TREM2 KO cells . Based on this, we find it unlikely that the higher sustained Ca2+ level in TREM2 KO cells is due to differences in Ca2+ clearance mechanisms, including Ca2+ pump activity. We did not attempt to record Icrac in iPSC microglia.We have not looked at the expression of SERCA at the protein level. However, we further examined our published RNAseq data on WT and TREM2 KO iPSC-microglia (PMID: 33097708) and compared the relative read counts of PMCA and SERCA isoforms that are known to be expressed in WT and TREM2 KO microglia and found no significant differences. Data for the relative expression of relevant Ca2+ off/on protocol with ADP  as they have done with thapsigargin. In Figure 2B, ADP was applied in the absence of external Ca2+, but sadly those recordings did not subsequently replenish Ca2+ to gauge Ca2+ entry. One can rationalize the result of 2-APB , a non specific channel blocker that also inhibits IP3Rs. However, the fact that low concentrations of Gd3+  reduce the initial Ca2+ release peak, suggests that there is a contribution of Ca2+ entry to this initial response. Also, it would have been helpful to include in Figure 1 Suppl1 the control (non KO cells) in these experiments for reference.– It is quite surprising that the authors did not opt to use the CaWe thank the reviewer for these comments and suggestions. We have accordingly performed new experiments and address this under Essential revision comment no. 3a.Additional thoughts to the attention of the authors:2+ might lead to a "spill" outside the restricted Ca2+ nanodomain, thus disrupting polarity. Do the authors have any evidence of altered location of leading-edge vs trailing edge proteins or focal adhesion proteins in TREM2 KO cells?– The authors have speculated on the fact that increased cytosolic Ca2+ signals play a role in generating cell polarity and regulating membrane protrusion and retraction in some migratory cell-types (PMID: 25977921). Local Ca2+ pulses have been detected at the leading edge of migrating fibroblasts in response to PDGF gradients (PMID: 19118385), and in migrating sheets of endothelial cells in response to a scratch wound (PMID: 24463606). More recently, localized Ca2+ signaling has been proposed to correlate with process extension in murine microglia based on intravital imaging, though it’s role in chemotaxis was not explored (PMID: 32716294). We have now rephrased the relevant sentence in the Discussion section (lines 360-363) to make it clear that we are speculating on the possible mechanism by which excessive Ca2+ signaling can impede gradient sensing. Reflecting the uncertainties, we have removed Figure 8 considering it may be overly speculative.No, we did not investigate the interesting question regarding the polarity of membrane proteins or focal adhesion proteins in TREM2 KO cells. Ca– Have the authors performed RNAseq on WT control and TREM2 KO cells? If so, does pathway analysis shows changes in the Cell motility genes between these two groups? This might offer some interesting clues to pursue in future studies.12 and P2RY13 receptors and were part of what stimulated our work presented here. However, some other chemotactic molecules including CCL2 and CCL3 are also included in this reciprocal gene list and warrant further study.The authors have performed and previously published RNA-seq on WT and TREM2 KO cells . Using gene ontology analysis, the authors did find differences in motility including “regulations of natural killer cell chemotaxis”, “Positive regulation of cell migration”, and “regulation of smooth muscle cell migration”. Even in the more selective gene ontology of genes which change in opposite directions after TREM2 KO and TREM2 antibody stimulation, we find “positive regulation of leukocyte chemotaxis” as one of the most significant gene family suggesting differences in motility. These gene lists included P2RYReviewer #3 (Recommendations for the authors):2+ responses to ADP stimulation in TREM2KO cells, and that this leads to a defect in turning behaviour and hence no directed migration to a chemotactic signal.This study by Jairaman et al. describes how iPSC-derived microglia exhibit exaggerated cytosolic Ca2+ responses to ADP and then extend their findings to cell movement and directed migration. Given the substantial body of evidence linking microglia to the pathogenesis of Alzheimer's disease, and the role for TREM2, the work by Jairaman et al. is of translational significance. As an aside, the introduction of the calcium-sensitive reporter Salsa6F is a welcome new tool in the arsenal for recording cytosolic calcium. Overall, this is an elegant, novel and important study. Nevertheless, I have a few comments/suggestions.Overall, the experiments are well conducted, carefully controlled and the findings are new and exciting. The authors nicely dissect out the underlying molecular basis for the exaggerated CaWe thank the reviewer for the positive and insightful comments. We have done new experiments to address specific suggestions as outlined below.2+ plateau to ADP in TREM2KO cells is due to enhanced Ca2+ release from the stores ie greater store depletion. Evidence is presented that maximal SOCE is not compromised in TREM2KO cells, that store content under resting conditions is unaffected, InsP3R activities are similar. But the authors do not demonstrate that store content falls more following ADP receptor stimulation in the KO cells. This could be shown by directly measuring ER calcium to ADP in WT and KO cells or by applying ADP in Ca-free solution and then assessing store content with an ionomycin pulse. The ionomycin response should be smaller in the TREM2KO cells, after ADP exposure.1. The authors argue that the increased CaWe thank the reviewer for suggesting the ionomycin pulse experiment. We have accordingly performed new experiments, and address this under Essential revision comment# 3b.2+ release from the ER. However, the increase in P2Y12 protein levels is modest at best, and the increase for P2y13 is less than 2-fold. The authors' point would be strengthened by showing InsP3 levels are increased in the KO cells compared with WT ones, for the same dose of ADP. Single cell InsP3 probes are available. Alternatively, a population measurement could be carried out.2. The authors suggest that an increase in P2Y12 and P2Y13 receptor expression in the KO cells accounts for the increased Ca12 receptors, which confounds our ability to accurately compare P2Y receptor activity. Although the antibodies against P2Y12 and P2Y13 receptors were found to be nonspecific , we now include flow cytometry data showing increased PM expression of P2Y12 receptor in live TREM2 KO cells ; these data complement the functional assays and transcriptomic data  showing >2 fold increase in P2Y12 and P2Y13 receptor RNA expression in TREM2 KO microglia. These differences are likely to be biologically relevant for downstream signaling, given the positive feedback mechanisms and signal amplification associated with GPCR signaling (PMID: 29074251). Increased protein expression of P2Y12 and P2Y13 receptors has also been reported in -/-Trem2 mice and in a mouse AD model, and we now include this point in the Introduction section of the manuscript (line 87-89).While we agree that the suggested experiment with InsP3 probe would further bolster our conclusion, we have noted in essential revision comment no. 4 that transfection of iPSC-microglia shifts them from a resting to a highly activated state leading to downregulation of P2Y2+-permeable channels could contribute to the Ca2+ plateau. The authors should therefore knock down Orai1 or at least use more selective inhibitors such as Synta66 or the GSK compound.3. The evidence for an involvement of SOCE is based on two rather non-specific inhibitors (2-APB and Gd3+). P2YRs, like other GPCRs, activate TRPC proteins and these CaWe thank the reviewer for this suggestion and have performed additional pharmacological experiments and CRISPRbased Orai1 KO to address this point in Figure 3 and Figure 3—figure supplement 1. The results confirm involvement of Orai channels in SOCE activated by store-depletion with TG and ADP.4. The authors should include some controls to show the P2Y antibodies are indeed specific. For example, they could use a cell line that lacks P2Y12/13. In Figure 2I, J, what happens to the ADP response when both P2Y inhibitors are present at the same time? It would be important to show that the combination of P2Y12 and P2Y13 suppress ADP responses fully.2+ responses to ADP in the presence of both P2RY12 and P2Y13 inhibitors. This combination inhibited the ADP Ca2+ response completely in both WT and TREM2 KO microglia .Per reviewer suggestion, we examined Ca5. The authors should add a justification for the concentrations of agonists they have used . A dose-response curve is included for ADP but a comment on the doses selected would be helpful.12 receptors, but microglial cells experience a range of concentrations depending on the pathophysiological context, and depending on distance from the injury site. We now include a sentence in the Introduction (line 54) about purinergic concentrations in the brain.Extracellular concentration of purinergic signals range from hundreds of nanomolar to μM levels, and are shaped by a variety of factors including baseline secretion, extent of local tissue damage and the prevalence of ectonucleotidases that cleave purinergic ligands (PMID: 18302942). Studies in microglial field have often used tens of μM ADP  to study the biology of P2Y2+ influx in TREM2 KO cells leads to microglia motility and process extension to a greater extent than WT cells. This is attributed to the increased SOCE. If so, then one might expect raising external Ca2+ in WT cells to have the same effect. Is this the case?6. It is nicely shown that Ca2+ concentration in WTs to test whether that results in a higher magnitude of cell speed and process extension (compared to TREM2 KOs). Our data in Figure 5 and Figure 5 figure supplement 1 shows that Ca2+ influx pathways are required for optimal cell motility and process dynamics in iPSC microglia in general . We should also note that the effects of TREM2 deletion on process branching/ lengthening is not as drastic as its effects on cell motility. We have addressed this particular point in response to comments from reviewer No. 1 under Essential revision, comment no. 9.We have not examined effects of increasing the extracellular Ca2+ signal leads to a chemotaxis defect? The authors' data would suggest that chemotaxis might have a bell-shaped dependence on cytosolic Ca2+, with too little or too much impeding migration towards a cue.7. Is anything known mechanistically how the exaggerated Ca2+ signals in this process remains poorly understood. Please also see our earlier response (comment No. 5 by reviewer# 2) on how excessive cytosolic Ca2+ may disrupt cell polarity. The histogram in Figure 6G comparing the percent of fast-moving cells as a function of cytosolic Ca2+ further indicate that there may indeed be a bell-shaped effect with regard to effects of Ca2+ on instantaneous cell speeds in TREM2 KO cells. This is reflected in the chemotaxis assay in Figure 7B and C in which reducing ADP signaling in TREM2 KO cells rescues the defect in chemotaxis. It is possible that high cytosolic Ca2+ serves as a temporary STOP signal in microglia similar to its effects on T cells. We note this point in the Discussion (lines 386-388) and speculate that TREM2 KO cells may be more subject to this effect with ADP, given the higher expression of P2RY12 and P2Y13 receptors.The fundamental issue in chemotaxis is how shallow gradients of a chemokine translates to steep polarization of signaling proteins within the cell. This is generally thought to occur downstream of the chemokine receptor and upstream of actin cytoskeleton (PMID: 33990789), but the specific role of Ca2+ rise or via a global cytosolic Ca elevation?8. Do the authors know whether the effects of SOCE are mediated through a local Ca2+ signals in regulating microglial cell motility in the current study but hope to explore this aspect of Ca2+ signaling as part of a follow up study.We have not looked at role of local vs. global Ca9. Perhaps I missed something but Figure 1 S2(D) seems to show a significant decrease in STIM1 levels in the KO cells.We thank the reviewer for this comment. This point was made by other reviewers as well and needs clarification. We have addressed this under Essential revision comment no. 2.10. The authors present SOCE as the peak signal remaining after 5 minutes. It is not clear whether the value has been subtracted from the pre-stimulation levels. In some graphs, the signal after 5 minutes looks the same as the resting level, but the bar charts show higher values.2+ response and Ca2+ at 5 minutes. The data-points  in the bar-graphs are all baseline subtracted, as described in the legend. The Y-axis is now labelled “Fluo-4/Fura-Red Ratio (baseline subtracted)” to make this point clear. We also note that the bar-graph summary includes data from multiple experiments, while the average traces are from a single imaging run.We assume the reviewer is referring to the traces and bar-graphs in current Figure 1B, D, E and F showing both peak Ca


The use of innovative technologies has revolutionized cancer research in recent years, and in the field of pediatric oncohematology, great results have been achieved. However, within this context, acute myeloid leukemia still represents a considerable challenge for clinicians, as frequent cases of relapse or refractory disease, especially in specific subgroups, remain present. With this review, the authors aim to recapitulate the main features of this extremely heterogeneous malignancy, by highlighting the concept of clonal evolution and how, thanks also to the impact of new high throughput techniques, this could be exploited to deepen the current knowledge on molecular mechanisms driving this disease. Overall, this study will seek to pave the way for making new tools available to further improve diagnosis and treatment protocols in pediatric patients.Despite improvements in therapeutic protocols and in risk stratification, acute myeloid leukemia (AML) remains the leading cause of childhood leukemic mortality. Indeed, the overall survival accounts for ~70% but still ~30% of pediatric patients experience relapse, with poor response to conventional chemotherapy. Thus, there is an urgent need to improve diagnosis and treatment efficacy prediction in the context of this disease. Nowadays, in the era of high throughput techniques, AML has emerged as an extremely heterogeneous disease from a genetic point of view. Different subclones characterized by specific molecular profiles display different degrees of susceptibility to conventional treatments. In this review, we describe in detail this genetic heterogeneity of pediatric AML and how it is linked to relapse in terms of clonal evolution. We highlight some innovative tools to characterize minor subclones that could help to enhance diagnosis and a preclinical model suitable for drugs screening. The final ambition of research is represented by targeted therapy, which could improve the prognosis of pediatric AML patients, as well as to limit the side toxicity of current treatments. Pediatric acute myeloid leukemia (AML), with an incidence of approximately seven occurrences per 1 million children annually ,3,4,5. TAML is a heterogeneous disease, from clinical behavior to morphology, immunophenotyping and genetic abnormalities, due to the abnormal proliferation of myeloid progenitor cells in the bone marrow . It has PML) gene with the retinoic acid receptor alpha (RARA), in acute promyelocytic leukemia, which represent ~5% of pediatric AML [Regardless of its heterogeneity, some characteristic chromosomal changes and molecular lesions are recurrent in pediatric AML . These itric AML . This sutric AML , thanks  AML [/L .RUNX1-RUNX1T1 and CBFB-MYH11, respectively.Other subtypes of pediatric AML, characterized by a favorable prognosis, are those involving core binding factor (CBF), accounting for approximately 20–25% of pediatric AML cases , such asKMT2A is a histone methyltransferase that is rearranged in 35–60% of infants and 10–15% of children and adolescents [CBFA2T3-GLIS2, caused by inv(16) (p13.3q24.3), which is present in 15–20% of pediatric non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL) and about 7–8% of pediatric AML with normal karyotype [NUP98) and over 30 different fusion partners, the most common of which are KDM5A and NDS1, can be found in 4–9% of pediatric AML [DEK-NUP214, present in just 1–2% of pediatric AML [MDS1 and EVI1 complex locus (MECOM) [lescents , and it lescents . It reprlescents . A subtyed by inv  .NPM1) which encodes for a chaperon protein normally localized predominantly in the nucleolus, however, when mutated, it is aberrantly localized in the cytoplasm, causing the activation of oncogenes. This is rarely altered in pediatric AML [FLT3-ITD mutations [C/EBPA), present in 5–10% of pediatric AML cases [FLT3) is a common gene involved in pediatric AML, whose frequency is age-related, being rare in infants and rising as age increases. FLT3 is essential for the proliferation, survival and differentiation of stem/progenitor cells. The most common type of mutation in FLT3 is represented by internal tandem duplication (ITD) mutations, but point mutations are also frequent, and both these lesions lead to the constitutive activation of this gene [WT1 gene is expressed in CD34+ hematopoietic stem progenitors and is crucial for the regulation of normal growth and development. Among pediatric AML cases, 15% are characterized by inactivating mutations of WT1 [FLT3-ITD mutations are also present [TET2, IDH1 and IDH2 are much less prevalent compared to adult AML, characterizing only 1–2% of pediatric patients [NRAS, KRAS, CBL, GATA2, SETD2 and PTPN11 are more common in younger patients. Moreover, MYC alterations were identified as exclusive in children, suggesting different leukemogenesis mechanisms in children compared to adults [With the advent of large-scale genomic approaches, several molecular aberrations have been likewise identified, such as the gene nucleophosmin 1  family, BCOR and BCORL1, EZH2. ASXL1 and ASXL2 mutations have been found in pediatric de novo AML, mainly in patients with t [ASXL3 mutation has been described by Masetti et al. [ASXL2 were lower than others, suggesting that this mutation is a secondary event. BCOR and its homologue BCORL1 suppress gene transcription through epigenetic mechanisms [BCORL1 has been shown to be a tumor suppressor gene, and, as SMC3, BCORL1 appears to be mutated at an early stage in leukemic cells [EZH2 is one of the most commonly mutated genes in pediatric Down syndrome AMKL, and it encodes for a subunit of a complex responsible for methylation [Among the epigenetic regulators, mutations have been found in the Additional Sex combs-Like  , and an 1 , and achanisms . BCORL1 ic cells . EZH2 ishylation . NRAS, KRAS, PTPN11, and of tyrosine kinases such as KIT and FLT3. NRAS mutations have been found to be relapse specific, like those in CREBBP, a coactivator of several hematopoietic transcription factors [Farrar et al. [PTPN11 mutations, lost at relapse when they appeared as subclonal variants at diagnosis. Masetti et al. [PTPN11 mutation gained at relapse (MF of 31.9%), suggesting its role in increasing cell proliferation and/or survival. As previously described, FLT3 is essential for proliferation, survival and differentiation of stem/progenitor cells. Masetti et al. [FLT3-TKD-mutated subclone present at diagnosis (MF of 3.4%) underwent expansion at relapse (MF of 13.3%), leading to increased cell proliferation and/or survival, and this mutation, together with NRAS and KIT mutations, was also described as a secondary event contributing to disease progression by Shiba et al. [Signaling pathway mutations include mutations of genes involved in Ras pathways such as  factors . Farrar r et al.  describei et al.  found a i et al.  showed ta et al. . C/EBPA and WT1 mutations exhibit different patterns of recurrency in the study presented by Masetti et al. A highly penetrant biallelic mutation of C/EBPA was revealed, and in one patient, a homozygous non-frameshift insertion was present both at diagnosis and relapse in the majority of the tumor-cell population (MF > 80%). On the contrary, WT1 mutations appeared highly unstable, two different frameshift insertions were detected mainly at diagnosis (MF of 27.6% and 13.9), while a single-nucleotide variant was detected only at relapse (MF of 40%). SETD2 is a methyltransferase involved in the recruitment of mismatch repair (MMR) machinery, and its mutation results in a loss of function of the methyltransferase activity, leading to the build-up of several subclonal mutations and consequently, the increased plasticity and adaptability of leukemia cells, due to the failure of DNA repair [SETD2 gene has been described both at diagnosis (MF of 32.5%) and at relapse (MF of 31.7%) [TYK2 gene is a member of the Janus tyrosine kinases (JAK) family involved in cell growth, differentiation and survival. Masetti et al. [TYK2 both at diagnosis (MF of 43%) and at relapse (MF of 14.9%), which causes a hyperactivation of the TYK2 pathway, resulting in aberrant cell survival through the upregulation of BCL2 (anti-apoptotic protein). SALL1 is a member of the transcriptional network that regulates stem cell pluripotency [In addition, also genes that do not belong to these groups are frequently mutated. The previously described ipotency ; it has van Galen et al. refined and established a short-read and nanopore sequencing method to detect genetic aberrations in individual cells [FLT3 genotypes within the same patient. For instance, an in-depth analysis revealed that four different FLT3 alleles co-exist and they were referred to distinct subclones; the one with FLT3-ITD contained more primitive cells, while another with FLT3-TKD contained more differentiated cells. These variations might explain how AML differentiation is affected and likewise why it is correlated with a poor outcome [Even though limited literature is available in the pediatric context regarding clonal evolution studies, massive sequencing has already been performed on multiple pediatric leukemia patients samples, taken both at diagnosis and relapse, that allowed the broad characterization of major genetic lesions and subclonal leukemic populations, as described above ,34. Howeal cells . Eventua outcome ,37.Morita et al. introduced multiple single-cell genomics techniques to reveal the molecular architecture of a panel of 123 AML patients [NPM1, DNMT3A, FLT3, NRAS, KRAS, SRSF2 and TET2. The pair-wise analysis of mutation co-occurrence using single-cell data provided evidence of specific clonal relationship of AML driver mutations. Afterwards, they took advantage of a probabilistic model, single cell inference of tumor evolution (SCITE), to generate a phylogenic tree that recapitulated the evolutionary story of AML cells population [RAS (NRAS p.G12S or KRAS p.Q61H). In a PDX model derived from this sample, there was a considerably major expansion of the clone NRAS p.G12S. Interestingly, the same clonal evolution was observed in that specific AML patient after therapy, thus suggesting an important role for PDX in vivo studies, which reflects the actual assortment of leukemic cells [A recently published study by patients . Single-pulation . To furtic cells . NPM1 associated with lower CD34 expression, whereas TP53 with higher CD34 expression [FLT3, negative for common chromosomal inversions or translocations, in which Pereira et al. analyzed the immunophenotyping evolution from diagnosis until a third relapse by means of multiparametric flow cytometry [Moreover, another fundamental tool is represented by immunophenotypic analysis, performed in the aforementioned study by scDNA + protein-seq and then validated in multi-color flow cytometry. There is a clear association between some mutations and the expression of specific surface markers, for instance pression . The usepression . An addiytometry . At diagytometry . InteresAbelson et al. [FANC genes), telomere biology disorder , germline myeloid neoplasm-associated genes  and others such as C/EBPA and WT1, are associated with a predisposition to an earlier onset of AML and cases of familial AML [In addition, deep sequencing and new tools for analysis can help find predictive models of acute myeloid leukemia risk in healthy individuals, as demonstrated by the work of n et al.  in the an et al. . The timlial AML ,43,44. OChao et al. reprogrammed AML adult blasts with MLL rearrangement [MLL-r and KRAS mutation. The hematopoietic cells iPSC-derived with KRAS and MLL-r had different biological properties in terms of engraftment in NSG mice and, above all, in terms of response to different drugs. Indeed, the proliferation of this subclone is affected by means of a KRAS inhibitor as well as by ARA-c. The retrospective target screening of a patient’s blasts elucidated that at diagnosis, the KRAS clone was predominant, but it was not detected at relapse. This was in agreement with the iPSC model, in which hematopoietic cells with KRAS mutation were responsive to ARA-c, while the hematopoietic cells with MLL-r and RAS wild type were resistant to chemotherapy. Moreover, the hematopoietic differentiation of iPSCs derived from AML patients confirmed that these cells clustered in a different way in terms of gene expression and methylation, compared to the hematopoietic cells derived from control iPSCs, thus supporting the fact that the mutations present in AML-iPSC drive the leukemic phenotype only upon differentiation to hematopoietic cells (and not to other differentiated tissues) [As previously described, pediatric AML, both at diagnosis and relapse, is characterized by a prominent clone but also by minor subclones. As a consequence, molecular heterogeneity corresponds to different levels of sensitivity to conventional therapies. However, nowadays, it is not always possible to easily handle enough patients’ primary blasts to perform comprehensive studies, thus motivating the development other innovative cells systems to do so. In the last 10 years, the advent of induced pluripotent stem cells (iPSCs) has offered a unique tool to isolate different subclones by reprogramming patients’ specific iPSCs. In this context, the reprogramming process of AML blasts can allow to isolate single clones that are useful to test different drugs. Indeed, for this purpose, angement . The tartissues) . Kotini et al. [The use of the iPSCs model to study clonal evolution can also be found in the research of i et al. . By repri et al. . Bertuccio et al. used a knock-in healthy donor iPSC with ETO2-GLIS2 under the control of a pan hematopoietic promotor [On the other hand, the improvement in genome editing knowledge allowed the possibility to engineer iPSCs derived from a healthy donor with onco-fusion genes. In this context, promotor . The difThe concept of clonal evolution has impacted in several ways the research approach in studying hematological diseases. The idea of a concrete cells’ heterogeneity, finely modulated at both chromatin, gene and protein level, that is able to give rise to specific paths of disease progression, has displaced the focus to the necessity of new methods to reach a deeper knowledge on how to treat a specific disease. Notably, this is an urgent need in pediatric hematological malignancies, in which, even if the treatment and survival rates have reached significant results, still frequent unclear cases of relapses and drug resistance are present. As previously reported, a huge amount of work lead to the discovery of most of the prominent genetic lesions in pediatric acute leukemias, and advanced high throughput technologies were fundamental to achieve this goal. Many innovative research works and tools, even if mostly considering adult samples, indeed highlighted the importance of different subclones in tuning AML from a preleukemic state to the overt disease. Furthermore, being able to identify and classify these subclones is also an essential issue to examine treatment responses, as different cell populations have exhibited a different degree of sensitivity and some of them might be the ones responsible for therapy resistance cases. In this context, the adoption of iPSC models in the research field has allowed to add a further means to unravel obscure mechanisms in clonal evolution and leukemic progression. Furthermore, this model is even more relevant for the pediatric cause, as it provides a way to analyze the whole process starting from embryonic-like cells development. The molecular mechanisms through which specific genetic lesions give rise to specific phenotypes  often take place in an early phase of differentiation, and stage-specific alterations contribute to distinct outcomes. Additional studies are needed to deepen the understanding of such mechanisms, which are of vital importance to immediately identify the disease at the diagnostic level, but also to precisely determine the proper effective treatment at clinical level. The future is, and has to be, oriented towards targeted therapy and tools that will help the clinics to easily manage pediatric patients, in order to help those cases which have to date been so difficult to interpret and to cure.