This month we look at the most exciting technological, methodological and conceptual advances in the field. If you also have a new exciting tech that you want to share, send us your preprint here, And maybe consider submitting it to Experimental Hematology as well: you will benefit from a fast editorial decision (5 days) and a smooth review process!
From the Simply Blood Community:
Epigenome editing of human hematopoietic stem cells enables sustained and reversible thrombosis prevention
https://www.biorxiv.org/content/10.64898/2026.03.27.714536v1
We establish DNA methylation-based epigenome editing in human hematopoietic stem cells to enable durable and reversible gene silencing. This approach enables long-term modulation of platelet function and establishes a platform for thrombosis prevention.
Social media handles: @lrbzldz.bsky.social
Technical advances
Versatile electroporation protocols enable reproducible CRISPR-RNP delivery across multiple primary mouse cells of the hematopoietic lineage
https://www.biorxiv.org/content/10.64898/2026.01.27.702081v1
Optimized ExPERT electroporation workflows achieve high-efficiency CRISPR-Cas9 delivery across three primary mouse hematopoietic cell types — CD8⁺ T cells, bone marrow-derived macrophages, and HSCs — while preserving viability, proliferation, and differentiation capacity. These findings establish a scalable, modular, and cost-effective non-viral genome editing platform, lowering barriers to genetic perturbation.
A comparison of long-read single-cell transcriptomic approaches
https://www.biorxiv.org/content/10.1101/2025.07.03.662955v1
A direct comparison of three commercial platforms using identical cDNA libraries shows concordant cell type identification across platforms, with shared limitations stemming from cDNA synthesis inefficiencies and read filtering. CRISPR-based depletion of highly expressed transcripts improved long-read data quality, and all platforms successfully enabled isoform-resolved analyses, including immunoglobulin reconstruction and alternative splicing detection in immune cells.
Isoform-Level Analysis of 10x Genomics Single-Cell cDNA Libraries from Cultured K562 Cells Using Long-Read Sequencing
https://www.biorxiv.org/content/10.1101/2025.08.12.668929v1
This study compares three long-read approaches — FLT-seq, SQK-PCS111, and EXP-PCA001 — for sequencing single-cell cDNA libraries, evaluating their efficiency in full-length cDNA enrichment, barcode identification, novel isoform detection, mutation calling, and transcript coverage profiling. Together, these benchmarks provide practical guidance for selecting the optimal ONT strategy for single-cell isoform-resolved transcriptomic studies.
Systematic fusion transcript discovery in mantle cell lymphoma using long-read sequencing
https://www.biorxiv.org/content/10.64898/2026.01.16.699780v1
Iso-Seq long-read sequencing of MCL cell lines uncovers thousands of novel transcripts including fusion transcripts among the longest in the MCL transcriptome. The majority of identified fusions appear intrachromosomal, and their biological significance remains largely unknown. A novel RBM15::LAMTOR5:AS fusion — between an m6A writer and a lncRNA — was validated and shown to increase upon CPSF73 inhibition, implicating read-through transcription in its biogenesis.
A Universal Duplex Sequencing Approach for Accurate Detection of Somatic Mutations
https://www.biorxiv.org/content/10.1101/2025.09.14.676103v1
UDSeq (Universal Duplex Sequencing) combines random fragmentation, efficient UMI ligation, and quantitative input control to achieve near-complete genome/exome coverage from as little as 100 pg DNA, with an estimated error rate of ~2.5×10⁻⁹ per base pair. Compared to prior duplex methods, UDSeq yields up to fourfold more usable duplex molecules while remaining cost-effective, and successfully captures exposure-specific mutational signatures across cell lines, rodent models, and cross-species samples.
Non-disruptive 3D profiling of combinations of epigenetic marks in single cells
https://www.biorxiv.org/content/10.1101/2025.06.13.659535v1
Epi-PHR is a novel image-based technology enabling locus-specific, high-resolution in situ detection of multiple epigenetic marks while preserving 3D genome organization in single cells. Applied to tissue and imprinted gene loci, it reveals allele-specific associations between epigenetic states and chromatin conformation.
A protocol for high-throughput microplate-based CUT&Tag
https://www.biorxiv.org/content/10.1101/2025.09.18.676951v1
Plate-CUT&Tag enables simultaneous processing of 96 CUT&Tag samples in a standard microplate without specialized equipment, matching the speed and data quality of conventional benchtop CUT&Tag. Validation in both cell lines and patient leukemia samples demonstrates its utility for large-scale preclinical and translational chromatin profiling studies.
Intravital imaging uncovers remodelling of humanised bone marrow-like niches
https://www.biorxiv.org/content/10.64898/2026.03.24.713949v1
The authors designed a platform enables real-time in vivo assessment of bone marrow niche dynamics. Using a titanium imaging window, this study performs longitudinal intravital imaging of human hematopoietic and mesenchymal stromal cells within an ectopically implanted humanized scaffold, revealing progressive vascularization by murine endothelial cells and ECM remodeling driven by stromal cell expansion.
Benchmarking three simple DNA staining-based image metrics for live-cell tracking of chromatin organization
https://www.biorxiv.org/content/10.64898/2026.03.30.715467v1
In this work, the authors extracted three image-derived metrics — CV, 1-Gini, and the newly introduced Diffuse Signal Index (DSI) — from routine live-cell DNA staining. This serve as fixation-free readouts of chromatin state, validated against Tn5-based accessibility measurements. Benchmarked in dHL-60 cells undergoing NETosis, DSI outperforms the others in discriminating NETing from non-NETing cells, providing a practical framework for real-time chromatin state monitoring without cell disruption.
The dry lab corner
IntegrateALL: an end-to-end RNA-seq analysis pipeline for multilevel data extraction and interpretable subtype classification in B-precursor ALL
https://www.biorxiv.org/content/10.1101/2025.09.25.673987v1
IntegrateALL is a Snakemake pipeline integrating expression-based classification, fusion/SNV calling, and virtual karyotyping — including the novel KaryALL classifier (accuracy 0.98) — to assign 26 WHO-defined B-ALL subtypes from RNA-seq data. Applied to 1,210 cases, it achieved unambiguous subtype assignment in 81.5%, and identified dual subtype-defining drivers in 2.6% of patients, revealing hierarchical oncogenic control. Validated across three independent cohorts, IntegrateALL provides a fully reproducible workflow for clinical and translational B-ALL molecular characterization.
Persistent hindrances to data re-use in single-cell genomics
https://www.biorxiv.org/content/10.1101/2025.10.02.680150v2
A systematic screening of GEO for human, mouse, and rat scRNA-seq studies reveals that only ~40% provided readily usable processed count data and fewer than 10% included cell-type annotations, severely limiting reuse potential. These findings expose critical gaps in current single-cell data sharing practices and highlight the urgent need for repositories to enforce stricter submission requirements for processed data and cell-type annotations.
Mapping transcriptional responses to cellular perturbation dictionaries with RNA fingerprinting
https://www.biorxiv.org/content/10.1101/2025.09.19.676866v1
The authors propose RNA fingerprinting, a statistical framework that learns denoised transcriptional signatures from perturbation dictionaries and probabilistically maps query cells to candidate perturbations, scaling to genome-wide screens and resolving combinatorial effects. Applied across diverse biological contexts, it identifies context-specific p53 regulators, characterizes drug mechanisms and off-target effects, and uncovers cytokine-driven B cell heterogeneity during influenza infection
A modular transcript enrichment strategy for scalable, atlas-aligned, and clonotype-resolved single-cell transcriptomics
https://www.biorxiv.org/content/10.64898/2026.02.06.703342v1
TRTL is a modular, targeted reverse transcription method that integrates into existing single-cell workflows, enabling user-defined transcript panel readout from tens to thousands of genes while retaining the ability to capture variable transcripts such as TCR and BCR sequences. Applied to mouse brain and T cell datasets, TRTL supports accurate cell type annotation at low sequencing depths and resolves dynamic T cell fate trajectories alongside clonotype profiling when combined with nuclear hashing-based multiplexing.
Modelling and theoretical biology
Statistical inference of the cellular origin of chronic myeloid leukemia using a discrete-parameter ABC–PMC framework
https://www.biorxiv.org/content/10.1101/2025.10.17.683025v1
Using a 27-compartment stochastic branching process model of hematopoiesis, combined with a discrete-parameter ABC-PMC algorithm, the authors consistently identify the hematopoietic stem cell compartment as the most probable origin of the BCR::ABL1 mutation in CML patients.
Theoretical estimates on the expected number of mutations for reconstructing clonal lineage trees
https://www.biorxiv.org/content/10.1101/2025.11.20.689642v1
This study establishes the theoretical and empirical basis for mutation subsampling as a strategy to scale clonal phylogenetic reconstruction, showing that modest numbers of mutations are sufficient to recover accurate clonal trees for typical clone counts. Theoretical bounds derived from common clonal lineage tree models are validated through simulations and real biological datasets, supporting subsampling as a robust and generalizable approach.
State-Dependent Regulatory Compression: Chromatin Geometry Gates Information Flow in Hematopoiesis
https://www.biorxiv.org/content/10.64898/2025.12.31.697240v1
Analysis of human bone marrow multiome data reveals that hematopoietic progenitors maintain conserved geometric boundaries while exhibiting significantly higher coupling efficiency between chromatin accessibility and transcription, reflecting distributed regulatory redundancy across multilineage programs rather than absence of control. Differentiated cells conversely display crystallized, pathway-specific regulatory channeling. These findings establish regulatory compression — informational channeling through selective pathways under geometric constraint — as an organizational principle of hematopoietic differentiation.
New experimental models
Integration of Hematopoietic and Thymus-like Niches in a Human iPSC-derived Bone Marrow Organoid
https://www.biorxiv.org/content/10.1101/2025.11.04.686649v1
Human iPSC-derived bone marrow organoids (iBMOs) faithfully reproduce native stromal and vascular architecture while supporting both robust T cell differentiation and dendritic cell output, functions normally requiring separate bone marrow and thymic environments. Upon engraftment in immunodeficient mice, iBMOs autonomously sustain human erythropoiesis and de novo bone formation, with single-cell transcriptomics revealing a hybrid stem/endothelial/stromal niche cluster underlying this dual functionality.
A humanized ossicle model of myelofibrosis reveals THPO-driven fibrosis, osteosclerosis and SPP1-dependent microenvironmental remodeling
https://www.biorxiv.org/content/10.64898/2026.03.12.711163v2
The authors present a human model of myelofibrosis: THPO-overexpressing human CD34⁺ cells transplanted into humanized bone marrow ossicles. They recapitulate key myelofibrosis features, including reticulin fibrosis, megakaryocyte clustering, myeloid skewing, and osteosclerosis. SPP1/OPN is identified as a key mediator of fibrotic niche remodeling — elevated in both the model and patient biopsies.
A patient derived xenograft repository capturing clinical and molecular heterogeneity of large B-cell lymphoma
https://www.biorxiv.org/content/10.64898/2026.01.19.700406v1
From the authors: Yang et al. describe X-LYMPH (Xenografts of Lymphoma), a publicly available and molecularly annotated PDX repository that captures the heterogeneity of large B-cell lymphoma. X-LYMPH includes models of chimeric antigen receptor T cell resistance, providing a shared foundation for mechanistic research and therapeutic development for lymphomas.
Leukemia stem cell expansion cultures reveal clonal drivers of leukemogenesis and therapy response
https://www.biorxiv.org/content/10.64898/2026.02.24.707683v1
Polymer-based Leukemic STem-cell Cultures (PLSTCs) achieve over 1000-fold enrichment of functional AML stem cells compared to traditional cultures, sustaining diverse self-renewing LSC states with stable heritable transcriptional programs. Clonal barcoding and dynamic state-fate analysis identify chemotherapy-resistant LSC clones primed for a fate-switch toward megakaryocytic-erythroid-like cells, while pooled CROPseq screening reveals chondroitin-sulfate synthesis as required for maintaining primitive LSC identity and leukemic recovery.
Development of a low-dose PBMC humanized mouse model using CD47;Rag2;IL2rγ triple KO mice: Enhanced leukocyte reconstitution and extended experimental window
https://www.biorxiv.org/content/10.64898/2026.03.25.714298v1
The authors present an improved murine model for xenograft studies. The novel NOD-CD47nullRag2nullIL-2rγnull (RTKO) strain engrafted with a low dose of PBMCs (3×10⁶ cells) achieves stable human leukocyte reconstitution with mild GvHD and a prolonged experimental window comparable to HSC-engrafted humanized mice. Combined with radiation tolerance conferred by the Rag mutation, this model offers a versatile, accessible platform for preclinical immuno-oncology and radiotherapy research.
Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells from Human Mobilized Peripheral Blood for Gene Therapy Applications
https://www.biorxiv.org/content/10.64898/2026.04.08.716064v1
From the authors: A mobilized peripheral blood HSC expansion protocol optimized for gene therapy allows robust polyclonal long-term engraftment of LV-transduced cells.
Modeling competitive transplantation using HLA-mismatched human hematopoietic stem cells
https://www.biorxiv.org/content/10.64898/2026.03.18.712629v1
A novel competitive transplantation method in NBSGW mice enables simultaneous engraftment and longitudinal tracking of HLA-mismatched human CD34+ donor grafts within a shared microenvironment using standard flow cytometry. This flexible platform accommodates different mouse models, conditioning strategies, and treatments.
Mitochondrial metabolic remodeling drives innate immune activation in Drosophila hemocytes
https://www.biorxiv.org/content/10.64898/2026.03.23.713618v1
Combined metabolic flux measurements and single-cell transcriptomics reveal that Drosophila hemocytes rely primarily on oxidative phosphorylation at homeostasis, while immune activation — particularly lamellocyte differentiation — drives enhanced mitochondrial respiration, structural network remodeling, and Drp1-dependent fission using glucose and trehalose as carbon sources. These findings establish mitochondrial metabolic reprogramming as a conserved feature of myeloid-like innate immune activation.
Lineage tracing and clonal evolution
Modeling mitochondrial inheritance enables high-precision single-cell lineage tracing in humans
https://www.biorxiv.org/content/10.64898/2026.02.12.705660v2
The authors present an updated version of their original mitochondrial lineage tracing technique: MitoDrift is a probabilistic framework integrating Wright-Fisher drift dynamics with sparse single-cell mtDNA measurements to produce confidence-refined lineage trees, outperforming existing methods in precision while maintaining high clonal recovery. Applied to human hematopoiesis, it reveals age-associated clonal diversity decline and links AP-1/stress programs to clonal expansions, while in multiple myeloma it captures therapy-driven clonal remodeling undetectable by copy number analysis
In vivo lineage tracing across human tissues using methylation barcodes in the protocadherin gene cluster
https://www.biorxiv.org/content/10.64898/2026.02.23.707349v1
In this work, the authors describe how stochastic methylation patterns in the protocadherin gene cluster serve as heritable, evolvable lineage barcodes across multiple non-neuronal tissues. It quantitatively recapitulates genetic clone sizes when tracked over a decade in serial samples. Critically, PCDH barcodes reveal cryptic clonal expansions invisible to standard driver-mutation sequencing and resolve subclonal architectures through continuous epimutation. This native, scalable barcoding system provides a driver-agnostic framework for reconstructing somatic evolution in human aging and cancer.
BloodVariome: a high-resolution atlas of inherited genetic effects in human immune cells
https://www.biorxiv.org/content/10.64898/2026.03.30.715213v1
BloodVariome integrates deep immunophenotyping of 1,533 traits across 127 immune cell populations in nearly 12,000 individuals, identifying 259 genetic associations — most undetectable by conventional bulk blood studies — with fine-grained compartmentalization restricted to single lineages or cell populations. By linking disease risk alleles to specific immune cell phenotypes, this atlas illuminates cellular mechanisms underlying autoimmunity, immunodeficiency, and hematologic malignancy while implicating novel regulators of immune development.
Scalable genotyping in fixed transcriptomes resolves clonal heterogeneity via single-cell sequencing
https://www.biorxiv.org/content/10.64898/2026.04.11.717967v1
The authors describe a novel assay for combined transcriptomics and genotyping in the same cell. GIFT uses a rationally designed gap-filling reaction to achieve >99% accurate detection of hundreds of somatic mutations alongside whole transcriptome profiles in single cells, including in FFPE tissue. Applied to 700,000 cells from 35 MPN donors, it reveals allelic dose-dependent interferon transcriptional programs and HSC priming linked to JAK2V617F, demonstrating its power for resolving genotype-to-phenotype relationships at population scale.
Blog post contributed by Alessandro Donada, PhD (Bluesky: @alessandrodonada.bsky.social) of the ISEH Publications Committee.
Please note that the statements made by Simply Blood authors are their own views and not necessarily the views of ISEH. ISEH disclaims any or all liability arising from any author's statements or materials.