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Clonal haematopoiesis of indeterminate potential (CHIP) is an aging-associated phenomenon that has recently been correlated with a broad spectrum of human diseases, including haematological malignancy, cytopenia, coronary heart disease, stroke, and overall mortality. CHIP is defined as a somatic variant in blood cells with an allele frequency (VAF) ≥ 0.02, however recent reports show smaller clones are associated with poorer clinical outcome. Error-corrected ultradeep next-generation sequencing (NGS) assays detecting variants < 0.02 VAF also have clinical value for monitoring measurable residual disease (MRD) for myeloid neoplasms. However, limited data are available on optimal parameters, limits of detection, and accuracy of ultra-sensitive detection. We investigated parameters to improve accuracy of Illumina sequencing-by-synthesis method, including read depth, input DNA quantity, and molecular barcoding-based data filtering, while adhering to clinical accreditation criteria. Validation data were generated from reference standards and reference samples from a clinically accredited pathology laboratory. Analytical range measurements included linearity and bias, and precision included repeatability, reproducibility and detection rate. The lower limit of detection was ≥ 0.004 (0.4%) at depth > 3,000 × . Trueness measured using reference standards demonstrated a sensitivity, specificity, positive and negative predictive values, and accuracy of 100%, including FLT3-ITD, and 100% concordance was achieved with reference samples for reported variants and absence of variants. Sequencing blood samples from 383 community-dwelling adults (mean depth 3758×) revealed 2,190 somatic variants/sample, > 99.9% were < 0.02 VAF. Our data including cost-benefit analysis enables pathology and research laboratories to make informed decisions for detection of CHIP (VAF ≥ 0.02), sub-CHIP (VAF 0.01-0.02) and MRD (VAF ≥ 0.004)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Tursky et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

High-risk childhood leukemia has a poor prognosis because of treatment failure and toxic side effects of therapy. Drug encapsulation into liposomal nanocarriers has shown clinical success at improving biodistribution and tolerability of chemotherapy. However, enhancements in drug efficacy have been limited because of a lack of selectivity of the liposomal formulations for the cancer cells. Here, we report on the generation of bispecific antibodies (BsAbs) with dual binding to a leukemic cell receptor, such as CD19, CD20, CD22, or CD38, and methoxy polyethylene glycol (PEG) for the targeted delivery of PEGylated liposomal drugs to leukemia cells. This liposome targeting system follows a "mix-and-match" principle where BsAbs were selected on the specific receptors expressed on leukemia cells. BsAbs improved the targeting and cytotoxic activity of a clinically approved and low-toxic PEGylated liposomal formulation of doxorubicin (Caelyx) toward leukemia cell lines and patient-derived samples that are immunophenotypically heterogeneous and representative of high-risk subtypes of childhood leukemia. BsAb-assisted improvements in leukemia cell targeting and cytotoxic potency of Caelyx correlated with receptor expression and were minimally detrimental in vitro and in vivo toward expansion and functionality of normal peripheral blood mononuclear cells and hematopoietic progenitors. Targeted delivery of Caelyx using BsAbs further enhanced leukemia suppression while reducing drug accumulation in the heart and kidneys and extended overall survival in patient-derived xenograft models of high-risk childhood leukemia. Our methodology using BsAbs therefore represents an attractive targeting platform to potentiate the therapeutic efficacy and safety of liposomal drugs for improved treatment of high-risk leukemia.

Pharmacological inhibition of MDM2/4, which activates the critical tumor suppressor p53, has been gaining increasing interest as a strategy for the treatment of acute myeloid leukemia (AML). While clinical trials of MDM2 inhibitors have shown promise, responses have been confined to largely molecularly undefined patients, indicating that new biomarkers and optimized treatment strategies are needed. We previously reported that the microRNA miR-10a is strongly overexpressed in some AML, and demonstrate here that it modulates several key members of the p53/Rb network, including p53 regulator MDM4, Rb regulator RB1CC1, p21 regulator TFAP2C, and p53 itself. The expression of both miR-10a and its downstream targets were strongly predictive of MDM2 inhibitor sensitivity in cell lines, primary AML specimens, and correlated to response in patients treated with both MDM2 inhibitors and cytarabine. Furthermore, miR-10a inhibition induced synergy between MDM2 inhibitor Nutlin-3a and cytarabine in both in vitro and in vivo AML models. Mechanistically this synergism primarily occurs via the p53-mediated activation of cytotoxic apoptosis at the expense of cytoprotective autophagy. Together these findings demonstrate that miR-10a may be useful as both a biomarker to identify patients most likely to respond to cytarabine+MDM2 inhibition and also a druggable target to increase their efficacy.

Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor-AB and 5-azacytidine into multipotent stem cells with stromal cell characteristics. We have now optimized culture conditions to reprogram human adipocytes into induced multipotent stem (iMS) cells and characterized their molecular and functional properties. Although the basal transcriptomes of adipocyte-derived iMS cells and adipose tissue-derived mesenchymal stem cells were similar, there were changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to muscle, bone, cartilage, and blood vessels, with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibers without ectopic tissue formation. Together, human adipocyte-derived iMS cells regenerate tissues in a context-dependent manner without ectopic or neoplastic growth., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Induced pluripotent stem cells (iPSCs) are an invaluable resource for the study of human disease. However, there are no standardized methods for differentiation into hematopoietic cells, and there is a lack of robust, direct comparisons of different methodologies. In the current study we improved a feeder-free, serum-free method for generation of hematopoietic cells from iPSCs, and directly compared this with three other commonly used strategies with respect to efficiency, repeatability, hands-on time, and cost. We also investigated their capability and sensitivity to model genetic hematopoietic disorders in cells derived from Down syndrome and β-thalassemia patients. Of these methods, a multistep monolayer-based method incorporating aryl hydrocarbon receptor hyperactivation ("2D-multistep") was the most efficient, generating significantly higher numbers of CD34 + progenitor cells and functional hematopoietic progenitors, while being the most time- and cost-effective and most accurately recapitulating phenotypes of Down syndrome and β-thalassemia., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

We present the largest comparative biogeographical analysis that has complete coverage of Australia's geography (20 phytogeographical subregions), using the most complete published molecular phylogenies to date of large Australian plant clades (Acacia, Banksia and the eucalypts). Two distinct sets of areas within the Australian flora were recovered, using distributional data from the Australasian Virtual Herbarium (AVH) and the Atlas of Living Australia (ALA): younger Temperate, Eremaean and Monsoonal biomes, and older southwest + west, southeast and northern historical biogeographical regions. The analyses showed that by partitioning the data into two sets, using either a Majority or a Frequency method to select taxon distributions, two equally valid results were found. The dataset that used a Frequency method discovered general area cladograms that resolved patterns of the Australian biomes, whereas if widespread taxa (Majority method, with >50% of occurrences outside a single subregion) were removed the analysis then recovered historical biogeographical regions. The study highlights the need for caution when processing taxon distributions prior to analysis as, in the case of the history of Australian phytogeography, the validity of both biomes and historical areas have been called into question., (© The Willi Hennig Society 2019.)

Myelodysplastic syndromes and chronic myelomonocytic leukemia are blood disorders characterized by ineffective hematopoiesis and progressive marrow failure that can transform into acute leukemia. The DNA methyltransferase inhibitor 5-azacytidine (AZA) is the most effective pharmacological option, but only ∼50% of patients respond. A response only manifests after many months of treatment and is transient. The reasons underlying AZA resistance are unknown, and few alternatives exist for non-responders. Here, we show that AZA responders have more hematopoietic progenitor cells (HPCs) in the cell cycle. Non-responder HPC quiescence is mediated by integrin α5 (ITGA5) signaling and their hematopoietic potential improved by combining AZA with an ITGA5 inhibitor. AZA response is associated with the induction of an inflammatory response in HPCs in vivo. By molecular bar coding and tracking individual clones, we found that, although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aberrant ERG (v-ets avian erythroblastosis virus E26 oncogene homolog) expression drives leukemic transformation in mice and high expression is associated with poor patient outcomes in acute myeloid leukemia (AML) and T-acute lymphoblastic leukemia (T-ALL). Protein phosphorylation regulates the activity of many ETS factors but little is known about ERG in leukemic cells. To characterize ERG phosphorylation in leukemic cells, we applied liquid chromatography coupled tandem mass spectrometry and identified five phosphorylated serines on endogenous ERG in T-ALL and AML cells. S283 was distinct as it was abundantly phosphorylated in leukemic cells but not in healthy hematopoietic stem and progenitor cells (HSPCs). Overexpression of a phosphoactive mutant (S283D) increased expansion and clonogenicity of primary HSPCs over and above wild-type ERG. Using a custom antibody, we screened a panel of primary leukemic xenografts and showed that ERG S283 phosphorylation was mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling and in turn regulated expression of components of this pathway. S283 phosphorylation facilitates ERG enrichment and transactivation at the ERG +85 HSPC enhancer that is active in AML and T-ALL with poor prognosis. Taken together, we have identified a specific post-translational modification in leukemic cells that promotes progenitor proliferation and is a potential target to modulate ERG-driven transcriptional programs in leukemia.

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell-free matrices from bone marrow cells that mimic 'human-body-niche-like' conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum-free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering-based biomimetic scaffolds for HSPC expansion for clinical applications., (Copyright © 2016 John Wiley & Sons, Inc.)

High expression of the ETS family transcription factor ERG is associated with poor clinical outcome in acute myeloid leukemia (AML) and acute T-cell lymphoblastic leukemia (T-ALL). In murine models, high ERG expression induces both T-ALL and AML. However, no study to date has defined the effect of high ERG expression on primary human hematopoietic cells. In the present study, human CD34+ cells were transduced with retroviral vectors to elevate ERG gene expression to levels detected in high ERG AML. RNA sequencing was performed on purified populations of transduced cells to define the effects of high ERG on gene expression in human CD34+ cells. Integration of the genome-wide expression data with other data sets revealed that high ERG drives an expression signature that shares features of normal hematopoietic stem cells, high ERG AMLs, early T-cell precursor-ALLs and leukemic stem cell signatures associated with poor clinical outcome. Functional assays linked this gene expression profile to enhanced progenitor cell expansion. These results support a model whereby a stem cell gene expression network driven by high ERG in human cells enhances the expansion of the progenitor pool, providing opportunity for the acquisition and propagation of mutations and the development of leukemia.

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell-free matrices from bone marrow cells that mimic 'human-body niche-like' conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum-free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering-based biomimetic scaffolds for HSPC expansion for clinical applications., (Copyright © 2014 John Wiley & Sons, Inc.)

Lineage-specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bone marrow failure and immunodeficiencies. The current methods for HSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell-free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell-free matrices from a murine bone marrow stromal cell line (MS-5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O₂ tensions. These acellular matrices were used as biological scaffolds for the lineage-specific expansion of magnetically sorted CD34⁺ cells and the results were evaluated by flow cytometry and colony-forming assays. We could get up to 80-fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering-based ex vivo HSPC expansion, and it may also provide insights into the constitution of the niche in which these cells reside in the bone marrow., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background Aims: Cord blood is considered to be a superior source of hematopoietic stem and progenitor cells for transplantation, but clinical use is limited primarily because of the low numbers of cells harvested. Ex vivo expansion has the potential to provide a safe, effective means of increasing cell numbers. However, an absence of consensus regarding optimum expansion conditions prevents standard implementation. Many studies lack clinical applicability, or have failed to investigate the combinational effects of different parameters., Methods: This is the first study to characterize systematically the effect of growth factor combinations across multiple oxygen levels on the ex vivo expansion of cord blood CD34(+) hematopoietic cells utilizing clinically approvable reagents and methodologies throughout., Results: Optimal fold expansion, as assessed both phenotypically and functionally, was greatest with thrombopoietin, stem cell factor, Flt-3 ligand and interleukin-6 at an oxygen level of 10%. With these conditions, serial expansion showed continual target population expansion and consistently higher expression levels of self-renewal associated genes., Conclusions: This study has identified optimized fold expansion conditions, with the potential for direct clinical translation to increase transplantable cell dose and as a baseline methodology against which future factors can be tested.

The type III transforming growth factor beta (TGFbeta) receptor (TbetaRIII) binds both TGFbeta and inhibin with high affinity and modulates the association of these ligands with their signaling receptors. However, the significance of TbetaRIII signaling in vivo is not known. In this study, we have sought to determine the role of TbetaRIII during development. We identified the predominant expression sites of TbetaRIII mRNA as liver and heart during midgestation and have disrupted the murine TbetaRIII gene by homologous recombination. Beginning at embryonic day 13.5, mice with mutations in TbetaRIII developed lethal proliferative defects in heart and apoptosis in liver, indicating that TbetaRIII is required during murine somatic development. To assess the effects of the absence of TbetaRIII on the function of its ligands, primary fibroblasts were generated from TbetaRIII-null and wild-type embryos. Our results indicate that TbetaRIII deficiency differentially affects the activities of TGFbeta ligands. Notably, TbetaRIII-null cells exhibited significantly reduced sensitivity to TGFbeta2 in terms of growth inhibition, reporter gene activation, and Smad2 nuclear localization, effects not observed with other ligands. These data indicate that TbetaRIII is an important modulator of TGFbeta2 function in embryonic fibroblasts and that reduced sensitivity to TGFbeta2 may underlie aspects of the TbetaRIII mutant phenotype.