Chronic myelomonocytic leukaemia (CMML) is a haematological malignancy characterised by overlapping myeloid dysplasia and proliferation with persisting monocytosis. While monocytes are the cardinal malignant cell type in CMML, as a stem cell neoplasm the disease clone comprises most lineages and differentiation stages, including granulocytes. To investigate the pathogenic contribution of granulocytes in CMML maintenance and progression, we performed phenotypic, transcriptomic and functional characterization of CMML granulocytes. Compared with healthy age-matched controls, CMML granulocytes exhibit defective maturation with reduced granularity and phagocytic capacity. Transcriptome analysis revealed activation of pathways linked to proliferation, Myc activity and inflammation. Notably, RETN , which encodes the inflammatory mediator resistin, was upregulated approximately 100-fold in CMML granulocytes; but not differentially expressed in CMML PBMNCs, sorted monocytes, or stem and progenitor cells compared to their healthy counterparts. Accordingly, resistin protein levels were 10-fold higher in plasma from CMML patients and higher plasma resistin levels correlate with poor overall survival and AML-free survival. Remarkably, exposure of healthy monocytes to exogenous recombinant resistin inhibited monocyte maturation and macrophage differentiation. Transcriptome analysis of resistin treated monocytes revealed that resistin induces gene signatures related to immune suppression and myeloid-derived suppressor cell phenotype. We found SEMA4A to be a downstream target of resistin and overexpressed in CMML monocytes. Consistent with known roles for SEMA4A, CMML patients displayed higher percentage of Tregs and elevated Th2/Th1 ratio compared with healthy controls and percentage of Tregs corresponding with associated resistin levels. Furthermore, we demonstrated that resistin directly skews the Th2/Th1 ratio via binding to monocytes. In conclusion, we showed that immature granulocytes in CMML produce high levels of resistin, which contributes to defective monocyte maturation and immune suppression.

Human cell reprogramming traditionally involves time-intensive, multistage, costly tissue culture polystyrene-based cell culture practices that ultimately produce low numbers of reprogrammed cells of variable quality. Previous studies have shown that very soft 2- and 3-dimensional hydrogel substrates/matrices (of stiffnesses ≤ 1 kPa) can drive ~2× improvements in human cell reprogramming outcomes. Unfortunately, these similarly complex multistage protocols lack intrinsic scalability, and, furthermore, the associated underlying molecular mechanisms remain to be fully elucidated, limiting the potential to further maximize reprogramming outcomes. In screening the largest range of polyacrylamide (pAAm) hydrogels of varying stiffness to date (1 kPa to 1.3 MPa), we have found that a medium stiffness gel (~100 kPa) increased the overall number of reprogrammed cells by up to 10-fold (10×), accelerated reprogramming kinetics, improved both early and late phases of reprogramming, and produced induced pluripotent stem cells (iPSCs) having more naïve characteristics and lower remnant transgene expression, compared to the gold standard tissue culture polystyrene practice. Functionalization of these pAAm hydrogels with poly-l-dopamine enabled, for the first-time, continuous, single-step reprogramming of fibroblasts to iPSCs on hydrogel substrates (noting that even the tissue culture polystyrene practice is a 2-stage process). Comparative RNA sequencing analyses coupled with experimental validation revealed that a novel reprogramming regulator, protein phosphatase and actin regulator 3, up-regulated under the gel condition at a very early time point, was responsible for the observed enhanced reprogramming outcomes. This study provides a novel culture protocol and substrate for continuous hydrogel-based cell reprogramming and previously unattained clarity of the underlying mechanisms via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes.
Copyright © 2024 Mohammad Mahfuz et al.

Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function1-8. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.
© 2023. The Author(s).

Cell reprogramming involves time-intensive, costly processes that ultimately produce low numbers of reprogrammed cells of variable quality. By screening a range of polyacrylamide hydrogels (pAAm gels) of varying stiffness (1 kPA – 1.3 MPa) we found that a gel of medium stiffness significantly increases the overall number of reprogrammed cells by up to ten-fold with accelerated reprogramming kinetics, as compared to the standard Tissue Culture PolyStyrene (TCPS)-based protocol. We observe that though the gel improves both early and late phases of reprogramming, improvement in the late (reprogramming prone population maturation) phase is more pronounced and produces iPSCs having different characteristics and lower remnant transgene expression than those produced on TCPS. Comparative RNA-Seq analyses coupled with experimental validation reveals that modulation of Bone Morphogenic Protein (BMP) signalling by a novel reprogramming regulator, Phactr3, upregulated in the gel at an earliest time-point without the influence of transcription factors used for reprogramming, plays a crucial role in the improvement in the early reprogramming kinetics and overall reprogramming outcomes. This study provides new insights into the mechanism via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes, opening new avenues for producing higher numbers of quality iPSCs or other reprogrammed cells at shorter timescales.

Human induced pluripotent stem cells (hiPSCs) can differentiate into cells of the three germ layers and are promising cell sources for regenerative medicine therapies. However, current protocols generate hiPSCs with low efficiency, and the generated iPSCs have variable differentiation capacity among different clones. Our previous study reported that MYC proteins (c-MYC and MYCL) are essential for reprogramming and germline transmission but that MYCL can generate hiPSC colonies more efficiently than c-MYC. The molecular underpinnings for the different reprogramming efficiencies between c-MYC and MYCL, however, are unknown. In this study, we found that MYC Box 0 (MB0) and MB2, two functional domains conserved in the MYC protein family, contribute to the phenotypic differences and promote hiPSC generation in MYCL-induced reprogramming. Proteome analyses suggested that in MYCL-induced reprogramming, cell adhesion-related cytoskeletal proteins are regulated by the MB0 domain, while the MB2 domain regulates RNA processes. These findings provide a molecular explanation for why MYCL has higher reprogramming efficiency than c-MYC.
© 2021. The Author(s).