Hematopoietic Regeneration Research Articles

Gut microbiota plays pivotal roles in benign and malignant hematopoiesis.

Accumulated evidence emerges that dynamic changes in human gut microbiota and microbial metabolites can alter the ecological balance of symbiotic hosts. The gut microbiota plays a role in various diseases through different mechanisms. More and more attention has been paid to the effects that human microbiota extends beyond the gut. This review summarized the current understanding of the roles that gut microbiota plays in hematopoietic regulation and the occurrence and development of benign and malignant hematologic diseases. The progress of the application of microbiota in treatment was discussed in order to provide new insights into clinical diagnosis and treatment in the future.

USP4 regulates ribosome biogenesis and protein synthesis for hematopoietic stem cell regeneration and leukemia progression.

Enhanced ribosome biogenesis and protein synthesis are required for cell proliferation. During hematopoietic regeneration, hematopoietic stem cells (HSCs) proliferate rapidly to replenish the hematopoietic system. How HSCs respond and regulate ribosome biogenesis and protein synthesis during regeneration remains unclear. Here, we analyzed the expression of a series of ubiquitin-specific-proteases (USPs) during HSC regeneration. We found USP4 expression is significantly increased in proliferating HSCs. Further functional and mechanistic investigations revealed a crucial regulatory function of USP4 in HSC regeneration and leukemia progression by modulating ribosome biogenesis and protein synthesis. USP4 deubiquitinates and stabilizes PES1 to facilitate ribosome biogenesis and protein synthesis in proliferative HSCs and leukemic cells. Usp4 deletion significantly decreases protein synthesis, proliferation and reconstitution capacity of HSCs. Usp4 inhibition suppresses ribosome biogenesis and proliferation of leukemic cells, and prolongs the survival of AML (Acute myeloid leukemia) mice. These findings provide a new insight into the response mechanism of ribosome biogenesis and protein synthesis in HSCs, and their contribution to leukemia progression.

A novel role of ETV6 as a pro-viral factor in host response by inhibiting TBK1 phosphorylation

E26-transforming specific (ETS) variant 6 (ETV6) is a transcription factor regulating the expression of interferon stimulating genes (ISGs) and involved in the embryonic development and hematopoietic regulation, but the role of ETV6 in host response to virus infection is not clear. In this study, we show that ETV6 was upregulated in DF-1 cells with poly(I:C) stimulation or IBDV, AIV and ARV infection via engagement of dsRNA by MDA5. Overexpression of ETV6 in DF-1 cells markedly inhibited IBDV-induced type I interferon (IFN-I) and ISGs expressions. In contrast, knockdown, or knockout of ETV6 remarkably inhibited IBDV replication via promoting IFN-I response. Furthermore, our data show that ETV6 negatively regulated host antiviral response to IBDV infection by interaction with TANK binding kinase 1 (TBK1) and subsequently inhibited its phosphorylation. These results uncovered a novel role of ETV6 as a pro-viral factor in host response by inhibiting TBK1 phosphorylation, furthering our understandings of RNA virus immunosuppression and providing a valuable clue to the development of antiviral reagents for the control of avian RNA virus infection.

Occupational exposure to arsenic and leukopenia risk: Toxicological alert.

Arsenic and its inorganic compounds affect numerous organs and systemic functions, such as the nervous and hematopoietic systems, liver, kidneys, and skin. Despite a large number of studies on arsenic toxicity, rare reports have investigated the leukopenia incidence in workers exposed to arsenic. In workplaces, the main source of workers' exposure is the contaminated air by the inorganic arsenic in mines, arsenic or copper smelter industries, and chemical factories. Erythropoiesis inhibition is one of the arsenic effects and it is related to regulatory factor GATA-1. This factor is necessary for the normal differentiation of early erythroid progenitors. JAK-STAT is an important intracellular signal transduction pathway responsible for the mediating normal functions of several cytokines related to cell proliferation and hematopoietic systems development and regulation. Arsenic inactivates JAK-STAT by inhibiting JAK tyrosine kinase and using the IFNγ pathway. The intravascular hemolysis starts after the absorption phase when arsenic binds to the globin of hemoglobin in erythrocytes and is transported into the body, which increases the oxidation of sulfhydryl groups in hemoglobin. So, this article intends to highlight the potential leukopenia risk via inhalation for workers exposed to arsenic and suggests a possible mechanism for this leukopenia through the JAK-signal transducer and activator of transcription (STAT) pathway inhibition.

Thrombopoietin improves the functions of bone marrow endothelial progenitor cells via METTL16/Akt signalling of haematological patients with chemotherapy-induced thrombocytopenia.

Bone marrow endothelial progenitor cells (BM EPCs) are crucial in supporting haematopoietic regeneration, while the BM EPCs of haematological patients with chemotherapy-induced thrombocytopenia (CIT) are unavoidably damaged. Therefore, the present study aimed to examine the effect of thrombopoietin (TPO) on the recovery of BM EPCs of CIT patients and to identify the underlying mechanisms. The cell functions were determined by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil)-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake and fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin-I (FITC-UEA-I) binding assay, as well as proliferation, migration and tube formation experiments. Endothelial cells were transfected with METTL16 lentivirus, followed by methylated RNA immunoprecipitation sequencing. Zebrafish with vascular defect was used as the invivo model. TPO significantly improved the quantity and functions of BM EPCs from CIT patients invitro and restored the subintestinal vein area of zebrafish with vascular defect invivo. Mechanically, TPO enhanced the BM EPC functions through Akt signal mediated by METTL16, which was downregulated in BM EPCs of CIT patients and involved in the regulation of endothelial functions. The present study demonstrates that TPO improves the recovery of BM EPCs from CIT patients with haematological malignancies via METTL16/Akt signalling, which provides new insights into the role of TPO in treating CIT in addition to direct megakaryopoiesis.

HLA-lacking clones in aplastic anaemia: Adaptive or maladaptive?

HLA loss represents the result of immune forces shaping bone marrow clonal dynamics in immune aplastic anaemia. Human leukocyte antigen (HLA)-deficient clones may rescue haematopoiesis by evading immune attacks, potentially guiding treatment strategies. Commentary on: Zaimoku etal. Haematopoietic regeneration by HLA-A*0206-deficient clones in severe aplastic anaemia without definitive immunosuppressive treatment. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19712.

Co-delivery of protopanaxatriol/icariin into niche cells restores bone marrow niches to rejuvenate HSCs for chemotherapy-induced myelosuppression

BackgroundUp to 80 % of chemotherapeutic drugs induce myelosuppression in patients. Chemotherapy not only impairs of hematopoietic stem cells (HSCs) but also damages bone marrow niches (vascular and endosteal). Current treatments for myelosuppression overlook these chemotherapy-induced damages to bone marrow niches and the critical role of niche restoration on hematopoietic regeneration. Ginsenoside protopanaxatriol (PPT) protects vascular endothelium from injury, while icariin (ICA) promotes osteogenic differentiation. The combination of PPT and ICA aims to restore damaged vascular and endosteal niches, thus rejuvenating HSCs for treating myelosuppression. PurposeThis study aims to develop effective, bone marrow niche-directed PPT/ICA therapies for treating chemotherapy-induced myelosuppression. Methods3D cell spheroids were used to investigate the effects of PPT/ICA on cell-cell interactions in vascular niches, osteogenesis, and extracellular matrix (ECM) secretion in endosteal niches. In vitro mimic niche models were designed to access the drug combination's efficacy in rejuvenating and mobilizing in HSCs within bone marrow niches. The delivery capability of PPT/ICA to key niche cell types (mesenchymal stromal cells (MSCs), endothelial cells (ECs), and osteoblasts (OBs)) via nanocarriers has been determined. DSS6 peptide-modified nanoparticles (DSS6-NPs) were prepared for specific co-delivery of PPT/ICA into key niche cell populations in vivo. ResultsPPT can prevent vascular niche injury by restoring vascular EC cell-cell adhesion and the intercellular interactions between ECs and MSCs in 5-fluorouracil (5-FU)-damaged cell spheroids. ICA repaired 5-FU-damaged endosteal niches by promoting osteogenesis and ECM secretion. The combination of PPT and ICA restores key HSC niche factor gene expressions, normalizing HSC differentiation and mobilization. The in vitro cellular uptake efficiency of nanocarriers in a mimic niche is positively correlated with their in vivo delivery into bone marrow niche cells. DSS6-NPs greatly enhance the delivery of PPT/ICA into MSCs and OBs within bone marrow niches. Co-loading of PPT/ICA into DSS6-NPs effectively repairs damaged bone marrow niches and promotes HSC rejuvenation in vivo. ConclusionThe combination of PPT and ICA effectively prevents injury to the vascular and endosteal niches, thereby promoting hematopoietic regeneration in the bone marrow. This study provides novel niche-directed PPT/ICA therapies for managing chemotherapy-induced myelosuppression.

Haematopoietic regeneration by HLA-A*0206-deficient clones in severe aplastic anaemia without definitive immunosuppressive treatment.

We describe the case of a 74-year-old man with severe aplastic anaemia who experienced persistent remission attributed to proliferation of HLA allele-deficient clones. Despite an initial worsening of pancytopenia with eltrombopag and ciclosporin treatment, gradual trilineage haematopoietic recovery occurred, with blood counts normalizing over 3 years. Flow cytometry and deep nucleotide sequencing revealed that haematopoiesis was primarily supported by several clones with somatic mutations that inactivated antigen presentation via HLA-A*0206. This suggests that monitoring haematopoietic regeneration by immune escape clones could be an alternative approach for immune aplastic anaemia patients who possess HLA allele-deficient clones and cannot tolerate standard therapy.

Mechanobiology and Primary Cilium in the Pathophysiology of Bone Marrow Myeloproliferative Diseases.

Philadelphia-Negative Myeloproliferative neoplasms (MPNs) are a diverse group of blood cancers leading to excessive production of mature blood cells. These chronic diseases, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), can significantly impact patient quality of life and are still incurable in the vast majority of the cases. This review examines the mechanobiology within a bone marrow niche, emphasizing the role of mechanical cues and the primary cilium in the pathophysiology of MPNs. It discusses the influence of extracellular matrix components, cell-cell and cell-matrix interactions, and mechanosensitive structures on hematopoietic stem cell (HSC) behavior and disease progression. Additionally, the potential implications of the primary cilium as a chemo- and mechanosensory organelle in bone marrow cells are explored, highlighting its involvement in signaling pathways crucial for hematopoietic regulation. This review proposes future research directions to better understand the dysregulated bone marrow niche in MPNs and to identify novel therapeutic targets.

Trim47 prevents hematopoietic stem cell exhaustion during stress by regulating MAVS-mediated innate immune pathway

The maintenance of hematopoietic stem cell (HSC) functional integrity is essential for effective hematopoietic regeneration when suffering from injuries. Studies have shown that the innate immune pathways play crucial roles in the stress response of HSCs, whereas how to precisely modulate these pathways is not well characterized. Here, we identify the E3 ubiquitin ligase tripartite motif-containing 47 (Trim47) as a negative regulator of the mitochondrial antiviral-signaling protein (MAVS)-mediated innate immune pathway in HSCs. We find that Trim47 is predominantly enriched in HSCs, and its deficiency impairs the function and survival of HSCs after exposure to 5-flurouracil (5-FU) and irradiation (IR). Mechanistically, Trim47 impedes the excessive activation of the innate immune signaling and inflammatory response via K48-linked ubiquitination and degradation of MAVS. Collectively, our findings demonstrate a role of Trim47 in preventing stress-induced hematopoietic failure and thus provide a promising avenue for treatment of related diseases in the clinic.

Spatial Transcriptomic Profiling Reveals Gene Expression Characteristics in Lymph Node-positive Breast Carcinoma.

Studies on the differences in gene expression characteristics according to lymph node metastasis in breast carcinoma are lacking. This study aimed to compare the spatially resolved transcriptomic profiles between node-positive and negative breast carcinomas. Eight patients with breast carcinoma were included, and digital spatial profiling and bioinformatic analysis were conducted to investigate the spatial transcriptomes. In the epithelial compartment, the top two most up-regulated genes were nuclear receptor subfamily 4 group A member 1 (NR4A1) and Jun proto-oncogene, activating protein-1 transcription factor subunit (JUN). The gene ontology (GO) enrichment analysis of the node-positive group revealed a significant up-regulation of genes associated with myeloid differentiation, mononuclear cell differentiation, and hematopoietic regulation. The gene set enrichment analysis (GSEA) revealed significant enrichment of gene sets associated with the regulation of inflammatory cytokines in both the epithelial and stromal compartments of the node-positive group. Our study highlights significant differences in gene expression profiles and spatially resolved transcriptional activities between node-positive and negative breast carcinomas. These findings underscore the importance of developing personalized treatment strategies for lymph node metastasis of breast carcinoma.

Mobilization dynamics of bone marrow hematopoietic stem cells during hematopoietic regeneration

Under stress hematopoiesis, previous studies have suggested the migration of hematopoietic stem cells (HSCs) from bone marrow (BM) to extramedullary sites such as the spleen. However, there is little direct evidence of HSC migration from the BM to the spleen. Here, we induced myeloablation via 5-fluorouracil (5-FU) and showed direct evidence of HSC migration from BM to spleen during hematopoietic regeneration via a photoconvertible fluorophore. Moreover, during steady state, HSCs preferentially migrated to BM rather than spleen, but during hematopoietic regeneration, HSCs preferred spleen as a migration site equivalently or greater. Furthermore, in the early phase, HSCs egressed from BM through the attenuated HSC retention. However, HSCs in the late phase gained significantly enhanced cell-autonomous motility, which was independent of chemotaxis. Collectively, HSC mobilization from BM, before the migration to the spleen, was dynamically changed from passive to active events during hematopoietic regeneration.

Silencing endomucin in bone marrow sinusoids improves hematopoietic stem and progenitor cell homing during transplantation.

Efficient homing of infused hematopoietic stem and progenitor cells (HSPCs) into the bone marrow (BM) is the prerequisite for successful hematopoietic stem cell transplantation. However, only a small part of infused HSPCs find their way to the BM niche. A better understanding of the mechanisms that facilitate HSPC homing will help to develop strategies to improve the initial HSPC engraftment and subsequent hematopoietic regeneration. Here, we show that irradiation upregulates the endomucin expression of endothelial cells in vivo and in vitro. Furthermore, depletion of endomucin in irradiated endothelial cells with short-interfering RNA (siRNA) increases the HSPC-endothelial cell adhesion in vitro. To abrogate the endomucin of BM sinusoidal endothelial cells (BM-SECs) in vivo, we develop a siRNA-loaded bovine serum albumin nanoparticle for targeted delivery. Nanoparticle-mediated siRNA delivery successfully silences endomucin expression in BM-SECs and improves HSPC homing during transplantation. These results reveal that endomucin plays a critical role in HSPC homing during transplantation and that gene-based manipulation of BM-SEC endomucin in vivo can be exploited to improve the efficacy of HSPC transplantation.

Charting epimutation dynamics in human hematopoietic differentiation.

DNA methylation plays a critical role in hematopoietic differentiation. Epimutation is a stochastic variation in DNA methylation that induces epigenetic heterogeneity. However, the effects of epimutations on normal hematopoiesis and hematopoietic diseases remain unclear. In this study, we developed a Julia package called EpiMut that enabled rapid and accurate quantification of epimutations. EpiMut was used to evaluate and provide an epimutation landscape in steady-state hematopoietic differentiation involving 13 types of blood cells ranging from hematopoietic stem/progenitor cells to mature cells. We showed that substantial genomic regions exhibited epigenetic variations rather than significant differences in DNA methylation levels between the myeloid and lymphoid lineages. Stepwise dynamics of epimutations were observed during the differentiation of each lineage. Importantly, we found that epimutation significantly enriched signals associated with lineage differentiation. Furthermore, epimutations in hematopoietic stem cells (HSCs) derived from various sources and acute myeloid leukemia were related to the function of HSCs and malignant cell disorders. Taken together, our study comprehensively documented an epimutation map and uncovered its important roles in human hematopoiesis, thereby offering insights into hematopoietic regulation.

BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation

Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity. BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.

Megakaryocytic IGF1 coordinates activation and ferroptosis to safeguard hematopoietic stem cell regeneration after radiation injury.

Hematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression, which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury, while if and how the niche is reshaped and regulates HSC regeneration are poorly understood. A mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number, distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry, immunofluorescence, colony assay and bone marrow transplantation, in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro, and was consolidated using megakaryocyte-specific knockout mice and transgenic mice. Megakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile, transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury, whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically, HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion, and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs, but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently, the delicate coordination between proliferation, mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly, punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury, representing a superior therapeutic approach for myelosuppression. Our study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.

From Marrow to Bone and Fat: Exploring the Multifaceted Roles of Leptin Receptor Positive Bone Marrow Mesenchymal Stromal Cells.

The bone marrow (BM) stromal cell microenvironment contains non-hematopoietic stromal cells called mesenchymal stromal cells (MSCs). MSCs are plastic adherent, form CFU-Fs, and give rise to osteogenic, adipogenic, chondrogenic progenitors, and most importantly provide HSC niche factor chemokine C-X-C motif ligand 12 (CXCL12) and stem cell factor (SCF). Different authors have defined different markers for mouse MSC identification like PDGFR+Sca-1+ subsets, Nestin+, or LepR+ cells. Of these, the LepR+ cells are the major source of SCF and CXCL12 in the BM microenvironment and play a major role in HSC maintenance and hematopoiesis. LepR+ cells give rise to most of the bones and BM adipocytes, further regulating the microenvironment. In adult BM, LepR+ cells are quiescent but after fracture or irradiation, they proliferate and differentiate into mesenchymal lineage osteogenic, adipogenic and/or chondrogenic cells. They also play a crucial role in the steady-state hematopoiesis process, as well as hematopoietic regeneration and the homing of hematopoietic stem cells (HSCs) after myeloablative injury and/or HSC transplantation. They line the sinusoidal cavities, maintain the trabeculae formation, and provide the space for HSC homing and retention. However, the LepR+ cell subset is heterogeneous; some subsets have higher adipogenic potential, while others express osteollineage-biased genes. Different transcription factors like Early B cell factor 3 (EBF3) or RunX2 help maintain this balance between the self-renewing and committed states, whether osteogenic or adipogenic. The study of LepR+ MSCs holds immense promise for advancing our understanding of HSC biology, tissue regeneration, metabolic disorders, and immune responses. In this review, we will discuss the origin of the BM resident LepR+ cells, different subtypes, and the role of LepR+ cells in maintaining hematopoiesis, osteogenesis, and BM adipogenesis following their multifaceted impact.

Establishment of human hematopoietic organoids for evaluation of hematopoietic injury and regeneration effect

BackgroundHuman hematopoietic organoids have a wide application value for modeling human bone marrow diseases, such as acute hematopoietic radiation injury. However, the manufacturing of human hematopoietic organoids is an unaddressed challenge because of the complexity of hematopoietic tissues.MethodsTo manufacture hematopoietic organoids, we obtained CD34+ hematopoietic stem and progenitor cells (HSPCs) from human embryonic stem cells (hESCs) using stepwise induction and immunomagnetic bead-sorting. We then mixed these CD34+ HSPCs with niche-related cells in Gelatin-methacryloyl (GelMA) to form a three-dimensional (3D) hematopoietic organoid. Additionally, we investigated the effects of radiation damage and response to granulocyte colony-stimulating factor (G-CSF) in hematopoietic organoids.ResultsThe GelMA hydrogel maintained the undifferentiated state of hESCs-derived HSPCs by reducing intracellular reactive oxygen species (ROS) levels. The established hematopoietic organoids in GelMA with niche-related cells were composed of HSPCs and multilineage blood cells and demonstrated the adherence of hematopoietic cells to niche cells. Notably, these hematopoietic organoids exhibited radiation-induced hematopoietic cell injury effect, including increased intracellular ROS levels, γ-H2AX positive cell percentages, and hematopoietic cell apoptosis percentages. Moreover, G-CSF supplementation in the culture medium significantly improved the survival of HSPCs and enhanced myeloid cell regeneration in these hematopoietic organoids after radiation.ConclusionsThese findings substantiate the successful manufacture of a preliminary 3D hematopoietic organoid from hESCs-derived HSPCs, which was utilized for modeling hematopoietic radiation injury and assessing the radiation-mitigating effects of G-CSF in vitro. Our study provides opportunities to further aid in the standard and scalable production of hematopoietic organoids for disease modeling and drug testing.

Abstract 4120: Adherent-to suspension transition by hematopoietic factors in metastatic dissemination of circulating tumor cells

Abstract Although metastasis is the foremost cause of cancer-related death, a specialized mechanism that reprograms anchorage dependency of solid tumor cells into circulating tumor cells (CTCs) during the metastatic cascade remains elusive. We discovered a biological phenomenon referred to as Adherent-to-Suspension Transition (AST) that reprograms adherent cells into suspension cells via aberrant induction of hematopoietic transcriptional regulators, which are hijacked by solid tumor cells to disseminate into CTCs. During dissemination, tumor microenvironment triggers epigenetic alteration of AST factors in the invasive front of solid tumors to evoke spontaneous cell-matrix dissociation, acquire anoikis resistance, and bypass immune surveillance of CTCs, however, in the absence of lineage differentiation and EMT. Using mouse models and de novo metastatic patient specimens, we uncovered how Adherent-Suspension Plasticity (ASP) dictates anchorage plasticity during the dissemination and colonization process within the metastatic cascade. Finally, we demonstrate therapeutic strategies that target AST factors to specifically abrogate CTC formation and suppress distant metastases. Together, these findings underscore an important early mechanism of how cancer cells spread and highlight novel targets for the development of anti-metastatic therapy. Furthermore, AST opens unexplored scenarios how reprogramming anchorage dependency orchestrates physiological events such as development, organ morphogenesis and immune cell activation. Citation Format: Hyun Woo Park. Adherent-to suspension transition by hematopoietic factors in metastatic dissemination of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4120.

Improving hematopoietic differentiation from human induced pluripotent stem cells by the modulation of Hippo signaling with a diarylheptanoid derivative

BackgroundThe diarylheptanoid ASPP 049 has improved the quality of adult hematopoietic stem cell (HSC) expansion ex vivo through long-term reconstitution in animal models. However, its effect on hematopoietic regeneration from human induced pluripotent stem cells (hiPSCs) is unknown.MethodWe utilized a defined cocktail of cytokines without serum or feeder followed by the supplementation of ASPP 049 to produce hematopoietic stem/progenitor cells (HSPCs). Flow cytometry and trypan blue exclusion analysis were used to identify nonadherent and adherent cells. Nonadherent cells were harvested to investigate the effect of ASPP 049 on multipotency using LTC-IC and CFU assays. Subsequently, the mechanism of action was explored through transcriptomic profiles, which were validated by qRT-PCR, immunoblotting, and immunofluorescence analysis.ResultThe supplementation of ASPP 049 increased the number of phenotypically defined primitive HSPCs (CD34+CD45+CD90+) two-fold relative to seeded hiPSC colonies, indicating enhanced HSC derivation from hiPSCs. Under ASPP 049-supplemented conditions, we observed elevated HSPC niches, including CD144+CD73− hemogenic- and CD144+CD73+ vascular-endothelial progenitors, during HSC differentiation. Moreover, harvested ASPP 049-treated cells exhibited improved self-renewal and a significantly larger proportion of different blood cell colonies with unbiased lineages, indicating enhanced HSC stemness properties. Transcriptomics and KEGG analysis of sorted CD34+CD45+ cells-related mRNA profiles revealed that the Hippo signaling pathway is the most significant in responding to WWTR1/TAZ, which correlates with the validation of the protein expression. Interestingly, ASPP 049-supplemented HSPCs upregulated 11 genes similarly to umbilical cord blood-derived HSPCs.ConclusionThese findings suggest that ASPP 049 can improve HSC-generating protocols with proliferative potentials, self-renewal ability, unbiased differentiation, and a definable mechanism of action for the clinical perspective of hematopoietic regenerative medicine.Graphical abstract