Activity Of Progenitor Cells Research Articles

Targeted blocking of CCR2 and CXCR2 improves the efficacy of transarterial chemoembolization of hepatocarcinoma

BackgroundTransarterial chemoembolization (TACE) has been shown to prolong survival in patients with unresectable hepatocellular carcinoma (HCC); however, the long-term survival remains dismal. Targeting macrophage and neutrophil infiltration is a promising strategy. The CCL2/CCR2 and CXCLs/CXCR2 axes are required for recruitment of macrophages and neutrophils, respectively, in HCC. We investigated the feasibility of CCL2/CCR2 and CXCLs/CXCR2 as therapeutic targets in combination with TACE for treating HCC.MethodsExpression of CCL2/CCR2 and CXCLs/CXCR2 was analyzed in the primary rat HCC model and one HCC cohort. The relationship between expression levels, neutrophil and macrophage infiltration, hepatocarcinogenesis progression in the rat model, and survival of HCC patients was assessed. The anti-tumor effects of blocking the CCL2/CCR2 and CXCLs/CXCR2 axes by CCR2 and CXCR2 antagonists in combination with TACE were evaluated in HCC rats. The numbers of macrophages, neutrophils, and hepatic progenitor cells were further determined to explore the underlying mechanisms.ResultsHigh macrophage and neutrophil infiltration and CXCL8 expression were associated with poor prognosis in the TCGA liver cancer dataset. High expression of CCL2/CCR2 and CXCL8/CXCR2 in clinical HCC specimens was associated with reduced survival. Expression of CCL2/CCR2 and CXCL1/CXCR2 was correlated with hepatocarcinogenesis progression in the primary rat HCC model. Blockade of CCL2/CCR2 and CXCLs/CXCR2 enhanced the anti-tumor effect of TACE treatment in this model. Blocking the CCL2/CCR2 and CXCLs/CXCR2 axes with CCR2 and CXCR2 antagonists in TACE-treated rats reduced macrophage and neutrophil infiltration and hepatic progenitor cell activation and thus overcame TACE resistance in HCC.ConclusionsThe results demonstrate the translational potential of immunotherapy targeting the CCL2/CCR2 and CXCLs/CXCR2 axes in combination with TACE therapy for the treatment of HCC.

Inhibition of Both IRAK1 and IRAK4 Is Required for Complete Suppression of NF-Kb Signaling across Multiple Receptor-Mediated Pathways in MDS and AML

There is increasing interest in the role of the IRAK kinases as targets in the treatment of MDS and AML (reviewed in J Bennett and DT Starczynowski, Curr Opin Hematol 2022). IRAK1 and IRAK4 lie downstream of multiple receptors that stimulate the canonical NF-κB signaling pathway upstream of TRAF6 via the myddosome complex. While TRAF6 is necessary to preserve the tonic NF-κB signaling that is required for hematopoietic stem cell (HSC) homeostasis (J Fang et. al. Cell Reports 2018), overactivity of the NF-κB signaling pathway has been implicated in both MDS and AML (reviewed in MCJ Bosman et. al., Crit Rev Oncol/Hematol 2016; JJ Trowbridge and DT Starczynowski, J Exp Med 2021). The requirement for both IRAK1 and IRAK4 in this pathway has been explored using both genetic and pharmacologic technologies. The use of genetic loss-of-function approaches revealed that deletion and/or inhibition of IRAK4 results in a compensatory increase in IRAK1 protein and activation (data unpublished), suggesting that high potency antagonism of both kinases will be required for optimal inhibition of NF-κB-mediated transcriptional responses in disease-propagating MDS and AML cells. In this report, we use a pharmacological approach in which a series of IRAK inhibitors of varying relative potency at IRAK1:IRAK4 were examined for both inhibition of NF-κB activation in AML cells and inhibition of leukemic progenitor cell function in vitro. We find that potency and efficacy for antagonism at NF-κB requires effective inhibition of both IRAK1 and IRAK4. We also find that the relative potency of NF-κB inhibition correlates with suppression of leukemic progenitor cell function in vitro, providing pharmacological validation that inhibition of both IRAK1 and IRAK4 are necessary for optimal inhibition of NF-κB and effect on leukemia progenitor cell function. We utilized an NF-κB reporter system expressed in human AML cells (THP1) to measure NF-κB dependent activation in response to a variety of IRAK4, IRAK1, or IRAK1/4 antagonists. The cells are highly responsive to both TLR agonists as well as to IL-1b, which allows for measurement of IRAK-mediated antagonism of multiple receptor-mediated pathways. Using the IRAK4-selective antagonists PF-06650833 and BAY 1834845 we find that both compounds fully suppress signaling through TLR2 (IC50vs. Pam3CSK4 = 7.2 and 150 nM, respectively). The IRAK4 antagonists inhibit signaling through the IL-1R with similar relative potency (IC50vs. IL-1b = 5.7 and 81 nM), but do not fully suppress signaling through this receptor (Span = 75% and 59%, respectively). Interestingly, the IRAK1 selective covalent inhibitor JH-X-119-01 does not inhibit signaling against either the TLR or the IL-1 receptor agonist. Together these data imply that neither IRAK4 nor IRAK1 inhibition alone is sufficient to fully inhibit NF-κB-mediated signaling through multiple receptor mediated pathways. For this reason, we then examined the activity of a series of IRAK4/IRAK1 inhibitors in AML cells. This allowed us to study the effect of adding in additional inhibitory activity at IRAK1 on the background of high potency IRAK4 inhibitors. Using two reference compounds with varying IRAK1:IRAK4 potency, we then compared the relative ability to inhibit TLR-agonist or IL-1b-agonist stimulated NF-κB activity in AML. KME-2780 has an IRAK1:IRAK4 potency ratio of 36 whereas KME-3859 has an IRAK1:IRAK4 potency ratio of 100. Unlike what we observed with the IRAK4-selective antagonists, both compounds can completely suppress NF-κB signaling through IL-1b, with relative potencies apparently determined by their activity at IRAK1: (IC50vs. Pam3CSK4 = 6.3 and 73.8 nM for KME- 2780 and KME-3859, respectively); (IC50vs. IL-1b = 9.3 and 227 nM for KME- 2780 and KME-3859, respectively). Finally, we examined the correlation between potency in the biochemical kinase assay at IRAK1 or IRAK4, activity in the NF-κB reporter assay, and leukemic progenitor cell activity in the colony forming assay for a series of KME compounds in AML and MDS cells. We find a correlation between kinase activity for both IRAK1 and IRAK4 and NF-κB activity that extends to the leukemia colony forming assays. This suggests that NF-κB signaling contributes to leukemia progenitor cell function and that optimal inhibition requires potent antagonism of both IRAK1 and IRAK4 in the setting of MDS and AML.

Doxorubicin-liposome combined with clodronate-liposome inhibits hepatocellular carcinoma through the depletion of macrophages and tumor cells

Macrophages in the liver have capacities of capturing and phagocytosing nanocarriers. Macrophages also play an important role in the inflammatory microenvironment and in the tumorigenesis, development and progression of hepatocellular carcinoma (HCC). Several studies have shown that depletion of macrophages is a viable strategy for drug delivery and tumor microenvironment regulation. We prepared liposomes containing doxorubicin and clodronate using an ammonium sulfate gradient and thin film hydration method. The repressive therapeutic effects of liposomes were compared by intrasplenic injection at different stages of a primary HCC model induced by diethylnitrosamine (DEN) in rats. Doxorubicin-liposome (DOX-LIP) and clodronate-liposome (CL-LIP) about 180–200 nm were successfully prepared and characterized. We found that DOX-LIP combined with CL-LIP could effectively inhibit the occurrence and development of liver cancer without major organ damage and side effects. The combination of doxorubicin and clodronate liposomes notably decreased hepatic CD68 + macrophages, enriched DOX in plasma and accumulated it for a long time in the liver and spleen, thus improving the tumor microenvironment, inhibiting the activation of hepatic progenitor cells (HPCs) and promoting the apoptosis of tumor cells, and finally producing the inhibitory and therapeutic effects of HCC in rats. Results of this study were expected to provide a new prospect for the chemotherapy of HCC.

A study of the molecular mechanism of quercetin and dasatinib combination as senolytic in alleviating age-related and kidney diseases.

Aging is a significant risk factor for the majority of prevalent human illnesses. The chance of having severe chronic conditions grows dramatically with advancing age. Indeed, more than 90% of people over 65 get at least one chronic disease, including diabetes, heart disease, malignancy, memory loss, and kidney disease, whereas more than 70% have two or more of these ailments. Mouse and human aging lead to increased senescent cells and decreased klotho concentrations. Mice lacking the protein α-klotho show faster aging, similar to human aging. α-Klotho upregulation extends life and slows or suppresses the onset of many age-related illnesses and kidney diseases. Like the consequences of α-klotho deficiency, senescent cell accumulation is linked to tissue dysfunction in various organs and multiple age-related kidney diseases. In addition, α-klotho and cell senescence are negatively and presumably mechanistically linked. Earlier research has demonstrated that klotho exerts its protective effects in age-related and kidney disease by interacting with Wnt ligands, serving as an endogenous antagonist of Wnt/β-catenin signaling. In addition, decreasing senescent cell burden with senolytics, a class of drugs that remove senescent cells selectively and extend the life span of mice. In this work, we are studying the molecular mechanism of the combination of quercetin and dasatinib as senolytic in easing age-related chronic renal illness by altering the level of klotho/Wnt/β-catenin. PRACTICAL APPLICATIONS: There is an inverse relationship between the onset and the development of age-related disorders and cellular senescence and Klotho. Earlier attempts to suppress transforming growth factor-beta 1 (TGF-β1) in kidney disease with anti-TGF-β1 antibodies were ineffective, and this should be kept in mind. Senolytic medications may benefit from targeting senescent cells, which enhances the protective factor α-klotho. In addition, our study provides a unique, translationally feasible route for creating orally active small compounds to enhance α-klotho, which may also be a valuable biomarker for age-related kidney disease. Additionally, other aspects of aging can be affected by senolytics, such as limiting age-related mitochondrial dysfunction, lowering inflammation and fibrosis, blunting reactive oxygen species (ROS) generation, decreasing deoxyribonucleic acid (DNA) damage, and reinforcing insulin sensitivity. Senolytic agents have been shown to increase adipose progenitor and cardiac progenitor cell activity in aging animals and animals with cellular senescence-related diseases, such as heart, brain, and kidney disease.

Impairment of muscular endothelial cell regeneration in dermatomyositis.

Inflammatory myopathies are heterogeneous in terms of etiology, (immuno)pathology, and clinical findings. Endothelial cell injury, as it occurs in DM, is a common feature of numerous inflammatory and non-inflammatory vascular diseases. Vascular regeneration is mediated by both local and blood-derived mechanisms, such as the mobilization and activation of so-called proangiogenic cells (PACs) or early endothelial progenitor cells (eEPCs). The current study aimed to evaluate parameters of eEPC integrity in dermatomyositis (DM), compared to necrotizing myopathy (NM) and to non-myopathic controls. Blood samples from DM and NM patients were compared to non-myositis controls and analyzed for the following parameters: circulating CD133+/VEGFR-2+ cells, number of colony-forming unit endothelial cells (CFU-ECs), concentrations of angiopoietin 1, vascular endothelial growth factor (VEGF), and CXCL-16. Muscle biopsies from DM and NM subjects underwent immunofluorescence analysis for CXCR6, nestin, and CD31 (PECAM-1). Finally, myotubes, derived from healthy donors, were stimulated with serum samples from DM and NM patients, subsequently followed by RT-PCR for the following candidates: IL-1β, IL-6, nestin, and CD31. Seventeen (17) DM patients, 7 NM patients, and 40 non-myositis controls were included. CD133+/VEGFR-2+ cells did not differ between the groups. Both DM and NM patients showed lower CFU-ECs than controls. In DM, intramuscular CD31 abundances were significantly reduced, which indicated vascular rarefaction. Muscular CXCR6 was elevated in both diseases. Circulating CXCL-16 was higher in DM and NM in contrast, compared to controls. Serum from patients with DM but not NM induced a profound upregulation of mRNS expression of CD31 and IL-6 in cultured myotubes. Our study demonstrates the loss of intramuscular microvessels in DM, accompanied by endothelial activation in DM and NM. Vascular regeneration was impaired in DM and NM. The findings suggest a role for inflammation-associated vascular damage in the pathogenesis of DM.

The role of P2Y12 in cardiovascular disease beyond atherothrombosis: P2Y12 signaling promotes emergency hematopoiesis after myocardial infarction

Abstract Background Adenosine diphosphate (ADP) plays a pivotal role in platelet activation. The purinergic ADP-receptor P2Y12 has therefore been targeted in the treatment of cardiovascular disease (CVD) to prevent atherothrombosis (1). Beyond P2Y12 expression on platelets, purinergic receptors have also been described on hematopoietic stem and progenitor cells (LSK) (2). After myocardial infarction (MI), accelerated LSK proliferation launches emergency hematopoiesis as the driving force behind the inflammatory response to MI, increasing inflammatory cell production in the bone marrow (BM) and providing leukocyte resupply for local cell recruitment to the infarct (3). The inflammatory cascade after MI covers intricate multilayered interactions between the injured myocardium and the hematopoietic BM that still remain to be fully elucidated and may unearth novel therapeutic strategies. Whereas P2X receptors have recently been found to be involved in cell trafficking (4), the role of P2Y receptors in the hematopoietic BM have not yet been characterized. Purpose This study aims to characterize the influence of P2Y12 signaling on emergency hematopoiesis and cardiac remodeling after MI. Methods Permanent coronary ligation was performed for MI to assess BM activation, inflammatory cell composition, cardiac remodeling and function in murine global and platelet-specific P2Y12 knockout models and under pharmacological P2Y12 inhibition with prasugrel using flow cytometry, qPCR, immunohistochemistry and echocardiography. In vitro studies including colony forming unit (CFU) assays and flow cytometry allowed for investigation of ADP-dependent effects on LSK cells and intracellular pathway analysis. Results We identified ADP as a danger signal for the hematopoietic BM, fueling emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. Detection of P2Y12 expression in LSK implicated a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 deficiency and P2Y12 inhibition with prasugrel decelerated emergency hematopoiesis and consecutively reduced the excessive inflammatory response to MI, translating to lower numbers of hematopoietic progenitors and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition ameliorated chronic adverse cardiac remodeling and preserved cardiac function after MI. Conclusion ADP-dependent P2Y12-mediated activation of hematopoietic stem and progenitor cells in the BM promotes emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel role of P2Y12 antagonists in CVD beyond atherothrombosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)

Systemic GDF11 replenishment ignites myocardial injury through diminishing anti-apoptotic activity of cardiac progenitor cells

Abstract Background Tissue damage due to acute myocardial infarction is caused by both the ischemic insult and subsequent reperfusion injury (I/R). Restoration of coronary blood flow accelerates cardiomyocyte death, a phenomenon referred to as reperfusion injury, the extent of which is partly modulated by cardiac progenitor cells (CPC). Development of novel therapies to reduce infarct size, the main determinant of outcome, represent a huge unmet medical need (1). Systemic levels of growth differentiation factor 11 (GDF11), a TGF-β superfamily member that shares 90% homology with myostatin, decline with age, and GDF11 replenishment by heterochronic parabiosis or systemic recombinant GDF11 (rGDF11) delivery was postulated to have rejuvenating effects (2). Purpose We aimed to probe the effects of systemic GDF11 replenishment on I/R injury and deepen insights into the molecular mechanisms involved. Methods We designed a vehicle-controlled study in which young (3–4 months) and old (22–24 months) C57Bl/6 mice were randomly assigned to either daily systemic rGDF11 or control treatment over 30 days before myocardial I/R injury was induced. Dissected hearts were subjected to in-depth profiling followed by IPA-guided -omics to identify key regulatory mechanisms. Finally, in vitro experiments on human CPCs and HL-1 cardiomyocytes were performed. Results Myocardial Gdf11 expression declined with age, whereas myostatin (Mstn) showed an opposing expression pattern (Fig. 1A), a trend similarly observed upon I/R (Fig. 1B). Surprisingly, after the 30-day study period (Fig. 1C), young and aged rGDF11-treated mice showed higher I/R-induced infarct size and serum cardiac troponin I levels than controls, despite comparable areas at risk (Fig. 1D). Importantly, while proxies of necroptosis/pyroptosis remained unchanged, rGDF11-treated animals showed reduced cardiomyocyte viability irrespective of their age (Fig. 2A). Targeted transcriptomics applied on cardiac tissues of both groups identified the CPC-marker Nkx2–5 to be differentially regulated (Fig. 2B-C), an expression pattern validated in an independent cohort at both mRNA and protein levels (Fig. 2D). In the adult myocardium, the expression of both Nkx2–5 and its cofactor Gata4 is mainly confined to CPCs; indeed, similar reductions in Nkx2–5 and Gata4 expression were observed in CPCs exposed to rGDF11 (Fig. 2E) which coincided with accelerated cardiomyocyte death if cultured in conditioned media obtained from CPCs treated with rGDF11 (Fig. 2F), pointing toward a paracrine signalling pathway. Conclusions Myocardial expression of GDF11 declines with age, and is blunted upon I/R injury, thereby opposing the expression pattern of myostatin. Surprisingly, however, systemic GDF11 replenishment by rGDF11 supplementation enhances rather than reduces myocardial infarct size through augmented apoptosis, a phenomenon mediated by diminished cardioprotective function of CPCs. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Foundation for Cardiovascular Research – Zurich Heart House

Prolonged FOS activity disrupts a global myogenic transcriptional program by altering 3D chromatin architecture in primary muscle progenitor cells

BackgroundThe AP-1 transcription factor, FBJ osteosarcoma oncogene (FOS), is induced in adult muscle satellite cells (SCs) within hours following muscle damage and is required for effective stem cell activation and muscle repair. However, why FOS is rapidly downregulated before SCs enter cell cycle as progenitor cells (i.e., transiently expressed) remains unclear. Further, whether boosting FOS levels in the proliferating progeny of SCs can enhance their myogenic properties needs further evaluation.MethodsWe established an inducible, FOS expression system to evaluate the impact of persistent FOS activity in muscle progenitor cells ex vivo. We performed various assays to measure cellular proliferation and differentiation, as well as uncover changes in RNA levels and three-dimensional (3D) chromatin interactions.ResultsPersistent FOS activity in primary muscle progenitor cells severely antagonizes their ability to differentiate and form myotubes within the first 2 weeks in culture. RNA-seq analysis revealed that ectopic FOS activity in muscle progenitor cells suppressed a global pro-myogenic transcriptional program, while activating a stress-induced, mitogen-activated protein kinase (MAPK) transcriptional signature. Additionally, we observed various FOS-dependent, chromosomal re-organization events in A/B compartments, topologically associated domains (TADs), and genomic loops near FOS-regulated genes.ConclusionsOur results suggest that elevated FOS activity in recently activated muscle progenitor cells perturbs cellular differentiation by altering the 3D chromosome organization near critical pro-myogenic genes. This work highlights the crucial importance of tightly controlling FOS expression in the muscle lineage and suggests that in states of chronic stress or disease, persistent FOS activity in muscle precursor cells may disrupt the muscle-forming process.

448 Physiological function of krox20 (Egr2) in epithelial stem cells

Krox20, a zinc finger-containing transcription factor, is well known for mediating stem and progenitor cell activation and differentiation in a variety of tissues. In a recent study, we reported for the first time a population of epithelial-derived Krox20-expressing keratinocytes in the hair follicle that ultimately terminally differentiate to form the structural component of the hair shaft. These Krox20-lineage cells in the hair follicle also mediate melanocyte differentiation via Stem Cell Factor production for hair pigmentation.

QS45. Parallels And Deviations in Mesenchymal Progenitor Cell Activation in Regenerative and Fibrotic Injuries

QS45. Parallels And Deviations in Mesenchymal Progenitor Cell Activation in Regenerative and Fibrotic Injuries

Cooperation between KDM6B overexpression and TET2 deficiency in the pathogenesis of chronic myelomonocytic leukemia.

Loss-of-function TET2 mutations are recurrent somatic lesions in chronic myelomonocytic leukemia (CMML). KDM6B encodes a histone demethylase involved in innate immune regulation that is overexpressed in CMML. We conducted genomic and transcriptomic analyses in treatment naïve CMML patients and observed that the patients carrying both TET2 mutations and KDM6B overexpression constituted 18% of the cohort and 42% of patients with TET2 mutations. We therefore hypothesized that KDM6B overexpression cooperated with TET2 deficiency in CMML pathogenesis. We developed a double-lesion mouse model with both aberrations, and discovered that the mice exhibited a more prominent CMML-like phenotype than mice with either Tet2 deficiency or KDM6B overexpression alone. The phenotype includes monocytosis, anemia, splenomegaly, andincreased frequencies and repopulating activity of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). Significant transcriptional alterations were identified in double-lesion mice, which were associated with activation of proinflammatory signals and repression of signals maintaining genome stability. Finally, KDM6B inhibitor reduced BM repopulating activity of double-lesion mice and tumor burden in mice transplanted with BM-HSPCs from CMML patients with TET2 mutations. These data indicate that TET2 deficiency and KDM6B overexpression cooperate in CMML pathogenesis of and that KDM6B could serve as a potential therapeutic target in this disease.

Metabolic control of progenitor cell propagation during Drosophila tracheal remodeling

Adult progenitor cells in the trachea of Drosophila larvae are activated and migrate out of niches when metamorphosis induces tracheal remodeling. Here we show that in response to metabolic deficiency in decaying tracheal branches, signaling by the insulin pathway controls the progenitor cells by regulating Yorkie (Yki)-dependent proliferation and migration. Yki, a transcription coactivator that is regulated by Hippo signaling, promotes transcriptional activation of cell cycle regulators and components of the extracellular matrix in tracheal progenitor cells. These findings reveal that regulation of Yki signaling by the insulin pathway governs proliferation and migration of tracheal progenitor cells, thereby identifying the regulatory mechanism by which metabolic depression drives progenitor cell activation and cell division that underlies tracheal remodeling.

A murine model of large-scale bone regeneration reveals a selective requirement for Sonic Hedgehog

Building and maintaining skeletal tissue requires the activity of skeletal stem and progenitor cells (SSPCs). Following injury, local pools of these SSPCs become active and coordinate to build new cartilage and bone tissues. While recent studies have identified specific markers for these SSPCs, how they become activated in different injury contexts is not well-understood. Here, using a model of large-scale rib bone regeneration in mice, we demonstrate that the growth factor, Sonic Hedgehog (SHH), is an early and essential driver of large-scale bone healing. Shh expression is broadly upregulated in the first few days following rib bone resection, and conditional knockout of Shh at early but not late post-injury stages severely inhibits cartilage callus formation and later bone regeneration. Whereas Smoothened (Smo), a key transmembrane component of the Hh pathway, is required in Sox9+ lineage cells for rib regeneration, we find that Shh is required in a Prrx1-expressing, Sox9-negative mesenchymal population. Intriguingly, upregulation of Shh expression and requirements for Shh and Smo may be unique to large-scale injuries, as they are dispensable for both complete rib and femur fracture repair. In addition, single-cell RNA sequencing of callus tissue from animals with deficient Hedgehog signaling reveals a depletion of Cxcl12-expressing cells, which may indicate failed recruitment of Cxcl12-expressing SSPCs during the regenerative response. These results reveal a mechanism by which Shh expression in the local injury environment unleashes large-scale regenerative abilities in the murine rib.

Identification of fibroblast progenitors in the developing mouse thymus.

ABSTRACTThe thymus stroma constitutes a fundamental microenvironment for T-cell generation. Despite the chief contribution of thymic epithelial cells, recent studies emphasize the regulatory role of mesenchymal cells in thymic function. Mesenchymal progenitors are suggested to exist in the postnatal thymus; nonetheless, an understanding of their nature and the mechanism controlling their homeostasis in vivo remains elusive. We resolved two new thymic fibroblast subsets with distinct developmental features. Whereas CD140αβ+GP38+SCA-1− cells prevailed in the embryonic thymus and declined thereafter, CD140αβ+GP38+SCA-1+ cells emerged in the late embryonic period and predominated in postnatal life. The fibroblastic-associated transcriptional programme was upregulated in CD140αβ+GP38+SCA-1+ cells, suggesting that they represent a mature subset. Lineage analysis showed that CD140αβ+GP38+SCA-1+ maintained their phenotype in thymic organoids. Strikingly, CD140αβ+GP38+SCA-1− generated CD140αβ+GP38+SCA-1+, inferring that this subset harboured progenitor cell activity. Moreover, the abundance of CD140αβ+GP38+SCA-1+ fibroblasts was gradually reduced in Rag2−/− and Rag2−/−Il2rg−/− thymi, indicating that fibroblast maturation depends on thymic crosstalk. Our findings identify CD140αβ+GP38+SCA-1− as a source of fibroblast progenitors and define SCA-1 as a marker for developmental stages of thymic fibroblast differentiation.

The role of phase separation in oncogenesis by fusion oncoproteins

NUP98 fusion oncoproteins (FOs) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid‐liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98‐HOXA9 (NHA9), retains a DNA‐binding homeodomain, whereas others harbor other types of DNA‐ or chromatin‐binding domains. NUP98 FOs have long been known to form puncta, but long‐standing questions are how nuclear puncta form, and how they drive leukemogenesis. We will discuss our studies of NHA9 condensates, showing that homotypic interactions and different types of heterotypic interactions are required to form nuclear puncta, which are associated with aberrant transcriptional activity and transformation of hematopoietic stem and progenitor cells. We also show that three other leukemia‐associated NUP98 FOs form nuclear puncta and transform hematopoietic cells. To extend our findings, we tested ~150 additional fusion oncoproteins associate with a wide range of human cancers for puncta formation in cells. We will discuss our observation that >50% of these formed cellular condensates, with this behavior for some linked with aberrant gene expression and cell transformation. Our findings indicate that LLPS is critical for leukemogenesis by NUP98 FOs and likely contributes to oncogenesis driven by many other fusion oncoproteins.

Differential responses of receptor tyrosine kinases C‐Met and EGFR in mice

In previous studies, we have demonstrated the critical roles of EGFR and HGFR (c‐Met) for liver regeneration and for conversion of hepatocytes to cholangiocytes by a “clean” partial hepatectomy model, in which hepatocytes and cholangiocytes grow rapidly and synchronously. In contrast, the vast majority of liver diseases involve a prolonged pathologic duration, therefore multiple types of cells participate and play different roles in the variant stages. However, it is not fully understood how EGFR and Met play their roles in this complicated setting. In this study, we explore the role of MET and EGFR signaling pathways in biliary injury after common bile duct ligation. Mice with inhibition of EGFR signaling alone (EGFRi) showed abolished hepatocyte proliferation and biliary duct cell proliferation (Ki67 immunochemistry staining) at 2, 5, and 7 days after BDL. However, Met knockout mice (KO) showed increased proliferation of cholangiocytes at 2, 5, and 7 days. Met KO mice subjected to BDL surgery had an increase of the CK19‐ marked ductular reaction at 2, 5, and 7 days. In contrast, EGFRi mice showed a reduced ductular reaction at the same time point. There was enhanced immunohistochemistry for Sox9 in EGFRi mice, but absence of its expression in Met KO mice at 7 days post‐BDL. From western blot results of total liver tissue lysate, we found increased EpCAM expression after BDL at 7 days in Met KO but not in EGFRi mice. In addition, we also observed that both p‐Merlin and cleaved p‐Mst1, components of the Hippo pathway, decreased only in Met knockout mice. Mice with combined disruption of Met and EGFR signaling became very ill by 24 hours and died at 1 to 2 days after bile duct ligation. From Kaplan‐Meier analysis, both EGFR inhibition and Met knockout mice had decreased survival rates starting at 11 days, though the survival rate of Met KO mice was overall much better than EGFRi mice by around 40%. We also observed that p‐Ezrin (Thr567) was downregulated in either Met KO or EGFRi mice, while the total Ezrin expression did not change.ConclusionsOur study demonstrates that both c‐Met and EGFR control biliary response to bile duct ligation. The cholangiocyte responses, however, were very different between these two receptors. Their combined disruption led to acute liver failure and early death following bile duct ligation. The receptor tyrosine kinases c‐Met and EGFR may play different roles in the biliary injury‐repair responses, including proliferation, trans‐differentiation, and bi‐potential progenitor cells activation.

An in silico model predicts the impact of scaffold design in large bone defect regeneration

Large bone defects represent a clinical challenge for which the implantation of scaffolds appears as a promising strategy. However, their use in clinical routine is limited, in part due to a lack of understanding of how scaffolds should be designed to support regeneration. Here, we use the power of computer modeling to investigate mechano-biological principles behind scaffold-guided bone regeneration and the influence of scaffold design on the regeneration process. Computer model predictions are compared to experimental data of large bone defect regeneration in sheep. We identified two main key players in scaffold-guided regeneration: (1) the scaffold surface guidance of cellular migration and tissue formation processes and (2) the stimulation of progenitor cell activity by the scaffold material composition. In addition, lower scaffold surface-area-to-volume ratio was found to be beneficial for bone regeneration due to enhanced cellular migration. To a lesser extent, a reduced scaffold Young's modulus favored bone formation. Statement of significance3D-printed scaffolds offer promising treatment strategies for large bone defects but their broader clinical use requires a more thorough understanding of their interaction with the bone regeneration process. The predictions of our in silico model compared to two experimental set-ups highlighted the importance of (1) the scaffold surface guidance of cellular migration and tissue formation processes and (2) the scaffold material stimulation of progenitor cell activity. In addition, the model was used to investigate the effect on the bone regeneration process of (1) the scaffold surface-area-to-volume ratio, with lower ratios favoring more bone growth, and (2) the scaffold material properties, with stiffer scaffold materials yielding a lower bone growth.

P2Y12-dependent activation of hematopoietic stem and progenitor cells promotes emergency hematopoiesis after myocardial infarction

Emergency hematopoiesis is the driving force of the inflammatory response to myocardial infarction (MI). Increased proliferation of hematopoietic stem and progenitor cells (LSK) after MI enhances cell production in the bone marrow (BM) and replenishes leukocyte supply for local cell recruitment to the infarct. Decoding the regulation of the inflammatory cascade after MI may provide new avenues to improve post-MI remodeling. In this study, we describe the influence of adenosine diphosphate (ADP)-dependent P2Y12-mediated signaling on emergency hematopoiesis and cardiac remodeling after MI. Permanent coronary ligation was performed to induce MI in a murine model. BM activation, inflammatory cell composition and cardiac function were assessed using global and platelet-specific gene knockout and pharmacological inhibition models for P2Y12. Complementary in vitro studies allowed for investigation of ADP-dependent effects on LSK cells. We found that ADP acts as a danger signal for the hematopoietic BM and fosters emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. We were able to detect P2Y12 in LSK, implicating a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 knockout and P2Y12 inhibitor treatment with prasugrel reduced emergency hematopoiesis and the excessive inflammatory response to MI, translating to lower numbers of downstream progeny and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition preserved cardiac function and reduced chronic adverse cardiac remodeling after MI. P2Y12-dependent signaling is involved in emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel, non-canonical value for P2Y12 antagonists beyond inhibition of platelet-mediated atherothrombosis.

Modulation of the Activity of Stem and Progenitor Cells by Immune Cells.

Numerous components of the immune system, including inflammatory mediators, immune cells and cytokines, have a profound modulatory effect on the homeostatic regulation and regenerative activity of endogenous stem cells and progenitor cells. Thus, understanding how the immune system interacts with stem/progenitor cells could build the foundation to design novel and more effective regenerative therapies. Indeed, utilizing and controlling immune system components may be one of the most effective approaches to promote tissue regeneration. In this review, we first summarize the effects of various immune cell types on endogenous stem/progenitor cells, focusing on the tissue healing context. Then, we present interesting regenerative strategies that control or mimic the effect of immune components on stem/progenitor cells, in order to enhance the regenerative capacity of endogenous and transplanted stem cells. We highlight the potential clinical translation of such approaches for multiple tissues and organ systems, as these novel regenerative strategies could considerably improve or eventually substitute stem cell-based therapies. Overall, harnessing the power of the cross-talk between the immune system and stem/progenitor cells holds great potential for the development of novel and effective regenerative therapies.

Liver regeneration and ethanol detoxification: A new link in YAP regulation of ALDH1A1 during alcohol-related hepatocyte damage.

Yes‐associated protein (YAP), a central effector in the Hippo pathway, is involved in the regulation of organ size, stem cell self‐renewal, and tissue regeneration. In this study, we observed YAP activation in patients with alcoholic steatosis, hepatitis, and cirrhosis. Accumulation of this protein in the nucleus was also observed in murine livers that were damaged after chronic‐plus‐single binge or moderate ethanol ingestion combined with carbon tetrachloride intoxication (ethanol/CCl4). To understand the role of this transcriptional coactivator in alcohol‐related liver injury, we knocked out the Yap1 gene in hepatocytes of floxed homozygotes through adeno‐associated virus (AAV8)‐mediated deletion utilizing Cre recombinase. Yap1 hepatocyte‐specific knockouts (KO) exhibited hemorrhage, massive hepatic necrosis, enhanced oxidative stress, elevated hypoxia, and extensive infiltration of CD11b+ inflammatory cells into hepatic microenvironments rich for connective tissue growth factor (Ctgf) during ethanol/CCl4‐induced liver damage. Analysis of whole‐genome transcriptomics indicated upregulation of genes involved in hypoxia and extracellular matrix (ECM) remodeling, whereas genes related to hepatocyte proliferation, progenitor cell activation, and ethanol detoxification were downregulated in the damaged livers of Yap1 KO. Acetaldehyde dehydrogenase (Aldh)1a1, a gene that encodes a detoxification enzyme for aldehyde substrates, was identified as a potential YAP target because this gene could be transcriptionally activated by a hyperactive YAP mutant. The ectopic expression of the human ALDH1A1 gene caused increase in hepatocyte proliferation and decrease in hepatic necrosis, oxidative stress, ECM remodeling, and inflammation during ethanol/CCl4‐induced liver damage. Taken together, these observations indicated that YAP was crucial for liver repair during alcohol‐associated injury. Its regulation of ALDH1A1 represents a new link in liver regeneration and detoxification.