Characterization Of Progenitor Cells Research Articles

Isolation and Characterization of Meniscus Progenitor Cells From Rat, Rabbit, Goat, and Human.

Meniscus progenitor cells (MPCs) have been identified as promising candidates for meniscus regeneration, and it is crucial for us to understand meniscus injury repair mechanism at the cellular level. In this study, we investigate the biological properties of MPCs isolated from different species using the differential adhesion to fibronectin (DAF) technique. We aim to characterize MPCs in different species and evaluate the feasibility of these models for future meniscal investigation. MPCs were isolated from freshly digested meniscus from rat, rabbit, goat, and human cells using DAF. Biological properties, including proliferation, colony-forming, multilineage differentiation, and migration abilities, were compared in MPCs and their corresponding mixed meniscus cell (MCs) population in each species. MPCs were successfully isolated by the DAF technique in all species. Rat MPCs appeared cobblestone-like, rabbit MPCs were more polygonal, goat MPCs had a spindle-shaped morphology, human MPCs appear more fibroblast-like. Compared with MCs, isolated MPCs showed progenitor cell characteristics, including multilineage differentiation ability and MSC (mesenchymal stem cells) markers (CD166, CD90, CD44, Stro-1) expression. They also highly expressed fibronectin receptors CD49e and CD49c. MPCs also showed greater proliferation capacity and retained colony-forming ability. Except for goat MPCs showed greater migration abilities than MCs, no significant differences were found in the migration ability between MPCs and MCs in other species. Our study shows that DAF is an effective method for isolating MPCs from rat, rabbit, goat, and human. MPCs in these species demonstrated similar characteristics, including greater proliferation ability and better chondrogenic potential.

Abstract 867: Molecular and functional characterization of melanocyte subpopulations in human epidermis based on single-cell RNA sequencing

Abstract The biological and molecular mechanisms that underpin the malignant transformation of normal melanocytes to melanomas are largely unknown. In part, this is due to the limited understanding of normal human melanocyte homeostasis and how melanocytes respond to oncogenic insults such as ultraviolet radiation (UVR). This is particularly true for interfollicular epidermal melanocytes, which have the highest levels of UVR exposure and from whence most melanomas are thought to arise. These knowledge gaps impede the development of strategies for active, targeted prevention of melanoma formation. We thus evaluated epidermal melanocytes transcriptionally, phenotypically and functionally after isolating them from human skin. Using single-cell RNA sequencing (scRNA-seq), we identified multiple transcriptionally distinct subpopulations within human epidermal melanocytes. RNA velocity analysis revealed subpopulations in different states of melanocytic differentiation, and immunohistochemistry staining demonstrated their distinct anatomical distribution throughout follicular and interfollicular epidermal compartments. Notably, one melanocyte subgroup, marked by increased expression of neurotrophic receptor tyrosine kinase 2 (NTRK2) and genes associated with ribosome biogenesis, exhibited molecular characteristics of progenitor cells. This subpopulation displayed human embryonic stem cell (hESC)-derived melanoblast markers, and their anatomical localization corresponded to that of intermediate melanocyte progenitors. NTRK2+ melanocytes demonstrated enhanced clonogenicity after UVR exposure in primary cell cultures and in ex vivo whole skin explants. In contrast, NTRK2- melanocytes were suppressed by UVR. Furthermore, scRNA-seq data of ex vivo melanocytes after UVR exposure revealed the upregulation of genes associated with cell proliferation within NTRK2-expressing melanocytes. In mouse back skin, the ratio of Ntrk2+ melanocytes increased within 24 hours of UVB irradiation, suggesting a proliferative response in these cells in vivo following UVR. We thus report the discovery in human epidermis of a putative melanocytic cell hierarchy, and of a candidate melanocyte progenitor subpopulation that responds proliferatively to UVR and is thus a candidate cell of origin of melanoma. Citation Format: Peinan Zhao, Fumihito Noguchi, Christopher Chew, Gamze Kuser Abali, Pacman Szeto, Youfang Zhang, Malaka Ameratunga, Isobel Leece, Jen G. Cheung, Miles Andrews, Nicholas C. Wong, Anthony T. Papenfuss, Mark Shackleton. Molecular and functional characterization of melanocyte subpopulations in human epidermis based on single-cell RNA sequencing [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 867.

State transition and intercellular communication of synovial fibroblasts in response to chronic and acute shoulder injuries unveiled by single-cell transcriptomic analyses

ABSTRACT Purpose We aimed to investigate the heterogeneity of synovial fibroblasts and their potential to undergo cell state transitions at the resolution of single cells. Materials and Methods We employed the single-cell RNA sequencing (scRNA-seq) approach to comprehensively map the cellular landscape of the shoulder synovium in individuals with chronic rotator cuff tears (RCTs) and acute proximal humerus fractures (PHFs). Utilizing unbiased clustering analysis, we successfully identified distinct subpopulations of fibroblasts within the synovial environment. We utilized Monocle 3 to delineate the trajectory of synovial fibroblast state transition. And we used CellPhone DB v2.1.0 to predict cell-cell communication patterns within the synovial microenvironment. Results We identified eight main cell clusters in the shoulder synovium. Unbiased clustering analysis identified four synovial fibroblast subpopulations, with diverse biological functions associated with protein secretion, ECM remodeling, inflammation regulation and cell division. Lining, mesenchymal, pro-inflammatory and proliferative fibroblasts subsets were identified. Combining the results from StemID and characteristic gene features, mesenchymal fibroblasts exhibited characteristics of fibroblast progenitor cells. The trajectory of synovial fibroblast state transition showed a transition from mesenchymal to pro-inflammatory and lining phenotypes. In addition, the cross talk between fibroblast subclusters increased in degenerative shoulder diseases compared to acute trauma. Conclusion We successfully generated the scRNA-seq transcriptomic atlas of the shoulder synovium, which provides a comprehensive understanding of the heterogeneity of synovial fibroblasts, their potential to undergo state transitions, and their intercellular communication in the context of chronic degenerative and acute traumatic shoulder diseases.

LRP11 promotes stem-like T cells via MAPK13-mediated TCF1 phosphorylation, enhancing anti-PD1 immunotherapy

BackgroundTumor-infiltrating T cells enter an exhausted or dysfunctional state, which limits antitumor immunity. Among exhausted T cells, a subset of cells with features of progenitor or stem-like cells has been...

CCRG-03. KAT5 REGULATES DEVELOPMENTAL TRANSITIONS AND CELLULAR HETEROGENEITY IN AN IN VIVO MODEL OF GLIOBLASTOMA

Abstract In solid tumors, G0-like states are likely critical for maintaining developmental hierarchies and cellular heterogeneity and promoting tumor growth/recurrence, yet little is known about tumor G0 states or regulation of their ingress/egress. To discover G0-like states and their regulators for glioblastoma (GBM), we previously performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) for genes that trap cells in G0 when inhibited. We identify the protein acetyltransferase KAT5 as a key regulator of GBM G0 ingress/egress. Here, we show in an in vivo GSC-derived orthotopic xenograft model that KAT5 regulates transcriptional, epigenetic, and proliferative heterogeneity impacting transitions into G0-like states. We show that KAT5 activity suppresses the emergence of non-dividing subpopulations with oligodendrocyte progenitor and radial glial cell characteristics. With regard to chromatin regulation, we show that loss of KAT5 activity alters sites of mixed epigenetic valency (which have both activating and repressive marks in bulk tumor populations) to promote emergence of OPC/RG G0-like populations in tumors. Our data reveal KAT5-associated transcriptional and epigenetic changes in tumors, which indicate that KAT5 activity biases cell state distribution away from OPC and RG-like subpopulations. In addition, loss of KAT5 resulted in a less invasive phenotype and prolonged survival of mice harboring orthotopic xenografts.

Loss of Function of SETD2 Tumor Suppressor in Chronic Myeloid Leukemia (CML) Progenitors Fosters Genomic Instability and Enhances Clonogenic Potential

BACKGROUND AND AIMS - Genomic instability is a hallmark of chronic myeloid leukemia (CML) cells since the chronic phase (CP) of the disease, and results in BCR::ABL1 mutations and/or additional genetic and genomic aberrations that may drive resistance to tyrosine kinase inhibitors (TKIs) and progression to blast crisis (BC). Genomic instability is also a feature of CML stem and progenitor cells and may contribute to their persistence. The SETD2 tumor suppressor codes for a protein that trimethylates histone H3 at lysine 36 (H3K36me3). In solid tumors, SETD2 loss of function has been shown to impair H3K36me3-mediated recruitment of DNA damage response components. We have recently reported that non genomic loss of function of SETD2 is a feature of BC CML and results from premature proteasome-mediated degradation of the SETD2 protein triggered by Aurora kinase A phosphorylation and MDM2 ubiquitination. In the present study, we aimed to assess SETD2/H3K36me3 status in CD34+ progenitors of CP CML patients (pts) and whether SETD2/H3K36me3 deficiency may play a role in genomic instability in CML models. METHODS - Western blotting (WB) was used to assess SETD2 protein expression and H3K36me3 as a surrogate marker of SETD2 function in the CD34+ cell fraction isolated from the bone marrow of 20 newly diagnosed CP CML pts and from a pool of healthy donors (HD). SETD2 forced expression in CD34+ progenitors from newly diagnosed CP CML pts and in the SETD2-deficient KCL22 cell line was performed by nucleofection. SETD2 knock-down in the SETD2-proficient LAMA84 cell line was performed by RNAi. Clonogenic capacity was evaluated by clonogenic assays. Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K36me3 was performed on an Illumina HiSeq2000 with a min 50 million 150-bp single-end reads per replicate. High resolution karyotyping wias perfomed with Cytoscan HD arrays. DNA damage and DNA repair activation were assessed in primary samples and cell lines by WB and immunofluorescence (IF) using antibodies specific for phospho-H2AX, mismatch repair (MMR) and homologous recombination repair (HR) proteins. RESULTS - WB demonstrated a marked down-modulation of SETD2 expression, paralleled by H3K36me3 deficiency, in the CD34+ cells of all newly diagnosed CP CML pts as compared to the total mononuclear fraction and to CD34+ cells from HDs. To investigate whether SETD2 loss affects the activation and proficiency of HR and MMR, we used chronic exposure to UV rays or a single exposure to hydrogen peroxide (1mM for 30 and 60 min). Both induced DNA damage in SETD2 siRNA-depleted LAMA84 cells. Compared to parental cells, cells silenced for SETD2 failed to activate the ATM-dependent repair pathway. Moreover, we found that ATM inactivation prevents H2AX foci formation, associated with a loss of RAD51 and RAD54 (HR) and MSH6 (MMR) repair foci. To confirm the hypothesis that SETD2 is a tumor suppressor implicated in maintaining genomic stability in CML, we transfected SETD2-deficient KCL22 cells with an ectopic SETD2 plasmid. SETD2 forced expression was able to restore DNA damage response, as demonstrated by WB and IF detection of ATM, p95 and H2AX phosphorylation, BRCA1, BRCA2 and CtIP expression and finally RAD51 and RAD54 localization on HR repair foci observed after UV or hydrogen peroxide exposure. High-resolution karyotyping after DNA damage showed increased rate of DNA breakpoints in SETD2-deficient cells, preferentially occuring at loci where H3K36me3 marks were lost as assessed by ChiP-seq. In line with the effects observed in cell line models, forced expression of SETD2 in CD34+ cells from 5 CP CML pts was found to restore proliferation control, since a >50% reduction in clonogenic potential was observed after nucleofection. CONCLUSIONS - Our findings demonstrate that SETD2 is a bona fide tumor suppressor in CML progenitors and establish a functional link between SETD2 loss of function and genomic instability. SETD2 inactivation may thus play a pivotal role in the harmful cascade of events that may foster drug resistance and ultimately lead to disease progression. Since SETD2 loss of function in CML is mediated by post-translation mechanisms, hence is reversible, therapeutic strategies aimed at interfering with these mechanisms may restore proliferation control and interrupt this cascade. Supported by AIRC IG 2019 (23001) and by Italian Ministry of Health, “Bando Ricerca Finalizzata 2016”, project GR-2016-02364880.

Astrocyte-like subpopulation of NG2 glia in the adult mouse cortex exhibits characteristics of neural progenitor cells.

Glial cells expressing neuron-glial antigen 2 (NG2), also known as oligodendrocyte progenitor cells (OPCs), play a critical role in maintaining brain health. However, their ability to differentiate after ischemic injury is poorly understood. The aim of this study was to investigate the properties and functions of NG2 glia in the ischemic brain. Using transgenic mice, we selectively labeled NG2-expressing cells and their progeny in both healthy brain and after focal cerebral ischemia (FCI). Using single-cell RNA sequencing, we classified the labeled glial cells into five distinct subpopulations based on their gene expression patterns. Additionally, we examined the membrane properties of these cells using the patch-clamp technique. Of the identified subpopulations, three were identified as OPCs, whereas the fourth subpopulation had characteristics indicative of cells likely to develop into oligodendrocytes. The fifth subpopulation of NG2 glia showed astrocytic markers and had similarities to neural progenitor cells. Interestingly, this subpopulation was present in both healthy and post-ischemic tissue; however, its gene expression profile changed after ischemia, with increased numbers of genes related to neurogenesis. Immunohistochemical analysis confirmed the temporal expression of neurogenic genes and showed an increased presence of NG2 cells positive for Purkinje cell protein-4 at the periphery of the ischemic lesion 12 days after FCI, as well as NeuN-positive NG2 cells 28 and 60 days after injury. These results suggest the potential development of neuron-like cells arising from NG2 glia in the ischemic tissue. Our study provides insights into the plasticity of NG2 glia and their capacity for neurogenesis after stroke.

Hey2 enhancer activity defines unipotent progenitors for left ventricular cardiomyocytes in juxta-cardiac field of early mouse embryo

The cardiac crescent is the first structure of the heart and contains progenitor cells of the first heart field, which primarily differentiate into left ventricular cardiomyocytes. The interface between the forming cardiac crescent and extraembryonic tissue is known as the juxta-cardiac field (JCF), and progenitor cells in this heart field contribute to the myocardium of the left ventricle and atrioventricular canal as well as the epicardium. However, it is unclear whether there are progenitor cells that differentiate specifically into left ventricular cardiomyocytes. We have previously demonstrated that an enhancer of the gene encoding the Hey2 bHLH transcriptional repressor is activated in the ventricular myocardium during mouse embryonic development. In this study, we aimed to investigate the characteristics of cardiomyocyte progenitor cells and their cell lineages by analyzing Hey2 enhancer activity at the earliest stages of heart formation. We found that the Hey2 enhancer initiated its activity prior to cardiomyocyte differentiation within the JCF. Hey2 enhancer-active cells were present rostrally to the Tbx5-expressing region at the early phase of cardiac crescent formation and differentiated exclusively into left ventricular cardiomyocytes in a lineage distinct from the Tbx5-positive lineage. By the late phase of cardiac crescent formation, Hey2 enhancer activity became significantly overlapped with Tbx5 expression in cells that contribute to the left ventricular myocardium. Our study reveals that a population of unipotent progenitor cells for left ventricular cardiomyocytes emerge in the JCF, providing further insight into the mode of cell type diversification during early cardiac development.

LncRNA PVT1 promotes strong stemness and endothelial progenitor cell characteristics in renal carcinoma stem cells.

Renal cancer stem cells (RCSCs) derived from clear cell renal cell carcinoma (ccRCC) tissues with higher microvessel density (MVD) have strong stemness and endothelial progenitor cells-like (EPCs-like) characteristics. A high level of lncRNA PVT1 expression is essential for simultaneously retaining strong RCSC stemness and EPCs-like characteristics. PVT1 binds with TAZ protein and prevents its phosphorylation, which promotes RCSC stemness. Moreover, RCSCs support endothelial differentiation and angiogenesis, which are mediated via the PVT1/miR-15b/KDR axis. This report provides insight into the determinants of RCSC impact on stemness and highlights the critical role of RCSC in angiogenesis. The presented findings suggest that targeting RCSC through PVT1 expression may be a new treatment strategy for ccRCC.

Pancreatic acinar heterogeneity hijacks carcinogenesis and homeostasis.

In this issue, Jiang and colleagues employ multiple lineage-tracing approaches to elaborate on the role of Tff2+ transit-amplifying progenitor cells in the pancreatic acinar compartment of mice. This work provides insights into the steady-state homeostasis and tumor-suppressive features of certain progenitor cells and presents findings on acinar cell heterogeneity.

HGG-18. SINGLE CELL RNA-SEQ ANALYSIS OF PEDIATRIC HIGH-GRADE GLIOMA PATIENT SAMPLES IDENTIFIES COMMON GLIAL AND IMMUNE CELL TYPES THAT HELP TO EXPLAIN HETEROGENEITY AND TUMORIGENESIS

Abstract BACKGROUND Pediatric high-grade gliomas (PHGG) are aggressive, undifferentiated central nervous system (CNS) tumors. PHGG comprises subtypes that differ phenotypically, histologically and by cellular composition. Despite intensive research, overall survival rates have remained for decades around 2% for diffuse midline glioma (DMG) and 20% for non-DMG PHGG. We hypothesized that PHGG’s heterogeneity, a key difficulty in devising therapies, results from functional and lineage differences in tumor cell types. METHODS Using 19 PHGG samples from our institution’s pediatric brain tumor bank, we constructed a single-cell RNA-Seq (scRNA-Seq) dataset that included tumor and immune cells, generated predicted cell types by mapping the scRNA-Seq data onto known cell types, and performed differential gene expression and geneset enrichment analyses. We acquired bulk RNA-Seq and DNA methylation data to characterize overall tumor properties. RESULTS Samples consisted of several principal tumor cell types, including cells with astrocyte characteristics, oligodendrocyte progenitor cell (OPC) characteristics, and cells that expressed OPC markers but had slight enrichment in mesenchymal and inflammatory gene expression (OPC-like/Mes cells). We also found a potential stemlike population that expressed neural stem cell markers. The predicted astrocytes, OPCs and OPC-like/Mes cells differed in their gene expression profiles, pathway enrichment, and tendency to proliferate as assessed from expression of cell cycle genes. A combined microglia/macrophage population, strongly enriched in mesenchymal and inflammatory gene expression, may serve to induce inflammatory gene expression in tumor cells. Key conclusions from the scRNA-Seq analysis were validated using multi-channel immunofluorescence staining. CONCLUSIONS PHGG comprises a small stemlike population and varying proportions of proliferating glial lineage tumor cells. CNS-resident microglia/macrophages may trigger mesenchymal gene expression in PHGG. We will use our results to investigate the roles of PHGG’s diverse tumor cell populations in resistance to radiotherapy and to develop treatments to target individual cell types comprising PHGG.

Epigenetic modulation inhibits epithelial-mesenchymal transition-driven fibrogenesis and enhances hepatic progenitor characteristics of chemically-derived hepatic progenitor cells

Epigenetic modulation inhibits epithelial-mesenchymal transition-driven fibrogenesis and enhances hepatic progenitor characteristics of chemically-derived hepatic progenitor cells

Phenotypic and Biomechanical Characteristics of Human Fetal Neural Progenitor Cells Exposed to Pesticide Compounds

Various forms of pesticides have been reported to be among the environmental toxicants, which are detrimental to human health. The active ingredients of these formulations can enter the human body through air, food, or water. Epidemiological studies suggest that these compounds strongly affect the developing brain in fetal and infant stages due to their ability to breach the underdeveloped blood–brain barrier. Since neural progenitor stem cells (NPCs) in the developing brain are the most vulnerable to these compounds, the mechanisms by which NPCs experience toxicity upon exposure to these chemicals must be investigated. Here, we assessed the viability of human fetal NPCs in 2D cultures in the presence of the active ingredients of six widely used pesticides using Live/Dead® and Hoechst staining. The IC50 values ranged from 4.1–201 μM. A significant drop in cell viability with increasing toxicant concentration (p < 0.01) was noted, with the order of toxicity being malathion < 4-aminopyridine < methoprene < prallethrin < temephos < pyriproxyfen. Changes in cellular biomechanical characteristics (Young’s modulus, tether force, membrane tension, and tether radius) were quantified using atomic force microscopy, whereas cell migration was elucidated over 48 h using a customized wound-healing assay. The Young’s modulus of fetal NPCs exposed to IC50/2 doses of these compounds was reduced by 38–70% and that of those exposed to IC50 doses was reduced by 71–80% (p < 0.001 vs. controls for both; p < 0.01 for IC50 vs. IC50/2 for each compound). Similar patterns were noted for tether forces and membrane tension in fetal NPCs. NPC migration was found to be compound type- and dose-dependent. These results attest to the significant detrimental effects of these compounds on various aspects of the human fetal NPC phenotype, and the utility of cell mechanics as a marker to assess developmental neurotoxicity.

Evaluation of Endothelial Progenitor Cell Characteristics as Clinical Biomarkers for Elderly Patients with Ischaemic Stroke.

Ageing impairs endothelial function and predisposes the person to ischaemic stroke (IS). Endothelial progenitor cells (EPCs) repair endothelial damage and induce post-ischaemic neovascularisation. Given the prevalence of IS in older population, this study explored whether changes in EPC number and function may reliably predict the type or outcome of stroke in patients ≥ 65years of age. For this, blood samples were collected once from healthy volunteers (HVs, n = 40) and four times (admission and days 7, 30 and 90 post-stroke) from participants with lacunar (n = 38) or cortical (n = 43) stroke. EPCs were counted with flow cytometry and defined as non-haematopoietic cells (CD45-) expressing markers for stemness (CD34 +), immaturity (CD133 +) and endothelial maturity (KDR +). Clonogenesis, tubulogenesis, migration and proliferation assays were performed as measures of EPC functionality. Biochemical profile of plasma inflammatory and angiogenic agents were studied using specific ELISAs. Primary outcome was disability or dependence on day 90 post-stroke, assessed by the modified Rankin Scale (mRS). Compared to HVs, EPC numbers were higher in stroke patients at all time points studied, reaching significance at baseline and day 30. No differences in EPC counts and functionality were observed between lacunar and cortical stroke groups at any time. Plasma endostatin, PDGF-BB, TNF-α and VEGF levels were higher in stroke patients vs HVs. Patient outcome, evaluated by mRS on day 90 post-stroke, did not correlate with EPC count or functionality. Baseline EPC counts may serve as a diagnostic marker for stroke but fail to distinguish between different stroke subtypes and predict post-stroke outcome.

Identification of IGF2 as Genomic Driver and Actionable Therapeutic Target in Hepatoblastoma.

Management of hepatoblastoma (HB), the most frequent pediatric liver cancer, is based on surgical resection and perioperative chemotherapy regimens. In this study, we aimed to identify actionable targets in HB and assess the efficacy of molecular therapies in preclinical models of HB. Paired tumor and adjacent tissues from 31 HBs and a validation set of 50 HBs were analyzed using RNA-seq, SNP and methylation arrays. IGF2 overexpression was identified as the top targetable HB driver, present in 71% of HBs (22/31). IGF2high tumors displayed progenitor cell features and shorter recurrence-free survival. IGF2 overexpression was associated in 91% of cases with fetal promoter hypomethylation, ICR1 deregulation, 11p15.5 loss of heterozygosity or miR483-5p overexpression. The antitumor effect of xentuzumab (a monoclonal antibody targeting IGF1/2) alone or in combination with the conventional therapeutic agent cisplatin was assessed in HB cell lines, in PDX-derived HB organoids and in a xenograft HB murine model. The combination of xentuzumab with cisplatin showed strong synergistic antitumor effects in organoids and in IGF2high cell lines. In mice (n=55), the combination induced a significant decrease in tumor volume and improved survival compared to cisplatin alone. These results suggest that IGF2 is an HB actionable driver and that, in preclinical models of HB, the combination of IGF1/2 inhibition with cisplatin induces superior antitumor effects than cisplatin monotherapy. Overall, our study provides a rationale for testing IGF2 inhibitors in combination with cisplatin in HB patients with IGF2 overexpression.

Isolation, characterization and differentiation of dermal papilla cells from Small-tail Han sheep

Dermal papilla cells (DPCs) are the key dermal component of the hair follicle that directly regulates hair follicle development, growth and regeneration. Successfully isolated and cultured DPCs from Small-tail Han sheep could provide a good model for the study of hair follicle development mechanism in vitro. DPCs were isolated using enzyme digestion and dissecting microscope from Small-tail Han sheep. Adherent cells were identified by cell characteristics, particular gene expression, differentiation capability to adipocyte and osteoblast using specific differentiation mediums. Additionally, flow cytometry was used to detect the cell cycle of DPCs. Cells originating from the dermal papilla showed the morphological appearance of mesenchymal cells (fibroblast-like cells). Purified DPCs were positive for α-SMA (α smooth muscle actin) and vimentin; in addition to their strong proliferation abilities in vitro, these DPCs can be differentiated into adipocyte and osteoblasts lineage under appropriate culture condition. DPCs were successfully isolated and subcultured from Small-tail Han sheep, which exhibited progenitor cell features and multiple differentiation potency. It provides a material for studying the molecular mechanism of hair follicle development and hair cycle, which will promote wool production in the future.

Site-specific characteristics of bone and progenitor cells in control and ovariectomized rats.

One-third of postmenopausal women experience at least one osteoporotic bone fracture in their lifetime that occurs spontaneously or from low-impact events. However, osteoporosis-associated jaw bone fractures are extremely rare. It was also observed that jaw bone marrow stem cells (BMSCs) have a higher capacity to form mineralized tissues than limb BMSCs. At present, the underlying causes and mechanisms of variations between jaw bone and limb bone during postmenopause are largely unknown. Thus, the objective of the current study was to examine the site-specific effects of estrogen deficiency using comprehensive analysis of bone quantity and quality, and its association with characterization of cellular components of bone. Nine rats (female, 6months old) for each bilateral sham and ovariectomy (OVX) surgery were obtained and maintained for 2months after surgery. A hemi-mandible and a femur from each rat were characterized for parameters of volume, mineral density, cortical and trabecular morphology, and static and dynamic mechanical analysis. Another set of 5 rats (female, 9months old) was obtained for assays of BMSCs. Following cytometry to identify BMSCs, bioassays for proliferation, and osteogenic, adipogenic, chondrogenic differentiation, and cell mitochondrial stress tests were performed. In addition, mRNA expression of BMSCs was analyzed. OVX decreased bone quantity and quality (mineral content, morphology, and energy dissipation) of femur while those of mandible were not influenced. Cellular assays demonstrated that mandible BMSCs showed greater differentiation than femur BMSCs. Gene ontology pathway analysis indicated that the mandibular BMSCs showed most significant differential expression of genes in the regulatory pathways of osteoblast differentiation, SMAD signaling, cartilage development, and glucose transmembrane transporter activity. These findings suggested that active mandibular BMSCs maintain bone formation and mineralization by balancing the rapid bone resorption caused by estrogen deficiency. These characteristics likely help reduce the risk of osteoporotic fracture in postmenopausal jawbone.

Abstract PO009: Identification of IGF2 as genomic driver and actionable therapeutic target in hepatoblastoma

Abstract Background and Aims: Management of hepatoblastoma (HB), the most frequent pediatric liver cancer, is based on surgical resection and perioperative chemotherapy regimens, commonly cisplatin. Here, we aimed to identify actionable targets in HB and assess the efficacy of molecular therapies in preclinical models of HB.Methods: Paired tumor and adjacent tissues from 31 resected HBs and a validation set of 50 HBs were analyzed at the transcriptomic, genomic and epigenomic level using RNAseq, SNP and methylation arrays, respectively. The main targetable driver in HB was identified by gene co-expression network analysis (GCN) and its overexpression was confirmed by qRT-PCR. The anti-tumor effect of driver inhibition with molecular therapies alone or in combination with cisplatin was assessed in cell lines, patient-derived HB organoids and in a HB xenograft murine model.Results: Seven network modules were significantly deregulated in tumors compared to non-tumoral samples, including IGF2 signaling pathway, cell cycle and survival and immune response. IGF2 overexpression (FC> 4 vs adjacent tissue) was identified as the top targetable HB driver (study cohort: 71%, 22/31; independent validation cohorts: 78% and 76%). IGF2high tumors displayed progenitor cell features and were significantly enriched in molecular classes with aggressive phenotypes and CTNNB1 mutations, while IGF2low tumors were enriched in inflammatory and TGF-β signaling. IGF2high tumors correlated with shorter recurrence-free survival after resection (median 34 months vs not reached for IGF2low; p = 0.02). IGF2 overexpression correlated in most cases (86%) with fetal promoter hypomethylation (50%), 11p15.5 loss of heterozygosity (LOH, 57%) or overexpression of miR483 (55%). Xentuzumab (anti-IGF1/2 mAb) alone or combined with cisplatin reduced proliferation and clonogenic capacity in IGF2high cell lines. The combination of xentuzumab and cisplatin exhibited synergistic effects in terms of cell viability in organoids derived from IGF2high human HBs. The combination treatment induced apoptosis and reduced IGF2 pathway activation in vitro. In mice (n = 13-14 per arm), this combination induced a significant decrease in the viable tumor volume (p < 0.01), extended survival compared to cisplatin alone (p < 0.05) and inhibited tumor angiogenesis (p < 0.05).Conclusion: IGF2 is an actionable driver in HB and its overexpression was associated with fetal promoter hypomethylation, LOH or miR483 overexpression. The combination of a mAb against IGF1/2 (xentuzumab) with cisplatin led to remarkable anti-tumoral effects in pre-clinical models, providing the rationale for exploring this regimen in IGF2high HB patients. Citation Format: Jordi Abril-Fornaguera, Laura Torrens, Juan Carrillo-Reixach, Alex Rialdi, Ugne Balaseviciute, Júlia Huguet-Pradell, Carla Montironi, Philipp Haber, Álvaro Del Río-Álvarez, Montserrat Domingo-Sàbat, Laura Royo, Nicholas Akers, Catherine E Willoughby, Judit Peix, Miguel Torres-Martin, Marc Puigvehi, Roser Pinyol, Stefano Cairo, Margaret Childs, Rudolf Maibach, Rita Alaggio, Piotr Czauderna, Bruce Morland, Bojan Losic, Vincenzo Mazzaferro, Ernesto Guccione, Daniela Sia, Carolina Armengol, Josep M Llovet. Identification of IGF2 as genomic driver and actionable therapeutic target in hepatoblastoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO009.

Receptor tyrosine kinase inhibitors negatively impact on pro-reparative characteristics of human cardiac progenitor cells

Receptor tyrosine kinase inhibitors improve cancer survival but their cardiotoxicity requires investigation. We investigated these inhibitors’ effects on human cardiac progenitor cells in vitro and rat heart in vivo. We applied imatinib, sunitinib or sorafenib to human cardiac progenitor cells, assessing cell viability, proliferation, stemness, differentiation, growth factor production and second messengers. Alongside, sunitinib effects were assessed in vivo. Inhibitors decreased (p < 0.05) cell viability, at levels equivalent to ‘peak’ (24 h; imatinib: 91.5 ± 0.9%; sunitinib: 83.9 ± 1.8%; sorafenib: 75.0 ± 1.6%) and ‘trough’ (7 days; imatinib: 62.3 ± 6.2%; sunitinib: 86.2 ± 3.5%) clinical plasma levels, compared to control (100% viability). Reduced (p < 0.05) cell cycle activity was seen with imatinib (29.3 ± 4.3% cells in S/G2/M-phases; 50.3 ± 5.1% in control). Expression of PECAM-1, Nkx2.5, Wnt2, linked with cell differentiation, were decreased (p < 0.05) 2, 2 and 6-fold, respectively. Expression of HGF, p38 and Akt1 in cells was reduced (p < 0.05) by sunitinib. Second messenger (p38 and Akt1) blockade affected progenitor cell phenotype, reducing c-kit and growth factor (HGF, EGF) expression. Sunitinib for 9 days (40 mg/kg, i.p.) in adult rats reduced (p < 0.05) cardiac ejection fraction (68 ± 2% vs. baseline (83 ± 1%) and control (84 ± 4%)) and reduced progenitor cell numbers. Receptor tyrosine kinase inhibitors reduce cardiac progenitor cell survival, proliferation, differentiation and reparative growth factor expression.

Abstract 4005: Identification of IGF2 as genomic driver and actionable therapeutic target in hepatoblastoma

Abstract Background and Aims: Management of hepatoblastoma (HB), the most frequent pediatric liver cancer, is based on surgical resection and perioperative platin-based regimens, commonly cisplatin. Here, we aimed to identify actionable targets in HB and assess the efficacy of molecular therapies in HB cell lines, organoids and murine models. Methods: Paired tumor and adjacent tissues from 31 resected HBs and a validation set of 50 HBs were analyzed at the transcriptomic, genomic and epigenomic level using RNAseq, SNP and methylation arrays, respectively. The main targetable driver in HB was identified by gene co-expression network analysis (GCN) and its overexpression was confirmed by qRT-PCR. The anti-tumor effect of driver inhibition with molecular therapies alone or in combination with cisplatin was assessed in cell lines, patient-derived HB organoids and in a HB xenograft murine model. Results: IGF2 overexpression (FC&amp;gt; 4 vs adjacent tissue) was identified as the top targetable HB driver (71%, 22/31). IGF2high tumors displayed progenitor cell features and were significantly enriched in molecular classes with aggressive phenotypes and CTNNB1 mutations, while IGF2low tumors were enriched in inflammatory and TGF-β signaling. IGF2high tumors correlated with shorter recurrence-free survival after resection (median 34 months vs not reached for IGF2low; p = 0.02). IGF2 overexpression correlated in most cases (86%) with fetal promoter hypomethylation (50%), 11p15.5 loss of heterozygosity (LOH, 57%) or overexpression of miR483 (55%). Xentuzumab (anti-IGF1/2 mAb) alone or combined with cisplatin reduced proliferation and clonogenic capacity in IGF2high cell lines. The combination of xentuzumab and cisplatin exhibited synergistic effects in terms of cell viability in organoids derived from IGF2high human HBs. In mice, this combination induced a significant decrease in the viable tumor volume and extended survival compared to cisplatin alone (n = 13-14 per arm, p = 0.04). Conclusion: IGF2 is an actionable driver in HB and its overexpression was associated with fetal promoter hypomethylation, LOH or miR483 overexpression. The combination of a mAb against IGF1/2 (xentuzumab) with cisplatin led to remarkable anti-tumoral effects in pre-clinical models, providing the rationale for exploring this regimen in IGF2high HB patients. Citation Format: Jordi Abril-Fornaguera, Laura Torrens, Juan Carrillo-Reixach, Alexander Rialdi, Ugne Balaseviciute, Carla Montironi, Philipp Haber, Álvaro Del Río-Álvarez, Montserrat Domingo-Sàbat, Laura Royo, Nicholas Akers, Catherine E. Willoughby, Judit Peix, Miguel Torres-Martin, Marc Puigvehi, Roser Pinyol, Stefano Cairo, Margaret Childs, Rudolf Maibach, Rita Alaggio, Piotr Czauderna, Bruce Morland, Bojan Losic, Vincenzo Mazzaferro, Ernesto Guccione, Daniela Sia, Carolina Armengol, Josep M. Llovet. Identification of IGF2 as genomic driver and actionable therapeutic target in hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4005.