Direct Reprogramming of Hepatocytes Into JAK/Stat-Dependent LGR5+ Liver Cells Able to Initiate Intrahepatic Cholangiocarcinoma.

Somatic cells that have been partially reprogrammed by the factors Oct4, Sox2, Klf4, and cMyc (OSKM) have been demonstrated to be potentially tumorigenic in vitro and in vivo due to the acquisition of cancer-associated genomic alterations and the absence of OSKM clearance over time. In the present study, we obtained partially reprogrammed, SSEA1-negative cells by transducing murine hepatocytes with Δ1Δ3-deleted adenoviruses that expressed the 4 OSKM factors. We observed that, under long-term 2D and 3D culture conditions, hepatocytes could be converted into LGR5-positive cells with self-renewal capacity that was dependent on 3 cross-signaling pathways: IL6/Jak/Stat3, LGR5/R-spondin, and Wnt/ß-catenin. Following engraftment in syngeneic mice, LGR5-positive cells that expressed the cancer markers CD51, CD166, and CD73 were capable of forming invasive and metastatic tumors reminiscent of intrahepatic cholangiocarcinoma (ICC): they were positive for CK19 and CK7, featured associations of cord-like structures, and contained cuboidal and atypical cells with dissimilar degrees of pleomorphism and mitosis. The LGR5+-derived tumors exhibited a highly vascularized stroma with substantial fibrosis. In addition, we identified pro-angiogenic factors and signaling pathways involved in neo-angiogenesis and vascular development, which represent potential new targets for anti-angiogenic strategies to overcome tumor resistance to current ICC treatments.

Interaction between AIF and CHCHD4 Regulates Respiratory Chain Biogenesis.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.

Deciphering Potential Molecular Signatures to Differentiate Acute Myeloid Leukemia (AML) with BCR::ABL1 from Chronic Myeloid Leukemia (CML) in Blast Crisis.

Acute myeloid leukemia (AML) with BCR::ABL1 has recently been recognized as a distinct subtype in international classifications. Distinguishing it from myeloid blast crisis chronic myeloid leukemia (BC-CML) without evidence of a chronic phase (CP), remains challenging. We aimed to better characterize this entity by integrating clonal architecture analysis, mutational landscape assessment, and gene expression profiling. We analyzed a large retrospective cohort study including CML and AML patients. Two AML patients harboring a BCR::ABL1 fusion were included in the study. We identified BCR::ABL1 fusion as a primary event in one patient and a secondary one in the other. AML-specific variants were identified in both. Real-time RT-PCR experiments demonstrated that CD25 mRNA is overexpressed in advanced-phase CML compared to AML. Unsupervised principal component analysis showed that AML harboring a BCR::ABL1 fusion was clustered within AML. An AML vs. myeloid BC-CML differential expression signature was highlighted, and while ID4 (inhibitor of DNA binding 4) mRNA appears undetectable in most myeloid BC-CML samples, low levels are detected in AML samples. Therefore, CD25 and ID4 mRNA expression might differentiate AML with BCR::ABL1 from BC-CML and assign it to the AML group. A method for identifying this new WHO entity is then proposed. Finally, the hypothesis of AML with BCR::ABL1 arising from driver mutations on a BCR::ABL1 background behaving as a clonal hematopoiesis mutation is discussed. Validation of our data in larger cohorts and basic research are needed to better understand the molecular and cellular aspects of AML with a BCR::ABL1 entity.

Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex.

One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using two-photon in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a "protracted" human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in "real time".

HLA-dependent heterogeneity and macrophage immunoproteasome activation during lung COVID-19 disease.

BACKGROUND: The worldwide pandemic caused by the SARS-CoV-2 virus is characterized by significant and unpredictable heterogeneity in symptoms that remains poorly understood. METHODS: Transcriptome and single cell transcriptome of COVID19 lung were integrated with deeplearning analysis of MHC class I immunopeptidome against SARS-COV2 proteome. RESULTS: An analysis of the transcriptomes of lung samples from COVID-19 patients revealed that activation of MHC class I antigen presentation in these tissues was correlated with the amount of SARS-CoV-2 RNA present. Similarly, a positive relationship was detected in these samples between the level of SARS-CoV-2 and the expression of a genomic cluster located in the 6p21.32 region (40 kb long, inside the MHC-II cluster) that encodes constituents of the immunoproteasome. An analysis of single-cell transcriptomes of bronchoalveolar cells highlighted the activation of the immunoproteasome in CD68 + M1 macrophages of COVID-19 patients in addition to a PSMB8-based trajectory in these cells that featured an activation of defense response during mild cases of the disease, and an impairment of alveolar clearance mechanisms during severe COVID-19. By examining the binding affinity of the SARS-CoV-2 immunopeptidome with the most common HLA-A, -B, and -C alleles worldwide, we found higher numbers of stronger presenters in type A alleles and in Asian populations, which could shed light on why this disease is now less widespread in this part of the world. CONCLUSIONS: HLA-dependent heterogeneity in macrophage immunoproteasome activation during lung COVID-19 disease could have implications for efforts to predict the response to HLA-dependent SARS-CoV-2 vaccines in the global population.

iPSC-Derived Hereditary Breast Cancer Model Reveals the BRCA1-Deleted Tumor Niche as a New Culprit in Disease Progression.

Tumor progression begins when cancer cells recruit tumor-associated stromal cells to produce a vascular niche, ultimately resulting in uncontrolled growth, invasion, and metastasis. It is poorly understood, though, how this process might be affected by deletions or mutations in the breast cancer type 1 susceptibility (BRCA1) gene in patients with a lifetime risk of developing breast and/or ovarian cancer. To model the BRCA1-deleted stroma, we first generated induced pluripotent stem cells (iPSCs) from patients carrying a germline deletion of exon 17 of the BRCA1 gene (BRCA1+/- who, based on their family histories, were at a high risk for cancer. Using peripheral blood mononuclear cells (PBMCs) of these two affected family members and two normal (BRCA1+/+) individuals, we established a number of iPSC clones via non-integrating Sendai virus-based delivery of the four OCT4, SOX2, KLF4, and c-MYC factors. Induced mesenchymal stem cells (iMSCs) were generated and used as normal and pathological stromal cells. In transcriptome analyses, BRCA1+/- iMSCs exhibited a unique pro-angiogenic signature: compared to non-mutated iMSCs, they expressed high levels of HIF-1α, angiogenic factors belonging to the VEGF, PDGF, and ANGPT subfamilies showing high angiogenic potential. This was confirmed in vitro through the increased capacity to generate tube-like structures compared to BRCA1+/+ iMSCs and in vivo by a matrigel plug angiogenesis assay where the BRCA1+/- iMSCs promoted the development of an extended and organized vessel network. We also reported a highly increased migration capacity of BRCA1+/- iMSCs through an in vitro wound healing assay that correlated with the upregulation of the periostin (POSTN). Finally, we assessed the ability of both iMSCs to facilitate the engraftment of murine breast cancer cells using a xenogenic 4T1 transplant model. The co-injection of BRCA1+/- iMSCs and 4T1 breast cancer cells into mouse mammary fat pads gave rise to highly aggressive tumor growth (2-fold increase in tumor volume compared to 4T1 alone, p = 0.01283) and a higher prevalence of spontaneous metastatic spread to the lungs. Here, we report for the first time a major effect of BRCA1 haploinsufficiency on tumor-associated stroma in the context of BRCA1-associated cancers. The unique iMSC model used here was generated using patient-specific iPSCs, which opens new therapeutic avenues for the prevention and personalized treatment of BRCA1-associated hereditary breast cancer.

Embryonic Program Activated during Blast Crisis of Chronic Myelogenous Leukemia (CML) Implicates a TCF7L2 and MYC Cooperative Chromatin Binding.

Chronic myeloid leukemia (CML) is characterized by an inherent genetic instability, which contributes to the progression of the disease towards an accelerated phase (AP) and blast crisis (BC). Several cytogenetic and genomic alterations have been reported in the progression towards BC, but the precise molecular mechanisms of this event are undetermined. Transcription Factor 7 like 2 (TFC7L2) is a member of the TCF family of proteins that are known to activate WNT target genes such as Cyclin D1. TCF7L2 has been shown to be overexpressed in acute myeloid leukemia (AML) and represents a druggable target. We report here that TCF7L2 transcription factor expression was found to be correlated to blast cell numbers during the progression of the disease. In these cells, TCF7L2 CHIP-sequencing highlighted distal cis active enhancer, such as elements in SMAD3, ATF5, and PRMT1 genomic regions and a proximal active transcriptional program of 144 genes. The analysis of CHIP-sequencing of MYC revealed a significant overlapping of TCF7L2 epigenetic program with MYC. The ß-catenin activator lithium chloride and the MYC-MAX dimerization inhibitor 10058-F4 significantly modified the expression of three epigenetic targets in the BC cell line K562. These results suggest for the first time the cooperative role of TCF7L2 and MYC during CML-BC and they strengthen previous data showing a possible involvement of embryonic genes in this process.

Global MicroRNA Profiling Uncovers miR-206 as a Negative Regulator of Hematopoietic Commitment in Human Pluripotent Stem Cells.

Although human pluripotent stem cells (hPSCs) can theoretically differentiate into any cell type, their ability to produce hematopoietic cells is highly variable from one cell line to another. The underlying mechanisms of this heterogeneity are not clearly understood. Here, using a whole miRNome analysis approach in hPSCs, we discovered that their hematopoietic competency was associated with the expression of several miRNAs and conversely correlated to that of miR-206 specifically. Lentiviral-based miR-206 ectopic expression in H1 hematopoietic competent embryonic stem (ES) cells markedly impaired their differentiation toward the blood lineage. Integrative bioinformatics identified a potential miR-206 target gene network which included hematopoietic master regulators RUNX1 and TAL1. This work sheds light on the critical role of miR-206 in the generation of blood cells off hPSCs. Our results pave the way for future genetic manipulation of hPSCs aimed at increasing their blood regenerative potential and designing better protocols for the generation of bona fide hPSC-derived hematopoietic stem cells.

iPSC-Derived Embryoid Bodies as Models of c-Met-Mutated Hereditary Papillary Renal Cell Carcinoma.

Hereditary cancers with cancer-predisposing mutations represent unique models of human oncogenesis, as a driving oncogenic event is present in germline. Currently, there are no satisfactory models to study these malignancies. We report the generation of IPSC from the somatic cells of a patient with hereditary c-met-mutated papillary renal cell carcinoma (PRCC). From these cells we have generated spontaneous aggregates organizing in structures which expressed kidney markers such as PODXL and Six2. These structures expressed PRCC markers both in vitro and in vivo in NSG mice. Gene-expression profiling showed striking molecular similarities with signatures found in a large cohort of PRCC tumor samples. This analysis, applied to primary cancers with and without c-met mutation, showed overexpression of the BHLHE40 and KDM4C only in the c-met-mutated PRCC tumors, as predicted by c-met-mutated embryoid bodies transcriptome. These data therefore represent the first proof of concept of "hereditary renal cancer in a dish" model using c-met-mutated iPSC-derived embryoid bodies, opening new perspectives for discovery of novel predictive progression markers and for drug-screening for future precision-medicine strategies.

Altered microtubule dynamics and vesicular transport in mouse and human MeCP2-deficient astrocytes.

Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder, characterized by normal post-natal development followed by a sudden deceleration in brain growth with progressive loss of acquired motor and language skills, stereotypic hand movements and severe cognitive impairment. Mutations in the methyl-CpG-binding protein 2 (MECP2) cause more than 95% of classic cases. Recently, it has been shown that the loss of Mecp2 from glia negatively influences neurons in a non-cell-autonomous fashion, and that in Mecp2-null mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern and greatly prolonged lifespan compared with globally null mice. We now report that microtubule (MT)-dependent vesicle transport is altered in Mecp2-deficient astrocytes from newborn Mecp2-deficient mice compared with control wild-type littermates. Similar observation has been made in human MECP2 p.Arg294* iPSC-derived astrocytes. Importantly, administration of Epothilone D, a brain-penetrant MT-stabilizing natural product, was found to restore MT dynamics in Mecp2-deficient astrocytes and in MECP2 p.Arg294* iPSC-derived astrocytes in vitro. Finally, we report that relatively low weekly doses of Epothilone D also partially reversed the impaired exploratory behavior in Mecp2(308/y) male mice. These findings represent a first step toward the validation of an innovative treatment for RTT.