Oudrhiri, N. | bioRxiv Science

bioRxiv2022

Modeling Global Genomic Instability in Chronic Myeloid Leukemia (CML) using patient-derived induced pluripotent stem cells (iPSC)

Using induced Pluripotent Stem Cell (iPSC) technology, it is now possible to reprogram the primary leukemic cells to pluripotency and generate a major source of stem cells. To determine the feasibility of modeling global genomic instability characterizing chronic myeloid leukemia (CML) progression towards blast crisis (BC), we have used a patient-specific iPSC line that has been submitted to in vitro mutagenesis with the mutagenic agent N-ethyl-N-nitrosourea (ENU). Increased genomic instability was validated using g-H2AX and micronuclei assays. As compared to non-mutagenized cultures, these iPSC generated an increased numbers of progenitors (x 5-Fold) which proliferated in liquid cultures with a blast cell morphology. CGH array analyses were performed using ENU-treated iPSC-derived hematopoietic cells in two different time points as compared to leukemic iPSC cultured without ENU. Several cancer genes were found to be involved in the ENU-treated cultures, some known to be altered in leukemia (BLM, IKZF1, NCOA2, ALK, EP300, ERG, MKL1, PHF6 and TET1). Remarkably, transcriptome geodataset GSE4170 (Radich et al., 2006) allowed us to associate 125 of 249 of the aberrations that we detected in CML iPSC, with genes already described during progression of CML to BC (p-value =9.43exp32, after ANOVA with 1000 permutations). 38 most predictive aberrations allowed perfect reclassification of BC and chronic phase samples by unsupervised classification. Among these candidates, eleven of them have been described in CML physiopathology and connected to tyrosine kinase inhibitor (TKI) resistance and genomic instability. Overall, these results demonstrated that we have generated for the first time to our knowledge, an in vitro genetic instability model in CML, reproducing genomic events described in patients with BC. This tool using patient-derived iPSC could be of interest to study CML progression, to discover novel targets and eventually to test novel therapies.

cancer biology↗

bioRxiv2020

Direct reprogramming of adult hepatocytes to generate LGR5+ endodermal progenitor

We successfully converted hepatocytes isolated from adult mice into expandable and stable leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)-positive endodermal progenitor cells (EndoPCs). This was accomplished in vitro through transient exposure to four transcriptional factors (OCT3/4, SOX2, KLF4, and cMYC) and STAT3 activators. EndoPCs were generated by a process that involved an epithelial-mesenchymal transition (EMT) without the use of any components of the canonical WNT/{beta}-catenin or LGR5/R-spondin signaling pathways. We showed that the proliferation and capacity for self-renewal of EndoPCs in 2D long-term culture were controlled by three interrelated signaling pathways: gp130/JAK/STAT3, LGR5/R-spondin, and WNT/{beta}-catenin. After long-term maintenance in two- and three-dimensional culture systems, EndoPCs were able to differentiate into liver-restricted lineages such as hepatocyte-like cells and bile duct-like structures in vitro and in vivo. After intra-muscular injection, EndoPCs generated macroscopically visible and well-vascularized liver-like tissue, which contained Alb+ liver parenchyma-like structures and substantial, KRT7/KRT19+ bile duct-like cell organizations. ConclusionWe have developed an efficient method for producing LGR5+ adult endodermal stem cells. These cells will be useful for the in vitro study of the molecular mechanisms of liver development and have important potential for therapeutic strategies, including approaches based on bioengineered liver tissue. These cells also open up new avenues for experiments focused on disease modeling, toxicology studies, and regenerative medicine applications.

developmental biology↗