Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity

Microenvironnement hématopoïétique du foie foetal (caractérisation et relation avec les cellules souches mésenchymateuses)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

E4F1: a novel candidate factor for mediating BMI1 function in primitive hematopoietic cells

The Polycomb group gene Bmi1 is essential for the proliferation of neural and hematopoietic stem cells. Much remains to be learned about the pathways involved in the severe hematopoietic phenotype observed in Bmi1 homozygous mutant mice except for the fact that loss of p53 or concomitant loss of p16(Ink4a) and p19(Arf) functions achieves only a partial rescue. Here we report the identification of E4F1, an inhibitor of cellular proliferation, as a novel BMI1-interacting partner in hematopoietic cells. We provide evidence that Bmi1 and E4f1 genetically interact in the hematopoietic compartment to regulate cellular proliferation. Most importantly, we demonstrate that reduction of E4f1 levels through RNA interference mediated knockdown is sufficient to rescue the clonogenic and repopulating ability of Bmi1(−/−) hematopoietic cells up to 3 mo post-transplantation. Using cell lines and MEF, we also demonstrate that INK4A/ARF and p53 are not essential for functional interaction between Bmi1 and E4f1. Together, these findings identify E4F1 as a key modulator of BMI1 activity in primitive hematopoietic cells

EPCR expression marks UM171-expanded CD34 cord blood stem cells.

A small subset of human cord blood CD34 cells express endothelial protein C receptor (EPCR/CD201/PROCR) when exposed to the hematopoietic stem cell (HSC) self-renewal agonist UM171. In this article, we show that EPCR-positive UM171-treated cells, as opposed to EPCR-negative cells, exhibit robust multilineage repopulation and serial reconstitution ability in immunocompromised mice. In contrast to other stem cell markers, such as CD38, EPCR expression is maintained when cells are introduced in culture, irrespective of UM171 treatment. Although engineered overexpression of EPCR fails to reproduce the effects of UM171 on HSC activity, its expression is required for the repopulating activity of human HSCs. Altogether, our results indicate that EPCR is a reliable and cell culture-compatible marker of UM171-expanded human cord blood HSCs

UBAP2L is amplified in a large subset of human lung adenocarcinoma and is critical for epithelial lung cell identity and tumor metastasis

International audienceThe ubiquitin-associated protein 2-like (UBAP2L) gene remains poorly studied in human and mouse development. UBAP2L interacts with the Polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and determines the activity of mouse hematopoietic stem cells in vivo Here we show that loss of Ubap2l leads to disorganized respiratory epithelium of mutant neonates, which die of respiratory failure. We also show that UBAP2L overexpression leads to epithelial-mesenchymal transition-like phenotype in a non-small cell lung carcinoma (NSCLC) cell line. UBAP2L is amplified in 15% of human primary lung adenocarcinoma specimens. Such patients express higher levels of UBAP2L and show a reduction in survival when compared with those who do not have this gene amplification. Supporting a possible role for UBAP2L in lung tumor progression, NSCLC cells engineered to express low levels of this gene produce much smaller tumors in vivo than wild-type control cells. Together, these results suggest that UBAP2L contributes to epithelial lung cell identity in mice and that it plays an important role in human lung adenocarcinoma.-Aucagne, R., Girard, S., Mayotte, N., Lehnertz, B., Lopes-Paciencia, S., Gendron, P., Boucher, G., Chagraoui, J., Sauvageau, G. UBAP2L is amplified in a large subset of human lung adenocarcinoma and is critical for epithelial lung cell identity and tumor metastasis

Define the surfaceome landscape of hematopoietic stem cells and pediatric leukemia specimens to improve the development of novel therapies for hematological diseases.

Background Information: Despite great advances in understanding the pathogenesis of various types of adult and childhood leukemias, there has been little progress in the development of new therapies and the 5-year survival rate remains low due to high incidence of relapse. Over the past decades, hematopoietic stem cell transplantation (HSCT) isolated from umbilical cord blood (CB) has evolved as a potent curative treatment intervention for patients with different type of blood disorders. Unfortunately, many patients are deprived of access to such therapies due to the low stem cell dose in CB units. The group of Dr. Guy Sauvageau and their collaborators have recently discovered a small-molecule, UM171, which promotes the ex vivo expansion of CB HSCs. However, the lack of reliable surface markers that can prospectively identify HSCs is still a major hurdle for the optimization of CB grafts. Purpose of the study: The aim of this collaborative research project is to identify novel and reliable HSC surface markers using a state-of-the-art surfaceomics approach. These results will help better purify and characterize HSCs in vitro, with the ultimate goal of developing novel strategies to achieve better ex vivo expansion of HSCs and generate optimized CB grafts. Methods: We have optimized and adapted a surface proteomic approach to simultaneously identify and quantify surface proteins in UM171-expanded CB cells. As a proof of concept experiment, we first used the hematopoietic cell line, OCI-AML5, which is responsive to UM171-induced cell expansion. To optimize this method for the analysis of CB cells, we performed a OCI-AML5 cell titration and determined the minimal number of hematopoietic cells required. Results: Using the recently discovered HSC marker, EPCR, we have performed a surfaceomics analysis of UM171-expanded CB HSCs and revealed the enrichment in more than 100 surface proteins in the CD34+EPCR+ population. In addition to well-known HSC markers (CD34, EPCR, CD133 and GPR56), we identified several surface proteins that may further subdivide the EPCR-positive population. To determine if these surface molecules are specific to HSC or other hematopoietic populations, their surface expression are being assessed by flow cytometry when antibodies are available. One such candidate could be GPA33, which appears to further subdivide the HSC population from CB cells. We are currently performing transplantation experiments in NSG mice to determine if GPA33 expression correlates with better engraftments. We are also producing unavailable antibodies against the other surface proteins to make possible their expression analysis via flow cytometry. Conclusion: Altogether, our published and preliminary data demonstrate the great potential of our surfaceomics method to identify novel and reliable HSC markers. The availability of the surfaceome landscape of HSCs will undeniably open new avenues for the optimization of HSCT. In addition, our potent surfaceomics approach will offer new opportunities for the development of antibody-based immunotherapies by uncovering targetable surface proteins from primary leukemia specimens

Expression of Pitx2 in stromal cells is required for normal hematopoiesis

Although the expression of Pitx2, a bicoid family homeodomain transcription factor, is highly regulated during hematopoiesis, its function during this process was not documented; we thus studied hematopoiesis in Pitx2-null mice. We found that Pitx2–/– embryos display hypoplastic livers with reduced numbers of hematopoietic cells, but these cells had normal hematopoietic potential, as evidenced by colony-forming assays, immature progenitor cell assays, and long-term repopulation assays. Because the microenvironment is also crucial to the development of normal hematopoiesis, we established Pitx2–/– and Pitx2+/+ stromas from fetal liver and studied their hematopoietic supportive capacity. We showed that the frequency of cobblestone area-forming cells was 4-fold decreased when using Pitx2–/– stromal cells compared with Pitx2+/+ stromal cells, whatever the Pitx2 genotype of hematopoietic cells tested in this assay. This defect was rescued by expression of Pitx2 into Pitx2–/– fetal liver stromal cells, demonstrating a major and direct role of Pitx2 in the hematopoietic supportive capacity of fetal liver stroma. Finally, we showed a reduced capacity of MS5 stromal cells expressing Pitx2 RNAi to support human hematopoiesis. Altogether these data showed that Pitx2 has major functions in the hematopoietic supportive capacity of fetal liver and adult bone marrow stromal cells

UM171 induces a homeostatic inflammatory-detoxification response supporting human HSC self-renewal.

Elucidation of the molecular cues required to balance adult stem cell self-renewal and differentiation is critical for advancing cellular therapies. Herein, we report that the hematopoietic stem cell (HSC) self-renewal agonist UM171 triggers a balanced pro- and anti-inflammatory/detoxification network that relies on NFKB activation and protein C receptor-dependent ROS detoxification, respectively. We demonstrate that within this network, EPCR serves as a critical protective component as its deletion hypersensitizes primitive hematopoietic cells to pro-inflammatory signals and ROS accumulation resulting in compromised stem cell function. Conversely, abrogation of the pro-inflammatory activity of UM171 through treatment with dexamethasone, cAMP elevating agents or NFkB inhibitors abolishes EPCR upregulation and HSC expansion. Together, these results show that UM171 stimulates ex vivo HSC expansion by establishing a critical balance between key pro- and anti-inflammatory mediators of self-renewal