Aurélie Baudet

RNAi screen identifies MAPK14 as a druggable suppressor of human hematopoietic stem cell expansion

We report on a forward RNAi screen in primary human hematopoietic stem and progenitor cells, using pooled lentiviral shRNA libraries deconvoluted by next generation sequencing. We identify MAPK14/p38α as a modulator of ex vivo stem cell proliferation and show that pharmacologic inhibition of p38 dramatically enhances the stem cell activity of cultured umbilical cord blood derived hematopoietic cells. p38 inhibitors should thus be considered in strategies aiming at expanding stem cells for clinical benefit.

Identification of the chemokine CCL28 as a growth and survival factor for human hematopoietic stem- and progenitor cells.

In an attempt to discover novel growth factors for hematopoietic stem and progenitor cells (HSPCs), we have assessed cytokine responses of cord blood (CB)-derived CD34(+) cells in a high-content growth factor screen. We identify the immunoregulatory chemokine (C-C motif) ligand 28 (CCL28) as a novel growth factor that directly stimulates proliferation of primitive hematopoietic cells from different ontogenetic origins. CCL28 enhances the functional progenitor cell content of cultured cells by stimulating cell cycling and induces gene expression changes associated with survival. Importantly, addition of CCL28 to cultures of purified putative hematopoietic stem cells (HSCs) significantly increases the ability of the cells to long-term repopulate immunodeficient mice compared with equivalent input numbers of fresh cells. Together, our findings identify CCL28 as a potent growth-promoting factor with the ability to support the in vitro and in vivo functional properties of cultured human hematopoietic cells.

Forward RNAi screens in human stem cells.

Identifying the genes and pathways that regulate self-renewal and differentiation in somatic stem cells is a central goal in stem cell and cancer biology. Here, we describe a method for RNAi-based screens in primary human hematopoietic stem and progenitor cells. These cells are suitable targets for complex, selection-based screens using pooled lentiviral shRNA libraries. The screening approach is a promising new tool to dissect regulatory mechanisms in hematopoietic and somatic stem cells, in general, and may be particularly useful to identify gene targets and modifiers that can be further exploited in strategies for ex vivo stem cell expansion.

FACS binding assay for analysing GDNF interactions.

Glial cell-line derived neurotrophic factor (GDNF) is a secreted protein with great therapeutic potential. However, in order to analyse the interactions between GDNF and its receptors, researchers have been mostly dependent of radioactive binding assays. We developed a FACS-based binding assay for GDNF as an alternative to current methods. We demonstrated that the FACS-based assay using TGW cells allowed readily detection of GDNF binding and displacement to endogenous receptors. The dissociation constant and half maximal inhibitory concentration obtained were comparable to other studies using standard binding assays. Overall, this FACS-based, simple to perform and adaptable to high throughput setup, provides a safer and reliable alternative to radioactive methods.

Transient inhibition of NF-κB signaling enhances ex vivo propagation of human hematopoietic stem cells

Despite extensive studies, defining culture conditions in which hematopoietic stem cells can be expanded ex vivo has been challenging. Here we show that chemical inhibition of the NF-κB signaling pathway leads to a significant improvement of hematopoietic stem cell function from ex vivo cultured human umbilical cord blood derived CD34+ cells. We found a distinct peak of activation of the NF-κB pathway shortly after cells were put in culture, and consequently inhibition of the pathway was both necessary and sufficient during the first 24 hours of culture where it reduced the levels of several pro-inflammatory cytokines. Taken together, NF-κB pathway inhibition facilitates propagation of hematopoietic stem cells in culture and may complement other strategies for hematopoietic stem cell expansion by relieving stress signals that are induced as an immediate response to culture initiation.

A new genetic tool to improve immune-compromised mouse models: Derivation and CRISPR/Cas9-mediated targeting of NRG embryonic stem cell lines

Development of human hematopoietic stem cells and differentiation of embryonic stem (ES) cells/induced pluripotent stem (iPS) cells to hematopoietic stem cells are poorly understood. NOD (Non‐obese diabetic)‐derived mouse strains, such as NSG (NOD‐Scid‐il2Rg) or NRG (NOD‐Rag1‐il2Rg), are the best available models for studying the function of fetal and adult human hematopoietic cells as well as ES/iPS cell‐derived hematopoietic stem cells. Unfortunately, engraftment of human hematopoietic stem cells is very variable in these models. Introduction of additional permissive mutations into these complex genetic backgrounds of the NRG/NSG mice by natural breeding is a very demanding task in terms of time and resources. Specifically, since the genetic elements defining the NSG/NRG phenotypes have not yet been fully characterized, intense backcrossing is required to ensure transmission of the full phenotype. Here we describe the derivation of embryonic stem cell (ESC) lines from NRG pre‐implantation embryos generated by in vitro fertilization followed by the CRISPR/CAS9 targeting of the Gata‐2 locus. After injection into morula stage embryos, cells from three tested lines gave rise to chimeric adult mice showing high contribution of the ESCs (70%–100%), assessed by coat color. Moreover, these lines have been successfully targeted using Cas9/CRISPR technology, and the mutant cells have been shown to remain germ line competent. Therefore, these new NRG ESC lines combined with genome editing nucleases bring a powerful genetic tool that facilitates the generation of new NOD‐based mouse models with the aim to improve the existing xenograft models.

Forward RNAi Screens in Human Hematopoietic Stem Cells

Identifying the genes and pathways that regulate self-renewal and differentiation in somatic stem cells is a central goal in stem cell and cancer biology. Here, we describe a method for RNA interference (RNAi)-based screens combined with next-generation sequencing (NGS) in primary human hematopoietic stem and progenitor cells (HSPCs). These cells are suitable targets for complex, selection-based screens using pooled lentiviral short hairpin RNA (shRNA) libraries. The screening approach presented in this chapter is a promising tool to dissect regulatory mechanisms in hematopoietic stem cells (HSCs) and somatic stem cells in general, and may be particularly useful to identify gene targets and modifiers that can be further exploited in strategies for ex vivo stem cell expansion.

Small molecule screen identifies differentiation-promoting compounds targeting genetically diverse acute myeloid leukaemia

Small molecule screen identifies differentiation-promoting compounds targeting genetically diverse acute myeloid leukaemia