Biology subjects

Larochelle, A.

Publications and source records attributed to Larochelle, A..

5 recordsLinked to original sources

bioRxiv2024

cMPL-Based Purification and Depletion of Human Hematopoietic Stem Cells: Implications for Pre-Transplant Conditioning

The transplantation of gene-modified autologous hematopoietic stem and progenitor cells (HSPCs) offers a promising therapeutic approach for hematological and immunological disorders. However, this strategy is often limited by the toxicities associated with traditional conditioning regimens. Antibody-based conditioning strategies targeting cKIT and CD45 antigens have shown potential in mitigating these toxicities, but their long-term safety and efficacy in clinical settings require further validation. In this study, we investigate the thrombopoietin (TPO) receptor, cMPL, as a novel target for conditioning protocols. We demonstrate that high surface expression of cMPL is a hallmark feature of long-term repopulating hematopoietic stem cells (LT-HSCs) within the adult human CD34+ HSPC subset. Targeting the cMPL receptor facilitates the separation of human LT-HSCs from mature progenitors, a delineation not achievable with cKIT. Leveraging this finding, we developed a cMPL-targeting immunotoxin, demonstrating its ability to selectively deplete host cMPLhigh LT-HSCs with a favorable safety profile and rapid clearance within 24 hours post-infusion in rhesus macaques. These findings present significant potential to advance our understanding of human hematopoiesis and enhance the therapeutic outcomes of ex vivo autologous HSPC gene therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/581887v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@10b265aorg.highwire.dtl.DTLVardef@102ec94org.highwire.dtl.DTLVardef@fa269dorg.highwire.dtl.DTLVardef@fd2583_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

cell biology↗

bioRxiv2023

Post-Transplant Administration of G-CSF Impedes Engraftment of Gene Edited Human Hematopoietic Stem Cells by Exacerbating the p53-Mediated DNA Damage Response

Granulocyte colony stimulating factor (G-CSF) is commonly used as adjunct treatment to hasten recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, the utility of G-CSF administration after ex vivo gene therapy procedures targeting human HSPCs has not been thoroughly evaluated. Here, we provide evidence that post-transplant administration of G-CSF impedes engraftment of CRISPR-Cas9 gene edited human HSPCs in xenograft models. G-CSF acts by exacerbating the p53-mediated DNA damage response triggered by Cas9- mediated DNA double-stranded breaks. Transient p53 inhibition in culture attenuates the negative impact of G-CSF on gene edited HSPC function. In contrast, post-transplant administration of G-CSF does not impair the repopulating properties of unmanipulated human HSPCs or HSPCs genetically engineered by transduction with lentiviral vectors. The potential for post-transplant G-CSF administration to aggravate HSPC toxicity associated with CRISPR-Cas9 gene editing should be considered in the design of ex vivo autologous HSPC gene editing clinical trials. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/547089v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1dae519org.highwire.dtl.DTLVardef@1f6ddacorg.highwire.dtl.DTLVardef@535cbaorg.highwire.dtl.DTLVardef@ecbeaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Modulation of WNT, Activin/Nodal and MAPK Signaling Pathways Increases Arterial Hemogenic Endothelium and Hematopoietic Stem/Progenitor Cell Formation During Human iPSC Differentiation

Several differentiation protocols enable the emergence of hematopoietic stem and progenitor cells (HSPCs) from human induced pluripotent stem cells (iPSCs), yet optimized schemes to promote the development of HSPCs with self-renewal, multilineage differentiation and engraftment potential are lacking. To improve human iPSC differentiation methods, we modulated WNT, Activin/Nodal and MAPK signaling pathways by stage-specific addition of small molecule regulators CHIR99021, SB431542 and LY294002, respectively, and measured the impact on hematoendothelial formation in culture. Manipulation of these pathways provided a synergy sufficient to enhance formation of arterial hemogenic endothelium (HE) relative to control culture conditions. Importantly, this approach significantly increased production of human HSPCs with self-renewal and multilineage differentiation properties, as well as phenotypic and molecular evidence of progressive maturation in culture. Together, these findings provide a stepwise improvement in human iPSC differentiation protocols and offer a framework for manipulating intrinsic cellular cues to enable de novo generation of human HSPCs with functionality in vivo. Significance StatementThe ability to produce functional HSPCs by differentiation of human iPSCs ex vivo holds enormous potential for cellular therapy of human blood disorders. However, obstacles still thwart translation of this approach to the clinic. In keeping with the prevailing arterial-specification model, we demonstrate that concurrent modulation of WNT, Activin/Nodal and MAPK signaling pathways by stage-specific addition of small molecules during human iPSC differentiation provides a synergy sufficient to promote arterialization of HE and production of HSPCs with features of definitive hematopoiesis. This simple differentiation scheme provides a unique tool for disease modeling, in vitro drug screening and eventual cell therapies.

cell biology↗

bioRxiv2022

Systematic Single Cell Pathway Analysis (SCPA) reveals novel pathways engaged during early T cell activation

Next generation sequencing technologies have revolutionized the study of T cell biology, capturing previously unrecognized diversity in cellular states and functions. Pathway analysis is a key analytical stage in the interpretation of such transcriptomic data, providing a powerful method for detecting alterations in important biological processes. Current pathway analysis tools are built on models developed for bulk-RNA sequencing, limiting their effectiveness when applied to more complex single cell RNA-sequencing (scRNA-seq) datasets. We recently developed a sensitive and distribution-free statistical framework for multisample distribution testing, which we implement here in the open-source R package Single Cell Pathway Analysis (SCPA). After demonstrating the effectiveness of SCPA over commonly used methods, we generate a scRNA-seq T cell dataset and characterize pathway activity over early cellular activation and between T cell populations. This revealed unexpected regulatory pathways in T cells, such as an intrinsic type I interferon system regulating T cell survival and a reliance on arachidonic acid metabolism throughout T cell activation. A systems level characterization of pathway activity in T cells across multiple human tissues also revealed alpha defensin expression as a hallmark of bone marrow derived T cells. Overall, our work here provides a widely applicable tool for single cell pathway analysis, and highlights unexpected regulatory mechanisms of T cells using a novel T cell dataset.

immunology↗

bioRxiv2019

Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system

One of the most promising objectives of clinical hematology is to derive engraftable autologous hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (iPSCs). Progress in translating iPSC technologies to the clinic relies on the availability of scalable differentiation methodologies. In this study, human iPSCs were differentiated for 21 days using STEMdiff, a monolayer-based approach for hematopoietic differentiation of human iPSCs that requires no replating, co-culture or embryoid body formation. Both monolayer and suspension cells were functionally characterized throughout differentiation. In the supernatant fraction, an early transient population of primitive CD235a+ erythroid cells first emerged, followed by hematopoietic progenitors with multilineage differentiation activity in vitro but no long-term engraftment potential in vivo. In later stages of differentiation, a nearly exclusive production of definitive erythroid progenitors was observed. In the adherent monolayer, we identified a prevalent population of mesenchymal stromal cells and limited arterial vascular endothelium (VE), suggesting that the cellular constitution of the monolayer may be inadequate to support the generation of HSCs with durable repopulating potential. Quantitative modulation of WNT/{beta}-catenin and activin/nodal/TGF{beta} signaling pathways with CHIR/SB molecules during differentiation enhanced formation of arterial VE, definitive multilineage and erythroid progenitors, but was insufficient to orchestrate the generation of engrafting HSCs. Overall, STEMdiff provides a clinically-relevant and readily adaptable platform for the generation of erythroid and multilineage hematopoietic progenitors from human pluripotent stem cells.\n\nHighlightsO_LIRobust, scalable and clinically-relevant monolayer-based culture system for hematopoietic differentiation of human iPSCs.\nC_LIO_LISuccessive emergence of primitive erythroid cells, definitive multilineage HSPCs and erythroid progenitors in the culture supernatant.\nC_LIO_LIAbundant mesenchymal cells and limited arterial vascular endothelium in the culture monolayer.\nC_LIO_LICHIR/SB molecules increase arterial vascular endothelium formation, suppress primitive hematopoiesis and promote definitive multilineage and erythroid progenitors.\nC_LI

developmental biology↗