Biology subjects

Forrester, L. M.

Publications and source records attributed to Forrester, L. M..

6 recordsLinked to original sources

bioRxiv2026

LIMK Inhibition and Metformin Block Mitochondrial Transfer Overcoming Macrophage Driven Therapy Resistance in Acute Myeloid Leukaemia

Chemoresistance is a major contributor to poor clinical outcomes in AML patients and can arise from interactions between AML cells and the bone marrow microenvironment (BME). How immune cells, particularly macrophages (M{varphi}s), facilitate this process requires better clarification. This study shows that M2-like M{varphi}s protect AML cells from apoptosis induced by daunorubicin (DNR) and cytarabine (Ara-C). This protection occurs via co-culture and is linked to enhanced mitochondrial transfer from M{varphi}s to AML cells. M{varphi}s interacted with AML cells via tunneling nanotube (TNT)-like structures. Furthermore, inhibition of mitochondrial transfer using cytochalasin B reduced the protective effect, indicating that mitochondria mediate this process. M{varphi}s transferred functional mitochondria to AML cells as evidenced by enhanced metabolic capacity and reduced reactive oxygen species levels in AML cells under chemotherapy stress. TH-257 (LIMK inhibitor) and metformin blocked mitochondrial transfer and M{varphi}-driven chemoprotection. Moreover, increased transcript expression levels of RhoC and cofilin correlate with inferior overall survival in AML patients. These findings suggest that M2-like M{varphi}s contribute to chemoresistance through TNT-mediated mitochondrial transfer and the LIMK-Cofilin pathway, identifying potential therapeutic targets to circumvent chemoresistance in AML.

cancer biology↗

bioRxiv2023

Modelling the Erythroblastic Island Niche of Dyserythropoietic Anaemia Type IV patients using Induced Pluripotent Stem Cells

Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. These patients present with a range of symptoms, including the persistence of nucleated red blood cells (RBCs) in the peripheral blood which reflects the known role for KLF1 within the erythroid cell lineage. The final stages of RBCs maturation and enucleation take place within the erythroblastic island (EBI) niche in close association with EBI macrophages. It is not known whether the detrimental effects of the E325K mutation in KLF1 are restricted to the erythroid lineage or whether deficiencies in macrophages associated with their niche also contribute to the disease pathology. To address this question, we generated an in vitro model of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from a CDA type IV patient as well as iPSCs genetically modified to express an KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2 protein showed significant deficiencies in the production of erythroid cells with associated disruption of some known KLF1 target genes. Macrophages could be generated from all iPSC lines but when the E325K-ERT2 fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle reduction in their ability to support RBC maturation was also associated with macrophages carrying the E325K-ERT2 transgene. Taken together these data support the notion that the clinically significant effects of the KLF1-E325K mutation are primarily associated with deficiencies in the erythroid lineage but that deficiencies in the niche might have the potential to exacerbate the condition. The strategy we describe provides a powerful approach to assess the effects of other mutations in KLF1 as well as other factors associated with the EBI niche.

cell biology↗

bioRxiv2022

Arterial cells support the development of human hematopoietic progenitors in vitro via secretion of IGFBP2.

Hematopoietic stem and progenitor cells develop from the hemogenic endothelium located in various sites during development, including the dorsal aorta from where Hematopoietic Stem Cells (HSCs) emerge. This process has proven especially challenging to recapitulate in vitro from pluripotent stem cells and further studies are needed to pinpoint the missing stimuli in vitro. Here, we compared iPSC-derived endothelial cells and in vivo HSC-primed hemogenic endothelium and identified 9 transcription factors expressed at significantly lower levels in cells generated in vitro. Using a novel DOX-inducible CRISPR activation system we induced the expression of those genes during in vitro differentiation. To study the phenotypical changes induced by the activation of target genes, we employed single cell RNA sequencing in combination with engineered gRNA that are detectable within the sequencing pipeline. Our data showed a significant expansion of arterial-fated endothelial cells associated with a higher in vitro progenitor activity. The expanded arterial cluster was marked by high expression of IGFBP2 and it was distinct from the hemogenic cluster that showed increased cell cycle progression. We demonstrated that the addition of IGFBP2 to differentiating PSCs resulted in a higher number of functional progenitors, identifying the supporting role of arterial cells play to the emergence of blood progenitors via IGFBP2 paracrine signalling.

developmental biology↗

bioRxiv2021

Endothelial-specific Gata3 expression is required for haematopoietic stem cell generation

To generate sufficient numbers of transplantable haematopoietic stem cells (HSCs) in vitro, a detailed understanding of how this process takes place in vivo is essential. The endothelial-to-haematopoietic transition (EHT), which culminates in the production of the first HSCs, is a highly complex process during which key regulators are switched on and off at precise moments and which is embedded into a myriad of microenvironmental signals from surrounding cells and tissues. We have previously demonstrated an HSC-supportive function for Gata3 within the sympathetic nervous system and the sub-aortic mesenchyme, but show here that it also plays a cell-intrinsic role during the EHT. It is expressed in haemogenic endothelial cells and early HSC precursors, where its expression correlates with a more quiescent state. Importantly, endothelial-specific deletion of Gata3 shows that it is functionally required for these cells to mature into HSCs, placing Gata3 at the core of the EHT regulatory network.

developmental biology↗

bioRxiv2020

Modulation of Aplnr signaling is required during the development and maintenance of the hematopoietic system

Apelin receptor (Aplnr/Agtrl1/Apj) marks a transient cell population during the differentiation of hematopoietic progenitor cells (HPCs) from pluripotent stem cells but the function of this signalling pathway during hematopoietic development both in vitro and in vitro is poorly understood. We generated an Aplnr-null mouse embryonic stem cell (mESC) line and demonstrated that they are significantly impaired in the production of HPCs indicating that the Aplnr pathway is required for their formation. Using Aplnr-tdTomato reporter mESCs we demonstrated that is expressed in a population of differentiating mesodermal cells committed to a hematopoietic and endothelial fate. Activation of this signaling pathway by the addition of the Apelin ligand to differentiating ESCs has no effect on the production of HPCs but the addition to ex vivo AGM cultures impaired the generation of long term reconstituting hematopoietic stem cells and appeared to drive myeloid differentiation. Taken together, our data suggest that the Aplnr pathway is required for the generation of cells that give rise to HSCs during development but its subsequent down regulation is required for their maintenance. HIGHLIGHTSO_LIHematopoietic differentiation is impaired in Aplnr-null ESCs C_LIO_LIAplnr-tdTomato reporter marks a subpopulation of ESC-derived mesoderm. C_LIO_LIAplnr signaling drives the maturation of lineage-committed myeloid progenitors C_LIO_LIIn AGM explant cultures HSC activity is reduced in the presence of Aplnr ligands. C_LI

developmental biology↗

bioRxiv2019

Single cell transcriptome analysis reveals markers of naive and lineage-primed hematopoietic progenitors derived from human pluripotent stem cells.

During embryogenesis the hematopoietic system develops through distinct waves that generate progenitors with increasing lineage potential, ultimately producing haematopoietic stem cells (HSCs). In vitro differentiation of human pluripotent stem cells (hPSCs) follows the early steps of haematopoietic development but the production of HSCs has proven more challenging. To study the dynamics and heterogeneity of hematopoietic progenitor cells generated in vitro from hPSCs, we performed RNA sequencing of over 10000 CD235a-CD43+single cells. We identified the transcriptome of naive progenitors and those primed toward erythroid, megakaryocyte and leukocyte lineages, and revealed their markers by clustering, trajectory analyses and functional assays. CD44 marks naive clonogenic progenitors that express the transcription factor, LMO4 and can be expanded upon BMP4 stimulation. Naive progenitors give rise to primed CD326+erythroid, ICAM2+CD9+megakaryocyte, and monocyte, neutrophil and eosinophil progenitors. We have generated an online dataset of human hematopoietic progenitors and their transcriptional remodelling upon lineage priming.

developmental biology↗