Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as ...short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage.
Research on patient-derived induced pluripotent stem cells (iPSCs) could immensely benefit from the implementation of CRISPR/Cas9 genome editing of iPSCs, creating unique opportunities such as the ...establishment of isogenic iPSC lines for disease modeling or personalized patient-specific drug screenings. Here we describe a stepwise protocol of safe, efficient, and selection-free CRISPR/Cas9-mediated gene correction or knockout in human iPSCs followed by 3D spin-embryoid body (EB)-based hematopoietic/neutrophilic iPSC-differentiation.
Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can progress to acute myeloid leukemia (CN/AML). Patient material to study leukemogenesis, especially ...hematopoietic progenitor cells (HPCs) is limited and hard to access. We have established a protocol for generation of HPCs from iPSCs followed by HPC expansion on Sl/Sl feeder cells expressing FLT3L. We performed drug treatment of iPSC-derived HPCs on feeder cells or under feeder-free conditions. Our protocol is also suitable for primary leukemia blasts.
For complete details on the use and execution of this protocol, please refer to Dannenmann et al. (2021), (2020), and (2019).
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•Differentiation of hiPSCs to CD34+CD45+ hematopoietic progenitor cells (HPCs)•Analysis of HPC differentiation potential by CFU-Assay•Expansion of iPSC-derived HPCs on Sl/Sl (FLT3L) feeder cells•Drug treatment of expanded HPCs on Sl/Sl (FLT3L) feeder cells and feeder-free
Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can progress to acute myeloid leukemia (CN/AML). Patient material to study leukemogenesis, especially hematopoietic progenitor cells (HPCs) is limited and hard to access. We have established a protocol for generation of HPCs from iPSCs followed by HPC expansion on Sl/Sl feeder cells expressing FLT3L. We performed drug treatment of iPSC-derived HPCs on feeder cells or under feeder-free conditions. Our protocol is also suitable for primary leukemia blasts.
In this chapter, we present an optimized CRISPR/Cas9 RNP nucleofection approach for gene knockout (KO) in hematopoietic stem and progenitor cells (HSPCs). With experimentally proved active ...locus-specific sgRNAs, we routinely reach over 80% gene KO in HSPCs, thus avoiding the need for cell sorting or enrichment of targeted cell population. Additionally, we provide a protocol for in vitro granulocytic differentiation of HSPCs after gene KO and detailed description of granulocyte function tests which can be applied to study the effects of a particular gene KO.
Mutations in the ELANE gene, encoding the neutrophil elastase (NE) protein, are responsible for most cyclic neutropenia (CyN) cases and approximately 25% of congenital neutropenia (CN) cases. In CN ...and in CyN, a median of 2.8% of CD34+ cells were early CD49f+ hematopoietic stem cells (eHSC) that did not express ELANE and thus escape from the unfolded protein response (UPR) caused by mutated NE. In CyN, the CD49f+ cells respond to granulocyte colony-stimulating factor (G-CSF) with a significant upregulation of the hematopoietic stem cell-specific transcription factors, C/EBPα, MLL1, HOXA9, MEIS1, and HLF during the ascending arm of the cycle, resulting in the differentiation of myeloid cells to mature neutrophils at the cycle peak. However, NE protein released by neutrophils at the cycle's peak caused a negative feedback loop on granulopoiesis through the proteolytic digestion of G-CSF. In contrast, in CN patients, CD49f+ cells failed to express mRNA levels of HSC-specific transcription factors mentioned above. Rescue of C/EBPα expression in CN restored granulopoiesis.
With an incidence of ~50%, the absence or reduced protein level of p53 is much more common than TP53 mutations in acute myeloid leukemia (AML). AML with FLT3-ITD (internal tandem duplication) ...mutations has an unfavorable prognosis and is highly associated with wt-p53 dysfunction. While TP53 mutation in the presence of FLT3-ITD does not induce AML in mice, it is not clear whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML. Here, we generated FLT3-ITD knock-in; p53 knockout (heterozygous and homozygous) double-transgenic mice and found that both alterations strongly cooperated in the induction of cytogenetically normal AML without increasing the self-renewal potential. At the molecular level, we found the strong upregulation of Htra3 and the downregulation of Lin28a, leading to enhanced proliferation and the inhibition of apoptosis and differentiation. The co-occurrence of Htra3 overexpression and Lin28a knockdown, in the presence of FLT3-ITD, induced AML with similar morphology as leukemic cells from double-transgenic mice. These leukemic cells were highly sensitive to the proteasome inhibitor carfilzomib. Carfilzomib strongly enhanced the activity of targeting AXL (upstream of FLT3) against murine and human leukemic cells. Our results unravel a unique role of p53 haploinsufficiency or loss in the development of FLT3-ITD + AML.
Severe congenital neutropenia (CN) is a rare heterogeneous group of diseases, characterized by a granulocytic maturation arrest. Autosomal recessive mutations in the HAX1 gene are frequently detected ...in affected individuals. However, the precise role of HAX1 during neutrophil differentiation is poorly understood. To date, no reliable animal model has been established to study HAX1-associated CN. Here we show that knockdown of zebrafish hax1 impairs neutrophil development without affecting other myeloid cells and erythrocytes. Furthermore, we have found that interference with the Hax1 function decreases the expression level of key target genes of the granulocyte-colony stimulating factor (G-CSF) signaling pathway. The reduced neutrophil numbers in the morphants could be reversed by G-CSF, which is also the main therapeutic intervention for patients who have CN. Our results demonstrate that zebrafish is a suitable model for HAX1-associated neutropenia. We anticipate that this model will serve as an in vivo platform to identify new avenues for developing tailored therapeutic strategies for CN patients, particularly for those individuals that do not respond to the G-CSF treatment.
Patients with the pre-leukemia bone marrow failure syndrome called severe congenital neutropenia (CN) have an approximately 15% risk of developing acute myeloid leukemia (AML; called here CN/AML). ...Most CN/AML patients co-acquire
CSF3R
and
RUNX1
mutations, which play cooperative roles in the development of AML. To establish an in vitro model of leukemogenesis, we utilized bone marrow lin
−
cells from transgenic C57BL/6-d715
Csf3r
mice expressing a CN patient–mimicking truncated
CSF3R
mutation. We transduced these cells with vectors encoding
RUNX1
wild type (WT) or
RUNX1
mutant proteins carrying the R139G or R174L mutations. Cells transduced with these
RUNX1
mutants showed diminished in vitro myeloid differentiation and elevated replating capacity, compared with those expressing WT
RUNX1
. mRNA expression analysis showed that cells transduced with the
RUNX1
mutants exhibited hyperactivation of inflammatory signaling and innate immunity pathways, including IL-6, TLR, NF-kappaB, IFN, and TREM1 signaling. These data suggest that the expression of mutated
RUNX1
in a
CSF3R
-mutated background may activate the pro-inflammatory cell state and inhibit myeloid differentiation.
Induced pluripotent stem cells (iPSCs) from patients with genetic disorders are a valuable source for in vitro disease models, which enable drug testing and validation of gene and cell therapies. We ...generated iPSCs from a severe congenital neutropenia (SCN) patient, who presented with a nonsense mutation in the glucose-6-phosphatase catalytic subunit 3 (G6PC3) gene causing profound defects in granulopoiesis, associated with increased susceptibility of neutrophils to apoptosis. Generated SCN iPSC clones exhibited the capacity to differentiate into hematopoietic cells of the myeloid lineage and we identified two cytokine conditions, i.e., using granulocyte-colony stimulating factor or granulocyte-macrophage colony stimulating factor in combination with interleukin-3, to model the SCN phenotype in vitro. Reduced numbers of granulocytes were produced by SCN iPSCs compared with control iPSCs in both settings, which reflected the phenotype in patients. Interestingly, our model showed increased monocyte/macrophage production from the SCN iPSCs. Most importantly, lentiviral genetic correction of SCN iPSCs with a codon-optimized G6PC3 transgene restored granulopoiesis and reduced apoptosis of in vitro differentiated myeloid cells. Moreover, addition of vitamin B3 clearly induced granulocytic differentiation of SCN iPSCs and increased the number of neutrophils to levels comparable with those obtained from healthy control iPSCs. In summary, we established an iPSC-derived in vitro disease model, which will serve as a tool to test the potency of alternative treatment options for SCN patients, such as small molecules and gene therapeutic vectors.
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