Name: flow cytometric analysis of mitochondrial reactive oxygen species in murine hematopoietic stem and progenitor cells and mll-af9 driven leukemia
Uploaded: 2019-09-05T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia at JoVE.com

This work was supported by The Fox Chase Cancer Center Board of Directors (DDM), the American Society of Hematology Scholar Award (SMS), American Cancer Society RSG (SMS) and the Department of Defense (Award#: W81XWH-18-1-0472).

All of the animal procedures described in this protocol have been approved by the Institutional Animal Care and Use committee (IACUC) at Fox Chase Cancer Center.
NOTE: The protocol workflow is divided into 4 parts as presented in Figure 1 and the required reagents are listed in the Table of Materials.
1. Bone Marrow (BM) Isolation
NOTE: MLL-AF9 leukemia mice we...

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Fluorogenic dyes that have been developed for the detection of ROS are frequently evaluated in fixed cells by microscopy or in live cells by flow cytometry22. Flow cytometric evaluation of mitochondrial ROS in BM cells using mitochondrial ROS fluorogenic dyes has two major advantages: 1) It is a fast and simple technique that is suitable for live cell analysis and 2) it allows for distinguishing and analyzing rare populations at the single-cell level in the BM using surface marker staining. The st...

Reactive Oxygen Species (ROS) are highly reactive molecules derived from molecular oxygen. The most well-defined cellular location of ROS production is the mitochondria, where electrons that pass through the electron transport chain (ETC) during oxidative phosphorylation (OXPHOS) are absorbed by molecular oxygen leading to the formation of a specific type of ROS called superoxides1. Through the actions of a series of enzymes, called superoxide dismutases or SODs, superoxides are converted into hydrogen peroxides, which are subsequently neutralized into water by enzymes such as catalase or glutathione peroxidases (GPX). Perturbations in ROS-regulatory mechanisms can lead to the excess production of ROS, often referred to as oxidative stress, which have harmful and potentially lethal cellular consequences such as macromolecule damage (i.e., DNA, protein, lipids). Moreover, oxidative stress is related to several pathologies, such as diabetes, inflammatory diseases, aging and tumors2,3,4. To maintain redox homeostasis and prevent oxidative stress, cells possess a variety of ROS-regulating mechanisms5.
Physiological levels of certain ROS are necessary for proper embryonic and adult hematopoiesis6. However, excess ROS is associated with DNA damage, cellular differentiation and exhaustion of the hematopoietic stem and progenitor pool. There is also evidence that alterations in redox biology may differ between leukemia and healthy cells. For example, ROS levels tend to be higher in acute myeloid leukemia (AML) cells relative to their healthy counterparts and other studies have suggested that leukemia stem cells maintain a low steady-state level of ROS for survival7,8. Importantly, strategies for therapeutically capitalizing on these redox differences have shown promise in several human cancer settings9,10. Therefore, assays that allow for the assessment of ROS levels in mouse models may improve our understanding of how these species contribute to cellular physiology and disease pathogenesis as well as potentially provide a platform for assessing the effectiveness of novel redox-targeting anti-cancer therapies.

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flow cytometric analysis of mitochondrial reactive oxygen species in murine hematopoietic stem and progenitor cells and mll-af9 driven leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

Presented is a method for analyzing ROS in the mitochondria of multiple healthy and MLL-AF9-expressing leukemia progenitor populations. Figure 1 displays a schematic view of the protocol workflow, which consists of 4 major steps: 1) BM isolation from mice; 2) Staining BM cells with a fluorogenic dye that recognizes mitochondrial ROS, particularly superoxides; 3) Surface marker antibody staining to delineate various healthy and leukemia hematopoietic populations; and 4) Flow cytometry acquisi...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia at JoVE.com

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Reactive oxygen species or ROS, are highly reactive oxygen species that are produced by cells, and influences their behavior. Though excess ROS can also cause widespread cellular havoc, and in severe cases, cell death. Thus, the maintenance of ROS homeostasis is of fundamental importance to cellular function and survival.The protocol presented here, focuses on measuring a specific type of ROS, that is generated by mitochondria, mainly as a metabolic by-product in live, normal, or healthy hematopoietic stem and progenitor cell populations, as well as in leukemia cells from the mouse model of blood cancer, Acute Myeloid Leukemia or AML. This protocol can also be used to evaluate how genetic manipulation, such as gene deletion or overexpression, impacts mitochondrial ROS in several healthy and malignant hematopoietic populations. And as a result, potentially provide insightful information about redux state and possibly the metabolism of the cells.This technique authorizes flow settle metric analysis of a protogenic probe to monitor mitochondrial ROS production in live healthy and malignant hematopoietic stem and progenitor sub-populations. This protocol is straightforward and can be completed in a few hours. Moreover, it is suitable for live cell analysis and allows for distinguishing and analyzing mitochondrial ROS in stem cell population in the bone marrow, using surface mark of staining.After collecting mononuclear bone marrow cells from wild tight mice and leukemic MLL-AF9 mice, according to the manuscript, aliquot 200 microliters of the cell suspension into each of nine single color control tubes. Aliquot the remaining cells into other experimental tubes, 200 microliters each. Then place into a centrifuge two experimental tubes containing bone marrow cells and leukemic cells respectively, and one control tube.Centrifuge at 300 times G for five minutes. Next, decant the supernatant, and re-suspend the cells in room temperature F-PBS with a live/dead cell stain, according to the manufacturer's instructions. Incubate on ice for 30 minutes.Afterwards, add 1 milliliter of room temperature F-PBS to the three live/dead stained tubes, and one single color control tube for mitochondrial ROS dye staining. Place the tubes into the centrifuge at 300 times G for five minutes at room temperature. Then, obtain a 5 millimolar mitochondrial ROS dye stock solution by re-suspending 50 micrograms of mitochondrial ROS dye in 13 microliters of DMSO.Then add 13 milliliters of room temperature F-PBS to the 13 microliter mitochondrial ROS dye to dilute to a final concentration of five micromolar. If necessary, add 13 microliters of 50 millimolar Verapamil to the solution to inhibit mitochondrial efflux pumps. Obtain the tubes from the centrifuge and aspirate off the wash of the live/dead cell stain.Add 200 microliters of F-PBS in the live/dead staining control and keep it in ice until ready to start the analysis. Add 200 microliters of the mitochondrial ROS dye stain containing Verapamil, to each experimental tube, as well as the mitochondrial ROS staining control tube. Vortex to mix and incubate for 10 minutes at 37 degrees Celsius in the dark.Then add 1 milliliter of room temperature F-PBS to the control and experimental tubes. Centrifuge five minutes at 300 times G at room temperature. Aspirate off the supernatent and wash the cells with an additional one milliliter of room temperature F-PBS.Centrifuge again for five minutes at 300 times G, at room temperature. First, prepare two antibody cocktails for healthy and leukemia bone marrow cells. Aspirate the supernatant from the experimental tube containing healthy bone marrow cells, and add 200 microliters of the antibody cocktail number one to the tube.Vortex to mix. Also prepare the single color control tubes by adding 200 microliters of F-PBS and one microliter of the corresponding antibody. Incubate for 60 minutes on ice in the dark.Aspirate the supernatant from the experimental tube containing leukemia bone marrow, and add 200 microliters of the antibody cocktail number two, to the tube. Vortex to mix. Incubate for 60 minutes on ice in the dark.After that, wash all the tubes with 1 milliliter of cold F-PBS and centrifuge for five minutes at 300 times G at room temperature. Re-suspend the cells in 500 microliters of cold F-PBS. Transfer the cell suspension into each flow cytometer tube with a 40 micrometer filter for excluding aggregates.First, insert one no stain control tube into the flow cytometry machine and start the acquisition to compensate the flow cytometry machine. Repeat for other control tubes. Afterwards, read the experimental tubes to set up the gates.To analyze the size and complexity of the cell population, first for healthy HSPC, set the forward scatter area and side scatter area plots populations. Press the Square Gate button, to gate out extraneous debris from the forward and side scatter plot. Then, on a forward scatter area and height plot, use a double discriminator to gate out doublets.Select live cells, lineage low cells, and the various healthy HSPC. For each population of interest, analyze the median fluorescence intensity of the TRPE channel in a histagram plot, to evaluate differences in the mitochondrial ROS signal. Repeat the same procedure of size analysis, gating and histogram plotting for leukemia populations.Bone marrow cells isolated from healthy mice were stained with a live/dead dye and a mitochondrial ROS dye, and subsequently stained with antibodies recognizing lineage markers:plus CD48, C-kit, Sca-1, CD34 and CD150. In addition, bone marrow cells from leukemia mice were also stained with CD45.2 to discriminate between MLL-AF9 leukemia cells from healthy recipient bone marrow cells stained with CD45.1. A comparison of mitochondrial ROS staining, between healthy CD48 negative LSK, and myeloid progenitors, shows that myeloid progenitors display significantly higher levels of mitochondrial ROS staining.Moreover, c-Kit high leukemia progenitors display significantly higher levels of mitochondrial ROS staining, compared to c-Kit intermediate low leukemia cells, healthy CD48 negative LSK and myeloid progenitors. C-Kit intermediate low leukemia cells, also displayed a significantly higher value compared to CD48 negative LSK cells, but not to myeloid progenitors. Mitochondrial ROS dyes are highly reactive and appropriate wash steps are needed to ensure that any excess of the dye has been removed before starting the following steps.There are several chemically distinct ROS fluorogenic dyes that can be used in this protocol, to achieve a bit of knowledge of the redux status in live hematopoietic cells. This technique has been extensively used in the literature, and may provide useful insights on the metabolic and signaling pathways that are differentially regulated in leukemia cells, if compared with their normal counterparts.

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Research

JoVE Journal

Cancer Research

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis.

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line ...