We would like to thank the members of the Institut Curie Flow Facility for their help with setting up the flow cytometry experiments. We also want to acknowledge the contributions of the other members of the Team Peri&#233;, during multiple discussions. We thank Dr. Julia Marchingo and Prof. Phil Hodgkin (Walter end Eliza Hall Institute of Medical Research) for sharing their protocol of the multiplexing of cell division dyes on lymphocytes. We thank the Saint Louis hospital cord blood biobank for providing the biological resources necessary for the development of this protocol. The study was supported by an ATIP-Avenir grant from CNRS and Bettencourt-Schueller Foundation (to L.P.), grants from the Labex CelTisPhyBio (ANR-10-LBX-0038) (to L.P. and A.D.), Idex Paris-Science-Lettres Program (ANR-10-IDEX-0001-02 PSL) (to L.P.), the Canceropole INCA Emergence (2021-1-EMERG-54b-ICR-1, to L.P.), and the ITMO MIIC grant (21CM044, to L.P.). As well as funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program ERC StG 758170-Microbar (to L.P.), A.D. was supported by a fellowship from Fondation de France.

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The authors declare no conflict of interest relevant to this work. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

The MultiGen assay is a high-throughput, easy to perform, and unexpensive assay, that has been instrumental to study lymphocyte23,24,35 and murine hematopoietic cells26,27. Here, we present a new development of the approach that allows to decipher ex vivo the early phase of human HSPC commitment, at the single-cell level using short term culture (Figure 6). Single-cell ex vivo culture systems are typically used to assess the long-term fate of HSPCs into mature cells, but some fates occur earlier than others36, potentially biasing the analysis toward fewer fates. In addition, these culture systems usually miss information about divisions during fate commitment. The first commitment steps have been shown to occur as early as at the start of the culture, sometimes without division26,37, making short term culture and tracking division essential to study early fate commitment. By simultaneously following the fate, division, and kinship, this assay allows to understand the role of the first division and fate decision in human HSPCs. Using the assay, it is possible to infer after how many divisions the commitment process occurs, the balance between self-renewal and differentiation for those early progenitors, and how those properties are inherited across generations. To our knowledge, this is the only assay that allows these types of measurements for human HSPCs, at single-cell resolution. In addition, using different combinations of cell division dyes, we increased the throughput of the analysis, making this assay a valuable tool for generating large datasets quickly. The dye combinations allow to follow several families in the same wells, increasing the number of cells available for analysis in short-term culture. The number of combinations could potentially be increased even more, via the addition of other dyes (e.g., yellow dye) or modifying the ratio of CFSE and CTV. However this reduces the number of other parameters that can be analyzed.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64918/64918fig06.jpg" /> 
Figure 6: Schematic representation of the protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64918/64918fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To perform the analysis successfully, due to the large number of wells and the reduced number of cells to analyze, it is necessary to run the flow cytometry analysis on an analyzer equipped with a plate reader. The new generation of bench analyzers is particularly adapted to this assay, as most of them have a smaller dead volume to reduce the percentage of cell loss. This in turn guarantees a higher efficiency in recovering the entirety of each well, prompting an efficiency estimated in the 70% range26. Estimating the cell loss during the flow cytometry acquisition is crucial for the analysis of each individual family. For example, assuming no cell death and counting the number of divisions, it is possible to estimate the number of cells per each family. Nevertheless, it is desirable to run some confirmatory experiments, particularly in the estimation of cell death in the tested culture conditions and measuring the recovery rate experimentally using a defined numbers of cells.
One of the crucial steps of this protocol is the peak assignment. As already mentioned, a good quality peak distribution is strongly dependent from the isolation of very narrow peaks at cell sorting. Nevertheless, it is still difficult to assign the correct number of divisions based uniquely on the distribution. As cell sorting and flow cytometry analysis are performed on two different machines, it is not possible to directly compare the intensity of each signal, so it could be difficult to know if the first peak observed on the right end of the histogram is peak 0 or peak 1. In this regard, few solutions are possible; one way is to perform an orthogonal experiment to accurately measure the number of divisions performed by these cells (e.g., live-cell imaging). Another possibility is to simply count the number of cells in the well under an inverted brightfield microscope, before running the flow cytometry analysis. This will infer an average number of divisions (assuming no cell death). Finally, a post-hoc solution for peak assignment is the detection of an unusual number of &#34;impossible families&#34;; these families are composed of a larger than possible number of cells per generation (e.g., five cells in generation 2, or two cells in generation 1 and one cell in generation 2). The possibility to exclude impossible families is coded in the statistical analysis step, and flags the impossible family. If the occurrence of these errors is too high, it is reasonable to assume that the peak assignment needs to be revised.
In this protocol, we included a few examples of data representation and analysis for the assay, as this has become an essential step in the generation and interpretation of large datasets38. The first example is the heatmap showing the totality of all analyzed cells, organized per family. This is an efficient tool to explore the general properties of the data and potential conclusions: are families composed by multiple cell types or do they tend to be homogeneous in composition? Are families spread over multiple generations, or are they mostly dividing the same number of times? This exploratory analysis needs to then be complemented with more specific plots and statistical testing. It can be used to quantitatively assess symmetric and asymmetric fate commitment, differentiation without division, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. It is fundamental, during the experimental planning, to set the cell culture length accordingly to the type of question asked; for example, for the first two questions (symmetric/asymmetric balance and differentiation without division), planning very short cultures steps allows the isolation of a large number of families that have performed only one or no divisions at all26. Conversely, longer experiments allow the exploration of the number of divisions required for a specific cell commitment, as they sample families at different stages of differentiation. Nevertheless, this method is not designed for long-term cultures (2-3 weeks), as cell dye dilution is not able to accurately track more than seven or eight divisions22. As a consequence, this tool is mostly adapted to study the early commitment of hematopoietic progenitors, and is not designed to make robust conclusions on the long term differentiation properties of these cells.
The statistical framework was developed specifically for the analysis of this type of data and based on the concept of permutations26. This was necessary because of the observation of a familial dependance on the cell type distribution and on the number of divisions performed. In other words, cells that are part of the same family are also more likely to display similar phenotypes and divide the same number of times. While an in-depth analysis is beyond the scope of this work, the provided set of statistical tests should be sufficient when assessing different conditions.
In conclusion, this protocol constitutes a valuable tool for assessing the cellular dynamics of hematopoietic stem and progenitor cells ex vivo, in a rapid and inexpensive way. Due to its flexibility and versatility regarding time point, culture conditions, and type of HSPCs analyzed, it allows to test a variety of experimental conditions. As a flow cytometry-based assay, it can be implemented in most labs, and it does not require extensive prior knowledge, making it a good candidate for screenings and pilot experiments.

The past decade was marked by the worldwide spreading of single-cell approaches to cellular and molecular biology. Following in the steps of single-cell genomics1,2, nowadays it is possible to study many components of a single cell (e.g., DNA, RNA, proteins), with novel single cell -omics techniques burgeoning every year. These techniques have shed light on old and new questions for the fields of immunology, neurobiology, oncology, and others, both using human and model organism cells3. By highlighting the differences between individual cells, single cell -omics prompted the definition of a new model of hematopoiesis, centered on the heterogeneity of hematopoietic stem and progenitor cells (HSPCs) and moving away from the classical model of discrete homogeneous populations4,5.
One of the few drawbacks of all the -omics techniques is the destruction of the cell of interest, precluding the possibility to assess its functionality. Conversely, other single-cell methods, such as single-cell transplantation assay and lineage tracing technologies, provide a readout of the functionality of the ancestor cell by assessing the fate of individual cells in vivo6,7. Lineage tracing technologies involves labeling the cell of interest with a heritable genetic7 or a fluorescent label8,9, allowing the fate of multiple single cells to be followed at the same time. However, the characterization of the starting cells is typically limited to a restricted number of parameters, such as the expression of a few surface proteins assessed by flow cytometry10. In addition, single-cell lineage tracing technologies require laborious detection of the cellular label, typically via DNA/RNA sequencing or imaging. This last point in particular limits the number of conditions that can be tested in a single experiment.
Another class of methods that are used to study the functionality of single cells are ex vivo cell culturing systems of single HSPCs. Easy to perform, these gold standard assays involve the sorting of individual cells into 96-wells cell culture vessels, and after culture, characterizing the cell progeny phenotype, typically by flow cytometry or morphological analysis. These assays have mostly been used to characterize the long-term differentiation of HSPCs into mature cells, typically after 2-3 weeks of culture11,12. Alternatively, they have been used to try to maintain and expand ex vivo HSPCs13,14,15,16,17,18, with the promise of medical benefit for human stem cell transplantation19. Lastly, they have been used to study the early commitment of HSPCs using short-term culture20, with the low number of cells generated in this culture being the main limiting factor. One drawback of these different kinds of ex vivo assays is that they only partially reflect the in vivo complexity; still, they are one of the rare ways to study human HSPC differentiation.
One missing piece of information from existing single-cell methods (single cell-omics, lineage tracing, and ex vivo culture) is the accurate detection of cell divisions, an essential parameter to consider when studying HSPC dynamics21. A simple way to assess the number of divisions via flow cytometry is the use of soluble &#34;protein dyes&#34;, like the 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE)22. These division dyes diffuse inside the cytoplasm of the stained cells, and diluted by half and passed to the two daughter cells at every cell division, allowing to enumerate up to 10 divisions. Combining several division dyes, it is possible to seed multiple individual progenitors in the same well, as each individual dye allows separation of the different descendants. This is the principle behind the use of cell dyes for multiplex clonal and division-tracking that was first introduced for murine lymphocytes23,24.
Here, we present the development of the MultiGen assay for use with murine and human HSPCs. It allows the testing of many single cells simultaneously for their properties of differentiation, division, and kinship ex vivo. This high-throughput, easy to perform, and inexpensive assay allows to measure the cellular phenotype, the number of divisions performed, and the cell kinship and clonal relationship with the other cells in the well, all at the same time. It can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions necessary for a given commitment fate. The protocol requires a fluorescence activated cell sorter (FACS) and a flow cytometer with a plate reader, plus the equipment necessary to perform cell culture. In addition to the technical protocol for execution of the assay on human HSPCs, we also provide the detailed analysis framework, including the statistical testing necessary to assess cellular properties related to the concept of cell family25. This protocol has already been used successfully to describe the murine HSPC compartment26,27.
The following protocol uses magnetically enriched CD34+ cells as the starting material28. In this way, it is possible to efficiently stain and isolate the human HSPCs from different blood sources (e.g., cord blood, bone marrow, peripheral blood). It is important not to discard the CD34- fraction, as it will be used as part of the protocol to set different types of experimental controls. The mentioned cell amounts and volumes can be scaled up or down, according to the experimental workflow and necessities. Similarly, the protocol can be adapted to the study of different types of progenitors, simply by modifying the antibodies used for the cell sorting and flow cytometry steps.

<table><tbody><tr><td>1.5 mL polypropylene microcentrifuge tubes</td><td>vWR</td><td>87003-294</td><td></td></tr><tr><td>15-mL polypropylene tubes</td><td>vWR</td><td>734-0451</td><td></td></tr><tr><td>50-mL polypropylene tubes</td><td>vWR</td><td>734-0448</td><td></td></tr><tr><td>96-well U-bottom culture plate&amp;nbsp;</td><td>Falcon</td><td>353077</td><td></td></tr><tr><td>Anti-human Lin APC</td><td>Thermo Fisher</td><td>22-7776-72</td><td>Dilution 1/40</td></tr><tr><td>ARIA III</td><td>BD</td><td></td><td>Can be replaced with any FACS sorter able to sort individual cells in 96-wells plate</td></tr><tr><td>Carboxyfluorescein succinimidyl ester (CFSE)</td><td>Life Technologies</td><td>C34570</td><td></td></tr><tr><td>Cell Trace Violet (CTV)&amp;nbsp;</td><td>Life Technologies</td><td>C34571</td><td></td></tr><tr><td>Compensation beads&amp;nbsp;</td><td>BD</td><td>552843</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium (DMEM)</td><td>Life Technologies</td><td>11320033</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Thermo Scientific</td><td>J62948-36</td><td>Prepare a solution 0.5 M, in sterilised water</td></tr><tr><td>FC block Fc1.3216</td><td>BD</td><td>564220</td><td>Dilution 1/50</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Dutscher</td><td>S1900-500C</td><td>Batch S00CH</td></tr><tr><td>FlowJo v10.8.1</td><td>BD</td><td></td><td></td></tr><tr><td>Mouse anti-human CD10 PerCP-5.5, clone HI10a</td><td>Biolegend</td><td>312216</td><td>Dilution 1/20</td></tr><tr><td>Mouse anti-human CD123 BUV395, clone 7G3</td><td>BD</td><td>564195</td><td>Dilution 1/15</td></tr><tr><td>Mouse anti-human CD34 APC-Cy7, clone 581</td><td>Biolegend</td><td>343513</td><td>Dilution 1/40</td></tr><tr><td>Mouse anti-human CD38 BV650, clone HB7</td><td>Biolegend</td><td>356620</td><td>Dilution 1/40</td></tr><tr><td>Mouse anti-human CD45RA AF700, clone HI100</td><td>BD</td><td>560673</td><td>Dilution 1/20</td></tr><tr><td>Mouse anti-human CD45RA PE-Cy7, clone HI100</td><td>BD</td><td>560675</td><td>Dilution 1/20</td></tr><tr><td>Mouse anti-human CD90 PE, clone 5E10</td><td>Biolegend</td><td>328110</td><td>Dilution 1/20</td></tr><tr><td>Phosphate Buffered Saline (PBS) 1X</td><td>Life Technologies</td><td>10010001</td><td></td></tr><tr><td>Python</td><td></td><td></td><td></td></tr><tr><td>R</td><td></td><td></td><td></td></tr><tr><td>Sterile 12x75 mm conical polypropylene tubes</td><td>Falcon</td><td></td><td></td></tr><tr><td>ZE5&amp;nbsp;</td><td>Biorad</td><td></td><td>Can be replaced with any flow cytometry analyzer equipped with a plate reader</td></tr><tr><td>&lt;strong&gt;Laboratory prepared&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell culture media</td><td></td><td></td><td>Depends from the specific experiment. Prepare fresh daily and store at +4 &amp;deg;C until use</td></tr><tr><td>DMEM + 10% FBS</td><td></td><td></td><td>Can be stored in sterile conditions, at +4 &amp;deg;C up to 1 year. To prepare 500 mL, add 50 mL of FBS to 450 mL DMEM</td></tr><tr><td>PBS 1X + EDTA 0.1%</td><td></td><td></td><td>Can be stored in sterile conditions, at room temperature, up to 1 year. To prepare 500 mL, add 3.42 mL of EDTA 0.5 M to 500 mL PBS 1X</td></tr><tr><td>Staining buffer</td><td></td><td></td><td>Can be stored in sterile conditions, at +4 &amp;deg;C up to 1 year. To prepare 500 mL, add 2 mL of EDTA 0.5 M and 1 mL FBS to 500 mL PBS 1X</td></tr></tbody></table>

simultaneous assessment of kinship, division number, and phenotype via flow cytometry for hematopoietic stem and progenitor cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Watch this Scientific Journal Video about Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells at JoVE.com

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

simultaneous-assessment-kinship-division-number-phenotype-via-flow

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.5K Views.  Institut Curie. Weinig methoden maken het mogelijk om snel de functionaliteit van hematopoietische voorlopercellen te beoordelen. Dit protocol meet de impact van de eerste celdelingen op het lot en de inzet van cellen. Dit protocol maakt het mogelijk om gelijktijdig deling en differentiatie te meten op eencellig niveau en met een hoge doorvoer zonder complexe en uitgebreide manipulatie te vereisen.Deze methode is geschikt voor elke hematopoietische populatie die in cultuur kan worden gehouden. Het kan...

Video: Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

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 ...

Cancer Research

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Genetics

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Developmental Biology

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Biology

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

Bioengineering

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

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