Name: simultaneous assessment of kinship, division number, and phenotype via flow cytometry for hematopoietic stem and progenitor cells
Uploaded: 2023-03-24T13:40:00
Duration: 10 min 20 s
Description: 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

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/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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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)&#160;</td>
			<td>Life Technologies</td>
			<td>C34571</td>
			<td></td>
		</tr>
		<tr>
			<td>Compensation beads&#160;</td>
			<td>BD</td>
			<td>552843</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;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&#160;</td>
			<td>Biorad</td>
			<td></td>
			<td>Can be replaced with any flow cytometry analyzer equipped with a plate reader</td>
		</tr>
		<tr>
			<td>Laboratory prepared</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 &#176;C until use</td>
		</tr>
		<tr>
			<td>DMEM + 10% FBS</td>
			<td></td>
			<td></td>
			<td>Can be stored in sterile conditions, at +4 &#176;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 &#176;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 ...

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