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• W3045603275 abstract "•Clonal lineage tracing approaches for studying hematopoiesis are described.•Single-cell transplantation assays and lentiviral barcoding technologies are discussed.•Sleeping Beauty barcoding for studying cell fate in native hematopoiesis is covered. The coordinated differentiation of hematopoietic stem and progenitor cells (HSPCs) into the various mature blood cell types is responsible for sustaining blood and immune system homeostasis. The cell fate decisions underlying this important biological process are made at the level of single cells. Methods to trace the fate of single cells are therefore essential for understanding hematopoietic system activity in health and disease and have had a major impact on how we understand and represent hematopoiesis. Here, we discuss the basic methodologies and technical considerations for three important clonal assays: single-cell transplantation, lentiviral barcoding, and Sleeping Beauty barcoding. This perspective is a synthesis of presentations and discussions from the 2019 International Society for Experimental Hematology (ISEH) Annual Meeting New Investigator Technology Session and the 2019 ISEH Winter Webinar. The coordinated differentiation of hematopoietic stem and progenitor cells (HSPCs) into the various mature blood cell types is responsible for sustaining blood and immune system homeostasis. The cell fate decisions underlying this important biological process are made at the level of single cells. Methods to trace the fate of single cells are therefore essential for understanding hematopoietic system activity in health and disease and have had a major impact on how we understand and represent hematopoiesis. Here, we discuss the basic methodologies and technical considerations for three important clonal assays: single-cell transplantation, lentiviral barcoding, and Sleeping Beauty barcoding. This perspective is a synthesis of presentations and discussions from the 2019 International Society for Experimental Hematology (ISEH) Annual Meeting New Investigator Technology Session and the 2019 ISEH Winter Webinar. Hematopoietic stem and progenitor cells (HSPCs) are responsible for the sustained production of mature blood cells during homeostasis and in response to hematopoietic stresses [1Seita J Weissman IL Hematopoietic stem cell: self-renewal versus differentiation.Wiley Interdiscip Rev Syst Biol Med. 2010; 2: 640-653Crossref PubMed Scopus (474) Google Scholar,2Eaves CJ Hematopoietic stem cells: concepts, definitions, and the new reality.Blood. 2015; 125: 2605-2613Crossref PubMed Scopus (304) Google Scholar]. Aberrant HSPC activity is also linked to a spectrum of hematological diseases, from clonal hematopoiesis to leukemias [3Luis TC Wilkinson AC Beerman I Jaiswal S Shlush LI Biological implications of clonal hematopoiesis.Exp Hematol. 2019; 77: 1-5Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 4Steensma DP Ebert BL Clonal hematopoiesis as a model for premalignant changes during aging.Exp Hematol. 2020; 83: 48-56Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 5Swierczek S Prchal JT Clonal hematopoiesis in hematological disorders: three different scenarios.Exp Hematol. 2020; 83: 57-65Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 6Horton SJ Huntly BJ Recent advances in acute myeloid leukemia stem cell biology.Haematologica. 2012; 97: 966-974Crossref PubMed Scopus (100) Google Scholar]. The classic models of hematopoiesis predict the existence of a homogeneous pool of multipotent and self-renewing hematopoietic stem cells (HSCs), which are equally contributing to the balanced production of all blood cell lineages through progressive lineage differentiation [7Laurenti E Göttgens B From haematopoietic stem cells to complex differentiation landscapes.Nature. 2018; 553: 418-426Crossref PubMed Scopus (307) Google Scholar,8Haas S Trumpp A Milsom MD Causes and consequences of hematopoietic stem cell heterogeneity.Cell Stem Cell. 2018; 22: 627-638Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar]. However, technology advances in the isolation of purified HSPC subsets and the capacity to investigate the behavior of single HSCs have revealed a high level of heterogeneity in their kinetics and patterns of reconstitution [1Seita J Weissman IL Hematopoietic stem cell: self-renewal versus differentiation.Wiley Interdiscip Rev Syst Biol Med. 2010; 2: 640-653Crossref PubMed Scopus (474) Google Scholar,2Eaves CJ Hematopoietic stem cells: concepts, definitions, and the new reality.Blood. 2015; 125: 2605-2613Crossref PubMed Scopus (304) Google Scholar,9Loughran S Haas S Wilkinson A Klein A Brand M Lineage commitment of hematopoietic stem cells and progenitors: insights from recent single cell and lineage tracing technologies.Exp Hematol. 2020 Jul 9; ([Online ahead of print.])https://doi.org/10.1016/j.exphem.2020.07.002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar]. This heterogeneity is reflected in variable degrees of self-renewal potential and distinct propensity to differentiate toward the different blood cell lineages. Accumulating evidence suggests that only a fraction of mouse HSCs generate balanced multilineage output. Instead, HSCs with biased and restricted lineage potentials have been identified [7Laurenti E Göttgens B From haematopoietic stem cells to complex differentiation landscapes.Nature. 2018; 553: 418-426Crossref PubMed Scopus (307) Google Scholar,8Haas S Trumpp A Milsom MD Causes and consequences of hematopoietic stem cell heterogeneity.Cell Stem Cell. 2018; 22: 627-638Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar,10Yamamoto R Wilkinson AC Nakauchi H Changing concepts in hematopoietic stem cells.Science. 2018; 362: 895-896Crossref PubMed Scopus (23) Google Scholar]. As the decision between self-renewal and differentiation is made by individual HSCs, examination of these processes requires analyses at the clonal level. In this perspective, we introduce and discuss the technical aspects of three powerful methodologies for single-cell lineage tracing studies in hematology research: single-cell transplantation, lentiviral barcoding, and Sleeping Beauty barcoding. This perspective represents a synthesis of presentations and discussions from the 2019 International Society for Experimental Hematology (ISEH) Annual Meeting New Investigator Technology Session and the 2019 ISEH Winter Webinar by Dr. Joana Carrelha, Dr. Dawn Lin, and Dr. Alejo Rodriguez-Fraticelli. The webinar is also available to watch online (https://www.iseh.org/page/ISEHWebinars). In vivo transplantation is a long-standing gold-standard technique for the study of self-renewal and differentiation of HSCs [11Weissman IL Shizuru JA. The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases.Blood. 2008 July 9; ([Online ahead of print.])https://doi.org/10.1182/blood-2008-08-078220Crossref Scopus (289) Google Scholar,12Wilkinson AC Igarashi KJ Nakauchi H Haematopoietic stem cell self-renewal in vivo and ex vivo.Nat Rev Genet. 2020 May 28; ([Online ahead of print.])https://doi.org/10.1038/s41576-020-0241-0Crossref PubMed Scopus (47) Google Scholar]. HSC frequency can be quantified with the limiting dilution transplantation assay (LDTA) [13Spangrude GJ Heimfeld S Weissman IL Purification and characterization of mouse hematopoietic stem cells.Science. 1988; 241: 58-62Crossref PubMed Scopus (2188) Google Scholar, 14Abramson S Miller RG Phillips RA The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems.J Exp Med. 1977; 145: 1567-1579Crossref PubMed Scopus (367) Google Scholar, 15Smith LG Weissman IL Heimfeld S Clonal analysis of hematopoietic stem-cell differentiation in vivo.Proc Natl Acad Sci USA. 1991; 88: 2788-2792Crossref PubMed Scopus (289) Google Scholar], which does not require flow cytometry or cell sorting. However, a more detailed analysis of the functional heterogeneity of HSCs has been achieved by sorting and transplanting highly purified single cells [15Smith LG Weissman IL Heimfeld S Clonal analysis of hematopoietic stem-cell differentiation in vivo.Proc Natl Acad Sci USA. 1991; 88: 2788-2792Crossref PubMed Scopus (289) Google Scholar,16Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242-245Crossref PubMed Scopus (1693) Google Scholar]. Large-scale studies of the in vivo outputs of single transplanted bone marrow (BM) HSCs [15Smith LG Weissman IL Heimfeld S Clonal analysis of hematopoietic stem-cell differentiation in vivo.Proc Natl Acad Sci USA. 1991; 88: 2788-2792Crossref PubMed Scopus (289) Google Scholar, 16Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242-245Crossref PubMed Scopus (1693) Google Scholar, 17Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar] have recently confirmed that the phenotypically defined HSC pool is substantially heterogeneous, but have also found that there is a specific number of distinct long-term engrafting HSC subtypes. In contrast to all conceivable stochastic variation, only a limited number of sustained lineage-biased and lineage-restricted HSC subtypes are actually observable upon transplantation, in addition to the classically defined multilineage HSCs [17Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 18Yamamoto R Wilkinson AC Ooehara J et al.Large-scale clonal analysis resolves aging of the mouse hematopoietic stem cell compartment.Cell Stem Cell. 2018; 22 (600–607.e604)Abstract Full Text Full Text PDF Scopus (80) Google Scholar, 19Carrelha J Meng Y Kettyle LM et al.Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.Nature. 2018; 554: 106-111Crossref PubMed Scopus (166) Google Scholar, 20Morita Y Ema H Nakauchi H Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.J Exp Med. 2010; 207: 1173-1182Crossref PubMed Scopus (300) Google Scholar, 21Sieburg HB Cho RH Dykstra B Uchida N Eaves CJ Müller-Sieburg CE The hematopoietic stem compartment consists of a limited number of discrete stem cell subsets.Blood. 2006; 107: 2311-2316Crossref PubMed Scopus (170) Google Scholar, 22Copley MR Beer PA Eaves CJ Hematopoietic stem cell heterogeneity takes center stage.Cell Stem Cell. 2012; 10: 690-697Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 23Benz C Copley MR Kent DG et al.Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs.Cell Stem Cell. 2012; 10: 273-283Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 24Müller-Sieburg CE Cho RH Karlsson L Huang JF Sieburg HB Myeloid-biased hematopoietic stem cells have extensive self-renewal capacity but generate diminished lymphoid progeny with impaired IL-7 responsiveness.Blood. 2004; 103: 4111-4118Crossref PubMed Scopus (178) Google Scholar]. Transplantation from adult mouse BM consists of five main steps (Figure 1): (1) BM harvest from donor mice, (2) sorting of HSCs by fluorescence-activated cell sorting (FACS), (3) mixture with unfractionated BM support cells, (4) injection of cells into preconditioned recipient mice, and (5) analysis of donor-derived cells in the recipients. Each of the main steps of this technique can be modified to some extent to fit different model systems and different experimental questions. This technique can be used not only with adult BM HSCs, but also with fetal liver and fetal BM HSCs [23Benz C Copley MR Kent DG et al.Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs.Cell Stem Cell. 2012; 10: 273-283Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar]. Expression of distinct alleles of the pan-hematopoietic marker CD45 in donor and recipient cells [13Spangrude GJ Heimfeld S Weissman IL Purification and characterization of mouse hematopoietic stem cells.Science. 1988; 241: 58-62Crossref PubMed Scopus (2188) Google Scholar,16Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242-245Crossref PubMed Scopus (1693) Google Scholar] and/or expression of distinct fluorescent reporters [17Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar,19Carrelha J Meng Y Kettyle LM et al.Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.Nature. 2018; 554: 106-111Crossref PubMed Scopus (166) Google Scholar] allows quantification by flow cytometry of the contribution of transplanted HSCs to the hematopoietic system of the recipient mouse, at multiple time points posttransplantation. The most typical and less invasive approach is the analysis of mature lineages in serial samples of peripheral blood, but donor-derived HSPCs can also be serially analyzed through BM aspirations. Serial sampling allows flexibility in experimental decisions based on the ongoing observation of donor-derived output, before more cell-destructive methods are used for terminal analysis. Different mouse lines with inducible or constitutive phenotypes can be used as donors and recipients in HSC transplantations. For example, mutant donors can be used to assess the impact of specific mutations on HSC function, Cre reporters can be used as donors to map the contribution of transplanted HSCs to specific lineages, and immune-compromised recipients can be used to improve engraftment. Not many donor and recipient phenotypes have yet been explored in single HSC transplantation studies, but a few fluorescent reporter lines have recently been used, such as Kusabira Orange (KuO) [17Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar,18Yamamoto R Wilkinson AC Ooehara J et al.Large-scale clonal analysis resolves aging of the mouse hematopoietic stem cell compartment.Cell Stem Cell. 2018; 22 (600–607.e604)Abstract Full Text Full Text PDF Scopus (80) Google Scholar], Vwf-GFP [25Sanjuan-Pla A Macaulay IC Jensen CT et al.Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.Nature. 2013; 502: 232-236Crossref PubMed Scopus (367) Google Scholar], Vwf-Tomato/Gata1-GFP [19Carrelha J Meng Y Kettyle LM et al.Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.Nature. 2018; 554: 106-111Crossref PubMed Scopus (166) Google Scholar], and Pdzk1ip1-GFP [26Sawai CM Babovic S Upadhaya S et al.Hematopoietic stem cells are the major source of multilineage hematopoiesis in adult animals.Immunity. 2016; 45: 597-609Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar]. However, mixed genetic backgrounds and immunological compatibility issues can lead to quantitatively and qualitatively reduced engraftment or even to total premature loss of engraftment. Single HSC transplantations are particularly sensitive to this issue, and therefore, the effects of new donor/recipient combinations should be carefully assessed. To maximize cell survival and increase the frequency of reconstituted mice in single HSC transplantations assays, processing and sorting of BM samples should be as simplified as possible, and the total time between donor BM harvest and single HSC injection should be minimized. A simple processing and sorting strategy can be very efficient when using a typical phenotypic definition of HSCs (e.g., Lineage−Sca1+cKit+CD150+CD48−CD34−). However, to ensure the feasibility of specific experimental setups, for example, when dealing with very rare phenotypic HSC subfractions, extra processing steps might be required before single-cell deposition, such as magnetic sorting with microbeads (cKit enrichment or Lineage depletion) or double-sorting with flow cytometry. Pre- or postsort culture of cells with media of different compositions might also be part of the experimental objectives [27Uchida N Dykstra B Lyons KJ Leung FYK Eaves CJ Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics.Exp Hematol. 2003; 31: 1338-1347Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar,28Dykstra B Kent D Bowie M et al.Long-term propagation of distinct hematopoietic differentiation programs in vivo.Cell Stem Cell. 2007; 1: 218-229Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar]. Overall, it must be considered that extra processing steps might affect HSC survival and homing ability. The chosen protocol should always be a reasonable compromise between the experimental objectives, the sorting efficiency and purity, and the quantity and quality of engraftment in recipient mice. A sorter with an automated cell deposition unit (ACDU) is required for deposition of single cells into U-bottom or V-bottom 96-well tissue culture plates. Most sorters use 0/32/16 as the default yield/purity/phase masks for single-cell deposition, but the less strict mode of 0/32/8 can be equally accurate for low- and mid-range sample concentrations, with the advantage of a significant reduction of the number of discarded target cells. It is possible to confirm single-cell deposition into wells using a brightfield microscope. However, in this technique it is very difficult to discern if lack of engraftment of a particular sorted cell was due to biological or technical factors, such as cell disruption or cell loss within the well or syringe. Single-cell FACS allows the use of index sorting [29Wilson NK Kent DG Buettner F et al.Combined single-cell functional and gene expression analysis resolves heterogeneity within stem cell populations.Cell Stem Cell. 2015; 16: 712-724Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar], meaning that the fluorescence intensity of each marker in the staining panel can be recorded for each individual sorted cell. This information can then be correlated with the functional output observed for each cell. HSCs are most commonly transplanted into recipient mice by intravenous injection, but intra-osseous injection can also be performed if HSC homing represents a significant variable within the experimental setting [30Zhan Y Zhao Y Hematopoietic stem cell transplant in mice by intra-femoral injection.Methods Mol Biol. 2008; 430: 161-169Crossref PubMed Google Scholar]. Direct deposition into the calvarium can also be performed for the purposes of live imaging [31Turcotte R Alt C Runnels JM et al.Image-guided transplantation of single cells in the bone marrow of live animals.Sci Rep. 2017; 7: 3875Crossref PubMed Scopus (7) Google Scholar]. Whole-body lethal irradiation of wild-type recipients is the most extensively used method for transplantation of mouse HSCs. Support BM cells must be injected together with the purified HSCs, as a means to provide radioprotective progenitors that ensure survival of the lethally irradiated recipients. A common approach in single HSC transplantations is to use 2–3 × 105 unfractionated BM cells from a wild-type mouse [17Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 18Yamamoto R Wilkinson AC Ooehara J et al.Large-scale clonal analysis resolves aging of the mouse hematopoietic stem cell compartment.Cell Stem Cell. 2018; 22 (600–607.e604)Abstract Full Text Full Text PDF Scopus (80) Google Scholar, 19Carrelha J Meng Y Kettyle LM et al.Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.Nature. 2018; 554: 106-111Crossref PubMed Scopus (166) Google Scholar], but the phenotype and dosage of these support BM cells can be modified according to the experimental purposes. Sublethally irradiated W41 mice [32Geissler EN McFarland EC Russell ES Analysis of pleiotropism at the dominant White-Spotting (W) locus of the house mouse: a description of ten new W alleles.Genetics. 1981; 97: 337-361PubMed Google Scholar] (the W41 mouse strain contains a spontaneous point mutation in the Kit gene that affects hematopoiesis [33Sharma Y Astle CM Harrison DE Heterozygous Kit mutants with little or no apparent anemia exhibit large defects in overall hematopoietic stem cell function.Exp Hematol. 2007; 35: 214-220Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]) have also been used as recipients in single-HSC transplantations [27Uchida N Dykstra B Lyons KJ Leung FYK Eaves CJ Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics.Exp Hematol. 2003; 31: 1338-1347Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 28Dykstra B Kent D Bowie M et al.Long-term propagation of distinct hematopoietic differentiation programs in vivo.Cell Stem Cell. 2007; 1: 218-229Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 29Wilson NK Kent DG Buettner F et al.Combined single-cell functional and gene expression analysis resolves heterogeneity within stem cell populations.Cell Stem Cell. 2015; 16: 712-724Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar]. Different methods of preconditioning recipient mice for transplantation can be considered [34Czechowicz A Kraft D Weissman IL Bhattacharya D Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches.Science. 2007; 318: 1296-1299Crossref PubMed Scopus (297) Google Scholar], and HSC engraftment can also be achieved without preconditioning [35Waskow C Madan V Bartels S Costa C Blasig R Rodewald HR Hematopoietic stem cell transplantation without irradiation.Nat Methods. 2009; 6: 267-269Crossref PubMed Scopus (70) Google Scholar], but these methods have not been used, and might not be feasible, for single HSCs. Single-HSC transplantation can require the use of large cohorts of recipient mice to obtain robust data. The exact number required depends on the experimental questions and, most importantly, the engraftment frequency obtained with a specific experimental setup. In general, the frequency of long-term engraftment of single adult mouse BM HSCs ranges from 30% to 60% [17Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar,19Carrelha J Meng Y Kettyle LM et al.Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.Nature. 2018; 554: 106-111Crossref PubMed Scopus (166) Google Scholar,36Kiel MJ Yilmaz OH Iwashita T Terhorst C Morrison SJ SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2357) Google Scholar]. Flow cytometry analysis of the output of engrafting HSCs is mostly unambiguous, but especially in single-HSC transplantations it is inevitable that some HSCs with extremely low levels of contribution might be misidentified as non-engrafting. Appropriate detection thresholds must be set for the analysis of the donor contribution to each lineage of interest. The background fluorescence levels of multiple non-transplanted control mice with the same phenotype as the experimental recipients represents a good measure for setting such a threshold. Identification of donor-derived cells based on positive markers (e.g., donor-derived cells are GFP+ in a GFP− recipient) is typically a more sensitive and accurate approach than the opposite (e.g., donor-derived cells are GFP− in a GFP+ recipient). Importantly, blood platelets often adhere to other cells during tissue processing. Although it might be impossible to completely avoid this problem, in blood leukocytes it can be reduced by taking steps such as collection of samples into heparin-treated or EDTA-treated tubes, separation of platelets from leukocytes by centrifugation, ammonium chloride incubation, Dextran-500 incubation, and increased percentages of serum and EDTA in the preparation media. When using as HSC donor a fluorescent reporter that labels platelets and also other cell types, it is important to be able to distinguish, for each population, the real donor-derived fluorescent signal of that population from the fluorescence signal of donor-derived platelets attached to non-donor-derived cells. Platelets can be identified in the flow cytometry analysis of other cell types by inclusion of platelet markers like CD41. Furthermore, the intensity of fluorescent reporter signals tends to be lower in platelets, and so limiting gating to cells with high fluorescence intensity can help to exclude platelet contamination from the analysis. The donor-derived compartments in each recipient of a single HSC encapsulate the activity of that single engrafting HSC, and thus, the properties of different HSC subtypes can be characterized. Donor-derived cells can be further tested in situ in particular experimental setups, but most commonly they can be sorted from multiple tissues of the recipient mice and tested with in vitro assays and in vivo serial transplantation assays. Generally, 16 weeks post-transplantation is considered to be the minimum time required to identify long-term HSC engraftment in a primary transplantation setting. However, even longer times, and additionally serial transplantation, might be required to study true long-term HSCs [37Benveniste P Frelin C Janmohamed S et al.Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential.Cell Stem Cell. 2010; 6: 48-58Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar]. Although it is not a high-throughput approach, single-HSC transplantation is a powerful method to study functional heterogeneity (Table 1). It allows quantification and qualification of the in vivo outputs of single HSCs at serial time points, and it can be flexibly combined with additional fate mapping techniques and multiple readout methods. This approach has been used to confirm improved purification of engrafting HSCs when using modified phenotypic definitions [29Wilson NK Kent DG Buettner F et al.Combined single-cell functional and gene expression analysis resolves heterogeneity within stem cell populations.Cell Stem Cell. 2015; 16: 712-724Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar] or new reporters [26Sawai CM Babovic S Upadhaya S et al.Hematopoietic stem cells are the major source of multilineage hematopoiesis in adult animals.Immunity. 2016; 45: 597-609Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar]. It has also allowed the detailed description of lineage-biased and lineage-restricted subsets of long-term engrafting HSCs that deviate from the classic definition of multilineage HSC [10Yamamoto R Wilkinson AC Nakauchi H Changing concepts in hematopoietic stem cells.Science. 2018; 362: 895-896Crossref PubMed Scopus (23) Google Scholar,17Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 18Yamamoto R Wilkinson AC Ooehara J et al.Large-scale clonal analysis resolves aging of the mouse hematopoietic stem cell compartment.Cell Stem Cell. 2018; 22 (600–607.e604)Abstract Full Text Full Text PDF Scopus (80) Google Scholar, 19Carrelha J Meng Y Kettyle LM et al.Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.Nature. 2018; 554: 106-111Crossref PubMed Scopus (166) Google Scholar]. Specific markers for the prospective purification of these lineage-biased and lineage-restricted HSC subsets are yet to be identified, and therefore, single HSC transplantation continues to be an important method of HSC isolation based on functional output, and it will continue to play a role in the study of the regulation of different HSC subtypes in normal and stress hematopoiesis. It is important to note that, even though transplantation assays reveal important information on the properties of HSCs, what is observed on transplantation might not reflect the typical output of HSCs in unperturbed hematopoiesis. To enhance our understanding of the functional heterogeneity and regulation of the HSC compartment, it is essential to combine observations from techniques that assay HSC behavior in both the transplantation setting and the native setting.Table 1Comparison of clonal methodsSingle-cell transplantationLentiviral barcodingEndogenous Sleeping Beauty barcodingRequirement for ex vivo manipulationYesYesNo" @default.
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• W3045603275 title "Single-cell lineage tracing approaches in hematology research: technical considerations" @default.
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• W3045603275 doi "https://doi.org/10.1016/j.exphem.2020.07.007" @default.
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