Table 1 |.

Genetic tools to track HSC activity

Tool	Description	Evidence	Refs.
Retrovirus-mediated gene transfer	Donor haematopoietic cells marked by retrovirus-mediated gene transfer are tracked following transplantation into recipient mice	The first definitive evidence of HSC multipotency	29
CD45 alleles	HSCs from a donor mouse are transplanted into recipient mice each carrying a different allele of the pan-leukocyte marker CD45 (either CD45.1 or CD45.2). These isoforms are detected using monoclonal antibodies. Clonal analysis is possible after single cell transplantation	The most widely used system for tracking HSC activity following transplantation, in bulk and clonal assays	9,30
Fluorescent protein transgenesa	Transgenic donor mice are engineered to constitutively express fluorescent protein(s) within haematopoietic cells, thereby enabling assessment of platelet, erythrocyte and leukocyte chimerism. Clonal analysis is possible after single cell transplantation	Assessment of platelet and erythrocyte lineages led to the identification of lineage biases and lineage-restricted cell types	13,34,35
Retroviral or lentiviral barcoding	HSCs are transduced ex vivo with a barcode derived from a retroviral or lentiviral library. Barcodes are detected by sequencing at multiple time points to track the HSCs over time	These methods enabled reconstitution dynamics of numerous HSCs to be tracked within a single recipient	39–41
In vivo lineage tracing	HSCs are barcoded and genetically tagged in vivo, e.g. using inducible CreER recombination. The Polylox system enables single HSCs to be individually tagged by one of several hundred thousand barcodes	These experiments found that adult HSCs play a minor role in day-to-day haematopoiesis at steady-state	43,51
Confetti and rainbow labelling	In vivo Cre recombination ‘shuffles’ a cassette of fluorescent protein genes, such that different clones express different fluorescent proteins. Although the number of distinguishable fluorescent proteins limits the number of clones that can be traced, the expression of fluorescent proteins enables prospective identification of clones	These methods have facilitated molecular studies of in vivo intraclonal HSC heterogeneity, including at the genetic and epigenetic levels	48,49
Transposon-based lineage tracing	HSB transposase labels HSCs clonally in situ	These methods identified HPCs rather than HSCs as major drivers of day-to-day hematopoiesis at steady-state	42
Dye-dilution assays	Transient expression of a histone H2B tagged with GFP (H2B-GFP) labels dormant HSCs, enabling assessment of their cell division kinetics throughout life	Functional HSCs retained the H2B-GFP label in vivo for >22 months, confirming their dormant state	50
Whole genome sequencing	Rare somatic mutations within an individual’s haematopoietic system can predict HSC activity longitudinally in native haematopoiesis	Modelling of these datasets allowed population dynamics of the human HSC compartment to be estimated	63

^aSuch as Kusabira orange or green fluorescent protein (GFP). HPC, haematopoietic progenitor cell; HSB, hyperactive Sleeping Beauty; HSC, haematopoietic stem cell.