Summary of multiple approaches to study the frequency and dynamics of HSC specification in mice, zebrafish, and humans
| Method . | Organism . | N of HSPC/HSPC precursors specified* . | Advantages . | Limitations . | Equipment/technical requirements . | References . |
|---|---|---|---|---|---|---|
| IAC cluster cartography | Mouse | 609 ± 84 c-Kit+ cells/E10.5 embryo 439 ± 87 c-Kit+ cells/E11.5 embryo | Direct morphologic evaluation; flexibility on use of additional markers | Lack of functional validation; single time point snapshot | Histology equipment; antibodies; confocal microscopy | 80 |
| Transplantation of fresh tissues | Mouse | 1-2 RUs/embryo at E10.5-E11.5; 60 RUs/ee at E12.5 | Well established; statistically robust and simple (Poisson statistics); commercial software available for LDA analysis | Does not score immature HSC precursors | Requires large cohorts of mice as transplant recipients; flow cytometry | 7,8,68,82,86 |
| Transplantation of cultured explants | Mouse | 60 RUs/ee between E9.5 and E12.5 | Allows for quantification of immature HSC precursor potential at early developmental time points (E8.5-E10.5) in murine embryos | Native development perturbed | Requires large cohorts of mice as transplant recipients; flow cytometry; explant culture | 94-98 |
| Marking of single blood precursors | Mouse | 333 E7.5-E8.5 blood precursors (Abcg2-CreERT2; Rosa26Lox-STOP-Lox-YFP) | Native development is preserved; simple math that assumes observed blood labeling is output of single precursor from which total n of precursors is extrapolated | Difficult to verify single precursor labeling; cannot determine upper bound for n of precursors | Abcg2-CreERT2; Rosa26Lox-STOP-Lox-YFP mouse model; single group of mice (≥5 mice); flow cytometry | 101 |
| MtMV of Confetti-reporter labeling | Mouse | 600 blood precursors (E7.5-E12.5) | Native development is preserved; quantitative; simple genetic mouse models; statistically robust and simple; based on Poisson statistics | Individual clones cannot be tracked; requires >3% of Confetti labeling and >500 cells examined; quantitative range is limited; commercial software for analyses not available | Cre recombinase mouse models; single group of mice (≥5 mice); flow cytometry with 405, 445, 488, 562, and 640 nm and specific band-pass filter configurations to discriminate Confetti colors | 17,92 |
| Zebrabow | Zebrafish | 20-26 lifelong precursors | Native development is preserved | Reliant on hue and saturation to identify individual clones | Driver and effector transgenic lines; color-clustering computational analysis platform; flow cytometry; confocal and inverted microscope | 11 |
| Hsp701 LASER induction | Zebrafish | 30 lifelong precursors | Native development is preserved | Reliant on labeling 1 cell, otherwise individual clones cannot be tracked | Micropoint microscope-targeted 440-nm laser; driver and effector transgenic lines; flow cytometry; confocal and inverted microscopes | 11 |
| Transposase-mediated tagging | Mouse | Not yet employed | Native development is preserved; single clones can be tracked; lineage relationships inferred | Complex genetic mouse model; bioinformatics expertise required to interpret data | Targeted sequencing; bioinformatics expertise | 104,105 |
| PolyLox-based barcoding | Mouse | Not yet employed | Native development is preserved; single clones can be tracked; lineage relationships inferred | Complex genetic mouse model; bioinformatics expertise required to interpret data; need to eliminate common rearrangements | Targeted sequencing; bioinformatics expertise | 112 |
| CRISPR-based barcoding | Mouse | Not yet employed | Native development is preserved; single clones can be tracked; lineage relationships inferred | Complex genetic mouse model; bioinformatics expertise required to interpret data | Targeted sequencing; bioinformatics expertise | 113-115 |
| Somatic mutations | Human | Native development is preserved | Costs limit n of analyzed clones; potential for confounding mutations during cell expansion; biased for sequencing clones able to expand in vitro; complex bioinformatics analyses | WGS platforms; bioinformatics expertise | 118 |
| Method . | Organism . | N of HSPC/HSPC precursors specified* . | Advantages . | Limitations . | Equipment/technical requirements . | References . |
|---|---|---|---|---|---|---|
| IAC cluster cartography | Mouse | 609 ± 84 c-Kit+ cells/E10.5 embryo 439 ± 87 c-Kit+ cells/E11.5 embryo | Direct morphologic evaluation; flexibility on use of additional markers | Lack of functional validation; single time point snapshot | Histology equipment; antibodies; confocal microscopy | 80 |
| Transplantation of fresh tissues | Mouse | 1-2 RUs/embryo at E10.5-E11.5; 60 RUs/ee at E12.5 | Well established; statistically robust and simple (Poisson statistics); commercial software available for LDA analysis | Does not score immature HSC precursors | Requires large cohorts of mice as transplant recipients; flow cytometry | 7,8,68,82,86 |
| Transplantation of cultured explants | Mouse | 60 RUs/ee between E9.5 and E12.5 | Allows for quantification of immature HSC precursor potential at early developmental time points (E8.5-E10.5) in murine embryos | Native development perturbed | Requires large cohorts of mice as transplant recipients; flow cytometry; explant culture | 94-98 |
| Marking of single blood precursors | Mouse | 333 E7.5-E8.5 blood precursors (Abcg2-CreERT2; Rosa26Lox-STOP-Lox-YFP) | Native development is preserved; simple math that assumes observed blood labeling is output of single precursor from which total n of precursors is extrapolated | Difficult to verify single precursor labeling; cannot determine upper bound for n of precursors | Abcg2-CreERT2; Rosa26Lox-STOP-Lox-YFP mouse model; single group of mice (≥5 mice); flow cytometry | 101 |
| MtMV of Confetti-reporter labeling | Mouse | 600 blood precursors (E7.5-E12.5) | Native development is preserved; quantitative; simple genetic mouse models; statistically robust and simple; based on Poisson statistics | Individual clones cannot be tracked; requires >3% of Confetti labeling and >500 cells examined; quantitative range is limited; commercial software for analyses not available | Cre recombinase mouse models; single group of mice (≥5 mice); flow cytometry with 405, 445, 488, 562, and 640 nm and specific band-pass filter configurations to discriminate Confetti colors | 17,92 |
| Zebrabow | Zebrafish | 20-26 lifelong precursors | Native development is preserved | Reliant on hue and saturation to identify individual clones | Driver and effector transgenic lines; color-clustering computational analysis platform; flow cytometry; confocal and inverted microscope | 11 |
| Hsp701 LASER induction | Zebrafish | 30 lifelong precursors | Native development is preserved | Reliant on labeling 1 cell, otherwise individual clones cannot be tracked | Micropoint microscope-targeted 440-nm laser; driver and effector transgenic lines; flow cytometry; confocal and inverted microscopes | 11 |
| Transposase-mediated tagging | Mouse | Not yet employed | Native development is preserved; single clones can be tracked; lineage relationships inferred | Complex genetic mouse model; bioinformatics expertise required to interpret data | Targeted sequencing; bioinformatics expertise | 104,105 |
| PolyLox-based barcoding | Mouse | Not yet employed | Native development is preserved; single clones can be tracked; lineage relationships inferred | Complex genetic mouse model; bioinformatics expertise required to interpret data; need to eliminate common rearrangements | Targeted sequencing; bioinformatics expertise | 112 |
| CRISPR-based barcoding | Mouse | Not yet employed | Native development is preserved; single clones can be tracked; lineage relationships inferred | Complex genetic mouse model; bioinformatics expertise required to interpret data | Targeted sequencing; bioinformatics expertise | 113-115 |
| Somatic mutations | Human | Native development is preserved | Costs limit n of analyzed clones; potential for confounding mutations during cell expansion; biased for sequencing clones able to expand in vitro; complex bioinformatics analyses | WGS platforms; bioinformatics expertise | 118 |
ee, embryo equivalent; IAC, intra-aortic cluster; LDA, limiting dilution analysis.
N of IAC cells, RUs, or lineage-traced cells.