The Principle of Inverse Correlation between Variance of FP% and precursor numbers.

(A) Schematic of the Confetti cassette at the mouse Rosa26 locus. Sequences of four fluorescence protein are interspaced by loxP sites. Upon Cre-expression, the Confetti cassette will recombine and express one of the four fluorescence proteins.

(B) The inverse relationship between variance of markers and precursor numbers. The distribution of FP% is determined in the progeny to estimate the number of precursors.

(C) Workflow to validate the correlation formula between variance of FP% and precursor numbers by a two-color cell system.

(D) BFP frequencies in wells seeded with 1 to 100,000 HL-60. Each dot represents a well. Two replicates were shown. Each replicate consists of at least 20 wells per seeded number. Error bars represent mean ± SD.

(E) The standard deviation of BFP% in wells seeded with various numbers of HL-60. Error bars represent mean ± SD. N = 3 replicates.

(F) The correlation between seeded numbers and standard deviation of BFP%. N = 3 replicates.

(G) The correlation between seeded numbers and the numbers estimated with standard deviation of BFP% and equation 2. N = 3 replicates except for seeded numbers 106 and 107 (grey circles). For 106 and 107, N= 1 replicates.

For (A-C), images were created with BioRender.com/f96e500.

Transplantation of Defined Number of Precursors

(A) Representative flow plots showing Confetti induction by HSC-Scl-CreERT in the LSK population.

(B) Schematic for transplantation of defined number of precursors. Briefly, 4×106 Confetti-induced WBM for “high precursor number” groups, or 0.25×106 WBM for “low precursor number” groups were transplanted into lethally irradiated recipient mice. Image was created with BioRender.com/f96e500.

(C) RFPConf% distribution in PB myeloid cells. Each dot represents one animal. Recipient sample size = 7-14 mice per replicate, N = 3 replicates per group. Error bars represent mean ± SD.

(D) The precursor numbers of B cells, T cells, and myeloid cells in recipient mice. Dash lines mark the level of historically estimated transplantable clone numbers. Each dot represents a precursor number calculated from multiple mice. Error bars represent mean ± SD.

(E) The correlation of precursor numbers calculated from RFPConf%, YFPConf% or CFPConf%. Each dot represents a precursor number calculated from multiple mice.

(F) The precursor number of BM MP and HSPC in recipient mice. Dash lines represent historically estimated transplantable clone numbers. Each dot represents a precursor number calculated from multiple mice. Error bars represent mean ± SD.

(G) The donor chimerism of recipient mice, and the CD45.2+ chimerism differences between high- and low- precursor number groups. Each solid dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals. Recipient sample size = 7-14 mice per replicate, N = 3 replicates for each precursor number group. Two-way ANOVA for paired samples was performed.

(H) The frequency of myeloid cells in the peripheral blood of recipients. Each dot represents one animal. Recipient sample size = 7-14 mice per replicate, N = 3 replicates for each precursor number group. Two-way ANOVA for paired samples was performed.

ns, non-significant, * p < 0.05, ** p < 0.001.

Quantification of active hematopoietic precursor at steady-state

(A) Experiment schematic for Confetti induction in adult-induced animals. Image was created with BioRender.com/f96e500.

(B) Confetti labeling in B cells, myeloid cells and BM HSPCs. Statistics for comparisons between labeling of month seven and other months were shown. One-way ANOVA for paired samples was performed.

(C) RFPConf% in PB myeloid cells and BM HSPCs. Statistics for comparisons between labeling of month seven and other months were shown. One-way ANOVA for paired samples was performed.

(D) The number of myeloid precursors and the average precursor number from month five to seven.

(E) The number of BM precursors. Paired permutation was performed.

(F) The frequency-to-clone ratio of bone marrow HSPCs. Paired permutation test was performed. Each solid dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals. Error bars represent mean ± SD. PB sample size = 14-22 mice per replicate; BM sample size = 8-10 mice per replicate; N = 4 replicates. ns, non-significant, * p < 0.05, ** p < 0.001.

Number of precursor post-5-FU treatment and along developmental ontogeny

(A) Experiment schematic for one-dose 5-FU treatment. Image was created with BioRender.com/f96e500.

(B) The number of BM precursors post-5FU treatment. Permutation test was performed. For UT, sample size = 8-10 mice per replicate; N = 4 replicates; for 5-FU, sample size = 6-8 mice per replicate; N = 5 replicates.

(C) Experiment schematic for Confetti induction at fetal stages. Image was created with BioRender.com/f96e500.

(D) The Confetti labeling of B cells, T cells and myeloid cells. Each dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals. Sample size = 4-11 mice per replicate; N = 5 replicates.

(E) The number of precursors in PB myeloid cells, B cells and T cells. Permutation test was performed. For E14.5 and adult-induction, N = 4 replicates; for E11.5, n = 3 replicates.

(F) The number of BM precursors. Each dots represent one precursor number. Permutation test was performed. For E14.5 and adult-induction, N = 4 replicates; for E11.5, N = 3 replicates.

(G) The relative fold change increase of precursor numbers from fetal- to adult-stage, as well as fold change of CRU and cell counts.

Error bars represent mean ± SD. ns, non-significant, * p < 0.05, ** p < 0.001.

Precursor numbers in a mouse model of FA

(A) Experimental workflow to generate Fancc+/+ and Fancc-/- mice, induce Confetti expression and track precursor number dynamics. Image was created with BioRender.com/f96e500.

(B) Confetti labeling in PB B cells and myeloid cells. Each dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals. Dashed lines connect the values for the same replicate.

(C) The number of PB myeloid precursor.

(D) The number of BM precursor. Permutation test was performed.

(E) Experiment schematic for competitive transplantation. Image was created with BioRender.com/f96e500.

(F) PB donor chimerism in recipient mice of young FA donors. Each dot represents one animal. Dashed lines connect the average donor chimerism of three replicates. For Fancc+/+ or Fancc+/-, n = 10 per replicate, N = 3 replicates; for Fancc-/-, N = 7-9 per replicate, N = 3 replicates. Two-way ANOVA was performed.

(G) The number of PB myeloid precursor post-transplantation of young FA donors. Dashed lines connect the average precursor numbers. Permutation test was performed. N = 3 for each group.

(H) PB donor chimerism recipient mice of aging FA donor cells. Each dot represents one animal. Dashed lines connect the average donor chimerism of three replicates. For both genotype, N = 10 per replicate, N = 3 replicates. Two-way ANOVA was performed.

(I) The number of PB B precursor post-transplantation of aging FA donors. Dashed lines connect the average precursor numbers. Permutation test was performed. N = 3 for each genotype.

For (A-C), Fancc+/+, sample size = 11-17 mice per replicate, N =5 replicates; Fancc+/-, sample size = 10-22 mice per replicate, N =7 replicates; Fancc-/-, sample size = 9-17 mice per replicate, N =4 replicates. For (D), Fancc+/+, sample size = 5-9 mice per replicate, N =5 replicates; Fancc+/-, sample size = 6-12 mice per replicate, N =5 replicates; Fancc-/-, sample size = 6-11 mice per replicate, N =4 replicates. Error bars represent mean ± SD. nd, not determined; ns, non-significant; * p < 0.05, ** p < 0.001. # represents lowest p value possible for permutation test.