Clonal Diversity As an Alternative Measure for Hematopoietic Reserve in Bone Marrow Failure

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome, typically presenting in early childhood. The pace of progression from diagnosis to symptomatic failure requiring therapy varies widely between patients, but peripheral blood cell counts, bone marrow cellularity and progenitor frequency measurement have proved poor predictors for the residual regenerative potential. Declining hematopoietic output in the context of aging or experimental stress leads to a decrease in clonal diversity. Here, we hypothesized that clonal diversity may also be an alternative measure for the residual hematopoietic functional reserve and developed an experimental platform to test our hypothesis. We crossed the previously described Confetti mouse model (Ganuza et al, NCB 2017) with a hematopoietic stem cell selective hSclCreERT Cre driver and the Fancc model of FA. We first optimized the clonal calculation formula to achieve an improved fit (R²: original 0.75, optimized: 0.96). We then performed a transplantation experiment with defined numbers of transplantable clones to directly validate performance (predicted: 250; original formula: 558, optimized formula: 354). Next, to determine homeostatic clonal diversity, we induced Confetti expression either in embryos at E14.5, or in adult mice at 2-month-old (n=30-, and ~60 mice, respectively) using the Confetti/hSclCreERT cross (no Fancc). In these cohorts we then measured the clonal diversity in peripheral blood myeloid and lymphoid subpopulations. We found that the number of B-cell clones marked by fetal induction was relatively stable before three-month of age (~7700 clones), followed by a decline to ~2700 clones at 7 months old. In contrast, T-cell clonal diversity marked by fetal induction was relatively stable for the observation period (~5100 to ~8000 clones). The number of granulocytes and myeloid clones at 7-month post-birth also decreased compared to that measured at one-month old (~6300 clones at one-month old; ~3600 clones at 7 months old). By contrast, clonal diversity and dynamics after adult induction differed substantially from fetal induction. Here we observed relatively greater clonal diversity at one-month post-induction in granulocytes and myeloid cells, which gradually declined to a stable level at 4-months post-induction. In contrast, clonal output measured from T-cell or B-cell was relatively stable throughout. We also examined the clonal diversity of stem and progenitor cells in the bone marrow (BM) at six months post-adult induction. In the adult induction cohort, we also found that the clonal diversity of granulocytes and myeloid cells in the peripheral blood was comparable with the clonal diversity of their progenitors in the BM (GMP and MPP3), suggesting peripheral blood clonal diversity as a reflection of the clonal diversity of the progenitors in the BM. Interestingly, we detected ~4500 clones in pre-pro-B cells, while only ~1300 clones in pro-B cells. This clonal contraction coincided with the stage when B-cell starts VDJ recombination. As a more conventional measure of residual activity, we next compared the frequency of stem and progenitor cell subpopulations in the BM with their clonal diversity and found that clonal diversity did not correlate with frequency. As an example, the frequency-to-clonal-diversity ratio of MPP2 progenitors is as low as LT-HSC even though MPP2 are much more highly proliferative, consistent with the notion that they contribute to downstream progenitor expansion, but are unable to symmetrically expand, thereby keeping their frequency low. Altogether, we have validated a system to measure clonal diversity and temporal dynamics in PB- and BM compartments. This provides us with reference metrics for clonal diversity studies in triple cross Confetti/hSclCreERT/Fancc^-/- mice currently underway. We predict our approach will reveal reduced clonality in Fancc^-/- mice and propose clonal diversity as a more accurate metric for functional reserve in BMF syndromes.

No relevant conflicts of interest to declare.