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X. Fan et al.
including some barcodes found contributing at high rela- tive levels to RBC compared to NRBC or to other lineag- es. Conversely, many clones found contributing to NRBC as well as BM or circulating myeloid cells (Gr and Mono) were undetectable or contributing at a fractionally much lower level in RBC. Overall, the Pearson correlations between RNA barcodes contributing to RBC versus myeloid cells were much lower than between the other lineages, including between NRBC and myeloid cells, and low even between NRBC and RBC (Figure 4D). These findings may result from legitimate HSPC clonal bias, i.e. HSPC clones that contribute in a highly-biased manner to the erythroid versus other lineages. But this seems unlike- ly, given the discrepancy in clonal patterns between NRBC and anucleate RBC. Instead, we suspect this reflects the marked transcriptional restriction and differ- ential RNA stability occurring in maturing RBC as they transition towards producing massive amounts of only a few proteins, most notably hemoglobin.37 A large fraction of vector insertion sites may be silenced transcriptionally or translationally during the final stages of RBC enucle- ation and release, making extrapolating clonal contribu- tions from RNA barcode expression problematic.
We were, however, able to use RNA barcode retrieval to track the stability of clonal contributions to circulating RBC over time, studying six macaques for intervals of up to 15 months. PB RBC RNA barcode analysis showed very stable barcode RBC contribution patterns over time in both young and old animals (Figure 5).
Erythropoietin stimulation does not alter erythroid clonal patterns
We investigated the impact of erythropoietic stress on erythroid clonal patterns. EPO is the key lineage-specific humoral regulator of mammalian erythropoiesis, and in the setting of anemia, levels increase to expand the ery- throid compartment. To assess whether lineage-biased clones could be recruited via lineage-specific stimulation, we administered a short course of high-dose EPO to achieve significant erythroid proliferation33-35 in barcoded monkey ZL40. BM and PB samples were collected from animal ZL40 at baseline 10.5 months following transplan- tation, at the peak of reticulocytosis on day 6 of EPO administration, and at recovery several months later, once blood counts and reticulocyte numbers had returned to baseline (Figure 6A and B).
The clonal patterns in BM NRBC were stable over time whether at baseline, peak or recovery following EPO, and matched those of BM monocytes and Gr (Figure 6C). The RNA barcode analysis of PB RBC RNA also showed an unchanged clonal pattern in response to EPO (Figure 6C). The results indicated that the EPO administration did not change the erythroid clonal output, it only stimulated the existing erythroid progenitor pool to produce more RBC, but without recruiting previously-quiescent HSPC clones to generate new erythroid cells.
Figure 5. Stability of erythroid clonal contributions over time. Heatmaps plotting barcode contributions to peripheral blood (PB) red blood cell (RBC) RNA over time in five young macaques and one aged (RQ3600) macaque, compared to DNA barcode from PB monocytes (Mono) and granulocytes (Gr). Each heatmap plots the top ten contributing clones in each sample across all samples, heatmaps were made as explained in Figure 2B. The color scale is on the right.
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haematologica | 2020; 105(7)