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X. Fan et al.
restricted LT-HSPC.16 However, the majority of single HSPC were multipotent.
Similar in vivo studies of human LT-HSPC properties via single cell transplantation or naïve hematopoietic tagging are not feasible, but the significant differences between human and rodent hematopoiesis make extrapolation dif- ficult.41 In vitro single cell CFU assays are the classic approach to study lineage relationships and search for intermediate progenitors in hematopoiesis.1,3 Notta et al.5 found that populations of human CD34+ HSPC sorted by previously-described erythroid and megakaryocytic line- age markers and grown in single cell culture did not reveal single progenitors with both erythroid and megakaryocyt- ic or erythroid and myeloid potential, in contrast to the presence of such progenitors in fetal liver and cord blood, thereby supporting the concept that both erythroid and megakaryocytic lineages emerge directly from multipo- tent HSC, at least postnatally. In contrast, several other groups did find appreciable numbers of single human pro- genitors with both erythroid and myeloid potential using a different culture system and lineage-defining antibodies.6,7
Our macaque barcoding approach provides a robust platform to quantitatively track the output of thousands of individual HPSC clones long-term over multiple lineag- es in vivo. The clonal landscape in NRBC closely matched that of other hematopoietic lineages sampled from the same BM locus at same time point, with a particularly close relationship between NRBC and Gr and monocytes, which are continuously produced from HSPC, in contrast to T cells that can clonally expand and renew peripherally. Our analysis of thousands of individual clones contribut- ing to purified nucleated erythroid precursors versus other lineages at the same time point and at the same marrow location did not uncover a measurable population of markedly erythroid-biased LT-HSPC in this post-trans- plantation model. Likewise, analysis of barcodes from hundreds of pooled myeloid and erythroid CFU grown from marrow post transplantation led to similar conclu- sions, although a small fraction of clones unique to CFU-E were identified; however, CFU-GM analyses also revealed unique clones. Given that engraftment in macaques results from many thousands of individual LT-HSPC,20 the appar- ent presence of lineage-restricted CFU was likely due to sampling bias or potentially differential clonal in vivo versus in CFU in vitro. However, we cannot rule out that these lin- eage-restricted clones detected only via CFU analysis rep- resent true myeloid or erythroid-restricted clones not detected in our NRBC analyses.
Our findings were confirmed at various time points from months to years post transplantation. Even in an aged macaque, previously shown to have long-term per- sistence of highly myeloid and lymphoid biased clones,22 we found no evidence for erythroid-biased clones not also biased towards myeloid output. A recent human lentiviral gene therapy study in patients with an immunodeficiency disorder used insertion site retrieval to map clonal rela- tionships between lineages, and also reported primarily shared clones contributing to erythroid and myeloid line- ages.42 Of note, our findings do not contradict the concept, derived from recent single cell gene expression studies, that erythroid pathways diverge very early from myeloid and lymphoid lineages, representing one of the first branches from differentiating LT-HSC. A multipotent LT- HSC marked by a barcode could produce daughters going
down both erythroid and non-erythroid pathways. Our results do suggest that no significant contributions from self-renewing or engrafting erythroid-restricted or highly biased progenitors could be detected, at least in this trans- plantation model. However, in several murine studies, the stress of transplantation or in vitro culture was shown to drive megakaryocytic-biased HSC to contribute to addi- tional lineages,16,17 thus our transplantation model may not reflect physiologic naïve hematopoiesis.
Since we have discovered that clonal output from indi- vidual HSPC can remain highly geographically restricted within the BM for up to years post transplantation,23 it may be difficult to study the entire clonal composition of NRBC or erythroid progenitors via marrow sampling alone. Thus we also analyzed the barcodes in circulating mature RBC and reticulocytes via retrieval of RNA bar- codes, given the lack of DNA in enucleated cells. Fractional contributions of DNA and RNA barcodes retrieved from the same nucleated sample of each lineage were compared and showed high correlation, suggesting the differentiation pathway for these lineages does not impact significantly on expression level of barcodes from our vector regardless of insertion site, due to expression of the marker gene and the barcode RNA from a strong constitutive viral promoter. However, RNA barcodes in circulating reticulocytes/mature RBC revealed a less diverse clonal pattern and major differences from clonal contribution patterns revealed in RNA of circulating myeloid and lymphoid cells, and even from NRBC con- currently sampled from the BM in animals many years post transplantation. Rather than implying erythroid line- age bias, we suspect these findings resulted from a major constriction of gene expression in anucleate erythroid cells, with marked silencing of many endogenous genes other than hemoglobin and red cell structural proteins. Bonafoux et al.43 reported globally skewed transcriptional activity follow erythroid differentiation, with major dif- ferences in gene expression between end stage anucleate erythroid cells and leukocytes, and even in comparison with earlier stage erythroid progenitors. More than 50% of transcripts in these end stage erythroid cells encoded globin. The fact that many viral integrations were likely also silenced and thus did not express the barcode is sup- ported by the observation that the GFP percentage in RBC was much lower than in Gr in all our macaques (Online Supplementary Figure S3). Despite the difficulties in comparing clonal contributions in RBC RNA to other lin- eages, sampling of PB RBC over prolonged periods of time allowed us to establish the long-term clonal stability of contributing HSPC to erythropoiesis for as long as four years post transplantation.
Given the advantage of our experimental models, we sought to answer some additional questions, including whether erythropoiesis at a clonal level will be stable under lineage-specific stimulation, or if lineage-specific clones might be recruited under this proliferative stress, as has been suggested for lineage-restricted megakaryocy- topoiesis.44 Thus, we tracked the clonal characteristics of hematopoiesis under stimulation by EPO, a key lineage- specific humoral regulator responsible for erythroid pro- genitor proliferation and accelerated maturation. We found no detectable impact on clonal contributions under EPO stimulation, suggesting that the pool of HSPC con- tributing to erythropoiesis does not shift between steady state and proliferative stress resulting from EPO stimula-
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haematologica | 2020; 105(7)