Clonal tracking of erythropoiesis in rhesus macaques

Abstract

T he classical model of hematopoietic hierarchies is being reconsidered on the basis of data from in vitro assays and single cell expression profiling. Recent experiments suggested that the erythroid lineage might differentiate directly from multipotent hematopoietic stem cells / progenitors or from a highly biased subpopulation of stem cells, rather than transiting through common myeloid progenitors or megakaryocyte-erythrocyte progenitors. We genetically barcoded autologous rhesus macaque stem and progenitor cells, allowing quantitative tracking of the in vivo clonal output of thousands of individual cells over time following transplantation. CD34 + cells were lentiviral-transduced with a high diversity barcode library, with the barcode in an expressed region of the provirus, allowing barcode retrieval from DNA or RNA, with each barcode representing an individual stem or progenitor cell clone. Barcode profiles from bone marrow CD45 -CD71 + maturing nucleated red blood cells were compared with other lineages purified from the same bone marrow sample. There was very high correlation of barcode contributions between marrow nucleated red blood cells and other lineages, with the highest correlation between nucleated red blood cells and myeloid lineages, whether at earlier or later time points post transplantation, without obvious clonal contributions from highly erythroid-biased or restricted clones. A similar profile occurred even under stressors such as aging or erythropoietin stimulation. RNA barcode analysis on circulating mature red blood cells followed over long time periods demonstrated stable erythroid clonal contributions. Overall, in this nonhuman primate model with great relevance to human hematopoiesis, we documented continuous production of erythroid cells from multipotent, non-biased hematopoietic stem cell clones at steady-state or under stress.

Panhematopoietic RNA barcoding enables kinetic measurements of nucleate and anucleate lineages and the activation of myeloid clones following acute platelet depletion

“…In this study, we have applied a novel RNA barcoding method to provide the quantitative and dynamic tracking of individual HSCs with regard to their lineage contribution and transcriptional signature during steady state and upon acute platelet depletion. In contrast to previous RNA barcoding studies of hematopoiesis [ 18 , 19 ], our approach labels both nucleated and anucleate PB lineages including platelets. We have validated cDNA to be a more universal approach compared to previously published DNA barcoding methods [ 17 ] and a refined platelet collection method enabled their kinetic clonal studies.…”

“…Recently, RNA-based barcoding has been applied in studies of the nucleated hematopoietic cells in the BM; however, the labeling protocol and cell isolation method applied did not effectively label or enrich platelets and erythrocytes [ 18 ]. Fan et al performed a clonal analysis of the erythroid lineage in macaque, including anucleate erythrocytes in the PB upon transplantation [ 19 ]. However, platelets, in spite of their clinical significance, abundance (up to 60% of HSC output [ 2 ], and the clear indications that their progenitors, megakaryocytes (MKs), are closely linked to HSCs [ 20 ], have so far been omitted from the clonal analysis.…”

Clonal Hematopoiesis Analyses in Clinical, Epidemiologic, and Genetic Aging Studies to Unravel Underlying Mechanisms of Age-Related Dysfunction in Humans

“…Non-human primates have a high similarity in hematopoietic stem and progenitor cell (HSPC) properties to humans. Screening a cohort of naturally aged macaques for somatic CHIP mutations identified predicted loss of function (LOF) mutations in 14/53 (27%) to date, with the most frequent mutations in genes most common in human CHIP ( Espinoza et al, 2019 ; Fan et al, 2020 ). A macaque model has been developed utilizing CRISPR/Cas9 technology to modify HSPCs from young adult macaques by delivering gRNAs targeting the three most frequent human CHIP genes along with Cas9 protein into HSPCs from three young adult macaques.…”

Parallel clonal and molecular profiling of hematopoietic stem cells using RNA barcoding

“…Cellular barcoding method was recently implemented for the clonal analysis of LT-HSC using genomic DNA [11][12][13]. The approach provided detailed information about LT-HSC subsets and clonal kinetics during haematopoiesis after transplantation [12,14], upon a challenge [15] or in aged animals [16]. Recently RNA-based barcoding has been applied for studies of the nucleated cells in the BM, however the labelling protocol applied did not effectively label platelets and erythrocytes [17], and thus a major component of HSC output was overlooked.…”