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X. Fan et al.
expression profiling analyzed by computation algorithms predicting differentiation trajectories have challenged the classical branching hematopoietic model in both rodents and humans. Adolffson and co-workers reported direct differentiation of murine megakaryocytic-erythroid line- ages from HSC/ MPP.4 Notta and co-workers analyzed human MPP subpopulations and demonstrated almost exclusively uni-lineage potential of single cells in vitro, sug- gesting that both erythroid and megakaryocytic lineages differentiate directly and separately from HSC/MPP.5 In vitro assays and single cell gene expression mapping of classical human MEP populations also suggested distinct erythroid and megakaryocytic pathways immediately downstream of multipotent progenitors, although other groups were able to purify rare bipotent progenitor cells.6,7 Both murine and human single-cell RNA-seq profiling of hematopoietic stem and progenitor cells (HSPC) uncov- ered very early transcriptional lineage priming immediate- ly downstream of HSC, imputing early branching towards individual hematopoietic lineages, and in some models the earliest branch being erythroid.8-13
In addition, large-scale optimized single cell murine transplantation assays have suggested that all long-term and self-renewing engrafting cells are not necessarily homogeneous or multipotent, with evidence for lineage- bias or even lineage-restriction. Dykstra and co-workers reported different classes of such cells with myeloid, or multipotent engraftment patterns long-term, maintained in secondary transplants, but did not examine erythroid or megakaryocytic lineages, given lack of expression of stan- dard congenic markers on these lineages.14 More recently, groups have devised strategies to allow tracking in all murine lineages, and uncovered megakaryocytic-restricted or highly-biased intermediate15 or long-term engrafting/self-renewing single cells.16 Use of an inducible transposon to create clonal tags in non-transplanted mice also uncovered a megakaryocyte-restricted differentiation pathway, and both clonal label propagation through vari- ous progenitor populations and gene expression profiling suggested that megakaryocyte-primed HSC are located at the top of the hematopoietic hierarchy.17 These powerful in vivo approaches are dependent on methodologies such as single cell transplantation, transposon activation or lin- eage tracing that are not feasible in humans or large ani- mals.
We have employed rhesus macaque (RM) HSPC autolo- gous transplantation combined with lentiviral genetic bar- coding to quantitatively track the in vivo clonal output of thousands of individual HSPC over time, in a model with great relevance to human hematopoiesis.18 Macaques and
humans have prolonged lifespans and similar HSPC cycling and dynamics.19 We previously demonstrated early lineage-restricted engraftment of short-term progenitors for several months, followed by stable very long-term out- put from engrafted multipotent HSPC, analyzing DNA barcodes from nucleated neutrophils and lymphoid line- ages, in the peripheral blood (PB) and BM.20,21 Persistent myeloid or B-cell lineage bias, although not complete lin- eage restriction, could be appreciated,20 and was increased in aged macaques.22 Peripheral maintenance and expan- sion of T-cell and mature natural killer (NK) clones was documented.23 We now apply this macaque model to examine the clonal ontogeny of the erythroid lineage at steady state post transplantation and under erythropoietic stimulation, employing both DNA and expressed RNA barcode analysis. Results in both young and aged macaques revealed closely shared clonal landscapes for erythropoiesis compared to myeloid and lymphoid lineag- es at both steady states following transplantation and under erythropoietic stress, and clonally-stable erythro- poiesis over time.
Methods
Autologous rhesus macaque transplantation
All experiments were carried out on protocols approved by the National Heart, Lung and Blood Institute (NHLBI) Animal Care and Use Committee, following institutional and Department of Health and Human Services guidelines. Details of peripheral blood HSPC mobilization, CD34+ purification, lentiviral transduction, and autologous transplantation following myeloablative (500 rads x2) total body irradiation have been published24 including details for the specific animals included in the current paper.20,21,23 Table 1 summarizes transplanted cell doses and length of follow up. Details of transplantation and the clonal patterns in non-erythroid lineages from animals ZH33, ZG66, ZJ31, ZK22, ZL40 and ZH19 have been previously reported.20,23,25,26
Barcoded library preparation, validation, transduction, and retrieval
The barcoded lentiviral vector consists of the backbone pCDH (Systems Biosciences) expressing the CopGFP marker gene fol- lowed by a 6 base pair (bp) library identifier and a 27 or 35bp highly diverse DNA barcode, flanked by polymerase chain reaction (PCR) amplification sites.27 Lentiviral vectors were produced using the χHIV packaging system optimized for RM HSPC transduction.28 CD34+ HSPC were transduced with high-diversity barcoded libraries ensuring that the majority of transduced HSPC contain only one barcode per cell and that each barcode uniquely defines a single HSPC, as described and validated.20,29 DNA from target cell
Table 1. Transplantation and follow-up characteristics of animals included in this study.
CD34+ transplant dose (millions)
CD34+ transplant dose/kg (millions) % GFP+ infused cells
Infused GFP+cells (millions) Follow-up time points (months)
ZH33
32
6.9
35%
11.1
46, 54.5, 60, 61
ZG66
48
8.5
35%
16.7
48, 53, 55.5
ZJ31
23
4.1
35%
8.0
3.5, 28, 31, 32, 33, 35
ZH19
48
7.1
23%
11.0
41, 44.5, 45.5, 48.5, 49
ZK22
82
7.2
31%
25.2
9, 11.5, 12.5, 15.5, 17.5
ZL40
57
8.1 22.5% 12.8 10.5, 12,
15.5
JD76 JM82
44.4 91
4.1 7.2 27.1% 34% 12.0 30.6
3.5 3.5
RQ3600
58.4
15.9 35% 20.4 46, 48, 49
Details of transplantation and the clonal patterns in non-erythroid lineages from animals ZH33, ZG66, ZJ31, ZH19, ZK22, ZL40 and RQ3600 been previously reported.20, 23, 25, 26
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haematologica | 2020; 105(7)