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Erythropoiesis at a clonal level
populations or cDNA reverse-transcribed from cellular RNA under- went low cycle PCR with primers bracketing the barcode followed by multiplex Illumina sequencing; see the Online Supplementary Appendix for details. Sequencing output was processed using cus- tom Python and R code to retrieve and quantitate barcode contri- butions (available at: www.github.com/dunbarlabNIH/), previously validated to closely reflect fractional contributions of each clone within polyclonal cellular populations.21
Hematopoietic cell purification and phenotypic analyses
0-15 mL BM aspirates were obtained from the posterior iliac crests or ischial tuberosities. BM and PB samples were separated into mononuclear cell (MNC) and granulocyte (Gr) fractions via centrifugation over a Ficoll gradient (MP Biomedicals). Red blood cells (RBC) were removed from the Gr pellet via red cell lysis with ACK buffer (Quality Biological). BM MNC were passed over an immunoselection column to purify CD34+ HSPC as described.24 CD34− MNC flowing through the column or PB MNC were stained with lineage-specific antibodies and sorted for CD45- CD71+ nucleated red blood cells (NRBC) as described,30 CD3+ T cells, CD20+ B cells, and CD3−CD20−CD14+ monocytes (Mono) as reported previously,23 using gating strategies shown in Online Supplementary Figure S1. The purity of sorted erythroid cells was validated by morphologic scoring of at least 500 cells on Wright’s stained and benzidine-stained cytospins. Erythroid cells constitut- ed at least 95% of sorted preparations. Monoclonal antibodies uti- lized are given in Online Supplementary Table S1.
Leukocyte depletion of peripheral blood was performed by fil- tration through a 10 mL syringe packed with 5 mL of cellulose fibers (Sigma-Aldrich) and fitted with two layers of WhatmanTM lens paper (GE Healthcare Life Sciences) covering the outlet.31 Before use, columns were rinsed with phosphate buffered saline (PBS) and then 5 mL whole blood was added and gently pressed to run through in droplets. Each product was checked for extent of CD45+ cell depletion by flow cytometry, and by morphologic scoring of at least 500 cells on a Wright’s stained smear. Each blood sample was >99%-depleted of non-erythroid cells.
Colony-forming unit assays
CD34+ cells were plated for colony-forming unit (CFU) assays according to the manufacturer’s (STEMCELL Technologies) instructions in two different methylcellulose formulations: one is MethoCult GF+H4435 complete methylcellulose medium contain- ing human IL-3, IL-6, SCF, G-CSF and GM-CSF to support forma- tion of myeloid CFU, the other is MethoCult H4230 methylcellu- lose medium supplemented with 3 IU/mL human erythropoietin (EPO) (PeproTech), 5 ng/mL rhesus IL-3 (R&D) and 100 ng/mL human SCF (Miltenyi Biotec), to support erythroid colony forma- tion.32 Cells were plated at 1000 cells/mL in H4435 medium or at 10,000 cells/ mL in EPO-supplemented H4230 medium, incubated at 37°C and 5% CO2. At day 12-14, colonies were enumerated and well-separated CFU were plucked individually for molecular analyses.
Erythropoietin treatment
Purified recombinant human EPO (PeproTech) was injected subcutaneously at 3000 U/kg for two doses 12 hours apart to stim- ulate macaque erythropoiesis33-35 in barcoded animal ZL40 at 12 months post transplantation. Baseline BM and PB samples were collected five weeks before EPO stimulation, and reticulocyte con- centrations in the PB were monitored every other day post EPO administration. When the reticulocytes rose to ≥8%, BM and PB samples were collected. Recovery samples were collected three months post EPO stimulation.
Results
Approaches for erythroid lineage tracking in the rhesus macaque model
In order to track clonal contributions to erythropoiesis in comparison to other lineages, CD34+ HSPC from seven young RM and one aged RM were transduced with high diversity barcoded lentiviral libraries under conditions favoring a single unique barcode marking individual HSPC and reinfused into the autologous RM following ablative total body irradiation (TBI); see Table 1 for a summary of transplantation and transduction parameters. Following engraftment, samples were obtained from BM and PB (Figure 1). As reported previously, short-lived, lineage- restricted progenitors contributed for the first 1-2 months, followed by stable highly polyclonal contributions to Gr, monocytes, B cells, T cells and CD56bright NK cells from long-lived, stable, multipotent long-term repopulating HSPC clones.21,23 In the current study, we designed assays to study clonal contributions to the erythroid lineage (Figure 1), with the anucleate state of mature circulating erythrocytes requiring design of alternative approaches for mapping of clonal contributions.
Clonal contributions to nucleated erythroid cells are shared with myeloid lineages
Nucleated RBC represent the final stage in marrow ery- throid differentiation, before nuclear extrusion and exit from the BM into the PB. NRBC can be identified and sort- ed to high purity based on absent/low expression of the pan leukocyte marker CD45, and high expression of the transferrin receptor CD7130,36 (Figure 2A). Unfortunately, RM-reactive antibodies recognizing other erythroid mark- ers, such as glycophorin, do not currently exist. We sorted NRBC from BM MNC based on a CD45–CD71+ pheno- type, with a starting population of 0.2-4.7%, and generat- ed NRBC preparations with high purity (>95%) as con- firmed by fluorescence-activated cell sorting (FACS), Wright-Giemsa staining and benzidine staining (Figure 2A and Online Supplementary Figure S4). Concurrent purified populations of NRBC, CD34+ HSPC, T cells, B cells, monocytes and Gr were isolated from 10-15 mL BM aspi- rates from four young adult rhesus monkeys (JD76, ZK22, ZH19 and ZH33, 7-10 years of age) 3.5-46 months post transplantation, and one aged rhesus monkey (RQ3600, 23 years of age), 48 months post transplantation and DNA was obtained for barcode retrieval. Obtaining enough BM prior to three months post transplantation was not feasi- ble due to low marrow cellularity during recovery from TBI.
As we reported recently,23 there is marked clonal geo- graphic segregation of the output from HSPC in the BM for at least six months post transplantation, followed by very gradual clonal mixing at different BM sites over sub- sequent months to years. Therefore, we analyzed the bar- code clonal pattern of all lineages from the same BM sam- ple, rather than comparing NRBC from one or a few BM sites to circulating myeloid and lymphoid cells. We visual- ized the contributions of the largest clones to each lineage mapped across all lineages in heat maps (Figure 2B) and analyzed Pearson correlations between all contributing clones (Figure 2C). At both early (3.5 months) and later time points up to several years post transplantation, clonal contributions were closely correlated between NRBC, monocytes and Gr (r values ranged from 0.68 to 0.95;
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