Page 61 - Haematologica - Vol. 105 n. 6 - June 2020

DKC1 drives upregulation of telomerase in normal human erythroblasts
nucleotides were verified by Sanger sequencing. Luciferase assays were performed using HEL 92.1.7 cells as described in the Online Supplementary Methods.
Statistics
All statistics were performed using GraphPad Prism 6.0b (La Jolla, CA, USA). Results were considered statistically significant when P<0.05.
Results
DKC1 is upregulated with erythroid lineage commitment
It was previously shown that telomerase activity is upregulated when CB-derived HSPC were switched to conditions promoting erythroid differentiation.14 To verify this finding in a pure population of erythroid cells, GLYA+ cells were sorted by FACS from cultures generated by ex vivo expansion of HSPC. CD34+ cells were first expanded in medium supplemented with STF for 1 week, then switched to medium containing SCF and erythropoietin (SE) for a further 2 weeks. FACS analysis using antibodies for GLYA and CD34 confirmed differentiation of HSPC and enrichment for GLYA+/CD34- erythroid cells (>80% of the viable population) at week 2 and week 3 (Figure 1A and Online Supplementary Figure S1). Erythroid cell popula-
AB
tions were further purified from week 2 cultures by FACS sorting cells based on either low or high expression of GLYA and lack of expression of the myeloid cell marker CD13. CD13+/GLYA- myeloid cells were also purified from the week 2 cultures for comparison with the ery- throid cells (Figure 1B). Telomerase activity was quantified in the FACS-sorted populations of GLYA+high, GLYA+low ery- throid cells and CD13+ myeloid cells, as well as CD34+ cells and an unpurified population of cells cultured in SE. The results demonstrated that telomerase activity was upregulated in GLYA+high erythroblasts relative to both uncultured CD34+ HSPC and unsorted SE-cultured cells (Figure 1C). In contrast, no significant telomerase activity was detected in CD13+ myeloid cells. These data confirm that telomerase activity was confined to the GLYA+ ery- throid subset of cells in the SE culture, and was downreg- ulated in differentiated myeloid cells.
To investigate the regulation of telomerase during ery- throid commitment, telomerase enzyme activity and expression of TERT, TERC and DKC1 was assessed at weekly time points over the 3-week culture period. As previously reported, telomerase was modestly upregulat- ed upon initial cytokine stimulation with STF,11,12 then fur- ther increased during the second week of culture after switching to SE (P<0.01) (Figure 2A).14 In parallel with the initial induction of telomerase activity, there was a meas- urable increase in TERT expression during the first week
C
Figure 1. Telomerase enzyme activity is upregulated in glycophorin A+ erythroblasts. Cord blood (CB)-derived CD34+ hematopoietic stem and progenitor cells (HSPC) were expanded for 7 days in medium supplemented with stem cell factor, thrombopoietin and Flt-3 ligand (STF), then cultured for an additional 2 weeks in medium with stem cell factor plus erythropoietin (SE). (A) Graphical representation of the proportions of CD34+ and glycophorin-positive (GLYA+) cells in the total viable cell fraction, shown as the mean ± standard error of the mean (SEM) calculated from nine independent CB cultures. (B) Cell populations enriched for erythroblasts with high or low GLYA expression, or CD13+ myeloid cells were isolated from SE cultures by fluorescence activated cell sorting (FACS). Panels show gates used for sorting cell subsets expressing low (1) or high (2) levels of GLYA and CD13 before and after FACS. (C) qTRAP measurement of telomerase enzyme activity in uncultured HSPC cells (CD34+), unfractionated cells from SE cultures (Unsorted) and FACS-sorted cell populations. Each symbol represents the mean from three measures of telom- erase activity in independent assays of cells from four CB cultures. *P<0.05, **P<0.01, Dunnett multiple comparisons test.
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