Page 64 - Haematologica April 2020

P. Balogh et al.
the importance of cell-intrinsic determinants, although micro-environmental factors also exert a critical influence.7-9
The transcription factor RUNX3 has been characterized as a participant in neural and lymphocyte development, TGFβ signaling, and solid tumor suppression.10-14 Several studies have also demonstrated its repression in aged nor- mal as well as tumor tissues, with the principal mecha- nism of inactivation being epigenetic alterations, particu- larly DNA methylation.15-18 Emerging data suggest a role in hematopoiesis, with zebrafish and murine loss of function studies revealing progenitor perturbations, although the extent of its role has remained unclear due to redundancy with Runx1.19-21 Most notably, induction of hematopoietic Runx3 deletion in mice elicited marrow changes similar to those reported with normal aging: increased marrow colony forming units (CFU) and increased peripheral blood mobilization of CFU by G-CSF treatment.5,22
This study shows RUNX3 to be expressed in murine and human HSPC, where it undergoes repression and epi- genetic modification during normal aging. HSPC levels of RUNX3 were found to determine developmental poten- tial, with deficiency restricting erythropoiesis at commit- ment and subsequent stages while fully permitting granu- lopoiesis. HSPC purified from patients with unexplained anemia of aging manifested RUNX3 deficiency and simi- lar developmental alterations. Changes in HSPC transcrip- tome due to RUNX3 deficiency suggest a role upstream of the erythroid master regulatory transcription factors KLF1 and GATA1.
Methods
Cell culture
Human CD34+ expansion medium consisted of Iscove's modi- fied Dulbecco's medium (IMDM) supplemented with bovine serum albumin, insulin and transferrin (BIT) 9500, and 100 ng/mL each of rhTPO, rhSCF, and rhFlt3-l, plus 10 ng/ml rhIL-3. Erythroid medium consisted of IMDM supplemented with BIT 9500, and 4.5 U/mL rhEPO and 25 ng/mL rhSCF. Megakaryocyte medium consisted of IMDM supplemented with BIT 9500, and 40 ng/mL rhTPO, 25 ng/mL rhSCF, and 20 ng/mL rhSDF1-alpha. Granulocyte medium consisted of IMDM supplemented with BIT 9500, and 25 ng/mL rhSCF, 10 ng/mL rhIL-3, and 20 ng/mL rhG- CSF. Colony formation assays were conducted using methylcellu- lose supplemented with 50 ng/mL rhSCF, 10 ng/mL rhIL-3, 20 ng/mL rhIL-6, 3 U/mL rhEPO, 20 ng/mL rhG-CSF, and 10 ng/mL rhGM-CSF.
Mass cytometry
Cells were stained for viability with 100 μM cisplatin, fixed with 1.6% paraformaldehyde, and stored at -80°C. Thawed sam- ples were barcoded, pooled, and surface stained at room temper- ature for 30 minutes (min). Cells were then permeabilized with methanol and stained for intercellular antigens for 1 hour (h) at room temperature. Next, cells were incubated with Fluidigm CellID Ir-Intercalator, re-suspended in water with normalization beads, and analyzed on a Fluidigm CyTOF 2. Data were bead- normalized and underwent barcode deconvolution using the debarcoding tool MATLAB standalone executable.23
Data were inverse hyberbolic sine-transformed using a co-fac- tor of 0.25. FlowSOM was used to construct a self-organizing map, and each cell was assigned a phenocode for every lineage
marker using flowType. Each grid point was then immunopheno- typed, and cell counts were tabulated to form a hierarchical count table. Differential abundance was tested for using edgeR with a quasi-likelihood framework as specified by the cydarTM package.
RNA-sequencing
RNA was extracted using the QIAgen RNeasy Plus Mini Kit, with added DNA digestion. Samples underwent ribosomal reduc- tion, and sequencing with 100bp, paired-end, and 50 million read- depth parameters on an Illumina HiSeq 2500 machine. Data were processed online at usegalaxy.org. Trimmomatic was used to eliminate low quality sequences from the reads, followed by alignment to the hg19 reference genome using HISAT2, and RmDup to eliminate PCR duplicates. Differential gene expression was assessed with both DESeq2 and Cufflinks tools. The Synergizer tool was used to convert UCSC gene identifiers into hgnc gene symbols.
Unexplained anemia of the elderly studies
Mononuclear cells were sorted phenotypically: HSC, Lin- CD34+CD38–CD904+ CD45RA–; MEP, Lin–CD34+CD38+CD123- CD45RA–. Colony assays were performed using complete methylcellulose with 12-14 days incubation. For microarray, RNA samples were quantified, subjected to reverse transcription, under- went two rounds of linear amplification, and biotinylated. 15 μg of RNA per sample was assayed using Affymetrix HG U133 Plus 2.0 microarrays. Data were analyzed using the gene expression commons platform.
Ethics statement
This study was reviewed and approved by the institutional review boards of the respective institutions and was conducted in accordance with the principles of the Declaration of Helsinki.
Data and software availability
RNA-sequencing accession numbers: GSE119264, GSE104406. Microarray accession number: GSE123991.
Results
Hematopoietic stem cell RUNX3 levels decline with aging
Prior reports have shown marrow-specific Runx3 knock- out to elicit aspects of the aging phenotype and to exag- gerate the myeloid skewing associated with aging.5,22 We therefore assessed RUNX3 expression in rigorously puri- fied human and murine hematopoietic stem cells. In humans, RNA-seq has been conducted on Lin–CD34+CD38– marrow cells from healthy young (18-30 years old) and aged (65-75) subjects (GSE104406). In mice, side population (SP) Lin–Sca+Kit+CD150+ marrow cells from young (4 months old) and aged (24 months) animals have undergone RNA-seq3 (GSE47819). Both datasets demonstrated HSC expression of RUNX3 with significant decreases associated with aging (Figure 1A and B). Human CD34+CD38+ later stage progenitors also showed diminished RUNX3 expression with aging, indicating that the changes are not HSC-restricted (Online Supplementary Figure S1A). Evidence for an aging-associated decline in progenitor protein levels was seen in human marrow sam- ples immunostained for RUNX3 (Online Supplementary Figure S1B). Analysis of murine bone marrow single-cell RNA-seq datasets24 (GSE89754) from animals with or without erythropoietin (EPO) treatment confirmed that
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