RNA-seq of healthy CD34+ HSPCs after co-culture with myelodyplastic syndrome patient-derived mesenchymal stromal cells

Cytopenia in at least one of the hematopoietic lineages (RBCs, WBCs or platelets) is a hallmark feature of MDS, indicating the lack of ability to support HSPC proliferation and the retention of their multilineage potential in the bone marrow. Here, wel investigate MDS-MSCs (with and without 5-azacitidine pre-treatment) as feeder layers for healthy CD34+ HSPCs. We performed RNA sequencing of co-cultured CD34+ HSPCs to compare their transcriptomic signatures after exposure to treated vs untreated MDS-MSCs. 5-azacitidine treated or MDS-MSCs were plated at a near confluent density of 2.0 × 104 cells/cm2 in DMEM with 10% FBS. 24 h later, healthy CD34+ HSPCs were seeded in contact with the MSC feeder layer at a density of 2.0 × 103 cells/cm2 in hematopoietic media and cultured for 14-21 days with replacement of media after 7 days. On the last day, CD34+ HSPCs were sorted for RNA extraction and subsequent sequencing.