Real-time tracking of RNA splicing in aged bone marrow hematopoietic stem and progenitor cells

Abstract Background RNA splicing is a critical regulator of hematopoietic stem and progenitor cell (HSPC) function and leukemogenesis. We previously identified differential exon usage characterized by increased exon skipping in both secondary AML and pediatric AML using RNA-sequencing in combination with our lentiviral dual fluorescent splicing reporter. Additionally, prior work demonstrated age-associated splice isoform expression in bone marrow (ABM) derived HSPCs (Crews…Jamieson, Cell Stem Cell 2016). This study expands on those findings by investigating exon usage specifically in aged bone marrow (ABM) using a 3D biosensing nanobioreactor system (Pham…Jamieson, bioRxiv 2024), which enables real-time confocal fluorescent tracking of RNA splicing in primary patient-derived cells. Methods Niche nanobioreactors were constructed from 2-port, fluoroethyl polymer (FEP) film bags that are gas permeable, chemically inert, and transparent for imaging. A porcine gelatin sponge (J&J MedTech) matrix was cut to size to function as a physical scaffold before resealing with a heat sealer under sterile conditions. Subsequently, these nanobioreactors were utilized in experiments involving ABM samples collected from routine knee and hip replacement surgeries under an IRB-approved protocol. Upon collection, mononuclear cells were isolated by Ficoll-Paque density centrifugation and subjected to CD34+ immunomagnetic bead selection. The CD34– bone marrow stromal cells were seeded directly into the nanobioreactor following magnetic bead selection. The CD34+ cells were lentivirally transduced with the dual fluorescent splicing reporter (van der Werf…Jamieson, Cell Reports Medicine 2023) and cultured for 48 hours then seeded into the same nanobioreactor as the autologous CD34– cell fraction in a 1:4 ratio. Results In ABM samples, CD34+ cells predominantly exhibited red fluorescence, consistent with exon inclusion. As a proof of concept, we co-transduced CD34+ cells with APOBEC3C overexpression, previously shown to increase exon skipping, and observed altered fluorescence patterns in real time via confocal microscopy, indicative of increased exon skipping. Notably, untransduced GFP–/RFP– cells were observed surrounding the GFP+/RFP+ CD34+ transduced cells, demonstrating the capacity of the nanobioreactor to support spatial resolution and cell-specific tracking. Conclusion We developed a 3D niche nanobioreactor system capable of live-cell tracking of RNA splicing events in primary HSPCs. Using this platform, we confirmed predominant exon inclusion in aged bone marrow and demonstrated that the dual fluorescent splicing reporter can detect exon skipping in response to APOBEC3C expression. Future work will compare ABM to young bone marrow (YBM) to assess the potential of this system as a tool to monitor age-related splicing changes and identify biomarkers of hematopoietic aging.