The curative benefits of autologous and allogeneic transplantation of hematopoietic stem cells (HSCs) have been proven in various diseases. However, the low number of true HSCs that can be collected from patients and the subsequent in vitro maintenance and expansion of true HSCs for genetic correction remains challenging. Addressing this issue, we here focused on optimizing culture conditions to improve ex vivo expansion of true HSCs for gene therapy purposes. In particular, we explored the use of epigenetic regulators to enhance the effectiveness of HSC-based lentiviral (LV) gene therapy. The histone deacetylase inhibitor quisinostat and bromodomain inhibitor CPI203 each promoted ex vivo expansion of functional HSCs, as validated by xenotransplantation assays and single-cell RNA sequencing analysis. We confirmed the stealth effect of LV transduction on the loss of HSC numbers in commonly used culture protocols, whereas the addition of quisinostat or CPI203 improved the expansion of HSCs in transduction protocols. Notably, we demonstrated that the addition of quisinostat improved the LV transduction efficiency of HSCs and early progenitors. Our suggested culture conditions highlight the potential therapeutic effects of epigenetic regulators in HSC biology and their clinical applications to advance HSC-based gene correction.

An effective immune system is characterized by a diverse immune repertoire. There is a strong demand for accurate and quantitative methods to assess the diversity of the immune repertoire for various (pre-)clinical applications, including the diagnosis and prognosis of primary immune deficiencies, or to assess the response to therapy. Current strategies for immune diversity assessment generally comprise the visual inspection of the length distribution of rearranged T- and B-cell receptors. Visual inspections, however, are prone to subjective assessments and thus lead to biases. Here, we introduce ImSpectR, a unified approach to quantify immunodiversity using either spectratype, repertoire sequencing or single cell RNA sequencing data. ImSpectR scores various types of deviations from the expected length distribution and integrates these into one measure, allowing for robust quantitative comparisons of immune diversity across individuals or conditions.