This repository contains the key scripts used to generate the final tables and figures featured in the accompanying manuscript:
**Zonari et al**, _Expansion of Hematopoietic Stem and Progenitor Cells from Human Mobilized Peripheral Blood for Gene Therapy_, XXX, 2025.
**Zonari E, Naldini MM, Barcella M, Volpin M, et al**, _Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells from Human Mobilized Peripheral Blood for Gene Therapy Applications_, 2026.
## Abstract
Ex vivo expansion of mobilized peripheral blood (mPB) hematopoietic stem cells (HSCs)
represents a promising approach to advance cell and gene therapy strategies yet is hampered by
loss of stem cell function when applying commonly used culture protocols. We performed an indepth
characterization of mPB expansion cultures by single cell RNA sequencing, which
highlighted differentiation trajectories with preservation of lineage fidelity in committed
progenitors. Defining a putative HSC cluster allowed an estimation of transduction efficiency in
ex vivo cultures, which correlated with long-term gene marking in xenografts and patients enrolled
in a gene therapy study. We then developed a clinically translatable, GMP-compliant process to
expand lentivirus (LV)-transduced HSCs from mPB of pediatric patients and adult donors, by
biologically informed protocol improvements of cytokine supplementation, media choice, timing
of LV transduction and combinations of small molecules preventing the activation of
differentiation programs. Our optimized process outperforms validated state-of-the-art cord blood
expansion protocols when applied to mPB. LV integration site analysis and genomic barcodebased
clonal tracking provided definitive proof for symmetric HSC self-renewal divisions
occurring during ex vivo culture. These results warrant clinical testing of this HSC
transduction/expansion process in an upcoming clinical gene therapy trial for autosomal recessive
osteopetrosis (EU CT 2024-518972-30), addressing the need of obtaining a high number of
functioning osteoclast progenitors for rapid bone marrow niche remodeling, where gene-corrected
HSC can engraft and allow long-term disease correction.
Ex vivo expansion of mobilized peripheral blood (mPB) hematopoietic stem cells
(HSCs) represents a promising approach to advance cell and gene therapy strategies
yet is hampered by loss of stem cell function when applying commonly used culture protocols. We performed in-depth characterization of mPB expansion cultures by
single cell RNA sequencing, which highlighted differentiation trajectories with
preservation of lineage fidelity in committed progenitors. Defining a putative HSC
cluster allowed an estimation of transduction efficiency in ex vivo cultures, which
correlated with long-term gene marking in xenografts and patients enrolled in a gene
therapy study. We then developed a clinically translatable, GMP-compliant process to
expand lentivirus (LV)-transduced HSCs from mPB of pediatric patients and adult
donors, by biologically informed protocol improvements of cytokine supplementation,
media choice, timing of LV transduction and combinations of small molecules
preventing the activation of differentiation programs. Our optimized process
outperforms validated state-of-the-art cord blood expansion protocols when applied to
mPB. LV integration site analysis and genomic barcode-based clonal tracking provided
definitive proof for symmetric HSC self-renewal divisions occurring during ex vivo
culture. These results warrant clinical testing of this HSC transduction/expansion
process in an upcoming clinical gene therapy trial for autosomal recessive
