A Comprehensive High Resolution Scrna-Seq Analytical Platform for Characterizing Human Hematopoietic Stem/Progenitor Cells States in Ex Vivo Drug Products
Authors: Baricordi Cristina , Loperfido Mariana , Yan Aimin , Barbarossa Luigi , Segura Mercedes , Golipour Azadeh , Mason Chris , Biasco Luca 
Published: February 2021

Human hematopoietic/stem progenitor CD34+ cells (HSPCs) are the core components of ex vivo lentiviral gene therapy (GT) for the treatment of rare monogenic diseases. Assessment of CD34+ composition before and after genetic modification as well as in vivo upon transplantation in the patient is a critical parameter to establish the efficacy of GT. We recently showed how the currently available immunophenotyping markers for the identification of HSPC subpopulations do not allow a clear-cut identification of the diversity of cell states within the CD34+ compartment. Furthermore, due to the instability of such markers upon in vitro manipulation, immunophenotyping does not offer a reliable assay to measure the HSPC content within the CD34+ Drug Product (DP). We have combined immunophenotyping, FACS sorting, and selective in vitro manipulation conditions with single-cell RNA-Sequencing (scRNA-Seq) capturing a picture of unprecedent clarity of CD34+ cells dynamics before and after expansion and genetic modification.
Using the 10X Chromium System we firstly generated a comprehensive baseline map of CD34+ cell states (total of 77,692 single cells) from the 3 main sources of human HSPCs: bone marrow (BM), mobilized peripheral blood (G-CSF+plerixafor mobilization, MPB) and cord blood (CB). The combination of unsupervised clustering and expression of lineage-associated gene signatures allowed the identification of 11 cell states within these bulk CD34+ populations, and unveiled key differences in their composition depending on different cells' origin.
We then investigated the dynamic changes of MPB CD34+ cells, the most advanced and clinically relevant HSPC cell source, upon ex vivo manipulation. To this aim we cultured bulk CD34+ cells and 7 FACS-sorted CD34+ subpopulations independently for 40 hours in presence of SCF, IL3, FLT3-L and TPO. Classical immunophenotyping of the CD34+ population before and after culture showed a substantial enrichment of cells with a CD34+CD38- profile suggesting a selective in vitro maintenance and expansion of primitive progenitors. However, immunophenotyping of individually cultured HSPC subsets revealed that the major factor contributing to this observation is instead the progressive loss of CD38 expression by CD38+ committed progenitors. Thus, the currently available CD34+ immunophenotyping characterization is not designed to provide an accurate assessment of the true DP composition before infusion into patients.
By means of scRNA-Seq analysis of 81,126 single cells we were able to identify with high granularity the changes in cell state of each HSPC subset during culture. Firstly, the combined scRNA-seq analysis on sorted HSPC subsets at baseline allowed to draw the highest resolution map of MPB CD34+ cell states available to date (total of 24,736 single cells) and allowed the identification of 7 novel transcriptional states which are independent from the original cell fractionation based on the known CD34+ surface markers. Using these transcriptional signatures we interrogated the dynamic changes of each HSPC subset looking for transcriptional divergences/similarities between the baseline and the end of culture cell states. We could identify the trajectories of each subpopulation towards different hematopoietic differentiation stages upon in vitro manipulation. As expected HSCs and MPPs were the only subsets which maintained after culture a fraction of cells retaining the original primitive transcriptional signature. We believe that in this way we could quantify with unprecedented accuracy the putative fraction of cells in the DP that preserved a multipotent long-term potential vs the one that progressed towards myeloid differentiation identifying in the process variable degrees of cell maturation.
We are currently exploring specific molecular signatures within the HSC compartment, which remained stable before and after culture, for the identification of novel more reliable markers of human HSC to be validated in vitro and in vivo.
Overall our analytical platform provides the basis for unravelling and comparing the impact of multiple conditions of cell cultures and gene modification on the HSPC DP. To our knowledge this constitutes the most advanced suite for the comprehensive characterization of CD34+ cells states, with potential applications spanning manufacturing, pre-clinical and clinical stages.
CB and ML: equal contribution

Disclosures
Baricordi: AVROBIO Inc: Current equity holder in publicly-traded company, Other: Trainee. Loperfido:AVROBIO Inc: Current equity holder in publicly-traded company, Other: Trainee. Yan:AVROBIO Inc: Current Employment. Barbarossa:AVROBIO Inc: Other: Trainee. Segura:AVROBIO Inc: Current Employment. Golipour:AVROBIO Inc: Current Employment, Current equity holder in publicly-traded company. Mason:AVROBIO Inc: Current Employment, Current equity holder in private company. Biasco:AVROBIO Inc: Current Employment, Current equity holder in publicly-traded company.