Introduction of the lentiviral vector LV platform has spurre

Introduction of the lentiviral vector (LV) platform has spurred applications of gene therapy based on the transplantation of ex-vivo-engineered, autologous hematopoietic stem and progenitor cells (HSPCs) (Naldini, 2015). Recent clinical trials for patients affected by primary immunodeficiencies, hemoglobinopathies, or inborn errors of metabolism have shown high levels of gene transfer into HSPCs, which were stably maintained in multiple hematopoietic lineages until the latest follow-up, reaching up to 9 years in the earliest trial (Cartier et al., 2009; Aiuti et al., 2013; Biffi et al., 2013; Hacein-Bey Abina et al., 2015; Sessa et al., 2016). The post-transplant hematopoiesis reconstituted by polyclonal, gene-marked HSPCs has provided substantial and sustained therapeutic benefit to most treated patients to date. Contrary to the gene therapy trials performed with gamma-retroviral vectors, no adverse events related to insertional mutagenesis of semi-randomly integrating LVs have been reported to date, even though substantial integration loads, typically ranging over 5–20 million integrations per kg body weight, have now been infused into >150 patients. The side effects reported in these gene therapy trials are typically related to the conditioning regimen and include mucositis and temporary bone marrow (BM) aplasia. Trials employing full myeloablation and BM-derived transduced CD34+ cells often showed more prolonged grade 4 neutropenia and thrombocytopenia than allogeneic BM transplantation, despite administering at least similar doses of CD34+ cells/kg (Sessa et al., 2016). Delayed recovery may be caused by the ex vivo culture of the cell therapy product, which typically lasts more than 60 hr (Aiuti et al., 2013; Biffi et al., 2013). Indeed, experimental evidence has accumulated that cultured HSPCs progressively lose engraftment potential by recruitment into orexin and loss of adhesion molecules, thus impeding their homing into the niche and driving lineage commitment and differentiation (Glimm et al., 2000; Kallinikou et al., 2012; Larochelle et al., 2012). This notion contrasts with recent reports on successful ex vivo cord blood (CB) expansion leading to accelerated hematologic recovery in patients (reviewed in Kiernan et al., 2016). Differences among HSPC sources (CB versus BM or mobilized peripheral blood [mPB]) may contribute to diverging outcomes, and a complete understanding is key to harnessing emerging CB expansion protocols for ex vivo gene transfer procedures, which utilize BM or mPB HSPCs. Moreover, CD34+ HSPCs comprise a heterogeneous mixture of progenitors at various stages of lineage commitment, the composition of which changes according to age, cell source, and mobilization procedure, and studies investigating the impact of ex vivo culture on defined subpopulations are lacking. Only a minute fraction of these CD34+ cells corresponds to long-term (LT) hematopoietic stem cells (HSCs). Limiting-dilution transplants into immunodeficient mice indicate that no more than 0.1% of lineage-negative CB cells (50%–75% CD34+) engraft longterm (McDermott et al., 2010). In line with an even lower HSC frequency in BM or mPB CD34+ cells, capture/re-capture statistics performed on longitudinally sampled LV integration sites from patients treated by gene therapy indicate that ∼0.01% of the infused CD34+ cells contribute to long-term hematopoiesis (Aiuti et al., 2013; Biffi et al., 2013; Biasco et al., 2015). These data indicate that there is a substantial margin to more precisely tailor gene transfer to LT-HSCs as opposed to the bulk of CD34+ cells, adapting ex vivo manipulation specifically to the requirements of the therapeutically relevant cell subsets. Several landmark studies have identified surface markers that allow prospective isolation of functionally diverse HSPC subsets (Majeti et al., 2007; Notta et al., 2011). However, most of these studies were done on CB cells that did not undergo ex vivo culture, making the results not necessarily representative of the cells typically used in HSPC gene therapy trials. Furthermore, most studies functionally validating HSC markers employed binary sorting gates (markerpositive versus markernegative). Given that antibody staining for many HSPC markers, such as CD38, CD49f, and CD90, results in a gradient of cells with increasing antigen density rather than clearly segregating two populations, large proportions of HSPCs with an intermediate phenotype have not been analyzed in these functional assays.