Circulating hematopoietic stem/progenitor cell subsets contribute to human hematopoietic homeostasis.

ABSTRACT: In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPCs) are present in the peripheral blood, but their contribution to human hematopoiesis remain unsolved. By integrating advanced immunophenotyping, single-cell transcriptional and functional profiling, and integration site (IS) clonal tracking, we unveiled the biological properties and the transcriptional features of human cHSPC subpopulations in relationship to their bone marrow (BM) counterpart. We found that cHSPCs reduced in cell count over aging and are enriched for primitive, lymphoid, and erythroid subpopulations, showing preactivated transcriptional and functional state. Moreover, cHSPCs have low expression of multiple BM-retention molecules but maintain their homing potential after xenotransplantation. By generating a comprehensive human organ-resident HSPC data set based on single-cell RNA sequencing data, we detected organ-specific seeding properties of the distinct trafficking HSPC subpopulations. Notably, circulating multi-lymphoid progenitors are primed for seeding the thymus and actively contribute to T-cell production. Human clonal tracking data from patients receiving gene therapy (GT) also showed that cHSPCs connect distant BM niches and participate in steady-state hematopoietic production, with primitive cHSPCs having the highest recirculation capability to travel in and out of the BM. Finally, in case of hematopoietic impairment, cHSPCs composition reflects the BM-HSPC content and might represent a biomarker of the BM state for clinical and research purposes. Overall, our comprehensive work unveiled fundamental insights into the in vivo dynamics of human HSPC trafficking and its role in sustaining hematopoietic homeostasis. GT patients' clinical trials were registered at ClinicalTrials.gov (NCT01515462 and NCT03837483) and EudraCT (2009-017346-32 and 2018-003842-18).

Early bone marrow alterations in patients with adenosine deaminase 2 deficiency across disease phenotypes and severities.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is a complex monogenic disease caused by recessive mutations in the ADA2 gene. DADA2 exhibits a broad clinical spectrum encompassing vasculitis, immunodeficiency, and hematological abnormalities. Yet, the impact of DADA2 on the bone marrow (BM) microenvironment is largely unexplored. OBJECTIVE: This study comprehensively examined the BM and peripheral blood of pediatric and adult patients with DADA2 presenting rheumatologic/immunologic symptoms or severe hematological manifestations. METHODS: Immunophenotyping of hematopoietic stem cells (HSCs), progenitor cells, and mature cell populations was performed for 18 patients with DADA2. We also conducted a characterization of the mesenchymal stromal cells (MSCs). RESULTS: Our study revealed a significant decrease in primitive HSCs and progenitor cells, alongside their reduced clonogenic capacity and multilineage differentiation potential. These BM defects were evident in patients with both severe and non-severe hematological manifestations, including pediatric patients, demonstrating that BM disruption can emerge silently and early on, even in patients who do not show obvious hematological symptoms. Beyond stem cells, there was a reduction in mature cell populations in the BM and peripheral blood, affecting myeloid, erythroid, and lymphoid populations. Furthermore, BM MSCs in DADA2 patients exhibited reduced clonogenic and proliferation capabilities and were more prone to undergo cellular senescence marked by elevated DNA damage. CONCLUSION: Our exploration into the BM landscape of DADA2 patients sheds light on the critical hematological dimension of the disease and emphasizes the importance of vigilant monitoring, even in the case of subclinical presentation.

Transcriptomic analysis of BM-MSCs identified EGR1 as a transcription factor to fully exploit their therapeutic potential.

Bone marrow-mesenchymal stromal cells (BM-MSCs) are key components of the BM niche, where they regulate hematopoietic stem progenitor cell (HSPC) homeostasis by direct contact and secreting soluble factors. BM-MSCs also protect the BM niche from excessive inflammation by releasing anti-inflammatory factors and modulating immune cell activity. Thanks to these properties, BM-MSCs were successfully employed in pre-clinical HSPC transplantation models, increasing the rate of HSPC engraftment, accelerating the hematological reconstitution, and reducing the risk of graft failure. However, their clinical use requires extensive in vitro expansion, potentially altering their biological and functional properties. In this work, we analyzed the transcriptomic profile of human BM-MSCs sorted as CD45-, CD105+, CD73+, and CD90+ cells from the BM aspirates of heathy-donors and corresponding ex-vivo expanded BM-MSCs. We found the expression of immune and inflammatory genes downregulated upon cell culture and selected the transcription factor EGR1 to restore the MSC properties. We overexpressed EGR1 in BM-MSCs and performed in vitro tests to study the functional properties of EGR1-overexpressing BM-MSCs. We concluded that EGR1 increased the MSC response to inflammatory stimuli and immune cell control and potentiated the MSC hematopoietic supportive activity in co-culture assay, suggesting that the EGR1-based reprogramming may improve the BM-MSC clinical use.

A GLB1 transgene with enhanced therapeutic potential for the preclinical development of ex-vivo gene therapy to treat mucopolysaccharidosis type IVB.

Mucopolysaccharidosis type IVB (MPSIVB) is a lysosomal storage disorder caused by ß-galactosidase (ß-GAL) deficiency characterized by severe skeletal and neurological alterations without approved treatments. To develop hematopoietic stem progenitor cell (HSPC) gene therapy (GT) for MPSIVB, we designed lentiviral vectors (LVs) encoding human ß-GAL to achieve supraphysiological release of the therapeutic enzyme in human HSPCs and metabolic correction of diseased cells. Transduced HSPCs displayed proper colony formation, proliferation, and differentiation capacity, but their progeny failed to release the enzyme at supraphysiological levels. Therefore, we tested alternative LVs to overexpress an enhanced ß-GAL deriving from murine (LV-enhGLB1) and human selectively mutated GLB1 sequences (LV-mutGLB1). Only human HSPCs transduced with LV-enhGLB1 overexpressed ß-GAL in vitro and in vivo without evidence of overexpression-related toxicity. Their hematopoietic progeny efficiently released ß-GAL, allowing the cross-correction of defective cells, including skeletal cells. We found that the low levels of human GLB1 mRNA in human hematopoietic cells and the improved stability of the enhanced ß-GAL contribute to the increased efficacy of LV-enhGLB1. Importantly, the enhanced ß-GAL enzyme showed physiological lysosomal trafficking in human cells and was not associated with increased immunogenicity in vitro. These results support the use of LV-enhGLB1 for further HSPC-GT development and future clinical translation to treat MPSIVB multisystem disease.

Hematopoietic reconstitution dynamics of mobilized- and bone marrow-derived human hematopoietic stem cells after gene therapy.

Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n = 7), bone marrow (n = 5) or the combination of the two sources (n = 1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study (NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome.