Mucopolysaccharidosis type IVA (MPSIVA) is a lysosomal storage disease (LSD) caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which causes the accumulation of keratan sulphate (KS) and chondroitin sulphate (CS). Patients with MPSIVA typically present with severe skeletal and joint disorders, which are not addressed by conventional therapies. Currently, no animal model accurately replicates the human disease, hindering the development of novel therapeutic interventions. To overcome this limitation, we established, by CRISPR-Cas9 technology, a Galns-/- mouse model that expresses a non-functional enzyme and accumulates CS and KS in the urine, plasma and distinct tissues, and glycosaminoglycans in the spleen. The mice exhibit shortened long bones, trabecular bone alterations and skeletal abnormalities in the growth plate. Additionally, we observed increased levels of inflammatory and oxidative markers in visceral organs and plasma. Our newly developed model of MPSIVA demonstrates clear and quantifiable signs of skeletal alterations, providing novel means of assessment of the safety and efficacy of innovative therapies, including hematopoietic stem and progenitor cell gene therapy, which has recently been shown to provide a beneficial effect on skeletal alterations in Hurler syndrome.

[This corrects the article DOI: 10.3389/fgene.2022.1056114.].

Background: Deficiency of adenosine deaminase 2 (DADA2) is complex monogenic disease caused by recessive mutations in the ADA2 gene. DADA2 exhibits a broad clinical spectrum encompassing vasculitis, immunodeficiency, and hematologic 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 with rheumatologic/immunologic symptoms or severe hematologic 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 mesenchymal stromal cells. 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 nonsevere hematologic manifestations, including pediatric patients, demonstrating that BM disruption can emerge silently and early on, even in patients who do not show obvious hematologic 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 mesenchymal stromal cells in patients with DADA2 exhibited reduced clonogenic and proliferation capabilities and were more prone to undergo cellular senescence marked by elevated DNA damage. Conclusions: Our exploration into the BM landscape of patients with DADA2 sheds light on the critical hematologic dimension of the disease and emphasizes the importance of vigilant monitoring, even in the case of subclinical presentation. (J Allergy Clin Immunol 2025;155:616-27.)

Mucopolysaccharidosis type I (MPS-I) is a rare pediatric disease caused by mutations in the α-L-iduronidase (IDUA) gene encoding for a lysosomal enzyme involved in glycosaminoglycan metabolism. While newborns with the severe Hurler variant are usually asymptomatic at birth, progressive disease manifestations emerge early in life. Since previous studies on lentiviral vector gene therapy (GT) in Hurler patients have demonstrated superior metabolic correction and early beneficial clinical effects, we investigated whether applying this GT approach during the neonatal period could be effective in preventing disease pathology before it becomes irreversible. Thus, newborn MPS-I mice were transplanted with affected bone marrow-derived progenitor cells transduced with an IDUA-encoding lentiviral vector. Treated animals displayed increased IDUA levels, significantly reducing substrate accumulation in analyzed organs, indicating metabolic correction. Skeletal manifestations, typically resistant to conventional therapies, showed improvements at radiographic and histological levels post-treatment. Additionally, a decrease in brain cortex vacuolization and inflammation suggested neurological amelioration. Overall, this study provides a proof of principle demonstrating the effectiveness of neonatal ex vivo GT in MPS-I mice and supports its potential for further optimization at the pre-clinical level.

Necrotizing enterocolitis (NEC) presents a life-threatening intestinal emergency primarily affecting premature infants in neonatal intensive care units. This disease is a significant cause of morbidity and mortality in such newborns. NEC involves inflammation, bacterial overgrowth, and cell death affecting a portion of the bowel wall, commonly the distal ileum. Despite advances in neonatal care, the pathogenesis of NEC remains not fully understood. Although its pathogenesis remains not fully elucidated, the upregulation of Toll-like receptor 4 in the premature intestinal epithelium is recognized as a key factor contributing to epithelial barrier dysfunction. Recent studies have explored the potential of mesenchymal stromal cells (MSCs) in NEC management. MSCs are up-and-coming candidates for preclinical NEC models as they possess anti-inflammatory and immune modulatory properties, which reduce inflammation, help increase intestinal integrity, and help tissue repair. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) have proven impactful in most experimental settings, mitigating injury from NEC and facilitating intestinal development. While MSC therapies hold promise, challenges remain regarding inconsistent isolation and expansion of these cells, variable differentiation, and possible tumorigenicity in vivo. As a result, the focus has been drawn to MSC-derived secretome, especially exosomes, as a novel cell-free therapeutic. These bioactive molecules transported by exosomes can reduce inflammation and facilitate tissue repair, providing a safer and more plausible alternative to treating NEC. Further research is needed to standardize secretome production and evaluate its clinical efficacy and safety. This review aims to provide a comprehensive overview of the mechanisms of action and the available research on human (h)BM-MSCs to support the development of studies that may prevent and/or treat the disease.

The hematopoietic stem cell and multipotent progenitor (HSC/MPP) pool dynamically responds to stress to adapt blood output to specific physiological demands. In β-thalassemia (Bthal), severe anemia and ineffective erythropoiesis generate expansion of erythroid precursors and a chronic stress status in the bone marrow (BM) microenvironment. However, the response to the BM altered status at the level of the HSC/MPP compartment in terms of lineage commitment has not been investigated. Bulk and single-cell RNA-sequencing reveal that Bthal HSCs/MPPs are expanded and activated with enhanced priming along the whole Ery differentiation trajectory. Consistently, HSC/MPP showed an altered TGFβ expression and autophagy transcriptional signatures along with a declined dormancy state. We discovered that the altered TGFβ signaling fosters the Ery potential of HSCs by reducing their autophagic levels, and in vivo stimulation of autophagy is sufficient to rescue the imbalance of the HSC compartment. Our findings identify the interplay between TGFβ and HSC autophagy as a key driver in the context of non-malignant hematopoiesis.

Mucopolysaccharidosis type IVB (MPSIVB) is a lysosomal storage disorder caused by β-galactosidase (β-GAL) deficiency characterized by severe skeletal and neurological alterations without approved treatments. To develop hematopoietic stem progenitor cell-gene therapy (HSPC-GT) for MPSIVB, we designed lentiviral vectors (LVs) encoding human β-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 β-GAL deriving from murine (LV-enhGLB1) and human selectively mutated GLB1 sequences (LV-mutGLB1). Only human HSPCs transduced with LV-enhGLB1 overexpressed β-GAL in vitro and in vivo without evidence of overexpression-related toxicity. Their hematopoietic progeny efficiently released β-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 β-GAL contribute to the increased efficacy of LV-enhGLB1. Importantly, the enhanced β-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.

Mucopolysaccharidosis type IVB (MPSIVB) is a lysosomal storage disorder caused by (3-galactosidase ((3 (3-GAL) deficiency fi ciency 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 (3-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 (3-GAL deriving from murine (LV-enhGLB1) and human selectively mutated GLB1 sequences (LV-mutGLB1). Only human HSPCs transduced with LVenhGLB1 overexpressed (3-GAL in vitro and in vivo without evidence of overexpression-related toxicity. Their hematopoietic progeny efficiently fi ciently released (3-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 (3-GAL contribute to the increased efficacy fi cacy of LV-enhGLB1. Importantly, the enhanced (3-GAL enzyme showed physiological lysosomal trafficking fi cking in human cells and was not associated with increased immunogenicity in vitro. These results support the use of LVenhGLB1 for further HSPC-GT development and future clinical translation to treat MPSIVB multisystem disease.

Stress conditions affect hematopoietic stem cell (HSC) fate and blood lineage output, indicating that hematopoiesis is flexible to adapt to different stimuli. In β-thalassemia (Bthal), the severe anemia and the ineffective erythropoiesis generate a chronic stress status of the bone marrow (BM) microenvironment which resulted altered both in cellular and molecular components, affecting HSC biology. Our aim is to understand how this stress microenvironment have an impact on primitive HSC influencing their lineage commitment and the hematopoietic process. Transcriptome profile, from bulk RNAseq and scRNAseq, highlighted that Bthal HSCs/MPPs (hematopoietic stem cells/multipotent progenitors), differently from healthy donor (HD) ones, are in an activated state with an enhanced erythroid (Ery) priming along the Ery differentiation trajectory. Specific pathways contributing to maintain a quiescent state, such as TGFβ pathway, were deregulated thus suggesting a reduced long-term repopulating potential. Along with this finding, an enrichment of transcriptional processes and a low degree of dormancy compatible with a fast kinetic of quiescence exit were evident. Immunophenotype and functional studies showed an altered primitive compartment composition with an increased MPP frequency in Bthal patients compared to HDs, marked by an enhanced Ery potential. In vivo studies showed that an altered balance of TGFβ/BMP signals in the BM microenvironment fosters the Ery potential of HSCs by reducing their autophagic activity. In vivo targeting Bthal HSCs with specific drug rebalances their lineage cell fate. Our data demonstrate for the first time that stress signals in chronic anemia reduce HSC dormancy and autophagy, promoting a skewed and accelerated Ery lineage commitment. This concept of hematopoiesis opens the way for a deeper knowledge of HSC biology in non-malignant conditions.

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.