We previously showed correction of sickle cell anemia (SCA) in mice utilizing a lentiviral vector (LV) expressing human γ-globin. Herein, we made a G16D mutation in the γ-globin gene to generate the G16D mutation (GbGM) LV to increase fetal hemoglobin formation. We also generated an insulated version of this LV, GbGMI, inserting a 36-bp insulator from the Foamy virus in the long terminal repeats of the LV. Preclinical batches of GbGM and GbGMI LV showed both were highly efficacious in correcting SCA in mice, with sustained gene transfer in primary transplanted SCA mice and high hematopoietic stem cell (HSC) transduction in colony-forming unit-spleen in secondary transplanted mice. CRISPR-mediated targeting of the proviruses into the LMO2 proto-oncogene showed remarkably reduced LMO2 activation by both insulated and uninsulated LV, compared to the SFFV γ-RV vector targeted to the same locus. We therefore used the GbGM LV to perform preclinical human CD34+ gene transfer. We assessed gene transfer and engraftment of human HSCs in two immunocompromised mouse models: persistent stable GbGM-transduced cell engraftment was comparable to that of untransduced cells with no detrimental effects on hematopoiesis up to 20 weeks post transplant. These robust preclinical studies in mouse and human HSCs allowed its translation into a clinical trial.
© 2025 The Authors.

Mesenchymal stromal cells (MSCs) exert broad therapeutic effects across a range of inflammatory diseases. Their mechanism of action has largely been attributed to paracrine signalling, orchestrated by an array of factors produced by MSCs that are collectively termed the "secretome". Strategies to enhance the release of these soluble factors by pre-exposure to inflammatory cytokines, a concept known as "licensing", is thought to provide a means of enhancing MSC efficacy. Yet, recent evidence shows that intravenously infused MSCs entrapped within the lungs undergo apoptosis, and their subsequent clearance by host phagocytes is essential for their therapeutic efficacy. We therefore sought to clarify the mechanisms governing regulated cell death in MSCs and how exposure to inflammatory cytokines impacts this process. Our results show that MSCs are relatively resistant to cell death induced via the extrinsic pathway of apoptosis, as well as stimuli that induce necroptosis, a form of regulated inflammatory cell death. Instead, efficient killing of MSCs required triggering of the mitochondrial pathway of apoptosis, via inhibition of the pro-survival proteins MCL-1 and BCL-XL. Apoptotic bodies were readily released by MSCs during cell disassembly, a process that was inhibited in vitro and in vivo when the apoptotic effectors BAK and BAX were genetically deleted. Licensing of MSCs by pre-exposure to the inflammatory cytokines TNF and IFN-γ increased the sensitivity of MSCs to intrinsic apoptosis in vitro and accelerated their in vivo clearance by host cells within the lungs after intravenous infusion. Taken together, our study demonstrates that inflammatory "licensing" of MSCs facilitates cell death by increasing their sensitivity to triggers of the intrinsic pathway of apoptosis and accelerating the kinetics of apoptotic cell disassembly.
© 2025. The Author(s).

Deficiency of surfactant protein-C (SPC) increases susceptibility to lung infections and injury, and suppressed expression of SPC has been associated with the severity of acute respiratory distress syndrome (ARDS). Alveolar type 2 epithelial cells (AT2) are critical for maintenance and repair of the lung. However, the role of the SPC in the regulation of AT2 cell lineage and the underlying mechanisms are not completely understood.
This study aimed to investigate the mechanisms by which SPC regulates AT2 lineages. Sftpc-/- mice were used to model the SPC deficiency in ARDS patients. We utilized three-dimensional (3D) organoids to compare AT2 lineage characteristics between wild type (WT) and Sftpc-/- mice by analyzing AT2 proliferation, alveolar type 1 cells (AT1) differentiation and CD74 expression, using colony-formation assay, immunofluorescence, flow cytometry, and immunoblots.
The results showed that Sftpc-/- mice demonstrated a reduced AT2 cell population. Influenza A virus subtype H1N1 (H1N1) infected Sftpc-/- mice demonstrated reduced AT2 proliferation and AT1 differentiation. Western blot indicated elevated levels of CD74 protein in AT2 cells of Sftpc-/- mice. Colony-forming efficiency was significantly attenuated in AT2 cells isolated from Sftpc-/- mice compared to the WT controls. Podoplanin (PDPN, a marker of AT1 cells) expression and transient cell count significantly increased in Sftpc-/- organoids. Moreover, siRNA-mediated gene silencing of CD74 in AT2 cells significantly increased AT2 proliferation and AT1 differentiation in Sftpc-/- organoids.
This study suggests that SPC regulates AT2 lineage in vitro and in vivo. The SPC might influence AT2 lineage during the lung epithelium repair by activating signaling mechanism involving CD74 receptor.

Alveolar type 2 epithelial (AT2) cells synthesize surfactant protein C (SPC) and repair an injured alveolar epithelium. A mutated surfactant protein C gene (SftpcL184Q, Gene ID: 6440) in newborns has been associated with respiratory distress syndrome and pulmonary fibrosis. However, the underlying mechanisms causing Sftpc gene mutations to regulate AT2 lineage remain unclear. We utilized three-dimensional (3D) feeder-free AT2 organoids in vitro to simulate the alveolar epithelium and compared AT2 lineage characteristics between WT (C57BL/6) and SftpcL184Q mutant mice using colony formation assays, immunofluorescence, flow cytometry, qRT-PCR, and Western blot assays. The AT2 numbers were reduced significantly in SftpcL184Q mice. Organoid numbers and colony-forming efficiency were significantly attenuated in the 3D cultures of primary SftpcL184Q AT2 cells compared to those of WT mice. Podoplanin (PDPN, Alveolar type 1 cell (AT1) marker) expression and transient cell count was significantly increased in SftpcL184Q organoids compared to in the WT mice. The expression levels of CD74, heat shock protein 90 (HSP90), and ribosomal protein S3A1 (RPS3A1) were not significantly different between WT and SftpcL184Q AT2 cells. This study demonstrated that humanized SftpcL184Q mutation regulates AT2 lineage intrinsically. This regulation is independent of CD74, HSP90, and RPS3A1 pathways.

Organoid models have become an integral part of the research methodology in the lung field. These systems allow for the study of progenitor and stem cell self-renewal, self-organization, and differentiation. Distinct models of lung organoids mimicking various anatomical regions of mature lungs have emerged in parallel to the increased gain of knowledge regarding epithelial stem and progenitor cell populations and the corresponding mesenchymal cells that populate the in vivo niche. In the distal lung, type 2 alveolar epithelial cells (AEC2s) represent a stem cell population that is engaged in regenerative mechanisms in response to various insults. These cells self-renew and give rise to AEC1s that carry out gas exchange. Multiple experimental protocols allowing the generation of alveolar organoids, or alveolospheres, from murine lungs have been described. Among the drawbacks have been the requirement of transgenic mice allowing the isolation of AEC2s with high viability and purity, and the occasional emergence of bronchiolar and bronchioalveolar organoids. Here, we provide a refined gating strategy and an optimized protocol for the generation of alveolospheres from wild-type mice. Our approach not only overcomes the need for transgenic mice to generate such organoids, but also yields a pure culture of alveolospheres that is devoid of bronchiolar and bronchioalveolar organoids. Our protocol contributes to the standardization of this important research tool.