Human pluripotent stem cells (hPSCs) can be used to investigate hematopoietic development and have the potential to advance cell-based therapies and to facilitate developmental biology studies. However, efficient ex vivo differentiation into hematopoietic lineages, including red blood cells (RBCs) of the erythroid lineage and immune cells such as macrophages of the myeloid lineage, is hampered by the need for precise temporal regulation of cytokines and growth factors. In this study, we developed an optimized protocol for hematopoietic lineage specification from hPSCs by fine-tuning the temporal dynamics of cytokine and growth factor applications. Prolonged mesodermal specification in the absence of hemogenic cytokines significantly enhanced the generation of hematopoietic progenitors (CD34＋CD45＋) with robust functional potential. Early administration of interleukin (IL)-3 during hematopoietic specification promoted progenitor expansion and maturation. Supplementation of bone morphogenetic protein 4 at the hematopoietic maturation stage enhanced the differentiation efficiency and preferentially drove myeloid lineage commitment toward macrophages at the expense of erythroid differentiation. The timing of erythropoietin administration was important in erythroid lineage commitment, and delayed treatment (day 10) enhanced erythroblast expansion and RBC production. By contrast, the timing of IL-6, GM-CSF, and M-CSF exposure did not significantly affect macrophage differentiation efficiency, suggesting that myeloid lineage specification follows a default pathway under optimized differentiation conditions. These findings suggest a refined, time-controlled strategy for directing hematopoietic differentiation from hPSCs, and provide insight into therapeutic blood cell production, regenerative medicine, and ex vivo modeling of hematopoietic disorders.

Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing β-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.
© 2024. The Author(s).

Iron availability is a critical factor for both bacteria and humans, and its availability significantly influences host-pathogen dynamics. As Mtb has coevolved with the human race, Mtb relentlessly tries to exploit iron from the tightly regulated iron machinery of host. Sirtuins are evolutionary conserved NAD + -dependent deacetylases involved in various cellular processes including infection. Notably, the cytosolic protein, Sirtuin 2 regulates cellular iron homeostasis in hepatocytes and after Mtb infection, SIRT2 translocates to the nucleus leading to decreased protective immune response. However, the underlying mechanism as to how Mtb exploits SIRT2 for iron acquisition remains unknown. In the current study, we observe that the decreased bacillary load in SIRT2 inhibited or knock down cells is due to low availability of iron to the bacilli. Inhibition or knockdown of SIRT2 in Mtb infected cells displays differential modulation of iron import and export proteins suggesting ongoing tussle by host to limit the bioavailability of iron to pathogen. More specifically, by flow cytometry analysis, we show significant upregulation of cell surface Apo Tf and GAPDH in infected SIRT2 inhibited macrophages. Thus, in SIRT2 depleted state, we delineate a different mechanism of iron export occurring through Apo Tf and GAPDH during infection in contrast to the classical iron exporter Fpn1. Collectively, our findings showed the importance of SIRT2-mediated iron regulation in Mtb pathogenesis and can encourage designing of novel host-targeted therapeutics.

The erythrocyte silent Duffy blood group phenotype in Africans is thought to confer resistance to Plasmodium vivax blood-stage infection. However, recent studies report P. vivax infections across Africa in Fy-negative individuals. This suggests that the globin transcription factor 1 (GATA-1) SNP underlying Fy negativity does not entirely abolish Fy expression or that P. vivax has developed a Fy-independent red blood cell (RBC) invasion pathway. We show that RBCs and erythroid progenitors from in vitro differentiated CD34 cells and from bone marrow aspirates from Fy-negative samples express a functional Fy on their surface. This suggests that the GATA-1 SNP does not entirely abolish Fy expression. Given these results, we developed an in vitro culture system for P. vivax and show P. vivax can invade erythrocytes from Duffy-negative individuals. This study provides evidence that Fy is expressed in Fy-negative individuals and explains their susceptibility to P. vivax with major implications and challenges for P. vivax malaria eradication.
Copyright © 2023. Published by Elsevier Inc.

Phenotypic drug discovery (PDD) enables the target-agnostic generation of therapeutic drugs with novel mechanisms of action. However, realizing its full potential for biologics discovery requires new technologies to produce antibodies to all, a priori unknown, disease-associated biomolecules. We present a methodology that helps achieve this by integrating computational modeling, differential antibody display selection, and massive parallel sequencing. The method uses the law of mass action-based computational modeling to optimize antibody display selection and, by matching computationally modeled and experimentally selected sequence enrichment profiles, predict which antibody sequences encode specificity for disease-associated biomolecules. Applied to a phage display antibody library and cell-based antibody selection, ∼105 antibody sequences encoding specificity for tumor cell surface receptors expressed at 103-106 receptors/cell were discovered. We anticipate that this approach will be broadly applicable to molecular libraries coupling genotype to phenotype and to the screening of complex antigen populations for identification of antibodies to unknown disease-associated targets.
© 2023 The Author(s).