The Hippo-YAP/TAZ-TEAD axis promotes multipotency in bone marrow stromal cells.

The mechanisms by which bone marrow stromal cells (BMSCs) maintain multilineage potency in vitro remain elusive. To identify the transcriptional regulatory circuits that contribute to BMSC multipotency, we performed paired single-nucleus multiomics of the expansion of freshly isolated BMSCs and of BMSCs undergoing tri-lineage differentiation. By computationally reconstructing the regulatory programs associated with initial stages of differentiation and early expansion, we identified the TEAD family of transcription factors, which is inhibited by Hippo signaling, as highly active in the BMSC in vitro multipotent state.

Neuronal nitric oxide synthase is required for erythropoietin stimulated erythropoiesis in mice.

Erythropoietin (EPO), produced in the kidney in a hypoxia responsive manner, is required for red blood cell production. In non-erythroid tissue, EPO increases endothelial cell production of nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) that regulates vascular tone to improve oxygen delivery. This contributes to EPO cardioprotective activity in mouse models.

Quantitative Craniofacial Analysis and Generation of Human Induced Pluripotent Stem Cells for Muenke Syndrome: A Case Report.

In this case report, we focus on Muenke syndrome (MS), a disease caused by the p.Pro250Arg variant in fibroblast growth factor receptor 3 (FGFR3) and characterized by uni- or bilateral coronal suture synostosis, macrocephaly without craniosynostosis, dysmorphic craniofacial features, and dental malocclusion. The clinical findings of MS are further complicated by variable expression of phenotypic traits and incomplete penetrance.

Lineage-specific differentiation of osteogenic progenitors from pluripotent stem cells reveals the FGF1-RUNX2 association in neural crest-derived osteoprogenitors.

Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported.

Gene Editing in Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is an immune deficiency characterized by defects in the production of microbicidal reactive oxygen species (ROS) by the phagocytic oxidase (phox) enzyme complex in neutrophils. We have previously described targeted gene editing strategies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases for gene targeting with homology-directed repair in CGD patient stem cells to achieve functional restoration of expression of phox genes and NADPH oxidase activity in differentiated neutrophils. In this chapter, we describe detailed protocols for targeted gene editing in human-induced pluripotent stem cells and hematopoietic stem cells and for subsequent differentiation of these stem cells into mature neutrophils, as well as assays to characterize neutrophil identity and function including flow cytometry analysis of neutrophil surface markers, intracellular staining for phox proteins, and analysis of ROS generation.

Gene-edited pseudogene resurrection corrects p47-deficient chronic granulomatous disease.

Pseudogenes are duplicated genes with mutations rendering them nonfunctional. For single-gene disorders with homologous pseudogenes, the pseudogene might be a target for genetic correction. Autosomal-recessive p47-deficient chronic granulomatous disease (p47-CGD) is a life-threatening immune deficiency caused by mutations in , a gene with 2 pseudogenes, and .

Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction.

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene, resulting in absent or defective gp91 protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91 expression and ROS production in iPSC-derived granulocytes.

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers.

Nonhuman primate (NHP) induced pluripotent stem cells (iPSCs) offer the opportunity to investigate the safety, feasibility, and efficacy of proposed iPSC-derived cellular delivery in clinically relevant in vivo models. However, there is need for stable, robust, and safe labeling methods for NHP iPSCs and their differentiated lineages to study survival, proliferation, tissue integration, and biodistribution following transplantation. Here we investigate the utility of the adeno-associated virus integration site 1 (AAVS1) as a safe harbor for the addition of transgenes in our rhesus macaque iPSC (RhiPSC) model.

Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis.

In vitro generation of mature neutrophils from human induced pluripotent stem cells (iPSCs) requires hematopoietic progenitor development followed by myeloid differentiation. The purpose of our studies was to extensively characterize this process, focusing on the critical window of development between hemogenic endothelium, hematopoietic stem/progenitor cells (HSPCs), and myeloid commitment, to identify associated regulators and markers that might enable the stem cell field to improve the efficiency and efficacy of iPSC hematopoiesis. We utilized a four-stage differentiation protocol involving: embryoid body (EB) formation (stage-1); EB culture with hematopoietic cytokines (stage-2); HSPC expansion (stage-3); and neutrophil maturation (stage-4).

An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease.

There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD.

Generation of functionally mature neutrophils from induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are pluripotent stem cells established from somatic cells. The capability of iPSCs to differentiate into any mature cell lineage under the appropriate conditions allows for modeling of cell processes as well as disease states. Here, we describe an in vitro method for generating functional mature neutrophils from human iPSCs.

Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells.

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34(+) hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34(+) HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60(+), yielding 5.