Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production.

Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-ϕ activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34(+) HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.

Fluorinated Silane-Modified Filtroporation Devices Enable Gene Knockout in Human Hematopoietic Stem and Progenitor Cells.

Intracellular delivery technologies that are cost-effective, non-cytotoxic, efficient, and cargo-agnostic are needed to enable the manufacturing of cell-based therapies as well as gene manipulation for research applications. Current technologies capable of delivering large cargoes, such as plasmids and CRISPR-Cas9 ribonucleoproteins (RNPs), are plagued with high costs and/or cytotoxicity and often require substantial specialized equipment and reagents, which may not be available in resource-limited settings. Here, we report an intracellular delivery technology that can be assembled from materials available in most research laboratories, thus democratizing access to intracellular delivery for researchers and clinicians in low-resource areas of the world. These filtroporation devices permeabilize cells by pulling them through the pores of a cell culture insert by the application of vacuum available in biosafety cabinets. In a format that costs less than $10 in materials per experiment, we demonstrate the delivery of fluorescently labeled dextran, expression plasmids, and RNPs for gene knockout to Jurkat cells and human CD34+ hematopoietic stem and progenitor cell populations with delivery efficiencies of up to 40% for RNP knockout and viabilities of >80%. We show that functionalizing the surfaces of the filters with fluorinated silane moieties further enhances the delivery efficiency. These devices are capable of processing 500,000 to 4 million cells per experiment, and when combined with a 3D-printed vacuum application chamber, this throughput can be straightforwardly increased 6-12-fold in parallel experiments.

Lipid-Bicelle-Coated Microfluidics for Intracellular Delivery with Reduced Fouling.

Innovative technologies for intracellular delivery are ushering in a new era for gene editing, enabling the utilization of a patient's own cells for stem cell and immunotherapies. In particular, cell-squeezing platforms provide unconventional forms of intracellular delivery, deforming cells through microfluidic constrictions to generate transient pores and to enable effective diffusion of biomolecular cargo. While these devices are promising gene-editing platforms, they require frequent maintenance due to the accumulation of cellular debris, limiting their potential for reaching the throughputs necessary for scalable cellular therapies. As these cell-squeezing technologies are improved, there is a need to develop next-generation platforms with higher throughput and longer lifespan, importantly, avoiding the buildup of cell debris and thus channel clogging. Here, we report a versatile strategy to coat the channels of microfluidic devices with lipid bilayers based on noncovalent lipid bicelle technology, which led to substantial improvements in reducing cell adhesion and protein adsorption. The antifouling properties of the lipid bilayer coating were evaluated, including membrane uniformity, passivation against nonspecific protein adsorption, and inhibition of cell attachment against multiple cell types. This surface functionalization approach was applied to coat constricted microfluidic channels for the intracellular delivery of fluorescently labeled dextran and plasmid DNA, demonstrating significant reductions in the accumulation of cell debris. Taken together, our work demonstrates that lipid bicelles are a useful tool to fabricate antifouling lipid bilayer coatings in cell-squeezing devices, resulting in reduced nonspecific fouling and cell clogging to improve performance.

Precision Medicine in Pediatric Neurooncology: A Review.

Central nervous system tumors are the leading cause of cancer related death in children. Despite much progress in the field of pediatric neurooncology, modern combination treatment regimens often result in significant late effects, such as neurocognitive deficits, endocrine dysfunction, secondary malignancies, and a host of other chronic health problems. Precision medicine strategies applied to pediatric neurooncology target specific characteristics of individual patients' tumors to achieve maximal killing of neoplastic cells while minimizing unwanted adverse effects. Here, we review emerging trends and the current literature that have guided the development of new molecularly based classification schemas, promising diagnostic techniques, targeted therapies, and delivery platforms for the treatment of pediatric central nervous system tumors.

HIF1α-induced PDGFRß signaling promotes developmental HSC production via IL-6 activation.

Hematopoietic stem cells (HSCs) have the ability to both self-renew and differentiate each of the mature blood cell lineages and thereby reconstitute the entire blood system. Therefore, HSCs are therapeutically valuable for treatment of hematological malignances and bone marrow failure. We showed recently that transient glucose elevation elicited dose-dependent effects on HSCs through elevated metabolic activity and subsequent reactive oxygen species-mediated induction of Hypoxia-Inducible Factor 1α (Hif1α). Platelet-Derived Growth Factor B (pdgfb), a Hif1α-target, and its receptor, pdgfrb, were significantly upregulated in response to metabolic stimulation. Although the function of PDGF signaling is well established in vascular development, its role in hematopoiesis is less understood. Exposure to either a pan-PDGF inhibitor or a PDGFRß-selective antagonist in the context of Hif1α stimulation blocked elevations in hematopoietic stem and progenitor cell (HSPC) formation as determined by runx1;cmyb whole-mount in situ hybridization (WISH) and HSPC-reporter flow cytometry analysis. Similar results were observed for morpholino (MO) knockdown of pdgfrb or dominant-negative pdgfrb expression, indicating that PDGFRß signaling is a key downstream mediator of Hif1α-mediated induction of HSPCs. Notably, overexpression of Pdgfb ligand enhanced HSPC numbers in the aorta-gonado-mesonephros (AGM) at 36 hours postfertilization (hpf) and in the caudal hematopoietic tissue at 48 hpf. A survey of known PDGF-B/PDGFRß regulatory targets by expression analysis revealed a significant increase in inflammatory intermediates, including Interleukin 6 (IL-6) and its receptor (IL-6R). MO-mediated knockdown of il6 or chemical inhibition of IL-6R antagonized the effect of Pdgfb overexpression. Furthermore, epistatic analysis of IL-6/IL-6R function confirmed activity downstream of Hif1α. Together, these findings define a Hif1α-regulated signaling axis mediated through PBFGB/PDGFRß and IL-6/IL-6R that acts to control embryonic HSPC production.

Developmental Vitamin D Availability Impacts Hematopoietic Stem Cell Production.

Vitamin D insufficiency is a worldwide epidemic affecting billions of individuals, including pregnant women and children. Despite its high incidence, the impact of active vitamin D3 (1,25(OH)D3) on embryonic development beyond osteo-regulation remains largely undefined. Here, we demonstrate that 1,25(OH)D3 availability modulates zebrafish hematopoietic stem and progenitor cell (HSPC) production. Loss of Cyp27b1-mediated biosynthesis or vitamin D receptor (VDR) function by gene knockdown resulted in significantly reduced runx1 expression and Flk1+cMyb+ HSPC numbers. Selective modulation in vivo and in vitro in zebrafish indicated that vitamin D3 acts directly on HSPCs, independent of calcium regulation, to increase proliferation. Notably, ex vivo treatment of human HSPCs with 1,25(OH)D3 also enhanced hematopoietic colony numbers, illustrating conservation across species. Finally, gene expression and epistasis analysis indicated that CXCL8(IL-8) was a functional target of vitamin D3-mediated HSPC regulation. Together, these findings highlight the relevance of developmental 1,25(OH)D3 availability for definitive hematopoiesis and suggest potential therapeutic utility in HSPC expansion.