Developmental Vitamin D Availability Impacts Hematopoietic Stem Cell Production

SUMMARY 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

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

Cell Squeezing Devices for Intracellular Delivery

Cost-effective, efficient, vector-free, and nontoxic intracellular delivery technologies are required for a broad range of clinical and laboratory applications. In the context of the recent revolution in precise genomic editing enabled by the discovery and engineering of editing machinery (e.g., CRISPR/Cas9, base editors, etc.), introducing biomolecular payloads into target cells with high efficiency and minimal disruption to cell homeostasis has been become paramount. Methods for cell permeabilization that employ mechanical deformation have gained popularity as they may be less disruptive to cell biology than (traditional) electroporation. However, these methods require acquisition of specialized equipment and extensive training, which can be a hurdle for laboratories, especially those located in low resource areas of the world. Here, we report the development of a table-top “filtroporation” cell-processing technology that is efficient, non-toxic, scalable, and that does not require additional costly specialized materials or equipment. This platform utilizes commercially available poly(ethylene terephthalate) cell culture inserts and laboratory house vacuum. As cells are passed through the inserts’ pores, they become transiently permeabilized. Our studies show that this platform can deliver CRISPR/Cas9 ribonucleoproteins (RNPs) to effect gene knockout in CD34+ hematopoietic stem and progenitor cells (HSPCs) with high efficiency and minimal toxicity targeting the genomic locus relevant for treatment of β hemoglobinopathies, beta globin (HBB). We performed RNA-Seq and compared our results to electroporation, showing that filtroporation induces less apoptosis and inflammation, and preserves stem cell self-renewal potential. Additionally, we studied the membrane repair kinetics following filtroporation-induced permeabilization and found that the repair process occurs within 30 s of treatment. Our knockout studies revealed that membrane repair is, at least partly, mediated by inward scission of the membrane, suggesting that pores formed on the surface of treated cells are in the 50 – 100 nm range. The commercially available membranes employed for filtroporation are limited by low porosity (<1%) and the paucity of pore diameters available from commerical sources. We expand the reach of filtroporation by manufacturing custom membranes by microfabrication. We retain full control over pore diameter, distribution, and membrane thickness, enabling customization to apply filtration-mediated cell permeabilization to any cell type, as well as to scale up for clinical applications. We report our efforts to improve upon existing protocols for manufacturing of low- and high-porosity parylene C films

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

Exploring the Bottom-Up Growth of Anisotropic Gold Nanoparticles from Substrate-Bound Seeds in Microfluidic Reactors

We developed an unconventional seed-mediated in situ synthetic method, whereby gold nanostars are formed directly on the internal walls of microfluidic reactors. The dense plasmonic substrate coatings were grown in microfluidic channels with different geometries to elucidate the impacts of flow rate and profile on reagent consumption, product morphology, and density. Nanostar growth was found to occur in the flow-limited regime and our results highlight the possibility of creating shape gradients or incorporating multiple morphologies in the same microreactor, which is challenging to achieve with traditional self-assembly. The plasmonic-microfluidic platforms developed herein have implications for a broad range of applications, including cell culture/sorting, catalysis, sensing, and drug/gene delivery.The authors acknowledge the use of instruments at the Electron Imaging Center for NanoMachines supported by NIH (1S10RR23057) and CNSI at UCLA and technical assistance by Ivo Atanasov. We also thank Ms. Lisa Kawakami for the fabrication of the channel masters. G.A.V.-W. thanks the UCLA graduate division for funding through the University of California Office of the President Dissertation Year Fellowship. N.C. acknowledges support from the National Institute of Biomedical Imaging and Bioengineering (R00EB028325). L.S. is supported by the 2020 Postdoctoral Junior Leader-Incoming Fellowship by “la Caixa” Foundation (ID 100010434, code LCF/BQ/PI20/11760028) and by a 2022 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation. S.J.J. acknowledges support from the National Institutes of Health (NIH) Common Fund through a NIH Director’s Early Independence Award, Grant DP5OD028181. S.J.J. and G.A.V.-W. acknowledge support through a Scholar Award from the Hyundai Hope on Wheels Foundation (20193309).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Exploring the Bottom-Up Growth of Anisotropic Gold Nanoparticles from Substrate-Bound Seeds in Microfluidic Reactors

We developed an unconventional seed-mediated in situ synthetic method, whereby gold nanostars are formed directly on the internal walls of microfluidic reactors. The dense plasmonic substrate coatings were grown in microfluidic channels with different geometries to elucidate the impacts of flow rate and profile on reagent consumption, product morphology, and density. Nanostar growth was found to occur in the flow-limited regime and our results highlight the possibility of creating shape gradients or incorporating multiple morphologies in the same microreactor, which is challenging to achieve with traditional self-assembly. The plasmonic-microfluidic platforms developed herein have implications for a broad range of applications, including cell culture/sorting, catalysis, sensing, and drug/gene delivery

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