Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture

Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60–90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27–90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.National Institutes of Health (U.S.) (Grant EB000244)National Institutes of Health (U.S.) (Grant DE016561)Alnylam Pharmaceuticals (Firm

OrganoidChip facilitates hydrogel-free immobilization for fast and blur-free imaging of organoids

Organoids are three-dimensional structures of self-assembled cell aggregates that mimic anatomical features of in vivo organs and can serve as in vitro miniaturized organ models for drug testing. The most efficient way of studying drug toxicity and efficacy requires high-resolution imaging of a large number of organoids acquired in the least amount of time. Currently missing are suitable platforms capable of fast-paced high-content imaging of organoids. To address this knowledge gap, we present the OrganoidChip, a microfluidic imaging platform that incorporates a unique design to immobilize organoids for endpoint, fast imaging. The chip contains six parallel trapping areas, each having a staging and immobilization chamber, that receives organoids transferred from their native culture plates and anchors them, respectively. We first demonstrate that the OrganoidChip can efficiently immobilize intestinal and cardiac organoids without compromising their viability and functionality. Next, we show the capability of our device in assessing the dose-dependent responses of organoids’ viability and spontaneous contraction properties to Doxorubicin treatment and obtaining results that are similar to off-chip experiments. Importantly, the chip enables organoid imaging at speeds that are an order of magnitude faster than conventional imaging platforms and prevents the acquisition of blurry images caused by organoid drifting, swimming, and fast stage movements. Taken together, the OrganoidChip is a promising microfluidic platform that can serve as a building block for a multiwell plate format that can provide high-throughput and high-resolution imaging of organoids in the future.This article is published as Moshksayan, K., Harihara, A., Mondal, S. et al. OrganoidChip facilitates hydrogel-free immobilization for fast and blur-free imaging of organoids. Sci Rep 13, 11268 (2023). https://doi.org/10.1038/s41598-023-38212-8. Posted with permission

Discovery and Validation of Immunological Biomarkers in Milk for Health Monitoring of Dairy Cows - Results from a Multiomics Approach

<p>At onset of milk production and in early lactation highly producing dairy cows are most susceptible for inflammatory diseases due to functional suppression of immune cells. Intensive supervision of the animals is essential and implementation of new technologies to on-farm routines will be the next step to provide automation and improvement of herd health monitoring programs. Objective of our study was to identify and validate immunological biomarkers in milk that indicate extra-mammary inflammatory diseases to characterize the general health status of highly-producing dairy cows. In total 89 healthy and 75 diseased animals (German Holstein cows) were included. Diseases were distinguished by either systemic (extra-mammary) occurrence or those affecting the mammary gland (mastitis) and further classified by their severity. For protein biomarker discovery we used a top-down approach to narrow down a broad range of secreted gene products of the milk cell transcriptome (microarray) and proteome to a few promising candidates which were validated using real-time PCR and ELISA. The most promising biomarker candidates were statistically evaluated. Receiver operating characteristic analysis revealed haptoglobin, secretory component, lactoferrin and vascular endothelial growth factor showing the highest discriminatory capability for diseased vs. healthy cows. Values for sensitivity at a specificity of 94% were 82% for haptoglobin, 59% for secretory component, 55% for lactoferrin and 67% for vascular endothelial growth factor. Statistical evaluation by multinomial logistic regression and k-nearest neighbor method confirmed haptoglobin as the best single-use biomarker. In combination with secretory component or lactoferrin an increase in overall sensitivity or specificity, depending on the classification method, could be achieved. The application of the validated health biomarkers in combination with an easy high-throughput detection system would offer a solution to adapt dairy herd management to changing requirements on animal welfare, farming efficiency, milk supply and food safety in modern agriculture.</p&gt

The Display of Single-Domain Antibodies on the Surfaces of Connectosomes Enables Gap Junction-Mediated Drug Delivery to Specific Cell Populations

Gap junctions, transmembrane protein channels that directly connect the cytoplasm of neighboring cells and enable the exchange of molecules between cells, are a promising new frontier for therapeutic delivery. Specifically, cell-derived lipid vesicles that contain functional gap junction channels, termed Connectosomes, have recently been demonstrated to substantially increase the effectiveness of small molecule chemotherapeutics. However, because gap junctions are present in nearly all tissues, Connectosomes have no intrinsic ability to target specific cell types, which potentially limits their therapeutic effectiveness. To address this challenge, here we display targeting ligands consisting of single-domain antibodies on the surfaces of Connectosomes. We demonstrate that these targeted Connectosomes selectively interact with cells that express a model receptor, promoting the selective delivery of the chemotherapeutic doxorubicin to this target cell population. More generally, our approach has the potential to boost cytoplasmic delivery of diverse therapeutic molecules to specific cell populations while protecting off-target cells, a critical step toward realizing the therapeutic potential of gap junctions