Alleviating Cell Lysate-Induced Inhibition to Enable RT-PCR from Single Cells in Picoliter-Volume Double Emulsion Droplets.

Microfluidic droplet assays enable single-cell polymerase chain reaction (PCR) and sequencing analyses at unprecedented scales, with most methods encapsulating cells within nanoliter-sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) allows sorting droplets with commercially available fluorescence-activated cell sorter (FACS) machines, making it possible to isolate single cells based on phenotypes of interest for downstream analyses. However, sorting DE droplets with standard cytometers requires small droplets that can pass FACS nozzles. This poses challenges for molecular biology, as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, which can be efficiently alleviated through heat lysis. We further show that the magnitude of inhibition depends on the cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most mouse and human cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL DE droplets with fluorescence quantifiable via FACS. These results open up new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet-based single-cell analysis technologies.

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection.

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating the measurement of expression kinetics in both isolated cells and whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a foundation for the development and expansion of transgenic techniques in this unique model system.

Comparisons of cell proliferation and cell death from tornaria larva to juvenile worm in the hemichordate Schizocardium californicum.

BACKGROUND: There are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in the development of indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when and where the tissue that will give rise to the adult originates. How do the processes of cell division and cell death redesign the body plans of indirect developers? In this study, we present patterns of cell proliferation and cell death during larval body plan development, metamorphosis, and adult body plan formation, in the hemichordate Schizocardium californium (Cameron and Perez in Zootaxa 3569:79-88, 2012) to answer these questions. RESULTS: We identified distinct patterns of cell proliferation between larval and adult body plan formation of S. californicum. We found that some adult tissues proliferate during the late larval phase prior to the start of overt metamorphosis. In addition, using an irradiation and transcriptomic approach, we describe a genetic signature of proliferative cells that is shared across the life history states, as well as markers that are unique to larval or juvenile states. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis. CONCLUSIONS: Cell proliferation during the development of S. californicum has distinct patterns in the formation of larval and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferated and gave rise to the larvae and juveniles have a genetic signature that suggested a heterogeneous pool of proliferative progenitors, rather than a set-aside population of pluripotent cells. Taken together, we propose that the gradual morphological transformation of S. californicum is mirrored at the cellular level and may be more representative of the development strategies that characterize metamorphosis in many metazoan animals.

Mapping single-cell atlases throughout Metazoa unravels cell type evolution.

Comparing single-cell transcriptomic atlases from diverse organisms can elucidate the origins of cellular diversity and assist the annotation of new cell atlases. Yet, comparison between distant relatives is hindered by complex gene histories and diversifications in expression programs. Previously, we introduced the self-assembling manifold (SAM) algorithm to robustly reconstruct manifolds from single-cell data (Tarashansky et al., 2019). Here, we build on SAM to map cell atlas manifolds across species. This new method, SAMap, identifies homologous cell types with shared expression programs across distant species within phyla, even in complex examples where homologous tissues emerge from distinct germ layers. SAMap also finds many genes with more similar expression to their paralogs than their orthologs, suggesting paralog substitution may be more common in evolution than previously appreciated. Lastly, comparing species across animal phyla, spanning sponge to mouse, reveals ancient contractile and stem cell families, which may have arisen early in animal evolution.

Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS.

In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput screening via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5-25 µm in diameter) within highly monodisperse double emulsions (35 µm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS screening of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional phenotyping.

Neural Progenitor Cells Alter Chromatin Organization and Neurotrophin Expression in Response to 3D Matrix Degradability.

Neural progenitor cells (NPCs) are promising therapeutic candidates for nervous system regeneration. Significant efforts focus on developing hydrogel-based approaches to facilitate the clinical translation of NPCs, from scalable platforms for stem cell production to injectable carriers for cell transplantation. However, fundamental questions surrounding NPC-hydrogel interactions remain unanswered. While matrix degradability is known to regulate the stemness and differentiation capacity of NPCs, how degradability impacts NPC epigenetic regulation and secretory phenotype remains unknown. To address this question, NPCs encapsulated in recombinant protein hydrogels with tunable degradability are assayed for changes in chromatin organization and neurotrophin expression. In high degradability gels, NPCs maintain expression of stem cell factors, proliferate, and have large nuclei with elevated levels of the stemness-associated activating histone mark H3K4me3. In contrast, NPCs in low degradability gels exhibit more compact, rounded nuclei with peripherally localized heterochromatin, are non-proliferative yet non-senescent, and maintain expression of neurotrophic factors with potential therapeutic relevance. This work suggests that tuning matrix degradability may be useful to direct NPCs toward either a more-proliferative, stem-like phenotype for cell replacement therapies, or a more quiescent-like, pro-secretory phenotype for soluble factor-mediated therapies.

Mechanically resolved imaging of bacteria using expansion microscopy.

Imaging dense and diverse microbial communities has broad applications in basic microbiology and medicine, but remains a grand challenge due to the fact that many species adopt similar morphologies. While prior studies have relied on techniques involving spectral labeling, we have developed an expansion microscopy method (µExM) in which bacterial cells are physically expanded prior to imaging. We find that expansion patterns depend on the structural and mechanical properties of the cell wall, which vary across species and conditions. We use this phenomenon as a quantitative and sensitive phenotypic imaging contrast orthogonal to spectral separation to resolve bacterial cells of different species or in distinct physiological states. Focusing on host-microbe interactions that are difficult to quantify through fluorescence alone, we demonstrate the ability of µExM to distinguish species through an in vitro defined community of human gut commensals and in vivo imaging of a model gut microbiota, and to sensitively detect cell-envelope damage caused by antibiotics or previously unrecognized cell-to-cell phenotypic heterogeneity among pathogenic bacteria as they infect macrophages.

Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling.

Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from ∼0.5 to 50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodelling to facilitate cadherin-mediated cell-cell contact and promote ß-catenin signalling. In two additional hydrogel systems, permitting NPC-mediated matrix remodelling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodelling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D.

Nanoengineered Osteoinductive and Elastomeric Scaffolds for Bone Tissue Engineering.

Synthesis and fabrication of porous and elastomeric nanocomposite scaffolds from biodegradable poly(glycerol sebacate) (PGS) and osteoinductive nanosilicates is reported. Nanosilicates are mineral-based two-dimensional (2D) nanomaterials with high surface area which reinforced PGS network. The addition of nanosilicates to PGS resulted in mechanically stiff and elastomeric nanocomposites. The degradation rate and mechanical stiffness of nanocomposite network could be modulated by addition of nanosilicates. Nanocomposite scaffolds supported cell adhesion, spreading, and proliferation and promoted osteogenic differentiation of preosteoblasts. The addition of nanosilicates to PGS scaffolds increased alkaline phosphatase (ALP) activity and production of matrix mineralization. In vivo studies demonstrated biocompatibility and biodegradability of nanocomposite scaffolds. Overall, the combination of elasticity and tailorable stiffness, tunable degradation profiles, and the osteoinductive capability of the scaffolds offer a promising approach for bone tissue engineering.