Purification and characterization of human neural stem and progenitor cells.

The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.

Cell surface fluctuations regulate early embryonic lineage sorting.

In development, lineage segregation is coordinated in time and space. An important example is the mammalian inner cell mass, in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the fetus). While the molecular requirements have been well studied, the physical mechanisms determining spatial segregation between EPI and PrE remain elusive. Here, we investigate the mechanical basis of EPI and PrE sorting. We find that rather than the differences in static cell surface mechanical parameters as in classical sorting models, it is the differences in surface fluctuations that robustly ensure physical lineage sorting. These differential surface fluctuations systematically correlate with differential cellular fluidity, which we propose together constitute a non-equilibrium sorting mechanism for EPI and PrE lineages. By combining experiments and modeling, we identify cell surface dynamics as a key factor orchestrating the correct spatial segregation of the founder embryonic lineages.

Adhesion-Based Self-Organization in Tissue Patterning.

Since the proposal of the differential adhesion hypothesis, scientists have been fascinated by how cell adhesion mediates cellular self-organization to form spatial patterns during development. The search for molecular tool kits with homophilic binding specificity resulted in a diverse repertoire of adhesion molecules. Recent understanding of the dominant role of cortical tension over adhesion binding redirects the focus of differential adhesion studies to the signaling function of adhesion proteins to regulate actomyosin contractility. The broader framework of differential interfacial tension encompasses both adhesion and nonadhesion molecules, sharing the common function of modulating interfacial tension during cell sorting to generate diverse tissue patterns. Robust adhesion-based patterning requires close coordination between morphogen signaling, cell fate decisions, and changes in adhesion. Current advances in bridging theoretical and experimental approaches present exciting opportunities to understand molecular, cellular, and tissue dynamics during adhesion-based tissue patterning across multiple time and length scales.

Intelligent Image-Activated Cell Sorting.

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

Pooled Genome-Scale CRISPR Screens in Single Cells.

Assigning functions to genes and learning how to control their expression are part of the foundation of cell biology and therapeutic development. An efficient and unbiased method to accomplish this is genetic screening, which historically required laborious clone generation and phenotyping and is still limited by scale today. The rapid technological progress on modulating gene function with CRISPR-Cas and measuring it in individual cells has now relaxed the major experimental constraints and enabled pooled screening with complex readouts from single cells. Here, we review the principles and practical considerations for pooled single-cell CRISPR screening. We discuss perturbation strategies, experimental model systems, matching the perturbation to the individual cells, reading out cell phenotypes, and data analysis. Our focus is on single-cell RNA sequencing and cell sorting-based readouts, including image-enabled cell sorting. We expect this transformative approach to fuel biomedical research for the next several decades.

Glycan Masking Focuses Immune Responses to the HIV-1 CD4-Binding Site and Enhances Elicitation of VRC01-Class Precursor Antibodies.

An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-2∗02, the germline VRC01 VH segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G+ B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design.

Measurement of adhesion and traction of cells at high yield reveals an energetic ratchet operating during nephron condensation.

Developmental biology-inspired strategies for tissue-building have extraordinary promise for regenerative medicine, spurring interest in the relationship between cell biophysical properties and morphological transitions. However, mapping gene or protein expression data to cell biophysical properties to physical morphogenesis remains challenging with current techniques. Here, we present multiplexed adhesion and traction of cells at high yield (MATCHY). MATCHY advances the multiplexing and throughput capabilities of existing traction force and cell-cell adhesion assays using microfabrication and a semiautomated computation scheme with machine learning-driven cell segmentation. Both biophysical assays are coupled with serial downstream immunofluorescence to extract cell type/signaling state information. MATCHY is especially suited to complex primary tissue-, organoid-, or biopsy-derived cell mixtures since it does not rely on a priori knowledge of cell surface markers, cell sorting, or use of lineage-specific reporter animals. We first validate MATCHY on canine kidney epithelial cells engineered for rearranged during transfection (RET) tyrosine kinase expression and quantify a relationship between downstream signaling and cell traction. We then use MATCHY to create a biophysical atlas of mouse embryonic kidney primary cells and identify distinct biophysical states along the nephron differentiation trajectory. Our data complement expression-level knowledge of adhesion molecule changes that accompany nephron differentiation with quantitative biophysical information. These data reveal an "energetic ratchet" that accounts for spatial trends in nephron progenitor cell condensation as they differentiate into early nephron structures, which we validate through agent-based computational simulation. MATCHY offers semiautomated cell biophysical characterization at >10,000-cell throughput, an advance benefiting fundamental studies and new synthetic tissue strategies for regenerative medicine.

Cellular segregation in cocultures is driven by differential adhesion and contractility on distinct timescales.

Cellular sorting and pattern formation are crucial for many biological processes such as development, tissue regeneration, and cancer progression. Prominent physical driving forces for cellular sorting are differential adhesion and contractility. Here, we studied the segregation of epithelial cocultures containing highly contractile, ZO1/2-depleted MDCKII cells (dKD) and their wild-type (WT) counterparts using multiple quantitative, high-throughput methods to monitor their dynamical and mechanical properties. We observe a time-dependent segregation process governed mainly by differential contractility on short (<5 h) and differential adhesion on long (>5 h) timescales. The overly contractile dKD cells exert strong lateral forces on their WT neighbors, thereby apically depleting their surface area. Concomitantly, the tight junction-depleted, contractile cells exhibit weaker cell-cell adhesion and lower traction force. Drug-induced contractility reduction and partial calcium depletion delay the initial segregation but cease to change the final demixed state, rendering differential adhesion the dominant segregation force at longer timescales. This well-controlled model system shows how cell sorting is accomplished through a complex interplay between differential adhesion and contractility and can be explained largely by generic physical driving forces.

Cell sorting in vitro and in vivo: How are cadherins involved?

Animal tissues are composed of heterogenous cells, and their sorting into different compartments of the tissue is a pivotal process for organogenesis. Cells accomplish sorting by themselves-it is well known that singly dispersed cells can self-organize into tissue-like structures in vitro. Cell sorting is regulated by both biochemical and physical mechanisms. Adhesive proteins connect cells together, selecting particular partners through their specific binding properties, while physical forces, such as cell-cortical tension, control the cohesiveness between cells and in turn cell assembly patterns in mechanical ways. These processes cooperate in determining the overall cell sorting behavior. This article focuses on the 'cadherin' family of adhesion molecules as a biochemical component of cell-cell interactions, addressing how they regulate cell sorting by themselves or by cooperating with other factors. New ideas beyond the classical models of cell sorting are also discussed.

Self-organized collective cell behaviors as design principles for synthetic developmental biology.

Over the past two decades, molecular cell biology has graduated from a mostly analytic science to one with substantial synthetic capability. This success is built on a deep understanding of the structure and function of biomolecules and molecular mechanisms. For synthetic biology to achieve similar success at the scale of tissues and organs, an equally deep understanding of the principles of development is required. Here, we review some of the central concepts and recent progress in tissue patterning, morphogenesis and collective cell migration and discuss their value for synthetic developmental biology, emphasizing in particular the power of (guided) self-organization and the role of theoretical advances in making developmental insights applicable in synthesis.

Multiomics Characterization of a Less Invasive Microfluidic-Based Cell Sorting Technique.

Fluorescence-activated cell sorting (FACS) is a specialized technique to isolate specific cell subpopulations with a high level of recovery and accuracy. However, the cell sorting procedure can impact the viability and metabolic state of cells. Here, we performed a comparative study and evaluated the impact of traditional high-pressure charged droplet-based and microfluidic chip-based sorting on the metabolic and phosphoproteomic profile of different cell types. While microfluidic chip-based sorted cells more closely resembled the unsorted control group for most cell types tested, the droplet-based sorted cells showed significant metabolic and phosphoproteomic alterations. In particular, greater changes in redox and energy status were present in cells sorted with the droplet-based cell sorter along with larger shifts in proteostasis. 13C-isotope tracing analysis on cells recovering postsorting revealed that the sorter-induced suppression of mitochondrial TCA cycle activity recovered faster in the microfluidic chip-based sorted group. Apart from this, amino acid and lipid biosynthesis pathways were suppressed in sorted cells, with minimum impact and faster recovery in the microfluidic chip-based sorted group. These results indicate microfluidic chip-based sorting has a minimum impact on metabolism and is less disruptive compared to droplet-based sorting.

Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohn's disease associated adherent-invasive Escherichia coli infection.

Bacterial infection is a dynamic process resulting in a heterogenous population of infected and uninfected cells. These cells respond differently based on their bacterial load and duration of infection. In the case of infection of macrophages with Crohn's disease (CD) associated adherent-invasive Escherichia coli (AIEC), understanding the drivers of pathogen success may allow targeting of cells where AIEC replicate to high levels. Here we show that stratifying immune cells based on their bacterial load identifies novel pathways and therapeutic targets not previously associated with AIEC when using a traditional homogeneous infected population approach. Using flow cytometry-based cell sorting we stratified cells into those with low or high intracellular pathogen loads, or those which were bystanders to infection. Immune cells transcriptomics revealed a diverse response to the varying levels of infection while pathway analysis identified novel intervention targets that were directly related to increasing intracellular AIEC numbers. Chemical inhibition of identified targets reduced AIEC intracellular replication or inhibited secretion of tumour necrosis factor alpha (TNFα), a key cytokine associated with AIEC infection. Our results have identified new avenues of intervention in AIEC infection that may also be applicable to CD through the repurposing of already available inhibitors. Additionally, they highlight the applicability of immune cell stratification post-infection as an effective approach for the study of microbial pathogens.

MitoTracker Red for isolation of zone-specific hepatocytes and characterization of hepatic sublobular metabolism.

BACKGROUND: The liver lobule is divided into three zones or regions: periportal (PP or Zone 1) that is highly oxidative and active in ureagenesis, pericentral (PC or Zone 3) that is more glycolytic, and midzonal (MZ or Zone 2) with intermediate characteristics. AIM: Our goal was to isolate and metabolically characterize hepatocytes from specific sublobular zones. METHODS: Mice were administered rhodamine123 (Rh123) or MitoTracker Red (MTR) prior to intravital imaging, liver fixation, or hepatocyte isolation. After in vivo MTR, hepatocytes were isolated and sorted based on MTR fluorescence intensity. Alternatively, E-cadherin (Ecad) and cytochrome P450 2E1 (CYP2E1) immunolabeling was performed in fixed liver slices. Ecad and CYP2E1 gene expression in sorted hepatocytes was assessed by qPCR. Oxygen consumption rates (OCR) of sorted hepatocytes were also assessed. RESULTS: Multiphoton microscopy showed Rh123 and MTR fluorescence distributed zonally, decreasing from PP to PC in a flow-dependent fashion. In liver cross-sections, Ecad was expressed periportally and CYP2E1 pericentrally in association with high and low MTR labeling, respectively. Based on MTR fluorescence, hepatocytes were sorted into PP, MZ, and PC populations with PP and PC hepatocytes enriched in Ecad and CYP2E1, respectively. OCR of PP hepatocytes was ∼4 times that of PC hepatocytes. CONCLUSIONS: MTR treatment in vivo delineates sublobular hepatic zones and can be used to sort hepatocytes zonally. PP hepatocytes have substantially greater OCR compared to PC and MZ. The results also indicate a sharp midzonal demarcation between hepatocytes with PP characteristics (Ecad) and those with PC features (CYP2E1). This new method to sort hepatocytes in a zone-specific fashion holds the potential to shed light on sublobular hepatocyte metabolism and regulatory pathways in health and disease.

Raman micro-spectroscopy as a tool to study immunometabolism.

In the past two decades, immunometabolism has emerged as a crucial field, unraveling the intricate molecular connections between cellular metabolism and immune function across various cell types, tissues, and diseases. This review explores the insights gained from studies using the emerging technology, Raman micro-spectroscopy, to investigate immunometabolism. Raman micro-spectroscopy provides an exciting opportunity to directly study metabolism at the single cell level where it can be combined with other Raman-based technologies and platforms such as single cell RNA sequencing. The review showcases applications of Raman micro-spectroscopy to study the immune system including cell identification, activation, and autoimmune disease diagnosis, offering a rapid, label-free, and minimally invasive analytical approach. The review spotlights three promising Raman technologies, Raman-activated cell sorting, Raman stable isotope probing, and Raman imaging. The synergy of Raman technologies with machine learning is poised to enhance the understanding of complex Raman phenotypes, enabling biomarker discovery and comprehensive investigations in immunometabolism. The review encourages further exploration of these evolving technologies in the rapidly advancing field of immunometabolism.

Isolation of stage-specific spermatogenic cells by dynamic histone incorporation and removal in spermatogenesis.

Due to the lack of an efficient in vitro spermatogenesis system, studies on mammalian spermatogenesis require the isolation of specific germ cell populations for further analyses. BSA gradient and elutriation have been used for several decades to purify testicular germ cells; more recently, flow cytometric cell sorting has become popular. Although each method has its advantages and disadvantages and is used depending on the purpose of the experiment, reliance on flow cytometric cell sorting is expected to be more prevalent because fewer cells can be managed. However, the currently used flow cytometric cell sorting method for testicular germ cells relies on karyotypic differences via DNA staining. Thus, it remains challenging to separate post-meiotic haploid cells (spermatids) according to their differentiation stage despite significant variations in morphology and chromatin state. In this study, we developed a method for finely separating testicular germ cells using VC mice carrying fluorescently tagged histones. This method enables the separation of spermatogonia, spermatocytes, and spermatids based on the intensity of histone fluorescence and cell size. Combined with a DNA staining dye, this method separates spermatids after elongation according to each spermiogenic stage. Although the necessity for a specific transgenic mouse line is less versatile, this method is expected to be helpful for the isolation of testicular germ cell populations because it is highly reproducible and independent of complex cell sorter settings and staining conditions.

Evaluation of single-cell sorting accuracy using antibody-derived tag-based qPCR.

Single-cell sorting (index sorting) is a widely used method to isolate one cell at a time using fluorescence-activated cell sorting (FACS) for downstream applications such as single-cell sequencing or single-cell expansion. Despite widespread use, few assays are available to evaluate the proteomic features of the sorted single cell and further confirm the accuracy of single-cell sorting. With this caveat, we developed a novel assay to confirm the protein expression of sorted single cells by co-staining cells with the same marker using both antibody-derived tags (ADTs) and fluorescent antibodies. After single-cell sorting, we amplified the oligo of the ADT reagent as a surrogate signal for the protein expression using multiplex TaqMan™ qPCR on sorted cells. This assay is not only useful for confirming the identity of a sorted single cell but also an efficient method to profile proteomic features at the single-cell level. Finally, we applied this assay to characterize protein expression on whole cell lysate. Because of the sensitivity of the TaqMan™ qPCR, we can detect protein expression from a small number of cells. In summary, the ADT-based qPCR assay developed here can be utilized to confirm single-cell sorting accuracy and characterizing protein expression on both single cells and whole cell lysate.

The leaf idioblastome of the medicinal plant Catharanthus roseus is associated with stress resistance and alkaloid metabolism.

Catharanthus roseus leaves produce a range of monoterpenoid indole alkaloids (MIAs) that include low levels of the anticancer drugs vinblastine and vincristine. The MIA pathway displays a complex architecture spanning different subcellular and cell type localizations, and is under complex regulation. As a result, the development of strategies to increase the levels of the anticancer MIAs has remained elusive. The pathway involves mesophyll specialized idioblasts where the late unsolved biosynthetic steps are thought to occur. Here, protoplasts of C. roseus leaf idioblasts were isolated by fluorescence-activated cell sorting, and their differential alkaloid and transcriptomic profiles were characterized. This involved the assembly of an improved C. roseus transcriptome from short- and long-read data, IDIO+. It was observed that C. roseus mesophyll idioblasts possess a distinctive transcriptomic profile associated with protection against biotic and abiotic stresses, and indicative that this cell type is a carbon sink, in contrast to surrounding mesophyll cells. Moreover, it is shown that idioblasts are a hotspot of alkaloid accumulation, suggesting that their transcriptome may hold the key to the in-depth understanding of the MIA pathway and the success of strategies leading to higher levels of the anticancer drugs.

Clinical grade multiparametric cell sorting and gene-marking of regulatory T cells.

BACKGROUND AIMS: Regulatory T cells (Tregs) are the main mediators of peripheral tolerance. Treg-directed therapy has shown promising results in preclinical studies of diverse immunopathologies. At present, the clinical applicability of adoptive Treg transfer is limited by difficulties in generating Tregs at sufficient cell dose and purity. METHODS: We developed a Good Manufacturing Practice (GMP) compliant method based on closed-system multiparametric Fluorescence-Activated Cell Sorting (FACS) to purify Tregs, which are then expanded in vitro and gene-marked with a clinical grade retroviral vector to enable in vivo fate tracking. Following small-scale optimization, we conducted four clinical-scale processing runs. RESULTS: We showed that Tregs could be enriched to 87- 92% purity following FACS-sorting, and expanded and transduced to yield clinically relevant cell dose of 136-732×106 gene-marked cells, sufficient for a cell dose of at least 2 × 106 cells/kg. The expanded Tregs were highly demethylated in the FOXP3 Treg-specific demethylated region (TSDR), consistent with bona fide natural Tregs. They were suppressive in vitro, but a small percentage could secrete proinflammatory cytokines, including interferon-γ and interleukin-17A. CONCLUSIONS: This study demonstrated the feasibility of isolating, expanding and gene-marking Tregs in clinical scale, thus paving the way for future phase I trials that will advance knowledge about the in vivo fate of transferred Tregs and its relationship with concomitant Treg-directed pharmacotherapy and clinical response.

Advances in high-throughput screening approaches for biosurfactants: current trends, bottlenecks and perspectives.

The market size of biosurfactants (BSs) has been expanding at an extremely fast pace due to their broad application scope. Therefore, the re-construction of cell factories with modified genomic and metabolic profiles for desired industrial performance has been an intriguing aspect. Typical mutagenesis approaches generate huge mutant libraries, whereas a battery of specific, robust, and cost-effective high-throughput screening (HTS) methods is requisite to screen target strains for desired phenotypes. So far, only a few specialized HTS assays have been developed for BSs that were successfully applied to obtain anticipated mutants. The most important milestones to reach, however, continue to be: specificity, sensitivity, throughput, and the potential for automation. Here, we discuss important colorimetric and fluorometric HTS approaches for possible intervention on automated HTS platforms. Moreover, we explain current bottlenecks in developing specialized HTS platforms for screening high-yielding producers and discuss possible perspectives for addressing such challenges.

High-Throughput Single-Cell Metabolic Labeling, Sorting, and Sequencing of Active Antibiotic-Resistant Bacteria in the Environment.

Active antibiotic-resistant bacteria (ARB) play a major role in spreading antimicrobial resistance (AMR) in the environment; however, they have remained largely unexplored. Herein, we coupled bio-orthogonal noncanonical amino acid tagging with high-throughput fluorescence-activated single-cell sorting (FACS) and sequencing to characterize the phenome and genome of active ARB in complex environmental matrices. Active ARB, conferring resistance to six antibiotics throughout wastewater treatment, were distinguished and quantified. The percentage and concentration of active ARB ranged from 0.28% to 45.3% and from 1.1 × 104 to 2.09 × 107 cells/mL, respectively. Notably, the final effluents retained up to 4.79 × 104 cells/mL of active ARB. Targeted FACS and genomic sequencing revealed a distinct taxonomic composition of active ARB compared with that of the overall population. The coexistence of antibiotic resistome and mobilome in active ARB was also identified, including three high-quality metagenomic assembly genomes assigned to pathogenic bacteria, highlighting the substantial health risks due to their activity, phenotypic resistance, mobility, and pathogenicity. This study advances our understanding of previously overlooked active ARB in the environment by linking their resistance phenotype to their genotype. This high-throughput method will enable efficient quantitative surveillance of active AMR, providing valuable insights into risk control and management.