Cell Biology

In this article, we provide a method to isolate embryonic melanoblasts from reporter mouse strains. The mice from which these cells are isolated are bred into the ROSA26^mT/mG reporter background, which results in green fluorescent protein (GFP) expression in the targeted melanoblast population. These cells are isolated and purified by fluorescence-activated cell sorting using GFP fluorescence. We also provide a method to culture the purified melanoblasts for further analysis. This method yields > 99% purity melanoblasts specifically targeted, and can be used for a variety of studies, including gene expression, clonogenic experiments, and biological assays, such as viability, capacity for directional migration, or differentiation into melanin-producing melanocytic cells.

Graphical overview

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient–derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34⁺ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41⁺ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders.

Graphical abstract

Schematic representation of the isolation of CD34⁺ progenitor cells and subsequent TPO-independent megakaryocyte differentiation

Limbal mesenchymal stromal cells (LMSC), a cellular component of the limbal stem cell niche, have the capability of determining the fate of limbal epithelial progenitor cells (LEPC), which are responsible for the homeostasis of corneal epithelium. However, the isolation of these LMSC has proven to be difficult due to the small fraction of LMSC in the total limbal population, and primary cultures are always hampered by contamination with other cell types. We recently published the efficient isolation and functional characterization of LMSC from the human corneal limbus using CD90 as a selective marker. We observed that flow sorting yielded a pure population of LMSC with superior self-renewal capacity and transdifferentiation potential, and supported the maintenance of the LEPC phenotype. Here, we describe an optimized protocol for the isolation of LMSC from cadaveric corneal limbal tissue by combined collagenase digestion and flow sorting with expansion of LMSC on plastic.

Graphical abstract:

Cholangiocytes are epithelial cells lining the intrahepatic and extrahepatic bile ducts. Cholangiocytes perform key physiological functions in the liver. Bile synthesized by hepatocytes is secreted into bile canaliculi, further stored in the gallbladder, and finally discharged into the duodenum. Due to liver injury, biliary epithelial proliferate in response to endogenous or exogenous signals leading to cholangiopathies, inflammation, fibrosis, and cholangiocarcinoma. Cholangiocytes exhibit anatomical and functional heterogeneity, and understanding such diversified functions will potentially help in finding effective therapies for various cholestatic liver diseases. To perform such functional studies, effective cholangiocyte isolation and culture procedures are needed. This protocol will aid in easy isolation and expansion of cholangiocytes from the liver.

Hypnozoites are dormant liver-stage parasites unique to relapsing malarial species, including the important human pathogen Plasmodium vivax, and pose a barrier to the elimination of malaria. Little is known regarding the biology of these stages, largely due to their inaccessible location. Hypnozoites can be cultured in vitro but these cultures always consist of a mixture of hepatocytes, developing forms, and hypnozoites. Here, using a GFP-expressing line of the hypnozoite model parasite Plasmodium cynomolgi, we describe a protocol for the FACS-based isolation of malarial hypnozoites. The purified hypnozoites can be used for a range of ‘-omics’ studies to dissect the biology of this cryptic stage of the malarial life cycle.

Various stem cells have been found to be dependent on mitochondrial energetics. The role of mitochondria in regulating the self-renewal of normal stem cells and stem-like tumor initiating cells (TICs) is increasingly being appreciated. We proposed that TIC populations have a sub population of cells that are “primed” by mitochondria for self-renewal. Using ovarian cancer model, we have developed a protocol to identify and isolate these “primed” cells using Fluorescence-Assisted Cell Sorting (FACS). We combined live cell stains for a functional marker of TICs and for mitochondrial transmembrane potential to enrich TICs with higher mitochondrial potential that form in vitro spheroids 10-fold more than the other TICs with lower mitochondrial potential. This protocol can be directly used or modified to be used in various cell types. Thus, this protocol is anticipated to be invaluable for the basic understanding of mitochondrial and energetic heterogeneity within stem cell population, and may also prove valuable in translational studies in regenerative medicine and cancer biology.

With the advent of CRISPR-Cas and the ability to easily modify the genome of diverse organisms, rat models are being increasingly developed to interrogate the genetic events underlying mammary development and tumorigenesis. Protocols for the isolation and characterization of mammary epithelial cell subpopulations have been thoroughly developed for mouse and human tissues, yet there is an increasing need for rat-specific protocols. To date, there are no standard protocols for isolating rat mammary epithelial subpopulations. Analyzing changes in the rat mammary hierarchy will help us elucidate the molecular events in breast cancer, the cells of origin for breast cancer subtypes, and the impact of the tumor microenvironment. Here we describe several methods developed for 1) rat mammary epithelial cell isolation; 2) rat mammary fibroblast isolation; 3) culturing rat mammary epithelial cells; and characterization of rat mammary cells by 4) flow cytometric analysis; and 5) immunofluorescence. Cells derived from this protocol can be used for many purposes, including RNAseq, drug studies, functional assays, gene/protein expression analyses, and image analysis.

Most organs and tissues are composed of many types of cells. To characterize cellular state, various transcription profiling approaches are currently available, including whole-tissue bulk RNA sequencing, single cell RNA sequencing (scRNA-Seq), and cell type-specific RNA sequencing. What is missing in this repertoire is a simple, versatile method for bulk transcriptional profiling of cell types for which cell type-specific genetic markers or antibodies are not readily available. We therefore developed Probe-Seq, which uses hybridization of gene-specific probes to RNA markers for isolation of specific types of cells, to enable downstream FACS isolation and bulk RNA sequencing. We show that this method can enable isolation and profiling of specific cell types from mouse retina, frozen human retina, Drosophila midgut, and developing chick retina, suggesting that it is likely useful for most organisms.

The study of human neutrophils in vitro is challenging due to their short half-life and propensity for activation. However, with careful handling and manipulation in the laboratory, they can be a powerful tool to investigate immune responses in health and disease. Here we describe a method for the isolation of human neutrophils from peripheral blood samples, followed by a high-throughput screen to assess the efficacy of a library of compounds in inducing neutrophil apoptosis, which may have therapeutic potential in neutrophil-driven diseases. This protocol is based on previously-published neutrophil isolation methods utilizing Dextran sedimentation of red blood cells followed by the separation of granulocytes with plasma/Percoll discontinuous gradient centrifugation. Yields of ~1 x 10⁶ neutrophils per millilitre of blood, and purities of > 95% neutrophils are typical. Neutrophils are treated with a library of kinase inhibitors, followed by flow cytometry to assess the rate of neutrophil apoptosis. This protocol allows for the high-throughput screening of primary human immune cells to identify compounds with a potential to modify neutrophil function, and could be modified to assess other phenotypes if required.

Single cell RNA sequencing is a very powerful means for cellular heterogeneous studies and so becoming wildly utilized nowadays. To guarantee the success of such analysis, it is very important, though sometimes difficult, to obtain single cells suspension with high quality, especially from the primary solid organs like mammary glands. Digestion of mouse or human mammary glands with enzymes was previously described. However, the yield, viability, especially the separation degree of the cells have rarely been noticed in these studies. Here we described a detailed protocol for the single epithelial cells suspension preparation from mouse mammary glands, which could be applied for single cell RNA sequencing on different platforms. This protocol could be well adapted for dissociation of other solid organs and tumors, and the single cell suspension could be also used for many other experiments.