Cell Biology

Cellular senescence is a reprogrammed cell state triggered as an adaptative response to a variety of stresses, most often those affecting the genome integrity. Senescent cells accumulate in most tissues with age and contribute to the development of several pathologies. Studying molecular pathways involved in senescence induction and maintenance, or in senescence escape, can be hindered by the heterogeneity of senescent cell populations. Here, we describe a flow cytometry strategy for sorting senescent cells according to three senescence canonical markers whose thresholds can be independently adapted to be more or less stringent: (i) the senescence-associated-β-galactosidase (SA-β-Gal) activity, detected using 5-dodecanoylaminofluorescein Di-β-D-galactopyranoside (C₁₂FDG), a fluorigenic substrate of β-galactosidase; (ii) cell size, proportional to the forward scatter value, since increased size is one of the major changes observed in senescent cells; and (iii) cell granularity, proportional to the side scatter value, which reflects the accumulation of aggregates, lysosomes, and altered mitochondria in senescent cells. We applied this protocol to the sorting of normal human fibroblasts at the replicative senescence plateau. We highlighted the challenge of sorting these senescent cells because of their large sizes, and established that it requires using sorters equipped with a nozzle of an unusually large diameter: at least 200 µm. We present evidence of the sorting efficiency and sorted cell viability, as well as of the senescent nature of the sorted cells, confirmed by the detection of other senescence markers, including the expression of the CKI p21 and the presence of 53BP1 DNA damage foci. Our protocol makes it possible, for the first time, to sort senescent cells from contaminating proliferating cells and, at the same time, to sort subpopulations of senescent cells featuring senescent markers to different extents.

Graphical abstract

Cardiac fibroblasts are one of the major constituents of a healthy heart. Cultured cardiac fibroblasts are a crucial resource for conducting studies on cardiac fibrosis. The existing methods for culturing cardiac fibroblasts involve complicated steps and require special reagents and instruments. The major problems faced with primary cardiac fibroblast culture are the low yield and viability of the cultured cells and contamination with other heart cell types, including cardiomyocytes, endothelial cells, and immune cells. Numerous parameters, including the quality of the reagents used for the culture, conditions maintained during digestion of the cardiac tissue, composition of the digestion mixture used, and age of the pups used for culture determine the yield and purity of the cultured cardiac fibroblasts. The present study describes a detailed and simplified protocol to isolate and culture primary cardiac fibroblasts from neonatal murine pups. We demonstrate the transdifferentiation of fibroblasts into myofibroblasts through transforming growth factor (TGF)-β1 treatment, representing the changes in fibroblasts during cardiac fibrosis. These cells can be used to study the various aspects of cardiac fibrosis, inflammation, fibroblast proliferation, and growth.

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

The wing imaginal discs in Drosophila larvae are a pair of sac-like structures that later form the wings of the adult fly. During the past decades, wing discs have been used as a simple and accessible model system, for identifying genes and deciphering signaling cascades that play crucial roles in many aspects of development. In this protocol, we describe a simple method for preparing a cell suspension from wing discs (see Graphical abstract). This method can also be applied to the preparation of single-cell suspensions from other types of Drosophila tissues. When combined with genetic labeling, the dissociated cells are suitable for downstream analysis, such as flow cytometry. This method was recently used to isolate different populations of cells from Drosophila imaginal discs (Yang et al., 2022).

Graphical abstract:

Procedures to prepare a single-cell suspension from Drosophila imaginal discs.

Illustration of the main steps to dissect, temporarily store, and dissociate imaginal discs from Drosophila larvae. Refer to the Procedure section for detailed description of each step.

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.

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Superovulation is a method used to reduce the number of mice used per experiment by increasing the egg number. Conventionally, superovulation for obtaining mouse eggs involves the use of equine chorionic gonadotropin (eCG) for stimulation and human CG for induction. Female mice of the C57BL/6 inbred strain spontaneously ovulate approximately 10 eggs. The average number of eggs ovulated using the conventional superovulation method is approximately twice as high as that obtained by spontaneous ovulation. Here, we describe the conventional and non-conventional methods of intraperitoneal injection of superovulation reagents in mice and subsequent egg collection. The non-conventional superovulation method combining anti-inhibin serum (AIS) plus eCG for stimulation is more efficient than conventional superovulation. Appropriate intervals from each injection to sampling induce large numbers of high-quality eggs. Immediately after ovulation, eggs are surrounded by cumulus cells, forming an egg-cumulus complex. These cumulus cells are then removed from the egg-cumulus complex by treatment with hyaluronidase to obtain the exact number of eggs. This protocol is suitable for further manipulations such as intracytoplasmic sperm injection and cryopreservation of eggs, as well as for the analyses of responsivity to superovulation reagents in genetically modified mice obtained by genome editing.

Human adipose tissue-resident microvascular endothelial cells are not only garnering attention for their emergent role in the pathogenesis of obesity-related metabolic disorders, but are also of considerable interest for vascular tissue engineering due, in part, to the abundant, accessible, and uniquely dispensable nature of the tissue. Here, we delineate a protocol for the acquisition of microvascular endothelial cells from human fat. A cheaper, smaller, and simpler alternative to fluorescence-assisted cell sorting for the immunoselection of cells, our protocol adapts magnet-assisted cell sorting for the isolation of endothelial cells from enzymatically digested adipose tissue and the subsequent enrichment of their primary cultures. Strategies are employed to mitigate the non-specific uptake of immunomagnetic microparticles, enabling the reproducible acquisition of human adipose tissue-resident microvascular endothelial cells with purities ≥98%. They exhibit morphological, molecular, and functional hallmarks of endothelium, yet retain a unique proteomic signature when compared with endothelial cells derived from different vascular beds. Their cultures can be expanded for >10 population doublings and can be maintained at confluence for at least 28 days without being overgrown by residual stromal cells from the cell sorting procedure. The isolation of human adipose tissue-resident microvascular endothelial cells can be completed within 6 hours and their enrichment within 2 hours, following approximately 7 days in culture.

Graphical abstract:

The epidermis is the outermost layer of the skin. It is made up of mostly keratinocytes along with a small number of melanocytes and Langerhans cells. Melanocytes produce a pigment called melanin, which is transferred to the keratinocytes, and protects these cells from damage from UV radiation, as well as generating hair and skin colours. In this important relationship, keratinocytes exert control over melanocytes. Many questions regarding keratinocyte-melanocyte interactions have yet to be answered, and would benefit from study in model systems, to address diseases such as vitiligo and cutaneous melanoma. Most of the mouse is covered in fur and these areas lack the skin pigmenting inter-follicular epidermal (IFE) melanocytes. However, the mouse tail is pigmented analogously to human skin. Here, we present a method for isolating IFE melanocytes or keratinocytes expressing the tdTomato marker from the mouse tail, using fluorescence-activated cell sorting (FACS). The method involves firstly separating the tail skin epidermis from the dermis, and then digesting the epidermis to produce dissociated cells, which can then be sorted. These isolated cell populations can be studied using RNAseq or cultured in vitro. This protocol isolates IFE melanocytes or keratinocytes and immediately provides reasonable yields of cells, without the need to stain the cells for cell specific markers.

Skeletal stem cells residing in the suture mesenchyme are responsible for calvarial development, homeostatic maintenance, and injury-induced repair. These naïve cells exhibit long-term self-renewal, clonal expansion, and multipotency. They possess osteogenic abilities to regenerate bones in a cell-autonomous manner and can directly replace the damaged skeleton. Therefore, the establishment of reliable isolation and culturing methods for skeletal stem cells capable of preserving their stemness promises to further explore their use in cell-based therapy. Our research team is the first to isolate and purify skeletal stem cells from the calvarial suture and demonstrate their potent ability to generate bone at a single-cell level. Here, we describe detailed protocols for suture stem cell (SuSC) isolation and stemness maintenance in culture. These methods are extremely valuable for advancing our knowledge base of skeletal stem cells in craniofacial development, congenital deformity, and tissue repair and regeneration.

Osteoclast lineage cells (OLCs), including osteoclast precursors (OCPs) and mature osteoclasts (MOCs), participate in bone remodeling and mediate pathologic bone loss. Thus, it is essential to obtain OLCs for exploring their molecular features in both physiological and pathological conditions in vivo. However, the conventional protocols for obtaining OLCs ex vivo are not only time-consuming, but also unable to capture the cellular status of OLCs in vivo. In addition, the current antibody-based isolation approaches, such as fluorescence-/ magnetic-activated cell sorting, are not able to obtain pure osteoclasts because no unique surface antigen for osteoclasts has been identified. Here, we develop a rapid protocol for directly isolating OLCs from mouse bone marrow through magnetic-activated cell sorting (MACS). This protocol can rapidly enrich OCPs and MOCs, respectively, depending on the expression of the distinctive surface markers at their differentiation stages. It is optimized to isolate OLCs from four mice concurrently, of which sorting procedure could be completed within ~5 h.