细胞生物学

Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death. Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options. Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules. Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks. The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation. This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs. The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest.

Key features

• This protocol requires preexisting experience in hiPSC culturing for a successful outcome.

• The protocol relies on a small molecule differentiation scheme and an easy-to-follow methodology, which can be paused at several time points.

• The protocol generates >50 × 10⁶ astrocytes per differentiation, which can be cryopreserved at every passage, ensuring a large-scale experimental output.

Graphical overview

γδ T cells play a critical role in homeostasis and diseases such as infectious diseases and tumors in both mice and humans. They can be categorized into two main functional subsets: IFN-γ-producing γδT1 cells and IL-17-producing γδT17 cells. While CD27 expression segregates these two subsets in mice, little is known about human γδT17 cell differentiation and expansion. Previous studies have identified γδT17 cells in human skin and mucosal tissues, including the oral cavity and colon. However, human γδ T cells from peripheral blood mononuclear cells (PBMCs) primarily produce IFN-γ. In this protocol, we describe a method for in vitro expansion and polarization of human γδT17 cells from PBMCs.

Key Features

• Expansion of γδ T cells from peripheral blood mononuclear cells.

• Human IL-17A-producing γδ T-cell differentiation and expansion using IL-7 and anti-γδTCR.

• Analysis of IL-17A production post γδ T-cell expansion.

In the peripheral nervous system, Schwann cells are the primary type of glia; their in vitro differentiation and dedifferentiation system has not been described in detail in the literature. Thus, an in vitro differentiation and dedifferentiation system of rat Schwann cells is described in this protocol. These cultures and systems may be used to investigate the morphological and biochemical effects of drug interventions or lentivirus-mediated gene transfer on Schwann cells during differentiation or dedifferentiation.

Graphical abstract

Skeletal muscle stem cells differentiated from human-induced pluripotent stem cells (hiPSCs) serve as a uniquely promising model system for investigating human myogenesis and disease pathogenesis, and for the development of gene editing and regenerative stem cell therapies. Here, we present an effective and reproducible transgene-free protocol for derivation of human skeletal muscle stem cells, iMyoblasts, from hiPSCs. Our two-step protocol consists of 1) small molecule-based differentiation of hiPSCs into myocytes, and 2) stimulation of differentiated myocytes with growth factor-rich medium to activate the proliferation of undifferentiated reserve cells, for expansion and cell line establishment. iMyoblasts are PAX3⁺/MyoD1⁺ myogenic stem cells with dual potential to undergo muscle differentiation and to self-renew as a regenerative cell population for muscle regeneration both ex vivo and in vivo. The simplicity and robustness of iMyoblast generation and expansion have enabled their application to model the molecular pathogenesis of Facioscapulohumeral Muscular Dystrophy and Limb-Girdle Muscular Dystrophies, to both ex vivo and in vivo muscle xenografts, and to respond efficiently to gene editing, enabling the co-development of gene correction and stem cell regenerative therapeutic technologies for the treatment of muscular dystrophies and muscle injury.

Graphical abstract:

In mice, microglial precursors in the yolk sac migrate to the brain parenchyma through the head neuroepithelial layer between embryonic days 8.5 (E8.5)–E16.5 and acquire their unique identity with a ramified form. Based on the microglial developmental process, we dissected the neuroepithelial layer (NEL) of E13.5 mice, which is composed of microglial progenitor and neuroepithelial cells. The NEL was bankable and expandable. In addition, microglial precursors were matured according to NEL culture duration. The matured microglia (MG; CD11b-positive cells) were easily isolated from the cultured NEL using a magnetic-activated cell sorting system and named NEL-MG. In conclusion, we obtained higher yields of adult-like microglia (mature microglia: NEL-MG) compared to previous in vitro surrogates such as neonatal microglia and microglial cell lines.

Graphical abstract:

The isolation of intact single adult cardiomyocytes from model animals, mouse and rat, is an essential tool for cardiac molecular and cellular research. While several methods are reported for adult mouse cardiomyocyte isolation, the viability and yield of the isolated cells have been variable. Here, we describe step-by-step protocols for high viability and yield cardiomyocyte isolation from mouse and rat, based on the use of a stable pressure Langendorff perfusion system. After the animal is euthanized or terminally anesthetized, the heart is removed from the chest and subject to Langendorff perfusion. Then, the heart is digested by perfusion with collagenase and hyaluronidase. After thorough digestion, the cardiomyocytes are dispersed and gradually recovered, the extracellular Ca²⁺ concentration adjusted, and cells are then ready for use. This protocol will facilitate research that requires isolated adult mouse and rat cardiomyocytes.

Craniofacial anomalies (CFA) are a diverse group of deformities, which affect the growth of the head and face. Dysregulation of cranial neural crest cell (NCC) migration, proliferation, differentiation, and/or cell fate specification have been reported to contribute to CFA. Understanding of the mechanisms through which cranial NCCs contribute for craniofacial development may lead to identifying meaningful clinical targets for the prevention and treatment of CFA. Isolation and culture of cranial NCCs in vitro facilitates screening and analyses of molecular cellular mechanisms of cranial NCCs implicated in craniofacial development. Here, we present a method for the isolation and culture of cranial NCCs harvested from the first branchial arch at early embryonic stages. Morphology of isolated cranial NCCs was similar to O9-1 cells, a cell line for neural crest stem cells. Moreover, cranial NCCs isolated from a transgenic mouse line with enhanced bone morphogenetic protein (BMP) signaling in NCCs showed an increase in their chondrogenic differentiation capacity, suggesting maintenance of their in vivo differentiation potentials observed in vitro. Taken together, our established method is useful to visualize cellular behaviors of cranial NCCs.

As a model to interrogate human macrophage biology, macrophages differentiated from human induced pluripotent stem cells (hiPSCs) transcend other existing models by circumventing the variability seen in human monocyte-derived macrophages, whilst epitomizing macrophage phenotypic and functional characteristics over those offered by macrophage-like cell lines (Mukherjee et al., 2018). Furthermore, hiPSCs are amenable to genetic manipulation, unlike human monocyte-derived macrophages (MDMs) (van Wilgenburg et al., 2013; Lopez-Yrigoyen et al., 2020), proposing boundless opportunities for specific disease modelling.

We outline an effective and efficient protocol that delivers a continual production of hiPSC-derived-macrophages (iMACs), exhibiting human macrophage surface and intracellular markers, together with functional activity.

The protocol describes the resuscitation, culture, and differentiation of hiPSC into mature terminal macrophages, via the initial and intermediate steps of expansion of hiPSCs, formation into embryoid bodies (EBs), and generation of hematopoietic myeloid precursors.

We offer a simplified, scalable, and adaptable technique that advances upon other protocols, utilizing feeder-free conditions and reduced growth factors, to produce high yields of consistent iMACs over a period of several months, economically.

Macrophages are key cells in the innate immune system and play a role in a variety of diseases. However, macrophages are terminally differentiated and difficult to manipulate genetically via transfection or through CRISPR-Cas9 gene editing. To overcome this limitation, we provide a simplified protocol for the generation of mouse embryonic stem cells-derived macrophages (ESDM). Thus, genetic manipulation can be performed using embryonic stem cells, selecting for the desired changes, and finally producing macrophages to study the effects of the previous genetic manipulation. These studies can contribute to many areas of research, including atherosclerosis and inflammation. Production of ESDM has been previously achieved using embryoid body (EB) intermediates. Here, we optimized the EB method using a simplified medium, reducing the number of recombinant proteins and medium recipes required. Our EB-based differentiation protocol consists of three stages: 1) floating EB formation; 2) adherence of EBs and release of floating macrophage progenitors; and, 3) terminal differentiation of harvested macrophage progenitors. The advantages of this protocol include achieving independent floating EBs in stage 1 by using a rocker within the tissue culture incubator, as well as the exclusion of small EBs and cell clusters when harvesting macrophage progenitors via cell filtration.

Adipocytes exhibit different morphological and functional characteristics, depending on their anatomical location, developmental origin, and stimulus. While white adipocytes tend to accumulate energy as triglycerides, brown and beige adipocytes tend to direct carbon sources to fuel thermogenesis. White and beige adipocytes originate from common progenitor cells, which are distinct from brown adipocyte precursors. Having a method to study white vs. beige vs. brown adipocyte differentiation may help to unveil the mechanisms driving distinct adipogenic programs. Preadipocytes can be cultured and differentiated in vitro using a combination of compounds to stimulate adipogenesis. Here, we describe and compare protocols designed to stimulate adipocyte differentiation and induce brown/beige-like or white-like characteristics in differentiating adipocytes. The protocols consist in exposing murine preadipocytes to pharmacological stimuli aimed at triggering adipogenesis and inducing (or not) a thermogenic gene expression program. After 8 days of differentiation with a pro-browning cocktail, immortalized preadipocytes isolated from interscapular brown fat (9B cells) or inguinal white fat (9W cells) from the same mouse expressed higher levels of brown/beige adipocyte markers (e.g., Ucp1) and pan-adipocyte differentiation markers (e.g., Pparg, Cebpa and aP2) when compared to the same cells differentiated with a cocktail that lacked brown/beige adipogenic inducers (i.e., rosiglitazone, T3, and indomethacin). Consistent with a higher thermogenic potential of brown vs. beige adipocytes, differentiated 9B cells expressed higher Ucp1 levels than differentiated 9W cells. This simple protocol may help researchers to understand mechanisms of adipogenesis and how adipocytes become thermogenic.