Stem Cell

The liver is an essential organ that is involved in the metabolism, synthesis, and secretion of serum proteins and detoxification of xenobiotic compounds and alcohol. Studies on liver diseases have largely relied on cancer-derived cell lines that have proven to be inferior due to the lack of drug-metabolising enzymes. Primary human hepatocytes are considered the gold-standard for evaluating drug metabolism. However, several factors such as lack of donors, high cost of cells, and loss of polarity of the cells have limited their widescale adoption and utility. Stem cells have emerged as an alternative source for liver cells that could be utilised for studying liver diseases, developmental biology, toxicology testing, and regenerative medicine. In this article, we describe in detail an optimised protocol for the generation of multicellular 3D liver organoids composed of hepatocytes, stellate cells, and Kupffer cells as a tractable robust model of the liver.

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME’s intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche.

Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors’ identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions.

Stem cell spheroids are rapidly becoming essential tools for a diverse array of applications ranging from tissue engineering to 3D cell models and fundamental biology. Given the increasing prominence of biotechnology, there is a pressing need to develop more accessible, efficient, and reproducible methods for producing these models. Various techniques such as hanging drop, rotating wall vessel, magnetic levitation, or microfluidics have been employed to generate spheroids. However, none of these methods facilitate the easy and efficient production of a large number of spheroids using a standard 6-well plate. Here, we present a novel method based on pellet culture (utilizing U-shaped microstructures) using a silicon mold produced through 3D printing, along with a detailed and illustrated manufacturing protocol. This technique enables the rapid production of reproducible and controlled spheroids (for 1 × 10⁶ cells, spheroids = 130 ± 10 μm) from human induced pluripotent stem cells (hIPSCs) within a short time frame (24 h). Importantly, the method allows the production of large quantities (2 × 10⁴ spheroids for 1 × 10⁶ cells) in an accessible and cost-effective manner, thanks to the use of a reusable mold. The protocols outlined herein are easily implementable, and all the necessary files for the method replication are freely available.

Key features

• Provision of 3D mold files (STL) to produce silicone induction device of spheroids using 3D printing.

• Cost-effective, reusable, and autoclavable device capable of generating up to 1.2× 10⁴ spheroids of tunable diameters in a 6-well plate.

• Spheroids induction with multiple hIPSC cell lines.

• Robust and reproducible production method suitable for routine laboratory use.

Graphical overview

Spheroid induction process following the pellet method on molded silicon discs

Here, we describe immunofluorescent (IF) staining assay of 3D cell culture colonoids isolated from mice colon as described previously. Primary cultures developed from isolated colonic stem cells are called colonoids. Immunofluorescence can be used to analyze the distribution of proteins, glycans, and small molecules—both biological and non-biological ones. Four-day-old colonoid cell cultures grown on Lab-Tek 8-well plate are fixed by paraformaldehyde. Fixed colonoids are then subjected to antigen retrieval and blocking followed by incubation with primary antibody. A corresponding secondary antibody tagged with desired fluorescence is used to visualize primary antibody–marked protein. Counter staining to stain actin filaments and nucleus to assess cell structure and DNA in nucleus is performed by choosing the other two contrasting fluorescences. IF staining of colonoids can be utilized to visualize molecular markers of cell behavior. This technique can be used for translation research by isolating colonoids from colitis patients’ colons, monitoring the biomarkers, and customizing their treatments.

Key features

• Analysis of molecular markers of cell behavior.

Protocol to visualize proteins in 3D cell culture.

• This protocol requires colonoids isolated from mice colon grown on matrigel support.

• Protocol requires at least eight days to complete.

Graphical overview

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

The blastocysts consist of dozens of cells of three distinct lineages: epiblast (Epi), trophoblast (TB), and primitive endoderm (PrE). All embryonic and extraembryonic tissues are derived from Epi, TB, and PrE. Stem cell lines representing preimplantation Epi and TB have been established and are known as embryonic stem cells (ESCs) and trophoblast stem cells (TSCs). Extraembryonic endoderm cells (XENCs) constitute a cell line that has been established from PrE. Although in vivo, PrE gives rise to visceral endoderm (VE), parietal endoderm (PE), and marginal zone endoderm (MZE); XENCs, on blastocyst injection into chimeras, primarily contribute to the distal region of PE. Here, we provide a comprehensive protocol for the establishment of fully potent primitive endoderm stem cell (PrESC) lines. PrESCs are established and maintained on mouse embryonic fibroblast (MEF) feeder cells in a serum-free medium supplemented with fibroblast growth factor 4 (FGF4), heparin, CHIR99021, and platelet-derived growth factor-AA (PDGF-AA). PrESCs co-express markers indicative of pluripotency and endoderm lineage commitment, exhibiting characteristics akin to those of PrE. On transplantation of PrESCs into blastocysts, they demonstrate a high efficiency in contributing to VE, PE, and MZE. PrESCs serve as a valuable model for studying PrE, sharing similarities in gene expression profiles and differentiation potential. PrESCs constitute a pivotal cornerstone for in vitro analysis of early developmental mechanisms and for studies of embryo reconstitution in vitro, particularly in conjunction with ESCs and TSCs.

Key features

• Establishment and maintenance of primitive endoderm stem cell (PrESCs) capable of recapitulating the developmental prowess inherent to PrE.

• Offering a source of PrE lineage for embryo-like organoid reconstitution studies.

Graphical overview

Human induced pluripotent stem cells (hiPSCs) hold immense promise in regenerative medicine as they can differentiate into various cell lineages, including adipocytes, osteoblasts, and chondrocytes. Precisely guiding hiPSC-derived mesenchymal progenitor cells (iMSCs) towards specific differentiation pathways is crucial for harnessing their therapeutic potential in tissue engineering, disease modeling, and regenerative therapies. To achieve this, we present a comprehensive and reproducible protocol for effectively differentiating iMSCs into adipocytes and osteoblasts. The differentiation process entails culturing iMSCs in tailored media supplemented with specific growth factors, which act as cues to initiate adipogenic or osteogenic commitment. Our protocol provides step-by-step guidelines for achieving adipocyte and osteoblast differentiation, ensuring the generation of mature and functional cells. To validate the success of differentiation, key assessment criteria are employed. For adipogenesis, the presence of characteristic lipid droplets within the iMSC-derived cells is considered indicative of successful differentiation. Meanwhile, Alizarin Red staining serves as a marker for the osteogenic differentiation, confirming the formation of mineralized nodules. Importantly, the described method stands out due to its simplicity, eliminating the need for specialized equipment, expensive materials, or complex reagents. Its ease of implementation offers an attractive advantage for researchers seeking robust and cost-effective approaches to derive adipocytes and osteoblasts from iMSCs. Overall, this protocol establishes a foundation for exploring the therapeutic potential of hiPSC-derived cells and advancing the field of regenerative medicine.

Key features

• iMSC derivation in this protocol uses embryonic body formation technique.

• Adipogenesis and osteogenesis protocols were optimized for human iPSC-derived iMSCs.

• Derivation of iMSC from hiPSC was developed in a feeder-free culture condition.

• This protocol does not include human iPSC reprogramming strategies.

Graphical overview

Brain organoids have been widely used to study diseases and the development of the nervous system. Many reports have investigated the application of brain organoids, but most of these models lack vascular structures, which play essential roles in brain development and neurological diseases. The brain and blood vessels originate from two different germ layers, making it difficult to induce vascularized brain organoids in vitro. We developed this protocol to generate brain-specific blood vessel and cerebral organoids and then fused them at a specific developmental time point. The fused cerebral organoids exhibited robust vascular network-like structures, which allows simulating the in vivo developmental processes of the brain for further applications in various neurological diseases.

Key Features

• Culturing vascularized brain organoids using human embryonic stem cells (hESCs).

• The new approach generates not only neural cells and vessel-like networks but also brain-resident microglia immune cells in a single organoid.

Graphical overview

Workflow and timeline for vessel organoid and vascularized brain organoid generation. (By Figdraw, ID: RTIURffccf)

Induced pluripotent stem cells (iPSCs) generated from human sources are valuable tools for studying skeletal development and diseases, as well as for potential use in regenerative medicine for skeletal tissues such as articular cartilage. To successfully differentiate human iPSCs into functional chondrocytes, it is essential to establish efficient and reproducible strategies that closely mimic the physiological chondrogenic differentiation process. Here, we describe a simple and efficient protocol for differentiation of human iPSCs into chondrocytes via generation of an intermediate population of mesenchymal progenitors. These methodologies include step-by-step procedures for mesenchymal derivation, induction of chondrogenic differentiation, and evaluation of the chondrogenic marker gene expression. In this protocol, we describe the detailed procedure for successful derivation of mesenchymal progenitor population from human iPSCs, which are then differentiated into chondrocytes using high-density culture conditions by stimulating with bone morphogenetic protein-2 (BMP-2) or transforming growth factor beta-3 (TGFβ-3). The differentiated iPSCs exhibit temporal expression of cartilage genes and accumulation of a cartilaginous extracellular matrix in vitro, indicating successful chondrogenic differentiation. These detailed methodologies help effective differentiation of human iPSCs into the chondrogenic lineage to obtain functional chondrocytes, which hold great promise for modeling skeletal development and disease, as well as for potential use in regenerative medicine for cell-based therapy for cartilage regeneration.

Key features

• Differentiation of human iPSCs into chondrocytes using 3D culture methods.

• Uses mesenchymal progenitors as an intermediate for differentiation into chondrocytes.