Stem Cell

Bovine muscle satellite cells (MuSC) and fibro-adipogenic progenitor cells (FAP) are muscle resident stem cells that are responsible for postnatal muscle growth, intramuscular fat deposition, and extracellular matrix generation. These cells are of increasing interest for the cultivated meat community due to their ability to generate all the major components of meat; additionally, these cells are of interest to conventional animal science research to elucidate mechanisms to improve meat quality. To use these cells for these goals, efficient and accurate cell isolation, culture, and differentiation are essential to evaluate their cell fate decisions and behaviors. In this protocol, we detail a simultaneous isolation of both MuSCs and FAPs with multiple intermediate stopping points, allowing for flexibility for day-of time constraints. We also detail improved growth conditions to maximize cell expansion and procedures to assess cell differentiation. This protocol provides a flexible isolation procedure that is compatible with sampling in modern slaughterhouses or from biopsies. Additionally, the differentiation procedures provide improved differentiation but still allow in vitro treatment and assessment.

Adipogenic differentiation efficiency remains highly variable across laboratories and cellular models, underscoring a critical need for a robust and standardized protocol. Here, we describe an optimized and highly effective protocol for inducing adipogenesis in multiple models, including murine 3T3-L1 preadipocytes, stromal vascular fraction (SVF) from neonatal and adult mice, and human adipose-derived stem cells (hADSCs). Systematic optimization was performed on key parameters such as initial cell confluence, induction timing, inducer composition, and culture surface coating. We show that high cell density, rosiglitazone supplementation, and an extended primary induction phase combine to promote lipid accumulation. Notably, we introduce a crucial modification—prolonged low-dose insulin stimulation during the maintenance phase—that is essential for the efficient differentiation of adult SVF. Furthermore, when applied to hADSCs, the protocol consistently induced robust adipogenesis, confirming its cross-species applicability. Taken together, this comprehensive and reproducible protocol serves as a valuable tool for advancing in vitro adipogenesis research.

Adipose cells vary functionally, with white adipocytes storing energy and brown/beige adipocytes generating heat. Mouse and human subcutaneous white adipose tissue (WAT)-derived stromal vascular fraction (SVF) provides mesenchymal stem cells (MSCs) that can be differentiated into thermogenic adipocytes using pharmacological cocktails. After six days of browning induction, these cells exhibited significant upregulation of thermogenic markers (UCP1, Cidea, Dio2, PRDM16) along with adipogenic genes (PPARγ, aP2), showing enhanced thermogenic potential. This in vitro system offers a practical platform to study adipogenesis and thermogenic regulation.

Musculoskeletal pathologies present challenges in athletic horses, often leading to functional impairment. The slow or limited regenerative capacity of bone, joint, and tendon/ligament injuries, coupled with the limitations of conventional treatments, highlights the need for innovative therapies such as ortho-biologics and mesenchymal stem/stroma cells. Traditional 2D cell culture systems with fetal bovine serum (FBS) fail to replicate the complexity of the in vivo environment, whereas 3D cultures more accurately mimic native tissue architecture and cell–cell interactions. This study describes a novel method for isolating muscle-derived progenitor cells in a 3D environment using an autologous plasma-based gel and an innovative cell retrieval solution. The cultured cells exhibit immunomodulatory effects on T lymphocytes, trilineage differentiation potential, and immunophenotypic characteristics consistent with conventional mesenchymal stem/stromal cells. This streamlined 3D culture technique offers a promising platform for generating minimally manipulated autologous cell products tailored for equine regenerative medicine.

Bottom-up tissue engineering using cell spheroids offers many advantages in recapitulating native cell–cell and cell–matrix interactions. Many tissues, such as cartilage, bone, cardiac muscle, intestine, and neural tissues, have been tissue-engineered using cell spheroids. However, previous methods for spheroid assembling, such as mold casting, hydrogel-based bioprinting, or needle array, either lack control over final tissue geometry or face challenges in scalability and throughput. In this protocol, we describe a robust and scalable tissue engineering method for assembling cell spheroids into a thin, planar spheroid sheet. The spheroids are sandwiched between two flexible meshes held by a frame, facilitating uniform spheroid fusion while ensuring nutrient exchange and ease of handling. We demonstrate this method by producing thin cartilage tissue from human mesenchymal stem cells undergoing chondrogenic differentiation. This approach offers a practical platform for producing thin membrane-like tissue constructs for many research and therapeutic applications.

Three-dimensional (3D) human brain tissue models derived from induced pluripotent stem cells (iPSCs) have transformed the study of neural development and disease in vitro. While cerebral organoids offer high structural complexity, their large size often leads to necrotic core formation, limiting reproducibility and challenging the integration of microglia. Here, we present a detailed, reproducible protocol for generating multi-cell type 3D neurospheres that incorporate neurons, astrocytes, and optionally microglia, all derived from the same iPSCs. While neurons and astrocytes differentiate spontaneously from neural precursor cells, generated by dual SMAD-inhibition (blocking BMP and TGF-b signaling), microglia are generated in parallel and can infiltrate the mature neurosphere tissue after plating neurospheres into 48-well plates. The system supports a range of downstream applications, including functional confocal live imaging of GCaMP6f after adeno-associated virus (AAV) transduction of neurospheres or immunofluorescence staining after fixation. Our approach has been successfully implemented across multiple laboratories, demonstrating its robustness and translational potential for studying neuron–glia interactions and modeling neurodegenerative processes.

Adult muscle stem cells (MuSCs) are the key cellular source for regenerating skeletal muscle in vertebrates. MuSCs are typically identified in skeletal muscle by the expression of the paired box protein 7 (PAX7) protein. Here, we developed a combined RNA fluorescent in situ hybridization (FISH) using RNAscope technology and an immunofluorescence (IF) protocol for the simultaneous detection of Pax7 mRNA and PAX7 protein in individual MuSCs in vivo. Interestingly, we show that while most PAX7⁺ (protein) MuSCs express Pax7 mRNA, there is a subset of Pax7⁺ (mRNA) cells that do not express PAX7 protein. Altogether, we developed a combined FISH/IF protocol that allows for the co-detection of mRNA and protein in MuSCs in vivo, a strategy that can be applied to any target gene. The functional significance of the Pax7-expressing subset of cells lacking PAX7 protein prior to injury remains unknown.

Adipocytes are endocrine cells that function as the main energy storage in our body. They are commonly used in clinical procedures, including their removal via liposuction and transplantation in plastic surgery. Building on this, adipocytes can be used for ex vivo cellular manipulations, enabling therapeutic modifications that can provide beneficial clinical outcomes after transplantation. Here, we provide a detailed protocol on how to modify adipocytes and adipose organoids using CRISPR activation (CRISPRa), a technology termed adipose manipulation transplantation (AMT).

Well-differentiated airway epithelial cultures are commonly used to study airway stem cell lineages, ion and fluid transport, respiratory virus infection and replication, and disease mechanisms in vitro. This culture model involves the isolation and expansion of airway stem cells followed by their differentiation at an air–liquid interface (ALI), a process that has been previously documented in humans and mice. Domestic ferrets (Mustela putorius furo) have gained considerable importance in respiratory disease research due to their notable susceptibility to these conditions and their anatomical similarities to humans. Here, we present a comprehensive description of the isolation and culture of stem/progenitor cells from the ferret airway, along with a protocol for their differentiation at the ALI. Our findings have demonstrated that this ferret culture system not only supports the differentiation of the predominant airway epithelial cell types but also facilitates the generation of rare airway epithelial subpopulations, including pulmonary ionocytes, tuft cells, and pulmonary neuroendocrine cells. Additionally, we provide a detailed procedure for measuring transepithelial ion transport relevant to airway diseases, particularly cystic fibrosis. The ability to isolate and culture ferret airway stem cells, combined with ALI differentiation and functional assessment of transepithelial ion transport, offers a powerful platform for evaluating genetic and pharmacologic interventions related to cystic fibrosis.

The fatal motor neuron (MN) disease amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of the phrenic MNs (phMNs) controlling the activity of the diaphragm, leading to death by respiratory failure. Human experimental models to study phMNs are lacking, hindering the understanding of the mechanisms of phMN degeneration in ALS. Here, we describe a protocol to derive phrenic-like MNs from human induced pluripotent stem cells (hiPSC-phMNs) within 30 days. During spinal cord development, phMNs emerge from specific MN progenitors located in the dorsalmost MN progenitor (pMN) domain at cervical levels, under the control of a ventral-to-dorsal gradient of Sonic hedgehog (SHH) signaling and a rostro-caudal gradient of retinoic acid (RA). The method presented here uses optimized concentrations of RA and the SHH agonist purmorphamine, followed by fluorescence-activated cell sorting (FACS) of the resulting MN progenitor cells (MNPCs) based on a cell-surface protein (IGDCC3) enriched in hiPSC-phMNs. The resulting cultures are highly enriched in MNs expressing typical phMN markers. This protocol enables the generation of hiPSC-phMNs and is highly reproducible using several hiPSC lines, offering a disease-relevant system to study mechanisms of respiratory MN dysfunction. While the protocol has been validated in the context of ALS research, it can be adopted to study human phrenic MNs in other research fields where these neurons are of interest.