The aim of this study was to develop an improvised protocol for ex vivo expansion of caprine satellite cells (SCs) using growth factors and their cryopreservation. The SCs were isolated from muscle tissue using collagenase type-I for enzymatic digestion. In vitro culture of SCs was done using Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum (FBS) and 15% horse serum. Immunofluorescence and PCR confirmed positive expression of PAX7 and MyoD, and negative expression of MyHC. Supplementation with 10 ng/ml basic fibroblast growth factor (bFGF) + 100 ng/ml insulin-like growth factor-1 (IGF-1) in the basal culture medium (DMEM + 2% FBS) showed similar results as control (routine culture medium). Subsequently, SCs were successfully cryopreserved using 10% dimethyl sulfoxide and 40% FBS with post-thaw viability of 74.72%. We have successfully established a protocol for the isolation, culture, and cryopreservation of goat SCs. Caprine SCs can be successfully expanded in vitro using growth factors (10 ng/ml bFGF and 100 ng/ml IGF-1) with minimum supplemental FBS in basal culture medium. Additionally, investigations could assess the viability and integrity of cryopreserved goat SCs post-thaw, ensuring that their proliferative capabilities remain intact. © 2025 Wiley Periodicals LLC. Basic Protocol: Isolation, culture, cost-effective expansion, and cryopreservation of caprine satellite cells.
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become a powerful source for the in vitro modeling of cardiac diseases and various other essential applications, including cardiotoxicity screening and regenerative cell replacement therapies. Although many differentiation protocols have been developed to generate cardiomyocytes from human pluripotent stem cells, these protocols are costly and complex, requiring expensive and often unnecessary components (e.g., B27 medium supplement). In addition, the use of animal-derived growth factors limits their use for regenerative medicine purposes. To address these issues, herein, we have developed an efficient, cost-effective, and protein-free hPSC-CM protocol using only two components: DMEM/F12 basal medium and l-ascorbic acid 2-phosphate. By eliminating xenobiotic and complex components, the efficiency of directed differentiations is increased, the variability between cardiac differentiations is decreased, and the scalability of cell production is enhanced. Adaptation of this efficient, low-cost, and user-friendly cardiac differentiation protocol will enrich the utility and applicability of hPSC-CMs in drug discovery, cell therapies, tissue engineering, disease modeling, precision medicine, and cardiac regenerative medicine. © 2025 Wiley Periodicals LLC. Basic Protocol 1: hPSC cell culture Basic Protocol 2: hPSC-CM differentiation Basic Protocol 3: Characterization of hPSC-CMs by immunofluorescence (IF) imaging.
Human induced pluripotent stem cell (hiPSC)-based disease modeling can be successfully recapitulated to mimic disease characteristics across various human pathologies. Glaucoma, a progressive optic neuropathy, primarily affects the retinal ganglion cells (RGCs). While multiple groups have successfully generated RGCs from non-diseased hiPSCs, producing RGCs from glaucomatous human samples holds significant promise for understanding disease pathology by revealing patient-specific disease signatures. Given that keratocytes originate from the neural crest and previous reports suggest that ocular fibroblasts from glaucomatous donors carry pathogenic signatures, it is highly plausible that these signatures imprinted within the keratocytes will also be present in the derived RGCs. Thus, we aimed to generate RGCs from both glaucomatous and non-glaucomatous donor keratocytes and validate disease-specific signatures in 3D retinal organoids and in isolated RGCs. Our protocol describes the generation of iPSCs from keratocytes of both glaucomatous and non-glaucomatous donors, followed by their differentiation into retinal organoids. Subsequent isolation and culturing of RGCs were performed. Disease signatures in the RGCs were validated in both 3D retinal organoids (ROs) and 2D RGC cultures, and glaucomatous RGCs in 3D and 2D cultures demonstrated increased cleaved CASP3 and significant RGC loss, indicating disease imprints in the hiPSC-derived RGCs. This model offers a venue and high throughput platform for studying glaucomatous disease pathology and holds significant potential for drug discovery using RGCs derived from human donors. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing of keratocytes from human cadaveric donors Basic Protocol 2: Reprogramming donor keratocytes into iPSCs Basic Protocol 3: Evaluation of chromosomal loss during reprogramming in iPSCs by karyotyping Basic Protocol 4: Generation of 3D ROs Basic Protocol 5: Dissociation and culturing of RGCs from 3D ROs Support Protocol 1: Immunostaining for phenotypic characterization of cells Support Protocol 2: Sectioning of 3D ROs and immunostaining Support Protocol 3: Western blotting for cleaved CASP3 and THY1.
Bone marrow adipose tissue (BMAT) has garnered significant attention due to its critical roles in leukemia pathogenesis, cancer metastasis, and bone marrow failure. BMAT is a metabolically active, distinct tissue that differs from other fat depots. Marrow adipocytes, closely interacting with hematopoietic stem/progenitor cells and osteoblasts, play a pivotal role in regulating their functions. However, standardized methods for isolating and defining human BMAT (hBMAT) remain unclear. In animal models, BMAT is commonly isolated directly from the bone marrow through flushing, enzymatic digestion, or mechanical disruption. In humans, BMAT isolation often involves the adipogenic induction of bone marrow mesenchymal stem/stromal cells (BM-MSCs) derived from bone marrow aspirates. However, this approach reflects cellular responses to chemical stimuli and may not accurately represent in vivo differentiation potential. Similarly, BMAT obtained from hip or knee replacement surgeries might not reflect basal physiological conditions due to inflammatory influences. Here, we describe a direct method for culturing BMAT from the fatty layer of bone marrow aspirates obtained from healthy transplant donors. This protocol employs centrifugation and washing steps using basic laboratory equipment, offering simple and non-enzymatic approach. For validation, isolated cells are characterized according to the International Society for Cell & Gene Therapy (ISCT) criteria. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human BMAT-MSCs from the fatty layer of the bone marrow Basic Protocol 2: Culture expansion, trypsinization, and cryopreservation of BMAT-MSCs Support Protocol 1: Immunophenoypic characterization of human BMAT-MSCs by flow cytometry Support Protocol 2: In vitro characterization of multilineage differentiation potential of human BMAT-MSCs Support Protocol 3: Further characterization of gene expression in human BMAT-MSCs using qRT-PCR.
Astrocytes are key regulators of central nervous system (CNS) homeostasis, and their dysfunction is implicated in neurological and neurodegenerative disorders. Here, we describe a two-step protocol to generate astrocytes from human induced pluripotent stem cells (hiPSCs) using a bankable neural progenitor cell (NPC) intermediate, followed by low-density passaging and overexpression of the gliogenic transcription factor NFIA. A bankable NPC intermediate allows for facile differentiation into both purified neuronal and astrocyte cell types in parallel from the same genetic background, depending on the experimental needs. This article presents a protocol to generate NPCs from hiPSCs, which are then differentiated into hiPSC-derived astrocytes, termed iAstrocytes. The resulting iAstrocytes express key markers of astrocyte identity at transcript and protein levels by bulk RNA-Seq and immunocytochemistry, respectively. Additionally, they respond to the inflammatory stimuli poly(I:C) and generate waves of calcium activity in response to either physical activity or the addition of ATP. Our approach offers a simple and robust method to generate and characterize human astrocytes, which can be used to model human disease affecting this cell type. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of hiPSCs to NPCs Basic Protocol 2: Differentiation of NPCs into iAstrocytes Support Protocol 1: Molecular validation of iAstrocytes Support Protocol 2: Calcium imaging-based validation of iAstrocyte function Support Protocol 3: Differentiation of NPCs into neurons.
