Pluripotent Stem Cell Culture

Adult stem-cell epithelial organoid culture is a three-dimensional culture method in which adult epithelial stem cells or isolated epithelial crypts self-organize into organ-like epithelial structures that retain stem-cell activity and generate differentiated epithelial lineages (). In the intestinal model, Lgr5-positive crypt base columnar cells are adult stem cells that generate all intestinal epithelial lineages, and single Lgr5-positive cells or isolated crypts can form long-term crypt–villus organoids in extracellular matrix culture when supplied with epithelial niche signals (). Organoid growth reflects epithelial stem-cell survival, self-renewal, proliferation, and multilineage differentiation; the main readouts are organoid-forming efficiency, growth, morphology, passaging capacity, marker expression, and lineage composition ().

This protocol maintains and expands human pluripotent stem cells under feeder-free, chemically defined conditions using E8 medium and vitronectin-coated culture surfaces; the readout is sustained adherent colony growth with undifferentiated morphology and retained pluripotency-marker expression during serial passaging. E8-based hPSC culture relies on defined soluble factors and matrix-dependent adhesion rather than feeder cells; vitronectin supports hPSC attachment through integrin-mediated interactions, and EDTA passaging dissociates colonies as small aggregates without enzymatic digestion, centrifugation, or routine ROCK-inhibitor treatment.

Mesenchymal stromal/stem cells are isolated by their ability to adhere to tissue-culture plastic, expand as fibroblast-like colonies, and retain defined MSC identity after expansion; the accepted identity readout is plastic adherence, expression of CD105/CD73/CD90, absence of major hematopoietic markers including CD45/CD34/CD14 or CD11b/CD79α or CD19/HLA-DR, and in-vitro osteogenic, adipogenic, and chondrogenic differentiation.

Human iPSC reprogramming converts somatic cells into pluripotent cells by introducing defined transcription factors; classic human studies used OCT3/4, SOX2, KLF4, and c-MYC, or OCT4, SOX2, NANOG, and LIN28, and judged reprogramming by embryonic-stem-cell-like morphology, pluripotency-marker expression, normal karyotype, and differentiation into derivatives of the three germ layers. This protocol is framed around non-integrating reprogramming culture, with Sendai virus, episomal plasmids, or synthetic modified mRNA as literature-supported delivery options; Sendai virus is an RNA vector reported to avoid host-genome integration, episomal vectors can generate integration-free human iPSCs, and modified mRNA can reprogram human cells while avoiding genomic modification.

Embryoid bodies are three-dimensional aggregates formed from ESCs or iPSCs in non-adherent suspension conditions, and EB formation is used to initiate spontaneous differentiation and assess differentiation potential across ectodermal, mesodermal, and endodermal lineages. EB readouts include aggregate formation, EB size and morphology, lineage-marker expression by immunostaining or qPCR, and downstream lineage differentiation outcomes.

This protocol describes hPSC-derived cerebral organoid generation using embryoid body formation, neural induction, extracellular-matrix-supported neuroepithelial expansion, and long-term 3D culture under static, spinning, or air-liquid interface conditions; the readouts are organoid morphology, neuroepithelial organization, neural progenitor and neuronal marker expression, cortical-region identity, axon outgrowth, and functional neural activity when maturation assays are included.

Directed neural induction of hPSCs uses inhibition of BMP and TGF-β/Activin/Nodal SMAD signaling to bias pluripotent cells toward neuroectoderm; the readout is emergence of neural plate/rosette-like cells and neural progenitor markers such as PAX6, SOX1/SOX2, and Nestin, followed by expandable NPCs that can generate neuronal lineages.