Current Protocols in Stem Cell Biology最新文献

Esophageal cancers comprise adenocarcinoma and squamous cell carcinoma, two distinct histologic subtypes. Both are difficult to treat and among the deadliest human malignancies. We describe protocols to initiate, grow, passage, and characterize patient-derived organoids (PDO) of esophageal cancers, as well as squamous cell carcinomas of oral/head-and-neck and anal origin. Formed rapidly (<14 days) from a single-cell suspension embedded in basement membrane matrix, esophageal cancer PDO recapitulate the histology of the original tumors. Additionally, we provide guidelines for morphological analyses and drug testing coupled with functional assessment of cell response to conventional chemotherapeutics and other pharmacological agents in concert with emerging automated imaging platforms. Predicting drug sensitivity and potential therapy resistance mechanisms in a moderate-to-high throughput manner, esophageal cancer PDO are highly translatable in personalized medicine for customized esophageal cancer treatments. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Generation of esophageal cancer PDO

Basic Protocol 2: Propagation and cryopreservation of esophageal cancer PDO

Basic Protocol 3: Imaged-based monitoring of organoid size and growth kinetics

Basic Protocol 4: Harvesting esophageal cancer PDO for histological analyses

Basic Protocol 5: PDO content analysis by flow cytometry

Basic Protocol 6: Evaluation of drug response with determination of the half-inhibitory concentration (IC₅₀)

Support Protocol: Production of RN in HEK293T cell conditioned medium

Monocytes and macrophages are essential for immune defense and tissue hemostasis. They are also the underlying trigger of many diseases. The availability of robust and short protocols to induce monocytes and macrophages from human induced pluripotent stem cells (hiPSCs) will benefit many applications of immune cells in biomedical research. Here, we describe a protocol to derive and functionally characterize these cells. Large numbers of hiPSC-derived monocytes (hiPSC-mono) could be generated in just 15 days. These monocytes were fully functional after cryopreservation and could be polarized to M1 and M2 macrophage subtypes. hiPSC-derived macrophages (iPSDMs) showed high phagocytotic uptake of bacteria, apoptotic cells, and tumor cells. The protocol was effective across multiple hiPSC lines. In summary, we developed a robust protocol to generate hiPSC-mono and iPSDMs which showed phenotypic features of macrophages and functional maturity in different bioassays. © 2020 The Authors.

Basic Protocol 1: Differentiation of hiPSCs toward monocytes

Support Protocol 1: Isolation and cryopreservation of monocytes

Support Protocol 2: Characterization of monocytes

Basic Protocol 2: Differentiation of different subtypes of macrophages

Support Protocol 3: Characterization of hiPSC-derived macrophages (iPSDMs)

Support Protocol 4: Functional characterization of different subtypes of macrophages

The homeostatic proliferation-differentiation gradient in the esophageal epithelium is perturbed under inflammatory disease conditions such as gastroesophageal reflux disease and eosinophilic esophagitis. Herein we describe the protocols for rapid generation (<14 days) and characterization of single-cell-derived, three-dimensional (3D) esophageal organoids from human subjects and mice with normal esophageal mucosa or inflammatory disease conditions. While 3D organoids recapitulate normal epithelial renewal, proliferation, and differentiation, non-cell autonomous reactive epithelial changes under inflammatory conditions are evaluated in the absence of the inflammatory milieu. Reactive epithelial changes are reconstituted upon exposure to exogenous recombinant cytokines. These changes are modulated pharmacologically or genetically ex vivo. Molecular, structural, and functional changes are characterized by morphology, flow cytometry, biochemistry, and gene expression analyses. Esophageal 3D organoids can be translated for the development of personalized medicine in assessment of individual cytokine sensitivity and molecularly targeted therapeutics in esophagitis patients © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Generation of esophageal organoids from biopsy or murine esophageal epithelial sheets

Basic Protocol 2: Propagation and cryopreservation of esophageal organoids

Basic Protocol 3: Harvesting of esophageal organoids for RNA isolation, immunohistochemistry, and evaluation of 3D architecture

Basic Protocol 4: Modeling of reactive epithelium in esophageal organoids.

Pluripotent stem cell (PSC) cultures are subjected to selective pressures that can result in acquisition and expansion of recurrent genetic abnormalities at any time. These recurrent abnormalities enhance the variant cells harboring them with a competitive advantage over wild-type cells. Variant cells can eventually supplant wild-type cells entirely and become fixed in culture. Such variants can impact the efficacy of PSCs in research and clinical applications. Therefore, routine genomic characterization is required for reliable and effective use of PSCs. In this article we describe the capabilities and limitations of several assays commonly used for assessing PSC genomic stability. Based on this analysis, we provide a recommendation for integrating assays into a comprehensive testing regimen that maximizes coverage while minimizing cost. © 2020 by John Wiley & Sons, Inc.

Genome editing has become one of the most powerful tools in present-day stem cell and regenerative medicine research, but despite its rapid acceptance and widespread use, some elements of the technology still need improvement. In this unit, we present data regarding the use of a new, more efficient type of transcription activator-like effector nuclease (TALEN) for gene editing. Our group has generated bicistronic genes in which classical TALEN coding sequences are linked by 2A elements to different reporter molecules, such as fluorochromes (TALEN-F) or membrane receptors (TALEN-M). This structure results in two proteins transcribed from the same transcript, of which the second (the reporter) can be used as the target for selection by fluorescence-assisted cell sorting (FACS) or magnetic-activated cell sorting (MACS). The application of these new TALEN genes allows a rapid enrichment of cells in which both members of the TALEN pair are active, thus eliminating the need for lengthy selection in culture and laborious characterization of a large number of clones. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Generation of new TALENs

Basic Protocol 2: Genome editing using TALEN-F

Alternate Protocol 1: Generation of TALEN-M

Support Protocol 1: mRNA in vitro transcription (IVT) of TALEN-T2A-reporter expression vector

Alternate Protocol 2: Editing of primary T cells using TALEN-M

Basic Protocol 3: Verifying gene editing

Support Protocol 2: Rapid expansion protocol for edited T-cells

Translating human induced pluripotent stem cell (hiPSC)–derived cells and tissues into the clinic requires streamlined and reliable production of clinical-grade hiPSCs. This article describes an entirely animal component–free procedure for the reliable derivation of stable hiPSC lines from donor peripheral blood mononuclear cells (PBMCs) using only autologous patient materials and xeno-free reagents. PBMCs are isolated from a whole blood donation, from which a small amount of patient serum is also generated. The PBMCs are then expanded prior to reprogramming in an animal component–free erythroblast growth medium supplemented with autologous patient serum, thereby eliminating the need for animal serum. After expansion, the erythroblasts are reprogrammed using either cGMP-grade Sendai viral particles (CytoTune™ 2.1 kit) or episomally replicating reprogramming plasmids (Epi5™ kit), both commercially available. Expansion of emerging hiPSCs on a recombinant cGMP-grade human laminin substrate is compatible with a number of xeno-free or chemically defined media (some available as cGMP-grade reagents), such as E8, Nutristem, Stemfit, or mTeSR Plus. hiPSC lines derived using this method display expression of expected surface markers and transcription factors, loss of the reprogramming agent–derived nucleic acids, genetic stability, and the ability to robustly differentiate in vitro to multiple lineages. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Isolating peripheral blood mononuclear cells using CPT tubes

Support Protocol 1: Removal of clotting factors to produce serum from autologous plasma collected in Basic Protocol 1

Basic Protocol 2: PBMC expansion in an animal-free erythroblast expansion medium containing autologous serum

Basic Protocol 3: Reprogramming of expanded PBMCs with Sendai viral reprogramming particles

Alternate Protocol: Reprogramming of expanded PBMCs with episomal plasmids

Basic Protocol 4: Picking, expanding, and cryopreserving hiPSC clones

Support Protocol 2: Testing Sendai virus kit–reprogrammed hiPSC for absence of Sendai viral RNA

Support Protocol 3: Testing Epi5 kit–reprogrammed hiPSC for absence of episomal plasmid DNA

Support Protocol 4: Assessing the undifferentiated state of human pluripotent stem cell cultures by multi-color immunofluorescent staining and confocal imaging

Support Protocol 5: Coating plates with extracellular matrices to support hiPSC attachment and expansion

The complex program of mouse development entails specification of the embryonic epiblast (Epi) as well as the extra-embryonic trophectoderm (TE) and primitive endoderm (PrE). These three lineages of mouse blastocyst can be modeled in vitro using stem cells derived from primary tissues. In these cultures, cells self-renew while retaining their developmental potential if put back into a developing embryo. Indeed, embryonic stem cells (ESC), when injected into a blastocyst, readily contribute to all embryonic lineages. Similarly, trophoblast stem cells (TSCs) will give rise to all TE-derived trophoblast lineages, and extraembryonic endoderm cells (XEN) will contribute to the PrE-derived yolk sack. These model systems are a powerful tool to study early development, lineage specification, and placenta formation. Only recently reproducible and chemically defined culture systems of these cells have been described. This overview discusses such novel methods for culturing ESC/TSC/XEN, as well as their molecular signatures and developmental potential. Recent strides in expanding the developmental potential of stem cells as well as achieving models more reminiscent of their in vivo counterparts are discussed. Finally, such in vitro stem cells can self-assemble into structures resembling embryos when used in novel 3D-culture systems. This article discusses the strengths and limitations of such “synthetic embryos” in studying developmental processes. © 2020 by John Wiley & Sons, Inc.

TP53 point mutations are found in 50% of all cancers and seem to play an important role in cancer pathogenesis. Thus, human induced pluripotent stem cells (hiPSCs) overexpressing mutant TP53 are a valuable tool for the generation of in vitro models of cancer stem cells or for in vivo xenograft models. Here, we describe a protocol for the alteration of gene expression in hiPSCs via overexpression of a mutant form of the TP53 (R249S) gene using lentiviral transduction. A high amount of TP53 protein is detected 1 week after transduction and antibiotic selection. Differentiation of transduced hiPSCs gives insight into better understanding cancer formation in different tissues and may be a useful tool for genetic or pharmacologic screening assays. © 2019 The Authors.

Basic Protocol 1: Production and concentration of third-generation lentivirus

Support Protocol 1: Cloning of gene of interest into modulation vector

Support Protocol 2: Preparation of DMEM GlutaMAX™ with 10% fetal bovine serum and 1% penicillin-streptomycin

Basic Protocol 2: Transduction of human induced pluripotent stem cells and selection of positively transfected cells

Support Protocol 3: Preparation of Matrigel^®-coated plates

Support Protocol 4: Preparation of mTeSR™1 medium

Cover: In Fukunaga et al. (https://doi.org/10.1002/cpsc.100), the image shows immunohistochemistry for CX26 and SOX2 in mouse cochlea and differentiated cells from 2D culture. (A-C) Staining for CX26 (green), SOX2 (red), and DAPI (blue) in 16-week mouse cochlea. Heterogeneous SOX2-positive regions include inner sulcus cells (ISC). IHC, inner hair cells. (D-I) Staining for CX26 (green), SOX2 (red), and DAPI (blue) in 2D culture at day 15. Heterogeneous SOX2-positive regions include iCx26GJCs. (G-I) show magnifications of boxed regions in (D-F), respectively. Arrowheads point to GJPs. Scale bars: 20 µm (D-F), 10 µm (A-C), and 5 µm (G-I). Parts of the figure are reproduced from Fukunaga et al. (2016) with permission from Elsevier.

Skeletal muscle tissue regeneration requires quiescent satellite cell activation, proliferation, and differentiation. Regenerative capacity of satellite cells can be studied in vitro by differentiating under low-serum conditions (2% to 5%) to form multinucleated myotubes. Myotubes are fixed and stained, and indices of differentiation are quantified. Jenner and Giemsa stains are typically used for myotube staining; however, this staining process can be variable depending on factors such as stain pH, staining time, and time since stain preparation. This article includes protocols for myoblast isolation, proliferation, and differentiation in vitro; Jenner-Giemsa staining; HEMA 3 staining; and quantification. Representative images using each staining method and quantification are included. The protocols identify critical steps and considerations for cell culture and each staining method and provide an even simpler alternative to Jenner-Giemsa staining. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Primary myoblast isolation

Alternate Protocol 1: Plating cryopreserved myoblasts

Basic Protocol 2: Myoblast passage and expansion

Basic Protocol 3: Myoblast differentiation

Basic Protocol 4: HEMA 3 staining

Alternate Protocol 2: Jenner-Giemsa staining

Basic Protocol 5: Quantification of myotube density

Basic Protocol 6: Quantification of fusion index

Basic Protocol 7: Quantification of myotubes per field