Current Protocols in Stem Cell Biology最新文献

We have developed an organotypic culture system that allows the production of bone tissue features on a centimeter scale. A composite, calcium phosphate–strained fibrin gel system is able to organize itself in the presence of osteoblastic cells, creating basic hierarchical units as seen in vivo, and can be modified to produce a range of other tissues that require such directional structuring. Constructs evolve over time into multi-compositional structures containing a high mineral content and terminally differentiated, osteocyte-like cells. These tissues can be cultured over extended durations (exceeding 1 year) and are responsive to a variety of chemical and biological agents. The platform can reduce the number of animals used in experimentation by acting as an intermediate stage in which more personalized research conditions can be generated. We provide a thorough description of the protocol used to successfully culture and modify this system, as well as guidance on compositional characterization. © 2019 by John Wiley & Sons, Inc.

This unit describes protocols for isolating subpopulations of extracellular vesicles (EVs) purified from human adipose tissue–derived mesenchymal stromal cells by density gradient centrifugation and for characterizing them by flow cytometry (FCM). Determining the optimal strategy for isolating EVs is a critical step toward retrieving the maximal amount while ensuring the recovery of different vesicular subtypes. The first protocol details density gradient centrifugation to isolate both exosomes and microvesicles. In the second protocol, characterization of EV subpopulations by FCM is depicted, taking advantage of non-conventional modalities, in accordance with the latest technical indications. The procedures described here can be easily reproduced and can be employed regardless of the cell type used to obtain EVs. © 2019 by John Wiley & Sons, Inc.

This article describes a protocol for isolating human spermatogonial stem cells (hSSCs) from human testis tissues, which can be utilized for further characterization. Human testis tissues are first subjected to mechanical separation, enzymatic digestion, and physical filtering, yielding a single testicular cell suspension. SSCs or other cell types of interest can be sorted out via different enrichment approaches using selected cell surface markers (e.g., magnetic-activated cell sorting, MACS). Those enriched cells can further be used for different purposes, including cell culture, RNA sequencing, and other genomics-based profiling, which allow for further characterization of their properties. © 2019 by John Wiley & Sons, Inc.

We have established an in vitro model of the human congenital heart defect (CHD)–associated mutation NKX2.5 R141C. We describe the use of the hanging drop method to differentiate Nkx2.5^R141C/+ murine embryonic stem cells (mESCs) along with Nkx2.5^+/+ control cells. This method allows us to recapitulate the early stages of embryonic heart development in tissue culture. We also use qRT-PCR and immunofluorescence to examine samples at different time points during differentiation to validate our data. The in vivo model is a mouse line with a knock-in of the same mutation. We describe the isolation of RNA from embryonic day 8.5 (E8.5) embryos and E9.5 hearts of wild-type and mutant mice. We found that the in vitro model shows reduced cardiomyogenesis, similar to Nkx2.5^R141C/+ embryos at E8.5, indicating a transient loss of cardiomyogenesis at this time point. These results suggest that our in vitro model can be used to study very early changes in heart development that cause CHD. © 2018 by John Wiley & Sons, Inc.

Macrophages play important roles in many diseases. We describe a protocol and the associated resources for the differentiation of human induced pluripotent stem cell-derived macrophages (IPSDM) and their applications in understanding human macrophage physiology and relevant diseases. The protocol uses an embryoid body–based approach with a combination of serum-free condition for hematopoiesis specification, followed by adherent culture with serum and M-CSF for myeloid expansion and macrophage maturation. The protocol produced an almost pure culture of CD45⁺/CD18⁺ macrophages yielding up to 2 × 10⁷ cells per 6-well plate of iPSCs within 24 days, demonstrating high efficiency, purity, and scalability. The IPSDM and monocyte-derived macrophages (HMDM) cultured in the same medium were compared at morphological, functional and transcriptomic levels by RNA-sequencing. IPSDM and HMDM showed broadly similar profiles of coding transcriptome, alternative splicing events, and long noncoding RNAs, with advantages and successful applications in disease modeling using patients-derived and CRISPR-edited iPSC lines. © 2018 by John Wiley & Sons, Inc.

Mesenchymal stem cells (MSCs) are of great interest due to their properties of immune modulation, tissue regeneration, and multipotent differentiation. Future developments of clinical applications, however, require a higher yield of MSCs, lower number of passages of cells in culture, and shorter time from harvest to use. Optimization and standardization of techniques for mesenchymal adipose tissue–derived stem cell isolation offers solutions to current bottlenecks as a larger amount of MSCs can be isolated. These improvements result in shorter expansion time, fewer passages, less donor material needed, and higher MSC yield. This paper describes an MSC isolation method combining enzymatic digestion with mechanic disruption. This protocol is a standardized and easy-to-implement method for reaching significantly higher MSC yields compared to conventional enzymatic isolation protocols. Based on the results presented, we hypothesize that the combined enzymatic and mechanical method increases the surface area of the adipose tissue, facilitating digestion by enzymes. This approach reduces the amount of adipose tissue and in vitro expansion time needed to reach sufficient amounts of MSCs for clinical purposes. Importantly, the method does not require increased amounts of collagenase, nor does it impair the viability or differentiability of the MSCs. Using this protocol increases MSC yield by a factor of three. As a consequence, these results indicate that the physiological concentration of MSCs in adipose tissue is higher than previously assumed. © 2018 by John Wiley & Sons, Inc.

Organoids—or pluripotent stem cell–derived in vitro-grown simplified mini organs—have become a tremendously important model to study human organ development and disease. To restrict the noise inherent to the heterogeneous cell mixtures derived from organoid cultures, we developed a new technique of fluorescence-assisted cell sorting (FACS) of virus-infected cerebral organoid cultures. This method still includes the advantage of growing cells in a more natural environment than traditional cell culture, but now renders samples suitable for downstream cell type-specific multi-omics analyses. The protocol starts from stem cell-derived mature brain organoids and includes steps for: preparing the culture for viral infection, production of the viral stocks, FACS sample preparation, and gating and sorting implementation. The protocol has been developed for Zika virus infection, but can be extrapolated to other viruses or fluorescent marker expression as illustrated in an alternate protocol using a single-cycle lentivirus expressing a fluorescent reporter protein. © 2018 by John Wiley & Sons, Inc.

Human PSCs offer tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR-Cas9 technology has expanded this potential by providing a valuable tool for engineering or correcting disease-associated mutations. Because of the high efficiency with which CRISPR-Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele, without indel formation on the non-targeted allele. To overcome this obstacle, we describe the use of two oligonucleotides, one expressing the sequence change, with the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodology to make this protocol efficient with small numbers of cells and to limit the amount of labor-intensive clone passaging. This protocol provides a streamlined and technically simple approach for generating valuable tools to model human disease in stem cells. © 2018 by John Wiley & Sons, Inc.