Current Protocols in Stem Cell Biology最新文献

Our laboratory and others have developed protocols to generate glucose-responsive stem cell–derived β cells in vitro. The cells resulting from these protocols could supplement or replace the use of human cadaveric islets for cell-based therapy for diabetes. The combination of an unlimited supply of pluripotent stem cell–derived β cells and gene-editing approaches will facilitate numerous in vitro studies not possible with cadaveric islets. Here, we describe a protocol for fluorescent labeling and isolation of stem cell–derived β cells. This purification of SC-β cells is based on intracellular zinc content and is a simple method to complement other approaches for generating and assaying these cells. © 2019 The Authors.

Basic Protocol: Fluorescent labeling and isolation of stem cell-derived β cells

Mutation of the gene GJB2, encoding connexin 26 (CX26; also known as gap junction beta 2), is the most frequent cause of hereditary deafness worldwide. CX26 is expressed in cochlear nonsensory cells, such as cochlear supporting cells, and forms gap junction plaques (GJPs) at cell-cell borders. Cochlear CX26-GJP-forming cells (Cx26GJCs) are thought to be an important therapeutic target for treatment of hereditary deafness. Nevertheless, the generation of Cx26GJCs—such as cochlear supporting cells—from embryonic stem/induced pluripotent stem (ES/iPS) cells has not been reported to date. Here, we detail a novel strategy for differentiating iPS cells into functional Cx26GJCs such as are found in cochlea. Several assays to characterize the phenotype of iPS-derived Cx26GJCs are described, including qRT-PCR, immunohistological analysis, morphological analysis, a scrape-loading and dye transfer assay, and calcium imaging. This in vitro model has applications in the establishment of inner-ear cell therapies and in drug screening to target GJB2-related hearing loss. © 2019 by John Wiley & Sons, Inc.

Basic Protocol: Induction of mouse stem cells to create CX26-GJP-forming cells

Support Protocol 1: Maintenance and passage of mouse induced pluripotent stem cells

Support Protocol 2: Screening for high GJB2 and GJB6 expression in SFEBq culture using quantitative real-time PCR

Support Protocol 3: Characterization of cells at different stages of differentiation by immunostaining

Support Protocol 4: Ultrastructural analyses of cells at different stages of CX26-GJP-forming cell induction

Support Protocol 5: Functional analyses of stem cell–derived CX26-GJP-forming cells

In the 20 years since the first human pluripotent stem cell (hPSC) lines were established, there have been a plethora of protocols developed that allow us to generate a wide range of human cell types in vitro. Efforts to achieve a greater degree of specificity and efficiency in generating desired cell types have resulted in increasingly complex approaches. The magnitude and timing of signals has become key, and the concept of a “fully defined” system is a forever sought-after goal with shifting goalposts. This overview discusses two related approaches that can be used to deliver a tightly regulated, intermediate-strength signal, and which can also manage the impact of endogenous signaling variation and enable a switch away from bovine serum albumin–containing medium to a better-defined system without suffering a subsequent loss of robustness or efficiency. The approaches, referred to as top-down inhibition and baseline activation, were developed to deliver intermediate levels of BMP and WNT signaling during neural crest induction from hPSC, but could be applied to a variety of other signals and differentiation systems. © 2019 by John Wiley & Sons, Inc.

Morphogens are biological molecules that alter cellular identity and behavior across both space and time. During embryonic development, morphogen spatial localization can be confined to small volumes in a single tissue or permeate throughout an entire organism, and the temporal effects of morphogens can range from fractions of a second to several days. In most cases, morphogens are presented as a gradient to adjacent cells within tissues to pattern cell fate. As such, to appropriately model development and build representative multicellular architectures in vitro, it is vital to recapitulate these gradients during stem cell differentiation. However, the ability to control morphogen presentation within in vitro systems remains challenging. Here, we describe an innovative platform using channels patterned within thick, three-dimensional hydrogels that deliver multiple morphogens to embedded cells, thereby demonstrating exquisite control over both spatial and temporal variations in morphogen presentation. This generalizable approach should have broad utility for researchers interested in patterning in vitro tissue structures. © 2019 by John Wiley & Sons, Inc.

Cover: In Chichagova et al. (https://doi.org/10.1002/cpsc.95), the images show developing retinal organoids.

Sympathetic neurons are crucial for maintenance of body homeostasis and regulation of all organs. Diseases can arise from malfunction of sympathetic neurons, including malignancies, hypertension, and genetic disorders. Human pluripotent stem cells (hPSCs) allow modeling of human diseases and the in-depth study of pathologies of specific cell types associated with such disorders. Advances in the ability to differentiate hPSCs in vitro has allowed the generation of specific cell types such as sympathetic neurons, which provides the novel opportunity to study diseases affecting the sympathetic nervous system in the human context. Here, we compare selected recent publications that have achieved the goal of generating sympathetic neurons from hPSCs. We discuss strengths and weaknesses of each approach and debate future improvements and the next steps for using these neurons to better our understanding of sympathetic neuron disorders and their treatments. © 2019 by John Wiley & Sons, Inc.

Human pluripotent stem cell (hPSC) derivatives are valuable for a variety of research applications and have the potential to revolutionize approaches to personalized medicine. However, differentiation efficiency varies among cell lines and protocols. Therefore, methods to reliably determine cell type identity in cultures of hPSC derivatives in a manner that is consistent among laboratories are needed. While flow cytometry is apt for routine assessment of population heterogeneity, standardized protocols are not available for most cell types. This article describes a workflow for establishing a fit-for-purpose protocol for flow cytometric analysis of hPSC derivatives. Based on the application of this workflow, a standard operating procedure (SOP) was developed for the analysis of cardiac troponin in hPSC-derived cardiomyocytes (hPSC-CM). Throughout the article, important concepts related to antibody validation and gating strategies are presented to enable users to properly validate any antibody of interest and develop a rigorous SOP for their experimental needs. © 2019 by John Wiley & Sons, Inc.

We previously established a two-step protocol for differentiation of human pluripotent stem cells (hPSCs) into trophoblasts, using a StemPro-based minimal medium (EMIM) with bone morphogenetic protein-4 (BMP4). This protocol was suboptimal, resulting in induction of mixed mesoderm and trophoblast markers. Furthermore, adapting hPSCs to StemPro has proven difficult, and prolonged culture in this medium has been shown to promote genomic instability. Therefore, we moved on to the use of new media, including E8, and most recently, StemFlex, for rapid adaptation from feeder to non-feeder conditions. In the new protocol, we have incorporated the WNT inhibitor IWP2 into the first step, resulting in uniform differentiation of hPSCs into cytotrophoblast (CTB)-like cells, without induction of the mesoderm lineage. We also show that, at the end of the second step, there are distinct populations of terminally differentiated multinucleated human chorionic gonadotropin (hCG)-producing syncytiotrophoblast (STB) and HLAG⁺ extravillous trophoblast (EVT)-like cells. © 2019 by John Wiley & Sons, Inc.

This unit describes a protocol for generating retinal organoids that contain all major retinal cell types and are responsive to light from human pluripotent stem cells (hPSCs). hPSCs are differentiated in 96-well plates to allow large-scale production of organoids that could be used for multiple applications, including study of human retinal development, disease modeling, and compound screening. The differentiation approach is based on the knowledge that insulin-like growth factor 1 signaling together with retinoic acid and triiodothyronine is important for retinal development. After 22 weeks in culture, the organoids form a thick layer of neuroepithelium containing photoreceptors and bipolar, horizontal, amacrine, Müller, and retinal ganglion cells. Differentiation progress can be tracked by morphological observations and protein localization, as detected with immunocytochemistry. © 2019 by John Wiley & Sons, Inc.

This article describes a screening platform to identify compounds that affect human embryonic vascular development. The procedure comprises the generation of human embryonic-like endothelial cells (ECs) from human pluripotent stem cells (hPSCs) and subsequent maturation under arterial flow conditions; the use of these cells for the high-throughput screening of small molecules that specifically inhibit the survival of embryonic-like ECs; the confirmation of the hits in embryonic-like ECs cultured under flow shear stress; and final validation in mouse embryonic ECs. The embryonic-like ECs express embryonic genes including DLL1, EPHB2, LYN, TEK, ID1, NRP2, CAST, FLT1, IGF1, DKK3, NIN, LEF1, and SORBS3. The entire screening procedure (without the validation step) can be completed within 1 month. This platform is an alternative/complement to standard animal protocols for assessing the effects of chemicals on embryonic vascular development. © 2019 by John Wiley & Sons, Inc.