Current Protocols in Stem Cell Biology最新文献

Mesenchymal stem/stromal cells (MSCs) provide therapeutic effects in many diseases. Contrary to initial hypotheses, they act in a paracrine rather than a cellular manner. To this end, extracellular vesicles (EVs) have been found to mediate the therapeutic effects, even when harvested from MSC-conditioned cell culture supernatants. Lacking self-replicating activity and being so small that MSC-EV preparations can be sterilized by filtration, EVs provide several advantages as therapeutic agents over cellular therapeutics. At present, methods allowing EV preparation from larger volumes are scarce and regularly require special equipment. We have developed a polyethylene glycol−based precipitation protocol allowing extraction of EVs from several liters of conditioned medium. MSC-EVs prepared with this method have been successfully applied to a human graft-versus-host disease patient and to several animal models. Although the method comes with its own limitations, it is extremely helpful for the initial evaluation of EV-based therapeutic approaches. Here, we introduce the technique in detail and discuss all critical steps. © 2020 The Authors.

Basic Protocol 1: Preparation of MSC-conditioned medium for scaled MSC-EV production

Basic Protocol 2: PEG precipitation OF MSC-EV from MSC-conditioned medium

Current Protocols in Stem Cell Biology is publishing a special collection of eight articles from members of the PluriCore Network in the German Stem Cell Network. © 2020 Wiley Periodicals LLC.

Keratinocytes, as a primary somatic cell source, offer exceptional advantages compared to fibroblasts, which are commonly used for reprogramming. Keratinocytes can beat fibroblasts in reprogramming efficiency and reprogramming time and, in addition, can be easily and non-invasively harvested from human hair roots. However, there is still much to know about acquiring keratinocytes and maintaining them in cell culture. In this article, we want to offer readers the profound knowledge that we have gained since our initial use of keratinocytes for reprogramming more than 10 years ago. Here, all hints and tricks, from plucking the hair roots to growing and maintaining keratinocytes, are described in detail. Additionally, an overview of the currently used reprogramming methods, viral and non-viral, is included, with a special focus on their applicability to keratinocytes. This overview is intended to provide a brief but comprehensive insight into the field of keratinocytes and their use for reprogramming into induced pluripotent stem cells (iPSCs). © 2020 The Authors.

Translating basic biological knowledge into applications remains a key issue for effectively tackling neurodegenerative, neuroinflammatory, or neuroendocrine disorders. Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) still poses a demanding challenge for drug development targeting central nervous system diseases. Validated in vitro models of the BBB could facilitate effective testing of drug candidates targeting the brain early in the drug discovery process during lead generation. We here review the potential of mono- or (isogenic) co-culture BBB models based on brain capillary endothelial cells (BCECs) derived from human-induced pluripotent stem cells (hiPSCs), and compare them to several available BBB in vitro models from primary human or non-human cells and to rodent in vivo models, as well as to classical and widely used barrier models [Caco-2, parallel artificial membrane permeability assay (PAMPA)]. In particular, we are discussing the features and predictivity of these models and how hiPSC-derived BBB models could impact future discovery and development of novel CNS-targeting therapeutics. © 2020 The Authors.

The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors.

Basic Protocol 1: Expansion of hiPSCs

Basic Protocol 2: Cell banking of hiPSCs

Support Protocol 1: Pluripotency assessment by flow cytometry

Support Protocol 2: Thawing control: Viability and sterility

Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT-PCR

Support Protocol 4: Identity: Short tandem repeat analysis

Advances in human pluripotent stem cell (hPSC) techniques have led them to become a widely used and powerful tool for a vast array of applications, including disease modeling, developmental studies, drug discovery and testing, and emerging cell-based therapies. hPSC workflows that require clonal expansion from single cells, such as CRISPR/Cas9-mediated genome editing, face major challenges in terms of efficiency, cost, and precision. Classical sub-cloning approaches depend on limiting dilution and manual colony picking, which are both time-consuming and labor-intensive, and lack a real proof of clonality. Here we describe the application of three different automated cell isolation and dispensing devices that can enhance the single-cell cloning process for hPSCs. In combination with optimized cell culture conditions, these devices offer an attractive alternative compared to manual methods. We explore various aspects of each device system and define protocols for their practical application. Following the workflow described here, single cell−derived hPSC sub-clones from each system maintain pluripotency and genetic stability. Furthermore, the workflows can be applied to uncover karyotypic mosaicism prevalent in bulk hPSC cultures. Our robust automated workflow facilitates high-throughput hPSC clonal selection and expansion, urgently needed in the operational pipelines of hPSC applications. © 2020 The Authors.

Basic Protocol: Efficient automated hPSC single cell seeding and clonal expansion using the iotaSciences IsoCell platform

Alternate Protocol 1: hPSC single cell seeding and clonal expansion using the Cellenion CellenONE single-cell dispenser

Alternate Protocol 2: hPSC single cell seeding and clonal expansion using the Cytena single-cell dispenser

Support Protocol 1: Coating cell culture plates with Geltrex

Support Protocol 2: hPSC maintenance in defined feeder-free conditions

Support Protocol 3: hPSC passaging in clumps

Support Protocol 4: Laminin 521 coating of IsoCell plates and 96-well/384-well plates

Support Protocol 5: Preparation of medium containing anti-apoptotic small molecules

Support Protocol 6: 96- and 384-well target plate preparation prior to single cell seeding

Support Protocol 7: Single cell dissociation of hPSCs

Support Protocol 8: IsoCell-, CellenONE-, and Cytena-derived hPSC clone subculture and expansion

Structures resembling whole organs, called organoids, are generated using pluripotent stem cells and 3D culturing methods. This relies on the ability of cells to self-reorganize after dissociation. In combination with certain supplemented factors, differentiation can be directed toward the formation of several organ-like structures. Here, a protocol for the generation of retinal organoids containing all seven retinal cell types is described. This protocol does not depend on Matrigel, and by keeping the organoids single and independent at all times, fusion is prevented and monitoring of differentiation is improved. Comprehensive phenotypic characterization of the in vitro–generated retinal organoids is achieved by the protocol for immunostaining outlined here. By comparing different stages of retinal organoids, the decrease and increase of certain cell populations can be determined. In order to be able to detect even small differences, it is necessary to quantify the immunofluorescent signals, for which we have provided a detailed protocol describing signal quantitation using the image-processing program Fiji. © 2020 The Authors.

Basic Protocol 1: Differentiation protocol for 3D retinal organoids

Basic Protocol 2: Immunostaining protocol for cryosections of retinal organoids

Support Protocol: Embedding and sectioning protocol for 3D retinal organoids

Basic Protocol 3: Quantitation protocol using Fiji

We describe a protocol for efficient generation of human-induced pluripotent stem cells (hiPSCs) from urine-derived cells (UDCs) obtained from adult donors using self-replicative RNA containing the reprogramming factors OCT3/4, SOX2, KLF4, GLIS1, and c-MYC (ReproRNA-OKSGM). After electroporation, transfection efficiency is quantified by measuring OCT3/4-expressing UDCs using flow cytometry and should be ≥0.1%. hiPSC colonies emerge within 3 weeks after transfection and express multiple pluripotency markers. Moreover, the UDC-derived hiPSCs are able to differentiate into cells of all three germ layers and display normal karyotypes. ReproRNA-OKSGM is available commercially and only requires a single transfection step so that the protocol is readily accessible, as well as straightforward. In addition to a detailed step-by-step description for generating clonal hiPSCs from UDCs using ReproRNA-OKSGM, we provide guidance for basic pluripotency characterization of the hiPSC lines. © 2020 The Authors.

Basic Protocol: Reprogramming of urine-derived cells using ReproRNA-OKSGM

Support Protocol 1: Determination of the pluripotency status of hiPSCs by flow cytometry

Support Protocol 2: Characterization of functional pluripotency of hiPSCs

Great progress has been made with protocols for the differentiation and functional application of hPSC-cardiomyocytes (hPSC-CMs) in recent years; however, the cryopreservation and recovery of hPSC-CMs still presents challenges and few reports describe in detail the protocols and general workflow. In order to facilitate cryopreservation and recovery of hPSC-CMs for a wide range of applications, we provide detailed information and step-by-step protocols. The protocols are simple and use common reagents. They are comprised of a fast dissociation, cryopreservation using standard equipment, and gentle recovery following thawing. We discuss various features of the protocols, as well as their utilization in the context of common hPSC-CM differentiation and application workflows. Finally, we compare two proprietary and two common in-house formulations of cryopreservation media used for hPSC-CMs, and despite differences in their price and composition find broadly similar recovery rates and cellular function after thawing. © 2019 The Authors.

Basic Protocol 1: Dissociation and cryopreservation of hPSC-CMs

Basic Protocol 2: Thawing and recovery of cryogenically frozen hPSC-CMs

CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. However, limitations regarding the delivery and efficacy of these technologies restrict their application in primary cells. This article describes a protocol for penetrant, reproducible, and fast CRISPR-Cas9 genome editing in cell cultures derived from primary cells. The protocol employs transient nucleofection of ribonucleoprotein complexes composed of chemically synthesized 2′-O-methyl-3′phosphorothioate-modified single guide RNAs (sgRNAs) and purified Cas9 protein. It can be used both for targeted insertion-deletion mutation (indel) formation at up to >90% efficiency (via use of a single sgRNA) and for targeted deletion of genomic regions (via combined use of multiple sgRNAs). This article provides examples of the nucleofection buffer and programs that are optimal for patient-derived glioblastoma (GBM) stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs), but the protocol can be readily applied to other primary cell cultures by modifying the nucleofection conditions. In summary, this is a relatively simple method that can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells such as many cancer stem-like cells. © 2020 Wiley Periodicals LLC

Basic Protocol: Cas9:sgRNA ribonucleoprotein nucleofection for insertion-deletion (indel) mutation and genomic deletion generation in primary cell cultures