Protocols

Embryoid body formation from human pluripotent stem cells in chemically defined E8 media

Yongshun Lin, Guokai Chen

Embryoid bodies (EB) are the three-dimensional aggregates formed in suspension by pluripotent stem cells (PSC), including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). EB differentiation is a common platform to generate specific cell lineages from PSCs. However, most EB formation protocols contain undefined components, such as Fetal Bovine Serum (FBS), Knock-Out Serum Replacement (KOSR) or albumin product. These animal-sourced components significantly limit the application of EB formation to generate potentially clinically relevant cell products. At the same time, the undefined composition of the above components can lead to inconsistent outcomes in experiments due to batch differences in their production. This inconsistency also severely affects researchers’ ability to further improve procedure and its final products. Meanwhile, unlike their mouse counterparts, human PSCs usually cannot survive in suspension unless in aggregates or under ROCK inhibitor treatment. As such, it is essential that EB formation and further differentiation can be conducted in chemically defined, animal product-free conditions. This will allow better consistency as well as an easier route to translate into clinically relevant production. We previously developed a fully chemically defined medium Essential 8 (or E8) for the maintenance and expansion of human pluripotent stem cells in the clinical grade environment. In this protocol, we describe a set of optimized procedures to produce EBs from human PSCs in E8 or E8-based media.

Genome editing in human pluripotent stem cells

Derek T. Peters, Chad A. Cowan, Kiran Musunuru

Genome editing is used to make targeted modifications to the genome of eukaryotic cells. There are many potential applications of genome editing in human pluripotent stem cells (hPSCs) including the generation of knockout and reporter cell lines. This protocol describes a system for efficient genome editing in hPSCs using engineered transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeat (CRISPR) technology.

Splitting hPSCs with Dispase

Mark J. Tomishima

This protocol outlines the techniques used to routinely passage hPSCs in the SKI Stem Cell Research Facility at Sloan-Kettering. We prefer to culture our cells on mouse embryo fibroblasts (MEFs) but occasionally grow the cells feeder free for particular applications, such as nucleofection, viral transduction or karyotyping.

Splitting hESC/hiPSC lines with EDTA in feeder-free conditions

Guokai Chen

Individualization leads to severe cell death in human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). This phenomenon leads to difficulties in handling cells in applications such as passaging, cryopreservation and transfection, and the treatment with ROCK inhibitors has been found to effectively improve cell survival.

Splitting hESC/hiPSC lines on MEF using Accutase

Hyesoo Kim

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are known to be vulnerable to apoptosis upon various technical manipulation, such as single cell dissociation, freezing and thawing, etc., which hinder their use for clonal isolation in gene transfer, differentiation and FACS cell sorting.

General spinfection

W. Travis Berggren, Margaret Lutz, and Veronica Modesto

This protocol was developed in the Salk STEM Cell Core to enable researchers to consistently and reproducibly produce reprogrammed iPS cells, the initial idea came via word of mouth reports of its effectiveness to increase the efficiency of viral transduction. It has most commonly been used on retrovirus and lentivirus factors. Initial evaluation of this method showed 5–10× increase in transduction efficiency.

Assessment of human pluripotent stem cells with PluriTest

Franz-Josef Müller, Björn Brändl, Jeanne F. Loring

Assessing pluripotency in human cells is inherently an intractable problem. In animal systems, pluripotency can be verified through direct means: pluripotent stem cells can be introduced into an developing embryo and thus the cellular developmental potential of any given in vitro preparation can be directly determined by observing the amount of chimaerism or viability of organisms partially or fully derived from in vitro stem cells.

Conditioning pluripotent stem cell media with mouse embryonic fibroblasts (MEF-CM)

Mark J. Tomishima

There are a number of methods to expand human pluripotent stem cells (hPSCs) without feeders. I prefer to culture my hPSCs with mouse embryonic fibroblasts (MEFs) but there are occasions when feeder free growth is required (e.g., nucleofection, viral transduction or karyotyping to name a few).

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