Stem Cell

Human induced pluripotent stem (iPS) cell lines harboring mutations in disease-related genes serve as invaluable in vitro models for unraveling disease mechanisms and accelerating drug discovery efforts. Introducing mutations into iPS cells using traditional gene editing approaches based on the CRISPR-Cas9 endonuclease often encounters challenges such as unintended insertions/deletions (indels) and off-target effects. To address these limitations, we present a streamlined protocol for introducing highly accurate gene mutations into human iPS cells using prime editing, a “search-and-replace” genome-editing technology that combines unwanted indel-minimized CRISPR-Cas9 nickase with reverse transcriptase. This protocol encompasses the design of prime editing guide RNAs (pegRNAs) required for binding and replacement at target loci, construction of prime editor and pegRNA expression vectors, gene transfer into iPS cells, and cell line selection. This protocol allows for the efficient establishment of disease-associated gene variants within 6–8 weeks while preserving critical genomic context.

The advent of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has marked a significant advancement in genetic engineering technology. However, the editing of induced pluripotent stem cells (iPSCs) with CRISPR presents notable challenges in ensuring cell survival and achieving high editing efficiency. These challenges become even more complex when considering the specific target site. P53 activation as a result of traditional CRISPR editing can lead to apoptosis, potentially worsening cell health or even resulting in cell death. Mitigating this apoptotic response can enhance cell survival post-CRISPR editing, which will ultimately increase editing efficiency. In our study, we observed that combining p53 inhibition with pro-survival small molecules yields a homologous recombination rate of over 90% when using CRISPR in human iPSCs. This protocol significantly streamlines the editing process and reduces the time and resources necessary for creating isogenic lines.

Gene-edited human pluripotent stem cells provide attractive model systems to functionally interrogate the role of specific genetic variants in relevant cell types. However, the need to isolate and screen edited clones often remains a bottleneck, in particular when recombination rates are sub-optimal. Here, we present a protocol for flexible gene editing combining Cas9 ribonucleoprotein with donor templates delivered by adeno-associated virus (AAV) vectors to yield high rates of homologous recombination. To streamline the workflow, we designed a modular system for one-step assembly of targeting vectors based on Golden Gate cloning and developed a rapid protocol for small-scale isolation of AAV virions of serotype DJ. High homology-directed repair (HDR) rates in human pluripotent stem cells (hPSCs), ~70% in ACTB and ~30% in LMNB1, were achieved using this approach, also with short (300 bp) homology arms. The modular design of donor templates is flexible and allows for the generation of conditional and/or complex alleles. This protocol thus provides a flexible and efficient strategy workflow to rapidly generate gene-edited hPSC lines.

Induced pluripotent stem cells (iPSCs) hold significant promise for numerous applications in regenerative medicine, disease modeling, and drug discovery. However, the conventional workflow for iPSC generation, with cells grown under two-dimensional conditions, presents several challenges, including the need for specialized scientific skills such as morphologically assessing and picking colonies and removing differentiated cells during the establishment phase. Furthermore, maintaining established iPSCs in three-dimensional culture systems, while offering scalability, necessitates an enzymatic dissociation step for their further growth in a complex and time-consuming protocol. In this study, we introduce a novel approach to address these challenges by reprogramming somatic cells grown under three-dimensional conditions as spheres using a bioreactor, thereby eliminating the need for two-dimensional culture and colony picking. The iPSCs generated in this study were maintained under three-dimensional conditions simply by transferring spheres to the next bioreactor, without the need for an enzymatic dissociation step. This streamlined method simplifies the workflow, reduces technical variability and labor, and paves the way for future advancements in iPSC research and its wider applications.

Human induced pluripotent stem cells (hiPSCs) have been extensively used in the fields of developmental biology and disease modeling. CRISPR/Cas9 gene editing in iPSC lines often has a low frequency, which hampers its application in precise allele editing of disease-associated single nucleotide polymorphisms (SNPs), especially those in the noncoding parts of the genome. Here, we present a unique workflow to engineer isogenic iPSC lines by SNP editing from heterozygous to homozygous for disease risk alleles or non-risk alleles using a transient and straightforward transfection-based protocol. This protocol enables us to simultaneously obtain pure and clonal isogenic lines of all three possible genotypes of a SNP site within about 4 to 5 weeks.

Neuronal processes have an RNA composition that is distinct from the cell body. Therefore, to fully understand neuronal biology in health and disease we need to study both somas, dendrites and axons. Here we describe a detailed protocol of a newly refined method, Axon-seq, for RNA sequencing of axons (and dendrites) grown in isolation using single microfluidic devices. We also detail how to generate motor neurons from mouse and human pluripotent stem cells for sequencing, but Axon-seq is applicable to any neuronal cell. In Axon-seq, the axons are recruited through a growth factor gradient, lysed and directly processed to cDNA without RNA isolation. A careful bioinformatic step ensures that any soma-contaminated samples are easily identified and removed.

Shuttling of proteins between different cellular compartments controls their proteostasis and can contribute in some cases to regulate their activity. Biochemical analysis of chromatin-bound proteins, such as transcription factors, is often difficult because of their low yield and due to the interference from nucleic acids. This protocol describes a method to efficiently fractionate cells combined with a mechanical (i.e., sonication) or an enzymatic treatment (i.e., benzonase) that facilitates analysis of chromatin-bound protein extracts by Western blot analysis or by protein pull-down assays. This approach can be valuable to enrich a particular protein within a particular subcellular fraction either to study specific post-translational modification patterns or to identify specific protein-protein interactions.

Neuronal electrical properties are often aberrant in neurological disorders. Human induced pluripotent stem cells (hiPSCs)-derived neurons represent a useful platform for neurological disease modeling, drug discovery and toxicity screening in vitro. Multi-electrode array (MEA) systems offer a non-invasive and label-free platform to record neuronal evoked-responses concurrently from multiple electrodes. To better detect the neural network changes, we used the Axion Maestro MEA platform to assess neuronal activity and bursting behaviors in hiPSC-derived neuronal cultures. Here we describe the detailed protocol for neuronal culture preparation, MEA recording, and data analysis, which we hope will benefit other researchers in the field.