Stress-free cell aggregation by using the CEPT cocktail enhances embryoid body and organoid fitness.

Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.

Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail.

Human pluripotent stem cells (hPSCs) are inherently sensitive cells. Single-cell dissociation and the establishment of clonal cell lines have been long-standing challenges. This inefficiency of cell cloning represents a major obstacle for the standardization and streamlining of gene editing in induced pluripotent stem cells for basic and translational research. Here we describe a chemically defined protocol for robust single-cell cloning using microfluidics-based cell sorting in combination with the CEPT small-molecule cocktail. This advanced strategy promotes the viability and cell fitness of self-renewing stem cells. The use of low-pressure microfluidic cell dispensing ensures gentle and rapid dispensing of single cells into 96- and 384-well plates, while the fast-acting CEPT cocktail minimizes cellular stress and maintains cell structure and function immediately after cell dissociation. The protocol also facilitates clone picking and produces genetically stable clonal cell lines from hPSCs in a safe and cost-efficient fashion. Depending on the proliferation rate of the clone derived from a single cell, this protocol can be completed in 7-14 d and requires experience with aseptic cell culture techniques. Altogether, the relative ease, scalability and robustness of this workflow should boost gene editing in hPSCs and leverage a wide range of applications, including cell line development (e.g., reporter and isogenic cell lines), disease modeling and applications in regenerative medicine.

A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells.

Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.

Generating Neural Stem Cells from iPSCs with Dopaminergic Neurons Reporter Gene.

Genetic reporters offer attractive approaches to investigate gene expression, gene function, and spatiotemporal assessment in vitro and in vivo. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of the dopamine neurotransmitter, and thus it has been used as a reliable marker for dopaminergic neurons in vitro and in vivo. Herein we describe a method for making iPSC lines with TH-green fluorescent protein reporter gene using CRISPR/Cas9 technique.

Reference Transcriptome for Deriving Marmoset Induced Pluripotent Stem Cells.

Limited access to primary tissue from various nonhuman primate (NHP) species represents a significant unmet need that hampers progress in understanding unique cellular diversity and gene regulation of specific tissues and organs in stem cell translational research. Most comparative biology studies have been limited to using postmortem tissue usually frozen specimens with limited utility for research. The generation of induced pluripotent stem cell (iPSC) lines from somatic cells, such as adult skin or blood cells, offers an alternative to invasive and ethically controversial interventions for acquiring tissue. Pluripotent iPSCs have virtually an unlimited capacity to proliferate and differentiate into all cell types of the body. We are generating high-quality validated NHP iPSC lines to offer to scientific community and facilitate their research programs. We use the non-integrative episomal vector system to generate iPSCs from NHP skin biopsies. In this chapter we describe the validation of NHP iPSC lines by confirming pluripotency and their propensity to differentiate into all three germ layers ectoderm, mesoderm, and endoderm according to established standards and measurable limits for a set of marker genes incorporated into a scorecard.

Isolation and Differentiation of Self-Renewable Neural Stem Cells from Marmoset-Induced Pluripotent Stem Cells.

Neural stem cells (NSCs) are multipotent and self-renewing precursor cells that give rise to all cell types of the central nervous system (CNS). They can be used for modeling CNS in vitro, for developmental studies and for cell replacement therapies. NSCs can be derived from pluripotent stem cells through differentiation using specific growth factors. Nonhuman primates (NHP) are critical preclinical models for translational research. Induced pluripotent stem cells (iPSCs) can be generated from NHP for the purposes of allogenic or autologous cell replacement studies. Here, we describe the derivation of NSCs from NHP iPSCs.

Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage.

Humans and nonhuman primates (NHP) are similar in behavior and in physiology, specifically the structure, function, and complexity of the immune system. Thus, NHP models are desirable for pathophysiology and pharmacology/toxicology studies. Furthermore, NHP-derived induced pluripotent stem cells (iPSCs) may enable transformative developmental, translational, or evolutionary studies in a field of inquiry currently hampered by the limited availability of research specimens. NHP-iPSCs may address specific questions that can be studied back and forth between in vitro cellular assays and in vivo experimentations, an investigational process that in most cases cannot be performed on humans because of safety and ethical issues. The use of NHP model systems and cell specific in vitro models is evolving with iPSC-based three-dimensional (3D) cell culture systems and organoids, which may offer reliable in vitro models and reduce the number of animals used in experimental research. IPSCs have the potential to give rise to defined cell types of any organ of the body. However, standards for deriving defined and validated NHP iPSCs are missing. Standards for deriving high-quality iPSC cell lines promote rigorous and replicable scientific research and likewise, validated cell lines reduce variability and discrepancies in results between laboratories. We have derived and validated NHP iPSC lines by confirming their pluripotency and propensity to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm) according to standards and measurable limits for a set of marker genes. The iPSC lines were characterized for their potential to generate neural stem cells and to differentiate into dopaminergic neurons. These iPSC lines are available to the scientific community. NHP-iPSCs fulfill a unique niche in comparative genomics to understand gene regulatory principles underlying emergence of human traits, in infectious disease pathogenesis, in vaccine development, and in immunological barriers in regenerative medicine.

A more efficient method to generate integration-free human iPS cells.

We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match ∼20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.

Generation of mouse-induced pluripotent stem cells with plasmid vectors.

Reprogramming of somatic cells into pluripotent stem cells has been reported by introducing a combination of several transcription factors (Oct3/4, Sox2, Klf4 and c-Myc). The induced pluripotent stem (iPS) cells from patient's somatic cells could be a useful source for drug discovery and cell transplantation therapies. However, to date, most iPS cells were made using viral vectors, such as retroviruses and lentiviruses. Here we describe an alternative method to generate iPS cells from mouse embryonic fibroblasts (MEFs) by continual transfection of plasmid vectors. This protocol takes around 2 months to complete, from MEF isolation to iPS cell establishment. Although the reprogramming efficiency of this protocol is still low, the established iPS cells are most likely free from plasmid integration. This virus-free technique reduces the safety concern for iPS cell generation and application, and provides a source of cells for the investigation of the mechanisms underlying reprogramming and pluripotency.

Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.

Induced pluripotent stem (iPS) cells can be generated from somatic cells by the introduction of Oct3/4 (also known as Pou5f1), Sox2, Klf4 and c-Myc, in mouse and in human. The efficiency of this process, however, is low. Pluripotency can be induced without c-Myc, but with even lower efficiency. A p53 (also known as TP53 in humans and Trp53 in mice) short-interfering RNA (siRNA) was recently shown to promote human iPS cell generation, but the specificity and mechanisms remain to be determined. Here we report that up to 10% of transduced mouse embryonic fibroblasts lacking p53 became iPS cells, even without the Myc retrovirus. The p53 deletion also promoted the induction of integration-free mouse iPS cells with plasmid transfection. Furthermore, in the p53-null background, iPS cells were generated from terminally differentiated T lymphocytes. The suppression of p53 also increased the efficiency of human iPS cell generation. DNA microarray analyses identified 34 p53-regulated genes that are common in mouse and human fibroblasts. Functional analyses of these genes demonstrate that the p53-p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation.