Efficient translation of induced pluripotent stem cells (iPSCs) depends on implementing advanced strategies that ensure optimal self-renewal and functional differentiation. Unlike mouse cells, human iPSCs are highly sensitive and prone to undergo cellular stress and apoptosis even during routine cell passaging. Improving cell culture conditions specifically for human stem cells (e.g. media formulations, coating substrates) has been a daunting challenge for the field for more than 20 years. The NCATS Stem Cell Translation Laboratory (SCTL) addressed the cell viability problem by performing innovative high-throughput combinatorial screening of nearly 16,000 compounds in 1536-well format. We discovered that the combination of Chroman 1, Emricasan, Polyamines, and Trans-ISRIB (CEPT) dramatically improves cell survival of iPSCs as well as differentiated cell types (e.g., neurons, cardiomyocytes, and hepatocytes). Mechanistically, the CEPT cocktail prevents several stress mechanisms that otherwise compromise cell structure and function and improves routine cell passaging, biobanking of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and establishment of genome-edited new iPSC lines. Thus, CEPT represents a unique polypharmacology strategy for comprehensive cytoprotection, providing a new rationale for efficient and safe utilization of iPSCs. The protocols and technologies that are being established by SCTL are firmly based on this chemical platform and should enable robust regenerative medicine applications. To learn more about CEPT, read our article published in Nature Methods or go to our iPSC Portal Resources page for the CEPT cocktail protocol and supporting whole-exome sequencing and qHTS datasets.
The overarching objective of these collaborative projects is to delineate and control the process by which iPSCs exit the pluripotent state and differentiate into the three primary germ layers (ectoderm, mesoderm, and endoderm) as well as trophectoderm. SCTL scientists systematically study the underlying cell signaling pathways and gene regulatory networks that govern cell fate commitment and enable stepwise cell differentiation similar to principles of developmental biology. We use this understanding coupled with state-of-the-art high-throughput chemical screening, functional genomics, single-cell analysis, and functional characterizations to establish reproducible, scalable, and translation-grade protocols that are freely available to the research community. We are currently focusing on the efficient generation and functional characterization of specific cell populations representing the central nervous system (e.g., astrocytes, cortical neurons, and hypothalamic neurons), neural crest lineage (e.g., nociceptors and Schwann cells), mesoderm (cardiomyocytes), endoderm (hepatocytes), and various cell types of the placenta (e.g., cytotrophoblast, syncytiotrophoblast, and self-renewing trophoblast stem cells). Following publication, user-friendly differentiation protocols will be added to our iPSC Portal Resources page.
Manual culture of iPSCs is variable and labor-intensive, posing significant challenges for high-throughput applications. To address this problem, the SCTL developed and tested automated cell culture methods that enable standardization, biomanufacturing or large cell numbers, and increases the efficiency of collaborative projects. Using a robotic platform system (Sartorius CompacT SelecT™), SCTL scientists automated all essential steps of iPSC culture and directed differentiation (e.g., neurons, cardiomyocytes, and hepatocytes) under chemically defined conditions. This approach allows rapid and standardized manufacturing of billions of iPSCs that can be produced in parallel from up to 90 different patient-and disease-specific cell lines. Overall, non-stop robotic iPSC culture can increase scientific rigor and productivity to overcome major obstacles in the translation of iPSCs into therapies. To learn more about robotic iPSC culture, read our preprint available on bioRxiv. Once the article is published, the supporting protocol and datasets will be shared on our iPSC Portal Resources page.
As part of the NCATS Division of Preclinical Innovation, the SCTL has access to advanced high-throughput and high-content screening, multi-omics technologies, and functional cell characterization methods that are being used in an integrated fashion. The setup is ideal for discovery biology and generation of foundational datasets and resources to enable translational work. Depending on projects, quantitative high-throughput screening is carried out using NCATS’ large chemical libraries or genome-wide functional screens enabling systematic gene knockdown studies. Next-generation sequencing using latest technologies (Illumina NovaSeq™ 6000), various single-cell analysis platforms (e.g., 10X Genomics Chromium, Fluidigm Hyperion™, and Fluidigm Helios™), and high-throughput electrophysiology (Axion Biosystems Maestro APEX™) generate unique datasets, which will be shared with the scientific community. Once SCTL publications are available online, the supporting omics data (e.g., transcriptome, proteome), qHTS, and other data sets will be made available to the public in interactive and/or raw formats. To address the issues of reproducibility that have been a significant obstacle in the stem cell field, SCTL will also provide detailed protocols for researchers seeking to replicate or expand upon our work. To see all available protocols and datasets, visit the iPSC Portal Resources page.
As part of the NCATS mission, SCTL scientists study important translational problems and develop new therapeutic strategies for public health emergencies. Current projects are focused on identifying new drugs and drug combinations of already approved drugs that can be used against Zika virus and SARS-CoV-2 (drug repurposing). In multiple collaborative projects, the SCTL is also working on projects that utilize iPSC technology to fight the opioid crisis and develop non-addictive pain drugs as part of the NIH Helping to End Addiction Long-termSM (HEAL) initiative.