Automated human induced pluripotent stem cell culture and sample preparation for 3D live-cell microscopy.

To produce abundant cell culture samples to generate large, standardized image datasets of human induced pluripotent stem (hiPS) cells, we developed an automated workflow on a Hamilton STAR liquid handler system. This was developed specifically for culturing hiPS cell lines expressing fluorescently tagged proteins, which we have used to study the principles by which cells establish and maintain robust dynamic localization of cellular structures. This protocol includes all details for the maintenance, passage and seeding of cells, as well as Matrigel coating of 6-well plastic plates and 96-well optical-grade, glass plates.

Integrated intracellular organization and its variations in human iPS cells.

Understanding how a subset of expressed genes dictates cellular phenotype is a considerable challenge owing to the large numbers of molecules involved, their combinatorics and the plethora of cellular behaviours that they determine. Here we reduced this complexity by focusing on cellular organization-a key readout and driver of cell behaviour-at the level of major cellular structures that represent distinct organelles and functional machines, and generated the WTC-11 hiPSC Single-Cell Image Dataset v1, which contains more than 200,000 live cells in 3D, spanning 25 key cellular structures. The scale and quality of this dataset permitted the creation of a generalizable analysis framework to convert raw image data of cells and their structures into dimensionally reduced, quantitative measurements that can be interpreted by humans, and to facilitate data exploration.

A comprehensive analysis of gene expression changes in a high replicate and open-source dataset of differentiating hiPSC-derived cardiomyocytes.

We performed a comprehensive analysis of the transcriptional changes occurring during human induced pluripotent stem cell (hiPSC) differentiation to cardiomyocytes. Using single cell RNA-seq, we sequenced > 20,000 single cells from 55 independent samples representing two differentiation protocols and multiple hiPSC lines. Samples included experimental replicates ranging from undifferentiated hiPSCs to mixed populations of cells at D90 post-differentiation.

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation.

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization.

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20.

Fate of iPSCs derived from azoospermic and fertile men following xenotransplantation to murine seminiferous tubules.

Historically, spontaneous deletions and insertions have provided means to probe germline developmental genetics in Drosophila, mouse and other species. Here, induced pluripotent stem cell (iPSC) lines were derived from infertile men with deletions that encompass three Y chromosome azoospermia factor (AZF) regions and are associated with production of few or no sperm but normal somatic development. AZF-deleted iPSC lines were compromised in germ cell development in vitro.

Short-term effects of whole-body exposure to (56)fe ions in combination with musculoskeletal disuse on bone cells.

Space travel and prolonged bed rest cause bone loss due to musculoskeletal disuse. In space, radiation fields may also have detrimental consequences because charged particles traversing the tissues of the body can elicit a wide range of cytotoxic and genotoxic lesions. The effects of heavy-ion radiation exposure in combination with musculoskeletal disuse on bone cells and tissue are not known.