Derek T. Peters

Highly multiplexed subcellular RNA sequencing in situ

Transcripts Visualized in Situ Despite advances, current methods for single-cell sequencing are unable to resolve transcript location within the cell, so Lee et al. (p. 1360, published online 27 February) developed a method of fluorescent in situ RNA sequencing (FISSEQ) that works in vivo to show messenger RNA localization within cells. The method amplifies complementary DNA targets by rolling circle amplification, and then in situ cross-linking locks amplicons to produce ample, highly localized templates for three-dimensional sequencing. The technique was tested in fibroblasts to reveal the differences between individual cells during wound repair. Reads of cellular RNA transcripts demonstrate spatial expression differences during simulated wound healing. Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids.

Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology, including disease phenotypes after genome editing. In three-dimensional cultures, epiblast-stage hPSCs form spheroids surrounding hollow, amniotic-like cavities. GSK3β inhibition differentiates spheroids into segmented, nephron-like kidney organoids containing cell populations with characteristics of proximal tubules, podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes, and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids, indicating that they are tissue specific. Our findings establish a reproducible, versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications.

Efficient culturing and genetic manipulation of human pluripotent stem cells.

Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs.

Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells

ABSTRACT Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. Highlighted article: Expression of cell surface protein ASGR1 can be used to isolate a subpopulation of more mature, homogenous hepatocyte-like cells derived during differentiation from pluripotent stem cells.

Interrogation of the Atherosclerosis-Associated SORT1 (Sortilin 1) Locus With Primary Human Hepatocytes, Induced Pluripotent Stem Cell-Hepatocytes, and Locus-Humanized Mice

Objective— The noncoding single-nucleotide polymorphism rs12740374 has been hypothesized to be the causal variant responsible for liver-specific modulation of SORT1(sortilin 1) expression (ie, expression quantitative trait locus) and, by extension, the association of the SORT1 locus on human chromosome 1p13 with low-density lipoprotein cholesterol levels and coronary heart disease. The goals of this study were to compare 3 different hepatocyte models in demonstrating that the rs12740374 minor allele sequence is responsible for transcriptional activation of SORT1 expression. Approach and Results— We found that although primary human hepatocytes of varied rs12740374 genotypes strongly replicated the SORT1 expression quantitative trait locus observed previously in whole-liver samples, a population cohort of induced pluripotent stem cell–derived hepatocyte-like cells poorly replicated the expression quantitative trait locus. In primary human hepatocytes from multiple individuals heterozygous at rs12740374, we used CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats–associated 9) to specifically target the rs12740374 minor allele sequence ex vivo, resulting in a reproducible reduction in SORT1 expression. We generated a locus-humanized transgenic mouse with a bacterial artificial chromosome bearing the human SORT1 locus with the rs12740374 minor allele. In this mouse model, we used CRISPR-Cas9 to target the rs12740374 minor allele sequence in the liver in vivo, resulting in a substantial reduction of hepatic SORT1 expression. Conclusions— The rs12740374 minor allele sequence enhances SORT1 expression in hepatocytes. CRISPR-Cas9 can be used in primary human hepatocytes ex vivo and locus-humanized mice in vivo to interrogate the function of noncoding regulatory regions. Induced pluripotent stem cell–derived hepatocyte-like cells experience limitations that prevent faithful modelling of some hepatocyte expression quantitative trait loci.