Single-Cell Gene Expression Analysis of a Human ESC Model of Pancreatic Endocrine Development Reveals Different Paths to β-Cell Differentiation

The production of insulin-producing β cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny, we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or β-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state, a principle that could be relevant to other systems. Notably, activation of the key β-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional β cells

A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors

Standardized USER fusion Flexible Assembly Sequence Tags (FASTs).

<p>FW: forward; RV: reverse.</p>a<p>All color codes match <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096693#pone-0096693-g003" target="_blank">Figure 3</a>.</p

Elements included in the pBASE and pFAST vector platforms.

<p>α-2,6 ST: N-terminal targeting signal of beta-galactoside alpha-2,6-sialyltransferase; β-1,4 GT: N-terminal targeting signal of beta-1,4-galactosyltransferase; c-Ha-ras: C-terminal targeting signal of c-Ha-ras p21 protein; COX-VIII: N-terminal targeting signal of cytochrome c oxidase subunit VIII; CRT: N-terminal targeting signal of calreticulin; ER: endoplasmic reticulum; GalNAcT1: N-terminal targeting signal of N-acetylgalactosaminyltransferase; hIFN-γ: human Interferon-gamma; NLS: C-terminal nuclear localization sequence; PTS1: C-terminal peroxisomal target signal 1; TGN: <i>trans-</i>Golgi network.</p

Plasmids applied as PCR templates in this study.

<p>AmpR: ampicillin resistance gene; BGH: bovine growth hormone; CMV: cytomegalovirus; DHFR, dihydrofolate reductase; eCFP: enhanced cyan fluorescent protein; eGFP: enhanced green fluorescent protein; eYFP: enhanced yellow fluorescent protein; hGH: human growth hormone; HygR: hygromycin resistance gene; IRES: internal ribosomal entry site; mCherry: monomeric Cherry fluorescent protein; NeoR: neomycin resistance gene; pA: polyadenylation signal; PGK: phosphoglycerate kinase-1; SEAP: secreted alkaline phosphatase; SV40: Simian virus 40.</p

Confocal laser microscopy of fixed U-2-OS cells transiently transfected with pFAST-vectors.

<p>Confocal laser microscopy of fixed U-2-OS cells transiently transfected with control and pFAST-vectors 48h after transfection with (<b>A</b>) pC1_ccdB as negative control, (<b>B</b>) pFAST1-eGFP, (<b>C</b>) pFAST2-eYFP, (<b>D</b>) pFAST3-eCFP, and (<b>E</b>) pFAST4-mCherry. (<b>A1</b>–<b>E1</b>) microscopy with fluorescence filters. (<b>A2</b>–<b>E2</b>) nuclei stained with DAPI (dark blue). (<b>A3</b>–<b>E3</b>) merged pictures.</p

Confocal laser microscopy of U-2-OS cells expressing localized fluorescent proteins.

<p>Confocal laser microscopy of fixed U-2-OS cells transiently expressing fluorescent proteins localized to major cellular compartments. Shown are representative images of eYFP or eGFP detected 48 hours after transfection: (<b>A1-3</b>) pFAST6-eYFP::NLS, (<b>B1-3</b>) pFAST5-eGFP::PTS1, (<b>C1-3</b>) pFAST37-eGFP::c-Ha-Ras, (<b>D</b>) pFAST57-CRT::eGFP::KDEL, (<b>E</b>) pFAST58-COXVIII::eGFP, (<b>F</b>) pFAST59-GalNAcT1::eGFP, (<b>G</b>) pFAST61-b1,4GT::eGFP, (<b>H</b>) pFAST56-a-2,6ST-eGFP. (<b>A1</b>–<b>H1</b>) microscopy with fluorescence filters. (<b>A2</b>–<b>H2</b>) nuclei stained with DAPI (dark blue). (<b>A3</b>–<b>H3</b>) merged pictures.</p

Amounts of SEAP in extracted media of HEK293 cells.

<p>SEAP: secreted alkaline phosphatase.</p><p>*Negative control was a plasmid expressing a fluorescent protein, but not SEAP.</p

FAST-mediated vector assembly.

<p>Construction of vector types (<b>A</b>–<b>C</b>) requires the same three steps: <b>1:</b> Preparation of building blocks with appropriate FASTs by PCR or annealing of complementary oligonucleotides. <b>2:</b> USER fusion and hybridization: the USER enzyme and all building blocks are mixed in one reaction. <b>3: </b><i>E. coli</i> transformation with the USER cloning reaction mix. (<b>A</b>) Insertion of a promoter-GOI-terminator expression cassette in an <i>E. coli</i> vector backbone. (<b>B</b>) Assembly illustrated with five elements: the expression cassette as three building blocks, an interchangeable selection marker, and a vector backbone. (<b>C</b>) Similar to (<b>B</b>), but with seven building blocks including C- and N-terminal tags. The N- and C-terminal tag can either be a reporter, a fusion protein, a localization sequence or an epitope tag. GOI: gene of interest; N-tag: N-terminal sequence tag; C-tag: C-terminal sequence tag; P, promoter; and T, terminator.</p