Yuhong Pang

Microfluidic single-cell whole-transcriptome sequencing.

Significance RNA sequencing of single cells enables measurement of biological variation in heterogeneous cellular populations and dissection of transcriptome complexity that is masked in ensemble measurements of gene expression. The low quantity of RNA in a single cell, however, hinders efficient and consistent reverse transcription and amplification of cDNA, limiting accuracy and obscuring biological variation with high technical noise. We developed a microfluidic approach to prepare cDNA from single cells for high-throughput transcriptome sequencing. The microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.

Deletion of Shp2 in the Brain Leads to Defective Proliferation and Differentiation in Neural Stem Cells and Early Postnatal Lethality

ABSTRACT The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.

Role of Gab2 in mammary tumorigenesis and metastasis

Overexpression of the adaptor/scaffolding protein Gab2 has been detected in primary human breast cancer cells and cell lines, although its functional significance in breast carcinogenesis is not fully understood. Here, we show a requirement for Gab2 in promoting mammary tumor metastasis. Although Gab2 expression levels were elevated in mammary tumors induced by the Neu (ErbB-2) oncogene, homozygous deletion of Gab2 in mice had only a modest effect on the initiation of Neu-induced mammary tumors. Notably, ablation of Gab2 severely suppressed lung metastasis. Gab2-deficient cancer cells displayed normal Akt activities, and their proliferative rate in vitro was similar to control cells. However, Gab2−/− cancer cells exhibited decreased migration and impaired Erk activation, and the defects were rescued by re-introduction of Gab2 into Gab2−/− cells. These findings suggest that although Gab2 overexpression may confer growth advantage to tumor cells, the functional requirement for Gab2 in mammary tumor initiation/growth may be dispensable, and that Gab2 may have a prominent role in promoting mammary tumor metastasis.

A conserved mechanism for control of human and mouse embryonic stem cell pluripotency and differentiation by shp2 tyrosine phosphatase.

Recent studies have suggested distinctive biological properties and signaling mechanisms between human and mouse embryonic stem cells (hESCs and mESCs). Herein we report that Shp2, a protein tyrosine phosphatase with two SH2 domains, has a conserved role in orchestration of intracellular signaling cascades resulting in initiation of differentiation in both hESCs and mESCs. Homozygous deletion of Shp2 in mESCs inhibited differentiation into all three germ layers, and siRNA-mediated knockdown of Shp2 expression in hESCs led to a similar phenotype of impaired differentiation. A small molecule inhibitor of Shp2 enzyme suppressed both hESC and mESC differentiation capacity. Shp2 modulates Erk, Stat3 and Smad pathways in ES cells and, in particular, Shp2 regulates BMP4-Smad pathway bi-directionally in mESCs and hESCs. These results reveal a common signaling mechanism shared by human and mouse ESCs via Shp2 modulation of overlapping and divergent pathways.

WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aging analysis.

Caenorhabditis elegans is a leading model organism for studying the basic mechanisms of aging. Progress has been limited, however, by the lack of an automated system for quantitative analysis of longevity and mean lifespan. To address this barrier, we developed ‘WormFarm’, an integrated microfluidic device for culturing nematodes. Cohorts of 30–50 animals are maintained throughout their lifespan in each of eight separate chambers on a single WormFarm polydimethylsiloxane chip. Design features allow for automated removal of progeny and efficient control of environmental conditions. In addition, we have developed computational algorithms for automated analysis of video footage to quantitate survival and other phenotypes, such as body size and motility. As proof‐of‐principle, we show here that WormFarm successfully recapitulates survival data obtained from a standard plate‐based assay for both RNAi‐mediated and dietary‐induced changes in lifespan. Further, using a fluorescent reporter in conjunction with WormFarm, we report an age‐associated decrease in fluorescent intensity of GFP in transgenic worms expressing GFP tagged with a mitochondrial import signal under the control of the myo‐3 promoter. This marker may therefore serve as a useful biomarker of biological age and aging rate.

Quantitative study of the dynamic tumor-endothelial cell interactions through an integrated microfluidic coculture system.

The interaction between tumor and endothelial cells is crucial to cancer metastasis and angiogenesis. We developed a novel microfluidic device to assess the cell-cell interaction quantitatively at the single cell resolution. This integrated chip offers 16 coculture experiments in parallel with controllable microenvironments to study interactions between cells dynamically. We applied this approach to model the tumor invasion using Hela cells and human umbilical vein endothelial cells (HUVECs) and monitored the migration of both. We observed the retreatment of HUVECs upon the approach of Hela cells during coculture, indicating that the interaction between two cells was mediated by soluble factors. This interaction was further analyzed through quantitatively processing the phase-contrast microscopic time-lapse images of each individual coculture chamber. We also confirmed this paracrine effect by varying the frequency of medium change. This microfluidic technique is highly controllable, contamination free, fully automatic, and inexpensive. This approach not only offers a unique way to quantitatively study the interaction between cells but also provides accurate spatial-temporal tunability of microenvironments for cell coculture. We believe this method, intrinsically high-throughput and quantitative, will greatly facilitate the study of cell-cell interactions and communications.

The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin–cholesterol domains

According to the raft hypothesis, sphingolipid–cholesterol (CHOL) microdomains are involved in numerous cellular functions. Here, we have prepared liposomes to simulate the lipid composition of rafts/caveolae using phosphatidylchone, sphingomyelin (SPM)–CHOL in vitro. Experiments of both 1,6‐diphenyl‐1,3,5‐hexatriene and merocyanine‐540 fluorescence showed that a phase transition from ld to lo can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca2+‐ATPase (PMCA), in lipid rafts (lo phase). Three complementary approaches to characterize the physical appearance of PMCA were employed in the present study. Tryptophan intrinsic fluorescence increase, fluorescence quenching by both acrylamid and hypocrellin B decrease, and MIANS fluorescence decrease, indicate that the conformation of PMCA embedded in lipid lo phase is more compact than in lipid ld phase. Also, our results showed that PMCA activity decreased with the increase of SPM–CHOL content, in other words, with the increase of lo phase. This suggests that the specific domains containing high SPM–CHOL concentration are not a favorable place for PMCA activity. Finally, a possible explanation about PMCA molecules concentrated in caveolae/rafts was discussed.

Openly Accessible Microfluidic Liquid Handlers for Automated High-Throughput Nanoliter Cell Culture

Cell culture is typically performed in Petri dishes, with a few million cells growing together, or in microwell plates with thousands of cells in each compartment. When the throughput of each experiment, especially of screening based assays, is increased, even using microliter solution per well will cost a considerable amount of cells and reagents. We took a rational approach to reduce the volume of each cell culture chamber. We designed and fabricated a poly(dimethylsiloxane) based liquid pipet chip to deliver and transfer nanoliter (50-500 nL) samples and reagents with high accuracy and robustness. A few tens to a few hundreds of cells can be successfully seeded, transferred, passaged, transfected, and stimulated by drugs on a microwell chip using this pipet chip automatically. We have used this system to test the cell growth dynamically, observed the correlation between the culture conditions and cell viabilities, and quantitatively evaluated cell apoptosis induced by cis-diammineplatinum(II) dichloride (cisplatin). This system shows great potential to facilitate large-scale screening and high-throughput cell-array based bioassays with the volume of each individual cell colony at the nanoliter level.

H3K4me3 epigenomic landscape derived from ChIP-Seq of 1 000 mouse early embryonic cells

Epigenetic regulation is crucial to the establishment and maintenance of the identity of a cell. Recent studies suggest that transcription is implemented amongst a mixture of various histone modifications [1]. It has also been recognized that to interrogate function of genetic information, comprehensively systematic profiling of the epigenome in multiple cell stages and types is required [2]. Chromatin immunoprecipitation (ChIP) has become one of the most critical assays to investigate the complex DNA-protein interactions [3]. Combined with profiling technologies such as microarrays (ChIP-on-chip) or high-throughput sequencing (ChIP-Seq), this assay becomes a great tool to study the epigenetic regulatory networks in cells [4-6]. However, the ChIP process produces limited amount of DNA due to the low yield of antibody pull-down, DNA damage during fragmentation and cleavage of DNA-protein complex, and complicated downstream analysis [7]. The conventional approaches have to consume a considerable amount of samples, typically 10 6-10 7 cells, to overcome this low-yield issue and obtain reliable results [5]. This limitation also restricts ChIP applications from precious primary tissue samples such as early embryonic cells or rare tumor stem cells. ChIP-Seq, compared with ChIP-on-Chip, deeply sequences the target DNA fragments and generates highly comprehensive data with higher resolution, fewer ar-tifacts, greater coverage and larger dynamic range [6]. Although recent application of automated microfluidic ChIP (AutoChIP) was successfully performed using 2 000 cells through locus-specific analysis by qPCR [8], such assays do not achieve the comprehensiveness afforded by DNA sequencing approaches. Recently, several approaches have been developed to perform ChIP-Seq using as low as 10 000 or even only 5 000 cells [7, 9-11, 14]. However, all of these methods rely on ChIP reactions in tens of microliters and preamplification of ChIP product before sequencing library preparation, either through linear amplification (by in vitro transcription) or exponential amplification (by PCR), both of which potentially introduce significant bias. Adli et al. [7] reported a modified protocol to realize the ChIP-Seq using 10 000 cells by revising the random primers used in amplification to reduce the primer self-annealing, with an optimized PCR condition to cover the GC-rich regions. Ng et al. [14] developed another protocol to perform ChIP-Seq of H3K4me3 modification using 10 000 mouse primor-dial germ cells, requiring pre-amplification before the sequencing library preparation. Sachs et al. [15] reported a chromatin immunoprecipitation study with low number of cells without pre-amplification, however, it needs at least 50 000 cells as starting material. Here …

Identification of kinship and occupant status in Mongolian noble burials of the Yuan Dynasty through a multidisciplinary approach

The Yuan Dynasty (AD 1271–1368) was the first dynasty in Chinese history where a minority ethnic group (Mongols) ruled. Few cemeteries containing Mongolian nobles have been found owing to their tradition of keeping burial grounds secret and their lack of historical records. Archaeological excavations at the Shuzhuanglou site in the Hebei province of China led to the discovery of 13 skeletons in six separate tombs. The style of the artefacts and burials indicate the cemetery occupants were Mongol nobles. However, the origin, relationships and status of the chief occupant (M1m) are unclear. To shed light on the identity of the principal occupant and resolve the kin relationships between individuals, a multidisciplinary approach was adopted, combining archaeological information, stable isotope data and molecular genetic data. Analysis of autosomal, mitochondrial and Y-chromosomal DNA show that some of the occupants were related. The available evidence strongly suggests that the principal occupant may have been the Mongol noble Korguz. Our study demonstrates the power of a multidisciplinary approach in elucidating information about the inhabitants of ancient historical sites.