Single-Nucleus RNA-Seq of Differentiating Human Myoblasts Reveals the Extent of Fate Heterogeneity
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Published online 26 August 2016 Nucleic Acids Research, 2016, Vol. 44, No. 21 e158 doi: 10.1093/nar/gkw739 Single-nucleus RNA-seq of differentiating human myoblasts reveals the extent of fate heterogeneity Weihua Zeng1,2, Shan Jiang1,2, Xiangduo Kong3, Nicole El-Ali1,2, Alexander R. Ball, Jr3, Christopher I-Hsing Ma3, Naohiro Hashimoto4, Kyoko Yokomori3,* and Ali Mortazavi1,2,* 1Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697-2300, USA, 2Center for Complex Biological Systems, University of California Irvine, Irvine, CA 92697-2280, USA, 3Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, CA 92697-1700, USA and 4Department of Regenerative Medicine, National Center for Geriatrics and Gerontology, 7–430 Morioka, Oobu, Aichi 474–8522, Japan Received February 25, 2016; Revised August 09, 2016; Accepted August 12, 2016 ABSTRACT INTRODUCTION Myoblasts are precursor skeletal muscle cells that Approximately 40% of the human body consists of skeletal differentiate into fused, multinucleated myotubes. muscle (1). The minimum functional unit of skeletal mus- Current single-cell microfluidic methods are not op- cle is the multinucleated myotube, which originates from timized for capturing very large, multinucleated cells fusing myoblasts. Muscle cell differentiation (myogenesis) such as myotubes. To circumvent the problem, we entails activation of muscle-specific transcription network governed by four partially-redundant muscle-specific reg- performed single-nucleus transcriptome analysis. ulatory factors (MRFs) (Myf5, MyoD1, Myogenin and Using immortalized human myoblasts, we performed MRF4/Myf6) working together with E proteins and MEF2 RNA-seq analysis of single cells (scRNA-seq) and family members (2). Molecular and genetic experiments in single nuclei (snRNA-seq) and found them compara- vivo in mice, chicken and Drosophila over the last decades ble, with a distinct enrichment for long non-coding have uncovered the genetic and epigenetic networks critical RNAs (lncRNAs) in snRNA-seq. We then compared for skeletal muscle differentiation (3–5). In vitro, the precise snRNA-seq of myoblasts before and after differen- process of myogenesis has been extensively studied using the tiation. We observed the presence of mononucle- murine immortalized C2C12 myoblast cell line as a model ated cells (MNCs) that remained unfused and ana- system because of its high proliferation and differentiation lyzed separately from multi-nucleated myotubes. We capacities (for examples of genome-wide studies, see (6–9) found that while the transcriptome profiles of my- among many others). However, similar progress in human myogenesis has been limited by the lack of a convenient hu- oblast and myotube nuclei are relatively homoge- man myogenesis model system and the high heterogeneity neous, MNC nuclei exhibited significant heterogene- of primary human muscle cells. ity, with the majority of them adopting a distinct mes- Scalable single-cell RNA sequencing (scRNA-seq) using enchymal state. Primary transcripts for microRNAs platforms such as Fluidigm (10,11) and drop-seq (12,13)has (miRNAs) that participate in skeletal muscle differen- revolutionized the study of cell-to-cell transcriptome het- tiation were among the most differentially expressed erogeneity. These methods have enabled large-scale charac- lncRNAs, which we validated using NanoString. Our terization of cell-to-cell heterogeneity and distinct subpop- study demonstrates that snRNA-seq provides reli- ulations of cells in tissues as well as in the defined inter- able transcriptome quantification for cells that are mediary states during cell differentiation (10–14). In partic- otherwise not amenable to current single-cell plat- ular, methods such as Monocle (15) have been developed forms. Our results further indicate that snRNA-seq to deconvolve time courses of development in differenti- ating cells where collected time points can consist of mix- has unique advantage in capturing nucleus-enriched tures of cells in different states or stages of differentiation. lncRNAs and miRNA precursors that are useful in The power of Monocle was demonstrated elegantly using mapping and monitoring differential miRNA expres- human skeletal myogenesis on the Fluidigm platform, in sion during cellular differentiation. which primary human myoblasts were differentiated into myocytes and myotubes (15). However, a critical limitation *To whom correspondence should be addressed. Tel: +1 949 824 6762; Fax: +1 949 824 4709; Email: [email protected] Correspondence can also be addressed to Kyoko Yokomori. Tel: +1 949 824 8215; Fax: +1 949 824 2688; Email: [email protected] C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] e158 Nucleic Acids Research, 2016, Vol. 44, No. 21 PAGE 2 OF 13 of the Fluidigm microfluidic chips is the size restrictions on Separation of myotubes and MNCs, and single nucleus ex- cells that can be captured for single-cell analysis. Mature traction multinucleated myotubes, which are typically longer than In order to separate MNCs from multinucleated myotubes, 100m, are too large to be captured on the largest-size chip 6 cm tissue culture plates were washed with PBS twice fol- designed for cells up to 30m. This issue limited the pre- lowed by the addition of 1.5 ml trypsin/EDTA (Invitrogen). vious analysis to unfused cells or smaller, less mature my- We monitored the plate with a microscope at room tem- otubes. A potential solution to this problem is to isolate cell perature and tapped the plate repeatedly. After 1–2 min, nuclei and perform RNA-seq on single nuclei as has previ- the MNCs detached first, and we took the supernatant ously been done manually in a dozen neurons (16). and washed the plate gently with PBS twice. The PBS was We report the successful use of single-nucleus RNA se- combined with the supernatant to recover MNCs. My- quencing (snRNA-seq) to analyze gene expression profiles otubes were still attached on the plate and we used a cell of large myotubes compared to myoblasts and differentiated scraper to scrape them out. After the cells were lifted by MNCs on the Fluidigm platform using the immortalized trypsin/EDTA or cell scraper, they were pelleted at 1000 x human Hu5/KD3 myoblast cell line (KD3). This line was ◦ g, 4 C for 5 min. The cell membrane was ruptured with cold derived from a single clone of abdominal muscle cells that cell lysis buffer (10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 3 were immortalized using hTERT with p16INK4a-resistant mM MgCl , 0.005% IGEPAL CA-630) for 4–5 min and the R24C mutant CDK4 (mtCDK4) and cyclin D1 (17). KD3 2 lysis efficiency was checked by Trypan blue (Lonza) stain- exhibits a normal karyotype and the phenotypic character- ◦ ing. The nuclei were then pelleted at 500 × g, 4 C for 5 min istics of its primary parent cells (including doubling time) and resuspended in cold PBS buffer. We used less IGEPAL as well as the ability to undergo myogenic terminal differ- CA-630 than usual for cell lysis in order to preserve intact entiation in vitro and in vivo with no evidence of transfor- MNC nuclei. We found that the KD3 myotube nuclei re- mation (i.e. anchorage-independent growth in vitro or tu- covery efficiency after lysis is 50–60%, compared to 70–80% mor formation in vivo)(17). KD3 has all of the attributes from myoblast or MNC, but believe that the amount of for serving as an ideal system to study human myogene- IGEPAL will need to be optimized for specific cell samples. sis using functional genomics. Comparison of scRNA-seq Suspended nuclei were passed through Falcon 40 m cell and snRNA-seq in myoblasts indicate that we can recover Strainer (Fisher Scientific) to remove cell debris. comprehensive single-nucleus transcriptomes that are com- parable to single-cell transcriptome with higher enrichment of long-noncoding RNAs (lncRNAs) in snRNA-seq. We Single nucleus capture, RNA-seq library construction and se- then compared single-nuclei from myoblasts as well as my- quencing otubes and MNCs that did not fuse after 72 h in differen- tiation medium. We found relative uniformity of transcrip- Single cells and nuclei were isolated using the Fluidigm C1 tional profiles in myoblast and myotube nuclei. In contrast, System. We used the largest integrated fluidic circuits (IFCs) greater heterogeneity was observed in the MNC nuclei, with of 17–25 m for KD3 cells. Single nuclei C1 runs were com- the majority of them showing a non-myoblast, mesenchy- pleted using the smallest IFC (5–10 m). Cells were loaded mal profile, providing the evidence for alternative differen- at an approximate concentration of 400 cells/linatotal tiation. We also analyzed the expression of lncRNAs and of 50 l. Each individual capture site was visualized to en- found that some of them represent primary miRNAs (pri- sure single cell or single nucleus capture. After visualization, miRNAs) that are differentially expressed between the three the IFC was loaded with Clontech SMARTer kit lysis, RT, samples. Corresponding miRNA expression was validated and PCR amplification reagents. Lysis, RT and PCR am- using the NanoString platform. Thus, snRNA-seq has dis- plification was completed overnight and harvested the fol- tinct advantages in characterizing nuclear lncRNAs and is lowing day. After harvesting, all cDNA was normalized be- highly instrumental in deconvolving fate heterogeneity dur- tween 0.1 and 0.3 ng/l and libraries were constructed us- ing myoblast differentiation. ing Illumina’s Nextera XT library prep kit per Fluidigm’s protocol. Constructed libraries were multiplexed and puri- fied using AMPure beads.