Thursday, May 25, 2017

Dissecting biological systems at the level of single cells

A major focus of technology development over the past years has been on increasing sensitivity to work with low cell numbers. The ultimate goal for many cell biologists is to assay cells individually. The desire to do so is in part fueled by the increasing appreciation of extensive heterogeneity in many tissues. For example, tumors are very heterogeneous, and often include not only different tumor cell clones, but also differentiated cells, infiltrating T-cells, macrophages, and fibroblasts. In the hematopoietic system, it has become apparent that even the most sophisticated flow cytometry sorting scheme has limits: highly purified hematopoietic stem cells still exhibit heterogeneous behaviors when assessed using single-cell transplantation assays (Dykstra et al., 2007; Kiel et al., 2005). In this blog, we will outline some of the most exciting developments and state-of-the-art technologies that stand to transform our understanding of tissue organization.

Next Generation Sequencing of single cells
Many single-cell assays start with isolation of individual cells. There are several ways to approach this. The most widely used method is fluorescence-activated cell sorting (FACS), in which single cells can be deposited in 96- or 384-well plates using a flow cytometer (Figure 1, panel A). For example, Smart-Seq2 deposits cells directly into lysis buffer, followed by enzymatic reactions to reverse transcribe and amplify the mRNA (Picelli et al., 2014). Smart-Seq was one of the first single-cell RNA-seq protocols and has the distinct advantage of capturing whole transcripts, as opposed to other technologies that are 3’ biased. Microfluidics can also be used to capture cells in tiny droplets, followed by molecular barcoding of molecules in the droplet (Klein et al., 2015; Macosko et al., 2015) (Figure 1, panel B). Microfluidics enables processing of thousands of cells per experiment, resulting in much lower cost and labor; however, current protocols only capture the 3’ end of the transcript. For example, the companies Fluidigm, 10X Genomics and 1CellBio all offer single-cell RNA-seq technologies based on microfluidics. Recently, researchers at MIT have isolated single cells in slides with ~86,000 subnanoliter wells (Gierahn et al., 2017) (Figure 1, panel C). Single cells are captured together with beads, followed by sealing using semipermeable membranes, cell lysis and hybridization of the mRNA to barcoded oligos on the beads. This technology has the potential to further reduce cost and increase accessibility of single-cell RNA-seq, but is currently not offered by a commercially available platform. Following cell isolation and capturing of nucleic acids of interest, the material needs to be amplified using enzymatic reactions. RNA-seq generally starts with reverse transcription of RNA into DNA, whereas genomic DNA protocols, such as ATAC-seq and ChIP-seq, begin with amplification using PCR or T7 RNA polymerase (Buenrostro et al., 2017; van Galen et al., 2016; Rotem et al., 2015). Illumina adapter ligation, Nextera or PCR are the most common approaches to prepare the DNA for Next Generation Sequencing.

Figure 1: Single cell isolation for nucleic acid sequencing can be performed using flow cytometry (A), microfluidics (B) or subnanoliter wells (C).
 
Data analysis
With single-cell sequencing technologies, vast amounts of data are generated, and data analysis presents a formidable challenge. Demultiplexing, alignment, quality checks and duplicate filters can influence all downstream steps of the analysis. Some technologies incorporate unique molecular identifiers (UMIs), which tells you whether two similar sequencing reads were derived from the same or different starting molecules. Some protocols include linear amplification by T7 RNA polymerase, which can increase sensitivity, but can affect how duplicate sequencing reads should be collapsed. Setting a minimum number of detected transcripts is a common method to filter low-quality cells, but may inadvertently exclude cells that have less mRNA (such as hematopoietic stem cells). After quality filtering, cells are often clustered using dimensionality reduction methods such as Principal Component Analysis (PCA) or t-Distributed Stochastic Neighbor Embedding (t-SNE). These methods can be influenced by artifacts such as read depth and batch effects, that have to be carefully controlled. Considering these variables before starting a single-cell RNA-seq project is essential (Grün and van Oudenaarden, 2015). Many laboratories are working on the computational challenges in analyzing single-cell data, and investigators such as Dana Pe’er, Peter Kharchenko and John Marioni have published packages that can help with analysis.

Single-cell imaging
Since measuring averages of heterogeneous populations often mask unique properties of rare cell types, such as adult hematopoietic stem cells, it is evident that single-cell analysis is a prerequisite for unbiased understanding of cellular and molecular behavior (Schroeder, 2011). The single-cell sequencing approaches mentioned above have significantly improved our understanding of cellular and molecular heterogeneity. However, there is another layer when studying biological processes lasting days/weeks (i.e. lineage commitment of embryonic or adult stem cells); the temporal dynamics. Over a given timeframe, both developmental and cell-cycle stage might influence the profile of individual cells. Since high-throughput sequencing requires lysing cells prior to downstream analysis, such methods are limited to a static picture of cell’s properties, and therefore lack temporal resolution. Live-cell imaging, ideally in an in vivo setting, could provide such data. Advances in non-invasive in vivo imaging allowed observation of entire zebrafish embryos for periods up to 2.5 days (Keller et al., 2010). Unfortunately, technical challenges such as optical tissue properties (most embryos are less transparent than zebrafish), size, accessibility to relevant structures (i.e. bone marrow imaging) and inability to long-term immobilize living animals pose significant obstacles. This limits in vivo imaging to few compatible tissues and to short time frames, and thus to events with rapid kinetics. In vitro time-lapse imaging offers an attractive, but technically challenging alternative, requiring expertise in a number of hardware and software components. It enables monitoring fates and dynamic molecular properties of individual cells and their progeny before, during and after a certain change occurs for periods up to 2 weeks (Kokkaliaris et al., 2016). Specialized software can then be used to attribute specific properties to individual cells and reconstruct the kinship within a colony in multidimensional lineage trees post-acquisition (Skylaki et al., 2016). When coupled with endpoint gene expression methods, it can retrospectively identify cell-state transitions (Hormoz et al., 2016). However, in vitro time-lapse imaging is currently limited to tracking few proteins simultaneously and cannot substitute the need for observing biological phenomena in their physiological environment. Although its usability depends on the biological question, in vitro time-lapse imaging can be a powerful approach for high throughput screenings or monitoring signaling dynamics over time.

Conclusions
Recent advances in single-cell analysis have significantly improved our understanding of cell behavior in homeostasis and disease. Sequencing RNA or DNA from single cells poses great engineering and computer science challenges. The innovations in this field are fast-paced, and future breakthroughs will enable higher capture efficiencies of molecules within cells, with robust protocols that are accessible to more investigators. Such technologies are already contributing to a reconsideration of the hematopoietic hierarchy (Nestorowa et al., 2016; Paul et al., 2015; Villani et al., 2017; etc.). Coupling gene-expression assays with in situ live-cell imaging adds another dimension enabling detection of local differences between similar or anatomically distinct regions of the same tissue (Silberstein et al. 2016). As methods become more sophisticated and multiplexed, unraveling complex tissues at the level of cellular resolution will make a lasting contribution to our understanding of biological systems in health and disease.




Peter van Galen, PhD
Publications Committee Member
Massachusetts General Hospital
Broad Institute of MIT and Harvard, USA
Konstantinos D. Kokkaliaris, PhD
Publications Committee Member
Department of Biosystems Science & Engineering, ETH Zurich, Switzerland

Citations
Buenrostro, J.D., Corces, R., Wu, B., Schep, A.N., Lareau, C., Majeti, R., Chang, H., and Greenleaf, W. (2017). Single-cell epigenomics maps the continuous regulatory landscape of human hematopoietic differentiation.

Dykstra, B., Kent, D., Bowie, M., McCaffrey, L., Hamilton, M., Lyons, K., Lee, S.-J., Brinkman, R., and Eaves, C. (2007). Long-term propagation of distinct hematopoietic differentiation programs in vivo. Cell Stem Cell 1, 218–229.

van Galen, P., Viny, A.D., Ram, O., Ryan, R.J.H., Cotton, M.J., Donohue, L., Sievers, C., Drier, Y., Liau, B.B., Gillespie, S.M., et al. (2016). A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes. Mol. Cell 61, 170–180.

Gierahn, T.M., Wadsworth, M.H., 2nd, Hughes, T.K., Bryson, B.D., Butler, A., Satija, R., Fortune, S., Love, J.C., and Shalek, A.K. (2017). Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput. Nat. Methods.

Grün, D., and van Oudenaarden, A. (2015). Design and Analysis of Single-Cell Sequencing Experiments. Cell 163, 799–810.

Hormoz, S., Singer, Z.S., Linton, J.M., Antebi, Y.E., Shraiman, B.I., and Elowitz, M.B. (2016). Inferring Cell-State Transition Dynamics from Lineage Trees and Endpoint Single-Cell Measurements. Cell Syst 3, 419–433.e8.

Keller, P.J., Schmidt, A.D., Santella, A., Khairy, K., Bao, Z., Wittbrodt, J., and Stelzer, E.H.K. (2010). Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat. Methods 7, 637–642.

Kiel, M.J., Yilmaz, O.H., Iwashita, T., Yilmaz, O.H., Terhorst, C., and Morrison, S.J. (2005). SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109–1121.

Klein, A.M., Mazutis, L., Akartuna, I., Tallapragada, N., Veres, A., Li, V., Peshkin, L., Weitz, D.A., and Kirschner, M.W. (2015). Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161, 1187–1201.

Kokkaliaris, K.D., Drew, E., Endele, M., Loeffler, D., Hoppe, P.S., Hilsenbeck, O., Schauberger, B., Hinzen, C., Skylaki, S., Theodorou, M., et al. (2016). Identification of factors promoting ex vivo maintenance of mouse hematopoietic stem cells by long-term single-cell quantification. Blood 128, 1181–1192.

Macosko, E.Z., Basu, A., Satija, R., Nemesh, J., Shekhar, K., Goldman, M., Tirosh, I., Bialas, A.R., Kamitaki, N., Martersteck, E.M., et al. (2015). Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell 161, 1202–1214.

Nestorowa, S., Hamey, F.K., Pijuan Sala, B., Diamanti, E., Shepherd, M., Laurenti, E., Wilson, N.K., Kent, D.G., and Göttgens, B. (2016). A single cell resolution map of mouse haematopoietic stem and progenitor cell differentiation. Blood.

Paul, F., Arkin, Y. ’ara, Giladi, A., Jaitin, D.A., Kenigsberg, E., Keren-Shaul, H., Winter, D., Lara-Astiaso, D., Gury, M., Weiner, A., et al. (2015). Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors. Cell 163, 1663–1677.

Picelli, S., Faridani, O.R., Björklund, A.K., Winberg, G., Sagasser, S., and Sandberg, R. (2014). Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181.

Rotem, A., Ram, O., Shoresh, N., Sperling, R.A., Goren, A., Weitz, D.A., and Bernstein, B.E. (2015). Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat. Biotechnol. 33, 1165–1172.

Schroeder, T. (2011). Long-term single-cell imaging of mammalian stem cells. Nat. Methods 8, S30–S35.

Silberstein, Lev et al. 2016. Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators. Cell Stem Cell 19(4): 530–43.

Skylaki, S., Hilsenbeck, O., and Schroeder, T. (2016). Challenges in long-term imaging and quantification of single-cell dynamics. Nat. Biotechnol. 34, 1137–1144.

Villani, A.-C., Satija, R., Reynolds, G., Sarkizova, S., Shekhar, K., Fletcher, J., Griesbeck, M., Butler, A., Zheng, S., Lazo, S., et al. (2017). Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science 356.

9 comments:

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    Replies
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  2. Hi Everyone
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  3. Am writing this article to thank Dr. Keke Odin for the wondrous miracle that he did for me because he helped me recently to bring back my Ex Wife. Thank you sir for your genuine spells. This is really incredible,I have never experienced anything like this in my life. Before i met you Sir, i have tried every possible means that i could to get my wife back, but i actually came to realize that nothing was working out for me, and that my wife had developed lot of hatred for me.. I thought there was no hope to reunite with my wife. But when i read good reviews about how Dr. Keke help others get back there ex lovers, make others to win big on lottery, cure of any sickness. I decided to give it a try and i did everything that he instructed me and i Trusted him and followed his instructions just as he guaranteed me in 48 hours, and that was exactly when my wife called me and come back to me.. I'm so happy for the good work you did for me. We are more contented now than ever. Everything looks perfect and so natural! Thank you so much Sir for your authentic and indisputable spells. Email him now for help greatkekespelltemple@gmail.com or call +1 386-336-9876 . Thanks Sir for your help. once again his email address is
    greatkekespelltemple@gmail.com
    call him on: +1 386-336-9876


    Am writing this article to thank Dr. Keke Odin for the wondrous miracle that he did for me because he helped me recently to bring back my Ex Wife. Thank you sir for your genuine spells. This is really incredible,I have never experienced anything like this in my life. Before i met you Sir, i have tried every possible means that i could to get my wife back, but i actually came to realize that nothing was working out for me, and that my wife had developed lot of hatred for me.. I thought there was no hope to reunite with my wife. But when i read good reviews about how Dr. Keke help others get back there ex lovers, make others to win big on lottery, cure of any sickness. I decided to give it a try and i did everything that he instructed me and i Trusted him and followed his instructions just as he guaranteed me in 48 hours, and that was exactly when my wife called me and come back to me.. I'm so happy for the good work you did for me. We are more contented now than ever. Everything looks perfect and so natural! Thank you so much Sir for your authentic and indisputable spells. Email him now for help greatkekespelltemple@gmail.com or call +1 386-336-9876 . Thanks Sir for your help. once again his email address is
    greatkekespelltemple@gmail.com
    call him on: +1 386-336-9876

    ReplyDelete
  4. Thanks to Jai Mata Sunlight for helping bring back my boyfriend I already lost for another lady. And the most painful part of it was that, they were happy and while I was in pain. And they were planing to get married. Before I contact Maa Sunlight for help with all hope of getting him back was lost. I really can’t help thinking with those days I will see him hanging around with her…then he sees me and behave like he never knew me. I feel like killing myself but thanks also to my best friend (Margaret) who pitifully connected me to the great Maa Osa Sunlight. The great mother of love. The Queen Of The Universe. The Mother Of The World. All over the world, if you search on magazines, Internets lots of people testifying for her goodness she have done. And I was asking myself, where have I been that I didn’t know her all this while?… I am indeed grateful for all wonders she did for me, for bringing him back with such a desperation that Is like a…between life, and death ,for him and until he sees me he will not have rest of mind. After I told her my problems, and she requested few things from me of which I provided. That night the lovespell was performed and Maa Sunlight told to shower but not use soap before I leave the next day for work. I didn’t doubt but I was thinking how that could bring him? so I did it and was wait for result…thinking lot of things in my mind. many ways he could show up, and if he’d whether he’d leave again? but I never knew she has a very unique special ways of bringing him. But to my greatest surprise he came with a such a lovely way that strengthen me with soo much believe that yes the words of the goddess were true and not just an ordinary words. And now we are living together in peace and in happiness. And is not that she hurt any of them…they had a minor argument and both decided to break up the spell was controlling them. And that was how she brought him to me and before he came back to me..maa sunlight already told me that he is on the way coming just like she was remoting him spiritually. Awesome powers I have never seen, or read about. Through this I figure, this life. This very earth we are is govern and be ruled with powers. Do you need help? I will help you by leaving her email below so you contact her with your and get helped. Sunlightmata@gmail.com pls when contact her, pls be serious and obedient for to be able to help because if you are not, she would and she might not help thinking you are not serious. Thanks to you mother again glory be unto your name.

    ReplyDelete
  5. I want to thank Dr.Kala for bringing back my ex husband, we broke up for more than 9 months and he told me that he never want to see me in his life again. I love him so much and he was my first and only love, I was confused and depressed due to the love i had for him.I did everything i could to have him come back to me but all went in vain. I search for tips i could use to get him back online and i saw so many testimonies about Dr Kala and how he help people to get back lost love and i decide to give him a try, i did not believe in spell casting i just want to try it may be it would work out for me. I contacted Dr.Kala for help on his email (kalalovespell@gmail.com), he told me that he have to cast a love spell on him, i told him to start it and after 2 days as Dr Kala told me,my husband called me and started to apologize for leaving me and also he told me that he still love me.He return to me and beg me to forgive him which i did and accepted him. I was very happy and i thank Dr.Kala for helping me get back my ex husband to my LIFE. His spell is the the greatest all over the world, it was the love spell he cast on my husband that make him to come back to me. All you ladies who want their ex husband back i want you to contact Dr.Kala at the following email address and get all your problem solve..No problem is too big for him to solve..you can contact him via Email: KALALOVESPELL@GMAIL.COM or you can call and WhatsApp him on +2347051705853

    ReplyDelete
  6. This is certainly a shocking and a genuine Testimony. I visited a forum here on the internet on the 8TH OF APRIL 2017, And I saw a marvelous testimony of this powerful great spell caster called DR Omoluyi on the forum. I never believed it, because I've never heard nor learnt anything about magic before. Not a soul would have been able to influence me about spells, not until i visited ( doctoromoluyispelltemple@gmail.com ) and behold he did it for me and restored my marriage of 6 years back to me and brought my spouse back to me within 12 to 16 hours just as I have read on the forum.. I was truly astonished and shocked when my husband knelt down begging me for forgiveness to accept him back. I am really short of expressions, and I don't know how much to convey my appreciation to you DR Omoluyi, you are a God sent to me and my entire family. Visit him today via Email and Website on ( doctoromoluyispelltemple@gmail.com ) or [ http://www.doctorokpamenspells.com ]. Am Lori Dante by name.

    ReplyDelete