Now that you know what my team does, I will present you my project here. Question The aim of the project is to track proliferation of single stem cells in vivo, using fluorescent proliferation dyes. The two questions leading the experiment are: 1. What is the level of concordance of stem cell division ? (i.e. do both daughter cells divide the same way, synchronous, and does each daughter cell divide as often?) 2. How many times does each injected stem cell divide ? Hematopoietic stem cells (HSCs) differentiation has already been studied at a population scale with fluorescent dyes, but following single cells is new. Fluorescent cell staining dyes Before going into the details of my project, I’ll give you some important information about the staining dyes. The most well-known of them is CFSE (carboxyfluorescein succinimidyl ester) and it’s often used in immunology. It binds covalently to intracellular amine-containing molecules[1], resulting in homogeneously labeled cells. When a labeled cell divides, it gives 1/2 of its fluorescence to each daughter cell. Then they give 1/4th of the fluorescence to their daughter cells and so on. Theoretically, these dyes allow detection of up to 8 cell divisions before the fluorescence is too low to discern from background (when the dye is too diluted to detect its fluorescence). It’s a great tool to follow cell proliferation: as the cells have different amounts of fluorescence given their number of division, it’s possible to observe how many times they have divided (Figure 1). Here's an example of an experiment with CFSE labeled cells. Each peak represents a population of cells. The more they divide, the more the fluorescence decreases, that’s why the undivided fraction (population of undivided cells) peak is the highest. Method 1- Multiplex label on Jurkat cells Before working with HSCs, it’s easier to use a cell line. A cell line offers 2 main advantages: it’s easy to use because its cells are immortal and proliferate constantly. What’s more, we don’t have to kill a mouse each time we need cells. That’s why we developed the staining method using Jurkat cells, a human T lymphocyte line, stemming from a young boy who had T cell leukaemia. To follow single stained stem cells, each cell has to be labeled differently. We have 6 different dyes: CTY (CellTrace Yellow), CFSE, CMAC (CellTracker Blue CMAC), CellTracker Red CMPTX, CTFR (CellTrace Far Red) and CTV (CellTrace Violet). However, 6 different colors is not enough: if we inject more than 6 stem cells in a mouse, we can get more information by sacrificing only one animal. That’s why we first decided to combine the 6 colors 2 by 2 to get more unique stains. For now on, I will call this “multiplex label”[2] and here is what my 96 well plate looked like: The population on the top right is positive for Far Red and Violet. With this method, we can get 22 different combinations out of only 6 stains, which is a good start but not enough yet. 2- Sequential label on Jurkat cells, over time So we thought about some more combinations. To get them, a second layer of label is added to the multiplex label, the “sequential label”[3]. So we selected 2 colors, Celltrace Violet (CTV) and Celltrace Far Red (CTFR), and labeled the cells in 4 different concentrations: CTV 1:2000 CTFR 1:2000 CTV 1:2000, CTFR 1:4000 CTV 1:4000, CTFR 1:2000 After this first labelling, I quenched (stopped) the staining and pooled everything: here there are 4 different types of labeled cells in only one tube. Then, I can use these cells (represented by pink dots on the figure) to make a multiplex label: As you can see of this figure, there is a new population. Both cell subsets on the top right are double positive for Far Red and Violet, but at different concentrations. As the sequential label gives 4 different combinations and the second label 11, their combination gives us 4x11= 44 different combinations with only one mouse, while we would need 44 mice if we had one color !! Once the labelling technique was working well, I started following the labeled Jurkat cells over time, to see if I could see some proliferation going and for how long we could detect fluorescence. 3- Single HSCs label in vitro This is the stage I am right now. Here I’m doing the same thing I did with Jurkat cells, but on HSCs taken from murine bone marrow. Then, they must be activated to proliferate and grow in medium in vitro. After a couple of days, I will analyse the sample using flow cytometry and see if the cells have proliferated and how many times each cell divided. 4- Single HSCs label in vitro The last part of the project will be to do the same in vivo. First, take stem cells from a donor mouse, label them and inject the 44 differently labeled cells in another mouse. Before the injection, the mouse will be sub lethally (twice less than lethal) irradiated to kill half of its immune system (so that our HSCs can proliferate). After a few days the mouse is sacrificed, the bone we injected the cells in is sampled and analysed to find back our cells and see how they divided. If you’re a bit confused, here’s a short recap of my project: References:
[1] Parish CR (December 1999). "Fluorescent dyes for lymphocyte migration and proliferation studies". Immunology and Cell Biology. 77 (6): 499–508. doi:10.1046/j.1440-1711.1999.00877.x. PMID 10571670 [2] Andersen, Rikke Sick et al. "Parallel Detection Of Antigen-Specific T Cell Responses By Combinatorial Encoding Of MHC Multimers". Nature Protocols 7.5 (2012): 891-902. [3] Marchingo, J. M. et al. "T-Cell Stimuli Independently Sum To Regulate An Inherited Clonal Division Fate". Nature Communications 7 (2016): 13540. Credits: Fig.1: Oostendorp, Robert A. J., Julie Audet, and Connie J. Eaves. "High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo." Blood 95.3 (2000): 855-862. Black6 mouse: https://www.jax.org/strain/003548 Bone and syringe: https://thenounproject.com Everything else: my skills
9 Comments
Gaspard Baudrin
13/3/2017 07:33:13 am
Hello Noémie,
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Noémie Paillon
13/3/2017 08:50:56 am
Hello Gaspard !
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Gaspard Baudrin
13/3/2017 09:29:52 am
I would like to thank you for your fast and clear answer !
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Noémie Paillon
13/3/2017 09:34:45 am
Ok then maybe not...I'll see !
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Margaux
14/3/2017 05:17:20 am
Hi Noémie!
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Noémie Paillon
14/3/2017 08:51:07 am
Hello Margaux !
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margaux
14/3/2017 09:13:30 am
Very clear indeed!
Cécile
16/3/2017 05:34:07 am
Very interesting blog post , this technique is so clever!
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Noémie Paillon
16/3/2017 09:29:47 am
Heeello Cécile
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