Quantification of Mouse Hematopoietic Progenitors’ Formation Using Time-lapse Microscopy and Image Analysis

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Mar 2018



In vitro differentiation of mouse embryonic stem cells (mESCs) towards blood cells constitutes a well-established system to study the endothelial-to-hematopoietic transition (EHT) at the onset of blood development. Assessing the emergence of small non-adherent round blood cells in the culture without disturbing it is essential to evaluate the progression of EHT and also to test conditions potentially enhancing or repressing this process. Here, we describe how to quantify the formation of mouse hematopoietic progenitors during EHT in normal conditions or following over-expression of eight essential transcription factors using time-lapse microscopy and image analysis.

Keywords: Hematopoietic progenitors (造血祖细胞), Embryonic stem cells (胚胎干细胞), Differentiation (分化), Inducible cell line (可诱导细胞系), Hemangioblast culture (成血管细胞培养), Microscopy (显微技术), Time-lapse imaging (延时成像), Round cell counting (圆形细胞计数)


The first hematopoietic stem and progenitor cells (HSPCs) emerge from endothelial cells in the large arteries of the mouse embryo (de Bruijn et al., 2000; Zovein et al., 2008; Chen et al., 2009;). This evolutionarily conserved event is called endothelial to hematopoietic transition (EHT). As a result of EHT, endothelial cells lose their specific markers, start to express hematopoietic genes, gain round morphology and eventually detach from the endothelial layer (Boisset et al., 2010; Kissa and Herbomel, 2010; Rybtsov et al., 2014). These specific series of events are used to detect EHT activity and monitor the progress of hematopoietic progenitor formation. The widely used method is to quantify endothelial and hematopoietic marker expressing cells. This can be done by gene expression analysis at the single-cell level (Bergiers et al., 2018). Also, protein expression change can be used to assess EHT progression by flow cytometry analysis of endothelial markers such as VE-Cadherin, CD31 and hematopoietic markers such as CD41, CD45, CD43. For spatial information, the same markers can be used for immunofluorescent staining on the fixed tissue. However, all of these techniques require harvesting of the examined cells and therefore represent a single time-point. Besides, the switch between endothelial and hematopoietic gene expressions occurs gradually and in a heterogeneous way so that a marker analysis of a specific time-point is not enough to follow the transition. To complement the gene/protein expression based detection methods, there is a need of a protocol to monitor morphological changes and quantify the round hematopoietic cell progenitors arising through EHT.

To study the hematopoietic cell formation dynamics in vitro, researchers developed embryonic stem cell differentiation protocol (Keller et al., 1993; Kennedy et al., 1997; Sroczynska et al., 2009). The protocol follows the developmental stages of hematopoiesis and recapitulates EHT in vitro (Choi et al., 1998; Nishikawa et al., 1998; Palis et al., 1999). Briefly, mouse embryonic stem cells are cultured to form embryoid bodies containing the in vitro equivalent of hemangioblast, or blast colony forming cells (BL-CFCs), which is then isolated by cell sorting. Those cells grow and give rise to smooth muscle, endothelial and hematopoietic cells (Keller et al., 1993; Faloon et al., 2000). Time-lapse imaging capturing the BL-CFC culture allows us to visualize cells transitioning into hematopoietic progenitors in real-time. Unlike the endothelial and smooth muscle cells, the cells undergoing EHT become round and bud-off from the endothelial cell layer (Lancrin et al., 2009). Here, by combining in vitro time-lapse imaging of an adherent BL-CFC culture with automatic image analysis we introduce a simple and efficient method to quantify those round cells during a culture period, which gives a direct measure of the number of cells undergoing EHT. This protocol enables us to easily test novel parameters affecting EHT rate such as over-expression of certain transcription factors (Bergiers et al., 2018) or testing pathway inhibiting small molecules in the culture media (Vargel et al., 2016). Below, we describe the details of the time-lapse microscopy of BL-CFC culture and image analysis to assess the number of cells underwent EHT.

Materials and Reagents

  1. Haemacytometre cover slips (Roth, catalog number: L189.1)
  2. BD Falcon Conical Tubes, Polypropylene, 15 ml, high-clarity, dome-seal screw cap (BD Biosciences, catalog number: 352096)
  3. BD Falcon Conical Tubes, Polypropylene, 50 ml, high-clarity, flat-top screw cap (BD Biosciences, catalog number: 352070)
  4. Costar® 6-well cell culture multiple well plate, flat bottom, with lid (Corning, catalog number: 3506)
  5. Millex-GP Syringe Filter Unit, 0.22 μm, polyethersulfone, 33 mm, gamma sterilized (Merck Millipore, catalog number: SLGP033RS)
  6. Mμlti®-safety microcentrifuge tubes, SafeSeal® Tubes (Carl Roth, catalog number: 7080.1)
  7. Stericup GP 0.2 μm/150 ml (Merck-Millipore, catalog number: SCGPU01RE)
  8. Stericup GP 0.2 μm/500 ml (Merck-Millipore, catalog number: SCGPU05RE)
  9. TipOne® 10 μl Graduated Filter Tip (Sterile), Refill (Starlab, catalog number: S1121-2710)
  10. TipOne® 1,000 μl Graduated Filter Tip (Sterile), Refill (Starlab, catalog number: S1122-1730)
  11. TipOne® 20 μl Graduated Filter Tip (Sterile), Refill (Starlab, catalog number: S1120-1710)
  12. TipOne® 200 μl Graduated Filter Tip (Sterile), Refill (Starlab, catalog number: S1120-8710)
  13. i8TFs mESC line (Bergiers et al., 2018)
  14. D4T endothelial cells (Choi et al., 1998)
  15. Ascorbic acid (Sigma, catalog number: A4544)
  16. Bovine Serum Albumin (BSA) (Sigma, catalog number: A9418)
  17. Doxycycline (Sigma, catalog number: D9891)
  18. Fetal bovine serum (FBS) (PAA, catalog number: A15-102)
  19. Gelatin (BDH, catalog number: 440454B)
  20. IL-6 (R and D, catalog number: 406 ML)
  21. IMDM (Lonza, catalog number: BE12-726F)
  22. L-glutamine (Gibco, catalog number: 25030-024)
  23. Monothioglycerol (MTG) (Sigma, catalog number: M6145)
  24. OxoidTM Phosphate Buffered Saline (PBS) Tablets (Thermo Scientific, catalog number: BR0014G)
  25. Transferrin (Roche, catalog number: 10652202001)
  26. VEGF (R and D, catalog number: 293-VE)
  27. Distilled Water (Thermo Scientific, GibcoTM, catalog number: 15230188)
  28. 0.1% gelatin solution (see Recipes)
  29. 10 mg/ml doxycycline stock solution (see Recipes)
  30. 5 mg/ml ascorbic acid stock solution (see Recipes)
  31. D4T endothelial cell supernatant (see Recipes)
  32. PBS + 0.1% BSA solution (see Recipes)
  33. 10 μg/ml VEGF stock solution (see Recipes)
  34. 10 μg/ml IL6 stock solution (see Recipes)
  35. Conditioned IMDM (see Recipes)
  36. IMDM + 20% FBS (see Recipes)
  37. MTG dilution (see Recipes)
  38. BL-CFC culture medium (see Recipes)


  1. Falcon® 10 ml Serological Pipet, Polystyrene, 0.1 Increments, Individually Packed, Sterile (Corning, catalog number: 357551)
  2. Falcon® 2 ml Aspirating Pipet, Polystyrene, without Graduations, Individually Wrapped, Sterile (Corning, catalog number: 357558)
  3. Falcon® 5 ml Serological Pipet, Polystyrene, 0.1 Increments, Individually Packed, Sterile (Corning, catalog number: 357543)
  4. 37 °C, 5% CO2 cell culture incubator (Thermo Scientific, model: Series II Water Jacket)
  5. Biosafety cabinet (Tissue culture hood) (Thermo Scientific, model: MSC Advantage)
  6. Brightfield inverted Microscope (Leica, model: DMIL LED Inverted)
  7. Centrifuge (Eppendorf, model: 5810 R)
  8. Computer (Dell Precision T3500)
  9. IncuCyte HD (Essen Biosciences)
  10. Neubauer counting chamber improved (Roth, catalog number: T729.1)
  11. Pipetman P10, 1 to 10 μl (Gilson, catalog number: F144802)
  12. Pipetman Starter Kit (P20, P200, P1000) (Gilson, catalog number: F167300)
  13. Pipette controller, PIPETBOY acu 2 (VWR, catalog number: 612-0928)
  14. Standard -20 °C freezer (LIEBHERR, model: LCv 4010 MediLine)
  15. Standard -80 °C freezer (Heraeus, model: HFU586 Top Freeze)
  16. Standard fridge (LIEBHERR, model: LKUexv 1610 MediLine)
  17. Vacuum pump BVC control (Vacuubrand, catalog number: 20727200)


  1. CellProfiler (Kamentsky et al., 2011; www.cellprofiler.org)
  2. Fiji (Schindelin et al., 2012; http://fiji.sc/Fiji)
  3. IncuCyte software (Essen Biosciences, 2011A Rev2)


Prior to the start of the BL-CFC culture, ESCs are differentiated according to previously described protocols (Sroczynska et al., 2009). Flk1+ cells are isolated using magnetic sorting following the manufacturer’s instructions (Miltenyi Biotech). The procedure described below has been optimized for adherent BL-CFC culture allowing the use of time-lapse microscopy. The quantities and volumes listed were further adapted for the comparison of two culture conditions: with or without doxycycline.
Note: Before starting, prepare working solutions as described in “Recipes”. Careful, BL-CFC culture medium should be prepared fresh.

Day 0

  1. Put 1 ml/well of 0.1% gelatin solution (see Recipes) into eight wells of Costar® 6-well cell culture plates (one plate of four wells for each line) and leave at room temperature (RT) for at least 20 min.
  2. For each line, introduce 0.425 million Flk1+ cells obtained after embryoid body differentiation of ESCs and counted using a Neubauer counting chamber (Sroczynska et al., 2009) into a 15-ml Falcon tube containing 5 ml of IMDM + 20% FBS.
  3. Centrifuge at 290 x g for 5 min.
  4. For each line, remove the supernatant using an aspirating pipet connected to the vacuum pump BVC control, gently tap the bottom of the tube to loosen the cell pellet and resuspend into 8.5 ml of BL-CFC culture medium.
    Note: Always use individual pipets and tips for each cell line to avoid cross-contamination.
  5. Aspirate the gelatin from the pre-coated Costar® 6-well cell culture plate wells using an aspirating pipet.
  6. Resuspend and evenly distribute 2 ml/well of cell suspension into four gelatin-coated wells for each cell lines.
    Note: This means that 0.1 million Flk1+ cells are added per well.
  7. Agitate the plates parallel to the bench doing vertical and horizontal small jolting movements.
    Note: Careful not to do circular movements which would result in the formation of a vortex and, consequently, in all the cells going to the center of the well.
  8. Place the plate into a 37 °C, 5% CO2 cell culture incubator for 24 h.
  9. Keep the BL-CFC culture medium in the fridge for the doxycycline dilution on the next day.

Note: The differentiation rate can be affected by cell confluence. Make sure the cells are equally spread across the surface of the wells to insure that the emergence of round blood cells will occur at the same rate everywhere in the wells.

Day 1
  1. Prepare 0.2 μg/μl doxycycline dilution by adding 4 μl of 10 mg/ml doxycycline stock solution (see Recipes) to 196 μl of BL-CFC culture medium kept in the fridge from the day before. Mix well.
  2. Take out the culture plates from the incubator and check under the microscope that the wells are all similar to each other.
  3. Add 10 μl of 0.2 μg/μl doxycycline dilution to each +dox condition well (two wells for each line at a final doxycycline concentration of 1 μg/ml). Add the doxycycline directly in the center of the wells and mix directly after by agitating the culture plates carefully.
  4. Add 10 μl of the BL-CFC culture medium to each -dox condition well as control (two wells for each line).
  5. Put back the two plates inside the incubator and place them carefully into the IncuCyte HD microscope.
    Note: Don’t write on top of the wells and avoid splashes of the medium on the lid to prevent malfunctioning of the IncuCyte HD as it takes pictures from the top.
  6. While the lid condensation is going away, set up the IncuCyte HD device using the IncuCyte software on the computer:
    1. Connect to the device and log in.
    2. Click on “Schedule Upcoming Scans” in the Task List panel on the left (see Figure 1 A).
    3. In the Drawer Setup window, select an empty vessel on the screen, right-click on the location where you inserted your first plate and click on “New” (see Figure 1).
    4. Keep this vessel selected and select the following settings in the Scan Setup tab, on the left side of the drawer map (see Figure 1 B):
      Tray Type: Microplates
      Vessel Type: 6-well Corning
      Scan Type: Phase Contrast
      Scan Pattern: Select the appropriate scan pattern or create one clicking on “Edit Scan Patterns” on the bottom left of the window (see Figure 1 C). Select 16 images/well for each 6-well.
      Note: The IncuCyte can take up to 121 images per well in a 6-well plate format. However, by choosing this option, we would have increased the time of imaging considerably, therefore leading to time points spaced by at least two hours instead of the 15 min needed to capture the morphological changes occurring during the BL-CFC culture. 
    5. With the vessel selected, go to the Properties tab (see Figure 1 D) and fill the following fields (see Figure 2 A):
      Label: Name of your experiment/vessel type
      Cell type: Exact name of the cell line/cell type used
      Passage: Number of passages

      Figure 1. IncuCyte setup

    6. Click on “Plate Map” (see Figure 2 B) and in the new window set up the plate map of your experiment (see Figure 3):
      1. Add a new compound and name it “+dox” (see Figure 3 A).
      2. Select the +dox compound, the wells that you treated with doxycycline and click on “Add +dox” (see Figure 4 A).
      3. Select the concentration and click on OK.
      4. Click on OK.

      Figure 2. IncuCyte Properties setup

      Figure 3. IncuCyte Plate Map initial setup

      Figure 4. IncuCyte Plate Map setup

    7. Repeat the setup of the vessel (from Day1 Steps 6c-6f) for your second plate.
    8. Right-click on the upper timeline (see Figure 2) and click on “Set intervals”.
    9. Set intervals every 15 min starting from time 0, for a total of 24 h.
      Note: Even with a total of 24 h, the Incucyte device will scan your vessels every 15 min until you manually remove your vessels from the software.
    10. Check the grey bars to make sure they are not overlapping (see Figure 5 A), check that IncuCyte device is not scanning (see Figure 5 B) and click on “Apply” (see Figure 5 C).

      Figure 5. Set Intervals

    Note: When the plate is put in the incubator, the difference of temperature between the room and the incubator generates water condensation on the lid (as an example, see Figure 6 A). Wait at least 30 min before initiating the imaging so that the microscope can focus properly on the cells.
  7. Come back after 15 min to check that everything is working and switch off the computer if you wish.

Day 3
  1. After 48-h treatment, click on “Schedule Upcoming Scans” in the Task List panel of the IncuCyte software.
  2. Right-click on the upper timeline and select “Delete Intervals”.
  3. Click on “Apply” to stop the scanning by IncuCyte.
  4. Take out your plates and proceed with flow cytometry analysis according to standard protocols.

Data analysis

  1. Export your images from the IncuCyte software:
    1. Click on “Find Scanned Vessels” in the Task List panel of the IncuCyte software (see Figure 6 B).
    2. Select one of your cell line vessels (see Figure 6 C) and click on “View Vessel” button in the lower right-hand corner of the screen (see Figure 6 D).

      Figure 6. View Vessels

    3. Check that the scans are fine (no dust, condensation, etc.).
    4. Selecting “Export Movie or Image Set…” from the “Utilities” pull down menu (see Figure 7 A).

      Figure 7. Export image set

    5. In the new window, select the appropriate time frame (see Figure 8 A), the wells corresponding to one condition (see Figure 8 B), all images (see Figure 8 C), select the sequence type labeled “set of individual images” (see Figure 8 D) and export phase-contrast original image (.JPEG).
    6. Specify the destination folder and the prefix of the files that will be generated (see Figure 8 E).
      Note: Create one folder per condition per cell line.
    7. Click on “Export … files” (see Figure 8 F).

      Figure 8. Final step of export image set

    8. Repeat Steps 1a-1h for all the vessels and conditions to be analyzed (see Supplemental File 1: Example dataset of IncuCyte images for the i8TF cell line for an example dataset).

  2. Analyze your images using CellProfiler software to get the number of round cells for each time-point, cell line and condition:
    1. Install CellProfiler2.2.0, which can be downloaded from here: http://cellprofiler.org/previous_releases/
      Note: You need Java as well: http://cellprofiler.org/releases/.
    2. Open CellProfiler and load the CellProfiler pipeline (see Supplemental File 2: CellProfiler pipeline for round cell counting) using File > Import > Pipeline from File… in the main menu of CellProfiler.
    3. Drag and drop the whole IncuCyte export destination folder into the File list area of the Images module (see Figure 9 A).
    4. Click on “Analyze images” on the bottom left to start processing (see Figure 9 B). 
    Note: The pipeline has been developed to automatically generate an output folder named as the input folder with "-- analyzed" appended. The output folder will be generated in the folder containing the input folder. If you want to modify this, simply change the Output File Location to Default Output Folder in the SaveImages and ExportToSpreadsheet Analysis modules, and change the output folder name by selecting “View output settings” in the Output panel of the main window.

    Figure 9. Image module of CellProfiler

    1. Use the Browser to access the output “image.txt” file in the output folder (as an example, see Supplemental File 3: CellProfiler output files for the example dataset) and open it with Excel or in R to visualize the results as graphs. In the output .txt file, the Count_RoundCells column contains the round cell numbers. The ImageNumber column contains the order in which the images were analyzed by CellProfiler. The Metadata_condition column contains the prefix given while exporting the images from the IncuCyte software. The Metadata_day column contains the day in which the images were taken. The Metadata_filename column contains the file names of the images analyzed such as exported by the IncuCyte software. The Metadata_foldername column corresponds to the input folder. The Metadata_position column contains the position of the images inside the well. The Metadata_time column contains the time in which the images were taken. The Metadata_well column contains the name of the well from which the images were taken. Each row corresponds to one image.
    2. See Figures 10 and 11, and Supplemental File 3: CellProfiler output files for the example dataset for the analysis output of the example dataset.
      1. The Metadata extraction described above relies on the IncuCyte file naming scheme. If this naming scheme is changing, e.g., due to version updates by IncuCyte, or because you are using a different microscope, it will not work. In such cases please contact us and we will help you adapting the Metadata extraction inside CellProfiler.
      2. In the output folder, CellProfiler also generates copies of the individual images with all counted round cells marked by a yellow dot (Figure 10).

      Figure 10. IncuCyte images before and after CellProfiler analysis

      Figure 11. Graph showing the number of round cells over time as calculated by CellProfiler 

  3. Generate movies from your images using Fiji software to illustrate differences between round cell emergence rates and highlight other morphological variations:
    1. Install Fiji software, which can be downloaded from here: http://fiji.sc/Fiji.
    2. Open Fiji.
    3. In your Browser, sort the IncuCyte exported files by Name, select all the files corresponding to the images taken from one frame of one condition for one cell line. By clicking on the last image, drag and drop all the files in Fiji. Let Fiji open the files without changing the order of the opened windows.
    4. To create a stack, select Image > Stacks > Images to Stack and then click on OK in the new window.
    5. Check the proper alignment of the stack slices by clicking on the play icon on the bottom left of the image. If the movie is readable, go directly to Step 3h.
    6. To align the slices, select Plugins > Image Stabilizer. In the new window, insert the following values:
      Transformation: Translation
      Maximum Pyramid Levels: 1
      Template Update Coefficient (0-1): 0.90
      Maximum Iterations: 200
      Error Tolerance: 0.0000001
      Select Output to a New Stack and click on OK.
    7. Check the slice alignment of the stabilized stack by clicking on the play icon on the bottom left of the image. If the movie is readable, go to Step 3h otherwise repeat Step 3f until all the slices are properly aligned (up to 3-4 times).
      h. Save the movie by selecting File > Save As > AVI…. In the new window, select JPEG Compression and 10 fps as Frame Rate.
    8. See Videos 1 and 2 based on the example dataset.

      Video 1. BL-CFC culture in normal conditions. The video is a 48-h-time-lapse microscopy analysis of BL-CFC culture in absence of doxycycline.

      Video 2. BL-CFC culture following over-expression of eight key transcription factors. The video is a 48-h-time-lapse microscopy analysis of BL-CFC culture in presence of doxycycline, i.e., following the over-expression of eight key transcription factors (Runx1, Cbfb, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2).


Note: Prepare solutions 1-9 in advance.

  1. 0.1% gelatin solution (stored at 4 °C)
    0.2 g of gelatin
    200 ml of PBS
    Note: Dissolve the powder, sterile filter with Stericup GP 0.2 μm/500 ml, aliquot and store at 4 °C.
  2. 10 mg/ml doxycycline stock solution (stored at -20 °C)
    10 mg of doxycycline
    1 ml of sterile distilled water
    Note: Dissolve the powder, aliquot and store at -20 °C. Always freshly thaw an aliquot.
  3. 5 mg/ml ascorbic acid stock solution (stored at -20 °C)
    0.5 g of ascorbic acid
    100 ml of distilled and sterile water
    Note: Dissolve the powder, sterile filter with a Stericup GP 0.2 μm/150ml, aliquot and store at -20 °C. Always freshly thaw an aliquot.
  4. D4T endothelial cell supernatant (stored at -20 °C)
    Prepare D4T endothelial cell supernatant following the procedure described by Choi and colleagues (Choi et al., 1998).
    Note: Prepare, aliquot and store at -20 °C. Once thawed, can be stored at 4 °C and used up to one month after.
  5. PBS + 0.1% BSA solution
    0.05 g of BSA
    50 ml of PBS
    Note: Dissolve, sterile filter using Millex-GP Syringe Filter, aliquot and store at -20 °C.
  6. 10 μg/ml VEGF stock solution (stored at -80 °C)
    Dissolve a 10 μg vial in 1 ml of sterile PBS + 0.1% BSA solution
    Note: Dissolve, aliquot and store at -80 °C. Once thawed, can be stored at 4 °C and used up to one month after.
  7. 10 μg/ml IL6 stock solution (stored at -80 °C)
    Dissolve a 10 μg vial in 1 ml of sterile PBS + 0.1% BSA solution
    Note: Dissolve, aliquot and store at -80 °C. Once thawed, can be stored at 4 °C and used up to one month after.
  8. Conditioned IMDM (stored at 4 °C)
    1 bottle of IMDM
    5 ml of L-glutamine
    5 ml of Penicillin-streptomycin
  9. IMDM + 20% FBS (stored at 4 °C)
    30 ml of FBS
    120 ml of conditioned IMDM
    Note: Sterile filter using StericupTM 150 ml bottle.
  10. MTG dilution (to be prepared fresh)
    13 μl of MTG
    1 ml of conditioned IMDM
    Note: Careful, MTG is viscous. Mix well after dilution.
  11. BL-CFC culture medium (to be prepared fresh)
    14.49 ml of conditioned IMDM
    2 ml of FBS
    0.2 ml of L-glutamine
    0.12 ml of Transferrin
    0.06 ml of MTG dilution
    0.1 ml of 5 mg/ml acid ascorbic stock solution
    3 ml of D4T endothelial cell supernatant
    0.01 ml of 10 μg/ml VEGF stock solution
    0.02 ml of 10 μg/ml IL6 stock solution
    Note: Prepare fresh in a 50 ml Falcon tube and sterile filter using Millex-GP Syringe Filter.


The EMBL Interdisciplinary Postdocs (EIPOD) Initiative (Post-doc fellowship) funded Isabelle Bergiers. The European Molecular Biology Laboratory has funded this work.

Competing interests

The authors do not have any conflicts of interests or competing interests.


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  18. Zovein, A. C., Hofmann, J. J., Lynch, M., French, W. J., Turlo, K. A., Yang, Y., Becker, M. S., Zanetta, L., Dejana, E., Gasson, J. C., Tallquist, M. D. and Iruela-Arispe, M. L. (2008). Fate tracing reveals the endothelial origin of hematopoietic stem cells. Cell Stem Cell 3(6): 625-636.


小鼠胚胎干细胞(mESCs)向血细胞的体外分化构成了一个成熟的系统,用于研究血液发育开始时的内皮 - 造血转换(EHT)。 评估培养物中小的非粘附性圆形细胞的出现而不干扰它对于评估EHT的进展以及测试可能增强或抑制该过程的条件是至关重要的。 在这里,我们描述了如何量化在正常条件下EHT期间或使用延时显微镜和图像分析过度表达八种必需转录因子后小鼠造血祖细胞的形成。
【背景】第一个造血干细胞和祖细胞(HSPCs)从小鼠胚胎的大动脉中的内皮细胞中出现(de Bruijn et al。,2000; Zovein et al。, 2008; Chen et al。,2009;)。这种进化上保守的事件称为内皮细胞向造血转化(EHT)。作为EHT的结果,内皮细胞失去其特异性标记物,开始表达造血基因,获得圆形形态并最终从内皮层分离(Boisset et al。,2010; Kissa and Herbomel,2010; Rybtsov et al。,2014)。这些特定系列事件用于检测EHT活性并监测造血祖细胞形成的进展。广泛使用的方法是量化内皮细胞和造血标记物表达细胞。这可以通过单细胞水平的基因表达分析来完成(Bergiers et al。,2018)。此外,蛋白质表达变化可用于通过内皮标志物(例如VE-钙粘蛋白,CD31)和造血标志物(例如CD41,CD45,CD43)的流式细胞术分析来评估EHT进展。对于空间信息,相同的标记可用于固定组织上的免疫荧光染色。然而,所有这些技术都需要收获检查的细胞,因此代表单个时间点。此外,内皮细胞和造血基因表达之间的转换逐渐地并且以异质方式发生,使得特定时间点的标记分析不足以跟随转变。为了补充基于基因/蛋白质表达的检测方法,需要一种方案来监测形态变化并量化通过EHT产生的圆形造血细胞祖细胞。

为了研究体外造血细胞形成动力学,研究人员开发了胚胎干细胞分化方案(Keller et al。,1993; Kennedy et al。 ,1997; Sroczynska et al。,2009)。该方案遵循造血发育阶段并在体外概括EHT (Choi et al。,1998; Nishikawa et al。,1998; Palis et al。,1999)。简而言之,培养小鼠胚胎干细胞以形成含有体外等效的成血成血管细胞或胚细胞集落形成细胞(BL-CFC)的胚状体,然后通过细胞分选将其分离。这些细胞生长并产生平滑肌,内皮细胞和造血细胞(Keller et al。,1993; Faloon et al。,2000)。捕获BL-CFC培养物的延时成像使我们能够可视化实时转变为造血祖细胞的细胞。与内皮细胞和平滑肌细胞不同,经历EHT的细胞变成圆形并从内皮细胞层发芽(Lancrin 等人,,2009)。在这里,通过将粘附BL-CFC培养物的体外延时成像与自动图像分析相结合,我们引入了一种简单有效的方法来量化培养期间的圆形细胞,从而直接测量进行EHT的细胞数量。该协议使我们能够轻松测试影响EHT速率的新参数,例如某些转录因子的过表达(Bergiers et al。,2018)或抑制培养基中小分子的测试途径(Vargel 等人,2016)。下面,我们描述了BL-CFC培养的延时显微镜和图像分析的细节,以评估进行EHT的细胞数量。

关键字:造血祖细胞, 胚胎干细胞, 分化, 可诱导细胞系, 成血管细胞培养, 显微技术, 延时成像, 圆形细胞计数


  1. Haemacytometre封面单(Roth,目录号:L189.1)
  2. BD Falcon锥形管,聚丙烯,15 ml,高透明度,圆顶密封螺帽(BD Biosciences,目录号:352096)
  3. BD Falcon锥形管,聚丙烯,50 ml,高透明度,平顶螺旋盖(BD Biosciences,目录号:352070)
  4. Costar ® 6孔细胞培养多孔板,平底,带盖(Corning,目录号:3506)
  5. Millex-GP注射器过滤装置,0.22μm,聚醚砜,33 mm,γ灭菌(Merck Millipore,目录号:SLGP033RS)
  6. Mμlti® - 安全微量离心管,SafeSeal ®管(Carl Roth,目录号:7080.1)
  7. Stericup GP0.2μm/ 150 ml(Merck-Millipore,目录号:SCGPU01RE)
  8. Stericup GP0.2μm/ 500 ml(Merck-Millipore,目录号:SCGPU05RE)
  9. TipOne ®10μl渐变过滤器吸头(无菌),替换物(Starlab,目录号:S1121-2710)
  10. TipOne ®1,000μl渐变过滤器吸头(无菌),替换物(Starlab,目录号:S1122-1730)
  11. TipOne ®20μl渐变过滤嘴(无菌),补充液(Starlab,目录号:S1120-1710)
  12. TipOne ®200μl渐变过滤器吸头(无菌),替换物(Starlab,目录号:S1120-8710)
  13. i8TFs mESC系列(Bergiers et al。,2018)
  14. D4T内皮细胞(Choi et al。,1998)
  15. 抗坏血酸(西格玛,目录号:A4544)
  16. 牛血清白蛋白(BSA)(西格玛,目录号:A9418)
  17. 强力霉素(西格玛,目录号:D9891)
  18. 胎牛血清(FBS)(PAA,目录号:A15-102)
  19. 明胶(BDH,目录号:440454B)
  20. IL-6(R和D,目录号:406 ML)
  21. IMDM(Lonza,目录号:BE12-726F)
  22. L-谷氨酰胺(Gibco,目录号:25030-024)
  23. Monothioglycerol(MTG)(Sigma,目录号:M6145)
  24. Oxoid TM 磷酸盐缓冲盐水(PBS)片剂(Thermo Scientific,目录号:BR0014G)
  25. 转铁蛋白(罗氏,目录号:10652202001)
  26. VEGF(R和D,目录号:293-VE)
  27. 蒸馏水(Thermo Scientific,Gibco TM ,目录号:15230188)
  28. 0.1%明胶溶液(见食谱)
  29. 10 mg / ml多西环素原液(见食谱)
  30. 5 mg / ml抗坏血酸原液(见食谱)
  31. D4T内皮细胞上清液(见食谱)
  32. PBS + 0.1%BSA溶液(参见食谱)
  33. 10μg/ ml VEGF原液(参见食谱)
  34. 10μg/ ml IL6原液(参见配方)
  35. 条件IMDM(见食谱)
  36. IMDM + 20%FBS(见食谱)
  37. MTG稀释(参见食谱)
  38. BL-CFC培养基(见食谱)


  1. Falcon ® 10 ml血清移液管,聚苯乙烯,0.1增量,单独包装,无菌(Corning,目录号:357551)
  2. Falcon ® 2毫升吸气管,聚苯乙烯,无刻度,单独包装,无菌(康宁,目录号:357558)
  3. Falcon ® 5 ml血清移液管,聚苯乙烯,0.1增量,单独包装,无菌(Corning,目录号:357543)
  4. 37°C,5%CO 2 细胞培养箱(Thermo Scientific,型号:Series II Water Jacket)
  5. 生物安全柜(组织培养罩)(Thermo Scientific,型号:MSC Advantage)
  6. 明场倒置显微镜(徕卡,型号:DMIL LED倒置)
  7. 离心机(Eppendorf,型号:5810 R)
  8. 电脑(Dell Precision T3500)
  9. IncuCyte HD(埃森生物科学)
  10. Neubauer计数室改进(Roth,目录号:T729.1)
  11. Pipetman P10,1至10μl(Gilson,目录号:F144802)
  12. Pipetman入门套件(P20,P200,P1000)(Gilson,产品目录号:F167300)
  13. 移液器控制器,PIPETBOY acu 2(VWR,目录号:612-0928)
  14. 标准-20°C冰箱(LIEBHERR,型号:LCv 4010 MediLine)
  15. 标准-80°C冰箱(Heraeus,型号:HFU586 Top Freeze)
  16. 标准冰箱(LIEBHERR,型号:LKUexv 1610 MediLine)
  17. 真空泵BVC控制(Vacuubrand,目录号:20727200)


  1. CellProfiler(Kamentsky et al。,2011; www.cellprofiler.org
  2. 斐济(Schindelin et al。,2012; http://fiji.sc/Fiji
  3. IncuCyte软件(Essen Biosciences,2011A Rev2)


在BL-CFC培养开始之前,根据先前描述的方案区分ESC(Sroczynska 等人,,2009)。按照制造商的说明书(Miltenyi Biotech),使用磁性分选分离Flk1 + 细胞。下面描述的程序已针对贴壁BL-CFC培养进行了优化,允许使用延时显微镜。列出的数量和体积进一步适应两种培养条件的比较:有或没有多西环素。


  1. 将1 ml /孔的0.1%明胶溶液(参见配方)放入8个Costar ® 6孔细胞培养板的孔中(每个培养板一个,每个培养皿4个孔)并在室温下放置(RT )至少20分钟。
  2. 对于每个品系,引入胚胎干细胞胚胎体分化后获得的0.425百万Flk1 + 细胞,并使用Neubauer计数室(Sroczynska et al。,2009)计数为15- ml含有5毫升IMDM + 20%FBS的Falcon管。
  3. 在290 x g 离心5分钟。
  4. 对于每一行,使用连接到真空泵BVC对照的吸移管移除上清液,轻轻敲打管底部以松开细胞沉淀并重悬于8.5ml BL-CFC培养基中。
  5. 使用抽吸吸管从预涂的Costar ® 6孔细胞培养板孔中吸出明胶。
  6. 将2ml /孔的细胞悬浮液重悬并均匀分布到每个细胞系的四个明胶包被的孔中。
    注意:这意味着每孔添加了10万个Flk1 + 细胞。
  7. 平行于工作台搅拌板,进行垂直和水平的小振动。
  8. 将板置于37℃,5%CO 2 细胞培养箱中24小时。
  9. 将BL-CFC培养基保存在冰箱中,以便在第二天进行强力霉素稀释。


  1. 通过将4μl10mg/ ml多西环素储备溶液(参见配方)加入到前一天保存在冰箱中的196μlBL-CFC培养基中,制备0.2μg/μl多西环素稀释液。好好混合。
  2. 从培养箱中取出培养皿,在显微镜下检查孔是否彼此相似。
  3. 向每个+ dox 条件下加入10μl0.2μg/μl多西环素稀释液(最终多西环素浓度为1μg/ ml时每个细胞系两个孔)。将多西环素直接添加到孔的中心,然后通过仔细搅拌培养板直接混合。
  4. 向每个 - dox 条件中加入10μlBL-CFC培养基作为对照(每个品系两个孔)。
  5. 将两块板放回培养箱内,小心地放入IncuCyte HD显微镜中。
    注意:请勿在井顶上书写并避免盖子上的介质飞溅,以防止IncuCyte HD从顶部拍摄时出现故障。
  6. 当盖子冷凝消失时,使用计算机上的IncuCyte软件设置IncuCyte HD设备:
    1. 连接到设备并登录。
    2. 单击左侧任务列表面板中的“安排即将进行的扫描”(参见图1A)。
    3. 在Drawer Setup窗口中,选择屏幕上的空容器,右键单击插入第一个板的位置,然后单击“New”(参见图1)。
    4. 选中此容器并在抽屉映射左侧的扫描设置选项卡中选择以下设置(参见图1B):
      注意:IncuCyte每孔最多可拍摄121张图像,采用6孔板格式。但是,通过选择此选项,我们可以大大增加成像时间,从而导致时间点间隔至少两个小时,而不是捕获BL-CFC培养期间发生的形态变化所需的15分钟。&nbsp; < / EM>
    5. 选择容器后,转到Properties选项卡(参见图1 D)并填写以下字段(参见图2A):

      图1. IncuCyte设置

    6. 点击“Plate Map”(参见图2B),在新窗口中设置实验的板图(参见图3):
      1. 添加一个新化合物并将其命名为“+ dox ”(参见图3A)。
      2. 选择+ dox 化合物,用强力霉素处理的孔,然后单击“Add + dox”(参见图4A)。
      3. 选择浓度,然后单击“确定”。
      4. 单击“确定”。

      图2. IncuCyte Properties设置

      图3. IncuCyte Plate Map初始设置

      图4. IncuCyte Plate Map设置

    7. 为第二块板重复设置容器(从第1天第6c-6f步骤开始)。
    8. 右键单击上面的时间轴(参见图2),然后单击“设置间隔”。
    9. 从时间0开始每15分钟设置一次间隔,总共24小时。
    10. 检查灰色条以确保它们不重叠(参见图5A),检查IncuCyte设备是否未扫描(参见图5B)并单击“应用”(参见图5 C)。


  7. 15分钟后回来检查一切是否正常,如果您愿意,请关闭计算机。

  1. 处理48小时后,单击IncuCyte软件的“任务列表”面板中的“安排即将到来的扫描”。
  2. 右键单击上方时间轴,然后选择“删除间隔”。
  3. 单击“应用”以停止IncuCyte的扫描。
  4. 取出平板,按照标准方案进行流式细胞仪分析。


  1. 从IncuCyte软件导出图像:
    1. 单击IncuCyte软件的Task List面板中的“Find Scanned Vessels”(参见图6B)。
    2. 选择一个细胞系血管(参见图6 C),然后单击屏幕右下角的“查看血管”按钮(参见图6 D)。


    3. 检查扫描是否正常(没有灰尘,冷凝,等。)。
    4. 从“Utilities”下拉菜单中选择“Export Movie or Image Set ...”(参见图7A)。


    5. 在新窗口中,选择适当的时间范围(见图8A),对应于一个条件的井(见图8B),所有图像(见图8C),选择标记为“单个图像集”的序列类型(见图8D)并输出相位对比原始图像(.JPEG)。
    6. 指定目标文件夹和将生成的文件的前缀(参见图8E)。
    7. 单击“导出...文件”(参见图8F)。


    8. 对所有要分析的容器和条件重复步骤1a-1h(参见补充文件1:示例数据集的i8TF单元格行的IncuCyte图像的示例数据集)。

  2. 使用CellProfiler软件分析您的图像,以获得每个时间点,细胞系和条件的圆形细胞数:
    1. 安装CellProfiler2.2.0,可以从这里下载: http://cellprofiler.org/previous_releases/
      注意:您也需要Java: http://cellprofiler.org/releases/
    2. 打开CellProfiler并加载CellProfiler管道(请参阅补充文件2:用于圆形细胞计数的CellProfiler管道)使用文件&gt;导入&gt; CellProfiler主菜单中的File ... 管道。
    3. 将整个IncuCyte导出目标文件夹拖放到 Images 模块的文件列表区域(参见图9A)。
    4. 点击左下方的“分析图像”开始处理(参见图9 B)。&nbsp;
    注意:管道已经开发为自动生成一个名为输入文件夹的输出文件夹,其中附加了“ - analyze”。输出文件夹将在包含输入文件夹的文件夹中生成。如果要修改此项,只需在SaveImages和ExportToSpreadsheet分析模块中将输出文件位置更改为默认输出文件夹,然后通过在主窗口的“输出”面板中选择“查看输出设置”来更改输出文件夹名称。 >

    图9. CellProfiler的图像模块

    1. 使用浏览器访问输出文件夹中的输出“image.txt”文件(作为示例,请参阅补充文件3:CellProfiler输出示例数据集的文件)并使用Excel或R打开它以将结果可视化为图形。在输出.txt文件中, Count_RoundCells 列包含圆形单元格编号。 ImageNumber 列包含CellProfiler分析图像的顺序。 Metadata_condition 列包含从IncuCyte软件导出图像时给出的前缀。 Metadata_day 列包含拍摄图像的日期。 Metadata_filename 列包含所分析图像的文件名,例如由IncuCyte软件导出的文件名。 Metadata_foldername 列对应于输入文件夹。 Metadata_position 列包含井内图像的位置。 Metadata_time 列包含拍摄图像的时间。 Metadata_well 列包含从中拍摄图像的井的名称。每行对应一个图像。
    2. 参见图10和11,并补充文件3:CellProfiler输出示例数据集的分析输出的示例数据集的文件
      1. 上述元数据提取依赖于IncuCyte文件命名方案。如果这个命名方案正在改变,例如,由于IncuCyte的版本更新,或者因为您使用的是不同的显微镜,它将无法工作。在这种情况下,请联系我们,我们将帮助您在CellProfiler中调整元数据提取。
      2. 在输出文件夹中,CellProfiler还会生成各个图像的副本,所有计算的圆形单元格都用黄点标记(图10)。


      图11.显示CellProfiler&nbsp; 计算的圆形细胞数随时间变化的图表

  3. 使用斐济软件从图像生成电影,以说明圆形细胞出现率之间的差异,并突出显示其他形态变异:
    1. 安装斐济软件,可以从这里下载: http://fiji.sc/Fiji
    2. 打开斐济。
    3. 在浏览器中,按名称对IncuCyte导出的文件进行排序,选择与从一个条件的一个帧中获取的图像对应的所有文件。通过单击最后一个图像,拖放斐济的所有文件。让斐济打开文件而不改变打开的窗口的顺序。
    4. 要创建堆栈,请选择 Image&gt;堆栈&gt;图像到堆栈,然后在新窗口中单击“确定”。
    5. 单击图像左下角的 play 图标,检查堆栈切片的正确对齐方式。如果电影可读,请直接进入步骤3h。
    6. 要对齐切片,请选择插件&gt;图像稳定器。在新窗口中,插入以下值:
    7. 单击图像左下角的 play 图标,检查稳定堆栈的切片对齐情况。如果电影是可读的,请转到步骤3h,否则重复步骤3f,直到所有切片都正确对齐(最多3-4次)。
      H。选择文件&gt;保存电影;另存为&gt; AVI .... 在新窗口中,选择JPEG压缩和10 fps作为帧速率。
    8. 根据示例数据集查看视频1和2。



  1. 0.1%明胶溶液(储存在4°C)
    注意:将粉末无菌过滤器与Stericup GP0.2μm/ 500 ml溶解,等分试样并在4°C下储存。
  2. 10 mg / ml多西环素原液(-20°C保存)
  3. 5 mg / ml抗坏血酸原液(-20°C保存)
    注意:将粉末无菌过滤器用Stericup GP0.2μm/ 150ml溶解,等分并储存在-20°C。总是新鲜解冻等分试样。
  4. D4T内皮细胞上清液(保存在-20°C)
    按照Choi及其同事描述的程序制备D4T内皮细胞上清液(Choi et al。,1998)。
  5. PBS + 0.1%BSA溶液
  6. 10μg/ ml VEGF原液(储存于-80°C)
    将10μg小瓶溶于1ml无菌PBS + 0.1%BSA溶液中 注意:溶解,等分并储存在-80°C。解冻后,可以在4°C下储存,并在使用后一个月内使用。
  7. 10μg/ ml IL6储备液(储存于-80°C)
    将10μg小瓶溶于1ml无菌PBS + 0.1%BSA溶液中 注意:溶解,等分并储存在-80°C。解冻后,可以在4°C下储存,并在使用后一个月内使用。
  8. 有条件的IMDM(存储在4°C)
    5毫升青霉素 - 链霉素
  9. IMDM + 20%FBS(储存在4°C)
    注意:无菌过滤器使用Stericup TM 150毫升瓶。
  10. MTG稀释(待新鲜制备)
  11. BL-CFC培养基(待新鲜制备)
    0.01 ml的10μg/ ml VEGF原液
    注意:使用Millex-GP注射器过滤器在50 ml Falcon管和无菌过滤器中新鲜制备。


EMBL跨学科博士后(EIPOD)倡议(博士后奖学金)资助了Isabelle Bergiers。欧洲分子生物学实验室资助了这项工作。




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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bergiers, I., Tischer, C., Bölükbaşı, Ö. V. and Lancrin, C. (2019). Quantification of Mouse Hematopoietic Progenitors’ Formation Using Time-lapse Microscopy and Image Analysis. Bio-protocol 9(1): e3137. DOI: 10.21769/BioProtoc.3137.
  2. Bergiers, I., Andrews, T., Vargel Bolukbasi, O., Buness, A., Janosz, E., Lopez-Anguita, N., Ganter, K., Kosim, K., Celen, C., Itir Percin, G., Collier, P., Baying, B., Benes, V., Hemberg, M. and Lancrin, C. (2018). Single-cell transcriptomics reveals a new dynamical function of transcription factors during embryonic hematopoiesis. Elife 7: e29312.