Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Podsumowanie

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Streszczenie

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation. We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.

We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry. Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters “aspect ratio” of a cell in the bright-field channel that aids in the recognition of elongated cells and “delta centroid XY Ter119/Draq5” that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate. The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

Wprowadzenie

Terminal differentiation within the erythroid lineage in mammals concludes with the dramatic process of enucleation, through which the orthochromatic erythroblast expels its membrane-encased nucleus (pyrenocyte)¹, generating a reticulocyte². The exact mechanism of this process, which is also the rate-limiting step of successful, large-scale, production of red blood cells in vitro, is not yet fully elucidated. The localization of key proteins and structures within enucleating erythroblasts relies on the use of fluorescent and electron microscopy^3-5. This tedious process typically results in the identification of a limited number of enucleation events and does not always allow meaningful statistical analysis. Expanding on a method of erythroblast identification described previously by McGrath et al.⁶, we have developed a novel approach of identifying and studying enucleation events by Multi-Spectral Imaging Flow Cytometry (multiparameter high-speed cell imaging in flow, a method that combines fluorescent microscopy with flow cytometry)⁷, which can provide a sufficient number of observations to obtain measurements and perform statistical analysis.

Here, we describe first two in vitro erythropoiesis culture methods used in order to synchronize erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.

Samples are run on an imaging flow cytometer and the collected cells are gated appropriately to identify orthochromatic erythroblasts⁶. Orthochromatic erythroblasts are then analyzed based on their aspect ratio, as measured in brightfield imaging, versus their value for the parameter delta centroid XY Ter119-DNA, which is defined as the distance between the centers of the areas stained for Ter119 and DNA, respectively. The population of cells with low aspect ratio and high delta centroid XY Ter119/DNA is highly enriched in enucleating cells. Using wild-type (WT) erythroblasts versus erythroblasts with Mx-Cre mediated conditional deletion of Rac1 on Rac2^-/- or combined Rac2^-/-; Rac3^-/- genetic background and this novel analysis protocol of multi-spectral imaging flow cytometry, we recently demonstrated that enucleation resembles asymmetric cytokinesis and that the formation of an actomyosin ring regulated in part by Rac GTPases is important for enucleation progression⁷.

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Protokół

1. Long-term In vitro Erythropoiesis Culture (Ex vivo Erythroid Differentiation Culture Protocol by Giarratana et al.⁸, Modified and Adapted for Mouse Cells)

This is a 3-step long-term in vitro erythropoiesis protocol. In the first step (days 0-4) 2 x 10⁵ cells/ml are placed in erythroblast growth medium supplemented with stem cell factor (SCF), interleukin-3 (IL-3), and erythropoietin (Epo). In the second step (days 5-6), cells are resuspended at 2 x 10⁵ cells/ml and co-cultured on adherent stroma cells (MS5) in fresh erythroblast growth medium supplemented only with Epo. In the third step (days 7-9), cells are cultured on a layer of MS-5 cells in fresh erythroblast growth medium without cytokines up to enucleation (Figure 1A).

All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Cincinnati Children’s Hospital Medical Center.

1. Harvest of bones and isolation of low-density bone marrow cells
   1. Add 2 ml sterile IMDM containing 2% fetal bovine serum (FBS) in a 15-ml conical tube and keep on ice.
   2. Euthanize a 2-6 month old wild type C57/BL6 mouse (along with or without genetically-targeted mouse of interest) following institution-approved protocol (e.g. CO₂ inhalation, followed by cervical dislocation).
   3. Isolate pelvic bones, femurs, and tibiae of both legs using forceps and scalpel, add them to the tube containing IMDM+2% FBS and keep on ice.
   4. Add 1 ml IMDM+2% FBS in a sterile flow-cytometry tube and flush bones using forceps and a tuberculin syringe with a 25-G x 5/8" needle. Flush IMDM+2% FBS through the bones a few times gently (by aspirating ~500 µl from the cell suspension and flushing it again through the bone), and collect the bone marrow cells into the flow-cytometry tube. Flushing is complete when bones appear white.
   5. Filter cell-suspension through a 40-μm cell strainer set on top of a 50-ml conical tube. Wash cell strainer with IMDM+2% FBS and using the same medium adjust final volume of cell suspension to 5 ml.
   6. Prepare low-density bone marrow cells (LDBM) by density gradient centrifugation: carefully layer the 5 ml of cell-suspension on 5 ml of density gradient cell separation medium 1.083 g/ml in a 15-ml tube and spin at 750 x g for 25 min at room temperature (RT) with no brake/low acceleration.
   7. Transfer supernatant (everything up to about the 2-ml mark of the 15-ml tube in order to acquire buffy coat) to a new 15-ml tube and perform one more wash with IMDM+2% FBS at 525 x g for 5 min at RT.
   8. Aspirate supernatant and lyse any remaining red blood cells (RBC) by suspending the pellet in 3 ml RBC lysis buffer for 5 min at RT. If a greater purity of erythroblasts is required at the final step (e.g. for biochemical studies), purify LDBM cells further at this step to Lin^neg cells by magnetic separation.
2. Ex vivo erythroid differentiation culture protocol starting from LDBM or Lin^neg cells (Figure 1A)
   1. Add 7 ml IMDM+2% FBS, spin at 525 x g for 5 min at RT and resuspend the pelleted cells in 2 ml erythroblast growth medium (EGM), consisting of:
      a. StemPro-34 medium, containing
      b. 2.6% StemPro-34 medium supplement,
      c. 20% BIT-9500,
      d. 900 ng/ml ferrous sulfate,
      e. 90 ng/ml ferric nitrate,
      f. 100 units/ml penicillin/streptomycin, 2 mM L-glutamine
      g. 10^-6 M hydrocortisone
      h. and freshly added cytokines:
      i) 100 ng/ml SCF,
      ii) 5 ng/ml IL-3, and
      iii) 3 IU/ml Epo.
   2. Count the cells using an automated cell counter or manually at a microscope using a hemocytometer.
   3. Plate in a 6-well cell-culture plate at a concentration of 5 x 10⁵ cells/well to a final volume of 2.5 ml in EGM (2 x 10⁵ LDBM cells/ml) and incubate at 37 °C (this is considered as day #0 of culture).
   4. Change medium by aspirating 1.5 ml of the supernatant, spinning at 525 x g for 5 min at RT, resuspending in 1.5 ml of fresh medium containing all 3 cytokines and adding back to the wells every day on days #2, 3, 4, to optimize proliferative phase. On day 3, remove all cells, count and split into appropriate number of wells in order to sustain well cell concentrations of ~2 x 10⁵ cells/ml. Maintain culture at 37 °C/5% CO₂.
   5. On day 4, plate MS5 cells (murine stromal cell line) to wells of a new cell-culture 6-well plate. The MS5 cell-culture medium is α-MEM containing 20% FBS, 100 units/ml penicillin/streptomycin, and 2 mM L-glutamine.
   6. On day 5, count number of cells (now significantly enriched in erythroblasts) in each well of the original culture plate using an automated cell counter or manually at a microscope using a hemocytometer.
   7. Lift all cells from each well and transfer to 15-ml conical tubes, spin down at 525 x g for 5 min at RT, aspirate supernatant and dissolve pellets with fresh EGM containing only Epo (3 IU/ml) to a concentration of 2 x 10⁵ cells/ml.
   8. Aspirate supernatant from the wells where MS-5 cells were plated the previous day (targeted to 70-80% confluency at the time of co-culture) and add the erythroid cells in these wells in EGM containing only Epo (although not their medium of choice, MS-5 cells survive well in EGM for several days).
   9. Change medium adding fresh EPO on day #6.
   10. Change medium on day #7, using EGM with no added cytokines. On days 7 through 9, test samples for enucleation with flow cytometry after staining with anti-Ter119 and Syto-16 daily and proceed to staining samples for Multi-Spectral Imaging Flow Cytometry (as detailed in section 3).
       Note: Before plating and on day 2, 4, 6, and 7 of culture, monitor cultured cells for erythroblast enrichment and differentiation with flow cytometry by assessing surface markers CD44 and Ter119 vs size (FSC).⁹ Alternatively CD71, Ter119, and FSC can also be used^10,11.

2. Fast Enucleation Assay, According to the Protocol Described by Yoshida et al.¹² with Modifications (Figure 1B)

1. Stress erythropoiesis induction and stroma cell preparation for in vitro erythropoiesis culture
   1. Anesthetize a 2-6 month old wild type C57/BL6 mouse per IACUC guidelines (e.g. with isoflurane solution). Ensure that the mouse has been adequately anesthetized by checking for absence of reflexive responses to a gentle hind-paw pinch and for regular respirations during the procedure. Use vet ophthalmic ointment on eyes to prevent dryness while under anesthesia.
   2. Induce stress erythropoiesis via tail bleeding to a final volume of 500 μl. Using an insulin syringe, inject equal volume of normal saline intraperitoneally to assure fluid resuscitation of the animal (hold animal in Trendelenburg position before injecting and inject in the lower abdomen so as not to damage internal organs).
   3. Do not leave the mouse unattended until it has regained motor control as indicated by the animal starting to move around the cage and being able to stand and walk without falling. The phlebotomized mouse is placed in a cage without other mice.
   4. After 2 days, plate MS-5 cells in wells of a 24-well cell-culture plate. Incubate MS-5 cells at 37 °C/5% CO₂ in MS5 cell medium (a-MEM containing 20% FBS, 100 units/ml penicillin/streptomycin, and 2 mM L-glutamine) with the goal to be 70-80% confluent in plate wells after 48 hr.
2. Harvest of spleen and processing of splenocytes
   1. Four days (96 hr) after stress erythropoiesis induction, euthanize previously bled mouse through IACUC-approved protocol (e.g. CO₂ inhalation followed by cervical dislocation).
   2. Harvest spleen and put it in a 15-ml conical tube containing IMDM 2% FBS and keep on ice.
   3. Back in the laboratory, in the tissue culture hood under sterile conditions, invert spleen-containing tube on a 40-μm cell strainer set on top of a 50-ml tube. Crush spleen using the plunger of a 5-ml plastic syringe.
   4. Wash cell strainer with IMDM+2% FBS and using the same medium, adjust the final volume of cell suspension to 5 ml.
   5. Carefully layer the 5 ml splenocyte suspension on 5 ml density gradient cell separation medium 1.083 g/ml in a 15-ml tube and spin at 750 x g for 25 min at RT with no brake/low acceleration.
   6. Transfer supernatant (solution down to about the 2-ml mark of the 15-ml tube in order to acquire buffy coat) to a new 15-ml tube and perform one more wash with IMDM+2% FBS at 525 x g for 5 min at RT.
   7. Aspirate supernatant and lyse red blood cells by suspending the pellet in 3 ml RBC lysis buffer for 5 min at RT.
   8. Add 7 ml IMDM+2% FBS to wash cells and to dilute and neutralize the RBC lysis buffer and spin at 525 x g for 5 min at 4 °C.
   9. Aspirate supernatant and suspend pelleted cells in 2 ml of erythroblast growth medium (EGM).
3. Culture of the isolated low-density splenocytes, enriched in erythroblasts, on plastic (first stage of fast in vitro erythropoiesis culture)
   1. Count cells on an automated cell counter or manually by a hemocytometer. Usual number of cells isolated per spleen at this stage is ~15 x 10⁶.
   2. Suspend cells further in EGM containing the cytokines (at final concentrations): SCF 50 ng/ml, IL-3 5 ng/ml, and Epo 2 U/ml.
   3. Plate 1-5 x 10⁶ cells in a final volume of 1 ml/well (same number of cells per well depending on total number of cells) of a 24-well cell-culture plate and incubate O/N at 37 °C/5% CO₂.
4. Culture of erythroblasts on MS5 cells (second stage of fast in vitro erythropoiesis culture
   1. Aspirate supernatant from the plastic well and lift the cells (highly enriched in erythroblasts) by adding 2 ml of cold 10 mM EDTA in PBS for 5 min, on ice to each well.
   2. Put cells in fresh tubes, wash once in EGM (without cytokines) and resuspend in the same.
   3. Plate 5 x 10⁵-1 x 10⁶ cells in a 1-2 ml volume to each MS5 cell-coated well of a 24-well plate. If pharmacological inhibitors are being used in the experiment, add them at appropriate concentrations now.
   4. Incubate for 6 to 8 hr (time guided by microscopic observation of approximately 30%-40% enucleation in the untreated WT sample). The binding of erythroblasts to MS-5 cells accelerates their enucleation.
   5. Lift cells from each well by adding 2 ml of cold PBS + 10 mM EDTA, for 5 min, on ice. Along with the erythroblasts, MS-5 cells will also be collected but these can later be easily excluded during flow cytometric analysis as FSC^hi Ter119^- cells.
   6. At this stage, cells can be fixed and stained for analysis by Multi-Spectral Imaging Flow Cytometry.

3. Staining of Erythroblasts for Localization of Intracellular Proteins or Lipid Rafts During Enucleation by Multi-spectral Imaging Flow Cytometry 

1. Wash cells in PBS, spin 525 x g for 5 min at RT and aspirate supernatant.
2. Fix cells by resuspending cell pellets in 500 μl of 3.7% formaldehyde in PBS for 15 min (fixation time may vary depending on the antigen being probed) and pipetting gently.
3. Transfer to 1.5-ml plastic centrifuge tubes and incubate for 15 min at RT.
4. Spin on a bench microcentrifuge 2,000 x g for 20 sec, aspirate supernatant and perform one wash by adding 500 μl PBS to each tube and pipetting gently.
5. Spin on a bench microcentrifuge 2,000 x g for 20 sec, aspirate supernatant and keep tubes on ice for at least 15 min. Permeablization steps 3.6-3.9 should be done quickly and efficiently, and following spinning/aspiration at RT, cell pellets should immediately be put back on ice, in order for cells to better retain their integrity.
6. Take acetone solutions out from -20 °C freezer and put on ice. Permeabilize cells by resuspending cell pellets first in 500 μl ice-cold 50% acetone (1:1 with dH₂O) and pipetting gently.
7. Spin on bench microcentrifuge 2,000 x g for 20 sec, aspirate supernatant and resuspend cell pellets in 500 μl ice-cold 100% acetone by pipetting gently.
8. Spin on bench microcentrifuge 2,000 x g for 20 sec, aspirate supernatant and resuspend cell pellets once more in 500 μl ice-cold 50% acetone by pipetting gently.
9. Spin on bench microcentrifuge 2,000 x g for 20 sec, aspirate supernatant and wash cells once in cold FACS buffer (PBS + 0.5% BSA) by pipetting gently.
10. Prepare labeling cocktail with antibodies or markers for the molecules of interest: 0.1 U/100 μl AF488-phalloidin for F-actin staining and 1 μl/100 μl Ter119-PECy7 for erythroid cell staining. Alternative or additional staining can also be done using AF-488-anti-β-tubulin antibody (1:50), AF-594–conjugated cholera toxin subunit B to label lipid rafts (1:200), anti-pMRLC (Ser19) primary antibody for the phosphorylated myosin regulatory light chain (1:50), followed by anti-rabbit AF-488–conjugated secondary antibody (1:400), and anti-γ-tubulin primary rabbit antibody (1:100) followed by anti-rabbit AF-555-conjugated secondary antibody (1:300).
11. Following supernatant aspiration, resuspend cell pellets in 100 μl of the marker cocktail, pipette gently and incubate for 30 min at RT.
12. Prepare FACS buffer containing 2.5 μM of the nuclear stain Draq5.
13. Wash cells in FACS buffer, spin down on bench microcentrifuge 2,000 x g for 20 sec, aspirate supernatant and resuspend in 60 μl FACS buffer containing Draq5.
14. Run samples on the imaging flow cytometer to collect at least 10,000 events per experiment, compensate the raw data files as previously published¹³, and analyze results as shown in Figure 2, using the analysis software specific to the imaging flow cytometer.

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Wyniki

First, cells are analyzed based on their Brightfield Aspect Ratio (the ratio of the length of their minor versus their major axis) and their Brightfield Area (indicative of their size). Events with a Brightfield Area value lower than 20 and higher than 200 are mostly debris and cell aggregates, respectively, and are excluded from the analysis (Figure 2A). Single cells (gate “R1”) are then analyzed based on their value for the Gradient RMS parameter, which indicates sharpness of image. Gate &#...

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Dyskusje

In recent years the study of erythroblast enucleation has gained increasing momentum since it is the step in in vitro erythropoiesis cultures that is most difficult to reproduce efficiently in order to achieve successful, large-scale production of red blood cells ex vivo. Up until recently, the study of erythroblast enucleation utilized mainly fluorescence microscopy and flow cytometry methods. Fluorescence microscopy methods, albeit helpful in identifying participating molecules, require...

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Materiały

Name	Company	Catalog Number	Comments
αMEM medium	CellGro	15-012-CV
IMDM medium	Hyclone (Thermo Scientific)	SH30228.01
Stempro-34 SFM	GIBCO (Life Tech)	10640
Stempro-34 nutrient supplement	GIBCO (Life Tech)	10641-025
Fetal Bovine Serum (FBS)	Atlanta Biologicals	512450
BIT9500	Stemcell Technologies	09500
Bovine Serum Albumin (BSA)	Fisher Scientific	BP-1600-100
Phosphate buffered saline (PBS)	Hyclone (Thermo Scientific)	SH30028.02
Penicillin/Streptomycin	Hyclone (Thermo Scientific)	SV30010
L-glutamine	Hyclone (Thermo Scientific)	SH30590.01
Isothesia (Isoflurane)	Butler-Schein	029405
Histopaque 1.083 mg/ml	Sigma	10831
BD Pharmlyse (RBC lysis buffer)	BD Biosciences	555899
Acetone	Sigma-Aldrich	534064
Formaldehyde	Fisher Scientific	BP 531-500
Hydrocortisone	Sigma	H4001
Stem Cell Factor (SCF)	Peprotech	250-03
Interleukin-3 (IL-3)	Peprotech	213-13
EPOGEN Epoetin Alfa (Erythropoietin, EPO)	AMGEN	available by pharmacy
CD44-FITC antibody	BD Pharmingen	553133
CD71-FITC antibody	BD Pharmingen	553266
Ter119-PECy7 antibody	BD Pharmingen	557853
Phalloidin-AF488	Invitrogen (Life Technologies)	A12379
β-tubulin-AF488 antibody	Cell Signaling	#3623
anti-rabbit AF488-secondary antibody	Invitrogen (Life Technologies)	A11008
anti-rabbit AF555-secondary antibody	Invitrogen (Life Technologies)	A21428
AF594-cholera toxin B subunit	Invitrogen (Life Technologies)	C34777
pMRLC (Ser19) antibody	Cell Signaling	#3671
γ-tubulin antibody	Sigma	T-3559
Syto16	Invitrogen (Life Technologies)	S7578
Draq5	Biostatus	DR50200
Ferrous sulfate	Sigma	F7002
Ferric nitrate	Sigma	F3002
EDTA	Fisher Scientific	BP120500
15-ml tubes	BD Falcon	352099
50-ml tubes	BD Falcon	352098
6-well plates	BD Falcon	353046
24-well plates	BD Falcon	351147
Flow tubes	BD Falcon	352008
Tuberculin syringe	BD	309602
Insulin syringe	BD	329461
Syringe needle 25-G 5/8	BD	305122
Capped flow tubes	BD	352058
40-μm cell strainer	BD Falcon	352340
Scalpel (disposable)	Feather	2975#21
FACS Canto Flow Cytometer	BD
ImagestreamX Mark II Imaging Flow Cytometer	AMNIS (EMD Millipore)
Image Data Exploration and Analysis Software (IDEAS) version 4.0 and up.	AMNIS (EMD Millipore)
Hemavet 950 Cell Counter	Drew Scientific	CDC-9950-002
NAPCO series 8000WJ Incubator	Thermo scientific
Allegra X-15R Centrifuge	Beckman Coulter	392932
Mini Mouse Bench centrifuge	Denville	C0801