Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

NOTE: After written consent, hBM samples were obtained during orthopedic surgery for hip replacement. Immediately after femoral neck osteotomy and before femoral reaming a 20 ml syringe containing 500 U.I. of heparin, was used to aspirate fresh BM. The protocol is to be considered widely applicable to any BM source.

1. Isolation of Human Bone Marrow Mononuclear Cells (hBM-MNCs)

1. Dilute 5 – 10 ml of fresh BM to 50 ml, applying Dulbecco's phosphate buffered saline solution (D-PBS) and mix by inversion. Equally distribute 25 ml in two new 50 ml conical tubes, add 25 ml of D-PBS to each tube and mix by inversion.
2. Allow the tubes to stand for 10 min at room temperature, for separation of mineral bone fragments and fat from solution.
3. Carefully remove floating fat with a sterile Pasteur pipette and filter cell suspension through 70 μm filters without disturbing the mineral bone fragment pellet.
4. Set four 50 ml tubes with 15 ml of medium for discontinuous density gradient centrifugation (1.077 g/ml). Make sure that this medium is at room temperature.
5. Gently lay 20 – 25 ml of diluted BM on top of density gradient medium. Perform this operation carefully, letting cell suspension trickle on the tube walls to prevent mixing layers.
6. Carry out density gradient centrifugation at 400 x g for 30 min at room temperature with brake disabled.
7. Collect the whitish ring of cells located between the two phases using a sterile Pasteur pipette and transfer it to a fresh 50 ml tube.
8. Wash cells with fresh culture medium: phenol red-free, low glucose (1,000 mg/l) Dulbecco's Modified Eagle Medium (DMEM), 10% (v/v) pooled human AB type serum (PhABS), 2 mM L-glutamine and antibiotics (DMEM/10% PhABS). Centrifuge at 400 x g for 5 min.
9. Aspirate supernatant and re-suspend pellet in 5 – 10 ml of fresh DMEM/10% PhABS.
10. Proceed to cell count. Determine the number of white blood cells by 1:1 dilution in trypan. Apply this to the hemocytometer, and observe under a phase contrast microscope. Exclude small and perfectly rounded erythrocytes and blue-stained dead cells from the cell count.
    NOTE: It is highly recommended to screen PhABS batches for their performance in MPC recovery. PhABS from USA sources have given best results while most sera of different origin resulted in MPC cultures with higher percentages of MSC-like cells.

2. Isolation of MPCs from hBM-MNCs

1. Set hydrophobic T-75 flasks with 15 ml of fresh DMEM/10% PhABS and let pH and temperature equilibrate by pre-incubation at 37 °C in 5% CO₂ for 30 min.
2. Seed 4 – 6 x 10⁷ hBM-MNCs per flask and incubate at 37 °C in 5% CO₂ for 48 hr.
3. Aspirate and discard medium and non-adherent cells from flasks. Add 15 ml of fresh DMEM/10% PhABS and incubate at 37 °C in 5% CO₂. Maintain cultures for 6 – 8 days, changing medium every 48 hr.
   OPTIONAL: To increase MPC yield, non-adherent cells from 2.3 could be re-plated in a new culture flask and maintained as described for the primary cultures.
4. Aspirate and discard medium from flasks, wash with fresh DMEM and add 2 ml of animal free protease detaching solution. Incubate at 37 °C for 5 – 15 min (avoid prolonged incubation).
5. Add 10 ml of fresh DMEM/10% PhABS, aspirate the cell suspension and centrifuge at 400 x g for 5 min
6. Aspirate and discard supernatant and re-suspend pellet in 1 – 2 ml of fresh DMEM/10% PhABS. Proceed to cell count as described in step 1.10.
   NOTE: Do not use trypsin/EDTA as detaching reagent. MPCs are trypsin resistant. Morphological screening of the cultures, before cell harvesting, is highly recommended in order to evaluate the presence of spindle-shaped MSC-like cells. In case considerable amount of MSC-like cells are detected, increase the purity of the cell product by selectively removing the contaminated cells. To do so, trypsin digestion can be carried out prior to MPC harvesting, by adding 2 ml of trypsin/EDTA 0.05% for 2 min. Wash cultures twice with 5 ml of DMEM/10% PhABS, then proceed to protease treatment as above.

3. Cell Characterization

1. Flow Cytometry
   1. Set up duplicate samples of 10⁵ freshly detached cells in washing solution: D-PBS supplemented with 0.5% (v/v) bovine serum albumin (BSA) and 0.02% (w/v) sodium azide. Centrifuge at 400 x g for 5 min.
      CAUTION: Sodium azide is toxic.
   2. Re-suspend pellets in 200 µl of washing solution and add anti-CD90, anti-CD45, anti-CD73 and anti-CD31 antibodies conjugated with florescent dyes ("TEST"); in parallel set up isotype controls ("CTRL").
      NOTE: Amounts of staining antibodies should be determined by titration or according to manufacturer's instructions.
   3. Incubate samples at 4 °C for 30 min.
   4. Centrifuge at 400 x g for 5 min. Re-suspend cells in 500 µl of washing solution and acquire at least 5 x 10⁴ events on multicolor flow cytometer ^{7 9 10}.
   5. Analyze results by dot-plots and use "CTRL" recorded events to set quadrants.
      NOTE: In order to define the culture as MPC culture the percentage of CD73^negCD90^negCD45⁺CD31⁺ cells should be over 95%. For some specific applications, i.e., gene expression analysis, the recovery cut-off has to be increased to 97 – 98%.
2. Nestin detection and F-actin organization analysis
   1. Plate freshly isolated MPCs on culture chamber slides (20,000/cm²). Allow cells to adhere by overnight incubation at 37 °C in 5% CO₂.
   2. Wash cells in washing solution and fix in 4% (w/v) para-formaldehyde at room temperature for 15 min. To remove fixative add washing solution, incubate for 2 min and pour.
   3. Repeat washing twice.
   4. Permeabilize cells in D-PBS supplemented with 0.05% (v/v) Triton X-100 for 15 min at RT.
   5. Stop reaction by protein free signal enhancer (30 min at RT) or standard blocking solution (D-PBS supplemented with 3% (w/v) BSA).
   6. Remove signal enhancer/blocking solution.
   7. Add 7 µg/ml (w/v) of anti-human nestin primary antibody and incubate at 4 °C overnight in a humidified chamber. In parallel, use isotypic control antibodies to evaluate not specific fluorescence signals.
   8. Wash slides by adding D-PBS, leave for 2 min and pour. Repeat twice.
   9. Add 2 µg/ml (w/v) of fluorescent dye conjugated secondary antibody and incubate at 4 °C for 1 hr in the dark.
   10. Wash slides as above.
   11. Add fluorescent Phalloidin (5 UI/ml), leave at RT for 30 min in the dark and wash 3 times in D-PBS.
   12. Remove the chamber walls and mount slides in aqueous mounting medium supplemented with antifade reagent and 4',6-diamidino-2-phenylindole (DAPI) for nuclei detection. Proceed to imaging ^{7 9 10}.

4. Mesengenic Differentiation of MPCs

1. Plate 2 x 10⁴/cm² freshly isolated MPCs in TC-treated T75 culture flasks and let cells adhere overnight in DMEM/10% PhABS at 37 °C in 5% CO₂.
2. Replace DMEM/10% PhABS with standard reduced serum medium, about 200 µl/cm², designed for mesenchymal stromal cell expansion (MSC-RS medium). Grow cells to confluence (P1-MSCs), typically from 7 to 10 days from the induction. Refresh medium every 2 days.
3. Aspirate and discard medium from flasks, wash with fresh MSC-RS medium and add 2 ml of animal free protease detaching solution. Incubate at 37 °C for 5 – 15 min (avoid prolonged incubation).
4. Add 10 ml of fresh MSC-RS medium, aspirate the cell suspension and centrifuge at 400 x g for 5 min
5. Aspirate supernatant and re-suspend pellet in 1 – 2 ml of fresh MSC-RS medium. Proceed to cell count as described in step 1.10
6. Proceed to sub-culture them by seeding 3 – 5 x 10³ cells/cm². Grow cells to confluence (P2-MSCs).
7. Harvest cells by protease digestion as described from step 4.3 to step 4.5 and proceed to characterization as described in section 3.
   NOTE: A percentage of CD73⁺CD90⁺CD45^negCD31^neg cells lower than 95% would indicate only partial differentiation and require a further culture passage.
8. Plate P2-MSCs at 2 x 10⁴/cm² in six well TC-treated plates and grow to confluence in MSC-RS medium.
9. Mark two wells as "No Diff" and refresh MSC-RS medium.
10. Mark two wells as "Osteo" and replace medium with 200 µl/cm² of standard osteogenic medium, specifically designed for MSC differentiation.
11. Mark two wells as "Adipo" and replace medium with 200 µl/cm² of standard adipogenic medium, specifically designed for MSC differentiation.
12. Maintain cultures at 37 °C in 5% CO₂ by changing the whole media each 48 hr.
    NOTE: It is highly recommended to use standard commercially available differentiating media for test reproducibility. After 2/3 weeks under differentiating conditions, calcium deposits appear in osteogenic induced cultures, while intracellular lipid droplets accumulation is evident in adipogenic induced cells.
13. Aspirate and discard culture media then wash with D-PBS.
14. Fix the cultures by adding 1 ml of 4% (w/v) para-formaldehyde for 15 min at room temperature.
15. To remove fixative add D-PBS, incubate for 2 min and pour. Repeat washing twice.
16. Stain one "No Diff" together with the two "Osteo" marked wells in hydroxyapatite specific fluorescent solution and one "No Diff" together with the two "Adipo" marked wells in 200 nM Nile Red solution. Incubate for 30 min at room temperature^11,12.
17. Remove staining solutions and wash in D-PBS twice.
18. Remove D-PBS, add D-PBS supplemented with 50% (v/v) glycerin and proceed to imaging^{7 9 10}.

5. MPC Spheroid Sprouting Assay

1. To produce 3D spheroids, lay 20 µl drops of freshly isolated MPC suspension (1.5 x 10⁴ cells/drop) on the inner surface of a Petri dish lid.
   NOTE: As handling drops could lead to their rupture, it is highly recommendable to lay them in excess.
2. Carefully use the lid to recap a Petri dish containing D-PBS to prevent hanging drops evaporation. Incubate at 37 °C in 5% CO₂ overnight to allow cells to aggregate in 3D spheroids.
3. Set a thick gel of murine extracellular matrix (ECM) proteins by adding 300 µl aliquots of standard ECM proteins in a pre-refrigerated 24-well culture plate, and incubate at 37 °C for 30 min.
4. Carefully tip over the Petri dish lid and gently pick up the spheroids using a sterile Pasteur pipette.
5. Lay spheroids onto the ECM protein gel, add 700 µl aliquots of standard VEGF-rich endothelial cell growth medium and incubate at 37 °C in 5% CO₂.
6. After 24 hr and at 7 days of culture, take pictures of the 3D cultures at 4X magnification power. Evaluate sprouting from spheroids applying image analysis software by measuring the radial distance between last invading cell and the spheroid edge. Repeat measures along at least 20 different directions. Mean distance is deemed as positive when 50 µm or over.