A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

The protocol follows the guidelines of National Center for Global Health and Medicine. All experimental procedures performed on mice are approved by the National Center for Global Health and Medicine animal experiment committee.
NOTE: An overview of the protocol is illustrated in Figure 1.

1. Lipid preparation

1. Dissolve the following lipids in methanol in glass tubes at the indicated concentrations: sodium palmitate, 16 mg/mL; sodium oleate, 30 mg/mL; and cholesterol, 4 mg/mL. Store the lipid solution at -30 °C and thaw the sample before use.
2. Mix the lipid solutions prepared in step 1.1 in a fresh glass tube at the doses necessary to obtain the final concentration of 100 µg/mL palmitate, 100 µg/mL oleate, and 20 µg/mL cholesterol. For instance, when preparing 10 mL of culture media, mix 62.5 µL of palmitate solution, 33 µL of oleate solution, and 50 µL of cholesterol solution in the glass tube.
3. Evaporate the methanol by passing nitrogen gas through the lipid solution (Figure 2A and 2B). If nitrogen gas is not available, pass air through the solution by using a pipette-aid.
4. Completely evaporate the remaining methanol by heating the glass tube in a water bath at 37 °C (Figure 2C).
   ​NOTE: The use of a high concentration of N₂ gas in confined space is potentially harmful. Although the volume used in the protocol to evaporate methanol is limited and thus is considered as safe, ventilating the room adequately and labeling areas of potentially high concentrations of N₂ gas is important should an unexpected gas leak occur.

2. Medium preparation

1. Prepare Dulbecco's modified Eagle medium (DMEM)/F12 medium (with HEPES and glutamine). Add penicillin and streptomycin sulfate to the medium to the final concentration of 50 units and 50 µg/mL, respectively. DMEM/F12 medium containing antibiotics can be stored at 4 °C for at least 2 months.
2. Add 4% w/v of bovine serum albumin (BSA) to Dulbecco's modified Eagle medium (DMEM)/F12 medium (with HEPES and glutamine).
3. Adjust the pH of the medium to pH 7.4-7.8 using NaOH solution, typically to pH 7.6.
4. Add the medium to the glass tube prepared in step 1. To achieve a solution with maximum solubility, addition of 3-15 mL medium is recommended.
5. Completely dissolve the lipids by sonication (optimal: more than 20 min of sonication). After the BSA and lipids are dissolved, the sample should be stored at -80 °C and used within 2 months. Thaw immediately before use.
6. Add 1/1,000 of the insulin, transferrin, sodium selenite, and ethanol amine (ITS-X) mixture to the DMEM/F12. Filter the mixed medium using a 0.22 µm filter (designated as "culture media"). Before use, add human stem cell factor (SCF) and human thrombopoietin (TPO) to the culture media at a final concentration of 3 ng/mL each. After adding the cytokines and ITS-X, the medium cannot be stored.
7. Staining buffer: Add 10% FCS to Ca- and Mg-free phosphate-buffered saline (PBS). This solution can be stored at 4 °C for 2 weeks.
8. Thawing media: Add 10% FCS to the DMEM/F12. This solution is single-use.
9. Cytokine stock solution: Dissolve each human SCF or human TPO in PBS (Ca- and Mg-free) at 20 µg/mL. This solution can be stored at -80 °C for at least one year without a loss of activity. Once thawed, store the stock solution at 4 °C and use within 1 month.
   ​NOTE: Examine the lot after every purchase to avoid variation in the contaminants in BSA. Culture HSCs at the same time with different batches of BSA and examine the phenotypic HSC number at day 7 as described below. If a BSA batch shows a low number or frequency of CD34+ cells (e.g. less than a half the number of input cells), avoid using this batch.

3. Preparation of human bone marrow CD34⁺ cells

1. Purchase human CD34⁺ bone marrow cells and store the cells in liquid nitrogen until use. Alternatively, bone marrow cells from a healthy volunteer or fresh cord blood cells can be used upon approval by the institute ethical committee and donor consent.
2. Warm 10 mL of thawing media in a 15 mL conical tube in a 37 °C water bath.
3. Thaw the frozen cells in vials in a 37 °C water bath within 2 min. After wiping the vial with 70% ethanol to remove contaminants, transfer the cells to the 15 mL conical tube containing the pre-warmed medium from step 3.2.
4. Centrifuge the 15 mL tube at 200 × g for 15 min at room temperature. Aspirate the supernatant carefully to keep the pellet intact (leaving < 50 µL of medium). Resuspend the cells with the remaining medium and transfer them to a 1.5 mL tube on ice.
   NOTE: Exposure to human-derived samples directly in the mucosa including the eye or wounded tissue may cause infection by known or unknown pathogens; thus, gloves and eyeglasses are recommended when handling human cells. Even if the purchased CD34⁺ cells test negative for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus 1 (HIV1), careful monitoring should be performed according to institutional guidelines if an exposure incidence occurs. Follow the institutional guidelines when disposing of materials exposed to human cells.

4. Sorting HSCs

1. Label the cells with fluorochrome-conjugated antibodies. Mix 50 µL of staining buffer plus 10 µL of anti-CD34-FITC, 2 µL of anti-CD38-PerCP-Cy5.5, 5 µL of anti-CD90-PE-Cy7, and 10 µL of anti-CD45RA-PE. Resuspend the cell pellet in the antibody mixture. Incubate the cells for 30 min at 4 °C in the dark.
2. Add 1 mL of staining buffer to wash the antibodies. Centrifuge the tubes at 340 × g for 5 min at 4 °C. Discard the supernatant.
3. Resuspend the cell pellet in 0.5 mL of staining buffer + 0.1% propidium iodide. Transfer the suspension into a 5 mL tube using a 40 µm filter.
4. Gate the phenotypic HSCs within the PI^–CD34⁺CD38^–CD90⁺CD45RA^– fraction using the FACS Aria-IIIu and sort the cells into a 1.5 mL tube filled with 500 µL of culture media (Figure 3). Using the gating strategy shown in Figure 3, ~10% of CD34⁺ cells are expected to be phenotypic HSCs. Record the sorted cell number to calculate the volume of media to resuspend the cells in step 5.3.
   NOTE: The gating strategy is performed as described previously¹⁶ while omitting the lineage marker staining given the low expression of lineage markers in the CD34+CD38- fraction from healthy individuals¹⁷ .
5. Centrifuge the sorted cells at 340 × g for 5 min at 4 °C and discard the supernatant. Carefully aspirate the supernatant to ensure that the pellet remains intact.
6. Store the sorted cells on ice until culture.
   ​NOTE: Fluorescence compensation of spectral overlap should be performed during the first experiment.

5. Cell culture

1. Transfer 200 µL of the culture media containing cytokines prepared in step 2 into flat-bottom 96-well plates.
2. To avoid evaporation of the medium, fill all unused wells with 100-200 µL of PBS.
3. Resuspend the sorted HSCs in culture media without cytokines at 60 cells/µL.
4. Aliquot 600 HSCs/well (10 µL of cell suspension) into each well. The cell number can be altered. Fewer than 300 cells will lead to larger technical variation, and thus more wells per condition are needed to detect biological differences. Culturing more than 1000 cells in a single well should be avoided because of cytokine/nutrient deprivation or accumulation of unfavorable cytokines/chemokines.
5. Culture the cells in a humidified multi-gas incubator at 37 °C in a 5% CO₂ and 1% O₂ atmosphere.
6. For cultures over 7 days, replacing half the media volume every 3-4 days is recommended. Carefully aspirate 100 µL of media by pipetting and add 100 µL of newly prepared culture media containing cytokines to each well. The culture media should be pre-warmed at 37 °C.

6. Analysis of marker phenotypes using flow cytometry

NOTE: Although the cells should be analyzed after 7 days of culture, the culture duration can be changed.

1. Discard 170 µL of the medium by using an 8-channel pipette.
2. Label the cells with the antibody mixture. Mix 0.5 µL of anti-CD34-FITC, 0.1 µL of anti-CD38-PerCP-Cy5.5, 0.25 µL of anti-CD90-PE-Cy7, 0.5 µL of anti-CD45RA-PE, and 9 µL of staining buffer per well. Add 10 µL of the mixture to the 96-well plate and incubate the cells for 30 min at 4 °C in the dark.
3. To wash the antibodies, add 100 µL of staining buffer to the wells and centrifuge the plates at 400 × g for 5 min at 4 °C using low acceleration and medium deceleration.
4. Carefully aspirate 100 µL of the supernatant to maintain the cells on the bottom of the wells, then resuspend the cells in 200 µL of staining buffer + 0.1% v/v of PI + 1% v/v of fluorescent microspheres (e.g., Flow-Check Fluorospheres).
5. Set up the flow cytometry instrument. Acquire data for the samples in fast mode with mixed sample mode on and uptake volumes of 100 µL.
6. Export the data in FCS format and analyze it using software such as FlowJo. Cell numbers can be determined using the fluorescent microsphere bead count. For instance, if the researcher adds 2000 beads per well and the bead count is 700, multiply the cell number of each fraction by 2000/700 to estimate the total cell number of the fraction in the well.
   ​NOTE: Fluorescent microspheres are toxic to cultured cells because of the presence of formaldehyde. This may induce cell death during analysis. Minimize the number of wells (<50 wells) analyzed continuously to avoid bias.

7. Transplantation of human HSCs

NOTE: Repopulation activity of cultured cells are validated by transplantation to immunodeficient mice. All procedures must be approved by animal experiment committees or their equivalents.

1. As donors, prepare a sufficient number of 8-12-week immunodeficient NOD-SCID-Il2rg-null (NOG) mice. As NOG mice are highly susceptible to infection, keep the breeding cages as clean as possible and feed the mice with a sterilized diet and water.
2. Culture an adequate number of HSCs for transplantation as described in step 5. For instance, when 5000 HSCs (day 0 equivalent) are transplanted to 6 recipient mice, culture 35,000-40,000 HSCs in 40 wells of a 96-well plate (200 µL of culture media per well) or in 8 wells of a 24-well plate (1 mL of culture media per well). Culture the cells for 2 weeks with half-media changes every 3-4 days in a humidified multi-gas incubator at 37 °C with 5% CO₂ and 1% O₂. The culture duration can be altered.
3. Irradiate NOG mice at 2.5 Gy at 6-24 h prior to transplantation.
4. Collect the cultured HSCs in 1.5 mL tubes. Centrifuge the tubes at 340 ×g for 5 min at 4°C.
5. Carefully aspirate the supernatants and resuspend the cells in sterile ice-cold staining buffer at a cell density of 5000 HSCs (day 0 equivalent) per 200 µL. Transfer this suspension to 3 mL polypropylene tubes. To sterilize the staining buffer, filter it using a 0.22 µm filter.
6. Before transplantation, anesthetize the mice by sevoflurane or isoflurane inhalation.
7. To transplant 5000 of cultured HSCs (day 0 equivalent), inject 200 µL of the cell suspension into the tail vein or retro orbital sinus of NOG mice irradiated in step 7.1 using a 1 mL syringe and 27-gauge needle. Freshly isolated or thawed bone marrow HSCs can be transplanted as a control. Gloves should be sterilized with 70% v/v ethanol between each procedure.

8. Analysis of the frequency of human-derived cells in the peripheral blood

1. To examine the repopulation of human cells, collect the peripheral blood at 1, 2, and 3 months after transplantation.
2. Before collecting the peripheral blood, anesthetize the mice by sevoflurane inhalation.
3. Collect 40-80 µL of peripheral blood from the retro-orbital sinus using heparinized glass capillary tubes and suspend this sample in 1 mL of PBS + heparin (1 U/mL) in 1.5 mL tubes.
4. Centrifuge the blood suspension at 340 × g for 3 min at 4 °C. Discard the supernatant and resuspend the pellet in 1 mL of PBS + 1.2% w/v dextran (200 kDa) for 45 min at room temperature.
5. Transfer the supernatant to another 1.5 mL tube and centrifuge at 340 × g for 3 min.
6. For red blood cell lysis, resuspend the cells in 0.17 M NH₄Cl for 5 min.
7. Centrifuge the cell suspension 340 × g for 3 min at 4 °C. Resuspend the cells in 50 µL of staining buffer containing 0.3 µL of anti-mouse Fc-block. Incubate this sample at 4 °C for 5 min.
8. Add the following antibodies for surface marker staining: 0.3 µL of mouse CD45-PE-Cy7, 0.3 µL mouse Ter-119-PE-Cy7, 0.3 µL of human CD45-BV421, 0.3 µL of human CD13-PE, 1.2 µL of human CD33-PE, 0.3 µL of human CD19-APC, 0.3 µL of human CD3-APC-Cy7. Incubate the cells at 4 °C for 15 min.
9. Wash once with 1 mL of staining buffer and centrifuge at 340 × g for 5 min at 4 °C.
10. Discard the supernatant and resuspend the cells in 200 µL of staining buffer + 0.1% v/v of PI.
11. Transfer the cell suspension to a 96-well flat-bottom plate and acquire data using the flow cytometer in fast mode with the mix sample mode on and an uptake volume of 100 µL.
12. Export the data in FCS format for analysis using software such as FlowJo.
13. Set up the flow cytometer. Acquire data for the samples in fast mode with the mix sample mode on and an uptake volume of 100 µL.
14. Export the data in FCS format for analysis using software such as FlowJo.