This protocol was developed for CD34⁺ HSCs derived from G-CSF mobilized peripheral blood. Differentiation and expansion into other blood cell lineages can be expected after a 14-day culturing period. This protocol may have to be modified when HSCs from other sources are used.

• Reagents and Materials
• StemXVivo Serum-Free HSC Base Media (R&D Systems, Catalog # CCM001)
• Iscove’s Modified Dulbecco’s Medium (IMDM) (Invitrogen, Catalog # 12440)
• Low Density Lipoproteins (LDL) (Calbiochem, Catalog # 437644)
• Human CD34⁺ HSCs
• Cytokines ( www.RnDSystems.com)
• Tissue culture plates (6-well plate; 24-well plate)
• Serological pipette
• Pipettes and pipette tips
• Hemocytometer
• Centrifuge
• Inverted microscope
• 37°C and 5% CO₂ incubator.

Procedure

1. Thaw the required volume of StemXVivo Serum-Free HSC Base Media at 2-8°C or room temperature. Mix the thawed media thoroughly using a serological pipette and warm the media in a 37°C incubator.
2. Cell Preparation
   1. Fresh cells
      1. Wash cells once at room temperature using 10 mL of IMDM.
      2. Centrifuge after wash for 10 minutes at 400 x g.
      3. Adjust the cell density to 1 x 10⁶ cells per mL in IMDM.
   2. Frozen cells
      1. Wash cells twice at room temperature using 10 mL of IMDM.
      2. Centrifuge after each wash for 10 minutes at 400 x g.
      3. Adjust the cell density to 1 x 10⁶ cells per mL in IMDM.
      
      Note:The CD34⁺ cells must be used immediately. Storage in medium without cytokines for over an hour may impair the ability of CD34⁺ cells to proliferate.
3. Add 40 ng/mL of LDL and the desired concentration of cytokines to the warmed StemXVivo Serum-Free HSC Base Media.
   Note:For optimal stability, do not pre-mix the LDL and cytokines with medium.
4. Add the appropriate volume of StemXVivo Serum-Free Base Media containing LDL and cytokines to the tissue culture flask, tissue culture plate or other cell culture containers. Add cells to a final density of 1 x 10⁴ cells per mL.
   

   Tissue Culture Plate	Suggested Culture Volume per Well
   24-well	1 mL
   6-well	4 mL

   Note: To avoid the evaporation of medium, place the tissue culture plate in a humidified container.
5. Incubate the cells in a 37°C, 5% CO 2 humidified incubator for 10-14 days. Change the media every four days by removing half of the media from the plate, and replenish with the same volume of fresh media supplemented with cytokines and LDL. If extensive cell expansion is observed, additional media (supplemented with cytokines and LDL) should be added to maintain the cells in the logarithm growth phase.
6. Optimal CD34⁺ cell expansion will be seen between day 10-14 for CD34⁺ cells derived from G-CSF mobilized peripheral blood. To determine the percentages of different hematopoietic progenitors in the final cell population, colony forming cell (CFC) assays using methylcellulose-based reagents (R&D Systems, Catalog # HSC001, HSC002, HSC003, HSC004, or HSC005) are recommended. Flow cytometry can also be used to identify cells based on their characteristic lineage-specific markers. For a complete list of cell characterization reagents, visit www.RnDSystems.com.
   

Representative Results

Figure 1 - Expansion of Total Nucleated Cells (TNC) and CD34+ cells from G-CSF mobilized CD34+ cells. Cells were cultured in the StemXVivo Serum-Free HSC Base Media supplemented with 100 ng/mL of recombinant human Flt-3 Ligand (R&D Systems, Catalog # 308-FKN) and 100 ng/mL of recombinant human Tpo (R&D Systems, Catalog # 288-TP). On day 0 (d10) and 14 (d14), cells were harvested and used to study the fold expansion of TNC (left panel) and CD34+ cells (right panel). Viable total nucleated cells were counted using a hemocytometer and the CD34+ expression on the expanded cells was analyzed by flow cytometry.