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Large numbers of erythroid cells can be generated by culturing CD34⁺ hematopoietic stem and progenitor cells (HSPCs) in StemSpan™ serum-free expansion medium, e.g, StemSpan™ SFEM II, supplemented with StemSpan™ Erythroid Expansion Supplement. The Erythroid Expansion Supplement contains the cytokines stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin (EPO), which together promote the proliferation and differentiation of HSPCs to generate thousands of erythroblasts per input CD34⁺ cell after 1 - 3 weeks of culture. Most of these culture-expanded erythroblasts express transferrin receptor (CD71) and glycophorin A (GlyA, CD235a), but express little or no hemoglobin (Hb). These cells have the capacity to mature into hemoglobinized normoblasts and reticulocytes when they are moved to appropriate culture conditions for maturation. One suggested maturation protocol is described below.

This protocol effectively promotes maturation and hemoglobinization of erythroblasts generated from cord blood or bone marrow CD34⁺ cells, but cell yields and viability can be variable between experiments. If desired, modifications of the maturation protocol may be made to include co-culture with stromal cells.^1-8

You will need:

• CD34⁺ cells (freshly isolated or cryopreserved from cord blood or bone marrow)
• StemSpan™ SFEM II Medium
• StemSpan™ Erythroid Expansion Supplement
• Erythropoietin (EPO)
• Human AB Serum
• Tissue culture plates
• General lab equipment (incubator, centrifuge, pipettes etc.)
• Anti-Human CD71 Antibody
• Anti-Human GlyA Antibody

Step 1. Expand Cells

Plate CD34⁺ cells at ~10,000 cells per mL in StemSpan™ SFEM II medium supplemented with Erythroid Expansion Supplement. Add fresh medium and supplement on day 4 and replate or dilute the cells in fresh medium and supplement every 3 - 4 days to ~1 x 10⁵ cells per mL. If maintained properly, cells will continue to proliferate and generate CD71⁺GlyA⁺ erythroblasts for > 2 weeks.

Step 2. Harvest and Wash Culture-Expanded Erythroblasts

Collect and count cells, while assessing viability, e.g., with trypan blue and a hemocytometer, or an automated cell counter. If desired, measure CD71 and GlyA expression using flow cytometry. Centrifuge for 5 - 10 minutes at 200 x g to pellet. Aspirate the supernatant. Wash once with either Iscove’s MDM or StemSpan™ medium (without added growth factors) to remove residual Erythroid Expansion Supplement or other additives. Cell yield can be variable, as it is highly dependent on the quality of the original CD34⁺ cell sample. A cell yield of at least 1000 CD71+GlyA+ cells per original CD34⁺ cell is typical after 2 weeks of culture, but it is possible to get as many as 100,000 or more erythroblasts per original CD34⁺ cell with high quality samples. If cells were maintained optimally during the expansion culture, viability should be high (~90%). Viability might be lower if cell growth was poor (< 1000 erythroblasts per original CD34⁺ cell) or if cultures were overgrown due to high plating density and/or suboptimal refeeding and replating.

Step 3. Replate in Maturation Conditions

Resuspend the cell pellet at a concentration of 5 - 10 x 10⁵ cells per mL in fresh SFEM II medium supplemented with 3% normal human AB serum and recombinant human EPO (at a concentration of 1 - 3 U/mL). The inclusion of human serum is important to maintain cell viability.

Step 4. Analyze Cells

After 4 - 7 days of culture, examine the cells for CD71 and GlyA expression by flow cytometry. To measure hemoglobinization, cells may be prepared on slides using a cytocentrifuge and stained with benzidine or a similar reagent. During maturation cell numbers do not increase significantly, viability can be variable and is typically lower than before maturation, cell size decreases, CD71 expression decreases but remains detectable on most cells, GlyA expression remains high or increases further, and cell pellets become visibly red due to the accumulation of Hb. Enucleated cells may be observed when cultures are viewed under a microscope, indicated by the presence of reticulocytes and pyrenocytes (the extruded nucleus encased in a thin cytoplasmic layer) (Figure 1).