Isolation and Co-culture of CD34+ Progenitor Cells

Mobilized CD34⁺ progenitor cells were isolated from leukapheresis
samples from 5 HD (male/female ratio: 3/2; median age: 34 years, range:
18–54 years) as previously described.^{18 (link)} CD34⁺progenitor cells were labeled using the human CD34 MicroBead Kit (Miltenyi
Biotec GmbH, Bergisch Gladbach, Germany) and purified in an AUTOMACs device.
The purity and viability of immunomagnetically sorted CD34⁺ cells
were determined by flow cytometry using fluorescein isothiocyanate
(FITC)-conjugated CD34 (11-0349-42, eBioscience Inc., San Diego, CA, USA)
and 7AAD.
These cells were then co-cultured with MSCs pretreated or not with EP. After
24 h of co-culture, 2 × 10³CD34⁺ were seeded in
methylcellulose semisolid MACS medium supplemented with stem cell factor
(SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF),
granulocyte colony-stimulating factor (G-CSF), IL-3, and IL-6 (Miltenyi
Biotec GmbH, Germany) and maintained for 14 days in a humidified atmosphere
at 37ºC with 5% CO₂. CFU-GM colonies were then scored with an
inverted microscope, as previously described.^{24 (link)}