BALAKUMARAN et al BMSC DEFECT IN DYSKERATOSIS CONGENITA 1 Supplementary Materials Bone marrow skeletal stem/progenitor cell defects in patients with dyskeratosis congenita and telomere biology disorders Arun Balakumaran, Prasun J. Mishra, Edyta Pawelczyk, Sayuri Yoshizawa, Brian Sworder, Natasha Cherman, Sergei A. Kuznetsov, Paolo Bianco, Neelam Giri, Sharon A. Savage, Glenn Merlino, Bogdan Dumitriu, Cynthia E. Dunbar, Neal S. Young, Blanche P. Alter, Pamela G. Robey Supplementary Methods Colony forming efficiency (CFE) assays To ascertain the approximate number of skeletal stem cells (SSCs) in bone marrow from normal donors and patients with bone marrow failure, bone fragments from surgical waste (normal donors) and when available, bone fragments from the Jamshidi needle used for aspiration (normal donors and patients) were processed as previously described. 1 The bone fragments in growth medium (α-MEM, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 20% lot-selected fetal bovine, non-heat inactivated serum, 10 -8 M dexamethasone and 10 -4 M ascorbic acid-2-phosphate) were gently scraped with a surgical blade and washed extensively, but gently, with the same medium to remove marrow. The cell suspensions were then passed through cell strainers (70 µm, Falcon) to yield single cell suspensions. Nucleated cells were counted via a hemocytometer, and T25 flasks were in plated in triplicate with 1x10 4 , 1x10 5 and 1x10 6 nucleated cells. The next day, the cultures were washed with nutrient medium to remove red blood cells and non-adherent nucleated cells, and re-fed with growth medium. After