LNK inhibition expands primary patient-derived FA HSPCs. (A) Experimental scheme of lentiviral transduction followed by CFC or xenotransplantation assay. Primary CD34⁺ HSPCs were isolated from BM aspirates of healthy individuals or FA patients and transduced with lentivirus-expressing shLuc or shLNK along with the mCherry marker. Transduced cells were either directly injected into NBSGW mice or plated for CFC assays after flow cytometric sorting of mCherry⁺ cells. (B) Healthy or FA HSPCs were sorted for mCherry positivity 48 hours posttransduction and plated onto semisolid methylcellulose culture media. CFU progenitors were enumerated 12 to 14 days later. Each symbol represents an individual plate; bars indicate mean values; error bars indicate plus or minus SD. *P < .05; **P < .01; ***P < .001 as determined by 2-tailed Student t tests. N = 2 healthy samples and 6 FA patient samples. (C) Transduced HSPCs from a FA patient (FA-D1 #600.01) were transplanted into NBSGW mice, and engraftment in the BM and spleen was assessed after 16 weeks. Percent mCherry⁺ in the engrafted hCD45⁺ BM and spleen is shown. N = 1 patient FA-D1 #600.01, 1 mouse per shRNA. Bars indicate percent mCherry⁺.