- مبلغ: ۸۶,۰۰۰ تومان
- مبلغ: ۹۱,۰۰۰ تومان
Xenograft assay allows functional analysis of leukemia-initiating cells of acute myeloid leukemia primary samples. However, 40% of samples derived from patients with better outcomes fail to engraft in immunodeficient mouse recipients when conventional protocols are followed. At diagnosis, the engraftment of intermediate-risk group samples cannot be anticipated. In this study, we decided to further explore the reasons for xenograft success and failure. No differences in extracellular phenotype, apoptosis, or cell cycle profile could distinguish samples that engraft (engrafter [E]) from samples that do not engraft (nonengrafter [NE]) in NSG mice. In addition, ex vivo long-term culture assay revealed, after 5 weeks, a lower content of leukemicLTC-initiating cells in the NE samples associated with a lower expansion rate capacity. Oneweek co-cultures with mesenchymal or osteoblastic or endothelial cells did not influence the proliferation rate, suggesting that E and NE samples are genuinely rapidly or slowly expanding independent of external cue. Engraftment success for some NE samples was consistently observed in recipient mice analyzed 6 months later than the conventional 3-month period. Eventually we implemented a flow cytometry-based assay, which allowed us to predict, in 1 week, the fast or delayed engraftment potential of a noncharacterized acute myeloid leukemia sample. This approach will be especially useful in selecting intermediate-risk-group patient samples and restricting the experimental duration to a 3-month period and, eventually, in reducing the number of animals and the cost and effort of unnecessary xenograft failures. © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. This is an open access article under the CC BY license.
AML cells were obtained after receipt of informed consent from St Bartholomew’s Hospital. Details of the patient samples are listed in Supplementary Table E1 (online only, available at www.exphem.org). Co-culture experiments were previously described . AML samples were collected at diagnosis, and mononucleated cells were isolated within 24 hours after collection by Ficoll-Paque Plus density gradient (GE Healthcare, France). Cord blood (CB) cells were obtained after receipt of informed consent from the Royal Free Hospital (UK). Both AML and CB sample collections were approved by the East London ethical committee and in accordance with the Declaration of Helsinki. Three to 5 different CB samples were pooled, and mononuclear cells were obtained by density centrifugation. Lineage markers expressing cells were depleted using StemSep columns and human progenitor enrichment cocktail (StemCell Technologies, Vancouver, BC, Canada). CD34+ CD38− cells (hematopoietic stem progenitor cells [HSPCs]) and CD34+ CD38+ cells (hematopoietic progenitor cells [HPCs]) were sorted on a MoFlo cell sorter (DakoCytomation Colorado, Fort Collins, CO, USA) or a BD FACS Aria (BD Biosciences, UK). Gates were set up to exclude nonviable cells and debris. Briefly, lineagedepleted recovered cells were washed twice and stained with antiCD34 Percp, anti-CD38 PE-cy7, AlexaFluor647-conjugated Annexin-V (Invitrogen), and DAPI (4’,6-diamidino-2-phenylindole). The purity of sorted fractions was assessed to ensure the sort quality. The stromal cell line mesenchymal MS-5 and the human osteosarcoma cell line SaOS-2 were obtained from the DSMZ cell bank (Braunschweig Germany) and maintained in Iscove’s modified Dulbecco’s medium (IMDM) containing 10% fetal calf serum (FCS) + 2 mmol/L L-glutamine or in McCoy’s 5a medium containing 15% FCS + 2 mmol/L L-glutamine, respectively.