Isolation of Leukemic Stem Cells by using a Lentiviral Reporter System
Translated title
Isolation af leukæmi stamceller ved brugen af et lentiviralt rapporter system
Author
Sørensen, Maja Balling Zacher
Term
4. term
Publication year
2020
Submitted on
2020-08-05
Pages
55
Abstract
Acute myeloid leukemia (AML) can be driven by leukemic stem cells (LSCs)—mutated blood stem or progenitor cells that initiate disease and often resist chemotherapy, leading to relapse. Identifying LSCs is difficult because their surface markers vary between patients. The ERG+85 enhancer/promoter is linked to endogenous ERG gene expression, which correlates with a stem‑like state. A reporter based on this element can therefore indicate cellular “stemness.” This thesis aimed to establish a lentiviral stemness reporter to identify LSC‑like cells in AML, enabling future testing of drugs that target them. A lentivirus carrying the pMIN‑ERG+85 vector was produced; it includes a constitutive green fluorescent protein (GFP) to mark infected cells and a blue fluorescent protein (BFP) under control of the ERG+85 enhancer/promoter to report stem‑like activity. Plasmids (pMIN‑ERG+85 or the control vector pMIN) were amplified in Escherichia coli and used to transfect HEK 293T cells for virus production. AML cell lines Kasumi‑1, KG‑1, and MOLM‑13 were transduced, infected cells were sorted by FACS and long‑term cultured, and fluorescence was repeatedly assessed by flow cytometry. Key findings were that polybrene improved transduction; Kasumi‑1 and MOLM‑13 were infectable whereas KG‑1 was not; and overall transduction was more efficient with pMIN than with pMIN‑ERG+85. Infected cells also showed higher BFP signal with the control pMIN than with pMIN‑ERG+85, suggesting leaky activity from the mCMV promoter, which complicates interpretation of the BFP readout. Due to time constraints, planned drug screening was not performed. Further work should address the BFP background and validate the reporter before using it for compound testing.
Akut myeloid leukæmi (AML) kan opstå fra leukæmistamceller (LSC’er) – muterede blodstam- eller forløberceller, som både kan starte sygdommen og gøre tilbagefald mere sandsynlige, fordi de ofte tåler kemoterapi bedre. At finde LSC’er er svært, da deres overflademarkører varierer mellem patienter. ERG+85‑enhancer/promoter er knyttet til cellers egne niveauer af ERG‑genet, som igen hænger sammen med en stamcelle‑lignende tilstand. Et rapportersystem baseret herpå kan derfor bruges til at indikere “stemness”. Formålet med specialet var at etablere en ny metode til at identificere LSC’er ved hjælp af et lentiviralt stamcelle‑reportersystem, så man senere kan teste stoffer, der målretter LSC’er i AML. Der blev produceret lentivirus indeholdende vektoren pMIN‑ERG+85, som har et konstant udtrykt grønt fluorescerende protein (GFP) til at markere inficerede celler og et blåt fluorescerende protein (BFP) under kontrol af ERG+85‑enhancer/promoter til at rapportere stamcelle‑lignende aktivitet. Plasmiderne (pMIN‑ERG+85 eller kontrolvektoren pMIN) blev først opformeret i Escherichia coli og derefter brugt til at transfikere HEK 293T‑celler for virusproduktion. AML‑cellelinjerne Kasumi‑1, KG‑1 og MOLM‑13 blev transduceret, inficerede celler blev sorteret med FACS og langtidskultiveret, og fluorescens blev gentagne gange målt med flowcytometri. Resultaterne viste, at polybrene øgede transduktionsraten; Kasumi‑1 og MOLM‑13 kunne inficeres, mens KG‑1 ikke kunne; og at transduktion generelt var mere effektiv med pMIN end med pMIN‑ERG+85. Desuden sås et højere BFP‑signal i kontrolvektoren pMIN end i pMIN‑ERG+85, hvilket tyder på “utæt” aktivitet fra mCMV‑promoteren og gør fortolkning af BFP‑signalet vanskeligere. På grund af tidsbegrænsninger blev planlagte lægemiddelscreeninger ikke gennemført. Yderligere arbejde bør fokusere på at løse BFP‑baggrundssignalet og validere reportersystemet, før det bruges til stofafprøvning.
[This apstract has been rewritten with the help of AI based on the project's original abstract]