title: Ex vivo assays to study self-renewal, long-term expansion, and leukemic transformation of genetically modified human hematopoietic and patient-derived leukemic stem cells.
abstract: With the emergence of the concept of the leukemic stem cell (LSC), assays to study them remain pivotal in understanding (leukemic) stem cell biology. Although the in vivo NOD-SCID or NSG xenotransplantation model is currently still the favored assay of choice in most cases, this system has some limitations as well such as its cost-effectiveness, duration, and lack of engraftability of cells from some acute myeloid leukemia (AML) patients. Here, we describe in vitro assays in which long-term expansion and self-renewal of LSCs isolated from AML patients can be evaluated. We have optimized lentiviral transduction procedures in order to stably express genes of interest or stably downmodulate genes using RNAi in primary AML cells, and these approaches are described in detail here. Also, we describe bone marrow stromal coculture systems in which cobblestone area-forming cell activity, self-renewal, long-term expansion, and in vitro myeloid or lymphoid transformation can be evaluated in human CD34(+) cells of fetal or adult origin that are engineered to express oncogenes. Together, these tools should allow a further molecular elucidation of derailed signal transduction in LSCs.
authors: Department of Experimental Hematology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700RB, Groningen, The Netherlands.
authors: Pallavi
authors: P
authors: Sontakke
authors: Marco
authors: M
authors: Carretta
authors: Marta
authors: M
authors: Capala
authors: Hein
authors: H
authors: Schepers
authors: Jan Jacob
authors: JJ
authors: Schuringa
mesh_terms: D000328:Adult; D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics / Q000378:metabolism; D001854:Bone Marrow Cells / Q000473:pathology; D002469:Cell Separation; D002471:Cell Transformation, Neoplastic; D018920:Coculture Techniques; D005312:Fetal Blood / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D015470:Leukemia, Myeloid, Acute / Q000473:pathology*; D051379:Mice; D014411:Neoplastic Stem Cells / Q000473:pathology*; D013997:Time Factors; D014161:Transduction, Genetic
chemical_list: D018952:Antigens, CD34