Establishing long-term cultures with self-renewing acute myeloid leukemia stem/progenitor cells.
OBJECTIVE
With the emergence of the concept of the leukemia stem cell, assays to study them remain pivotal in understanding (leukemic) stem cell biology.


METHODS
We have cultured acute myeloid leukemia CD34(+) cells on bone marrow stroma. Long-term expansion was monitored and self-renewal was addressed by replating of Leukemic-cobblestone area-forming cells (L-CAs). Also, lentiviral vectors were generated that could target L-CAs.


RESULTS
A strong expansion was observed in about 75% of the acute myeloid leukemia cases (n = 30) and long-term cultures could be maintained for up to 24 weeks on MS5 bone marrow stromal cells. Cells that were able to initiate leukemic cobblestone areas resided in the CD34(+) population and were absent from the CD34(-) population. Self-renewal within these L-CAs was determined by sequential passaging of these L-CAs onto new MS5 stromal layers, which resulted in the generation of second, third, and fourth L-CAs, which were able to sustain long-term expansion and generated high numbers of immature undifferentiated suspension cells. CD34(+) cells that were able to initiate long-term cultures all coexpressed MEIS1 and HOXA9, and expressed elevated BMI1 levels.


CONCLUSIONS
We present a novel long-term leukemic stem/progenitor assay in which new drugs can be tested and in which genes can be overexpressed or downmodulated using a lentiviral approach in order to obtain more insight into the process of leukemic transformation and self-renewal.
van Gosliga
Djoke
D
Department of Hematology, University Medical Center Groningen, Groningen, The Netherlands.
Schepers
Hein
H
Rizo
Aleksandra
A
van der Kolk
Dorina
D
Vellenga
Edo
E
Schuringa
Jan Jacob
JJ
D018952:Antigens, CD34; D001854:Bone Marrow Cells / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor / Q000378:metabolism / Q000473:pathology*; D015973:Gene Expression Regulation, Leukemic; D018398:Homeodomain Proteins / Q000096:biosynthesis; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism / Q000473:pathology*; D000075064:Myeloid Ecotropic Viral Integration Site 1 Protein; D009363:Neoplasm Proteins / Q000096:biosynthesis; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D009687:Nuclear Proteins / Q000096:biosynthesis; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis; D017154:Stromal Cells / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D014410:Tumor Stem Cell Assay
D018952:Antigens, CD34; C069077:BMI1 protein, human; D018398:Homeodomain Proteins; C000616391:MEIS1 protein, human; D000075064:Myeloid Ecotropic Viral Integration Site 1 Protein; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; C097899:homeobox protein HOXA9; D063150:Polycomb Repressive Complex 1