Efficient transient genetic labeling of human CD34+ progenitor cells for in vivo application.
Genetic labeling of human hematopoietic progenitor cells (HPC) and their consecutive fate-mapping in vivo is an approach to answer intriguing questions in stem cell biology. We recently reported efficient transient genetic labeling of human CD34+ HPC with the truncated low-affinity nerve growth factor receptor (DeltaLNGFR) for in vivo application. Here we investigate whether HPC labeling with DeltaLNGFR affects lineage-specific cell differentiation, whether DeltaLNGFR expression is maintained during lineage-specific cell differentiation and which leukemia cell line might be an appropriate cell culture model for human CD34+ HPC. Human CD34+ peripheral blood stem cells and various leukemia cell lines were characterized by immunophenotyping. Cells were transfected using nucleofection. Hematopoietic differentiation was studied by colony-forming assays. DeltaLNGFR expression was assessed using reverse transcription-PCR, immunofluorescence and flow cytometry. Nucleofection was efficient and did not significantly reduce hematopoietic cell differentiation. Mature myeloid cells (CD66b+) derived from human CD34+ HPC and Mutz2 cells maintained DeltaLNGFR expression at a high percentage (70 +/- 2% and 58 +/- 2%, respectively). Mutz2 cells may serve as an in vitro model for human myeloid HPC. The method described herein has been adopted to Good Manufacturing Practices (GMP) guidelines and is ready for in vivo application.
Wiehe
Juliane M I
JM
University of Ulm, Department of Internal Medicine II, Ulm, Germany.
Niesler
Carola
C
Torzewski
Jan
J
Zimmermann
Oliver
O
Wiesneth
Markus
M
Schmitt
Michael
M
Schwarz
Klaus
K
Döhner
Hartmut
H
Hombach
Vinzenz
V
Greiner
Jochen
J
D018952:Antigens, CD34 / Q000378:metabolism*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D005819:Genetic Markers / Q000235:genetics; D018922:HL-60 Cells; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D016130:Immunophenotyping; D020014:K562 Cells; D020932:Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D030781:Organisms, Genetically Modified; D023822:Tissue Engineering / Q000379:methods*; D014162:Transfection
D018952:Antigens, CD34; D005819:Genetic Markers; D020932:Nerve Growth Factor