Nucleofection, an efficient nonviral method to transfer genes into human hematopoietic stem and progenitor cells.
The targeted manipulation of the genetic program of single cells as well as of complete organisms has strongly enhanced our understanding of cellular and developmental processes and should also help to increase our knowledge of primary human stem cells, e.g., hematopoietic stem cells (HSCs), within the next few years. An essential requirement for such genetic approaches is the existence of a reliable and efficient method to introduce genetic elements into living cells. Retro- and lentiviral techniques are efficient in transducing primary human HSCs, but remain labor and time consuming and require special safety conditions, which do not exist in many laboratories. In our study, we have optimized the nucleofection technology, a modified electroporation strategy, to introduce plasmid DNA into freshly isolated human HSC-enriched CD34(+) cells. Using enhanced green fluorescent protein (eGFP)-encoding plasmids, we obtained transfection efficiencies of approximately 80% and a mean survival rate of 50%. Performing functional assays using GFU-GEMM and long-term culture initiating cells (LTC-IC), we demonstrate that apart from a reduction in the survival rate the nucleofection method itself does not recognizably change the short- or long-term cell fate of primitive hematopoietic cells. Therefore, we conclude, the nucleofection method is a reliable and efficient method to manipulate primitive hematopoietic cells genetically.
von Levetzow
Gregor
G
Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University Düsseldorf, D-40225 Düsseldorf, Germany.
Spanholtz
Jan
J
Beckmann
Julia
J
Fischer
Johannes
J
Kögler
Gesine
G
Wernet
Peter
P
Punzel
Michael
M
Giebel
Bernd
B
D000071916:AC133 Antigen; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D003114:Colony-Forming Units Assay; D018274:Electroporation / Q000379:methods; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D006023:Glycoproteins / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007962:Leukocytes / Q000166:cytology / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000032:analysis; D017154:Stromal Cells / Q000166:cytology; D014162:Transfection / Q000379:methods*
D000071916:AC133 Antigen; D000590:Amino Acid Chloromethyl Ketones; D015703:Antigens, CD; D018952:Antigens, CD34; D006023:Glycoproteins; D010455:Peptides; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D049452:Green Fluorescent Proteins