Lentiviral labeling reveals three germ layer differentiation potential of a single unrestricted somatic stem cell from human cord blood.
OBJECTIVE
Generation and expression of unrestricted somatic stem cells (USSC) from human cord blood as well as their in vitro functional characterization at the clonal level.


METHODS
USSC generation was initiated from fresh cord blood followed by lentiviral transfection and clone generation via limiting dilution. Individual clones were analyzed for lentiviral genomic integration patterns by ligation-mediated polymerase chain reaction. In vitro differentiation of clonal USSC was performed into mesodermal, endodermal, and ectodermal lineages according to our published protocols. Respective osteogenic, hepatic, and neuronal lineage-specification was documented by immunohistochemistry and tissue-specific protein expression was analyzed by Western blotting. MicroRNA expression analysis was achieved using the TaqMan microRNA Megaplex array.


RESULTS
Lentivirally labeled USSC cultures were successfully subjected to limiting dilution cloning. One clone containing a single lentiviral integration site was identified (clone 4) and used for further differentiation experiments. Ligation-mediated polymerase chain reaction results from mesodermally, endodermally, and ectodermally differentiated USSC clone 4 consistently showed only the primary single lentiviral integration site. Lineage-specific differentiation experiments were confirmed by morphology and cell-fate-specific monoclonal antibodies in immunocytochemistry. MicroRNA expression profiles did not reveal dramatic differences between clonal and nonclonal USSC.


CONCLUSIONS
The proof of the clonal existence of USSC is important for the assessment of biological properties unique for these unrestricted human stem cell candidates. As clones they can be subjected to advanced methods that enable defining of the multilayer nature of regulatory mechanisms through single-cell analysis.
Wernet
Peter
P
University Düsseldorf, Medical Faculty, Institute for Transplantation Diagnostics and Cell Therapeutics, Düsseldorf, NRW, Germany. mail: peter.wernet@uni-duesseldorf.de
Trapp
Thorsten
T
Zweigerdt
Robert
R
Mann
Jin
J
Trompeter
Hans-Ingo
HI
D015153:Blotting, Western; D002454:Cell Differentiation; D019070:Cell Lineage; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D004475:Ectoderm / Q000166:cytology / Q000378:metabolism; D004707:Endoderm / Q000166:cytology / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology*; D020869:Gene Expression Profiling; D005855:Germ Layers / Q000166:cytology* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D007763:Lac Operon / Q000235:genetics; D016086:Lentivirus / Q000235:genetics; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D035683:MicroRNAs; D001616:beta-Galactosidase