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. MATERIALS AND 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.
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. MATERIALS AND 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.
Authors: Hans-Ingo Trompeter; Hassane Abbad; Katharina M Iwaniuk; Markus Hafner; Neil Renwick; Thomas Tuschl; Jessica Schira; Hans Werner Müller; Peter Wernet Journal: PLoS One Date: 2011-01-20 Impact factor: 3.240