Erik A., Miljan, Ph.D.,
ReNeuron group plc,
ReNcell® VM and ReNcell® CX are two highly published neural stem cell lines derived from developing human brains. ReNcell® VM and CX cells are generated from the ventral mesencephalon and cortical regions of the brain, respectively, and transduced with the myc transcription factor. Both cell lines offer phenotype and genotype stability, in addition to the multipotential neuronal differentiation capacity, over long-term culture. This article describes the characteristics and differentiation of ReNcell® lines. Results of calcium and membrane potential changes in response to various ligands are also shown. The convenience to maintain in culture and flexibility to differentiate to individual scientists’ needs make ReNcell® lines the ideal platform for research and discovery.
Neural stem cells (NSCs) were first described in the rodent brain by Reynolds and Weis in 1992. Seven years later, the isolation of human NSCs was documented by Vescovi et al. Since those initial findings, NSCs have been valuable tools for neuroscience, signal transduction and developmental biology, to name but a few.
Early studies using human NSCs were limited by the short-term stability of genotype and phenotype in culture. However, immortalization of human NSCs with the myc transcription factor has proven highly effective at overcoming these challenges. ReNcell® VM and CX lines were immortalized using myc technology. Myc is believed to drive and sustain self-renewal and proliferation of the stem cell, thus keeping differentiation at bay until desired. An up-regulation of telomerase activity is observed in myc transduced ReNcell®, which lends itself to a stable genotype in culture. Traditionally thought of as a proto-oncogene, it now appears that myc may be a “stemness” gene. An exciting discovery was made when Takahashi and Yamanaka demonstrated that a fibroblast cell could be transformed into a stem cell by using only four genes: c-Myc, Oct4, Klf4, and Sox2. This finding has since been corroborated by independent research groups.
We recommend coating tissue culture plastic or glassware that are used to culture ReNcell® VM cells with laminin. Tissue culture flasks should be coated on the same day that the ReNcell® VM cells are thawed from liquid nitrogen or on the same day that the cells need to be passage. The following procedure is recommended:
Figure 1.ReNcell® VM Human Neural Progenitors
Figure 2. NSC Marker Expression.Both ReNcell® VM and CX human neural progenitors are grown as monolayers (A) and express NSC markers, Nestin (B, Red) and Sox-2 (B, Green). ReNcell® CX cells are able to differentiate into neurons expressing βIII-tubulin (C; Red) and glial cells expressing GFAP (D; Red). Dapi nuclear counterstain in Blue.
Figure 3. Electrophysiological properties of ReNcell® VM. A, B) Example of firing elicited by stepping the current command in 20 pA steps (400 ms duration) from a resting potential of ~-75 mV in ReNcell® VM 10 days after differentiation was started in control solution (A) and after adding 0.6 μM TTX (B). C, D) Same cell as in (A, B) responding with inward currents to incremental voltage steps (10 mV, from -60 mV to + 30 mV, 400 ms duration), before (C) and after (D) adding 0.6 μM TTX to the control solution. E) Average inward current-voltage relationships in control differentiated (solid squares) and after adding 0.6 μM TTX (open squares) and F) average steady-state current-voltage relationship for control (solid squares) and TTX treated (open squares).
Figure 4. Electrophysiological properties of ReNcell® CX. A, B) Example of ReNcell® CX 12 days after differentiation was started using the pre-Differentiated protocol, responding to current (A; from ~-80 mV) or voltage (B) steps (10 pA or mV increments, 400 ms). C) Average voltage-current relationship for the peak current elicited by stepping the voltage from -60 to + 30 mV in 10 mV increments for the ReNcell® CX with the preD (solid circles, n = 9) or the stdD (open circles, n = 7) protocols. D) Average IA peak current for the ReNcell® CX differentiated using preD (filled circles, n = 9) or the stdD (open circles, n = 7) protocols. Despite the apparent trend there is no significant difference between these groups due to the large scatter in the data.