ReNcell CX Research Articles

Dual Effects of Isoflurane on Neuronal Proliferation/Differentiation

Dual Effects of Isoflurane on Neuronal Proliferation/Differentiation

Evaluation of Biochemical Changes in Skin-Derived Mesenchymal Stem Cells duringIn VitroNeurodifferentiation by FT-IR Analysis

Recently FT-IR analysis has been employed to study changes in molecular signatures during embryonic stem cell differentiation. We were interested to find out whether FT-IR spectroscopy could be applied to analyze changes in human skin mesenchymal stem cell (S-MSC) biochemical profile duringin vitroneurodifferentiation. S-MSCs were propagated in serum-free medium with EGF and FGF-2 during six weeks. Neural progenitor cell line ReNcell CX (Millipore) was used as a reference cell line. Samples were collected each week and analyzed for neural marker nestin, tubulinβIII, GFAP, and CD271 expression. FT-IR analysis was carried out using microplate reader HTS-XT (Bruker, Germany). Despite the immunophenotype similarity, FT-IR spectroscopy revealed distinct profiles for S-MSC culture and ReNcell CX cells. FT-IR spectra analyses showed changes of protein and lipid concentration during neurodifferentiation and different carbohydrate composition in ReNcell CX and S-MSCs. It was possible to discriminate between S-MSC cultures at different time points during neurodifferentiation. The results of this study demonstrate that FT-IR spectroscopy is more sensitive than conventional immunophenotyping analysis and it has a great potential for the monitoring of the stem cell differentiation status.

Prosaposin in the secretome of marrow stroma‐derived neural progenitor cells protects neural cells from apoptotic death

Functionally, adult stem cells not only participate in replication and differentiation to various cell lineages, but also may be involved in rescuing cells from apoptosis. Identifying functional factors secreted by stem cells, as well as their target cells, may advance our understanding of stem cells' multifaceted physiologic functions. Here, we report that mouse bone marrow stromal cell-derived neuroprogenitor cells (mMSC-NPC) provide a protective function by secreting a key factor, prosaposin (PSAP), capable of rescuing mature neurons from apoptotic death. This factor is identified as the lead protein in the secretome of mMSC-NPC cultures by tandem mass spectroscopic profiling, and further validated by western blotting and immunocytochemistry. The secretome of MSC-NPC reduces toxin-induced cell death in cultures of rat pheochromocytoma neuronal cells, human ReNcell CX neurons, and rat cortical primary neurons; removal of PSAP by immunodepletion annuls this protective effect. This neuronal protection against toxin treatment was validated further by the recombinant PSAP peptide. Interestingly, the secretome of neuronal culture does not possess such a self-protective action. We suggest that upon injury, a subgroup of MSCs differentiates into neural/neuronal progenitor cells, and remains in this intermediate stem cell-like stage, defending injured neighboring mature neurons from apoptosis by secreting PSAP.

Development of a High-Throughput Screening Assay for Chemical Effects on Proliferation and Viability of Immortalized Human Neural Progenitor Cells

There is considerable public concern that the majority of commercial chemicals have not been evaluated for their potential to cause developmental neurotoxicity. Although several chemicals are assessed annually under the current developmental neurotoxicity guidelines, time, resource, and animal constraints prevent testing of large numbers of chemicals using this approach. Thus, incentive is mounting to develop in vitro methods to screen chemicals for their potential to harm the developing human nervous system. As an initial step toward this end, the present studies evaluated an automated, high-throughput method for screening chemical effects on proliferation and viability using ReNcell CX cells, a human neural progenitor cell (hNPC) line. ReNcell CX cells doubled in approximately 36 h and expressed the neural progenitor markers nestin and SOX2. High-throughput assays for cell proliferation (5-bromo-2'-deoxyuridine incorporation) and viability (propidium iodide exclusion) were optimized and tested using known antiproliferative compounds. The utility of this in vitro screen was evaluated further using a set of compounds containing eight known to cause developmental neurotoxicity and eight presumably nontoxic compounds. Six out of eight developmental neurotoxicants significantly inhibited ReNcell CX cell proliferation and/or viability, whereas two out of eight nontoxic chemicals caused only minimal effects. These results demonstrate that chemical effects on cell proliferation and viability can be assessed via high-throughput methods using hNPCs. Further development of this approach as part of a strategy to screen compounds for potential effects on nervous system development is warranted.

Differential development of neuronal physiological responsiveness in two human neural stem cell lines

BackgroundNeural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to neurodegenerative disease. Overexpression of the myc family transcription factors in human primary cells from developing cortex and mesencephalon has produced two stable multipotential NSC lines (ReNcell VM and CX) that can be continuously expanded in monolayer culture.ResultsIn the undifferentiated state, both ReNcell VM and CX are nestin positive and have resting membrane potentials of around -60 mV but do not display any voltage-activated conductances. As initially hypothesized, using standard methods (stdD) for differentiation, both cell lines can form neurons, astrocytes and oligodendrocytes according to immunohistological characteristics. However it became clear that this was not true for electrophysiological features which designate neurons, such as the firing of action potentials. We have thus developed a new differentiation protocol, designated 'pre-aggregation differentiation' (preD) which appears to favor development of electrophysiologically functional neurons and to lead to an increase in dopaminergic neurons in the ReNcell VM line. In contrast, the protocol used had little effect on the differentiation of ReNcell CX in which dopaminergic differentiation was not observed. Moreover, after a week of differentiation with the preD protocol, 100% of ReNcell VM featured TTX-sensitive Na+-channels and fired action potentials, compared to 25% after stdD. Currents via other voltage-gated channels did not appear to depend on the differentiation protocol. ReNcell CX did not display the same electrophysiological properties as the VM line, generating voltage-dependant K+ currents but no Na+ currents or action potentials under either stdD or preD differentiation.ConclusionThese data demonstrate that overexpression of myc in NSCs can be used to generate electrophysiologically active neurons in culture. Development of a functional neuronal phenotype may be dependent on parameters of isolation and differentiation of the cell lines, indicating that not all human NSCs are functionally equivalent.