In the present study, we generated several immortalized hSC lines that retain characteristics of primary SCs, including expression of glial cell markers and components of the ECM, and ability to myelinate rodent sensory neurons.
The ability of SCs to promote peripheral nerve regeneration has led to intensive research about their potential use for cell transplantation to support regeneration in the PNS and central nervous system (CNS). Indeed, SCs have been shown to promote axonal regeneration and remyelinate regrowing axons in various experimental paradigms of CNS and PNS injury [3
]. These studies support the idea that in humans, after nerve injury, autologous SCs could be obtained from healthy nerves, expanded ex vivo, and retransplanted at the sites of injury [28
However, SC auto-transplantation paradigm in humans has not been systematically pursued over the years and is still far from being introduced in clinical practice. One of the reasons is that function of SCs has been mostly studied in rodent models, which make it difficult to extrapolate the results to humans. In addition, SCs obtained from human donors are difficult to maintain in cell culture; low yields and poor proliferation rate result in overgrowth by fibroblasts [16
]. As a consequence, only intricate, time-consuming purification and expansion procedures allow establishing primary hSCs cultures in low numbers to study their functional characteristics [16
]. Procedures that have been proposed to increase purity and yield of harvested hSCs include magnetic-activated cell separation [32
], addition of growth and differentiation factors (neuregulin/forskolin) [13
], as well as modulation of signaling pathways such as extracellular signal-regulated kinase Erk1 and Erk2 [15
We sought to overcome drawbacks associated with culturing hSCs by generating a cell line that retains the properties of a primary hSC yet is easy to grow and propagate. By immortalizing fetal hSCs we were able to generate cell lines that meet these criteria. Since the essential function of SCs is their ability to myelinate axons, we further assessed the potential of our cell lines to myelinate rat sensory axons in vitro. Using similar in vitro models, it has been shown previously that embryonic and adult hSCs are able to myelinate rat axons [17
]. However, the amount of myelin is generally much less abundant compared with co-cultures with rSCs. In those studies, adult or embryonic hSCs were co-cultured with rat or human sensory neurons, which led to a sporadic myelination of solitary neurons. It has been suggested that hSCs may have negative effect on the health of neurons in this cell culture system, based on observations that the addition of human SC to neurons, regardless of human or rat, led to shrinkage of neuronal cell bodies and to a progressive loss of neurites [17
Consistent with those observations, our immortalized hSCs were able to myelinate rat axons; however, the amount of myelination was 100-fold less in comparison to cell culture-containing neonatal rSCs. Given the fact that 10,000 cells were initially added to the culture dish, <0.1% of SCs change to a myelinating phenotype. This, nevertheless, showed that our immortalization procedure did not inhibit the ability of cells to differentiate. One could argue that the low myelination potential is because the majority of SCs are unable to exit the cell cycle and continue to proliferate. However, since we did not observe any overgrowth of SCs in our co-culture system, we conclude that the immortalized SCs stop dividing when cultured in differentiation media and start to differentiate into nonmyelinating (majority) and myelinating (minority) phenotypes. Further, when assessed for proliferation capacity, hSCs had a marked decrease in BrdU incorporation when cultured with rat DRG neurons in myelinating media (Supplementary Fig. 1
). Further experiments would be needed to determine if changes in cell culture conditions could enhance the myelination by immortalized hSCs. However, the similarity between our results and from those studies with primary SCs favor the hypothesis that the intrinsic capacity of hSCs in this cell culture model is generally lower than those of the rSCs. In addition, when we compared the morphology of neurons cultured with either immortalized hSCs or rSCs, it turned out that the neurite density in cultures with hSCs was strikingly reduced and the average neuronal cell body diameter was significantly lower than those cultures with rSCs. These results confirm the published observations that human and rSCs have very divergent effects on rodent sensory neuronal health in vitro and may provide an explanation for the poor capability of immortalized hSCs to myelinate neurons in vitro.
Apart from studying myelination in vitro, immortalized cell lines may represent a useful tool for high-throughput drug screening to identify compounds that maybe therapeutically useful in peripheral neuropathies in which SC dysfunction plays a role in pathogenesis. Compared with rSC lines used for this purpose, immortalized hSC lines we developed likely offer an advantage as they may represent characteristics of the human primary SCs better. Further, unlike primary cells isolated from different sources, homogeneity of the immortalized hSCs is a major advantage to reduce well-to-well variability in high-throughput drug screening.
Since primary SCs are susceptible to apoptosis caused by H2
and, on the other hand, hTERT expression vectors could theoretically affect the response of these cells exposed to oxidative stress, we explored the utility of immortalized hSCs as a tool in high-throughput assays and developed a simple toxicity assay. Using a luciferase-based assay, we measured cellular ATP levels, which strongly correlate with the number of healthy cells [12
]. The advantage of the luciferase-based assay is that it is suitable for scaling up for high-throughput robotic assays because it does not involve any washing step, which often induces variability in such assays. As a proof-of-concept assay we chose to assess toxicity of H2
. Although short-term application of H2
may not fully replicate the slow, progressive oxidative stress that is seen in vivo, it has been used to model oxidative stress in a variety of degenerative and inflammatory diseases of the nervous system [34
]. Future studies can be directed at in vitro models that mimic pathogenic mechanisms in which SC pathology plays a role; for example, high glucose exposure can be used to mimic diabetes or reporter assays can be developed to enhance pmp22
gene expression in hereditary neuropathy with liability to pressure palsies.
The gene expression dataset generated from the immortalized hSC line will serve as a useful baseline for comparative studies for other types of immortalized cell lines and cells generated from stem cell populations [38
]. Highly expressed genes are listed in Supplementary Tables 1 and 2
, and the entire set of expressed genes is available upon request. Comparison of the gene expression profile of the hSC to human oligodendrocyte precursors or astrocytes reveals that although there are many similarities, key differences remain. Expression profile of genes unique to SCs can be used to validate cells generated from stem cells before widespread use in mechanistic or drug discovery studies.
In summary, we have developed several immortalized hSC lines, which show essential characteristics of primary hSCs, including their ability to myelinate. Our study supports the notion that immortalization techniques can be useful to generate human cell lines from cells that are considered difficult to immortalize such as SCs. In contrast to primary cell cultures, these cells tolerate easy cell culture conditions, which allow establishment of simple, time and cost efficient in vitro assays to screen large numbers of compounds for potential therapeutic use.