Isolation, neurotrophic potential and transfer-suitability of olfactory "Ensheathing" cells as a prerequisite for neuronal regeneration after spinal cord traumata

Status:

completed 06/2019

Aims:

A promising strategy to treat spinal cord injuries could be an autologous cell therapy. Due to the lifelong neurogenesis, the olfactory mucosa (OM) might be an attractive cell source. The neuroregenerative properties of olfactory multipotent stem cells (OSC) and olfactory ensheathing cells (OEC) have been successfully demonstrated in animal models. Recent studies have shown that cells with neuroregenerative properties are also found in the respiratory mucosa (RM), the so-called inferior turbinate stem cells (ITSC). Up to now, there are no precise and standard methods for cell isolation and characterization, which is a prerequisite for the therapeutical use and essential for quality and safety. The aim of this study was it to isolate OEC and to characterize these cells after subsequent purification as well as to determine their functionality. In addition, the behavior of the enriched cells was investigated in a plasma-clot-matrix of coagulated blood plasma, which can serve as a carrier-matrix for the cells in case of potential transplantation.

Activities/Methods:

Using enzymatic and mechanical methods, cells from both the olfactory epithelial cell layer and the lamina propria were successfully isolated, cultured and expanded. Furthermore, it was possible to isolate cells from the LP of the respiratory mucosa the so called ITSC, whereby these cells had a stronger and faster proliferation than the olfactory cells, which could be influenced by different stimuli. Cellular and functional differences between respiratory and olfactory mucosa could be observed clearly. For example, no vital, adherent cells could be isolated from the ET of RM. Only in the LP of the OM p75 (OEC marker) positive cells could be detected in young passages. However, both olfactory and respiratory cells from the lamina propria showed CD90 (stem cell marker) positive cells, whereby the percentage increased with increasing number of passages.

The purity and possible contamination with mucus-producing cells was examined by analysis of the expression of the mucin MUC5AC, as well as CD90, nestin, p75NTR and EpCAM. Mucin-positive cells could be detected immediately after isolation in both cell-fractions of the epithelial cell layer and the lamina propria of OM. However, during cultivation and passage of the cells these were lost. By performing the neurosphere assay, it was possible to purify olfactory stem cells from the epithelial cell layer of OM. These cells were positive for nestin and CD90 and could not differentiate into cells of the mesenchymal direction compared to bone-marrow stem cells (osteogenic and adipogenic differentiation). However, these purified stem cells displayed a neuronal-like morphology in neuronal differentiation medium and were positive for the neuronal marker ß-III Tubulin. The freshly isolated cell fractions from the lamina propria showed both p75NTR-positive olfactory ensheathing cells and CD90-positive mesenchymal stem cells. However, the share of p75NTR-positive cells decreased during further passages.

Results:

Via immunomagnetic purification, an enrichment of p75NTR positive/-CD90 negative cells could be achieved. The functional characterization of the purified cell fractions revealed that some of these cells were capable of phagocytosis, which is a typical characteristic of olfactory ensheathing cells. Analysis of cell culture supernatants from the olfactory ensheathing cell fractions using a human growth factor antibody array revealed no increased release of neurotrophic factors such as NGF, BDNF or NT-4/5 compared to pure cell culture medium. However, increased expression of insulin-like growth factor binding proteins was found in those cell culture supernatants. These conditioned cell culture supernatants caused a change in the morphology of neuronal embryonic stem cells, which had a neuron-like morphology and elongated cell processes. In a possible cell therapy, the autologous plasma clot represents a suitable cell carrier matrix. The analysis of the behavior of the isolated and purified cells in such a matrix showed that they were vital, proliferate and arranged three-dimensional. Physiologically, such a matrix would dissolve after a certain time, whereby the degradation of the plasma clot matrix could be observed in vitro after seven days.

In summary, olfactory ensheathing cells as well as olfactory stem cells and ITSC were successfully isolated, purified and characterized. However, it was found that a pure OEC cell fraction with a high cell yield could not be obtained. However, mucus-producing cells and epithelial cells were lost by cultivating the cells. The isolation and characterization of the OSC was very successful.

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