Neuronale Differenzierung caniner mesenchymaler Stammzellen aus dem Fettgewebe über den Zwischenschritt der Bildung dreidimensionaler Sphären
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Beschreibung
The application of regenerative therapies and especially cMSCs (canine Mesenchymal Stem Cells) holds great promise as treatment option of neurological diseases in canines. Nevertheless, the exact contribution of cMSCs concerning the recovery process still remains nearly unclear. In the present work it was investigated whether cMSCs possess neurological features following differentiation in this direction. On this basis, the generation of threedimensional spheres was induced in an intermediate stage prior to a final differentiation step. Formation of spheres should mimic the developement of neurospheres. The composition of the applied differentiation medium strongly corresponds to media which are used for cultivation of canine neuronal cells. This and the intermediate step of sphere-formation should create a roughly physiological milieu.
CMSCs were isolated from adipose tissue and subsequent to an effective characterisation the sphere-formation was initiated. During this neuronal pre-differentiation step cMSCS were cultured for three days in pre-diffententiation medium consisting of basic medium (DMEM/F-12), supplements (N2 and B-27) and growth factors (bFGF and EGF). On a trial basis the addition of heparin sodium was implemented. A crucial requirement for successful sphere-formation was the cultivation on an anti-adhesive surface in serum free medium. The spheres were investigated concerning their morphology and vitality to facilitate a comparision to neurospheres. The impact of different media compositions on sphere-formation was tested by metrical analyses like the number of cells per sphere and the amount and size of generated spheres.
Subsequently to the generation of spheres during pre-differentiation further neuronal differentiation was induced. To achieve this, the spheres were cultivated in differentiation medium on an adhesive surface for seven days. The medium contained the same basic medium and supplements as applied in the pre-differentiaion medium. Furthermore, 2.5 % fetal bovine serum (FBS) were added and growth factors were substituted by the neurotrophins NGF and BDNF. For attachement of spheres and migration of cells out of them the addition of FBS and cultivation on an adhesive surface were essential. After the final differentiation step the morphology of cells was assessed.
Undifferentiated cMSCs, spheres and cMSCs after four and seven days of further differentiation were analysed by RT-qPCR, immunofluorescence and western blot concerning their expression of neuronal markers.
In the following, the most relevant results are listed:
- The effective generation of spheres was only achieved if silicon pads were applied as anti-adhesive surface.
- An addition of FBS during pre-differentiation led to an attachement of spheres on the silicon pads.
- The addition of different supplements had no impact on the size or number of spheres.
- There was a positive correlation between the size of a sphere and its amount of cells.
- Proliferation of spheres took place especially during the first 48 hours of pre-differentiation.
- Spheres were vital, proliferating and contained only a very few dead cells.
- The spheres showed ultrastructural similarities to neurospheres.
- For adhesion of spheres and cell migration the addition of 2.5 % FBS and an adhesive surface couting with agarose were essential.
- After seven days of final neuronal differentiation the cells showed various morphology, which indicated that a beginning neuronal and glial differentiation took place.
- In the RT-qPCR analysed neuronal markers were also expressed by undifferentiated cells.
- There where remarks that expression of nestin was a feature of multipotent progenitor cells and suggested an enhanced cell proliferation. Thus, the role of nestin as a marker for neuronal progenitor cells is losing importance.
- The analysis of neuronal markers showed indications for a beginning neuronal differentiatin of cMSCs. It is possible that during differentiation a suppression of cells which are differentiating towards the neuronal direction by glial differentiating cells took place. Nevertheless, the positive signal for MAP2 in a few cells indicated ongoing neuronal differentiation. Frequently, discrepancies between results of RT-qPCR and immunofluorescences could be determined. These disparities might be explained by reduction of gene expression when there is a high degree of the protein inside the cell.
Finally, it was shown that the neuronal differentiated cMSCs revealed suggestions for a beginning and proceeding neuronal differentiation. In the course of this interindiviual differences between the donors were conspicuous. On day seven during the final differentiation step a few MAP2-positive cells with processes could be observed, but the majority of cells showed a positive signal for A2B5. Thus, it is plausible that glial differenciating cells were suppressing the cells which were differentiating in the neuronal direction during proceeding neuronal differentiation. Furthermore, the ultrastructural morphology, vitality and positive signal for nestin and β-III tubulin of spheres confirmed their proliferative neurosphere-like character. However, it is notable that the exclusive investigation of neuronal marker expression takes no account of other facets of neuronal identity like neural activity or the release of specific neurotransmitters. It is still questionable whether a complete differentiation of cMSCs into specific types of neurons, which need a special microenvironment, is possible. Nevertheless, this would not be required for therapeutical application of cMSCs, because application of undifferentiated and pre-differentiatied cells might have some advantages. In the future the role of MSCs as immunomodulatory operating “medicinal signaling cell” which supports regeneration of damaged tissues probably will be paramount.
Incidentially, a new method for induction of sphere-formation on silicon-pads was developed. Silicon-Pads are an easy and very effective possibility to culture cMSCs in a threedimensional manner. In the future this method could be useful for other applications, whenever a 3D-culture of cells is desired.
Autor*in
Laura Beate Heilen