Response to Referees
Manuscript
EJN-2007-03-12109
Title:
“Isolation, purification and expansion of myelination-competent, neonatal, mouse Schwann
Cells”,
Authors:
H. Honkanen, O. Lahti, M. Nissinen, R. Myllylä, S. Päiväläinen, M. H. Alanne, S. Kangas, S.
Peltonen, J. Peltonen and A. M. Heape.
Response to Referee 1
1. The most interesting information for the scientific community would be if this protocol could
achieve an as high efficient in vitro myelination capacity as rat dissociated cultures. … From
this work it does not become evident that these isolated Schwann cells are myelinating as
efficient in vitro as rat Schwann cells do.
The referee is right; our choice of images showed only the degree of maturation of the sheaths, not the
myelinating efficiency of the cocultures.
We have therefore added two immuno-cytochemistry images of two separate cultures (Figure 6, panels
E & F), which clearly illustrate the typically high yields of myelination achieved in the mouse pre-purified
DRG/Schwann cell model that we have developed as a complement of the present mouse Schwann cell
isolation method. These panels complement the phase-contrast image (of yet another coculture) already
presented in panel D, in which, for obvious reasons, only more mature sheaths can be seen. The
relevant paragraph in the Results section and Figure Legends have been modified slightly to take this
addition into account. As there was no change in the protocols employed, the Materials and Methods
section has been modified only to reflect the fact that the anti-MBP antibody was used alone (in Figure
6E).
In terms of numbers, we obtain 100-250 myelin sheaths/well in mouse 3-D cocultures performed in 96well culture plates, within 3 weeks under myelination-permissive conditions. This is now stated in the
Results section, and a short sentence has been added to the Materials and Methods section describing
how the sheaths were counted.
According to our experience of several years employing the rat model, these yields are at least as high
as those obtained in the rat DRG/Schwann cell coculture model. A sentence to this effect has been
added to the last paragraph of the discussion (Lines 3-4). This topic is discussed in detail in our upcoming paper (Päiväläinen et al. in preparation) describing the development of the mouse coculture
model.
2. There is a high need for a protocol with high in vitro myelination efficiency for mouse
Schwann cells, as it is possible with rat Schwann cells. The authors show briefly that these
purified Schwann cells are capable to myelinate drg cultures in vitro, but the degree of
myelination does not seem to be better than the usually used dissociated explant cultures
from E13 mouse drg's; a protocol which is much simpler than the one presented in this
manuscript.
As the referee correctly points out, the explant culture model is relatively simple compared to the
DRG/Schwann cell culture model, for which the purified Schwann cells are required. It allows good levels
of myelination and, more importantly, is applicable to mouse cells and has proved to be a valuable tool
for myelin biologists. However, the explant model cannot be used to perform mixed genotype cocultures,
in which, for example, the neurons purified from mice with one genotype (normal or mutant “x”) could be
cocultured with Schwann cells purified from mice with another genotype (mutant “y”, or normal), and vice
versa. The pre-purified DRG/Schwann cell coculture model, on the other hand, should be ideal for such
studies. It would not only allow to analyse the cell type-specificity of the expression of a given mutation
(spontaneous or bio-engineered), but would also allow to test the direct molecular and cellular effects of
different combinations of mutations on the axo-glial relationship (in a simplified biological context). No
mutant cross-breeding is necessary; it would be sufficient to simply “mix” the desired combination of cell
types, confident in the knowledge that all the cells of the same lineage (neurons or Schwann cells) carry
the same genotype. For this reason alone, the DRG/Schwann cell coculture has the potential of
becoming an extraordinarily powerful tool in studies of PNS axo-glial biology and inheritable pathologies.
However, for this potential to be fully exploited, it is necessary to adapt the model for use with mouse
cells, because rat mutants with compromised axo-glial function are very rare, while the diversity of
available mouse mutants is already high, and is increasing continually. This adaptation required solving
two problems: 1) obtaining sufficient amounts of myelination-competent mouse Schwann cells and, 2)
getting the letter to make myelin in culture. The current paper addresses, and solves, the first problem,
and illustrates briefly that the cells are indeed able to myelinate (efficiently) in culture. Our up-coming
paper (Päiväläinen et al. in preparation) addresses the second problem in more detail, and solves it. A
copy of the whole manuscript is attached.
It is this (rather lengthy) message that we wished to convey briefly in the Introduction and in the last
paragraph of the Discussion of our manuscript. As the latter is focused on solving the problem of the
Schwann cell isolation and growth, which can in fact be exploited for reasons other than myelination
studies, we feel that this paper is not the place to develop it much further (this is done in the up-coming
article dealing specifically with the mouse in vitro myelination model).
Response to Referee 2
1. I would appreciate a different presentation of the increased Schwann cell numbers in the
neuron-glia cultures. It is very difficult to judge from the phase contrast picture that SC
numbers are indeed increased. A stained culture might be more informative.
As suggested by the referee, we have repeated the coculture experiment using both phase contrast
microscopy and Hoechst nuclear staining. The phase-contrast images originally presented in Panels E &
F are now replaced with similar images of new cultures, performed under conditions identical to those
employed previously, and which we then fixed and stained with Hoechst nuclear stain (new panels G &
H). This clearly shows the increased cell number in the presence of the DRGs. We have enhanced the
contrast of the remaining phase-contrast images (panels A-D) of Figure 5 in order to facilitate the
viewing.