1Department of Biology, University of Dayton, 2Center for Tissue Regeneration and Engineering, University of Dayton
Bhavsar, R. B., Nakamura, K., Tsonis, P. A. A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt. J. Vis. Exp. (52), e2713, doi:10.3791/2713 (2011).
Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.
1. Iris Cell Culture
2. Aggregation of Iris Cells
3. Implantation of Aggregrated Cells
4. Embedding of Newt Eye
7. Representative Results:
This procedure of culturing newt iris cells has been utilized to study the regeneration potential of the dorsal and the ventral IPE cells. Moreover, it is also possible to study specific genes that contribute towards the lens regeneration mechanism in newt eye. When the cells have been cultured for 2 weeks (Figure 1) they can be transfected by genes to examine their function in lens regeneration. Of particular interest are genes that might induce the ventral iris. Since the ventral iris cells cannot transdifferentiate to lens (Figure 2) the inductive function of a candidate gene can be studied. In the past, using this technique we have shown that when six-3was over expressed in the presence of retinoic acid lens induction was observed from the ventral iris 6. In Figure 3we can see that the ventral iris aggregate gave rise to a fully grown and differentiated lens (arrowhead), not different than the host lens from the dorsal iris (arrow).
Figure 1. a) Dorsal iris pigmented epithelial cells cultured in vitro for a period of 2 weeks. b) Ventral iris pigmented epithelial cells cultured in vitro for a period of 2 weeks. Note that pigmentation persists to this stage in both dorsal and ventral iris cells.
Figure 2. Regeneration ability of cultured IPE cells. a) Lens induction from dorsal IPE cell aggregate (arrowhead). Host lens induction from dorsal iris (arrow), di: dorsal iris, vi: ventral iris, le: lens epithelium, lf: lens fibers. b) Absence of lens induction from ventral IPE cell aggregate (arrowhead). Host lens induction from dorsal iris (arrow).
Figure 3. Lens induction from ventral aggregate transfected with six-3 and treated with retinoic acid. Host lens induction from dorsal iris (arrow). Lens induction from ventral aggregate (arrowhead).
This protocol has established an in vitro system to study lens regeneration mechanisms in newts. Since the aggregates (either dorsal or ventral faithfully follow their in vivo behavior during regeneration this technique can alleviate the tremendous effort required for transgenesis in newts and can be used for gain of function as well as loss of function experiments 7,8,9 . Also, the aggregates or the irises as a whole can easily be treated with growth factors and examine their effects as described in this protocol. For example the role of BMP pathway has been studied using this technique 6.
No conflicts of interest declared.
This work was funded by an NIH grant Ey10540 to PAT.
|Ethyl 3-aminobenzoate methanesulfonic acid||Sigma-Aldrich||E-10521|
|Dispase||GIBCO, by Life Technologies||17105-041|
|Trypsin 1:250||GIBCO, by Life Technologies||27250018|
|L-15 ( Leibovitz)||Sigma-Aldrich||L-4386|
|Kanamycin Sulfate||GIBCO, by Life Technologies||100x, 15160|
|Fungizone (amphotericin B solution)||Sigma-Aldrich||A2942|
|24 well collagen coated plate||BD Biosciences||354408|