2. Culture of primary myogenic cells derived from adult muscle and electric organ of the gymnotiform S. macrurus Eric Archer

3. Introduction: Sternopygus macrurus –Weakly electric fish from South America –The electric organ is derived from skeletal muscle and is used for electro-sensing and communication –Possesses strong ability to regenerate

4. Regeneration: Regeneration is preceded by the formation of pool of mesenchymal stem cells known as a blastema The origin of blastema cells is a debated topic between groups studying regeneration A: Adult S. macrurus. B: Blastema growing at the end of the stump 1 week after tail amputation (arrow). C: Regeneration blastema is almost completely pigmented by 3 weeks after tail amputation. Tail will be whole in ~ 4 weeks

5. De-differentiation: This model suggests that fully differentiated adult cells can reverse the differentiation process, becoming more like stem cells osteogenesis

6. Adult stem cell recruitment: In the stem cell recruitment model, latent stem cells are activated in adult tissues Satellite cells are an example of a latent stem cell Satellite cells lie outside the cell membrane but contained within the basal lamina of a muscle fiber (A) and an electrocyte (B).

7. What do satellite cells do? Satellite cells are inactive in normal tissue Satellite cells fuse with a myofiber if it is injured as part of the cellular repair process. Satellite cells are the major mechanism of muscle regeneration in mammals

8. Why culture cells? Many experiments are difficult to perform in live animals, making an in vitro model a valuable tool for the study of regeneration Goal : Isolate and differentiate satellite cells from S. macrurus muscle and electric organ

9. Optimized culture protocol This protocol was modified from Fauconneau et. al. (2000) for the culture of satellite cells from rainbow trout. Skeletal muscle and EO were dissected from adult fish, and cut into 5mm 3 chunks before incubation overnight in growth medium (GM) (1) Tissue was mechanically dissociated by cutting with scissors to 1mm 3 pieces (2) 1212

10. Protocol (cont’d) Tissue pieces were treated with collagenase and trypsin for enzymatic dissociation, after which cells were triturated through a steel nozzle to further break up large chunks (3) Isolated cells were plated on collagen-coated wells and maintained in GM (4) 3434

11. Protocol (cont’d) After 1 day, cells were washed with GM to remove unattached cells from the well (5) Cells were passaged using 0.25% Trypsin/EDTA at approximately 75% confluence. Isolated cells at 160X magnification on Day 1 5

12. Characterization of isolated cells To determine if cells proliferate in our conditions, we used BrdU to label mitotically active cells BrdU is a thymidine analog and is incorporated into DNA during synthesis BrdU can be labeled with flourescent antibodies to show proliferating cells GM + BrdU Immunolabeling BrdU 12 Hours

13. Results of BrdU stain 40% of cells divided in 12 hours. Our growth conditions promote replication in cells isolated from skeletal muscle and electric organ BrdU Hoechst 33342 BrdU with Hoechst 33342

14. Further characterization Multiple cell types may be present in culture Can the isolated cells form differentiated muscle? Myogenic cell differentiation was induced by switching cells at 100% confluence in GM to differentiation media (DM) for 5 days Myogenic precursorsMyotubes 5 days GMDM

15. Results of MHC stain DNA (blue) stained by Hoechst 33342 Myosin heavy chain (green), a late muscle marker labeled by antibody staining

16. Staining with other muscle markers Phase contrast images show an elongated phenotype in cells labeled with muscle markers Many cells are also multinucleated (3 or more nuclei), a defining feature of myotubes in culture Titin Desmin MHC Marker with Hoechst 33342 Phase contrast images

17. Conclusions –We have established a protocol for the primary culture of muscle precursor cells from skeletal muscle and EO of S. macrurus. –Isolated cells differentiate to form multinucleated myotubes. –This is the first report of successful isolation of myogenic cells from muscle tissues of any electric fish.

18. Future directions –Further characterize isolated cells using specific antibody markers to determine the composition of primary cells. –Establish a cell line from S. macrurus myogenic precursor cells –Use this in vitro model system as a tool to investigate the cellular and molecular mechanisms of regeneration in S. macrurus.

19. With special thanks to: My partners in crime, Hyun-Jung Kim and Norma Escobedo Dr. Unguez and the rest of the Unguez lab Lori Weber from the Schuster Lab Dr. Johnson, Carol Turner, and Gail Freel with the NMSU MARC Program –This research was supported by NIH grants U56- CA96286, S06-GMO8136, RR16480, GM07667, and GM61222, as well as HHMI grant 52005881.