ES Cell Differentiation and Co-Culture Plating Protocol

ES Cell Differentiation:

1.         The ES cells were suspended in 5ml of DFK10 medium within a 60mm Petri dish for two days prior to treatment with sonic hedge hog (Shh) and retinoic acid (RA).

2.         In order to induce differentiation of the ES cells into motor neurons (MNs) the ES cells, which have formed embryoid bodies (EBs), were treated with 5μl of Shh and 5μl of RA for a final concentration of 1μM for both Shh and RA.

3.         Following a single treatment of Shh and RA the EBs were left for 5 days in order differentiate.  At the end of the 5 day period the cells expressing green fluorescent protein (GFP) were ready for implantation onto or into host tissue.  At this 5 day time point the upregulation of HB9, a protein that serves as a MN marker, was maximally expressed as well as other MN markers.

4.         At day 5 the EBs were plated onto cultured C2C12 cells (see protocol below.)  For plating the 12mm poly-D-lysine/lamanin coverslip with cultured C2C12 cells was transferred to a 35mm Petri dish in 3mm of DFK10 medium.  Then, 2 to 3 EBs were pipetted atop of the C2C12 tissue and were left to adhere to the tissue for an hour before being transported back to the incubator.

5.         Axon outgrowth was evident at 24 hours, maximally at 48 hours and tapers off at 72 hours. 

C2C12 Myoblast Differentiation:

1.         C2C12 cells (a cell line of chick myoblast cells - undifferentiated precursor to mouse muscle) were quickly thawed following storage in liquid nitrogen.

2.         The C2C12 cells were rinsed through a process of centrifugation, supernatant replacement and re-suspension of the cell pellet with fresh medium.  This rinsing process took place twice.  The centrifuge was run at 1000RPM for 3 minutes each time. 

3.         The C2C12 cells were plated into a 24 well plate containing 12mm round coverslips coated with poly-D-lysine/lamanin in order to provide a suitable substrate for adherence of the muscle tissue.  The C2C12 cells were plated within 500μl of C2C12 cell medium supplemented with 10% horse serum in order to facilitate differentiation of the myoblasts into muscle tissue.

4.         The cells were fed only after the first 3 days of plating and then once every two days thereafter.  The cells were allowed 10 days before the population has been sufficiently differentiated into muscle.  At this stage the muscle cells were ready to be plated upon.