PLAYABLE:
The Heroes:
Leonardo
Donatello
Michelangelo
Raphael
Splinter
Casey
April
Miyamoto Usagi (in a guest role from Stan Sakai's Usagi Yojimbo)
Utrom in a combat-oriented human robot
Master Yoshi (made for the origin chapters then used in original mode)
The C.O.W. Boys of Moo Mesa (guest appearance from the 2 crossover comic stories in the mid 2000's)
Traximus

The Villains:
Oroku Saki/Shredder
Baxter Stockman
Oroku Nagi (origin chapters, original mode)
Lord Hebi (Usagi Yojimbo)
Chief Terrorbull (C.O.W. Boys of Moo Mesa)
Emperor Zanramon
Lu Bu (guest appearance from Dynasty Warriors because one of the official TMNT characters is a guardian of time)
Tadakatsu Honda (See Lu Bu but Samurai Warriors instead)

Other:
Karai (she has been both enemy and ally, once donning the Shredder's armor)

NPC:
Allies:
The Utroms
The Foot Clan (at times, under Karai)

Enemies:
The Foot Clan (duh)
The Purple Dragons
The mob
Triceratons

The main plot would ideally be based mostly off the Mirage comics, following volumes 1 and 2, skipping the non-canon volume 3, and time skipping to volume 4 up to the point where Nickelodeon bought Mirage Studios and they end.

Maybe some guest appearance characters from the 2003 show and the first 2 movies would be ok.too.