Original concept art for a character who would eventually be remodeled to Isaac.
Isaac is a reference to the biblical story of the binding of Isaac, in which Abraham is commanded by God to sacrifice his only son, Isaac, to prove his loyalty and love.
"Then God said, 'Take your son, your only son, whom you love — Isaac — and go to the region of Moriah. Sacrifice him there as a burnt offering on a mountain I will show you.'" (Genesis 22:2)
In a response to TBoI Youtuber Sinvicta of whether there was any concept art of what Isaac's original design was, Edmund McMillen confirmed that Isaac's design was originally one of an Octorock while showing very similar features of how the gameplay would be modeled by posting an old concept art.
Edmund has stated that Isaac is left-handed, but in the opening cutscene, Isaac is holding a pencil with his right hand.
This could be a reference to the Catholic bias against left-handedness, stemming from Matthew 25:33 proclaiming that God will put those deserving to ascend to Heaven on his right-hand side and put those deserving to descend to Hell on his left-hand side. Isaac's mother could have forced him to learn things the "righty" way for fear of him being "evil."
Isaac is circumcised. This was confirmed by Edmund McMillen during a YouTube stream.
Isaac's voiceover in Rebirth was done by Tallulah Bossi, Matthias Bossi (Member of Ridiculon and voice of Dad/Narrator) and Carla Kihlstedt's (Musician and voice of Mom) daughter. For Repentance, the voice was done by their son, Viggo Bossi.