The implications of certain items, alongside their interactions with characters in the game, can create these.
The Egg Sack item. When collected, the pop-up in the corner of the screen says 'Daddy's Love'. What does it do? It means that Daddy Long Legs' shadow follows you and his feet will stomp on nearby enemies. Remembering that Isaac's own mother is out to get him, the idea of his father trying to protect him warms this troper's heart a little.
Mitigated a bit, though, by Word of God, who stated that Daddy Long Legs is not Isaac's father. Still nice that a boss would help you though.
This becomes somewhat humorous when you fight the actual boss himself with the item collected.
A bittersweet moment, given what happens during the main game, but in the 13th Ending and The Polaroid, we see Isaac's Mother cheerful and accepting of him.
In version 1.4 of Wrath of the Lamb, we finally have a not-chance-based way to obtain the Polaroid. Mom is keeping it.
God, or at least an Angel can appear and offer Isaac an item himself.
The Brother Bobby and Sister Maggy (likely Isaac's dead siblings) items as well as the Max's Head and Guppy's paw (Isaac's dead pets) items. It may not be much, but the thought of Isaac's loved ones still being able to help him even in death goes a long way to show that he isn't truly alone.
Meta example: there has been a new expansion for the original game, and it's free for the possessors of Wrath of the Lamb. Nice gift for those who stuck with the original edition.
On the birthday of Satoru Iwata, the daily run of Afterbirth has Isaac changed into a skin of Iwata.
at least one picture of promotional art has Isaac Bawling uncontrollably and wetting himself in the presence of several of the game's first bosses (Gemini, Monstro, Duke of Flies, Pin, a Gurgling, Fistula, Peep and Larry Jr.). Thing is, they aren't being menacing at all and actually look a little worried and concerned. even if this never happens in-game (unless the usable item's like Monstro's Tooth count as a real Heel–Face Turn scenerios) its kind of touching to see these monsters not torment Isaac for once.