Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

Table 1 Evidence discriminating between alternative hypotheses

Observations	Species	Ref.	Consistent with hypothesis?
			LESC	CESC	GBL
Holoclone-producing cells are present in limbus but not cornea	Human	[25]	++	±	±
Holoclone-producing cells are present in limbus and cornea	Pig	[1]	±	++	±
Production of clonogenic spheres from limbus and central cornea	Human	[29]	±	++	±
Limbal epithelial cells are superior to corneal epithelial cells for corneal repair	Human	[32,33]	++	+	±
The central corneal epithelium can maintain itself when isolated from the limbus Label-retaining cells are present in the limbus but not the corneal epithelium during normal homeostasis	Rabbit, mouse, human Mouse, rabbit	[1,39-42] [13,52-54,57]	+a ++	++ +	++ 0
After a 40-d chase 3H-TdR-labelled cells are present in both the limbus and the corneal epithelium during normal homeostasis	Rabbit	[2]	+b	+b	+b
Cells move centripetally from the periphery of the corneal epithelium during normal homeostasis	Human, mouse	[58,61-63]	++	±c	±c
Mosaic patterns change after birth and clones of labelled cells emerge from the limbus as radial stripes	Mouse	[27,72,77-79]	++	0	0
Distribution of rare stripes in corneas of KRT5-LacZ+/- transgenic mice	Mouse	[17]	++	±	±
Transplanted limbal tissue contributes to replacing experimentally debrided corneal epithelium	Rabbit, mouse	[1,76]	++	++	+
Transplanted limbal tissue does not contribute to the unwounded corneal epithelium	Mouse	[1]	0	++	++
More tumours arise from the limbal epithelium than corneal epithelium	Human	[80]	++d	+	+
Diverse cell types and blood supply makes limbus a likely stem cell niche	All species	[6]	++d	+	+
Distribution of markers associated with undifferentiated or stem cell phenotype	Human, mouse, rabbit	See Table 2	++	±	±
Lineage tracing studies show that limbal cells contribute to the unwounded corneal epithelium during normal homeostasis	Mouse	[64]	++	0	0

++: Expected for hypothesis; +: Consistent with hypothesis; ±: Not consistent with hypothesis unless specific assumptions are made or technical issues compromise the interpretation of the experiment; 0: Not consistent with hypothesis.

^aThis is compatible with the LESC hypothesis if self-maintenance of the central corneal epithelium is a back-up mechanism that is used when homeostasis is compromised and LESCs are unable to maintain the corneal epithelium.

^bThe chase period may not have been sufficient to identify label-retaining cells (see text).

^cThe CESC hypothesis, as originally stated[1], proposed that corneal epithelial cell movement was centrifugal but this assumption is not necessary. However, it requires ad hoc assumptions to account for centripetal movement across the full radius.

^dEvidence is circumstantial. LESC: Limbal epithelial stem cell.

Table 2 Examples of marker gene expression differences between the basal limbal and corneal epithelia during normal homeostasis identified by immunostaining

Positive markers			Negative markers
Marker	Species	Ref.	Marker	Species	Ref.
Integrin α9	Mouse, human	[3,105,106]	Keratin 3	Rabbit	[4]
∆Np631	Human	[3,107,108]	Keratin 3/Keratin 12	Human	[3,108]
∆Np63α	Human	[96]	NGF receptor (p75NTR)	Human	[3]
ABCG2	Human, rat, rabbit	[3,57,91-93,108]	Involucrin	Human	[3]
Vimentin	Human	[108]	Connexin 43	Human	[3,108]
Keratin 19	Human, mouse	[108,109]	E-cadherin	Human	[3]
Keratin 15	Human, mouse	[109]	Nestin	Human	[108]
N-cadherin	Human	[110]
Bmi1	Human	[111]
C/EBPδ	Human	[111]
ABCB5	Human, mouse	[54]

¹ΔNp63 is not expressed in the human basal corneal epithelium[107] but it is expressed in mouse and rat corneal epithelia[27,112,113]. Positive markers are expressed in basal limbal but not basal corneal epithelium. Negative markers are expressed in basal corneal but not basal limbal epithelium.