Location and Clonal Analysis of Stem Cells and Their Differentiated Progeny in the Human Ocular Surface

Abstract

We have analyzed the proliferative and differentiation potential of human ocular keratinocytes. Holoclones, meroclones, and paraclones, previously identified in skin, constitute also the proliferative compartment of the ocular epithelium. Ocular holoclones have the expected properties of stem cells, while transient amplifying cells have variable proliferative potential. Corneal stem cells are segregated in the limbus, while conjunctival stem cells are uniformly distributed in bulbar and forniceal conjunctiva. Conjunctival keratinocytes and goblet cells derive from a common bipotent progenitor. Goblet cells were found in cultures of transient amplifying cells, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45–50 cell doublings and at ∼15 cell doublings before senescence. Thus, the decision of conjunctival keratinocytes to differentiate into goblet cells appears to be dependent upon an intrinsic “cell doubling clock.” These data open new perspectives in the surgical treatment of severe defects of the anterior ocular surface with autologous cultured conjunctival epithelium.

Stem cells of the adult cornea: From cytometric markers to therapeutic applications

“…The peripherial cells formed meroclone colonies whose growth was stopped after a relatively small number of divisions (16)(17)(18). These cells are also referred to as transient amplifying cells (TACs).…”

“…Stem cells and their progeny exist millimeters apart in the human cornea (17). Limbal stem cells reside in the basal layer of the limbal epithelium and are interspersed with early transient amplifying cells (TACs).…”

Niche regulation of corneal epithelial stem cells at the limbus

“…For example, limbal SCs have the smallest cell size [5], are slow-cycling and hence label-retaining [6], and do not express markers destined for terminal differentiation such as cytokeratins 3 [1] and 12 [7][8][9], involucrin [10], and connexin 43 [11]. In contrast, the SC-containing limbal epithelium has a high proliferative potential in different cultures [12][13][14][15], and their in vitro proliferation is resistant to the inhibition by tumor-promoting phorbol esters [13,16,17]. Furthermore, limbal basal epithelial cells express cytokeratin 19 [18], and integrin a9 [10,19], and preferentially express such progenitor markers as p63 [20], especially its ∆Np63a isoform [21,22], Bcrp1/ABCG2 [10,[23][24][25], and N-cadherin [26].…”

Isoforms of ΔNp63 and the migration of ocular limbal cells in human corneal regeneration

“…TA cells that migrate from the limbus form the corneal epithelium (8). That the limbus is the site of stem cell precursors of the corneal epithelium is clear for several reasons: (i) the basal layer of the limbus lacks keratin 3 (a marker for corneal differentiation), whereas limbal suprabasal layers and all layers of the corneal epithelium express keratin 3 (9); (ii) the limbus contains slow-cycling cells and holoclone-forming cells, but the corneal epithelium does not (7,10); (iii) the corneal epithelial cells are not self-sustaining; they divide only a few times during their migration from the limbus to the central cornea (11); (iv) restoration of destroyed limbal͞corneal epithelium requires limbal transplantation (12) or grafts of autologous limbal cultures (13)(14)(15).…”