HC-HA/PTX3 from amniotic membrane reverts senescent limbal niche cells to Pax6+ neural crest progenitors to support limbal epithelial progenitors.

Quiescence and self-renewal of human corneal epithelial progenitor/stem cells (LEPC) are regulated by the limbal niche, presumably through close interaction with limbal (stromal) niche cells (LNC). Paired box homeotic gene 6 (Pax6), a conserved transcription factor essential for eye development, is essential for proper differentiation of limbal and corneal epithelial stem cells. Pax6 haploinsufficiency causes limbal stem cell deficiency, which leads to subsequent corneal blindness. We previously reported that serial passage of nuclear Pax6+ LNC resulted in the gradual loss of nuclear Pax6+ and neural crest progenitor status, the latter of which was reverted upon recovery of Pax6. These findings suggest Pax6 plays a pivotal role in supporting the self-renewal of LEPC in limbal niche. Herein, we show that HC-HA/PTX3, a unique matrix purified from amniotic membrane (AM) and consists of heavy chain 1of inter-α-trypsin inhibitor covalently linked to hyaluronic acid and complexed with pentraxin 3, is capable of reverting senescent LNC to nuclear Pax6+ neural crest progenitors that support self-renewal of LEPC. Such reversion is causally linked to early cell aggregation mediated by activation of C-X-C chemokine receptor type 4 (CXCR4)-mediated signaling followed by activation of bone morphogenetic protein (BMP) signaling. Furthermore, CXCR4-mediated signaling, but not BMP signaling, controls recovery of the nuclear Pax6+ neural crest progenitors. These findings not only explain why AM helps in vivo and ex vivo expansion of human LEPC, but they also illuminate the potential role of HC-HA/PTX3 as a surrogate matrix niche that complements stem cell-based therapies in regenerative medicine. © AlphaMed Press 2020 SIGNIFICANCE STATEMENT: The authors show that HC-HA/PTX3 purified from human amniotic membrane uniquely restores senescent limbal niche cells to nuclear Pax6+ neural crest progenitors to support self-renewal of limbal progenitor/stem cells. Such reprogramming is linked to early cell aggregation mediated by activation of C-X-C chemokine receptor type 4 (CXCR4) mediated signaling highlighted by transient nuclear translocation of CXCR4 followed by activation of bone morphogenetic protein signaling. This finding not only explains why amniotic membrane transplantation helps in vivo and ex vivo expansion of human limbal epithelial stem cells but also envisage that this new paradigm based on regenerative matrix HC-HA/PTX3 as a surrogate niche can set a new standard for regenerative medicine in and beyond ophthalmology.