Final Report Summary - EPI-PATHO-STEM (Study of epithelial stem cells, pathophysiology and therapy)
EPI-PATHO-STEM project aimed to study epithelial stem cell location, dynamics and molecular pathways in heath and pathology. As a model, we studied the stratified epithelium of the skin (epidermis) and the cornea. These closely related tissues which line the external surface of the mammalian body and eye are multi-layered tightly packed tissues providing protection against infections, injuries and water loss1–4. However, the cornea is a specialized transparent tissue that plays an optic role. Interestingly, the cornea becomes skin-like or may even grow hair in mice lacking DKK25 or NOTCH16 genes and in human pathology. Yet, the question of why corneal transparency is lost in various diseases is poorly understood. The accomplish the aims of EPI-PATHO-STEM, we used genetic mouse models for labelling and tracking the origin of stem/progenitor cells in living animals (articles 1-2 below), study the regulation of stem/progenitor cells extracted from cadaveric cornea by SOX2 gene (article 3), we have generated mouse strains that lack or over-express MIR184 gene and define the role of this gene in the epidermis (article 4), and finally, we discovered a new role for RAS genes in pluripotent stem cells (article 5).
A better understanding of epithelial stem cell fate decisions is essential for isolating them and optimizing their expansion ex vivo before transplanting them back onto skin burn patients and blind patients. Unfortunately, it is not possible yet to purify epithelial stem cells and their expansion ex vivo is associated with a rapid loss of stemness and multipotency. Consequently, skin appendages are not formed in such grafts. Therefore, the molecular cues that underlie stem cell fate decisions need to be unravelled.
We believe that EPI-PATHO-STEM addressed major questions in the field of stem cell biology revealed new signalling pathways that modulate stem cell self-renewal and differentiation and will bring insightful information of high clinical relevance to patients that suffer from stem cells deficiency diseases or diseases that involve imbalance between proliferation and differentiation. Among these pathologies are aniridia, ectodermal dysplasia, psoriasis, cancer and more. As detailed below, our findings shed light on major questions in the field and may lead to novel therapy in follow up studies. A summary of our research interest and of EPI-PATHO-STEM research is described here below and on our website https://shalomfe.net.technion.ac.il/.
(Article 1) Lineage tracing of stem and progenitor cells of the murine corneal epithelium7,8: Accumulating evidence supports the dogma that the corneal epithelium is regenerated by stem cells located exclusively in the limbal niche, at the corneal periphery. Accordingly, limbal stem cells (LSCs) give rise to progenitors that proliferate and migrate centripetally to repopulate the corneal epithelium, which has a short turnover. Moreover, LSC loss leads to corneal opacity and blindness while limbal grafting restores patients' vision. However, contradicting data suggested that the limbus does not participate in corneal homeostasis and that the cornea contains stem cells.
To address this burning question, we performed lineage tracing experiments using R26R-Confetti mice to follow K14+ limbal/corneal epithelial cells stochastically induced to express one out of four fluorescent genes. In homeostasis, radial limbal stripes of slow migrating cells proceeded towards the corneal center while, infrequently, slow cycling limbal clones resembling quiescent stem cells were observed. Additionally, rare corneal clones that did not migrate centripetally, but survived for over 4 months, were inspected. In contrast to limbal stripes, corneal clusters had minor contribution to tissue replenishment in homeostasis. Corneal cells, however, significantly contributed to mild wound repair while large limbal streaks appeared within a week following severe wounding that coincided with partial loss of corneal transparency. Altogether, this study suggests that the limbus plays a major role in corneal renewal, while the cornea has a long-term self-maintenance capability. This mouse model will allow for addressing key questions in corneal stem cell biology in the future.
(Article 2) Corneal committed cells restore the stem cell pool and tissue boundary following injury (submitted to publication). During morphogenesis, it is essential to preserve segregated cellular compartments to properly regulate cell fate decisions. While embryonic tissues possess extremely high plasticity and ability for tissue repair, the question of whether and how adult tissues cope with acute SC loss or boundary disruption has remained open.
The cornea serves as an excellent model for studying tissue bordering and SC biology. We discovered that K15-GFP transgene labels the murine corneal epithelial boundary and SC niche known as the limbus. K15-GFP+ basal epithelial cells expressed SC markers and were located at the margin site of corneal regeneration, as evident by lineage tracing. Remarkably, surgical deletion of the SC pool of the limbus was restored by corneal committed cells which underwent dedifferentiation into bona fide SCs. Notably, the recovered corneas displayed normal marker expression and appropriate dynamic of LSC regeneration. Interestingly, however, damage to the limbal stromal niche abolished K15-GFP recovery and led to loss of corneal transparency. We provide direct evidence for pathological wound healing by adjacent conjunctival tissue that was accompanied by neovascularization, loss of transparency and blindness. Altogether, this study indicates that committed corneal cells have large plasticity to dedifferentiate, repopulate the SC pool and correctly reform tissue boundary. By contrast, loss of SC and boundary of the cornea lead to impaired tissue functionality and pathology.
(Article 3) Sox2 controls stemness, proliferation and epigenetic landscape of corneal epithelial cells (under review). Sox2 has been extensively studied in the context of pluripotent stem cells. Interestingly, however, a major phenotype of patients that are carriers of point mutations in SOX2 gene, is eye absence or small eye coupled with corneal neovascularization, loss of corneal transparency and blindness. The latter symptoms is generally linked with stem cell (SC) failure. Yet, the expression pattern and function of Sox2 in the cornea remained unclear.
Here we report that Sox2 is expressed by stem and progenitor but not by differentiated cells of the corneal epithelium. Notably, Sox2+ SCs of the cornea displayed low epigenetic repressive marks while knockdown of Sox2 significantly enhanced this phenotype and reduced SC clonogenic potential. Moreover, Sox2 was required for cell proliferation while its repression resulted in accelerated cell differentiation.
Interestingly, we found that miR-450b which was reciprocally expressed with Sox2 in lens, neural and corneal lineages, is a direct repressor of Sox2. miR-450b repressed Sox2 protein expression, reduced stemness and induced differentiation of corneal epithelial cells. Altogether, we propose that Sox2 controls stemness, epigenetic state and proliferation while its repression by miR-450b induces differentiation. In light of these finding, we propose that Sox2 mutations may lead to SC failure and/or disturb the balance between cell proliferation and differentiation.
(Article 4) microRNA-184 induces a commitment switch to epidermal differentiation (revised version was submitted). miR-184 is an extremely highly evolutionary conserved microRNA (miRNA) from fly to human. The importance of miR-184 was underscored by the discovery that point mutations in miR-184 gene lead to corneal/lens blinding disease. However, miR-184-related function in vivo remained unclear. We discovered that miR-184 knockout mouse model displayed increased p63 expression in line with epidermal hyperplasia while forced expression of miR-184 by stem/progenitor cells enhanced Notch pathway and induced epidermal hypoplasia. In line, miR-184 reduced clonogenicity and accelerated differentiation of human epidermal cells. We showed that by directly repressing cytokeratin 15 (K15) and FIH1, miR-184 induces Notch activation and epidermal differentiation. The disease-causing miR-184C57U mutant failed to repress K15 and FIH1 and to induce Notch activation suggesting a loss-of-function mechanism. Altogether, we propose that, by targeting K15 and FIH1, miR-184 regulates the transition from proliferation to early differentiation, while miss-expression or mutation in miR-184 results in impaired homeostasis.
(5) RAS regulates the transition from naïve to primed pluripotent stem cells (under reviewing). Cancer cells and stem cells share several common features and signaling pathways. The transition from naïve to primed state of pluripotent stem cells is hallmarked by epithelial to mesenchymal transition, metabolic switch from oxidative phosphorylation to preferential usage of aerobic glycolysis, and dramatic changes in the epigenetic landscape. Since these changes are also hallmarks of neoplastic cell transformation, we hypothesized that oncogenic pathways may be involved in this process.
We discovered that the activity of RAS proteins is repressed in conditions that maintain naïve undifferentiated state of mouse embryonic stem cells (ESCs) and that by contrast, all three RAS isoforms are significantly activated upon early differentiation induced by either LIF withdrawal, embryoid body formation, or transition to the primed state. Forced expression of active RAS was sufficient to induce an exit from naïve state coupled with expression of repressive epigenetic marks and N-CADHERIN in expense of E- CADHERIN. By contrast, inhibition of RAS by short hairpin RNA or by a pharmacological RAS inhibitor, significantly attenuated differentiation. Altogether, this study indicates that RAS is located at a key junction of early ESC differentiation and that it controls key processes in priming of naïve cells.
A better understanding of epithelial stem cell fate decisions is essential for isolating them and optimizing their expansion ex vivo before transplanting them back onto skin burn patients and blind patients. Unfortunately, it is not possible yet to purify epithelial stem cells and their expansion ex vivo is associated with a rapid loss of stemness and multipotency. Consequently, skin appendages are not formed in such grafts. Therefore, the molecular cues that underlie stem cell fate decisions need to be unravelled.
We believe that EPI-PATHO-STEM addressed major questions in the field of stem cell biology revealed new signalling pathways that modulate stem cell self-renewal and differentiation and will bring insightful information of high clinical relevance to patients that suffer from stem cells deficiency diseases or diseases that involve imbalance between proliferation and differentiation. Among these pathologies are aniridia, ectodermal dysplasia, psoriasis, cancer and more. As detailed below, our findings shed light on major questions in the field and may lead to novel therapy in follow up studies. A summary of our research interest and of EPI-PATHO-STEM research is described here below and on our website https://shalomfe.net.technion.ac.il/.
(Article 1) Lineage tracing of stem and progenitor cells of the murine corneal epithelium7,8: Accumulating evidence supports the dogma that the corneal epithelium is regenerated by stem cells located exclusively in the limbal niche, at the corneal periphery. Accordingly, limbal stem cells (LSCs) give rise to progenitors that proliferate and migrate centripetally to repopulate the corneal epithelium, which has a short turnover. Moreover, LSC loss leads to corneal opacity and blindness while limbal grafting restores patients' vision. However, contradicting data suggested that the limbus does not participate in corneal homeostasis and that the cornea contains stem cells.
To address this burning question, we performed lineage tracing experiments using R26R-Confetti mice to follow K14+ limbal/corneal epithelial cells stochastically induced to express one out of four fluorescent genes. In homeostasis, radial limbal stripes of slow migrating cells proceeded towards the corneal center while, infrequently, slow cycling limbal clones resembling quiescent stem cells were observed. Additionally, rare corneal clones that did not migrate centripetally, but survived for over 4 months, were inspected. In contrast to limbal stripes, corneal clusters had minor contribution to tissue replenishment in homeostasis. Corneal cells, however, significantly contributed to mild wound repair while large limbal streaks appeared within a week following severe wounding that coincided with partial loss of corneal transparency. Altogether, this study suggests that the limbus plays a major role in corneal renewal, while the cornea has a long-term self-maintenance capability. This mouse model will allow for addressing key questions in corneal stem cell biology in the future.
(Article 2) Corneal committed cells restore the stem cell pool and tissue boundary following injury (submitted to publication). During morphogenesis, it is essential to preserve segregated cellular compartments to properly regulate cell fate decisions. While embryonic tissues possess extremely high plasticity and ability for tissue repair, the question of whether and how adult tissues cope with acute SC loss or boundary disruption has remained open.
The cornea serves as an excellent model for studying tissue bordering and SC biology. We discovered that K15-GFP transgene labels the murine corneal epithelial boundary and SC niche known as the limbus. K15-GFP+ basal epithelial cells expressed SC markers and were located at the margin site of corneal regeneration, as evident by lineage tracing. Remarkably, surgical deletion of the SC pool of the limbus was restored by corneal committed cells which underwent dedifferentiation into bona fide SCs. Notably, the recovered corneas displayed normal marker expression and appropriate dynamic of LSC regeneration. Interestingly, however, damage to the limbal stromal niche abolished K15-GFP recovery and led to loss of corneal transparency. We provide direct evidence for pathological wound healing by adjacent conjunctival tissue that was accompanied by neovascularization, loss of transparency and blindness. Altogether, this study indicates that committed corneal cells have large plasticity to dedifferentiate, repopulate the SC pool and correctly reform tissue boundary. By contrast, loss of SC and boundary of the cornea lead to impaired tissue functionality and pathology.
(Article 3) Sox2 controls stemness, proliferation and epigenetic landscape of corneal epithelial cells (under review). Sox2 has been extensively studied in the context of pluripotent stem cells. Interestingly, however, a major phenotype of patients that are carriers of point mutations in SOX2 gene, is eye absence or small eye coupled with corneal neovascularization, loss of corneal transparency and blindness. The latter symptoms is generally linked with stem cell (SC) failure. Yet, the expression pattern and function of Sox2 in the cornea remained unclear.
Here we report that Sox2 is expressed by stem and progenitor but not by differentiated cells of the corneal epithelium. Notably, Sox2+ SCs of the cornea displayed low epigenetic repressive marks while knockdown of Sox2 significantly enhanced this phenotype and reduced SC clonogenic potential. Moreover, Sox2 was required for cell proliferation while its repression resulted in accelerated cell differentiation.
Interestingly, we found that miR-450b which was reciprocally expressed with Sox2 in lens, neural and corneal lineages, is a direct repressor of Sox2. miR-450b repressed Sox2 protein expression, reduced stemness and induced differentiation of corneal epithelial cells. Altogether, we propose that Sox2 controls stemness, epigenetic state and proliferation while its repression by miR-450b induces differentiation. In light of these finding, we propose that Sox2 mutations may lead to SC failure and/or disturb the balance between cell proliferation and differentiation.
(Article 4) microRNA-184 induces a commitment switch to epidermal differentiation (revised version was submitted). miR-184 is an extremely highly evolutionary conserved microRNA (miRNA) from fly to human. The importance of miR-184 was underscored by the discovery that point mutations in miR-184 gene lead to corneal/lens blinding disease. However, miR-184-related function in vivo remained unclear. We discovered that miR-184 knockout mouse model displayed increased p63 expression in line with epidermal hyperplasia while forced expression of miR-184 by stem/progenitor cells enhanced Notch pathway and induced epidermal hypoplasia. In line, miR-184 reduced clonogenicity and accelerated differentiation of human epidermal cells. We showed that by directly repressing cytokeratin 15 (K15) and FIH1, miR-184 induces Notch activation and epidermal differentiation. The disease-causing miR-184C57U mutant failed to repress K15 and FIH1 and to induce Notch activation suggesting a loss-of-function mechanism. Altogether, we propose that, by targeting K15 and FIH1, miR-184 regulates the transition from proliferation to early differentiation, while miss-expression or mutation in miR-184 results in impaired homeostasis.
(5) RAS regulates the transition from naïve to primed pluripotent stem cells (under reviewing). Cancer cells and stem cells share several common features and signaling pathways. The transition from naïve to primed state of pluripotent stem cells is hallmarked by epithelial to mesenchymal transition, metabolic switch from oxidative phosphorylation to preferential usage of aerobic glycolysis, and dramatic changes in the epigenetic landscape. Since these changes are also hallmarks of neoplastic cell transformation, we hypothesized that oncogenic pathways may be involved in this process.
We discovered that the activity of RAS proteins is repressed in conditions that maintain naïve undifferentiated state of mouse embryonic stem cells (ESCs) and that by contrast, all three RAS isoforms are significantly activated upon early differentiation induced by either LIF withdrawal, embryoid body formation, or transition to the primed state. Forced expression of active RAS was sufficient to induce an exit from naïve state coupled with expression of repressive epigenetic marks and N-CADHERIN in expense of E- CADHERIN. By contrast, inhibition of RAS by short hairpin RNA or by a pharmacological RAS inhibitor, significantly attenuated differentiation. Altogether, this study indicates that RAS is located at a key junction of early ESC differentiation and that it controls key processes in priming of naïve cells.