We investigated which specific correlation exists between the endothelial cell density (ECD) of corneal endothelial cell (CEC) cultures and the features of the donor corneas from which they originate. CEC cultures were prepared from one donor cornea or by pooling together cells of more corneas from elderly donors with ECDs lower or higher than 2000 cells/mm². The ECDs of such primary cultures were evaluated and showed that that ECDs > 2000 cells/mm² can be obtained only when CECs are isolated from (1) corneas of young donors; (2) at least two elderly donor corneas (if ECD > 2000 cells/mm²), or three elderly donor corneas (if ECD < 2000 cells/mm²). Secondary cultures are all characterized by ECDs < 2000 cells/mm². Our study highlights the difficulties in obtaining cultures with ECDs > 2000 cells/mm². Even if achievable with corneas from young donors, this becomes a challenging task when corneas from elderly donors are used (i.e., the overall majority of those collected by eye banks) and particularly when corneas from elderly donors with ECD < 2000 cells/mm² are used. Pooling more of two corneas to obtain suitable CECs could technically overcome the problem. The above issues should be tackled appropriately before moving into clinical studies.

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is caused by heterozygous missense point mutations in the p63 gene, an important transcription factor during embryogenesis and for stem cell differentiation in stratified epithelia. Most of the cases are sporadic, related to de novo mutations arising during early-stage development. Familial cases show an autosomic dominant inheritance. The major cause of visual morbidity is limbal stem cell failure, which develops in the second to third decade of life. Patients often show ocular surface alterations, such as recurrent blepharitis and conjunctivitis, superficial microlesions of the cornea, and spontaneous corneal perforation and ulceration, leading to progressive corneal clouding and eventually visual loss. No definitive cures are currently available, and treatments to alleviate symptoms are only palliative. In this review, we will discuss the proposed therapeutic strategies that have been tested or are under development for the management of the ocular defects in patients affected by EEC syndrome: (i) gene therapy-based approaches by means of Allele-Specific (AS) siRNAs to correct the p63 mutations; (ii) cell therapy-based approaches to replenish the pool of limbal stem cells; and (iii) drug therapy to correct/bypass the genetic defect. However, as the number of patients with EEC syndrome is too limited, further studies are still necessary to prove the effectiveness (and safety) of these innovative therapeutic approaches to counteract the premature differentiation of limbal stem cells.

Human epithelial stem cells (ESCs) are characterized by long‐term regenerative properties, much dependent on the tissue of origin and varying during their lifespan. We analysed such variables in cultures of ESCs isolated from the skin, conjunctiva, limbus and oral mucosa of healthy donors and patients affected by ectrodactyly‐ectodermal dysplasia‐clefting syndrome, a rare genetic disorder caused by mutations in the p63 gene. We cultured cells until exhaustion in the presence or in the absence of DAPT (γ‐secretase inhibitor; N‐[N‐(3, 5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine T‐butyl ester). All cells were able to differentiate in vitro but exhibited variable self‐renewal potential. In particular, cells carrying p63 mutations stopped prematurely, compared with controls. Importantly, administration of DAPT significantly extended the replicative properties of all stem cells under examination. RNA sequencing analysis revealed that distinct sets of genes were up‐ or down‐regulated during their lifetime, thus allowing to identify druggable gene networks and off‐the‐shelf compounds potentially dealing with epithelial stem cell senescence. These data will expand our knowledge on the genetic bases of senescence and potentially pave the way to the pharmacological modulation of ageing in epithelial stem cells.

The development of fetal cardiac surgery, considered the ultimate goal in the treatment of congenital cardiac malformations, needs to be supported by detailed knowledge of the blood circulation in the fetal cardiovascular system. The hemodynamic behavior in distal territories is usually inferred from vessel resistance indices, which give limited physiological information. This study presents a mathematical model of the human fetal global cardiovascular system, developed to clarify the relationships and differences existing between upper and lower body circulation. We modelled the heart with two time-varying capacitances, each representing the respective ventricle's pressure-volume relationship. The fetal vascular system was represented using two six-element Windkessel models, for the upper and lower body respectively. We obtained the identification of the set of circuital and elastance function parameters of the model using Genetic Algorithms (GAs), which follow the laws of evolutionary theory. We compared the results of our numerical study on the model identified with data collected from measurements and literature, to validate the proposed global cardiovascular system model of the human fetus. This model is intended as an instrument to investigate the differences in blood distribution between the different vascular districts in the upper and lower fetal body and the role of the aortic isthmus, the small tract of vessel connecting upper and lower fetal vascular beds; it may also represent a useful tool in the assessment of dynamic balance during mechanical assistance of circulation.

Background/Aims: The Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) and Ankyloblepharon–ectodermal defect–cleft lip/palate (AEC) syndromes are rare autosomal dominant diseases caused by heterozygous mutations in the p63 gene. Patients are characterized by abnormalities of the skin, teeth, and hair and have limb defects, orofacial clefting and ectodermal dysplasia. In addition, they often show ocular surface alterations, leading to progressive corneal clouding and eventually blindness. Here, we present 8 cases describing patients affected by EEC (n = 6, with 5 sporadic and 1 familial cases) and AEC (n = 2, both sporadic cases) syndromes. We attempt to provide a description of the ocular disease progression over the years. Methods: Clinical examinations and monitoring of ocular parameters for the assessment of limbal stem cell deficiency were constantly performed on patients between 2009 and 2023. Quantitative data and comparison with existing cases described in the literature are reported. Results: The therapies supplied to patients were essential for the management of the symptoms, but unfortunately did not halt the progression of the pathology. Conclusions: A constant monitoring of the patients would help avoid the sudden worsening of symptoms. If the progression of the disease slows down, it would allow for the development of newer therapeutic strategies aimed at correcting the genetic defect.