Limbal stem cell therapy.

PURPOSE OF REVIEW: To highlight the progress and future direction of limbal stem cell (LSC) therapies for the treatment of limbal stem cell deficiency (LSCD). RECENT FINDINGS: Direct LSC transplantation have demonstrated good long-term outcomes. Cultivated limbal epithelial transplantation (CLET) has been an alternative to treat severe to total LSCD aiming to improve the safety and efficacy of the LSC transplant. A prospective early-stage uncontrolled clinical trial shows the feasibility and safety of CLET manufactured under xenobiotic free conditions. Other cell sources for repopulating of the corneal epithelium such as mesenchymal stem cells (MSCs) and induced pluripotent stem cells are being investigated. The first clinical trials of using MSCs showed short-term results, but long-term efficacy seems to be disappointing. A better understanding of the niche function and regulation of LSC survival and proliferation will lead to the development of medical therapies to rejuvenate the residual LSCs found in a majority of eyes with LSCD in vivo. Prior efforts have been largely focused on improving LSC transplantation. Additional effort should be placed on improving the accuracy of diagnosis and staging of LSCD, and implementing standardized outcome measures which enable comparison of efficacy of different LSCD treatments for different severity of LSCD. The choice of LSCD treatment will be customized based on the severity of LSCD in the future. SUMMARY: New approaches for managing different stages of LSCD are being developed. This concise review summarizes the progresses in LSC therapies for LSCD, underlying mechanisms, limitations, and future areas of development.

Single mRNA detection of Wnt signaling pathway in the human limbus.

Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their close microenvironment. It has been shown that Wnt signaling pathway is crucial for LSCs regulation. Previous differential gene profiling studies confirmed the preferential expression of specific Wnt ligands (WNT2, WNT6, WNT11, WNT16) and Wnt inhibitors (DKK1, SFRP5, WIF1, FRZB) in the limbal region compared to the cornea. Among all frizzled receptors, frizzled7 (Fzd7) was found to be preferentially expressed in the basal limbal epithelium. However, the exact localization of Wnt signaling molecules-producing cells in the limbus remains unknown. The current study aims to evaluate the in situ spatial expression of these 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7. Wnt ligands, DKK1, and Fzd7 expression were scattered within the limbal epithelium, at a higher abundance in the basal layer than the superficial layer. SFRP5 expression was diffuse among the limbal epithelium, whereas WIF1 and FRZB expression was clustered at the basal limbal epithelial layer corresponding to the areas of high levels of Fzd7 expression. Quantitation of the fluorescence intensity showed that all 4 Wnt ligands, 3 Wnt inhibitors (WIF1, DKK1, FRZB), and Fzd7 were highly expressed at the basal layer of the limbus, then in a decreasing gradient toward the superficial layer (P < 0.05). The expression levels of all 4 Wnt ligands, FRZB, and Fzd7 in the basal epithelial layer were higher in the limbus than the central cornea (P < 0.05). All 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7 were also highly expressed in the limbal stroma immediately below the epithelium but not in the corneal stroma (P < 0.05). In addition, Fzd7 had a preferential expression in the superior limbus compared to other limbal quadrants (P < 0.05). Taken together, the unique expression patterns of the Wnt molecules in the limbus suggests the involvement of both paracrine and autocrine effects in LSCs regulation, and a fine balance between Wnt activators and inhibitors to govern LSC fate.

Expansion of Human Limbal Epithelial Stem/Progenitor Cells Using Different Human Sera: A Multivariate Statistical Analysis.

Transplantation of human cultured limbal epithelial stem/progenitor cells (LESCs) has demonstrated to restore the integrity and functionality of the corneal surface in about 76% of patients with limbal stem cell deficiency. However, there are different protocols for the expansion of LESCs, and many of them use xenogeneic products, being a risk for the patients' health. We compared the culture of limbal explants on the denuded amniotic membrane in the culture medium-supplemental hormone epithelial medium (SHEM)-supplemented with FBS or two differently produced human sera. Cell morphology, cell size, cell growth rate, and the expression level of differentiation and putative stem cell markers were examined. Several bioactive molecules were quantified in the human sera. In a novel approach, we performed a multivariate statistical analysis of data to investigate the culture factors, such as differently expressed molecules of human sera that specifically influence the cell phenotype. Our results showed that limbal cells cultured with human sera grew faster and contained similar amounts of small-sized cells, higher expression of the protein p63α, and lower of cytokeratin K12 than FBS cultures, thus, maintaining the stem/progenitor phenotype of LESCs. Furthermore, the multivariate analysis provided much data to better understand the obtaining of different cell phenotypes as a consequence of the use of different culture methodologies or different culture components.

Monoubiquitination of RPN10 regulates substrate recruitment to the proteasome.

The proteasome recognizes its substrates via a diverse set of ubiquitin receptors, including subunits Rpn10/S5a and Rpn13. In addition, shuttling factors, such as Rad23, recruit substrates to the proteasome by delivering ubiquitinated proteins. Despite the increasing understanding of the factors involved in this process, the regulation of substrate delivery remains largely unexplored. Here we report that Rpn10 is monoubiquitinated in vivo and that this modification has profound effects on proteasome function. Monoubiquitination regulates the capacity of Rpn10 to interact with substrates by inhibiting Rpn10's ubiquitin-interacting motif (UIM). We show that Rsp5, a member of NEDD4 ubiquitin-protein ligase family, and Ubp2, a deubiquitinating enzyme, control the levels of Rpn10 monoubiquitination in vivo. Notably, monoubiquitination of Rpn10 is decreased under stress conditions, suggesting a mechanism of control of receptor availability mediated by the Rsp5-Ubp2 system. Our results reveal an unanticipated link between monoubiquitination signal and regulation of proteasome function.

Effect of Hypoxia-regulated Polo-like Kinase 3 (Plk3) on Human Limbal Stem Cell Differentiation.

Hypoxic conditions in the cornea affect epithelial function by activating Polo-like kinase 3 (Plk3) signaling and the c-Jun·AP-1 transcription complex, resulting in apoptosis of corneal epithelial cells. Hypoxic stress in the culture conditions also regulates limbal stem cell growth and fate. In this study, we demonstrate that there is a differential response of Plk3 in hypoxic stress-induced primary human limbal stem (HLS) and corneal epithelial (HCE) cells, resulting in different pathways of cell fate. We found that hypoxic stress induced HLS cell differentiation by down-regulating Plk3 activity at the transcription level, which was opposite to the effect of hypoxic stress on Plk3 activation to elicit HCE cell apoptosis, detected by DNA fragmentation and TUNEL assays. Hypoxic stress-induced increases in c-Jun phosphorylation/activation were not observed in HLS cells because Plk3 expression and activity were suppressed in hypoxia-induced HLS cells. Instead, hypoxic stress-induced HLS cell differentiation was monitored by cell cycle analysis and measured by the decrease and increase in p63 and keratin 12 expression, respectively. Hypoxic stress-induced Plk3 signaling to regulate c-Jun activity, resulting in limbal stem cell differentiation and center epithelial apoptosis, was also found in the corneas of wild-type and Plk3(-/-)-deficient mice. Our results, for the first time, reveal the differential effects of hypoxic stress on Plk3 activity in HLS and HCE cells. Instead of apoptosis, hypoxic stress suppresses Plk3 activity to protect limbal stem cells from death and to allow the process of HLS cell differentiation.

Ultrastructure of the posterior corneal stroma.

PURPOSE: To reinvestigate the ultrastructure of the posterior stroma of the human cornea and to correlate the findings with the stromal behavior after big-bubble creation. DESIGN: Observational consecutive 3-center case series. SPECIMENS: Fresh corneoscleral buttons from human donors (n = 19) and organ-cultured corneoscleral buttons (n = 10) obtained after Descemet's membrane endothelial keratoplasty. METHODS: Corneal specimens were divided into central (3 mm), mid peripheral (8 mm), and peripheral parts by trephination and processed for transmission electron microscopic and immunohistochemical analyses. A big bubble was created by air injection into the stroma of organ-cultured corneas before fixation. MAIN OUTCOME MEASURES: The distance of keratocytes to Descemet's membrane, number of collagen lamellae between keratocytes and Descemet's membrane, diameter and arrangement of collagen fibrils, thickness of stromal lamella created by air injection, and immunopositivity for collagen types III, IV, and VI. RESULTS: Stromal keratocytes were observed at variable distances from Descemet's membrane, increasing from 1.5 to 12 µm (mean, 4.97±2.19 µm) in the central, 3.5 to 14 µm (mean, 8.03±2.47 µm) in the midperipheral, and 4.5 to 18 µm (mean, 9.77±2.90 µm) in the peripheral regions. The differences in mean distances were significant (P < 0.0001). The number of collagen lamellae between Descemet's membrane and most posterior keratocytes varied from 2 to 10 and the diameter of collagen fibrils averaged 23.5±1.8 nm and corresponded with that of the remaining stroma. A thin layer (0.5-1.0 µm thick) of randomly arranged, unaligned collagen fibers, which was positive for collagen types III and VI, was observed at the Descemet-stroma interface. The residual stromal sheet separated by air injection in 8 of 10 donor corneas varied in thickness from 4.5 to 27.5 µm, even within individual corneas (≤3-fold), and was composed of 5 to 11 collagen lamellae that revealed keratocytes on their anterior surface and in between. CONCLUSIONS: Barring an anchoring zone of interwoven collagen fibers at the Descemet-stroma interface, the findings did not provide any evidence for the existence of a distinctive acellular pre-Descemet's stromal layer in the human cornea. The intrastromal cleavage plane after pneumodissection seems to be nonreproducibly determined by the intraindividually and interindividually variable distances of keratocytes to Descemet's membrane.

Late-onset rupture of an intracranial dermoid cyst: a case report.

BACKGROUND: Dermoid cysts are developmental abnormalities occurring between the third and fifth week of embryogenesis. These lesions can initially develop as intracranial or extracranial and persist throughout the patient's lifetime. While generally benign, their symptoms can be due to mass effect or local irritation secondary to rupture and release of contents, typically presenting as headaches and seizures. Intracranial dermoid cysts are rare and comprise less than 1% of all intracranial lesions, with rupture occurring approximately 0.18% of the time. CASE PRESENTATION: Our case describes a 42-year-old Hispanic female with a late-onset rupture of an intracranial dermoid cyst with associated new onset seizures. She underwent uncomplicated neurosurgical resection with mesh placement and was scheduled to follow-up as an outpatient. CONCLUSION: To avoid rupture and associated sequelae in future patients, we recommend considering a more invasive approach as the initial strategy if internal cysts are relatively accessible.

[Mixed adenoneuroendocrine carcinoma: case report]. / Carcinoma adenoneuroendocrino mixto: reporte de caso.

Introducción: Mixed adenoneuroendocrine carcinoma is a rare tumor of the gastrointestinal tract with double differentiation into adenomatous and neuroendocrine carcinoma, each component with at least 30%. Case report: A 60-year-old female with acute abdominal pain. Surgical treatment was decided, finding a tumor at the level of the cecum and ascending colon, a right hemicolectomy and ileostomy were performed. Discussion: Mixed adenoneuroendocrine carcinoma can appear in various organs. They are highly malignant tumors, with a high risk of metastasis. Conclusions: These tumors do not present symptoms or specific radiological or laboratory findings; diagnosis depends on postoperative histopathological and immunohistochemical studies.

Introducción: El carcinoma adenoneuroendocrino mixto es un tumor raro del tracto gastrointestinal con doble diferenciación en carcinoma adenomatoso y neuroendocrino, cada componente con al menos el 30%. Caso clínico: Mujer de 60 años con cuadro de dolor abdominal agudo. Se decide tratamiento quirúrgico, encontrando un tumor a nivel de ciego y colon ascendente, y se realizan hemicolectomía derecha e ileostomía. Discusión: El carcinoma adenoneuroendocrino mixto puede aparecer en diversos órganos. Son tumores muy malignos, con alto riesgo de metástasis. Conclusiones: Estos tumores no presentan síntomas ni hallazgos radiológicos o de laboratorio específicos; el diagnóstico depende de estudios histopatológicos e inmunohistoquímicos posoperatorios.

Presence of native limbal stromal cells increases the expansion efficiency of limbal stem/progenitor cells in culture.

Niche factors are important in the maintenance and regulation of stem cells. Limbal stromal cells are potentially a component of limbal stem cell (LSC) niche. We investigated the role of the limbal stromal cells in the ex vivo expansion of limbal stem/progenitor cells. Limbal epithelial cells were cultured as single-cell suspension and cell clusters from dispase II or collagenase A (ColA), or tissue explant. ColA isolated limbal stromal cells along with limbal epithelial cells. In the presence of limbal stromal cells, a higher absolute number of p63α(bright) cells (p < 0.05) and a higher proportion of K14 positive epithelial cells were obtained from both ColA and explant tissue cultures. Expansion of the stem/progenitor population from dispase isolation was more efficient in the form of cell clusters than single cell suspension based on the absolute number of p63α(bright) cells. Expansion of the stem cell population is similar in the single cell and cell cluster cultures that are derived from ColA isolation. Our finding suggests that limbal stromal cells and an intact cell-cell contact help to maintain LSCs in an undifferentiated state in vitro during expansion.

Ocular Surface Regeneration by Limbal Stem Cells Therapies: State of the Art, Challenges, and Perspectives.

Limbal stem cells (LSCs) are adult stem cells located at the limbus ensuring the continuous renewal of the corneal epithelium, critical to maintain an optimal visual function. Damages to the LSCs or their niche microenvironment lead to limbal stem cell deficiency (LSCD), a potentially blinding disease. Transplantation of LSCs as a treatment for severe to total LSCD has gained popularity since 1980s, owing to the clinical success of the first direct limbal autograft transplantation. Recent advances in the understanding of the LSCs' molecular identity and regulation have enabled preclinical and clinical advancements of promising LSCs therapies. However, lack of standardization of the diagnostic methods, staging of the disease severity, manufacturing process, and clinical outcome measures have hindered the advancement of the therapy. To move these therapies to the clinic, optimization and standardization of the diagnostic strategy, cell product manufacturing, and assessment of clinical efficacy with potency assays are key points to the development of customized therapies. Recent findings suggest that residual LSCs exist in eyes presenting with clinical signs of total LSCD, which opens new therapeutic strategies for eyes with partial LSCD. Prospective, randomized, multicentric controlled clinical trials are necessary to determine the efficacy of different LSCs therapies for different stages of LSCD using a set of standardized outcome measures.

Wnt activation as a potential therapeutic approach to treat partial limbal stem cell deficiency.

Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their niche involving numerous signaling pathways, such as Wnt. Wnt proteins are secreted morphogens that play critical roles in embryonic development, stem cell proliferation, self-renewal, tissue regeneration, and remodeling in adults. It has been shown that a small molecule Wnt mimic could improve LSCs expansion ex vivo. Damage to the LSCs and/or their niche can lead to limbal stem cell deficiency (LSCD), a condition that can cause corneal blindness and is difficult to treat. This study explored if repopulating residual LSCs in partial LSCD through Wnt activation could be a novel therapeutic approach. To mimic LSCD due to a chemical injury, single cultured LSCs were exposed to various concentrations of sodium hydroxide. A progressive loss of the LSCs phenotype was observed: the percentage of p63bright cells and cytokeratin (K)14+ cells decreased while the percentage of K12+ increased. Wnt activation was attained by treating the LSCs with lithium chloride (LiCl) and a small-molecule Wnt mimic, respectively. After 18 h of treatment, LSCs proliferation was increased, and the LSCs phenotype was recovered, while the untreated cells did not proliferate and lost their phenotype. The percentage of p63bright cells was significantly higher in the Wnt mimic-treated cells compared with untreated cells, while the percentage of K12+ cells was significantly lower. These findings suggest that local Wnt activation may rescue LSCs upon alkaline injury.

Septic Arthritis With Superimposed Acute Gouty Arthritis in a Rheumatoid Arthritis Patient.

Septic arthritis is a rare but serious complication of both rheumatoid and gouty arthritis and can lead to significant morbidity and even mortality. Here, we report a case of septic arthritis with bacteremia, monosodium urate crystals, and hyperuricemia in a 75-year-old male with long-standing rheumatoid arthritis. Arthrocentesis revealed gram-positive cocci representing group B streptococcus (Streptococcus agalactiae) infection and monosodium urate crystals. A diagnosis of septic arthritis with superimposed acute gouty arthritis was made and the patient was treated accordingly. Management included surgical irrigation and debridement, antibiotic therapy, and systemic glucocorticoids which resulted in a significant improvement in the patient's clinical status.

Changing characteristics of emergency surgery during COVID-19 pandemic: a retrospective cohort study.

BACKGROUND: In other countries, researchers have noticed diverse variations in the features of patients undergoing emergency surgery during the COVID-19 pandemic. In Mexico, there is not information about this issue. METHODS: Workers of the Mexican Government, who required emergency surgeries were studied by the general surgery service of a General Hospital administered by the Institute of Social Security and Services for State Workers Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado (ISSSTE), through the periods from March-August 2019 (non-exposed) and March-August 2020 (exposed). The analysis included: demographic data, laboratory information, post-operative diagnoses, symptoms' length, days of emergency stay, and post-operative stay. RESULTS: One hundred and ninety-three emergency surgeries were analyzed; 106 in 2019 and 87 in 2020 (a decrease of 18%). Throughout the pandemic, the number of days between the symptoms' onset and surgery was greater: 2019, 7.6 ± 4.6 days; 2020, 14 ± 6.7 days (p < 0.0001). In addition, cases of acute appendicitis decreased (2019-60.3%; 2020-42.5%), and those of acute calculous cholecystitis increased (2019-12.2%; 2020-24.1%). CONCLUSION: Through the COVID-19 pandemic, there were notable changes in the characteristics of Mexican Government's workers who warranted emergency surgery.

ANTECEDENTES: En otros países, han notado diversos cambios en las características de los pacientes sometidos a cirugía de emergencia durante la pandemia de COVID-19. En México no existe información sobre este tema. MÉTODO: Estudiamos a los trabajadores del gobierno mexicano que requirieron tratamiento quirúrgico de emergencia por el servicio de cirugía general de un Hospital General del Instituto de Seguridad y Servicios Sociales para los Trabajadores del Estado (ISSSTE), durante los periodos de marzo-agosto de 2019 (no expuestos) y marzo-agosto de 2020 (expuestos). El análisis incluyó: datos demográficos, datos de laboratorio, diagnósticos postoperatorios, duración de los síntomas, días de estancia en emergencias y estadía postoperatoria. RESULTADOS: Se analizaron 193 cirugías de emergencia; 106 en 2019 y 87 en 2020 (una disminución del 18%). En la pandemia, el número de días entre el inicio de los síntomas y la cirugía fue mayor: 2019, 7.6 ± 4.6 días; 2020, 14 ± 6.7 días (p < 0.0001). Además, disminuyeron los casos de apendicitis aguda (2019-60,3%; 2020-42,5%) y aumentaron los de colecistitis litiásica aguda (2019-12,2%; 2020-24,1%). CONCLUSIÓN: Durante la pandemia de COVID-19, hubo cambios notables en las características de los trabajadores del gobierno mexicano que ameritaron cirugías de emergencia.

Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid.

Mouse recipient cytoplasts for somatic cell nuclear transfer (SCNT) are routinely prepared by mechanical enucleation (ME), an invasive procedure that requires expensive equipment and considerable micromanipulation skills. Alternatively, oocytes can be enucleated using chemically assisted (AE) or chemically induced (IE) enucleation methods that are technically simple. In this study, we compared the reprogramming potential and developmental capacity of cloned embryos generated by ME, AE, and IE procedures and treated with the histone deacetylase inhibitor valproic acid. A rapid and almost complete deacetylation of histone H3 lysine 14 in the somatic nucleus followed by an equally rapid and complete re-acetylation after activation was observed after the injection of a cumulus cell nucleus into ME and AE cytoplasts. In contrast, histone deacetylation occurred at a much lower level in IE cytoplasts. Despite these differences, the cloned embryos generated from the three types of cytoplasts developed into blastocysts of equivalent total and inner cell mass mean cell numbers, and the rates of blastocyst formation and embryonic stem cell derivation were similar among the three groups. The cloned embryos produced from ME and AE cytoplasts showed an equivalent rate of full-term development, but no offspring could be obtained from the IE group, suggesting a lower reprogramming capacity of IE cytoplasts. Our results demonstrate the usefulness of AE in mouse SCNT procedures, as an alternative to ME. AE can facilitate oocyte enucleation and avoid the need for expensive microscope optics, or for potentially damaging Hoechst staining and u.v. irradiation, normally required in ME procedures.

An Incidental Discovery Following Hypertension and Headaches: A Pheochromocytoma Case Report.

Pheochromocytomas (PCC) are rare neuroendocrine tumors of the adrenal medulla that arise from chromaffin cells. These cells are neural crest derivatives and are innervated by the splanchnic nerve of the sympathetic nervous system which releases acetylcholine that in turn binds to nicotinic acetylcholine receptors of the adrenal medulla causing the release of catecholamines. The dopamine, norepinephrine, and epinephrine released from these tumors are responsible for the episodic hyperadrenergic symptoms seen in these cases such as hypertension, palpitations, and headaches. This case report discusses the incidental finding of a unilateral PCC in a 58-year-old woman who initially presented to our emergency department complaining of intermittent chest pain and headaches for two days.

The Role of Serial Imaging in Neurocysticercosis for Disease Resolution.

Neurocysticercosis (NCC), the most common parasitic infection of the CNS in humans, is a frequent cause of seizure disorders and epilepsy. The cystic larvae Taenia solium is endemic to developing countries where the population raises pigs as a reliable source of food, however, massive immigration has now forced the surge of the disease in developed areas making it a worldwide problem. Clinical presentation is affected by the size, number, and location of the lesions within the brain, with the most common manifestations being seizures, headaches, and increased intracranial pressure. The appearance of NCC on radiological imaging helps determine the stage of the disease, required for appropriate antiparasitic treatment. In this article, we detail the case of a patient who presented for recurrent seizures after reportedly undergoing treatment for NCC years prior.

Partial Thrombosis of Inferior Mesenteric Vein With Thrombophlebitis.

Inferior mesenteric vein thrombosis (IMVT) is a rare entity that can lead to a potentially lethal event unless recognized early in the disease. Although its prevalence is low, IMVT presents mainly in certain conditions such as in inflammatory processes like diverticulitis, arrhythmias, hypercoagulable states, connective tissue disorders, malignancy, or hereditary thrombophilias.  Mesenteric venous thrombophlebitis is a condition in which a blood clot in a vein causes inflammation and pain. It can appear in an acute or subacute manner that leads to acute mesenteric ischemia. The case of a 58-year-old male without a significant past medical history who presented with suprapubic abdominal pain secondary to a partial IMVT of unknown etiology with accompanying thrombophlebitis is discussed here.

Human limbal epithelial stem cell regulation, bioengineering and function.

The corneal epithelium is continuously renewed by limbal stem/progenitor cells (LSCs), a cell population harbored in a highly regulated niche located at the limbus. Dysfunction and/or loss of LSCs and their niche cause limbal stem cell deficiency (LSCD), a disease that is marked by invasion of conjunctival epithelium into the cornea and results in failure of epithelial wound healing. Corneal opacity, pain, loss of vision, and blindness are the consequences of LSCD. Successful treatment of LSCD depends on accurate diagnosis and staging of the disease and requires restoration of functional LSCs and their niche. This review highlights the major advances in the identification of potential LSC biomarkers and components of the LSC niche, understanding of LSC regulation, methods and regulatory standards in bioengineering of LSCs, and diagnosis and staging of LSCD. Overall, this review presents key points for researchers and clinicians alike to consider in deepening the understanding of LSC biology and improving LSCD therapies.

Establishment of mouse embryonic stem cells from isolated blastomeres and whole embryos using three derivation methods.

PURPOSE: the aim of the present study is to compare three previously described mouse embryonic stem cell derivation methods to evaluate the influence of culture conditions, number of isolated blastomeres and embryonic stage in the derivation process. METHODS: three embryonic stem cell derivation methods: standard, pre-adhesion and defined culture medium method, were compared in the derivation from isolated blastomeres and whole embryos at 4- and 8-cell stages. RESULTS: a total of 200 embryonic stem cell lines were obtained with an efficiency ranging from 1.9% to 72%. CONCLUSIONS: using either isolated blastomeres or whole embryos, the highest rates of mouse embryonic stem cell establishment were achieved with the defined culture medium method and efficiencies increased as development progressed. Using isolated blastomeres, efficiencies increased in parallel to the proportion of the embryo volume used to start the derivation process.

Role of Jagged1-mediated Notch Signaling Activation in the Differentiation and Stratification of the Human Limbal Epithelium.

The Notch signaling pathway plays a key role in proliferation and differentiation. We investigated the effect of Jagged 1 (Jag1)-mediated Notch signaling activation in the human limbal stem/progenitor cell (LSC) population and the stratification of the limbal epithelium in vitro. After Notch signaling activation, there was a reduction in the amount of the stem/progenitor cell population, epithelial stratification, and expression of proliferation markers. There was also an increase of the corneal epithelial differentiation. In the presence of Jag1, asymmetric divisions were decreased, and the expression pattern of the polarity protein Par3, normally present at the apical-lateral membrane of basal cells, was dispersed in the cells. We propose a mechanism in which Notch activation by Jag1 decreases p63 expression at the basal layer, which in turn reduces stratification by decreasing the number of asymmetric divisions and increases differentiation.