Human Oral Mucosa as a Potentially Effective Source of Neural Crest Stem Cells for Clinical Practice.

We report in this study on the isolation and expansion of neural crest stem cells (NCSCs) from the epithelium of oral mucosa (OM) using reagents that are GMP-certified and FDA-approved for clinical use. Characterization analysis showed that the levels of keratins K2, K6C, K4, K13, K31, and K15-specific to OM epithelial cells-were significantly lower in the experimental NCSCs. While SOX10 was decreased with no statistically significant difference, the earliest neural crest specifier genes SNAI1/2, Ap2a, Ap2c, SOX9, SOX30, Pax3, and Twist1 showed a trend in increased expression in NCSCs. In addition, proteins of Oct4, Nestin and Noth1 were found to be greatly expressed, confirming NCSC multipotency. In conclusion, our study showed that the epithelium of OM contains NCSCs that can be isolated and expanded with clinical-grade reagents to supply the demand for multipotent cells required for clinical applications in regenerative medicine. Supported by Emmaus Medical Inc.

Aldehyde Dehydrogenases Expression in Corneal Epithelial Cells with Limbal Stem Cell Deficiency.

PURPOSE: The purpose of the present study is to investigate the expression of aldehyde dehydrogenases (ALDHs) in rabbit corneas with limbal stem cell deficiency (LSCD) and corneas treated with cultured autologous oral mucosa epithelial cell sheet CAOMECS designed to reconstruct the ocular surface with LSCD. METHODS: New Zealand white rabbit autologous oral mucosal epithelial cells were isolated from a buccal biopsy and cultured to be grafted back onto corneas of rabbit model of LSCD. Immunofluorescent staining and Western blot analysis were used to compare the expression of ALDH1A1 and ALDH1A3 in healthy, LSCD-diseased, CAOMECS treated corneas. Human oral mucosal and corneal epithelial cells (OMECS and CECs) were cultured and treated with retinoic acid (RA) to further investigate the expression of ALDHs. RESULTS: In healthy corneas, ALDH1A1 and ALDH1A3 were markedly expressed in basal cells of corneal epithelium. In LSCD diseased corneas, ALDH1A1 and ALDH1A3 were markedly expressed in the conjunctivalized apical epithelial cells, the goblet cells, and the stroma. CAOMECS grafted corneas showed a decreased expression of ALDHs as compared to LSCD diseased corneas. Western blot analysis confirmed the up regulation of ALDH1A1 and ALDH1A3 expression in LSCD-diseased corneal epithelial cells. CAOMECS expressed low levels of ALDH1A1 and ALDH1A3, as compared to diseased CECs (D-CEC). When ALDH1A3 was up regulated by retinoic acid treatment in OMECS, Pax-6 expression was down regulated, suggesting a decrease in regenerative capacity when ALDH enzymes are up regulated. CONCLUSIONS: These findings report for the first time the up regulation of ALDH1A1 and ALDH1A3 in rabbit corneas with LSCD and document that CAOMECS grafting used to reconstruct corneal epithelium may reduce the expression levels of ALDH enzymes.

cG-CAOMECS-clinical-grade cultured autologous oral mucosal epithelial cell sheet.

The present study reports the feasibility and successful production of rabbit cG-CAOMECS, designed to reconstruct corneal epithelium of patients with bilateral limbal stem cell deficiency. To produce a safe, chemically defined and FDA compliant cG-CAOMECS, oral mucosal epithelial cells were isolated from a biopsy of rabbit buccal tissue and seeded on a cGMP-certified cell culture surface coated with GMP-grade extracellular matrix. A newly designed clinical-grade medium (KaFa™ medium) was utilized to carry out cell expansion. Detachment and harvesting of the produced cell sheet was accomplished using collagenase treatment. Live cell imaging and morphological analysis techniques were used to examine cell growth. Cells attached onto the surface and self-assembled into colony-forming units (CFUs). Microscopic examination showed that CFUs formed during the first 5 days, and basal monolayer cell sheet formed in less than 10 days. Cells expanded to form a multilayered epithelial cell sheet that was harvested after 17-19 days in culture. Immunostaining and Western blot analyses showed that deltaNp63 was expressed in the basal cells and K3/K12 was expressed in the apical cells, indicating the presence of corneal epithelial-like cells in the produced cell sheet. Adhesion molecules, E-cadherin, beta-catenin, and Cnx43 were also expressed and exhibited the epithelial integrity of the cell sheet. The expression of integrin-beta1 and beta4 confirmed that the collagenase treatment used for detaching and harvesting the cell sheet did not have adverse effects. Our results showed that the utilization of clinical-grade and FDA-approved reagents successfully supported the production of cG-CAMECS.

Clinical Trials of Limbal Stem Cell Deficiency Treated with Oral Mucosal Epithelial Cells.

The corneal surface is an essential organ necessary for vision, and its clarity must be maintained. The corneal epithelium is renewed by limbal stem cells, located in the limbus and in palisades of Vogt. Palisades of Vogt maintain the clearness of the corneal epithelium by blocking the growth of conjunctival epithelium and the invasion of blood vessels over the cornea. The limbal region can be damaged by chemical burns, physical damage (e.g., by contact lenses), congenital disease, chronic inflammation, or limbal surgeries. The degree of limbus damage is associated with the degree of limbal stem cells deficiency (partial or total). For a long time, the only treatment to restore vision was grafting part of the healthy cornea from the other eye of the patient or by transplanting a cornea from cadavers. The regenerative medicine and stem cell therapies have been applied to restore normal vision using different methodologies. The source of stem cells varies from embryonic stem cells, mesenchymal stem cells, to induced pluripotent stem cells. This review focuses on the use of oral mucosa epithelial stem cells and their use in engineering cell sheets to treat limbal stem cell deficient patients.

Vitrification and storage of oral mucosa epithelial cell sheets.

Shipping time and shipping delays might affect the quality of the stem cells based engineered "organs." In our laboratory, we have developed a limbal stem cell deficient (LSCD) rabbit model. To reverse the LSCD, we cultured oral mucosal epithelial cells for 2-3 weeks and engineered cultured autologous oral mucosa epithelial cell sheets (CAOMECS), which were grafted on the LSCD cornea. The purpose of this study was to vitrify CAOMECS and to store it until the CAOMECS can be grafted onto patients. CAOMECS were vitrified in LN2 for up to 204 days. We tested two different methods of vitrification with different solutions; however, CAOMECS were only viable when they were not stored in a vitrification solution; results were only reported from this CAOMECS. On the basis of hematoxylin and eosin staining, we showed that the CAOMECS morphology was well preserved after long-term storage in LN2 . Most of the preservation solutions maintained the CAOMECS phenotype (Ki67, proliferating cell nuclear antigen (PCNA), Beta-Catenin, ZO-1, E-Cadherin, CK3, CK4, CK13). The exception was the solution composed with ethylene glycol and Dimethyl sulfoxide (DMSO): this resulted in loss of DeltaN-p63 expression. DeltaN-p63 is an important marker for cell proliferation. The expression of proteins involved in cell-cell connection and the differentiation markers were maintained. Apoptosis was not detected in the thawed CAOMECS. We demonstrated that CAOMECS can be stored long-term in LN2 without affecting their morphology and phenotype.

Proteasomes in corneal epithelial cells and cultured autologous oral mucosal epithelial cell sheet (CAOMECS) graft used for the ocular surface regeneration.

PURPOSE: This study focuses on characterizing proteasomes in corneal epithelial cells (CEC) and in cultured autologous oral mucosal epithelial cell sheets (CAOMECS) used to regenerate the ocular surface. METHODS: Limbal stem cell deficiency (LSCD) was surgically induced in rabbit corneas. CAOMECS was engineered and grafted onto corneas with LSCD to regenerate the ocular surface. RESULTS: LSCD caused an increase in inflammatory cells in the ocular surface, an increase in the formation of immunoproteasomes (IPR), and a decrease in the formation of constitutive proteasome (CPR). Specifically, LSCD-diseased CEC (D-CEC) showed a decrease in the CPR chymotrypsin-like, trypsin-like and caspase-like activities, while healthy CEC (H-CEC) and CAOMECS showed higher activities. Quantitative analysis of IPR inducible subunit (B5i, B2i, and B1i) were performed and compared to CPR subunit (B5, B2, and B1) levels. Results showed that ratios B5i/B5, B2i/B2 and B1i/B1 were higher in D-CEC, indicating that D-CEC had approximately a two-fold increase in the amount of IPR compared to CAOMECS and H-CEC. Histological analysis demonstrated that CAOMECS-grafted corneas had a re-epithelialized surface, positive staining for CPR subunits, and weak staining for IPR subunits. In addition, digital quantitative measurement of fluorescent intensity showed that the CPR B5 subunit was significantly more expressed in CAOMECS-grafted corneas compared to non-grafted corneas with LSCD. CONCLUSION: CAOMECS grafting successfully replaced the D-CEC with oral mucosal epithelial cells with higher levels of CPR. The increase in constitutive proteasome expression is possibly responsible for the recovery and improvement in CAOMECS-grafted corneas.

Carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) for corneal epithelium reconstruction: a histological study.

This study investigates the therapeutic effects of carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) transplantation for experimentally induced severe rabbit limbal stem cell deficiency (LSCD). Buccal biopsies were performed and CAOMECS were cultured and transplanted onto diseased corneas. Six-month follow-up examinations indicated that three out of four corneas with CAOMECS grafts showed a decrease in superficial vascularization, while almost all the sham corneas did not show a similar decrease. H&E staining of corneas showed that CAOMECS transplantation reduced blood vessel invasion of central cornea, reduced lymphocyte infiltration and fibrotic tissue formation. DeltaNp63 stained markedly in the grafted cornea and to a lesser extent in the sham corneas. PCNA and Ki-67 staining were much greater in the sham corneas than in the grafted and normal corneas. K3 and K13 staining demonstrated that CAOMECS transplanted corneas had much more K3- and less K13- positive cells compared to the sham corneas. Muc5AC was decreased in the central region of grafted corneas. Very little alpha-smooth muscle actin (aSMA) staining was detected in grafted corneas, while there was a greater amount of aSMA staining in sham corneas. Staining for anti-angiogenic factor TIMP -3 was also increased, and pro-angiogenic factor MMP-3 was decreased in grafted corneas compared to sham corneas. Our results indicate that CAOMECS grafts resulted in improved epithelialization of the corneal surface and decreased vascularization and fibrosis of the diseased corneas.

L-glutamine therapy reduces endothelial adhesion of sickle red blood cells to human umbilical vein endothelial cells.

BACKGROUND: We have previously demonstrated that therapy with orally administered L-glutamine improves nicotinamide adenosine dinucleotide (NAD) redox potential of sickle red blood cells (RBC). On further analysis of L-glutamine therapy for sickle cell anemia patients, the effect of L-glutamine on adhesion of sickle RBC to human umbilical vein endothelial cells (HUVEC) was examined. METHODS: The first part of the experiment was conducted with the blood samples of the 5 adult sickle cell anemia patients who had been on L-glutamine therapy for at least 4 weeks on a dosage of 30 grams per day compared to those of patient control group. In the second part of the experiment 6 patients with sickle cell anemia were studied longitudinally. Five of these patients were treated with oral L-glutamine 30 grams daily and one was observed without treatment as the control. t-test and paired t-test were used for determination of statistical significance in cross-sectional and longitudinal studies respectively. RESULTS: In the first study, the mean adhesion to endothelial cells with the autologous plasma incubated cells were 0.97 +/- 0.45 for the treated group and 1.91 +/- 0.53 for the nontreated group (p < 0.02). Similarly with lipopolysaccharide (LPS) incubated cells the mean adhesion to endothelial cells were 1.39 +/- 0.33 for the treated group and 2.80 +/- 0.47 for the untreated group (p < 0.001). With the longitudinal experiment, mean decrease in the adhesion to endothelial cells was 1.13 +/- 0.21 (p < 0.001) for the 5 treated patients whereas the control patient had slight increase in the adhesion to endothelial cells. CONCLUSION: In these studies, oral L-glutamine administration consistently resulted in improvement of sickle RBC adhesion to HUVEC. These data suggest positive physiological effects of L-glutamine in sickle cell disease.

Inhibiting progression of coronary calcification using Aged Garlic Extract in patients receiving statin therapy: a preliminary study.

BACKGROUND: Aged Garlic Extract (AGE) reduces multiple cardiovascular risk factors, including blood pressure, cholesterol, platelet aggregation and adhesion, while stimulating nitric oxide generation in endothelial cells. However, no study has evaluated the ability of AGE to inhibit vascular calcification, a marker of plaque formation in human coronary arteries. OBJECTIVE: To assess the efficacy of Aged Garlic Extract (AGE) on changing the rate of atherosclerosis progression as compared to placebo. DESIGN: A placebo-controlled, double-blind, randomized pilot study to determine whether the atherosclerotic plaque burden detected by electron beam tomography (EBT) will change at a different rate under the influence of AGE as compared to placebo. Twenty-three patients were enrolled, and 19 patients completed the study protocol. AGE 4 ml or the equivalent amount of placebo was given to subjects. Duration of the study was 1 year. S-allylcysteine (SAC), one of the active compound of AGE, was measured in the blood as a compliance marker. RESULTS: The mean change of the calcium score (volumetric method) for the AGE group (n = 9) was 7.5 +/- 9.4% over 1 year. The placebo group (n = 10) demonstrated an average increase in calcium scores of 22.2 +/- 18.5%, significantly greater than the treated cohort (P = 0.046). There were no significant differences in individual cholesterol parameters or C reactive protein between the groups. In patients randomized to AGE, there was a nonsignificant trend for improving cholesterol/high-density lipoprotein ratio (P = 0.07) and homocysteine level (P = 0.08). CONCLUSIONS: This small pilot study indicates the potential ability of AGE to inhibit the rate of progression of coronary calcification, as compared to placebo over 1 year. Should these findings be extended and confirmed in larger studies, garlic may prove useful for patients who are at high risk of future cardiovascular events.

Changing pattern of AIDS: a bone marrow study.

We compared bone marrow findings in 2 groups of patients with AIDS during 2 different periods: group 1, n = 20; male/female ratio, 19/1; and group 2, n = 120; male/female ratio, 6/1. Bone marrow iron stores were decreased significantly in group 2 (P < .01), and the incidence of AIDS-related lymphomas was higher, with frequent bone marrow involvement. Two group 1 patients had Kaposi sarcoma, and a 21-month-old girl with transfusion-transmitted AIDS had Burkitt-like lymphoma. In group 2, 44 patients had a history of malignant neoplasms, including Kaposi sarcoma (10 cases), hematologic neoplasms (33 cases), and metastatic leiomyosarcoma (1 case). Of the 120 patients, 15 (12.5%) had bone marrow involvement by malignant neoplasms. The majority of the non-Hodgkin lymphomas were high-grade lymphomas. Patients with AIDS-related malignant neoplasms had higher CD4+ cell counts and viral loads than patients without malignant neoplasms (P < .01, P < .05, respectively). The finding of decreased iron stores in patients with AIDS might aid clinical management of their anemia. The increased incidence of malignant neoplasms, especially lymphomas, in recent years might be related to prolonged survival but with incomplete reconstitution of immune function after antiretroviral therapy.