Amniotic membrane extract eye drop promotes limbal stem cell proliferation and corneal epithelium healing

Objective: Human amniotic membrane [HAM] is used as a supporter for limbal stem cell [LSC] expansion and corneal surgery. The aim of study is to use HAM extracts from healthy donors to enhance proliferation of LSCs in vitro and in vivo

Materials and Methods: In this interventional experimental study, the effective and cytotoxic doses of the amniotic membrane extract eye drops [AMEED] was assessed by adding different concentrations of AMEED [0-2.0 mg/ml] to LSC cultures for 14 days. Subsequently, the expression levels of ATP-binding cassette sub-family G member 2 [ABCG2, a putative stem cell marker], cytokeratin 3 [K3, corneal maker], K12 and K19 [corneal-conjunctival cell makers] were assessed by real-time polymerase chain reaction [PCR]. In the second step, the corneal epithelium of 10 rabbits was mechanically removed, and the right eye of each rabbit was treated with 1 mg/ml AMEED [every 2 hours [group 1] or every 6 hours [group 2]]. The left eyes only received an antibiotic. The corneal healing process, conjunctival infection, degree of eyelid oedema, degree of photophobia, and discharge scores were evaluated during daily assessments. Finally, corneal tissues were biopsied for pathologic evidences

Results: In comparison to the positive control [10% foetal bovine serum [FBS]], 0.1-1 mg/ml AMEED induced LSC proliferation, upregulated ABCG2, and downregulated K3. There were no remarkable differences in the expression levels of K12 and K19 [P>0.05]. Interestingly, in the rabbits treated with AMEED, the epithelium healing duration decreased from 4 days in the control group to 3 days in the two AMEED groups, with lower mean degrees of eyelid oedema, chemosis, and infection compared to the control group. No pathologic abnormalities were observed in either of the AMEED groups

Conclusion: AMEED increases LSCs proliferation ex vivo and accelerates corneal epithelium healing in vivo without any adverse effects. It could be used as a supplement for LSC expansion in cell therapy

Healing effects of dried and acellular human amniotic membrane and mepitelas for coverage of skin graft donor areas; a randomized clinical trial

Objective: To compare the healing effects of dried and acellular human amniotic membrane and Mepitel for coverage of split-thickness graft donor site [STGDS]

Methods: Twenty patients who underwent STGDS regeneration surgery in identical anatomic regions were enrolled in this randomized controlled clinical trial conducted in Hazrate Fatemeh hospital [Iran]. Patients were randomly assigned in 3 groups of wound dressing; group A by Mepitel, group B AmiCare [Dried amniotic membrane] and group C OcuReg-A [Acellular amniotic membrane]. Re-epithelization rate [healing time], pain sensation, scar formation and infection rate were assessed till complete healing was achieved

Results: Our results showed no significant difference between Amicare, OcuReg-A and Mepitel in the features analyzed by us including: Re-epithelization rate [healing time] p=0.573, Pain sensation p=day 4[th]: 0.131, day 8[th]: 0.93 and day 12[th]: 0.365, Scar formation p>0.05 and Infection rate

Conclusion: Our findings confirmed the safety and efficacy of Ami Care [dried amniotic membrane] and OcuReg-A [Acellular amniotic membrane] in treatment of split-thickness donor site in comparison with Mepitel as a standard wound dressing. Trial registration number: IRCT201511118177N12

mediating role of early maladaptive schemas in the relation between personality and hardines

Objective: The purpose of this study was to determine the mediating role of the uncompromising initial schemas in the relationship between personality and hardiness

Method: The present study was a descriptive-correlational study. The statistical population included students from Payame Noor University of Qom. Based on the first stage sampling, the Faculty of Psychology was selected randomly and 550 persons were available as an example to the Big Five Factor Personality Inventory [Costa and McQuery, 1985], Early Maladaptive Schema Questionnaire [Yang, 1990] and, Personal Views Survey [PVS; Institute of Hardness, 1985] responded

Results: Path analysis showed that the research model had a good fit and 23% of the variance in the schema of separation and exclusion was explained through the neuroticism, extroversion, openness to experience, consensus, and taskability. Also, these veins and the separation and rejection schema had the ability to explain 30 percent of the toughness variance

Conclusion: The initialized schemas have a mediatorial role in relation to personality traits and hardiness. Regarding the findings, it can be argued that the personality tracks and the initialized schemas play an important role in predicting student hardiness

Umbilical cord blood platelet lysate as serum substitute in expansion of human mesenchymal stem cells

Objective: The diverse clinical applications for human mesenchymal stem cells [hM- SCs] in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate [UCB-PL] as a standard substitute for fetal bovine serum [FBS] and human peripheral blood-PL [PB-PL]

Materials and Methods: In this experimental study, platelet concentrates [PC] from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing [viral and microbial], total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS

Results: UCB-PL contained high levels of protein content, platelet-derived growth factor-AB [PDGF-AB], and transforming growth factor [TGF] compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70?C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages

Conclusion: PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy

STAT3 is overactivated in gastric cancer stem-like cells

Objective: Gastric cancer [GC] is widely associated with chronic inflammation. The pro inflammatory microenvironment provides conditions that disrupt stem/progenitor cell proliferation and differentiation. The signal transducer and activator of transcrip-tion-3 [STAT3] signaling pathway is involved in inflammation and also contributes to the maintenance of embryonic stem cell [ESCs] pluripotency. Here, we have investi-gated the activation status of STAT3 in GC stem-like cells [GCSLCs]

Materials and Methods: In this experimental research, CSLCs derived from the human GC cell line MKN-45 and patient specimens, through spheroid body formation, characterized and then assayed for the STAT3 transcription factor expression in mRNA and protein level further to its activation

Results: Spheroid cells showed higher potential for spheroid formation than the parental cells. Furthemore, stemness genes NANOG, c-MYC and SOX-2 were over expressed in spheroids of MKN-45 and in patient samples. In MKN-45 spheroid cells, epithelial mesenchymal transition [EMT] related markers CDH2, SNAIL2, TWIST and VIMENTIN were upregulated [P<0.05], but we observed no change in expression of the E-cadherin epithelial marker. These cells exhibited more resistance to docetaxel [DTX] when compared with parental cells [P<0.05] according to the MTS assay. Although immunostaining and Western blotting showed expression of the STAT3 protein in both spheroids and parents, the mRNA level of STAT3 in spheroids was higher than the parents. Nuclear translocation of STAT3 was accompanied by more intensive phospho-STAT3 [p-STAT3] in spheroid structures relative to the parent cells according to flow cytometry analysis [P<0.05]

Conclusion: The present findings point to STAT3 over activation in GCSLCs. Complementary experiments are required to extend the role of STAT3 in stemness features and invasion properties of GCSCs and to consider the STAT3 pathway for CSC targeted therapy

CD133 is not suitable marker for isolating melanoma stem cells from D10 cell line

Objective: Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells [CSCs] are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture

Materials and Methods: In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting [FACS] based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133+, CD133- and spheroid cells. Significant differences of the two experimental groups were compared using student's t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold

Results: Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133+ cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A compared to other groups [P<0.05]

Conclusion: Although CD133+ derived melanoma cells represented stemness features, our findings demonstrated that spheroid culture could be more effective method to enrich melanoma stem cells

In vitro differentiation of human umbilical cord blood CD133[+] cells into insulin producing cells in co-culture with rat pancreatic mesenchymal stem cells

Pancreatic stroma plays an important role in the induction of pancreatic cells by the use of close range signaling. In this respect, we presume that pancreatic mesenchymal cells [PMCs] as a fundamental factor of the stromal niche may have an effective role in differentiation of umbilical cord blood cluster of differentiation 133[+] [UCB-CD133[+]] cells into newly-formed beta-cells in vitro. This study is an experimental research. The UCB-CD133[+] cells were purified by magnetic activated cell sorting [MACS] and differentiated into insulin producing cells [IPCs] in co-culture, both directly and indirectly with rat PMCs. Immunocytochemistry and enzyme linked immune sorbent assay [ELISA] were used to determine expression and production of insulin and C-peptide at the protein level. Our results demonstrated that UCB-CD133[+] differentiated into IPCs. Cells in islet-like clusters with [out] co-cultured with rat pancreatic stromal cells produced insulin and C-peptide and released them into the culture medium at the end of the induction protocol. However they did not respond well to glucose challenges. Rat PMCs possibly affect differentiation of UCB-CD133[+] cells into IPCs by increasing the number of immature beta-cells

Differentiation potential of O Bombay human-induced pluripotent stem cells and human embryonic stem cells into fetal erythroid-like cells

There is constant difficulty in obtaining adequate supplies of blood components, as well as disappointing performance of "universal" red blood cells. Advances in somatic cell reprogramming of human-induced pluripotent stem cells [hiPSCs] have provided a valuable alternative source to differentiate into any desired cell type as a therapeutic promise to cure many human disease. In this experimental study, we examined the erythroid differentiation potential of normal Bombay hiPSCs [B-hiPSCs] and compared results to human embryonic stem cell [hESC] lines. Because of lacking ABO blood group expression in B-hiPSCs, it has been highlighted as a valuable source to produce any cell type in vitro. Similar to hESC lines, hemangioblasts derived from B-hiPSCs expressed approximately 9% KDR+CD31+ and approximately 5% CD31+CD34+. In semisolid media, iPSC and hESC-derived hemangioblast formed mixed type of hematopoietic colony. In mixed colonies, erythroid progenitors were capable to express CD71+GPA+HbF+ and accompanied by endothelial cells differentiation. Finally, iPS and ES cells have been directly induced to erythropoiesis without hemangioblast formation that produced CD71+HbF+erythroid cells. Although we observed some variations in the efficiency of hematopoietic differentiation between iPSC and ES cells, the pattern of differentiation was similar among all three tested lines

Purified protein fraction of garlic extract modulates cellular immune response against breast transplanted tumors in BALB/c mice model

Garlic [Allium sativum] has anti-inflammatory, anti-mutagenesis, and immunomodulatory properties that modulate anti-tumor immunity and inhibit tumor growth. In this study we have examined the effect of a protein fraction isolated from fresh garlic on anti-tumor response and intra-tumor lymphocyte infiltration. In this experimental study a protein fraction was purified from fresh garlic bulbs using ultra filtration, followed by chromatofocusing, and SDS-PAGE analysis. Anti-tumor activity was assessed by intra-tumor injection of the protein fraction and garlic extract, itself, into groups of 5 mice each. The percentage of peripheral blood and intra-tumor CD4[+] and CD8[+] cells were assessed by flow cytometry. Un-paired student's t test using the SPSS program was applied for all statistical analyses. Garlic extract included different type of proteins with different molecular weight. One of protein's fraction was immunomodeulator and was composed of three single polypeptides, with molecular masses of -10-13 kDa and different isoelectric points [pI]. These molecules augmented the delayed type hypersensitivity [DTH] response compared to the control group. Intratumor injection of the fraction provoked a significant increase in the CD8[+] subpopulation of T-lymphocytes, as well as a decrease in tumor size. The fraction increased peripheral blood CD8[+] T-lymphocytes in treated animals. The data confirms that protein fractions purified from fresh garlic bulbs augment CD8[+] T-cell infiltration into the tumor site, inhibiting tumor growth more efficiently than garlic extract. These findings provide a basis for further investigations on the purified polypeptide as a useful candidate for immunomodulation and tumor treatment

[In-vitro differentiation of human embryonic stem cells into hemangioblasts]

Human embryonic stem cells [hESCs] are capable of self-renewal and large-scale expansion. They also have the capacity to differentiate into a variety of cell types including liver, cardiac and neuron cells. However, it is not yet clear whether hESCs can differentiate to hemangioblasts under in-vitro conditions. Hemangioblasts are bipotential progenitors that can generate hematopoietic lineages and endothelial cells. The aim of this study was to identify the potential of human Royan H5 embryonic stem cells in differentiating into hemangioblast cells. HESCs were cultured at suspension system in DMEM/F12 supplemented with bFGF. 7-day old cells differentiated into blast cells under defined condition consisting of hematopoietic cytokines including BMP4, VEGF, etc. Blast cell markers kinase insert domain receptor [KDR], CD31, and CD34 were evaluated by flow cytometry and blast gene expressions [TAL-1, Runx-1 and CD34] were detected by qRT-PCR. Clonogenic assays were performed in semisolid medium by colony forming unit-assays. The hESCs [Royan H5] had the capacity of differentiating into hemangioblast cells. We could detect colonies that expressed 79% +/- 12.5 KDR+, 5.6% +/- 2.8 CD31[+]-CD34[+] and 6% +/- 2.12 KDR[+]-CD31[+] on day 8 in the hESCs. Up-regulation of TAL-1, Runx-1 and CD34 occurred during hemangioblast commitment [P

Mesenchymal stem cells derived from rat epicardial versus epididymal adipose tissue

Some investigation has indicated that adipose-derived stem cells possess different surface epitopes and differentiation potential according to the localization of fat pad from which the cells were derived. In the present study proliferation capacity and aging of such cells were explored. Adherent cells were isolated from the collagenase digests of adipose tissues excised from rat epicardial and epididymal regions and propagated with several subcultures. The cells were then investigated whether or not they were able to differentiate into bone, cartilage and adipose cell lineages. Studied cells from two adipose tissues were also compared with respect to their in vitro proliferation capacity. The presence of senescent cells in the culture was determined and compared using senescence-associated [SA] beta-galactosidase staining method. Successful differentiations of the cells were indicative of their mesenchymal stem cells [MSCs] identity. Epicardial adipose-derived cells tended to have a short population doubling time [45 +/- 9.6 hr] than the epididymal adipose-derived stem cells [69 +/- 16 hr, P< 0.05]. Colonogenic activity and the growth curve characteristics were all better in the culture of stem cells derived from epicardial compared to epididymal adipose tissue. Comparatively more percentage of senescent cells was present at the cultures derived from epididymal adipose tissue [P< 0.05]. Our data emphasize on the differences existed between the stem cells derived from adipose depots of different anatomical sites in terms of their proliferative capacity and in vitro aging. Such data can help understand varying results reported by different laboratories involved in adipose stem cell investigations

[Corneal differentiation following HSC70 and HSP72 expression in air-exposed limbal stem cells cultured on denuded amniotic membrane]

The aim of this study is to create an ex vivo model to examine the expression of two heat shock protein 70 [HSP70] family members, heat shock protein 72 [HSP72] and heat shock constitute protein 70 [HSC70], at the mRNA and protein levels in differentiating corneal cells from air exposed limbal stem cells. Limbal biopsies were cultured as explants on a cellular amniotic membrane for 14 days. The cells were then exposed to air for 16 extra additional days. The proposed expression of limbal stem cell markers [p63, ABCG2], corneal markers [K3/12, connexin 43], as well as HSP72 and HSC70 were analyzed by reverse transcription-polymerase chain reaction [RT-PCR] at the mRNA level, and by immunocytochemistry and flow cytometry at the protein level both pre and post air exposure. Fresh limbal and corneal tissues were used as control group. Air exposure decreased expression of p63 and increased expression of K3/ K12 indicating an increase in the number of corneal cells. Our data showed that HSP72 and HSC70 were expressed at the mRNA level before and after air exposure while their expression significantly increased post air exposure at the protein level. We assume HSC70 expression may be related to early and terminal stages of differentiation in cultured limbal stem cells. In addition, limbal stem cells were protected during normal development against oxidative stress thru increased HSP72 expression. These findings may have broader implications in development of therapeutic strategies for treating wound healing disorders by induction of HSPs

Immediate exposure to TNF-alpha activates Dendritic cells derived from non-purified cord blood mononuclear cells

Tumor necrosis factor alpha [TNF-alpha] is a primary mediator of immune regulation and might be required in the early stages of DC development from CD34[+] cells. However, details of optimal timing of exposure to TNF-alpha in DC development process in monocytes or non-purified hematopoitic cells are still lacking and clear benefits of this approach to the development of DCs remain to be validated. To evaluate the effect of early and late exposure to TNF-alpha on DC development from non-purified cord blood mononuclear cells. To define the effects of early exposure to TNF-alpha on cord blood mononuclear cells, we cultured UCB-MNC in the presence of SCF, Flt3L, GM-CSF and IL-4 for 14 days and matured them for an extra 4 days. TNF-alpha was added on day 0, 7 and 14 in TNF-alpha [+] group, and only on day 14 in TNF-alpha [-] group where it was used only as a maturation factor. Immediate exposure to TNF-alpha was shown to: [1] enhance the survival of cells in the first week of culture; [2] produce mature DCs with higher maturation markers [CD80, CD83, CD86 and HLA-DR]; and [3] increase secretion of IL-12 by mature DCs. In contrast, delayed exposure to TNF-alpha stimulate mature DCs with less purity producing a high level of IL-10 and a low level of IL-12. We developed a simple, easy and cost effective method to generate DCs from non-fractionating mononuclear cells in this study. Also we confirm the presence of a large number of functional DCs under inflammatory conditions, where local concentrations of TNF-alpha were high

effects of extracellular matrices and co-culture systems on cultured limbal stem cells

To study the effects of different matrices and co-culture systems on cultured limbal stem cells [LSCs]. Limbal explants were co-cultured with limbal fibroblasts [LF] and/or mouse embryonic fibroblasts [MEF] on filter inserts coated with amniotic membrane [AM], matrigel [MAT] and collagen type I [COL]. This study revealed that AM facilitated the cell migration and expansion significantly in comparison with other matrices. However, the gene expression profile of stemness markers of LSCs showed no significant differences among the experimental groups. The data indicated that at least in two-dimensional culture systems, the mentioned matrices have no significant effect on switching the expression of genes involved in differentiation process. In addition, the results of the two co-culture systems in case of different feeders, including MEF and LF were similar in growth rate and also preserving stemness quality of cultured limbal cells. To exclude the pollution of transplantable cultivated cells with probable mouse viruses, LF with human origin is recommended as feeder. Hence, limbal explants grown on AM in co-culture with LF will promise a quick and safe model for preparing undifferentiated epithelial sheets suitable for transplantation

Early results of autologous cultivated limbal stem cell transplantation in total limbal stem cell deficiency

To report the early results of transplantation of autologous limbal stem cells cultivated on amniotic membrane [AM] in patients with total unilateral limbal stem cell deficiency [LSCD]. Four eyes of 4 patients with total unilateral LSCD confirmed with impression cytology underwent transplantation of autologous limbal stem cell cultivated on AM. At each follow up visit, a complete eye examination with special attention to recurrence or regression of vascularization, corneal opacification, and epithelial defect healing was performed. Digital imaging was performed at each follow up visit. Impression cytology was repeated in all cases after surgery. The patients were followed for 5-13 months. Visual acuity improved in all cases. Decrease in corneal opacification and vascularization was obvious in 3 cases with coverage of the cornea with corneal epithelium. Sectoral conjunctivalization was evident in these 3 cases, however the corneas were ready for transplantation. The procedure failed in one case with total corneal conjunctivalization. Transplantation of autologous stem cells cultivated on AM seems to be an effective way for total LSCD. More definite judgment needs longer follow up together with long-term results of corneal transplantation in these patients