A novel method for a high enrichment of human corneal epithelial stem cells for genomic analysis

Understanding the molecular mechanisms that regulate the corneal epithelial stem cells (CESCs) in maintaining corneal homeostasis remains elusive largely due to the lack of a specific marker for their isolation. This study aims to enrich CESCs from human donor limbal epithelium and to evaluate the level of enrichment based on expression of ΔNp63α, a putative CESC marker. A two‐stage enrichment of CESCs was carried out. (a) The limbal basal epithelial cells were isolated by differential enzymatic treatment and five‐fold enrichment was achieved from 2% of CESCs present in the total limbal epithelium. The CESCs were quantified on the basis of two parameters—high expression of p63/ABCG2 and nucleus to cytoplasmic (N/C) ratio ≥0.7. (b) Cytospin smears of isolated basal cells were Giemsa stained and cells with N/C ratio ≥0.7 were separated by laser capture microdissection. This strategy resulted in an enrichment of CESCs to 78.57% based on two‐parameter analysis using p63 and 76.66% using ABCG2. RT‐PCR was carried out for ΔNp63 isoforms (α, β, and γ) and connexin‐43, with GAPDH for normalization. The expression of ΔNp63α was restricted to the enriched population of CESCs in contrast to its absence in limbal basal cells with N/C ratio <0.7 and CCECs. The unique expression of ΔNp63α and 5.9‐fold reduced connexin‐43 expression in the enriched population of CESCs indicates its high purity. Further analysis of these cells will help in elucidating the molecular mechanisms associated with stemness and also in identifying a specific marker for CESCs.     

Improvement of transfection with reprogramming factors in urinederived cells

Human-induced pluripotent stem cells (iPSCs) are an accessible source of adult-derived, patient-specific pluripotent stem cells for use in basic research, drug discovery, disease modeling, and stem cell therapy. Improving the accessibility of methods to obtain iPSCs regardless of the cell source can enhance their clinical application. Therefore, our purpose is to report a simple protocol to obtain iPS-like cells from urine-derived renal epithelial cells (RECs) using different extracellular matrices and transfection reagents. In this study, we began by culturing urine-derived cells from healthy donors to establish a primary culture of renal epithelial cells, followed by their characterization. Subsequently, we generated iPS-like cells by transfecting renal epithelial cells (RECs) with vectors expressing Oct4, Sox2, L-Myc, Lin-28, and Klf4, and we compared the efficacy of different extracellular matrices and transfection reagents. The resultant iPS-like cells showed a human embryonic stem cell-like morphology and expressed the specific pluripotency markers Oct3/4, Nanog, Lin28, and Klf4. We concluded that Lipofectamine Stem Cell transfection reagent is more effective than FuGENE in obtaining iPS-like cells under the conditions tested. Moreover, the three matrices are similar in their efficiency of obtaining iPS-like cells. This report provides an experimental protocol for obtaining and generating iPS-like cells from urine samples for further cell therapy research on different human diseases.     

Skin regeneration stimulation: the role of PCL‐platelet gel nanofibrous scaffold

Skin is the largest organ of the human body. Thus far, tissue engineering of skin has developed rapidly and has used many types of growth factors and nanofibrous scaffolds. In this study, we differentiated neonate keratinocytes for epithelialization on the polycaprolactone‐Platelet gel (PCL‐PG) scaffold. Fabricated PCL nanofibers prepared by electrospinning technology and coated by platelet gel. Subsequently, the structure of the scaffold was evaluated by SEM, FTIR‐ATR, contact angle and tensile test assays. After seeding the neonate keratinocytes on neat PCL and PCL‐PG scaffolds, the epidermal maturation was tested by detecting cytokeratin 10 and loricrin determinants by immunocytochemistry; moreover, keratinocyte genes such as keratin 14, keratin 10, and Involucrin were investigated by real‐time PCR. The results of MTT assay indicated an increase in cell viability and cell proliferation of neonate keratinocytes on PCL‐PG nanofiber scaffolds compared with PCL. RT‐PCR and immunocytochemical analysis showed better cell differentiation on the PCL‐PG scaffolds than neat PCL. Furthermore, SEM microscopy images demonstrated that neo‐keratinocytes enhance adhesion and proliferation on PCL‐PG nanofiber scaffolds. We found that PG increases biocompatibility and wettability of scaffold, cell adhesion, and expression of keratinocyte markers. Overall, this procedure is recommended to be employed in skin tissue engineering and wounds healing.     

High level of circPTN promotes proliferation and stemness in gastric cancer

Increasing evidence proves that circular RNAs (circRNAs) play an important role in regulating the biological behaviors of tumors. The central purpose of this research was to investigate the functions of circRNA in gastric cancer. The utilization of real-time PCR was to test circPTN expression in gastric cancer cells. Cell counting colony formation assays, CCK-8 assay, and EdU assay were used to investigate proliferation. Transwell assay was applied to investigate migration. We discovered that circPTN was highly expressed in gastric cancer cells. Low expression of circPTN inhibits gastric cancer cell proliferation and migration. Elevated expression of circPTN promotes gastric cancer cell proliferation and migration. Moreover, we discovered that circPTN could accelerate self-renewal and increase the expression of stemness markers. The results of our study suggested that a high level of circPTN expression promotes the proliferation and stemness of gastric cancer cells.     

Three‐dimensional Co‐culture of hepatic progenitor cells and mesenchymal stem cells in vitro and in vivo

Introduction: Here we co‐cultured hepatic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) to investigate whether the co‐culture environments could increase hepatocytes form. Methods: Three‐dimensional (3D) co‐culture model of HPCs and MSCs was developed and morphological features of cells were continuously observed. Hepatocyte specific markers Pou5f1/Oct4, AFP, CK‐18 and Alb were analyzed to confirm the differentiation of HPCs. The mRNA expression of CK‐18 and Alb was analyzed by RT‐PCR to investigate the influence of co‐culture model to the terminal differentiation process of mature hepatocytes. The functional properties of hepatocyte‐like cells were detected by continuously monitoring the albumin secretion using Gaussia luciferase assays. Scaffolds with HPCs and MSCs were implanted into nude mouse subcutaneously to set up the in vivo co‐culture model. Results: Although two groups formed smooth spheroids and high expressed of CK‐18 and Alb, hybrid spheroids had more regular structures and higher cell density. CK‐18 and Alb mRNA were at a relatively higher expression level in co‐culture system during the whole cultivation time (P < 0.05). Albumin secretion rates in the hybrid spheroids had been consistently higher than that in the mono‐culture spheroids (P < 0.05). In vivo, the hepatocyte‐like cells were consistent with the morphological features of mature hepatocytes and more well‐differentiated hepatocyte‐like cells were observed in the co‐culture group. Conclusions: HPCs and MSCs co‐culture system is an efficient way to form well‐differentiated hepatocyte‐like cells, hence, may be helpful to the cell therapy of hepatic tissues and alleviate the problem of hepatocytes shortage. Microsc. Res. Tech. 78:688–696, 2015. © 2015 Wiley Periodicals, Inc.     

Agmatine inhibits hypoxia-induced TNF-alpha release from cultured retinal ganglion cells

The effect of hypoxia on the release of tumor necrosis factor-alpha (TNF-alpha) in transformed rat retinal ganglion cells (RGCs) and the effect of agmatine on the hypoxia-induced production of TNF-alpha in RGCs were evaluated. RGCs were cultured under hypoxic conditions with 5% oxygen, with or without 100 microM agmatine. The expression levels of TNF-alpha and its receptor-1 (TNF-R1) were investigated by Western blot analysis. After 6 hours of hypoxia, we noted an increase in TNF-alpha production in RGCs. Agmatine significantly reduced TNF-alpha level after 12 hours of hypoxic treatment. The expression of TNF-R1 was not affected by the hypoxia or agmatine treatment. Our results show that agmatine inhibits the TNF-alpha production of RGCs in hypoxic condition. These results demonstrate a possible neuroprotective mechanism for agmatine against hypoxic damage in RGCs.     

Ultrastructural characteristics of ovine bone marrow‐derived mesenchymal stromal cells cultured with a silicon stabilized tricalcium phosphate bioceramic

Bioceramics are being used in experimental bone engineering application in association with bone marrow derived mesenchymal stem cells (BM‐MSCs) as a new therapeutic tool, but their effects on the ultrastructure of BM‐MSCs are yet unknown. In this study we report the morphological features of ovine (o)BM‐MSCs cultured with Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (SiTCP), able to promote the repair of induced bone defect in sheep model. oBM‐MSCs were isolated from the iliac crest, cultured until they reached near‐confluence and incubated with SiTCP. After 48 hr the monolayers were highly damaged and only few cells adhered to the plastic. Thus, SiTCP was removed, and after washing the cells were cultured until they became confluent. Then, they were trypsinizated and processed for transmission electron microscopy (TEM) and RT‐PCR analysis. RT‐PCR displayed that oBM‐MSCs express typical surface marker for MSCs. TEM revealed the presence of electron‐lucent cells and electron‐dense cells, both expressing the CD90 surface antigen. The prominent feature of electron‐lucent cells was the concentration of cytoplasmic organelles around the nucleus as well as large surface blebs containing glycogen or profiles of endoplasmic reticulum. The dark cells had a multilocular appearance by the presence of peripheral vacuoles. Some dark cells contained endocytic vesicles, lysosomes, and glycogen aggregates. oBM‐MSCs showed different types of specialized interconnections. The comparison with ultrastructural features of untreated oBM‐MSCs suggests the light and dark cells are two distinct cell types which were differently affected by SiTCP bioceramic. Skelite cultured ovine BM‐MSCs display electron‐dense and electron‐lucent cells which are differently affected by this bioceramic. This suggests that they could play a different role in bioceramic based therapy.     

Phenotypic,morphological, and metabolic characterization of vascular-spheres from human vascular mesenchymal stem cells

The ability to form spheroids under non-adherent conditions is a well-known property of human mesenchymal stem cells (hMSCs), in addition to stemness and multilineage differentiation features. In the present study, we tested the ability of hMSCs isolated from the vascular wall (hVW-MSCs) to grow as spheres, and provide a characterization of this 3D model. hVW-MSCs were isolated from femoral arteries through enzymatic digestion. Spheres were obtained using ultra-low attachment and hanging drop methods. Immunophenotype and pluripotent genes (SOX-2, OCT-4, NANOG) were analyzed by immunocytochemistry and real-time PCR, respectively. Spheres histological and ultrastructural architecture were examined. Cell viability and proliferative capacity were measured using LIVE/DEATH assay and ki-67 proliferation marker. Metabolomic profile was obtained with liquid chromatography–mass spectrometry. In 2D, hVW-MSCs were spindle-shaped, expressed mesenchymal antigens, and displayed mesengenic potential. 3D cultures of hVW-MSCs were CD44⁺, CD105^low, CD90^low, exhibited a low propensity to enter the cell cycle as indicated by low percentage of ki-67 expression and accumulated intermediate metabolites pointing to slowed metabolism. The 3D model of hVW-MSCs exhibits stemness, dormancy and slow metabolism, typically observed in stem cell niches. This culture strategy can represent an accurate model to investigate hMSCs features for future clinical applications in the vascular field.     

Bones Morphogenic Protein‐4 and retinoic acid combined treatment comparative analysis for in vitro differentiation potential of murine mesenchymal stem cells derived from bone marrow and adipose tissue into germ cells

Nowadays, infertility is no longer considered as an unsolvable disorder due to progresses in germ cells derived from stem lineage with diverse origins. Technical and ethical challenges push researchers to investigate various tissue sources to approach more efficient gametes. The purpose of the current study is to investigate the efficacy of a combined medium, retinoic acid (RA) together with Bone Morphogenic Protein‐4 (BMP4), on differentiation of Bone Marrow Mesenchymal Stem Cells (BMMSCs) and adipose‐derived mesenchymal stem cells (ADMSCs) into germ cells. Murine MSCs were obtained from both Bone Marrow (BM) and Adipose Tissue (AT) samples and were analyzed for surface markers to get further verification of their nature. BMMSCs and ADMSCs were induced into osteogenic and adipogenic lineage cells respectively, to examine their multipotency. They were finally differentiated into germ cells using media enriched with BMP4 for 4 days followed by addition of RA for 7 days (11 days in total). Analyzing of differentiation potential of BMMSCs‐ and ADMSCs were performed via Immunofluorescence, Flowcytometry and Real time‐PCR techniques for germ cell‐specific markers (Mvh, Dazl, Stra8 and Scp3). Mesenchymal surface markers (CD90 and CD44) were expressed on both BMMSCs and ADMSCs, while endothelial and hematopoietic cell markers (CD31 and CD45) had no expression. Finally, all germ‐specific markers were expressed in both BM and AT. Although germ cells differentiated from ADMSCs showed faster growth and proliferation as well as easy collection, they significantly expressed germ‐specific markers lower than BMMSCs. This suggests stronger differentiation potential of murine BMMSCs than ADMSCs.     

Role of spermatogonial stem cells extract in transdifferentiation of 5‐Aza‐2′‐deoxycytidine‐treated bone marrow mesenchymal stem cells into germ‐like cells

As one of the induced pluripotent stem cells (iPSCs) methods, spermatogonial stem cells (SSC_S) extract is considered as new approach in stem cell therapy of infertility. 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) inhibits methyltransferase enzyme, and induces gene reprogramming; herein, the effects of SSC_S extract incubation in 5‐aza‐dC‐treated bone marrow mesenchymal stem cells (BMMSCs) has been surveyed. BMMSCs were isolated from femurs of three to four weeks old male NMRI mice, and the cells at passage three were treated with 2 µM 5‐aza‐dC for 72 hours. SSCs were isolated, cultured, and harvested at passage three to collect SSC_S extract; BMMSCs were then incubated with SSC_S extract in the three time periods: 72 hours, one week and two weeks. There were five groups: control, sham, extract, 5‐aza‐dC and extract‐5‐aza‐dC. After one week of incubation, flow cytometry and real‐time polymerase chain reaction (PCR) exhibited high levels of expression for β1‐ and α6‐integrins and promyelocytic leukaemia zinc finger (PLZF) in extract and extract‐5‐aza‐dC groups (P < 0.05 vs. control and 5‐aza‐dC), and cells in these two groups had two forms of morphology, round and fusiform, similar to germ‐like cells. 5‐aza‐dC had no significant effects during the three time periods of evaluation. These data disclose the effectiveness of SSCs extract incubation in transdifferentiation of BMMSCs into germ‐like cells; this strategy could introduce a new approach for treatment of male infertility in clinic. Microsc. Res. Tech. 79:365–373, 2016. © 2016 Wiley Periodicals, Inc.     

Morphology and expression status investigations of specific surface markers on B‐cell chronic lymphocytic leukemia cells

The morphology of cells and expression status of specific surface markers [cluster of differentiation (CD)], such as CD5, CD19, CD20, CD38, and CD45, have long been considered as the essential indicators for the diagnosis and prognosis of B‐cell chronic lymphocytic leukemia (B‐CLL). Clinically, it is difficult to simultaneously obtain cell morphology and distribution of surface markers with flow cytometry, especially for some surrogate markers such as CD38. Here, as an alternative and complementary prognostic method, fluorescence microscopy and image processing method are introduced to directly visualize the cells from patients and to quantitatively determine the expression status of surface markers. In this study, the morphological parameters of B‐CLL cells were measured to establish the correlation between the cellular morphology and the surface marker expression. It was clear that the CD38+ and CD38? B‐CLL cells from the same CD38+ patients had hardly any size differences; however, an increase in perimeter was observed for CD38? patients. Moreover, the expression level of the receptors on the cell was independent of the cell size. There was no evidence showing that the expression intensities of CD19 and CD38 were related to each other for the CD38+ B‐CLL cells. On the same cells, CD5 was more selectively expressed on the cell membrane; however, the expression patterns suggested that the cell membrane of CD38? B‐CLL cells contained the least expression level of CD19. Microsc. Res. Tech. 76:1147–1153, 2013. © 2013 Wiley Periodicals, Inc.     

Chondrogenic differentiation of human adipose mesenchimal stem cells: Influence of a biomimetic gelatin genipin crosslinked porous scaffold

Human adipose derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with biomimetic materials. In this study, the chondrogenic potential of a porous gelatin based scaffold genipin (GNP) crosslinked was investigated in human mesenchymal stem cells obtained from adipose tissue. Cells were cultured up to 4 weeks on the scaffold and on monolayer, MTT assay was performed to evaluate cell viability, light, and transmission electron microscopy were carried out to demonstrate cell proliferation, scaffold adhesion, and cell colonization inside the porous architecture of the biomaterial. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan, and versican was investigated by Real Time PCR. Results showed an high cell viability, adhesion, and colonization of the scaffold. Real Time PCR data demonstrated an upregulation of all the chondrogenic markers analyzed. In conclusion, 3D gelatin GNP crosslinked porous scaffold provides an improved environment for chondrogenic differentiation of stem cells compared with cell monolayer culture system. Microsc. Res. Tech. 77:928–934, 2014. © 2014 Wiley Periodicals, Inc.     

Adrenomedullin functions as an important tumor survival factor in human carcinogenesis

Adrenomedullin (AM) is a pluripotent regulatory peptide initially isolated from a human pheochromocytoma (adrenal tumor) and subsequently shown to play a critical role in cancer cell division, tumor neovascularization, and circumvention of programmed cell death, thus it is an important tumor cell survival factor underlying human carcinogenesis. A variety of neural and epithelial cancers have been shown to produce abundant amounts of AM. Recent findings have implicated elevation of serum AM with the onset of malignant expression. In addition, patients with tumors producing high levels of this peptide have a poor prognostic clinical outcome. Given that most human epithelial cancers display a microenvironment of reduced oxygen tension, it is interesting to note that AM and several of its receptors are upregulated during hypoxic insult. The existence of such a regulatory pathway has been implicated as the basis for the overexpression of AM/AM-R in human malignancies, thereby generating a subsequent autocrine/paracrine growth advantage for the tumor cell. Furthermore, AM has been implicated as a potential immune suppressor substance, inhibiting macrophage function and acting as a newly identified negative regulator of the complement cascade, protective properties which may help cancer cells to circumvent immune surveillance. Hence, AM's traditional participation in normal physiology (cited elsewhere in this issue) can be extended to a primary player in human carcinogenesis and may have clinical relevance as a biological target for the intervention of tumor progression.     

Light exposure and cell viability in fluorescence microscopy

Test systems for measuring cell viability in optical microscopy (based on colony formation ability or lysosomal integrity) were established and applied to native cells as well as to cells incubated with fluorescence markers or transfected with genes encoding for fluorescent proteins. Human glioblastoma and Chinese hamster ovary cells were irradiated by various light doses, and maximum doses where at least 90% of the cells survived were determined. These tolerable light doses were in the range between 25 J cm^?2 and about 300 J cm^?2 for native cells (corresponding to about 250?3000 s of solar irradiance and depending on the wavelength as well as on the mode of illumination, e.g. epi‐ or total internal reflection illumination) and decreased to values between 50 J cm^?2 and less than 1 J cm^?2 upon application of fluorescent markers, fluorescent proteins or photosensitizers. In high‐resolution wide field or laser scanning microscopy of single cells, typically 10?20 individual cell layers needed for reconstruction of a 3D image could be recorded with tolerable dose values. Tolerable light doses were also maintained in fluorescence microscopy of larger 3D samples, e.g. cell spheroids exposed to structured illumination, but may be exceeded in super‐resolution microscopy based on single molecule detection.     

UCK2 promotes the proliferation,migration, and invasion of trophoblast cells in preeclampsia by activating the STAT3 pathway

Background: Preeclampsia (PE), characterized by hypertension and proteinuria, leads to serious maternal and infant complications. Uridine-cytidine kinase 2 (UCK2) belongs to the UCK family, a class of enzymes that catalyzes the conversion of uridine and cytidine to monophosphate form. However, the role of UCK2 in PE has not been reported. Methods: The expression of UCK2 was detected in the placenta of PE patients and N(ω)-nitro-L-arginine methyl esterinduced PE mouse model. Through forced up-regulation or down-regulation of UCK2 in vitro, we examined the effects of UCK2 on the proliferation, apoptosis, migration, and invasion of trophoblast cells. Stattic, the inhibitor of STAT3 pathway, was used to investigate whether the STAT3 pathway mediates the biological function of UCK2 in trophoblast cells. Results: The present study found that UCK2 showed low expression in the placenta of PE patients and PE mouse model. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and flow cytometry assays verified that up-regulation of UCK2 promoted the proliferation of trophoblast cells, while the silence of UCK2 suppressed cell proliferation. Besides, flow cytometry and TdT-mediated dUTP Nick-End Labeling assays demonstrated that knockdown of UCK2 resulted in apoptosis of trophoblast cells. The wound healing and transwell assays showed that the migration and invasion activities of the trophoblast cells were facilitated by the overexpression of UCK2 and were blocked by the silence of UCK2. Furthermore, the expression of phosphorylated STAT3 was increased with the upregulation of UCK2 and decreased with the inhibition of UCK2. When the STAT3 pathway was blocked by its inhibitor stattic, the promotion effects of UCK2 on trophoblast cells were suppressed. Conclusion: UCK2 promotes the proliferation, migration, and invasion of trophoblast cells, and these effects may be partly mediated by the activation of the STAT3 pathway.