miRNA let-7e targeting MMP9 is involved in adipose-derived stem cell differentiation toward epithelia

miRNA let-7e is involved in stem cell differentiation, and metalloproteinases are among its potential target genes. We hypothesized that the inhibitory action of let-7e on regulation of MMP9 expression could represent a crucial mechanism during differentiation of adipose-derived stem cells (ASCs). ASCs were differentiated with all-trans retinoic acid (ATRA) to promote differentiation, and the effect of let-7 silencing during differentiation was tested. Results indicate that ASCs cultured with ATRA differentiated into cells of the epithelial lineage. We found that ASCs cultured with ATRA or transfected with miRNA let-7e expressed epithelial markers such as cytokeratin-18 and early renal organogenesis markers such as Pax2, Wt1, Wnt4 and megalin. Conversely, the specific knockdown of miRNA let-7e in ASCs significantly decreased the expression of these genes, indicating its vital role during the differentiation process. Using luciferase reporter assays, we also showed that MMP9 is a direct target of miRNA let-7e. Thus, our results suggest that miRNA let-7e acts as a matrix metalloproteinase-9 (MMP9) inhibitor and differentiation inducer in ASCs.     

Conditioned medium from renal tubular epithelial cells initiates differentiation of human mesenchymal stem cells

Objectives: Mesenchymal–epithelial interactions play a pivotal role in tubular morphogenesis and in maintaining the integrity of the kidney. During renal repair, similar mechanisms may regulate cellular reorganization and differentiation. We have hypothesized that soluble factors from proximal tubular epithelial cells (PTC) induce differentiation of adipose-derived adult mesenchymal stem cells (ASC). This hypothesis has been tested using cultured ASC and PTC.
Material and methods: Conditioned medium was prepared from injured PTC and transferred to ASC cultures. ASC proliferation was analysed by a fluorometric and photometric assay. Signal transduction was analysed by phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2). Grade of ASC differentiation was assessed by morphological analysis and cell expression of characteristic markers.
Results: Conditioned medium significantly induced proliferation and phosphorylation of ERK1/ERK2 of ASC. After 12 days of incubation, cell morphology changed to an epithelial-like monolayer. Expression of cytokeratin 18 was induced by conditioned medium, while α-smooth muscle actin, CD49a and CD90 expression decreased. These alterations strongly indicate onset of the differentiation process to the epithelial lineage. In summary, soluble factors from PTC induce signal transduction and differentiation of ASC.
Conclusions: Our study shows that conditioned medium from renal tubular epithelial cells provides a convenient source of inductive signals to initiate differentiation of ASC towards epithelial lineage. We deduce that these interactions may play an important role during renal repair mechanisms.     

Mitogen limitation and bone morphogenetic protein-4 promote neurogenesis in SFME cells, an EGF-dependent neural stem cell line

Serum-free mouse embryo (SFME) cells are an epidermal growth factor (EGF)-dependent established line derived from brains of 16-d-old Balb/c mouse embryos. SFME cells grow indefinitely in serum-free medium without replicative senescence, chromosomal abnormalities, or malignant transformation. SFME cells express nestin, a neural stem cell marker, under serum-free conditions. Exposure to serum or transforming growth factor β (TGF-β) leads to a marked increase in differentiation toward the astrocytic lineage with expression of glial fibrillary acidic protein and other astrocyte markers. In this study, we show that treatment of SFME cells with bone morphogenetic protein-4 (BMP-4), another member of the TGF-β family, led to differentiation toward a neuronal lineage under conditions of low mitogenic stimulation (0.5 ng/mL) by EGF and fibroblast growth factor. Maximum mitogenic stimulation with 50 ng/mL EGF blocked the BMP-4 effect on neuronal differentiation, but did not block TGF-β-induced expression of markers of the astrocytic lineage. BMP-4 treatment also enhanced the activity of the neuron-specific enolase (NSE) promoter in SFME-NSE-lacZ cells that carry the gene for bacterial β-galactosidase under the control of the NSE promoter. Extended BMP-4 treatment caused SFME cells to express a neuronal phenotype synthesizing gamma-aminobutyric acid. These results indicate that SFME cells have the capacity to generate both neurons and astrocytes in vitro, which resemble the behavior of EGF-dependent multipotential stem cells in the central nervous system, and establish a relationship between effects of BMP-4 and degree of mitogenic stimulation by other peptide growth factors.     

Modulation of Intestinal Epithelial Cell Proliferation,Migration, and Differentiation In Vitro by Astragalus Polysaccharides

Previous studies have shown that Astragalus polysaccharides (APS) can be used to treat general gastrointestinal disturbances including intestinal mucosal injury. However, the mechanism by which APS mediate this effect is unclear. In the present study, the effects of APS on proliferation, migration, and differentiation of intestinal epithelial cells (IEC-6) were assessed using an in vitro wounding model and colorimetric thiazolyl blue (MTT) assays. The effect of APS on IEC-6 cell differentiation was observed using a light microscope and scanning electron microscope, and the expression of differentiation-specific markers of IEC-6 cells, such as cytokeratin 18 (CK18), alkaline phosphatase (ALP), tight junction protein ZO-2, and sucrase-isomaltase (SI), was determined by immunofluorescence assay (IFA) and real-time PCR. In addition, APS-induced signaling pathways in IEC-6 cells were characterized. Our results indicated that APS significantly enhance migration and proliferation of IEC-6 cells in vitro. APS-treated IEC-6 cells have numerous microvilli on their apical surface and also highly express CK18, ALP, ZO-2, and SI. Moreover, APS-treated IEC-6 cells, in which the activity and expression level of ornithine decarboxylase (ODC) were significantly elevated, also exhibited an increase in cellular putrescine, whereas no significant increase in TGF-β levels was observed. These findings suggest that APS may enhance intestinal epithelial cell proliferation, migration, and differentiation in vitro by stimulating ODC gene expression and activity and putrescine production, independent of TGF-β. Exogenous administration of APS may provide a new approach for modulating intestinal epithelial wound restitution in vivo.     

Bone marrow-derived mesenchymal stem cells differentiation into tubular epithelial-like cells in vitro

The possibility of differentiating bone marrow‐derived mesenchymal stem cells (BMSCs) into tubular epithelial‐like cells is explored in vitro. Purified BMSCs from Sprague–Dawley rats were obtained by density gradient centrifugation. Third generation BMSCs were divided into six groups and were cultured under different conditions. The expression of alkaline phosphatase and cytokeratin (CK)‐18 protein was detected through staining and immunocytochemistry, respectively, and the expression of E‐cadherin proteins was recorded through immunofluorescence. Some cells in ischemia/reperfusion (I/R), all‐trans retinoic acid (ATRA), epidermal growth factor (EGF) and bone morphogenetic protein‐7 (BMP‐7) groups turned positive, whereas the positive cells in the combined group significantly increased compared with the other groups. Compared with the control group, the positive expression rates of CK‐18 in the I/R, ATRA, EGF, BMP‐7 and the combined group were 11·50% ± 3·84%, 27·40% ± 2·70%, 29·60% ± 4·51%, 26·80% ± 5·00% and 44·00% ± 3·16%, respectively, and CK‐18 mRNA expression in the combined group was obviously higher than that in the other groups (P < 0·01). Immunofluorescence detection showed that E‐cadherin expression was not detectable in the control group, whereas the positive expression rates of E‐cadherin in the I/R, ATRA, EGF, BMP‐7 and the combined group were 6·75% ± 2·13%, 16·40% ± 2·69%, 18·25% ± 3·50%, 16·06% ± 2·00% and 30·26% ± 5·16%, respectively. The addition of ATRA, EGF and BMP‐7 induces BMSCs differentiation into tubular epithelial‐like cells in stimulated acute renal failure microenvironment in vitro. Copyright © 2011 John Wiley & Sons, Ltd.     

Epithelial differentiation of human adipose tissue-derived adult stem cells

Adult human stem cells are employed in novel treatments and bio-artificial devices. Recent studies have identified an abundant source of stem cells termed adipose-derived adult stem (ADAS)-cells in the subcutaneous adipose tissue. Under appropriate culture conditions ADAS-cells differentiate to various cell types, including chondrocytes, adipocytes, and smooth muscle cells. Aiming at epithelial differentiation this study investigated the effect of all-trans retinoic acid (ATRA) on human ADAS-cells. ATRA-induced cytokeratin 18 expression in ADAS-cells and nearly abolished vimentin expression as shown by Western blot. In immunofluorescence, the formation of keratin fibers in ATRA-treated ADAS-cells could be observed. The percentage of ADAS-cells being able to undergo epithelial differentiation as quantified by FACS-analysis was above 80%. Inhibition of cell growth by ATRA was shown using DAPI- and MTT-assays. ATRA can differentiate ADAS-cells toward the epithelial lineage. This finding, along with a previously described neural differentiation, shows that ADAS-cells have epithelial potential.     

Adenoviral gene transfer of BMP-7, Id2, or Id3 suppresses injury-induced epithelial-to-mesenchymal transition of lens epithelium in mice

We have examined the effect of adenovirus-mediated expression of bone morphogenic protein-7 (BMP-7) and inhibitors of differentiation 2 and 3 (Id2 and Id3) on injury-induced epithelial-to-mesenchymal transition (EMT) of lens epithelium in mice. Id2 and Id3 are known to be upregulated by BMP-7 and to antagonize Smad2/3 signaling. The Cre-LoxP system adenoviral gene transfer was used. Three microliters of adenoviral solution (2 x 10⁷ PFU/µl) were injected into the right lens of adult male C57BL/6 mice (n = 144) at the time of capsular injury induced using a hypodermic needle under both general and topical anesthesia. A mixture of Cre-adenovirus (Cre-Ad) and vector encoding mBMP-7, mId2, or mId3 was administered in a test group. Control lenses were treated with Cre-Ad alone. After healing intervals of 5 or 10 days, the animals were killed and then we performed histological processes or RNA extraction from the lens. RT-PCR, real-time RT-PCR, and immunohistochemistry showed expression of each introduced gene in the lens. Exogenous BMP-7 upregulated expression of Id2 and Id3 in injured lenses, and gene introduction of Id2 or Id3 also upregulated BMP-7 expression. Gene transfer of BMP-7, Id2, or Id3 delayed injury-induced EMT of the lens epithelial cells as evaluated by histology and expression patterns of -smooth muscle actin and collagens in association with reduction of Smad2 COOH-terminal phosphorylation. Gene transfer of BMP-7, Id2, or Id3 delayed injury-induced EMT of lens epithelial cells and subsequent sealing of the capsular break with fibrous tissue in mice. lens epithelial cell; bone morphogenic protein-7; inhibitor of differentiation; Smad     

Organotypic culture of human amnion cells in air-liquid interface as a potential substitute for skin regeneration

Background aimsThe aim of the present study was to evaluate the effects of air-liquid interface on the differentiation potential of human amnion epithelial cells (HAECs) to skin-like substitute in organotypic culture.MethodsHAECs at passage 1–2 were seeded onto a fibrin layer populated with human amnion mesenchymal cells to form the organotypic cultures. The organotypic HAECs were then cultured for 7, 14 and 21 d in two types of culture system: the submerged culture and the air-liquid interface culture. Cell morphogenesis was examined under the light and electron microscopes (transmission and scanning) and analyzed by immunohistochemistry.ResultsOrganotypic HAECs formed a single layer epithelium after 3 wk in submerged as well as air-liquid interface cultures. Ultrastructurally, desmosomes were observed in organotypic HAECs cultured in the air-liquid interface but not in the submerged culture. The presence of desmosomes marked the onset of early epidermal differentiation. Organotypic HAECs were positive against anti-CK18 and anti-CK14 in both the submerged and the air-liquid interface cultures. The co-expression of CK14 and CK18 suggested that differentiation of HAECs into skin may follow the process of embryonic skin development. However, weak expression of CK14 was observed after 2 and 3 wk of culture in air-liquid interface. CK10, involucrin, type IV collagen and laminin-5 expression was absent in organotypic HAECs. This observation reflects the initial process of embryonic epidermal differentiation and stratification.ConclusionsResults from the present study suggest that the air-liquid interface could stimulate early differentiation of organotypic HAECs to epidermal cells, with a potential use for skin regeneration.     

Retinoic Acid Improves Morphology of Cultured Peritoneal Mesothelial Cells from Patients Undergoing Dialysis

Patients undergoing continuous ambulatory peritoneal dialysis are classified according to their peritoneal permeability as low transporter (low solute permeability) or High transporter (high solute permeability). Factors that determine the differences in permeability between them have not been fully disclosed. We investigated morphological features of cultured human peritoneal mesothelial cells from low or high transporter patients and its response to All trans retinoic Acid (ATRA, vitamin A active metabolite), as compared to non-uremic human peritoneal mesothelial cells. Control cells were isolated from human omentum. High or low transporter cells were obtained from dialysis effluents. Cells were cultured in media containing ATRA (0, 50, 100 or 200 nM). We studied length and distribution of microvilli and cilia (scanning electron microscopy), epithelial (cytokeratin, claudin-1, ZO-1 and occludin) and mesenchymal (vimentin and α-smooth muscle actin) transition markers by immunofluorescence and Western blot, and transforming growth factor β1 expression by Western blot. Low and high transporter exhibited hypertrophic cells, reduction in claudin-1, occludin and ZO-1 expression, cytokeratin and vimentin disorganization and positive α-smooth muscle actin label. Vimentin, α-smooth muscle actin and transforming growth factor- β1 were overexpressed in low transporter. Ciliated cells were diminished in low and high transporters. Microvilli number and length were severely reduced in high transporter. ATRA reduced hypertrophic cells number in low transporter. It also improved cytokeratin and vimentin organization, decreased vimentin and α-smooth muscle actin expression, and increased claudin 1, occludin and ZO-1 expression, in low and high transporter. In low transporter, ATRA reduced transforming growth factor-β1 expression. ATRA augmented percentage of ciliated cells in low and high transporter. It also augmented cilia length in high transporter. Alterations in structure, epithelial mesenchymal markers and transforming growth factor-β1expression were differential between low and high transporter. Beneficial effects of ATRA were improved human peritoneal mesothelial cells morphology tending to normalize structures.     

Epigenetic regulation of bone morphogenetic protein-6 gene expression in breast cancer cells

Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Our previous research found that BMP-6 gene expression can be activated dose-dependently by estrogen in estrogen receptor positive (ER⁺) breast cancer cell line MCF-7, but not in ER negative (ER⁻) cell line MDA-MB-231. This experiment is designed to investigate the epigenetic regulatory mechanism of the BMP-6 gene expression in breast cancer cell lines MDA-MB-231, MCF-7 and T47D with regard to the methylation status in the 5′ flanking region of the human BMP-6 gene. The endogenous level of BMP-6 mRNA in ER⁻ cell line MDA-MB-231 was relatively lower than that in ER⁺ MCF-7 and T47D cell lines. After the treatment with 5-aza-2′-deoxycytidine (5-aza-dC, especially in the concentration of 10 μM), the BMP-6 mRNA expression in MDA-MB-231 was obviously up-regulated. However, 5-aza-dC treatment failed to regulate the expression of BMP-6 in MCF-7 and T47D cells. Using enzyme restriction PCR (MSRE-PCR), as well as bisulfite sequencing (BSG), methylation of human BMP-6 gene promoter was detected in MDA-MB-231; while in MCF-7 and T47D, BMP-6 gene promoter remained demethylated status. In 33 breast tumor specimens, promoter methylation of BMP-6 was detected by methylation-specific PCR, hypermethylation of BMP-6 was observed in ER negative cases (16 of 16 cases (100%)), while obviously lower methylation frequency were observed in ER positive cases (3 of 17 cases (18%)), indicating that BMP-6 promoter methylation status is correlated with ER status in breast cancer.