Differentiation of mouse embryonic stem cells into dental epithelial-like cells induced by ameloblasts serum-free conditioned medium

Embryonic stem cells (ESCs) possess an intrinsic self-renewal ability and can differentiate into numerous types of functional tissue cells; however, whether ESCs can differentiate toward the odontogenic lineage is still unknown. In this study, we developed an efficient culture strategy to induce the differentiation of murine ESCs (mESCs) into dental epithelial cells. By culturing mESCs in ameloblasts serum-free conditioned medium (ASF-CM), we could induce their differentiation toward dental epithelial cell lineages; however, similar experiments with the tooth germ cell-conditioned medium (TGC-CM) did not yield effective results. After culturing the cells for 14 days in the differentiation-inducing media, the expression of ameloblast-specific proteins such as cytokeratin (CK)14, ameloblastin (AMBN), and amelogenin (AMGN) was markedly higher in mESCs obtained with embryoid body (EB) formation than in mESCs obtained without EB formation. We observed that immunocompromised mice implanted with induced murine EBs (mEBs) showed tissue regenerative capacity and produced odontogenic epithelial-like structures, whereas those implanted with mSCE monolayer cells mainly formed connective tissues. Thus, for the first time, we report that ASF-CM provides a suitable microenvironment for inducing mESC differentiation along the odontogenic epithelial cell lineage. This result has important implications for tooth tissue engineering.     

CHIR-99021与FGF8协同诱导人表皮干细胞牙向分化

人表皮干细胞(human keratinocyte stem cells, hKSCs)可作为上皮源性的成体干细胞应用于牙齿再生,但是其诱导效率较低.本研究利用小分子化合物CHIR-99021提高hKSCs的Wnt/β-catenin信号活性,再与具有诱导成牙潜能的小鼠牙胚间充质重组,构建嵌合体,并移植裸鼠肾囊膜下培养20 d. 将嵌合体组织切片,并利用组织染色和免疫组化等方法鉴定牙齿结构. 结果显示,经FGF8诱导处理的hKSCs与小鼠牙胚间充质构成的嵌合体的成牙率为27.80%,其中成釉率仅为40.00%;经CHIR 99021诱导处理的hKSCs与小鼠牙胚间充质构成的嵌合体的成牙率仅为18.20%,其中成釉率高达100%;而CHIR 99021与FGF8协同作用,则进一步提高嵌合体成牙率至40.00%,其中成釉率也达75.00%. 进一步的研究发现,经CHIR-99021处理后,hKSCs的Wnt/β-catenin信号活性明显提高,同时FGF8的表达水平也显著上调. 以上结果表明,CHIR-99021可通过上调Wnt/β-catenin信号活性水平,同时促进FGF8表达,与FGF8协同,高效诱导hKSCs分化为具有分泌釉质功能的成釉质细胞. 研究结果对利用hKSCs作为上皮来源的成体细胞应用于人类牙齿再生的研究具有重要意义.     

The ultrastructure of human secretory ameloblasts

Summary The ultrastructure of secreting ameloblasts of deciduous teeth from a human foetus (crown-rump length 195 mm) was investigated. The ameloblasts demonstrate a formation of granules in a juxtanuclear Golgi complex. In the Tomes' process the granules are released either through the lateral plasma membrane into the intercellular space between the Tomes' processes or directly through the apical plasma membrane into the enamel.The human ameloblasts differ from non-human ameloblasts in having a non-oriented vesicular granular endoplasmic reticulum. Further, the majority of mitochondria are situated in the apical part of the ameloblast adjacent to the Tomes' process.We would like to thank chief-surgeon A. Christensen, Bispebjerg Hospital, Copenhagen for his help in acquiring foetal material. For technical assistance we would like to thank M. Balslev and U. Eberth, Anatomy Department A.     

A novel method to reconstruct epithelial tissue using high-purity keratinocyte lineage cells induced from human embryonic stem cells

The treatment of oral mucosa defect such as autologous oral mucosa caused by resection of oral mucosa carcinoma is still not ideal in clinical practice. However, Tissue engineering gives us the possibility to solve this problem. As we all know, Human embryonic stem cells (hESCs) have the ability to give rise to various cell types. We can take advantage of the totipotency of human embryonic stem cells to acquire keratinocytes. Directing the epithelial differentiation of hESCs can provide seed cells for the construction of epithelium tissue by tissue engineering. But, how to get high purity keratinocytes by induced stem cells then Applied to tissue engineering mucosa is an important challenge. We described a novel method to directly induce hESCs to differentiate into keratinocytes. Retinoic acid, ascorbic acid, and bone morphogenetic protein induced hESCs to differentiate into cells that highly expressed cytokeratin (CK)14. Our findings suggest that the retinoic acid, ascorbic acid and bone morphogenetic proteins induced hESCs to form high purity keratinocyte cell populations. In addition, we found that the highly pure keratinocyte populations reconstructed artificial tissue resembling epithelial tissue when inoculated in vitro on a biological scaffold.     

DNA-Mediated gene transfection into primary cultures of adult mouse keratinocytes

Summary An efficient and reproducible technique for the transfection of primary cultures of adult mouse keratinocytes has been developed. The procedure involves culturing the primary adult mouse epidermal cells at 32° C in an enriched media until they reach 70 to 95% confluency, followed by transfection with exogenous DNA in a low potassium environment. Using chloramphenicol acetyl transferase (CAT) transient gene expression assays and various strong viral promoter/CAT constructs, the transfection procedure was optimized for media formulation, plasmid DNA concentration, carrier DNA concentration, incubation temperature, incubation period, and cell density. Optimized parameters include the use of 6 μg plasmid DNA and 10 μg pUC19 carrier DNA per 60-mm tissue culture dish. Since primary keratinocytes undergo a well-characterized pattern of differentiation in vitro in response to extracellular calcium concentrations, this transfection procedure should provide a useful model in which to study both tissue- and differentiation-specific gene expression.     

Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells

Pluripotency of embryonic stem cells (ESCs) is maintained by the balancing of several signaling pathways, such as Wnt, BMP, and FGF, and differentiation of ESCs into a specific lineage is induced by the disruption of this balance. Sulfated glycans are considered to play important roles in lineage choice of ESC differentiation by regulating several signalings. We examined whether reduction of sulfation by treatment with the chemical inhibitor chlorate can affect differentiation of ESCs. Chlorate treatment inhibited mesodermal differentiation of mouse ESCs, and then induced ectodermal differentiation and accelerated further neural differentiation. This could be explained by the finding that several signaling pathways involved in the induction of mesodermal differentiation (Wnt, BMP, and FGF) or inhibition of neural differentiation (Wnt and BMP) were inhibited in chlorate-treated embryoid bodies, presumably due to reduced sulfation on heparan sulfate and chondroitin sulfate. Furthermore, neural differentiation of human induced pluripotent stem cells (hiPSCs) was also accelerated by chlorate treatment. We propose that chlorate could be used to induce efficient neural differentiation of hiPSCs instead of specific signaling inhibitors, such as Noggin.     

一种新的培养人胚胎干细胞的包被基质

目的开发一种新的培养人胚胎干细胞(hESCs)的包被基质,使hESCs的培养更加简便。方法用甲醇固定的小鼠胚胎成纤维细胞(MEF)作为包被基质,人胚胎干细胞系X-01在该基质上生长,每隔5~6 d传代一次,培养10代后,对人胚胎干细胞特性进行检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达和分化能力。结果 hESCs在新的基质上生长良好,经10次传代后仍能保持典型的hESCs克隆形态。碱性磷酸酶染色阳性,免疫荧光染色Oct4、SSEA4、Tra-1-60为阳性,体外分化可形成拟胚体。结论此种固定的基质可以大量制备,长期保存,并可以长期维持hESCs的未分化状态,为人胚胎干细胞的体外扩增探索出了一个新的途径。     

Flow cytometric sorting and analysis of human epidermal stem cell candidates

ESC (epidermal stem cells) play a central role in the regeneration of human epidermis. These cells are also responsible for wound healing and neoplasm formation. Efficient isolation of ESC allows their use in medicine and pharmacy as well as in basic science. Cultured keratinocytes and ESC may be used as biological dressing in burn injuries, chronic wounds and hereditary disorders. Therefore, the isolation and characterization of ESC have been goals in biomedical science. Here, we present a flow cytometric method for the isolation and analysis of human ESC candidates. The strategy presented for the isolation of ESC combines previously proposed enzymatic digestion and FACS‐sorting of the obtained cell suspension that utilizes morphological features, integrin‐β1 expression and Rh123 (Rhodamine 123) accumulation of the cells. We also performed a flow cytometric analysis of sorted cells using a cell tracer.     

Release of cytokines/chemokines and cell death in UVB-irradiated human keratinocytes, HaCaT

Ultraviolet (UV) B can lead to inflammatory responses such as sunburn, which involves the production of various inflammatory cytokines and chemokines, and the induction of cell death. Keratinocytes in the skin has one of the highest risks of exposure to UV. However, the detailed mechanisms underlying UVB irradiation-induced inflammation and cell death are not well known. Thus, we investigated the effect of UVB irradiation on the production of various cytokines/chemokines and the induction of cell death in UVB-irradiated human keratinocytes (HaCaT cells). We evaluated 11 cytokines/chemokines in cell culture supernatants from HaCaT cells exposed to 0-400 mJ/cm(2) UVB irradiation. UVB at a dose 400 mJ/cm(2) induced the release of various cytokines; interleukin (IL)-1beta, IL-6, IL-8, interferon (IFN)-gamma, granulocyte-colony stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, and tumor necrosis factor (TNF)-alpha. These results suggest that UVB irradiation-induced the release of several cytokines/chemokines and led to cell death in human keratinocytes. UV exposure may be associated with multiple physiological events in the human skin.     

Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic surface epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.     

The differentiation of human placenta-derived mesenchymal stem cells into dopaminergic cells in vitro

Mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after Parkinson’s disease. MSCs have significant advantages over other stem cell types, and greater potential for immediate clinical application. The aim of this study was to investigate whether MSCs from the human placenta could be induced to differentiate into dopaminergic cells. MSCs from the human placenta were isolated by digestion and density gradient fractionation, and their cell surface glycoproteins were analyzed by flow cytometry. These MSCs were cultured under conditions promoting differetiation into adipocytes and osteoblasts. Using a cocktail that includes basic fibroblast growth factor (bFGF), all trans retinoic acid (RA), ascorbic acid (AA) and 3-isobutyl-1-methylxanthine (IBMX), the MSCs were induced in vitro to become dopamine (DA) neurons. Then, the expression of the mRNA for the Nestin and tyrosine hydroxylase (TH) genes was assayed via RT-PCR. The expression of the Nestin, dopamine transporter (DAT), neuronal nuclear protein (NeuN) and TH proteins was determined via immunofluorescence. The synthesized and secreted DA was determined via ELISA. We found that MSCs from the human placenta exhibited a fibroblastoid morphology. Flow cytometric analyses showed that the MSCs were positive for CD44 and CD29, and negative for CD34, CD45, CD106 and HLA-DR. Moreover, they could be induced into adipocytes and osteocytes. When the MSCs were induced with bFGF, RA, AA and IBMX, they showed a change in morphology to that of neuronal-like cells. The induced cells expressed Nestin and TH mRNA, and the Nestin, DAT, NeuN and TH proteins, and synthesized and secreted DA. Our results suggest that MSCs from the human placenta have the ability to differentiate into dopaminergic cells.     

Increased strength in the Col-Tgel induces apoptosis in the human dental pulp stem cells: 3D culturing of human dental pulp stem cells at different strengths of collagen

Human dental pulp stem cells (HDPSCs) have great potential to be used in regenerative medicine. To use these stem cells effectively for this purpose, they should be grown in a 3D cell culture that mimics their natural niches instead of a 2D conventional cell culture. The aim of this study was to grow the HDPSCs in the 3D cell culture created by Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) in two different strengths to find a suitable 3D cell culture environment for these stem cells. Two stiffness of the 3D Col-Tgel were used to grow the HDPSCs: soft and medium matrix with strength of 0.9–1.5 kPa and 14–20 kPa, respectively. HDPSCs express markers similar to MSCs, therefore seven such markers were analyzed in the HDPSCs during their growth in the 2D and in the 3D soft and medium Col-Tgel. The CD105 and CD90 markers were significantly (p < 0.05) downregulated in HDPSCs cultured in both 3D cell culture conditions compared with HDPSCs in 2D cell culture. Furthermore, CD34 marker, a negative marker, expressed by a few cells in HDPSCs culture was upregulated (p < 0.05) in HDPSCs cultured in medium 3D Col-Tgel, indicating cells that expressing the marker grow better in medium 3D Col-Tgel. The apoptosis results revealed that HDPSCs in medium 3D Col-Tgel had the least number of live cells and a significantly (p < 0.05) higher early apoptosis rate compared to HDPSCs in 2D and 3D Col-Tgel medium. MTT analysis also showed a significant difference among the three cell culture conditions. We conclude that HDPSCs cultured on 3D soft Col-Tgel showed better proliferation than cells cultured in 3D medium gel. These results demonstrate that the ideal environment to grow HDPSCs in 3D is the soft Col-Tgel not medium Col-Tgel.     

Mouse 3T3 fibroblasts under the influence of fibroblasts isolated from stroma of human basal cell carcinoma acquire properties of multipotent stem cells

Background information. Multipotent mesenchymal stem cells can participate in the formation of a microenvironment stimulating the aggressive behaviour of cancer cells. Moreover, cells exhibiting pluripotent ESC (embryonic stem cell) markers (Nanog and Oct4) have been observed in many tumours. Here, we investigate the role of cancer‐associated fibroblasts in the formation of stem cell supporting properties of tumour stroma. We test the influence of fibroblasts isolated from basal cell carcinoma on mouse 3T3 fibroblasts, focusing on the expression of stem cell markers and plasticity in vitro by means of microarrays, qRT‐PCR (quantitative real‐time PCR) and immunohistochemistry. Results. We demonstrate the biological activity of the cancer stromal fibroblasts by influencing the 3T3 fibroblasts to express markers such as Oct4, Nanog and Sox2 and to show differentiation potential similar to mesenchymal stem cells. The role of growth factors such as IGF2 (insulin‐like growth factor 2), FGF7 (fibroblast growth factor 7), LEP (leptin), NGF (nerve growth factor) and TGFβ (transforming growth factor β), produced by the stromal fibroblasts, is established to participate in their bioactivity. Uninduced 3T3 do not express the stem cell markers and show minimal differentiation potential. Conclusions. Our observations indicate the pro‐stem cell activity of cancer‐associated fibroblasts and underline the role of epithelial—mesenchymal interaction in tumour biology.     

Activation of the FGF signaling pathway and subsequent induction of mesenchymal stem cell differentiation by inorganic polyphosphate

Inorganic polyphosphate [poly(P)] is a biopolymer existing in almost all cells and tissues, although its biological functions in higher eukaryotes have not been completely elucidated. We previously demonstrated that poly(P) enhances the function of fibroblast growth factors (FGFs) by stabilizing them and strengthening the affinity between FGFs and their cell surface receptors. Since FGFs play crucial roles in bone regeneration, we further investigated the effect of poly(P) on the cell differentiation of human stem cells via FGF signaling systems. Human dental pulp cells (HDPCs) isolated from human dental pulp show the characteristics of multipotent mesenchymal stem cells (MSCs). HDPCs secreted FGFs and the proliferation of HDPCs was shown to be enhanced by treatment with poly(P). Cell surface receptor-bound FGF-2 was stably maintained for more than 40 hours in the presence of poly(P). The phosphorylation of ERK1/2 was also enhanced by poly(P). The effect of poly(P) on the osteogenic differentiation of HDPCs and human MSCs (hMSCs) were also investigated. After 5 days of treatment with poly(P), type-I collagen expression of both cell types was enhanced. The C-terminal peptide of type-I collagen was also released at higher levels in poly(P)-treated HDPCs. Microarray analysis showed that expression of matrix metalloproteinase-1 (MMP1), osteopontin (OPN), osteocalcin (OC) and osteoprotegerin was induced in both cell types by poly(P). Furthermore, induced expression of MMP1, OPN and OC genes in both cells was confirmed by real-time PCR. Calcification of both cell types was clearly observed by alizarin red staining following treatment with poly(P). The results suggest that the activation of the FGF signaling pathway by poly(P) induces both proliferation and mineralization of stem cells.     

Cyclopamine treatment of human embryonic stem cells followed by culture in human astrocyte medium promotes differentiation into nestin- and GFAP-expressing astrocytic lineage

Human embryonic stem cells (hESCs) are able to differentiate into various cell types, including neuronal cells and glial cells. However, little information is available regarding astrocyte differentiation. This report describes the differentiation of hESCs into nestin- and GFAP-expressing astrocytes following treatment with cyclopamine, which is an inhibitor of Hedgehog (Hh) signaling, and culturing in human astrocyte medium (HAM). In hESCs, cyclopamine treatment suppressed the expression of Hh signaling molecules, the Hh signaling target gene, and ESC-specific markers. Clyclopamine also induced the differentiation of the cells at the edges of the hESC colonies, and these cells stained positively for the early neural marker nestin. Subsequent culturing in HAM promoted the expression of the astrocyte-specific marker GFAP, and these cells were also nestin-positive. These findings indicate that treatment with cyclopamine followed by culturing in HAM leads to the differentiation of hESCs into nestin- and GFAP-expressing astrocytic lineage.     

Serial cultivation of normal human keratinocytes: A defined system for studying the regulation of growth and differentiation

Summary We have developed a defined method for human epidermal keratinocyte culture. The minimally supplemented basal medium supported establishment of primary cultures from neonatal foreskin in a defined environment. It also supported serial cultivation and rapid expansion of cell number. Casein replaced serum for defined cryopreservation. Cells were serially cultivated in medium containing 0.08 mM calcium. The rate of cell division however remained high after addition of 1.8 mM calcium. The particulate transglutaminase activity of the cultures was low at confluence, even in the presence of 1.88 mM calcium, indicating an enrichment of the basal cell population. Culture with small amounts (0.3%) of chelated serum increased particulate transglutaminase activity approximately 2.2-fold in low calcium cultures and approximately 3.5-fold in high calcium cultures. A gradual reduction in growth rate of serum-treated cultures upon serial cultivation also indicated a depletion of cells with basal cell character. Bovine hypothalamic extract and cholera toxin were able to avert, in part, the differentiation-promoting effects of serum. Keratinocytes serially cultivated in the defined medium maintained the ability to develop normally into a morphologically differentiated epidermis.