Induction and differentiation of adipose-derived stem cells from human buccal fat pads into salivary gland cells

Atrophy or hypofunction of the salivary gland because of aging or disease leads to hyposalivation that affects patient quality of life by causing dry mouth, deterioration of mastication/deglutition, and poor oral hygiene status. Current therapy for atrophy or hypofunction of the salivary gland in clinical practice focuses on symptom relief using drugs and artificial saliva; therefore, there is still a need to develop new therapies. To investigate potential novel therapeutic targets, we induced the differentiation of salivary gland cells by co-culturing human adipose-derived stem cells isolated from buccal fat pads (hBFP-ASCs) with human salivary-gland-derived fibroblasts (hSG-fibros). We examined their potential for transplantation and tissue neogenesis. Following the culture of hBFP-ASCs and hSG-fibros, differentiated cells were transplanted into the submandibular glands of SCID mice, and their degree of differentiation in tissues was determined. We also examined their potential for functional tissue reconstitution using a three-dimensional (3D) culture system. Co-cultured cells expressed salivary-glandrelated markers and generated new tissues following transplantation in vivo. Moreover, cell reconstituted glandular structures in the 3D culture system. In conclusion, coculture of hSG-fibros with hBFP-ASCs led to successful differentiation into salivary gland cells that could be transplanted to generate new tissues.     

Isolation of tissue progenitor cells from duct-ligated salivary glands of swine

Tissue stem cells participate in the repopulation of tissue after injury. Tissue injury stimulates the normally quiescent tissue stem cells to differentiate and proliferate, in the process of replacing and/or repairing the damaged cells, and hence effecting tissue regeneration. The salivary glands retain the ability for frequent regeneration. Previously, we isolated progenitor cells from the injured salivary glands of mice and rats that differentiated into hepatic and pancreatic lineages. The isolated progenitors were CD49f-positive and intracellular laminin-positive, and proliferated on type I collagen while maintaining their multipotency. In this study, we analyzed the tissue stem cells induced by ligating the main excretory duct of the salivary gland in swine. After duct ligation of the gland, acinar cells receded due to apoptosis, and epithelial cells subsequently proliferated. We cultured cells obtained from the duct-ligated salivary gland and purified the cells by limited dilution. The isolated cells were positive for CD29, CD49f, intracellular laminin, AFP, CK19, CK18, and Thy-1(CD90), and weakly positive for c-Kit (CD117). After three-dimensional formation, the cells expressed insulin and albumin. We designated the cells as swine salivary gland-derived progenitor cells. Gene expression of insulin and albumin was significantly increased (five-fold) and that of insulin was also increased (3.8-fold) with differentiation medium with nicotinamide and/or GLP-1 treatment in spherical culture. The expressions of albumin and insulin were 1/10-fold and 1/4-fold compared to porcine hepatocytes and pancreatic endocrine cells. The differentiated SGP cells could release insulin, which were stimulated by glucose and potassium. These results indicate that swine SGP cells could differentiate into hepatocytes and beta-cells, functionally. Swine SGP cells were useful tools for therapy and analyzing endodermal regenerative models in large animals.     

Human salivary gland acinar cells spontaneously form three-dimensional structures and change the protein expression patterns

Applying tissue engineering principles to design an auto-secretory device is a potential solution for patients suffering loss of salivary gland function. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of reports on the in vitro cell biology and physiology of human salivary gland cells. This study used a low-calcium culture system to selectively cultivate human parotid gland acinar (PGAC) cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α-amylase) and function-related proteins (aquaporin-3, aquaporin-5, and ZO-1). Notably, when the cells reached confluence, three-dimensional (3D) cell aggregates were observed in crowded regions. These self-formed cell spheres were termed post-confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function-related proteins compared to the two-dimensional (2D) cells. Translocation of aquoporin-3 from cytosolic to alongside the cell boundaries, and of ZO-1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form PCSs without the help of 3D scaffolds and retain certain differentiation and polarity. This phenomenon implies that it is possible to introduce 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering.     

Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes.

When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non-stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigenesis. We have developed co-culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissue-culture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage-dependent growth, or as single-cell dispersions in soft agarose for assessment of anchorage-independent growth. Our procedures for assay of anchorage-independent growth allow us for the first time to detect and measure this transformation-defining characteristic in non-immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage-dependent growth of mammary epithelium, with cell number increasing as much as fifteenfold during a 6-day culture period. These same fetal tissues also stimulated anchorage-independent growth of the mammary epithelial cells, with colony-forming efficiencies of up to 40% in co-cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co-cultured epithelial cells were subsequently re-cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co-cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA), also caused anchorage-independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early-passage salivary mesenchyme cells was capable of stimulating growth under both anchorage-dependent and anchorage-independent conditions, confirming that these effects are mediated by a paracrine factor. The results indicate that stimulatory fetal mesenchymes produce soluble molecules that act analogously to transforming growth factors.     

Isolation, tissue localization, and cellular characterization of progenitors derived from adult human salivary glands

Progenitors that can transdifferentiate into cells with hepatic or pancreatic phenotypes can be isolated from experimentally injured salivary glands of rodents. In this study, we isolated progenitors from "uninjured" adult human salivary glands by fluorescence-activated cell sorting using anti-CD49f and anti-Thy-1 antibodies. The sorted cells that were contained in the CD49f+/Thy-1+ fraction showed good proliferation on type I collagen. Single purified progenitor cells in plate culture expressed intracellular laminin, CD49f, Thy-1, and NGF receptor p75 (p75(NGFR)). Immunohistological analysis revealed the expression of Thy-1 and p75(NGFR) in stromal cells in the periductal area of the salivary gland. Under overconfluent conditions in plate culture, cell clusters containing insulin and glucagon-positive cells were occasionally formed. In order to produce differentiated cell clusters with uniform quality, we used a spherical culture system. Autonomous differentiation of cells in clusters into insulin-positive cells was induced in the spherical culture system. We measured C-peptide to estimate the endogenously produced insulin content. The C-peptide content of the spheroid bodies was low (3.5 ng/mg of protein), and they simultaneously expressed the early islet differentiation factor Nkx6.1, proendocrine gene neurogenin3, and ductal cell marker cytokeratin19. The progenitors existing in the interstitium of the salivary gland were able to transdifferentiate into cells with a pancreatic endocrine phenotype.     

Serial cultivation of epithelial cells from human and macaque salivary glands

Summary To study the regulation of human salivary-type gene expression we developed cell culture systems to support the growth and serial cultivation of salivary gland epithelial and fibroblastic cell types. We have established 22 independent salivary gland epithelial cell strains from parotid or submandibular glands of human or macaque origin. Nineteen strains were derived from normal tissues and three from human parotid gland tumors. Both the normal and the tumor-derived salivary gland epithelial cells could be serially cultivated with the aid of a 3T3 fibroblast feeder layer in a mixture of Ham’s F12 and Dulbecco’s modified Eagle’s media supplemented with fetal bovine serum, calcium, cholera toxin, hydrocortisone, insulin, and epidermal growth factor. Salivary gland epithelial cells cultured under these conditions continued to express the genes for at least two acinar-cell-specific markers at early passages. Amylase enzyme activity was detected in conditioned media from cultured rhesus parotid epithelial cells as late as Passage 5. Proline-rich-protein-specific RNAs were detected in primary cultures of both rhesus and human parotid epithelial cells. Neither amylase enzyme activity nor PRP-specific RNAs were detected in fibroblasts isolated from the same tissues. In addition, salivary gland epithelial cells cultured under our conditions retain the capacity to undergo dramatic morphologic changes in response to different substrata. The cultured salivary gland epithelial cells we have established will be important tools for the study of salivary gland differentiation and the tissue-specific regulation of salivary-type gene expression.     

Practical choice for robust and efficient differentiation of human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have the distinct advantage of being able to differentiate into cells of all three germ layers. Target cells or tissues derived from hPSCs have many uses such as drug screening, disease modeling, and transplantation therapy. There are currently a wide variety of differentiation methods available. However, most of the existing differentiation methods are unreliable, with uneven differentiation efficiency and poor reproducibility. At the same time, it is difficult to choose the optimal method when faced with so many differentiation schemes, and it is time-consuming and costly to explore a new differentiation approach. Thus, it is critical to design a robust and efficient method of differentiation. In this review article, we summarize a comprehensive approach in which hPSCs are differentiated into target cells or organoids including brain, liver, blood, melanocytes, and mesenchymal cells. This was accomplished by employing an embryoid body-based three-dimensional (3D) suspension culture system with multiple cells co-cultured. The method has high stable differentiation efficiency compared to the conventional 2D culture and can meet the requirements of clinical application. Additionally, ex vivo co-culture models might be able to constitute organoids that are highly similar or mimic human organs for potential organ transplantation in the future.     

The interaction between co-cultured human nucleus pulposus cells and mesenchymal stem cells in a bioactive scaffold

Mesenchymal stem cells (MSCs) can differentiate into nucleus pulposus (NP) cells upon being co-cultured with NP cells. Important growth factors and morphogens secreted by MSCs during the differentiation process also enhance the biological properties of NP cells. In this study, the interactions between human NP cells and MSCs co-cultured in different cell-ratio (100% NP, 75% NP with 25% MSCs, 50% NP with 50% MSCs, 25% NP with 75% MSCs, and 100% MSCs) in a three-dimensional gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer scaffold were examined. Results showed that the cell proliferation was increased when NP and MSCs were co-cultured. Real-time PCR and immunohistochemical staining revealed that all co-culture groups produced type II collagen which represent normal NP cells but not type I collagen secreted by degenerated NP cells. FADD expression, which modulates cell survival and extracellular matrix homeostasis, was maintained in a stable status for co-cultured groups. The cultures containing 75% NP cells with 25% MSCs showed high level of collagen production and glycosaminoglycan content. Moreover, 75% NP cells with 25% MSCs had upregulated SOX9 that contributes to the improvement in type II collagen mRNA expression and protein production. These findings showed the NP/MSC cell-ratio influenced the cell functions dramatically. The co-culture of NP/MSC cells in a bioactive scaffold is a promising treatment for intervertebral disc diseases.     

HSG cells differentiated by culture on extracellular matrix involves induction of S-adenosylmethione decarboxylase and ornithine decarboxylase

The human salivary gland (HSG) epithelial cell line can differentiate when cultured on extracellular matrix preparations. We previously identified >30 genes upregulated by adhesion of HSG cells to extracellular matrix. In the current studies, we examined the role of one of these genes, the polyamine pathway biosynthetic enzyme S-adenosylmethionine decarboxylase (SAM-DC) and the related enzyme, ornithine decarboxylase (ODC), on HSG cell differentiation during culture on extracellular matrix. HSG cells cultured on fibronectin-, collagen I gel-, and Matrigel-coated substrates for 12-24 h upregulated SAM-DC and ODC mRNA expression and enzyme activity compared to cells cultured on non-precoated substrates. After 3-5 days, HSG cells grown on Matrigel- or collagen I gel-coated substrates acquired a differentiated phenotype: the cells showed changes in culture morphology and increased expression of salivary gland differentiation markers (vimentin, SN-cystatin, and alpha-amylase). Further, culturing the cells on substrates precoated with an anti-beta1-integrin-antibody promoted differentiation-like changes. HSG cells cultured on collagen I- or Matrigel-coated substrates rapidly entered the cell cycle but showed decreased cell proliferation at longer times. In contrast, cell proliferation was enhanced on fibronectin-coated substrates compared to cells on non-precoated substrates. Treatment with the polyamine synthesis inhibitors, difluoromethylornithine (DFMO), and methylglyoxal bis-(guanylhydrazone) (MGBG), inhibited cell proliferation and delayed (3)H-thymidine incorporation in HSG cells cultured on all of the substrates. Further, inclusion of DFMO and MGBG inhibited or delayed acquisition of the differentiated phenotype in HSG cells cultured on Matrigel- or collagen I gel-coated substrates. This suggests that the adhesion-dependent expression of SAM-DC and ODC contributes to extracellular matrix-dependent HSG cell differentiation.     

Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 × 10³, 1 × 10⁴ or 1 × 10⁵ cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-γ and TNF-α were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 × 10⁴ cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 × 10⁴ cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-γ-induced MHC II antigen expression in co-cultured ECs compared with single culture group (P < 0.05). TNF-α induced the expression of IL-6, IL-8 and sICAM in ECs. When co-cultured with Sertoli cells, their expressions were significantly lower than in the EC single culture group (P < 0.05). ECs co-cultured with Sertoli cells also did not significantly increase the stimulation index of spleen lymphocytes compared to the single culture group (P < 0.05). Our results suggested that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate post-transplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes.     

Inductive differentiation of two neural lineages reconstituted in a microculture system from Xenopus early gastrula cells.

Neural induction of ectoderm cells has been reconstituted and examined in a microculture system derived from dissociated early gastrula cells of Xenopus laevis. We have used monoclonal antibodies as specific markers to monitor cellular differentiation from three distinct ectoderm lineages in culture (N1 for CNS neurons from neural tube, Me1 for melanophores from neural crest and E3 for skin epidermal cells from epidermal lineages). CNS neurons and melanophores differentiate when deep layer cells of the ventral ectoderm (VE, prospective epidermis region; 150 cells/culture) and an appropriate region of the marginal zone (MZ, prospective mesoderm region; 5-150 cells/culture) are co-cultured, but not in cultures of either cell type on their own; VE cells cultured alone yield epidermal cells as we have previously reported. The extent of inductive neural differentiation in the co-culture system strongly depends on the origin and number of MZ cells initially added to culture wells. The potency to induce CNS neurons is highest for dorsal MZ cells and sharply decreases as more ventrally located cells are used. The same dorsoventral distribution of potency is seen in the ability of MZ cells to inhibit epidermal differentiation. In contrast, the ability of MZ cells to induce melanophores shows the reverse polarity, ventral to dorsal. These data indicate that separate developmental mechanisms are used for the induction of neural tube and neural crest lineages. Co-differentiation of CNS neurons or melanophores with epidermal cells can be obtained in a single well of co-cultures of VE cells (150) and a wide range of numbers of MZ cells (5 to 100). Further, reproducible differentiation of both neural lineages requires intimate association between cells from the two gastrula regions; virtually no differentiation is obtained when cells from the VE and MZ are separated in a culture well. These results indicate that the inducing signals from MZ cells for both neural tube and neural crest lineages affect only nearby ectoderm cells.     

Inhibition of Src and p38 MAP kinases suppresses the change of claudin expression induced on dedifferentiation of primary cultured parotid acinar cells

Sj?gren's syndrome and therapeutic radiation for head and neck cancers result in irreversible changes in the parenchyma of salivary glands, loss of acinar cells, prominence of duct cells, and fibrosis. To clarify mechanisms of salivary gland dysfunction, we identified a signaling pathway involved in the dedifferentiation of primary cultures of parotid acinar cells. We reported previously that the expression pattern of claudins changes during culture, is related to the three-dimensional organization of the cells, and reflects their ability to function as acinar cells. In this study, we found that this change of claudin expression is a process of dedifferentiation, because expression of other differentiation markers also changes during culture. The expression levels of claudins-4 and -6, cytokeratin 14, and vimentin are increased, and those of claudin-10, aquaporin 5, and amylase are decreased. Inhibitors of Src and p38 MAP kinases suppress these changes and increase the expression of acinar marker proteins. Differences in extracellular matrix components have no effect. Activation of p38 MAP kinase occurs during cell isolation from the parotid glands and is retained up to 6 h after the isolation. In contrast, activation of Src kinases does not increase during the cell isolation. The Src inhibitor PP1 suppresses the activation of p38 MAP kinase. Therefore, cellular stresses induced during cell isolation cause dedifferentiation and transition to duct-like cells through activation of p38 MAP kinase and constitutively active Src kinases.     

Effects of hepatocyte growth factor (HGF) and activin a on the morphogenesis of rat submandibular gland-derived epithelial cells in serum-free collagen gel culture

Summary To study the mechanisms of morphogenesis in salivary gland regeneration, we have established the RSMG-1 cell line derived from submandibular gland (SMG) of 10-wk-old Wistar female rats in serum-free culture. Our finding that RSMG-1 cells originated from duct cells was based on morphology and immunohistochemical results. In three-dimensional serum-free collagen gel culture, HGF induced branching morphogenesis of RSMG-1 cells. Histological examination revealed that HGF-induced branching structure exhibited well-formed lumina. This morphology closely resembles that found in vivo. The cells also expressed activin A. Exogenously added activin A at a high concentration reduced HGF-induced branching morphogenesis. These findings suggest that the morphogenesis of the salivary gland is modulated by HGF and activin A. Our results show that the RSMG-1 cell line may be useful in studies of salivary gland regeneration.     

Large-scale identification by shotgun proteomics of proteins expressed in porcine liver and salivary gland

Protein catalogs containing a large number of proteins expressed in a variety of organs can be powerful tools for stem-cell research, because this requires accurate knowledge about how cells differentiate. Salivary gland progenitor (SGP) cells are somatic stem cells isolated from the salivary gland that can differentiate into hepatic or pancreatic cell lineages. Their differentiation state has been assessed by the expression of major protein markers, but to use these cells in regenerative medicine, it will be necessary to establish additional means of quality assessment. We examined the use of shotgun proteomics for porcine salivary gland (a source of SGP cells) and liver (a destination of differentiated SGP cells) for determining the state of SGP cell differentiation. Protein complexes from each organ were digested into peptides and separated by two-dimensional liquid chromatography involving strong cation-exchange chromatography followed by reversed-phase liquid chromatography. The separated peptides were analyzed by on-line electrospray ionization tandem mass spectrometry using a quadrupole-time of flight mass spectrometer (ESI Q-TOF MS/MS), and the spectra obtained were processed to search peptides against a mammalian database for protein identification. Using this method, we identified 117 proteins in porcine salivary gland and 154 proteins in porcine liver. Of these, 72 and 109 were specific to salivary gland and liver, respectively, and some of these were previously shown to be organ specific. The current study can be utilized in the future as a basis to study the pattern of differentiation in protein expression by stem cells.     

Osteogenic differentiation of human periosteal-derived cells in a three-dimensional collagen scaffold

This study examined the osteogenic differentiation of cultured human periosteal-derived cells grown in a three dimensional collagen-based scaffold. Periosteal explants with the appropriate dimensions were harvested from the mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the cells were divided into two groups and cultured for 28 days. In one group, the cells were cultured in two-dimensional culture dishes with osteogenic inductive medium containing dexamethasone, ascorbic acid, and β-glycerophosphate. In the other group, the cells were seeded onto a three-dimensional collagen scaffold and cultured under the same conditions. We examined the bioactivity of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and measurements of the calcium content in the periosteal-derived cells of two groups. Periosteal-derived cells were successfully differentiated into osteoblasts in the collagen-based scaffold. The ALP activity in the periosteal-derived cells was appreciably higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The calcium level in the periosteal-derived cells seeded onto three-dimensional collagen scaffolds showed a 5.92-fold increase on day 7, 3.28-fold increase on day 14, 4.15-fold increase on day 21, and 2.91-fold increase on day 28, respectively, compared with that observed in two-dimensional culture dishes. These results suggest that periosteal-derived cells have good osteogenic capacity in a three-dimensional collagen scaffold, which provides a suitable environment for the osteoblastic differentiation of these cells.     

Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells

To examine the effects of somatic cell support on the cleavage and viability of fertilized sheep eggs, 434 pronucleate eggs were co-cultured for 3 or 6 days on oviduct cells or fibroblasts and 77 eggs were cultured in medium alone. During the first 3 days in culture 95% of the single-celled eggs cleaved regularly to non-compacted morulae on either of the feeder-layers but only 13% underwent similar regular cleavage in medium alone. Despite the identical cleavage rates in the co-culture groups, only 33% of embryos grown on fibroblasts as compared with 80% of embryos grown on oviduct cells were fully viable as judged by their ability to develop normally after transfer to recipient animals. The viability of embryos in the oviduct group was equal to that obtained after the direct transfer of morulae from donor to recipient sheep. After 6 days in culture 42% of embryos co-cultured with oviduct cells developed into expanded blastocysts as compared with only 4.5% cultured on fibroblasts. In both co-culture groups virtually all the remaining embryos blocked during the 4th cleavage. When transferred, 30% of blastocysts grown from the pronucleate stage on oviduct cells were viable. We conclude that: (1) during the first 3 days after fertilization cleavage will progress at a normal rate on different feeder-layers but oviduct cells appear to be required for the acquisition of full embryonic viability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse embryonic fibroblast (MEF)/BMP4-conditioned medium enhanced multipotency of human dental pulp cells

Dental pulp cells (DPCs) are valuable cell source for dental regeneration, albeit their application is restricted by limited pluripotency due to current culture condition. Mouse embryonic fibroblasts (MEFs) are served as feeder layer to maintain undifferentiated state of iPSCs and ESCs with long-term in vitro culture. Bone morphogenetic protein 4 (BMP4) plays an important role in the regulation of undifferentiated state and lineage commitment of cells through modulation of microenvironment. However, so far little was known how micro environment affect the multipotency of dental derived cells. To demonstrate the effect of optimized culture condition on multipotency of DPCs, cell proliferation and senescence of DPCs with MEF and/or rhBMP4-CM were examined by CCK8, telomerase activity and flow cytometry. Multilineage differentiation was detected by immunofluorescent staining, Real-time PCR and western blot. Expression of BMP4/NFATc1/LIF in the co-culture medium was evaluated by ELISA and expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs was detected by Real-time PCR. NFATc1 inhibitor INCA-6 was applied to DPCs with MEF and/or rhBMP4-CM, expression of NFATc1/Oct-4/Sox2/c-Myc was examined by Realtime PCR and western blot. Our results demonstrated that DPCs cultured with MEF and/or rhBMP4-CM showed increased cell proliferation, telomerase rate and multilineage differentiation capability. MEF-CM enhanced expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs through secretion of BMP4/NFATc1 in the culture medium. INCA-6 effectively restrained the MEF/BMP4-CM induced upregulation of Oct-4/Sox2/c-Myc/NFATc1 in DPCs. These resuts indicate that both MEF-CM and BMP4-CM provided similar efficient culture system to improve the multipotency of DPCs, which might contribute to the application of DPCs in dental regeneration.     

Human kallikrein 6 expression in salivary gland tumors.

Human kallikrein 6 (hK6), also known as zyme/protease M/neurosin), is expressed in many normal glandular tissues. The aim of this study was to determine whether hK6 is expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), using an immunohistochemical method. Pleomorphic adenomas (PA), adenoid cystic carcinomas, polymorphous low-grade adenocarcinomas, acinic cell carcinomas, mucoepidermoid carcinomas, and adenocarcinomas not otherwise specified of both minor and major salivary glands were examined. Cells lining duct-like structures and non-duct-like cells were scored. Only in PA of minor salivary gland origin was overall staining higher in duct-like than in non-duct-like cells. In all other tumors exhibiting both types of cells, hK6 staining was similar in both duct-like and non-duct-like cells. Tumors that exhibited non-duct-like cells only also exhibited cytoplasmic staining. Results of this study show that salivary gland tumors express hK6, apparently downregulated in comparison with normal salivary gland tissue, and that this expression is not specific for any of the tumors studied.