STRO-1 Positive Cells Research Articles

STRO-1 positive cell expansion during osteogenic differentiation: A comparative study of three mesenchymal stem cell types of dental origin

ObjectiveAlthough the osteogenic differentiation potential of mesenchymal stem cells of dental origin is well established, the roles of different marker proteins in this process remain to be clarified. Our aim was to compare the cellular and molecular changes, focusing in particular on mesenchymal stem cell markers, during in vitro osteogenesis in three dental stem cell types: dental follicle stem cells (DFSCs), periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs). DesignHuman DFSCs, PDLSCs and DPSCs were isolated, cultured and their osteogenic differentiation was induced for 3 weeks. Mineralization was assessed by von Kossa staining and calcium concentration measurements. The expression of mesenchymal and osteogenic markers was studied by immunocytochemistry and qPCR techniques. Alkaline phosphatase (ALP) activity and the frequency of STRO-1 positive cells were also quantified. ResultsThe three cultures all showed abundant mineralization, with high calcium content by day 21. The expression of vimentin and nestin was sustained after osteogenic induction. The osteogenic medium induced a considerable elevation of STRO-1 positive cells. By day 7, the ALP mRNA level had increased more than 100-fold in DFSCs, PDLSCs, and DPSCs. Quantitative PCR results indicated dissimilarities of osteoblastic marker levels in the three dental stem cell cultures. ConclusionsDFSCs, PDLSCs and DPSCs have similar functional osteogenic differentiation capacities although their expressional profiles of key osteogenic markers show considerable variations. The STRO-1 positive cell fraction expands during osteogenic differentiation while vimentin and nestin expression remain high. For identification of stemness, functional studies rather than marker expressions are needed.

Effect of photodynamic therapy on the morphological changes of periapical inflammation: An experimental study in rats.

We aimed to explore the morphological changes with photodynamic therapy (PDT) in experimentally produced periapical lesions in rats, and to investigate if PDT used as an adjunct to conventional root canal debridement helps to enhance healing the inflammatory response around root apex of rats. Sixty adult Sprague-Dawley male rats (70-90 days/140-160 g) were experimented. Pulp was removed from the mesial root of the first maxillary molar and left open to oral environment to allow the formation of periapical lesion. The rats were numbered and randomly divided into two groups: (i) PDT group (n = 30) received a single session of PDT with conventional debridement and, (ii) Control group (n = 30) received conventional debridement but with no PDT. PDT application involved the use of methylene blue photosensitizer for 5 min inside the root canals and irradiated with diode laser of 805 nm and 20 W power output for 90 s and sealed. After 4 weeks, the experimental rats were sacrificed by cervical dislocation. The maxillary first molar was then collected along with the surrounding tissue for further processing. Hematoxylin and eosin and immunohistochemical staining were used to observe the morphological effects. Proliferating Cell Nuclear Antigen (PCNA), STRO-1 and CD-44 were used as the primary antibodies for the immunohistochemical study. A reduction in inflammatory cells, which were mainly composed of lymphocytes, was observed in the periapical lesions after PDT. The number of PCNA-positive cells increased to approximately twice in the PDT as compared to the control group. These PCNA-positive cells included STRO-1 and CD-44 positive cells, indicating enhancement of wound healing and reduction in inflammatory cells. The findings of the present experimental study indicate that PDT application induced proliferation of PCNA-positive cells, which included STRO-1 and CD44-positive cells. This suggests that PDT may help to enhance healing periapical lesion, indicating the potential of PDT in the treatment of periapical periodontitis.

Effects of fibroblast growth factor-2 on cell proliferation of cementoblasts

Background/purposeFibroblast growth factor (FGF)-2 is known as a signaling molecule that induces tissue regeneration. Little is known about the effect of FGF-2 on cementoblasts for periodontal and periapical regeneration. The aim of this study was to investigate the effects of FGF-2 on murine immortalized cementoblast cell line (OCCM.30). Materials and methodsCell growth and proliferation was judged by using alamar blue reduction assay. Flow cytometry analysis was used to evaluate Stro-1 positive cells expression with or without FGF-2. Western blot was used to evaluate the expression of phosphorylated serine–threonine kinase Akt (p-Akt) and extracellular signal-regulated protein kinase (p-ERK) in cementoblasts. ResultsFGF-2 was found to increase cell growth in a dose-dependent manner (P<0.05). The concentration of 10 ng/mL FGF-2 enhanced cell proliferation in a time-dependent manner (P<0.05). In addition, 10 ng/mL FGF-2 significantly increased the number of Stro-1 positive cells in the first 24 hours (P<0.05). Moreover, 10 ng/mL FGF-2 was found to upregulate p-Akt and p-ERK in a time-dependent manner (P<0.05). ConclusionTaken together, FGF-2 could increase cementoblast growth, proliferation, and Stro-1 positive cells. These enhancements are associated with the upregulation of p-Akt and p-ERK expression. The application of FGF-2 may provide benefit for periodontal and periapical regeneration during the early phase of wound healing.

Cellular Aging promotes CEMP1 Expression in STRO-1 positive PDL cells

Cellular Aging promotes CEMP1 Expression in STRO-1 positive PDL cells

Bone tissue engineering using STRO-1 positive porcine dental germ stem cells encapsulated in a poly-ethylene glycol based hydrogel

Event Abstract Back to Event Bone tissue engineering using STRO-1 positive porcine dental germ stem cells encapsulated in a poly-ethylene glycol based hydrogel Görke Gürel Peközer1, 2*, Mustafa Ramazanoğlu3*, Karl Andreas Schlegel4* and Gamze Köse2* 1 Istanbul Technical University, Molecular Biology-Genetics and Biotechnology, Turkey 2 Yeditepe University, Genetics and Bioengineering, Turkey 3 İstanbul University, Oral Surgery, Turkey 4 Friedrich-Alexander-University of Erlangen-Nuremberg, Maxillofacial Surgery, Germany There are a large number of people suffering from an organ or tissue loss due to injury, infection or disease. Although therapies like surgical reconstruction, transplantation or usage of prosthesis have saved and improved countless lives, they remain imperfect solutions due to occurrence of infection, chronic irritation, donor shortages, tissue rejection, and longer term complications such as development of malignant tumour. Although bone has an inherent ability to heal without leaving a scar, critical size defects fail to heal without intervention. The implantation of an engineered scaffold material with osteogenic cells seems to be a promising approach to achieve an optimal bone healing in critical-sized defects, since cell-mediated osteogenesis, especially at the centre of the defect, would be beneficial when combined with the osteoconductive effect of the bone graft material[1]. Mesenchymal stem cells (MSCs) isolated from various sources can be used for this purpose. The isolation of MSCs from dental tissues is easy, cost effective and does not raise additional safety and ethical concerns since they are obtained during regular orthodontic procedures. In this study, domestic pig was used as model organism for the isolation and transplantation of TGSCs due to its anatomical, physiologic, and metabolic similarities with humans. Bone structure and size are also important for the choice of pig to study bone tissue engineering. STRO-1 is a useful MSC marker that distinguishes only a subpopulation of MSCs that shows characteristics like high clonogenicity and multipotency differentiating to fibroblasts, smooth muscle cells, adipocytes, osteoblasts, and chondrocytes[2]. In this study, mandibular third molar tooth germs were surgically removed from the jaws of 6-month-old domestic pigs. Porcine TGSCs (PTGSCs) were isolated by explant culture technique after mincing the entire tooth germ tissue, including the dental mesenchyme and its surrounding follicle. PTGSCs were characterized by flow cytometry and by osteogenic, chondrogenic and adipogenic differentiation. Passage 3 PTGSCs were then tagged with STRO-1 antibody. Cells that express STRO-1 were sorted as STRO-1(+) cells and cells that do not express STRO-1 were sorted as STRO-1(-) cells by FACS Aria III. STRO-1 (+), STRO-1 (-) and unsorted cells were compared for their proliferation capacity by MTS assay and osteogenic differentiation capabilities in vitro by a quantitative calcium deposition assay and von Kossa staining. STRO-1 (+) and unsorted cells were also used for healing of critical-size bone calvarial defects of pigs in vivo with the use of a novel poly-ethylene glycol based hydrogel (PEG-hydrogel / MX10) and Bone Ceramic as scaffold. According to in vitro studies, STRO-1(+) cells showed to be more clonogenic exhibiting higher proliferation rates compared to STRO-1(-) and unsorted cells, whereas unsorted cells performed better shown by calcium deposition and von Kossa staining when they were osteogenically induced indicating the importance of a heterogenous niche for their role in osteogenesis. According to in vivo studies, novel PEG-hydrogel and Bone Ceramic was found to be an effective carrier for stem cell transplantation. Although transplantation of pTGSCs with PEG-hydrogel and Bone Ceramic facilitated the healing of defects, STRO-1(+) cells did not perform better than unsorted cells. Furthermore, in vivo studies also revealed the importance of osteogenic induction prior to implantation of cells to defects since osteogenically induced groups of either STRO-1(+) or unsorted cells led to increased bone density compared to non-induced groups. PEG-hydrogel-Bone Ceramic combination and use of pTGSCs facilitated the healing of bone defect without any significant effect created by STRO-1(+) cells. Thus, STRO-1 sorting of pTGSCs is not an effective strategy to enhance the osteogenic capacity of TGSCs since they possibly perform their role when they are in interactions with other cells in a heterogenous niche. International Team for Implantation (ITI), Basel, Switzerland; The Scientific and Technological Research Council of Turkey (TUBITAK), Ankara, Turkey

STRO-1 positive pulmonary valve stem cells: preliminary report

STRO-1 positive pulmonary valve stem cells: preliminary report

Recombinant growth/differentiation factor-5 enhances cell proliferation and tendogenic differentiation of human periodontal ligament stem cells in vitro.

e22191 Background: To investigate the function of growth/differentiation factor-5 (GDF-5) on cell proliferation and tendogenic differentiation of human periodontal ligment stem cells (PDLSCs) in vitro. Methods: PDLSCs were obtained from human healthy periodontal ligaments of wisdom teeth. Immunofluorescence cytochemistry was used to detect the characters of mesenchymal stem cells (MSCs) in PDLSCs. Recombinant mouse GDF-5 (rmGDF-5) protein was used to stimulate the PDLSCs. Tetrazolium reduction assay was carried out for cell proliferation. Alkaline phosphatase (ALP) activity was measured by ALP activity assay and ALP staining. Real-time RT-PCR was performed to detect the gene expressions of aggrecan (ACAN), collagen, type III (COL3), scleraxis (SCX), decorin (DCN), tenomodulin (TNMD) and tenascin C (TNC) in PDLSCs after stimulated by rmGDF-5 protein. Results: 5.6% STRO-1 positive cells was detected in primary PDLSCs. The cell proliferation of PDLSCs was enhanced by rmGDF-5 in a dose-dependent manner at 0, 20, 100 and 500 ng/ml of concentration. The special markers of tendogenic differention, ACAN, COL3, SCX and TNMD were up-regulated at mRNA level after rmGDF-5 treatment for 1, 2 or 3 weeks, and the 20 ng/ml was the more effective than 100 ng/ml and 500 ng/ml for rmGDF-5 in PDLSCs. The expressions of TNC and DCN were increased at 4 or 8 hours after 20, 50 and 100 ng/m rmGDF-5 treatment. The ALP activity results showed that ALP activities were decreased paralleled with increased rmGDF-5 concentration. Conclusions: The rmGDF-5 could promote the cell proliferation and tendogenic differentiation of PDLSCs in vitro.

1050 HUMAN LIVER CARCINOMAS RECRUIT MESENCHYMAL STEM/STROMAL CELLS THAT CAN PROMOTE TUMOR GROWTH VIA PARACRINE SIGNALING

Background and Aims: Bone marrow mesenchymal stem/stromal cells (MSCs) can migrate to tumor sites and contribute to the tumor microenvironment. However, it is still hotly debated whether MSCs have a positive or negative effect on tumor growth. This study aims to investigate whether human liver carcinomas contain MSCs and whether MSCs may affect tumor growth. Methods: MSCs were cultured from surgical resected hepatocellular carcinoma (HCC) (n = 6) and liver metastatic colorectal tumor (LMCRC) (n = 7). Immunohistochemical staining of STRO-1 (the bestknown MSCs marker for in vivo detection) was performed in paraffin-embedded patient HCC and LM-CRC tissues (n = 24). The effects of MSCs on tumor growth were evaluated in immunedeficient mice. Results: Solid tumors formed in mice by subcutaneous engraftment of human hepatoma Huh7 cells were able to recruit MSCs. MSCs were also found in patient liver tumors (successfully cultured from 11 out of 13 liver tumors). Their MSC properties were characterized by adipocyte and osteocyte differentiation and common mesenchymal markers. Notably, in situ staining showed that STRO-1 positive cells are significantly enriched in the tumor, in particular the tumor-stromal region, compared with the adjacent area in HCC and LM-CRC tissues (n = 24, p < 0.01). In mice, coengraftment of Huh7 and MSCs resulted in significant larger tumors than engraftment of Huh7 alone (tumor weight 1.56±0.27 g Vs 0.44±0.19 g, Mean±SEM, n = 8, p < 0.01). Consistently, co-culturing Huh7 with irradiated MSCs significantly increased the number (196±29 Vs 123±36 clonies/5000 Huh7, Mean±SD, n = 5, p < 0.01) and the size (1329±258 Vs 570±155 pixels, n = 5, p < 0.01) of formed colonies. This effect was also observed by treatment of MSCs conditioned medium (MSC-CM), suggesting secreted tropic factors contributing to the tumor promoting effect. Genome-wide gene expression array and pathway analysis confirmed the upregulation of cell growth and proliferation-related processes and down-regulation of cell death-related pathways by treatment of MSC-CM in Huh7 cells. Conclusion: Human liver carcinomas recruit MSCs, which in turn can promote tumor growth. These results shed new light on the crosstalk between MSCs with liver cancer cells but also caution stem cell therapy of using MSCs for liver cancer and other liver diseases with high risk of developing malignancy.

Ibalance osteotomy in early treatment of osteoarthritis

Ibalance osteotomy in early treatment of osteoarthritis

Characterization of mesenchymal stem cells from human normal and hyperplastic gingiva

Human gingiva plays an important role in the maintenance of oral health and shows unique fetal-like scarless healing process after wounding. Here we isolate and characterize mesenchymal stem cells from human normal and hyperplastic gingival tissues (N-GMSC and H-GMSC, respectively). Immunocytochemical staining indicated that gingival lamina propria contained Stro-1 and SSEA-4 positive cells, implying existence of putative gingival MSC. Under attachment-based isolating and culturing condition, gingival MSC displayed highly clonogenic and long-term proliferative capability. By using single colony isolation and expansion approaches, we found both N-GMSC and H-GMSC possessed self-renewal and multipotent differentiation properties. N-GMSC and H-GMSC showed distinct immunoregulatory functions in a murine skin allograft setting via up-regulation of putative systemic regulatory T cells (Tregs). N-GMSC and H-GMSC were capable of regenerating collagenous tissue following in vivo transplantation, in which H-GMSC exhibited more robust regenerative capability. These findings suggest that gingival tissue contains tissue-specific mesenchymal stem cell population and is an ideal resource for immunoregulatory therapy due to its substantial availability and accessibility. In addition, gingival MSC over-activation may contribute to gingival hyperplastic phenotype.

Effect of bone morphogenetic protein-2 on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells in vitro

Objective To study the effects of the bone morphogenetic protein-2 (BMP-2) on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro. Methods hUCMSCs were cultured in vitro, and BMP-2 (20 mg/L) was added into the culture medium. The proliferation effect of BMP-2 on hUCMSCs was examined by MTT assay. The proportion of antigen-positive cells for STRO-1 was observed by flow cytometry (FCM). The osteopontin ( OPN), alkaline phosphatase (ALP) and type 1 collagen ( COL1 ) mRNA levels was detected by reverse transcription-polymerase chain reaction ( RT-PCR). The ALP activity was tested by ALP staining array. Von Kossa staining method was used to study the calcification effects. Results The proliferation rate was increased 1-, 3-,5-, 7-day BMP-2 (20 mg/L) groups and no BMP-2 group, but there was no statistically significant difference among groups (P ＞ 0. 05). When the concentration of serum in the culture medium decreased from 10% to 2%, the proliferation was just increasing 10% and no different between BMP-2 group and no BMP-2 group (P＞0.05). The percentages of STRO-1 positive cells was increasing from 25.1 ±4.0 to 51.1 ±6.4 after culture with BMP-2( P ＜ 0.01 ). The BMP-2 increased the ALP and COL1 gene expression, induced the OPN gene expression. A lot of hUCMSCs was positive in ALP staining array in BMP-2 medium. Many mineralized nodes was detected in the Von kossa staining method after culture 28 days in the BMP-2 medium.Conclusion BMP-2 can promote the osteogenic differentiation,and have a little effect on the prolferation of hUCMSCs. Key words: Bone morphogenetic protein-2; Umbilical cord mesenchymal stem cell; Osteogenic differntiation

Hyaluronan Mixed Esters of Butyric and Retinoic Acid Affording Myocardial Survival and Repair without Stem Cell Transplantation

Possible cardiac repair by adult stem cell transplantation is currently hampered by poor cell viability and delivery efficiency, uncertain differentiating fate in vivo, the needs of ex vivo cell expansion, and consequent delay in transplantation after the onset of heart attack. By the aid of magnetic resonance imaging, positron emission tomography, and immunohistochemistry, we show that injection of a hyaluronan mixed ester of butyric and retinoic acid (HBR) into infarcted rat hearts afforded substantial cardiovascular repair and recovery of myocardial performance. HBR restored cardiac [18F]fluorodeoxyglucose uptake and increased capillary density and led to the recruitment of endogenous Stro-1-positive stem cells. A terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay demonstrated that HBR-treated hearts exhibited a decrease in the number of apoptotic cardiomyocytes. In isolated rat cardiomyocytes and Stro-1 stem cells, HBR enhanced the transcription of vascular endothelial growth factor, hepatocyte growth factor, kdr, akt, and pim-1. HBR also increased the secretion of vascular endothelial growth factor and hepatocyte growth factor, suggesting that the mixed ester may have recruited both myocardial and Stro-1 cells also. An increase in capillarogenesis was induced in vitro with medium obtained from HBR-exposed cells. In the infarcted myocardium, HBR injection increased histone H4 acetylation significantly. Acetyl-H4 immunoreactivity increased in rat cardiomyocytes and Stro-1 cells exposed to HBR, compared with untreated cells. In conclusion, efficient cardiac regenerative therapy can be afforded by HBR without the need of stem cell transplantation or vector-mediated gene delivery.

Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

IntroductionRecent findings suggest that articular cartilage contains mesenchymal progenitor cells. The aim of this study was to examine the distribution of stem cell markers (Notch-1, Stro-1 and VCAM-1) and of molecules that modulate progenitor differentiation (Notch-1 and Sox9) in normal adult human articular cartilage and in osteoarthritis (OA) cartilage.MethodsExpression of the markers was analyzed by immunohistochemistry (IHC) and flow cytometry. Hoechst 33342 dye was used to identify and sort the cartilage side population (SP). Multilineage differentiation assays including chondrogenesis, osteogenesis and adipogenesis were performed on SP and non-SP (NSP) cells.ResultsA surprisingly high number (>45%) of cells were positive for Notch-1, Stro-1 and VCAM-1 throughout normal cartilage. Expression of these markers was higher in the superficial zone (SZ) of normal cartilage as compared to the middle zone (MZ) and deep zone (DZ). Non-fibrillated OA cartilage SZ showed reduced Notch-1 and Sox9 staining frequency, while Notch-1, Stro-1 and VCAM-1 positive cells were increased in the MZ. Most cells in OA clusters were positive for each molecule tested. The frequency of SP cells in cartilage was 0.14 ± 0.05% and no difference was found between normal and OA. SP cells displayed chondrogenic and osteogenic but not adipogenic differentiation potential.ConclusionsThese results show a surprisingly high number of cells that express putative progenitor cell markers in human cartilage. In contrast, the percentage of SP cells is much lower and within the range of expected stem cell frequency. Thus, markers such as Notch-1, Stro-1 or VCAM-1 may not be useful to identify progenitors in cartilage. Instead, their increased expression in OA cartilage implicates involvement in the abnormal cell activation and differentiation process characteristic of OA.

Localization of STRO-1, BMP-2/-3/-7, BMP receptors and phosphorylated Smad-1 during the formation of mouse periodontium

Bone morphogenetic proteins (BMPs) and BMP receptors (BMPRs) are known to regulate the development of calcified tissues by directing mesenchymal precursor cells differentiation. However, their role in the formation of tooth-supporting tissues remains unclear. We investigated the distribution pattern of STRO-1, a marker of mesenchymal progenitor cells and several members of the BMP pathway during the development of mouse molar periodontium, from the post-natal days 6 to 23 (D6 to D23). STRO-1 was mainly localized in the dental follicle (DF) at D6 and 13 then in the periodontal ligament (PDL) at D23. BMP-2 and -7 were detected in Hertwig's epithelial root sheath (HERS) and in DF, then later in differentiated periodontal cells. BMP-3 was detected after D13 of the periodontal development. BMPRs-Ib, -II, the activin receptor-1 (ActR-1) and the phosphorylated Smad1 were detected in DF and HERS at D6 and later more diffusely in the periodontium. BMPR-Ia detection was restricted to alveolar bone. These findings were in agreement with others data obtained with mouse immortalized DF cells. These results suggest that STRO-1 positive DF cells may be target of BMPs secreted by HERS. BMP-3 might be involved in the arrest of this process by inhibiting the signaling provided by cementogenic and osteogenic BMPs.

Isolation and characterization of multipotent human periodontal ligament stem cells

Periodontal ligament (PDL) repair is thought to involve mesenchymal progenitor cells capable of forming fibroblasts, osteoblasts and cementoblasts. However, full characterization of PDL stem cell (SC) populations has not been achieved. To isolate and characterize PDLSC and assess their capability to differentiate into bone, cartilage and adipose tissue. Human PDL cells were stained for STRO-1, FACS sorted and expanded in culture. Human bone marrow SC (BMSC) served as a positive control. PDLSC and BMSC were cultured using standard conditions conducive for osteogenic, chondrogenic and adipogenic differentiation. Osteogenic induction was assayed using alizarine red S staining and expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP). Adipogenic induction was assayed using Oil Red O staining and the expression of PPAR gamma 2 (early) and LPL (late) adipogenic markers. Chondrogenic induction was assayed by collagen type II expression and toluidine blue staining. Human PDL tissue contains about 27% STRO-1 positive cells with 3% strongly positive. In osteogenic cultures ALP was observed by day-7 in BMSC and day-14 in PDLSC. BSP expression was detectable by day-7; with more intense staining in PDLSC cultures. In adipogenic cultures both cell populations showed positive Oil Red O staining by day-25 with PPAR gamma 2 and LPL expression. By day-21, both BMSC and PDLSC chondrogenic induced cultures expressed collagen type II and glycosaminoglycans. The PDL contains SC that have the potential to differentiate into osteoblasts, chondrocytes and adipocytes, comparable with previously characterized BMSC. This adult PDLSC population can be utilized for potential therapeutic procedures related to PDL regeneration.

The odontogenic potential of STRO-1 sorted rat dental pulp stem cellsin vitro

The presence of heterogeneous cell populations in dental pulp may count for the considerable variation in the outcome of in vitro and in vivo experiments. Here, we intended to determine whether a minor cell sub-population of high proliferation and odontogenic potential existed among a larger compartment of perhaps more committed progenitors. In this study, the STRO-1 antigen, defining a mesenchymal stem cell or progenitor subpopulation, was used for separating rat dental pulp cells with fluorescence-activated cell sorting (FACS). Subsequently, the STRO-1 positive cells were tested for their ability to differentiate towards an odontoblast-like phenotype. Three cell populations (STRO-1 positive, STRO-1 negative, and non-sorted cells) were cultured in odontogenic medium containing dexamethasone and beta-glycerophosphate. Cultures were analyzed by light- and scanning electron microscopy (SEM), and assessed for proliferation, ALP activity, and calcium content. Results showed that the STRO-1 positive cell population was able to differentiate into the odontoblast phenotype, similar to the non-sorted population. The negative cells however showed a fibroblast-like phenotype. SEM and real-time PCR confirmed such results. In conclusion, the STRO-1 selection proved applicable for rat-derived material, to obtain a cell population which is more homogeneous. This positive cell fraction was capable of differentiating into the odontogenic pathway, whereas the negative fraction was not. However, the effect was not always advantageous, when compared to non-sorted cells.

Enrichment of osteogenic cell populations from rat bone marrow stroma

The presence of multiple cell types in bone marrow and their varying proportions from isolation to isolation may count for the considerable variation in the outcome of different experiments. The presence of these multiple subpopulations suggests a need for a method that can purify the osteogenic component, i.e. osteoprogenitors, from other components. The availabilities of monoclonal antibodies recognizing subpopulations of osteoblasts are providing means for antibody-based methods. The cell surface antigens STRO-1, ALP and HOP-26 were used for cell sorting experiments with fluorescence activated cell sorting (FACS). These cell populations were analyzed on differential gene expression, cell proliferation and differentiation into the osteoblastic lineage. The oligo-microarray results showed that only the ALP positive cell population expressed genes of the extracellular matrix; like different collagens, ECM-1 and matrix protease MMP-14. The real-time polymerase chain reaction (QPCR) results showed that STRO-1 and ALP positive cells had an upregulation in expression of lipoprotein lipase, osteocalcin, and collagen type I. Integrin beta-3 was only upregulated for ALP positive cells, while for these cells downregulation occurred for the genes myosine, alkaline phosphatase and integrin beta-1. HOP-26 positive cells showed an upregulation in collagen type I compared to control group. The DNA analysis revealed that the cells of the control group and the HOP-26 positive cells showed a 5 times higher cell growth compared to the STRO-1 and ALP positive cells. The alkaline phosphatase activity showed no activity for the control group. The STRO-1 and ALP positive cells had a higher activity compared to the HOP-26 positive. The calcium measurements revealed only for the control group calcium at day 24. Based on the results of our study, we conclude that the FACS method had no negative effect on the proliferating as well as differentiating response of the cells. Further, we conclude that by using an antibody-based cell selection method, different cell populations with different mRNA expression profiles and different osteogenic characteristics can be obtained.

The Involvement of Adult Stem Cells Originated from Bone Marrow in the Pathogenesis of Pterygia

Pterygium is a proliferative disease. Recent research has reported that stem cells are involved in the pathogenesis of various proliferative diseases, including solid tumors and diabetic proliferate vitreoretinopathy. In previous literature, we hypothesized that adult stem cells originated from bone marrow were involved in the pathogenesis of pterygium. We proved this by immunohistochemical staining with various stem cell markers. The staining showed adult stem cells in the pterygium. c-kit positive cells were observed primarily in the stroma, and some cells were also found in the basal epithelium. AC133 and CD34 positive cells were primarily found in the basal epithelium and were ovoid shaped, similar to the c-kit cells. However, some cells were found in vascular endothelium. STRO-1 positive cells were found mainly in the stroma and were spindle shaped. In recurrent pterygium, cells were more scattered and the expression pattern was denser. Therefore, we suggest a new theory of pterygium pathogenesis. Inflammation caused by environmental factors triggers the abnormal production of some growth factors and cytokines in order to recover from cellular damage. If these healing signals are excessive, limbal basal cells will be changed to abnormally-altered pterygial cells. The excessive wound healing process and remnant altered cells result in recurrence using the same mechanism.

A quantitative assay that evaluates the capacity of human stromal cells to support granulomonopoiesis in situ.

We describe an assay that makes possible the observation of granulomonocytic colonies grown on allogeneic stromal layers and the quantification of the stroma-adherent colony-forming cells (CFC). Stromal layers were generated from Stro-1 positive cells isolated from adherent layers of primary long-term marrow cultures using magnetic beads coated with the Stro-1 antibody. The stromal layers consisted mainly of myofibroblastic cells. Marrow fractions depleted of cells bearing receptors for soybean agglutinin (SBA) and enriched in CD34+ cells were obtained by panning. SBA-, CD34+ marrow cells were seeded onto stromal cells grown in 96-well plates. After four weeks, a mixture of cytokines was added (granulocyte-macrophage colony-stimulating factor [GM-CSF]: 25 U/ml, interleukin [IL]-3: 4 ng/ml, Steel factor: 5 ng/ml and growth factors provided by 3% conditioned medium from the 5637 cell line). Wells with large colonies (containing 10(3) to 10(4) cells) were scored after 14 days. Limiting dilution analysis of data revealed a Poisson distribution of the stroma-adherent CFC. There was an average of one stroma-adherent CFC per 167 CD34+ enriched marrow cells, which gave an estimated frequency of one CFC per 10(5) unfractionated bone marrow cells. Colonies contained cells that gave rise to CFU-GM after replating in agar (5-40 CFU-GM were provided per each stroma-adherent CFC), but not cells with self-renewal ability (as indicated by negative results after replating single colonies onto secondary adherent layers). Colonies usually formed from a cobblestone-area and developed in intimate contact with alpha SM actin positive stromal cells. Some of the stromal cells were located above granulocytic cells, corresponding to the description of "blanket cells." This assay should allow the study of colony-formation on marrow stroma without disrupting the hemopoietic niche.