Research progress on dedifferentiated fat cells and their application in oral and maxillofacial bone tissue engineering / 口腔疾病防治

@#The identification of suitable seed cells represents a critical scientific problem to be solved in the field of oral and maxillofacial bone tissue regeneration. The application of adipose-derived stem cells (ASCs) in tissue and organ repair and regeneration has been studied extensively. In recent years, dedifferentiated fat (DFAT) cells have also shown broad application prospects in the field of bone tissue engineering. DFAT cells express stem cell-related markers and have the potential to differentiate into adipocytes, osteoblasts, chondrocytes, nerve cells, cardiomyocytes and endothelial cells. In addition, DFAT cells also have the advantages of minimally invasive acquisition, strong proliferation and high homogeneity. Currently, all studies involving the application of DFAT cells in scaffold-based and scaffold-free bone tissue engineering can confirm their effectiveness in promoting bone regeneration. However, cytological research still faces some challenges, including relatively low cell culture purity, unclear phenotypic characteristics and undefined dedifferentiation mechanisms. It is believed that with the continuous development and improvement of isolation, culture, identification and directional induction of osteogenic differentiation methods, DFAT cells are expected to become excellent seed cells in the field of oral and maxillofacial bone tissue engineering in the future.

ALDH1A3 + INS + cells in non-diabetic subjects and risk for type 2 diabetes: Analysis of cross-sectional data / 中华内分泌代谢杂志

Objective:To investigate the correlations of β cell dedifferentiation in non-diabetic subjects with risk factors for type 2 diabetes mellitus(T2DM).Methods:Immunofluorescence staining with insulin and β cell dedifferentiated marker ALDH1A3 was used to evaluate the β cell dedifferentiation levels in 38 non-diabetic and 23 T2DM. Correlation analyses were performed between β cell dedifferentiation levels and available clinical parameters including age, body mass index, HbA 1C level, triglycerides, and cholesterol levels in non-diabetic subjects. Results:β cell dedifferentiation level defined by the positive expression of ALDH1A3 in β cells(ALDH1A3 + INS + cell proportion) was significantly elevated in T2DM subjects( P<0.001). In PreD subjects, ALDH1A3 + INS + cells proportion were decreased( P=0.050) and negatively correlated with HbA 1C( r=-0.44, P=0.006), but not with age and body mass index. The analysis of correlation with lipidemic parameters showed that ALDH1A3 + INS + cells proportion was positively correlated with plasma total cholesterol level( r=0.39, P=0.045), but not plasma total triglyceride. Conclusion:ALDH1A3 + INS + cells were found to be decreased in prediabetes, suggesting that there may be enhanced β-cell identity in prediabetes to compensate for insulin secretion requirements; ALDH1A3 + INS + cells were elevated in people with high plasma total cholesterol levels, suggesting that total cholesterol may be one of the factors that induce β-cell dedifferentiation.

The promotion of bone formation at the tendon-bone interface after ACL reconstruction with De-BMSCs transplantation and its mechanism / 中华骨科杂志

Objective:This study aimed to investigate the effect of differentiation osteogenic bone marrow mesenchymal stem cells (De-BMSCs) transplantation on the promotion of bone formation at the tendon-bone interface after anterior cruciate ligament reconstruction (ACLR), and further explored the molecular mechanism of the enhanced osteogenic effect of De-BMSCs.Methods:BMSCs from femur and tibia of New Zealand White rabbit were subjected to osteogenic induction and then cultured in no osteogenic factor medium; the obtained cell population was termed De-BMSCs. De-BMSCs were induced into osteo-, chondro-and adipo-differentiation in vitro to examine the characteristics of primitive stem cells. ACLR model with a semitendinosus tendon were performed in 48 adult rabbits, three groups were established: control group with alginate gel injectionat the tendon-bone interface, BMSCs group with the injection of alginate gel containing BMSCs, De-BMSCs group with the injection of alginate gel containing De-BMSCs. At 4 and 12 weeks after surgery, rabbits in each group were sacrificed to evaluate tendon-bone healing by histologic staining, micro-CT examination, and biomechanical test. During osteogenic differentiation of De-BMSCs, si-RNA of nuclear factor of activated T cells 2 (NFATc2) si-RNA of nuclear factor of activated T cells 1 (NFATc1) were used to verify the molecular mechanism of enhanced osteogenic effect of De-BMSCs.Results:De-BMSCs exhibited some properties similar to BMSCs including multiple differentiation potential and cell surface marker. At 4 weeks after surgery, the BV/TV value of the De-BMSCs group 0.36±0.01 was significantly higher than that of the control group 0.24±0.03 and BMSCs group 0.30±0.02 (all P<0.05), and the maximum load 40.34±1.19 N and stiffness 20.67±2.14 N/mm were significantly higher than those in the control group 14.88±2.74N, 8.67±2.19 N/mm and the BMSCs group 26.31±1.76 N, 13.81±2.14 N/mm (all P<0.05). At 12 weeks after surgery, the BV/TV value of the De-BMSCs transplantation group 0.47±0.02 was significantly higher than that of the control group 0.30±0.02 and the BMSCs group 0.35±0.03 (all P<0.05), and the maximum load 64.46±6.69 N and stiffness 25.18±3.11 N/mm were significantly higher than those in the control group 41.01±6.12 N, 11.59±2.54 N/mm and the BMSCs group 48.21±4.12 N, 15.89±2.94 N/mm (all P<0.05). During the osteogenic differentiation of De-BMSCs, the expressions of Nanog and NFATc1 were synergistically increased which promoted interaction of NFATc1 and Osterix ( P< 0.05), resulting in the increased expression of osteoblast marker genessuch as COL1A, OCN, OPN (all P< 0.05). Conclusion:De-BMSCs transplantation could promote bone formation at the tendon-bone interface after ACLR,Nanog/NFATc1/Osterix signaling pathway mediated the enhancement of the osteogenic differentiation effect of De-BMSCs.

Pancreatic β-cell dedifferentiation detected by flow cytometry / 中华内科杂志

Objective:To establish a method for detecting pancreatic β-cell dedifferentiation using flow cytometry.Methods:Experimental study. Min6 (mouse β cell line), αTC1-6 (mouse α cell line), HepG2 (human hepatocellular carcinoma cells) and mouse F9 cells (mouse teratocarcinoma cell) were cultured with conventional medium. Min6 cells were treated with interleukin-1β (IL-1β) in combined with tumor necrosis factor α (TNFα), or palmitic acid (PA) overnight and stained with anti-chromogranin A (ChgA), anti-insulin (Ins), anti-glucagon (Gcg), anti-SRY-box transcription factor 9 (Sox9) and anti-octamer binding transcription factor 4 (Oct4) antibodies, respectively. Flow cytometry was applied to detect the pression of ChgA, Ins, Gcg, Sox9, and Oct4 in the cells, respectively. Unpaired Student t test was used for statistical analysis. Results:Flow cytometry analyses showed that Ins and ChgA were highly expressed in Min6 cells, Gcg was highly expressed in αTC1-6, Sox9 was highly expressed in HepG2, and Oct4 was highly expressed in F9 cells, respectively (around 90%). Treatment of Min6 cells with IL-1β+TNFα significantly decreased Ins positive staining cells (92.775%±1.702% vs. 97.125%±0.246%, P=0.045), while increased Sox9 positive staining cells (41.675%±0.390% vs. 25.875%±3.348%, P=0.003). No significant changes in ChgA and Oct4 expression could be viewed (both P>0.05). PA treatment elevated the number of Gcg positive staining cells (54.500%±3.597% vs. 41.160%±3.007%, P=0.022). The levels of mRNA expression by qPCR of the above proteins were in consistent with the levels of protein expression by flow cytometry in Min6 cells. Conclusion:Flow cytometry can be used to detect proteins expressed in dedifferentiated models of β cells, which provides a new method for identify dedifferentiation of pancreatic β cells.

Research progress in thyroid cancer: dedifferentiation mechanisms and differentiation therapies / 中华核医学与分子影像杂志

Iodine accumulation represents a differentiation marker of thyroid cancer (TC) and a cornerstone of benefits from 131I therapy. However, dedifferentiation phenotypes occur in nearly 70% of recurrent or metastatic TCs driven by oncogenic mutations such as B-Raf proto-oncogene, serine/threonine kinase (BRAF), telomerase reverse transcriptase (TERT) promoters, and tumor proten p53 (TP53). Beyond genetic alterations, epigenetics, autophagy, tumor microenvironment and other pathways are also involved in the dedifferentiation of TC and the tolerance to 131I therapy. Targeting the above-mentioned pathways has potential to improve the malignant phenotype of TC and restore sensitivity to 131I therapy, which is of great clinical significance. Based on the relevant mechanisms of dedifferentiation, this paper elaborates on the progress of preclinical experiments and clinical studies related to differentiation therapies of TC.

Soft tissue myoepithelial carcinoma: is it a low-grade cancer? / Carcinoma mioepitelial de partes moles: um tumor de baixo grau de malignidade?

ABSTRACT Myoepithelial carcinoma is a rare neoplasm of very heterogeneous manifestation that represents a major challenge to the adoption of categorical prognostic criteria since, despite being classified as a low-grade cancer, it behaves very aggressively. While several studies have indicated complete surgical resection as the best treatment for myoepithelial carcinoma, adjuvant therapies have proven effective in preventing relapse. This study describes the case of a patient who developed myoepithelial carcinoma in the right thigh, affecting deep tissues, which were conducted as indicated in the current literature, however, presenting an unfavorable outcome. The available information comes from few studies; therefore, we emphasize the importance of gathering data on the subject, which are still scarce.

RESUMEN El carcinoma mioepitelial es una neoplasia rara, de manifestación muy heterogénea, que encierra un gran desafío para la adopción de criterios pronósticos categóricos, puesto que, a pesar de ser clasificado como tumor de bajo grado de malignidad, muestra comportamientos muy agresivos. Mientras varios estudios indican la resección quirúrgica completa como el mejor tratamiento para el carcinoma mioepitelial, terapias adyuvantes se han revelado efectivas para prevenir la recidiva. Este estudio describe el caso de una mujer que presentó carcinoma mioepitelial en muslo derecho, afectando partes profundas, lo cual fue conducido como indica la literatura actual, sin embargo, con un resultado desfavorable. Las informaciones disponibles son producto de pocos estudios, por ello, destacamos la importancia de reunir datos acerca del tema, que aún son escasos.

RESUMO O carcinoma mioepitelial é uma neoplasia rara, de manifestação muito heterogênea, que encerra um grande desafio na adoção de critérios prognósticos categóricos, uma vez que, apesar de ser classificado como tumor de baixo grau de malignidade, apresenta comportamentos muito agressivos. Enquanto vários estudos indicam a ressecção cirúrgica completa como o melhor tratamento para carcinoma mioepitelial, terapias adjuvantes têm-se revelado efetivas na prevenção da recidiva. Este estudo descreve o caso de uma paciente que desenvolveu carcinoma mioepitelial em coxa direita, acometendo partes profundas, o qual foi conduzido como indica a literatura atual, contudo, apresentando desfecho desfavorável. As informações disponíveis provêm de poucos estudos, logo, ressaltamos a importância de reunir dados a respeito do tema, que ainda são escassos.

Dedifferentiated liposarcoma with inflammatory myofibroblastic tumor-like features: a clinicopathological analysis of five cases / 中华病理学杂志

Objective@#To investigate the clinicopathological features, diagnosis and differential diagnosis of dedifferentiated liposarcoma (DDLPS) with inflammatory myofibroblastic tumor (IMT)-like features.@*Methods@#Five cases of DDLPS with IMT-like features were collected from the First Affiliated Hospital of Nanjing Medical University, the Affiliated Hospital of Nanjing University of Traditional Chinese Medicine and the First People′s Hospital of Qinzhou between 2013 and 2018. EnVision method and fluorescence in situ hybridization (FISH) were used to detect the immunophenotype of the tumor cells and the profile of MDM2 gene amplification respectively.@*Results@#All five cases were male and the median age was 61 (range 53 to 65) years. The clinical symptoms were mainly related to the space-occupying lesions. The tumors were located in duodenal mesentery (two cases), intestinal wall (one case), retroperitoneum (one case), and spermatic cord (one case). Grossly, the tumors were not well encapsulated, ranging from 3 to 13 cm (median 6.7 cm) in diameter, with tan to gray and firm cut surface. Histologically, the dedifferentiated component closely resembled inflammatory myofibroblastic tumor (IMT), with spindle/polygonal/stellate-shaped cells arranged in storiform, sheet-like, or random pattern, with varying degrees of chronic inflammation and fibrosis. All three major patterns seen in IMT (myxoid, cellular and hypocellular fibrous) were observed, the hypocellular fibrous pattern was the most common. Well-differentiated liposarcomatous component was found in the peripheral areas of all the tumors. One case had high grade dedifferentiated component. Four cases were strongly positive for MDM2 and p16. Two cases were positive for SMA, and one case was focally positive for desmin and one for CD34. None of the cases stained for ALK-1. FISH demonstrated MDM2 gene amplification in all five cases. Clinical follow-ups were available in all five cases and the interval ranged from 3 to 66 months (median 23 months). Two patients developed recurrences and one patient had metastasis. The remaining two patients were alive with no evidence of tumor recurrence at 3 and 14 months after surgery respectively.@*Conclusions@#DDLPS with IMT-like features is a more aggressive neoplasm than its histological mimic (IMT), and should not be misdiagnosed as other intermediate or low-grade malignant tumors, such as IMT, sclerosing liposarcoma, inflammatory liposarcoma, aggressive fibromatosis, solitary fibrous tumors, low-grade myofibroblastic sarcoma, and low-grade fibrosarcoma.

Pleomorphic and dedifferentiated leiomyosarcoma: a clinicopathologic analysis / 中华病理学杂志

Objective@#To investigate the clinicopathologic features, differential diagnosis and biological behavior of pleomorphic leiomyosarcoma (PLMS) and dedifferentiated leiomyosarcoma (DLMS).@*Methods@#Forty-nine cases were collected from November 2007 to December 2016, including eight that diagnosed at Fudan University Shanghai Cancer Center, and 41 consultation cases. The clinical findings and pathologic features were reviewed. Immunophenotype was obtained in 33 cases and follow-up information was available in 38 cases.@*Results@#There were 22 males and 27 females with ages ranging from 24 to 83 years (mean 52.5 years). Fifteen cases occurred in extremities, 14 in deep body cavity, 11 in the trunk, 4 in the head and neck, 2 in the bladder, and 1 each in the inguinal region, perineum and femoral vein, respectively. Tumor sizes ranged from 3 to 30 cm (mean 9.1 cm). The tumors were composed of at least small foci of typical leiomyosarcoma (LMS) and areas of high-grade pleomorphic/undifferentiated sarcoma. The typical LMS component showed the characteristic morphology of smooth muscle differentiation and was low to intermediate grade in most cases. Pleomorphic areas were mainly composed of atypical spindle and polygonal cells admixed with variable large, bizarre atypical cells and multinuclear giant cells, mostly mimicking undifferentiated pleomorphic sarcoma. The pleomorphic and leiomyosarcomatous areas were usually intermixed, but the demarcation may be distinct or gradual in some cases. The classical LMS component was positive for at least one myogenic marker: α-SMA in 97.0%(32/33), desmin in 72.7%(24/33), H-caldesmon in 90.9% (20/22), MSA in 14/16, and calponin in 15/15 of cases. The pleomorphic sarcoma component was reactive for at least one myogenic marker in 87.9% (29/33) of cases, usually showing focal and less intense immunoreactivity than classical LMS component: α-SMA was positive in 81.8%(27/33), desmin in 48.5%(16/33), H-caldesmon in 72.7% (16/22), MSA in 12/16, and calponin in 11/15 of cases. Based on staining for muscle markers in the pleomorphic component, 29 cases were designated as PLMS, 4 as DLMS. Ki-67 index ranged from 15% to 70% (mean 40%). Follow-up data was available in 38 cases (77.6%), of which 11 patients (28.9%) died of disease, 12 patients were alive with unresectable or recurrent disease, 14 patients were alive with no evidence of disease and another one died of unrelated cause. The median disease-free and overall survival was 6 and 10 months respectively. Twelve patients exhibited local recurrence and 11 developed metastases. The median interval to progression was 8 months.@*Conclusions@#The identification of areas of typical LMS is crucial for accurate diagnosis of PLMS and DLMS. Both PLMS and DLMS show more aggressive behavior and poorer prognosis than ordinary LMS.

Recent basic and genetic research progress of type 2 diabetes / 中华内分泌代谢杂志

In the past two years, a line of basic and genetic findings have been produced in the field of type 2 diabetes. Some evidence has suggested that mature β cell under long-term metabolic stress could de-differentiate into pre-endocrine cells and re-differentiate into α and PP endocrine cells. Several key factors were reported with genetic modified animal models in the past two years. A novel adipokine, Asprosin was found to control insulin resistance and food intake in both humans and mice. Additionally, researchers reported that gut microbiota was associated with the development of type 2 diabetes, and a few bacteria or certain enterotype could be valuable in the prediction of prevention and clinical intervention for diabetes. The genetic composition for missing heritability of type 2 diabetes and obesity was revealed with the next-generation sequencing strategy. Importantly, scientists at home and abroad made a significant progress in the field during the past few years, which should be reviewed here. (Chin J Endocrinol Metab, 2018, 34: 605-609)

Research progress of dedifferentiation and mechanism of beta cell in type 2 diabetes mellitus / 中国基层医药

Islet βcell dysfunction is one of the important links in the development of type 2 diabetes (T2DM).The latest research shows that the main cause of the continuous β-cell dysfunction under metabolic stress is mainly the dedifferentiation of β-cells,and become endocrine progenitor -like cells with multiple differentiation potentials.Studies have found that the process of dedifferentiation of pancreatic islet βcells is reversible.This finding has provided possibilities and new ideas for preventing and reversing the progressive decline of βcell function and delaying the occurrence of T2DM.

Roles of β-cell dedifferentiation in β-cell dysfunction / 中国病理生理杂志

β-cells may turn into other pancreatic islet cells through dedifferentiation.The dedifferentiation cells lose the ability of insulin secretion.Islet β-cell dedifferentiation is the important cause of β-cell dysfunction, which then leads to the occurrence of type 2 diabetes.Rescent research shows that many transcription factors play an important role in β-cell dedifferentiation,including forkhead box protein O1,NKX2.2,NKX6.1,MAFA and MAFB.In this paper, the influence factors of β-cell dedifferentiation and its mechanism were reviewed briefly.The discovery of β-cell dedifferen-tiation provides new thoughts and targets for prevention and treatment of type 2 diabetes.

Effects of basic fibroblast growth factor on chondrocyte phenotype and dedifferentiation in porous tantalum-chondrocyte composites in vitro / 第二军医大学学报

Objective To study the effect of basic fibroblast growth factor (bFGF) of different concentrations on phenotypes and dedifferentiation of rabbit chondrocytes in porous tantalum-chondrocyte composites in vitro, so as to provide theoretic basis for cartilage defect repair. Methods The articular chondrocytes from 3-week-old rabbit were cultured and identified by type IT collagen immunocytochemistry and Safranin 0 staining. The 3rd generation chondrocytes were implanted in the porous tantalum and was treated with bFGF of various concentrations. The bFGF-chondrocyte-porous tantalum composites (bFGF compostes) were then divided into 5 groups: group A (1 ng/mL bFGF composites), group B (10 ng/mL bFGF composites), group C (50 ng/mL bFGF composites), group D (chondrocyte-porous tantalum), and group E (pure chondrocyte). The proliferation of chondrocytes was measured by MTT and the cell morphology and growth were observed by scanning electron microscopy (SEM). Phenotypes and dedifferentiation (type I, II, IX, and X collagen) of the chondrocytes were detected by immunocytochemical method. Type II and X collagen mRNA was tested by real-time PCR. Results Type II collagen immunocytochemistry and Safranin 0 staining were positive, confirming that the cultured cells were chondrocytes. MTT results showed that chondrocyte proliferation in groups A, B, C, and D were significantly greater than that in group E CP

Dedifferentiated Extraskeletal Myxoid Chondrosarcoma of the Masticator Space: A Case Report

We describe a 69-year-old woman who presented with a dedifferentiated extraskeletal myxoid chondrosarcoma arising in the left masticator space. Computed tomography and magnetic resonance imaging revealed a 5 cm sized mass in the left masticator space. Histologically, the tumor consisted of two distinct areas. The less cellular area was a low-grade extraskeletal myxoid chondrosarcoma, composed of strands or cords of uniform spindle cells and abundant myxoid stroma. The more cellular, dedifferentiated area corresponded to a high grade myxofibrosarcoma, consisting of anaplastic tumor cells in myxoid stroma and geographic necrosis. The tumor cells of the former area were positive for S-100 protein, microtubule-associated protein-2 (MAP-2) and class III beta-tubulin, but negative for cytokeratin, smooth muscle actin, and desmin. The tumor cells in the latter, pleomorphic area showed MAP-2 and beta-tubulin immunoreactivity with a high Ki-67 labeling index. Based on its histologic and immunohistochemical features, the tumor was considered a dedifferentiated extraskeletal myxoid chondrosarcoma.

Dedifferentiated Extraskeletal Myxoid Chondrosarcoma of the Masticator Space: A Case Report

We describe a 69-year-old woman who presented with a dedifferentiated extraskeletal myxoid chondrosarcoma arising in the left masticator space. Computed tomography and magnetic resonance imaging revealed a 5 cm sized mass in the left masticator space. Histologically, the tumor consisted of two distinct areas. The less cellular area was a low-grade extraskeletal myxoid chondrosarcoma, composed of strands or cords of uniform spindle cells and abundant myxoid stroma. The more cellular, dedifferentiated area corresponded to a high grade myxofibrosarcoma, consisting of anaplastic tumor cells in myxoid stroma and geographic necrosis. The tumor cells of the former area were positive for S-100 protein, microtubule-associated protein-2 (MAP-2) and class III beta-tubulin, but negative for cytokeratin, smooth muscle actin, and desmin. The tumor cells in the latter, pleomorphic area showed MAP-2 and beta-tubulin immunoreactivity with a high Ki-67 labeling index. Based on its histologic and immunohistochemical features, the tumor was considered a dedifferentiated extraskeletal myxoid chondrosarcoma.

2-Deoxy-D-glucose regulates dedifferentiation through beta-catenin pathway in rabbit articular chondrocytes

2-deoxy-D-glucose (2DG) is known as a synthetic inhibitor of glucose. 2DG regulates various cellular responses including proliferation, apoptosis and differentiation by regulation of glucose metabolism in cancer cells. However, the effects of 2DG in normal cells, including chondrocytes, are not clear yet. We examined the effects of 2DG on dedifferentiation with a focus on the beta-catenin pathway in rabbit articular chondrocytes. The rabbit articular chondrocytes were treated with 5 mM 2DG for the indicated time periods or with various concentrations of 2DG for 24 h, and the expression of type II collagen, c-jun and beta-catenin was determined by Western blot, RT-PCR, immunofluorescence staining and immunohistochemical staining and reduction of sulfated proteoglycan synthesis detected by Alcain blue staining. Luciferase assay using a TCF (T cell factor)/LEF (lymphoid enhancer factor) reporter construct was used to demonstrate the transcriptional activity of beta-catenin. We found that 2DG treatment caused a decrease of type II collagen expression. 2DG induced dedifferentiation was dependent on activation of beta-catenin, as the 2DG stimulated accumulation of beta-catenin, which is characterized by translocation of beta-catenin into the nucleus determined by immunofluorescence staining and luciferase assay. Inhibition of beta-catenin degradation by inhibition of glycogen synthase kinase 3-beta with lithium chloride (LiCl) or inhibition of proteasome with z-Leu-Leu-Leu-CHO (MG132) accelerated the decrease of type II collagen expression in the chondrocytes. 2DG regulated the post-translational level of beta-catenin whereas the transcriptional level of beta-catenin was not altered. These results collectively showed that 2DG regulates dedifferentiation via beta-catenin pathway in rabbit articular chondrocytes.

Ectopic expression of cyclooxygenase-2-induced dedifferentiation in articular chondrocytes

Cyclooxygenase-2 (COX-2) is known to modulate bone metabolism, including bone formation and resorption. Because cartilage serves as a template for endochondral bone formation and because cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes (Ahrens et al., 1977; Sandell and Adler, 1999; Solursh, 1989), it is of interest to know whether COX-2 expression affect chondrocyte differentiation. Therefore, we investigated the effects of COX-2 protein on differentiation in rabbit articular chondrocyte and chick limb bud mesenchymal cells. Overexpression of COX-2 protein was induced by the COX-2 cDNA transfection. Ectopic expression of COX-2 was sufficient to causes dedifferentiation in articular chondrocytes as determined by the expression of type II collagen via Alcian blue staining and Western blot. Also, COX-2 overexpression caused suppression of SOX-9 expression, a major transcription factor that regulates type II collagen expression, as indicated by the Western blot and RT-PCR. We further examined ectopic expression of COX-2 in chondrifying mesenchymal cells. As expected, COX-2 cDNA transfection blocked cartilage nodule formation as determined by Alcian blue staining. Our results collectively suggest that COX-2 overexpression causes dedifferentiation in articular chondrocytes and inhibits chondrogenic differentiation of mesenchymal cells.

Isolation,cultivation and identification of dedifferentiation derived epithelial stem cells / 解放军医学杂志

Objective To introduce an efficient method to isolate and identify the dedifferentiation derived epidermal stem cells. Methods HEKa cells obtained from Casacade (USA) were induced to reverse their differentiated process and produce immature, stem-like cells-the dedifferentiation derived epidermal stem cells (dHEK cells)-by basic fibroblasts growth factors (bFGF) in vitro. Improved collagen Ⅳ-coated adhesion method was used to isolate and cultivate the dHEK cells. Subsequently, morphological features and phenotypic changes of these immature dHEK cells were immunochemically stained and studied with confocal microscopy and flow FACS array. Results Immunocytochemical analysis proved that dHEK cells and their subunits were positive for ?1 integrin, keratin 19 and 14, yet negative for the expression of keratin 10, which was considered as an established marker of differentiated cells. Double staining immunofluorescence demonstrated that markers such as ?6 integrin and CD71 were co-expressed in dHEK cells, and it was shown that there was important regional differences in the distribution of ?6 integrin and CD71 in dHEK cells and their subpopulations. More importantly, two-color flow cytometric analysis of ?6 integrin and CD71 consistently revealed two phenotypically discrete populations of cells in the isolated dHEK cells, i.e. a major subpopulation exhibiting high levels of both ?6 and CD71 expression (?6+CD71+ representing 66.97%?5.04% of the total cells) and a minor population characterized by high level of ?6 expression and low level of CD71 expression (?6+CD71- representing 24.52%?7.88% of the total cells) (n=3). Conclusions The dHEK cells isolated by collagen Ⅳ-coated adhesion method have some characteristics of native epidermal stem cells, and perhaps it may act as a new vehicle for non-genetic manipulation and therapy for both skin disorders and systemic deficiencies. Additionally, although viewed as an ideal substitute of ESCs, the safety of dHEK cells and their functional difference with native epidermal stem cells are still unclear, which need further research.