Investigation the effects of vitreous humor on proliferation and dedifferentiation of differentiated NTERA2 cells / Investigar os efeitos do humor vítreo na proliferação e desdiferenciação de células NTERA2 diferenciadas

Abstract Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.

Resumo Os mamíferos têm uma capacidade limitada de regenerar seus tecidos e órgãos. Um dos mecanismos associados à regeneração natural é a desdiferenciação. Várias moléculas pequenas, como vitamina C e fatores de crescimento, podem melhorar a eficiência da reprogramação. Neste estudo, as células NTERA2-D1 (NT2) foram induzidas à diferenciação (NT2-RA) com ácido retinóico (RA) 10-5 M por três dias e depois submetidas a várias quantidades de humor vítreo (VH). Os resultados mostram que a taxa de crescimento dessas células foi reduzida, enquanto essa taxa foi parcialmente restaurada após o tratamento com VH (NT2-RA-VH). A análise do ciclo celular com o método PI também mostrou que o número de células na fase S do ciclo celular nessas células estava aumentado. Os níveis de antígenos SSEA3 e TRA-1-81 em NT2-RA diminuíram, mas aumentaram em NT2-RA-VH a um nível semelhante ao das células NT2. O nível de SSEA1 teve um padrão oposto. A expressão do gene OCT4 diminuiu após o tratamento com AR, mas foi recuperado em células NT2-RA-VH. Em conclusão, sugerimos o VH como uma mistura potente para melhorar a reprogramação celular levando à desdiferenciação.

Lipossarcoma Bem Diferenciado do Retroperitônio com Desdiferenciação e Múltiplas Recidivas: Relato de Caso / Well-Differentiated Liposarcoma of the Retroperitoneum with Dedifferentiation and Multiple Recurrences: Case Report / Liposarcoma Bien Diferenciado de Retroperitoneo con Desdiferenciación y Múltiples Recurrencias: Informe de Caso

Introdução: Os lipossarcomas retroperitoneais são neoplasias mesenquimais raras, sendo mais comuns os bem diferenciados e os desdiferenciados. O subtipo bem diferenciado pode sofrer desdiferenciação para tumores de maior grau. São neoplasias difíceis de tratar cirurgicamente, pois apresentam altas taxas de recorrência local, alguns subtipos podem metastizar e são pouco sensíveis à radioterapia e à quimioterapia. Relato do caso: Paciente feminina, 45 anos, apresentou dor abdominal e massa abdominal palpável em 2017. Foi submetida à ressecção de lipossarcoma bem diferenciado de retroperitônio, sem intercorrências. Em 2020, manifestou dor abdominal e perda ponderal. A tomografia mostrou múltiplas massas volumosas abdominais, com biópsia sugestiva de lipossarcoma desdiferenciado. Foi submetida à radioterapia neoadjuvante e, em seguida, à ressecção cirúrgica das massas e ileocolectomia direita. Em 2022, apresentou quadro sugestivo de obstrução intestinal, sendo submetida à laparotomia que evidenciou intenso bloqueio de alças intestinais, fístula duodenal, tumor retroperitonial e peritonite fecal. Procedeu-se à ressecção de neoplasia retroperitoneal, ileostomia e rafia de fístula. O histopatológico mostrou lipossarcoma desdiferenciado recidivado. A paciente evoluiu com complicações operatórias e infecciosas, necessitando de cuidados intensivos e antibioticoterapia. Após melhora clínica, recebeu alta com dieta enteral e segue em acompanhamento ambulatorial. Conclusão: O lipossarcoma de retroperitônio pode sofrer desdiferenciação, recidivas multifocais e múltiplas recorrências, necessitando de várias abordagens cirúrgicas, o que aumenta a morbidade e o risco de complicações. A cirurgia com margens amplas continua sendo a principal modalidade terapêutica.

ABSTRACT Introduction: Retroperitoneal liposarcomas are rare mesenchymal neoplasms, with well-differentiated and dedifferentiated liposarcomas being most common. The well differentiated subtype can undergo dedifferentiation to higher grade tumors. These are difficult neoplasms to treat surgically because they have high rates of local recurrence, some subtypes can metastasize, and are poorly responsive to radiotherapy and chemotherapy. Case report: Female patient, 45 years old, presented abdominal pain and palpable abdominal mass in 2017. She underwent resection of well-differentiated liposarcoma of the retroperitoneum, without intercurrences. In 2020, she manifested abdominal pain and weight loss. Tomography showed multiple voluminous abdominal masses, with biopsy suggestive of dedifferentiated liposarcoma. The patient was submitted to neoadjuvant radiotherapy, followed by surgical resection of the masses and right ileocolectomy. In 2022, she presented symptoms suggestive of intestinal obstruction, and underwent laparotomy that revealed intense blockage of intestinal loops, duodenal fistula, retroperitoneal tumor, and fecal peritonitis. Retroperitoneal neoplasm resection, ileostomy and fistula closure were performed. Histopathology showed relapsed dedifferentiated liposarcoma. The patient evolved with operative and infectious complications, requiring intensive care and antibiotic therapy. After clinical improvement, the patient was discharged with enteral diet and continues under outpatient follow-up. Conclusion: Retroperitoneal liposarcoma may undergo multifocal dedifferentiation and recurrence, requiring several surgical approaches, increasing morbidity and the risk of complications. Wide margin surgery remains the main therapeutic modality.

Introducción: Los liposarcomas retroperitoneales son neoplasias mesenquimatosas raras, siendo los más comunes los liposarcomas bien diferenciados y desdiferenciados. El subtipo bien diferenciado puede sufrir desdiferenciación hacia tumores de mayor grado. Estas neoplasias son difíciles de tratar quirúrgicamente porque presentan altas tasas de recidiva local, algunos subtipos pueden hacer metástasis y responden mal a la radioterapia y la quimioterapia. Informe del caso: Mujer de 45 años, en 2017 presenta dolor abdominal y masa abdominal palpable. Fue sometida a la resección de un liposarcoma bien diferenciado del retroperitoneo, sin intercurrencias. En 2020, manifestó dolor abdominal y pérdida de peso. La tomografía mostró múltiples masas abdominales voluminosas, con biopsia sugestiva de liposarcoma desdiferenciado. Fue sometida a radioterapia neoadyuvante y luego a resección quirúrgica de las masas y a ileocolectomía derecha. En 2022, presentó síntomas de obstrucción intestinal y fue sometida a una laparotomía que reveló obstrucción de las asas intestinales, fístula duodenal, tumor retroperitoneal y peritonitis fecal. Se realizó la resección de la neoplasia retroperitoneal, la ileostomía y la fistulización. La histopatología mostró un liposarcoma desdiferenciado. La paciente evolucionó con complicaciones operatorias e infecciosas, requiriendo cuidados intensivos y terapia antibiótica. Tras la mejora clínica, la paciente fue dada de alta con dieta enteral y está en seguimiento. Conclusión: El liposarcoma retroperitoneal puede sufrir desdiferenciación multifocal y recurrencia, requiriendo varios a tratamientos quirúrgicos, aumentando la morbilidad y el riesgo de complicaciones. La cirugía con márgenes amplios sigue siendo la terapia principal.

Effects of rosuvastatin in homocysteine induced mouse vascular smooth muscle cell dedifferentiation and endoplasmic reticulum stress and its mechanisms / 中国应用生理学杂志

OBJECTIVE@#To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS).@*METHODS@#VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected.@*RESULTS@#Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01).@*CONCLUSIONS@#Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.

Metástasis pancreática, sitio inusual de diseminación por cáncer de tiroides. Documentada por PET CT/FDG y estudio histopatológico / Pancreatic metastasis, an unusual site for thyroid cancer spread. Documented by PET CT/FDG and histopathology

El cáncer diferenciado de tiroides (CDT) es la neoplasia endocrina maligna encontrada con mayor frecuencia, generalmente tiene un comportamiento lento en su evolución permitiendo realizar manejo quirúrgico y terapia ablativa con Iodo 131, lográndose la remisión completa en la gran mayoría de los casos. Un pequeño porcentaje de estas neoplasias presenta un comportamiento agresivo al registrar metástasis a distancia con localización principalmente en pulmón, hueso y cerebro y con focos de desdiferenciación celular, lo cual empobrece su pro nóstico y limita las opciones terapéuticas en este tipo de tumores. En el proceso de seguimiento del CDT, la tomografia por emisión de positrones con análogo de glucosa (PET/CT con F18-FDG) se ha constituido en una herramienta diagnóstica y pronóstica de imagen eficaz. Presentamos el caso de un paciente masculino de 50 años de edad con cáncer papilar de tiroides y enfermedad metastásica en páncreas, sitio inusual de diseminación para esta patología.

Differentiated Thyroid Cancer (DTC) is the most frequently found malignant endocrine neoplasia. It is usually slow developing, allowing for surgical management and ablation therapy with Iodine 131, achieving complete remission in the great majority of cases. However, a small percentage of tumours are aggressive, with distant metastases to lung, bone, brain, and foci of cellular dedifferentiation, which worsens prognosis and limits therapeutic options. In the monitoring process of the CDT, Positron emission tomography with glucose analogue (PET/CT with F18 - FDG) remains an important diagnostic and prognostic imaging tool. The case is presented of a 50 year old male patient with papillary thyroid cancer and metastatic disease in the pancreas, an unusual site in this disease.

Effect of JJYMD-C, a novel synthetic derivative of gallic acid, on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro

Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects.

Progress in dedifferentiated fat cells / 生物工程学报

When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to dedifferentiated fat (DFAT) cells. DFAT cells have many advantages compared with adipose-derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs). For example, DFAT cells are homogeneous and could be obtained from donors regardless of their age. Furthermore, DFAT cells also have the same multi-lineage potentials and low immunogenicity as ASCs. As an excellent source of seed cells for tissue engineering and stem cell transplantation, DFAT cells have better prospects in the treatment of many clinical diseases, such as bone defects, neurological diseases, ischemic heart disease and kidney disease. It is necessary to make more intensive studies of DFAT cells. This article summarizes progresses in the immunological characteristics, differentiation ability and potential clinical applications of DFAT cells.

Serum-free media for articular chondrocytes in vitro expansion / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>In vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a number of undesirable questions. In the present study, two kinds of defined, serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering.</p><p><b>METHODS</b>Bovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor. Expansion culture in a conventional 10% fetal bovine serum (FBS) medium served as control. Fibronectin coating was used to help cell adhesion in serum-free medium. Next, in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity. Cell pellets were expanded in different media to re-express the differentiated phenotype (re-differentiation) and to form cartilaginous tissue. The pellets were assessed by glycosaminoglycans contents, collagen II, collagen I and collagen X immunohistological staining.</p><p><b>RESULTS</b>Chondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium. In addition, chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I. Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium.</p><p><b>CONCLUSION</b>These findings provide alternative culture approaches for chondrocytes in vitro expansion, which may benefit the clinical use of autologous chondrocytes implantation.</p>

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.

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.

Transient lysosomal activation is essential for p75 nerve growth factor receptor expression in myelinated Schwann cells during Wallerian degeneration / 대한해부학회지

Myelinated Schwann cells in the peripheral nervous system express the p75 nerve growth factor receptor (p75NGFR) as a consequence of Schwann cell dedifferentiation during Wallerian degeneration. p75NGFR has been implicated in the remyelination of regenerating nerves. Although many studies have shown various mechanisms underlying Schwann cell dedifferentiation, the molecular mechanism contributing to the re-expression of p75NGFR in differentiated Schwann cells is largely unknown. In the present study, we found that lysosomes were transiently activated in Schwann cells after nerve injury and that the inhibition of lysosomal activation by chloroquine or lysosomal acidification inhibitors prevented p75NGFR expression at the mRNA transcriptional level in an ex vivo Wallerian degeneration model. Lysosomal acidification inhibitors suppressed demyelination, but not axonal degeneration, thereby suggesting that demyelination mediated by lysosomes may be an important signal for inducing p75NGFR expression. Tumor necrosis factor-alpha (TNF-alpha) has been suggested to be involved in regulating p75NGFR expression in Schwann cells. In this study, we found that removing TNF-alpha in vivo did not significantly suppress the induction of both lysosomes and p75NGFR. Thus, these findings suggest that lysosomal activation is tightly correlated with the induction of p75NGFR in demyelinating Schwann cells during Wallerian degeneration.

Cell reprogramming: control key genes to obtain needed cells / 中国医学科学院学报

Cell reprogramming is a progress in which the memory of a mature cell is erased and then the cell develops novel phenotype and function; ultimately, the fate of the cell changes. Cell reprogramming usually occurs at genes expression levels that no genomic DNA sequence change will be involved. By changing the programs of the genetic expressions of cells in terms of space and time, cell reprogramming alters the differentiation of cells and thus produces the required cells. Further research on cells reprogramming will elucidate the mechanisms that govern the cell development, and thus provides more information of the sources of seed cells used for regeneration medicine. More cells differentiated from many terminally differentiated cells will be obtained, which is extremely important for the understanding of molecular differentiation and for the development of cell replacement therapy. This article summarizes the classification, influencing factors, approaches and latest advances of cells reprogramming.

Generation and application of pluripotent stem cells from spermatogonial stem cells / 生物医学工程学杂志

Recent studies have confirmed that diverse adult tissue cells can be reprogrammed and induced to pluripotency, that is so-called induced pluripotent stem cells (iPS cells). But most of these dedifferentiated processes are induced by gene delivery with retroviral vectors. Some of the delivered genes are cancer causing. So, in current situation, these adult-derived embryonic stem-like cells cannot be used in clinical therapy to cure human diseases. Recently some articles that were published in the authoritative journals are receiving attentions. They show that, in mice and human, spermatogonial stem cells (SSCs) can be used for generating pluripotent stem cells without the exogenous genes and retroviruses, and they can also be used for autologous transplantation without ethical problems. These findings suggest that human SSCs may have considerable potential for cell-based, autologous organ regeneration therapy for various diseases. In this review, we describe and compare the methods that have been used to isolate, purificate and culture SSCs. We also describe the recent results in which SSCs can be transformed into pluripotent stem cells, and the pluripotent stem cells have potential applications in regenerative medicine and genetic medicine.

Dedifferentiated fat cells: an alternative source of adult multipotent cells from the adipose tissues / 国际口腔科学杂志·英文版

When adipose-derived stem cells (ASCs) are retrieved from the stromal vascular portion of adipose tissue, a large amount of mature adipocytes are often discarded. However, by modified ceiling culture technique based on their buoyancy, mature adipocytes can be easily isolated from the adipose cell suspension and dedifferentiated into lipid-free fibroblast-like cells, named dedifferentiated fat (DFAT) cells. DFAT cells re-establish active proliferation ability and undertake multipotent capacities. Compared with ASCs and other adult stem cells, DFAT cells showed unique advantages in their abundance, isolation and homogeneity. In this concise review, the establishment and culture methods of DFAT cells are introduced and the current profiles of their cellular nature are summarized. Under proper induction culture in vitro or environment in vivo, DFAT cells could demonstrate adipogenic, osteogenic, chondrogenic and myogenic potentials. In angiogenic conditions, DFAT cells could exhibit perivascular characteristics and elicit neovascularization. Our preliminary findings also suggested the pericyte phenotype underlying such cell lineage, which supported a novel interpretation about the common origin of mesenchymal stem cells and tissue-specific stem cells within blood vessel walls. Current research on DFAT cells indicated that this alternative source of adult multipotent cells has great potential in tissue engineering and regenerative medicine.

The cellular plasticity of human adipocytes / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the dedifferentiation phenomenon of human mature adipocytes cultured in vitro and to discuss the possibility of using dedifferentiation adipocytes (DA) as seed cells.</p><p><b>METHODS</b>Mature adipocytes and ASCs were harvested from human fat aspirates. Mature adipocytes were cultured and induced to DA by ceiling adherent culture method. Cell morphology were observed during the whole process. Viabilities of DA and ASCs were compared by MTT chromatometry and cell growth curves were drawn based on it. Cell surface markers of DA and ASCs were detected by flow cytometry. The adipogenic, osteogenic and chondrogenic ability of DA and ASCs were assessed by oil red O staining, alizarin bordeaux staining and alcian blue staining, respectively.</p><p><b>RESULTS</b>Human mature adipocytes can dedifferentiate into fibroblast-shaped DA. MTT chromatometry assay demonstrated that DA and ASCs both had strong reproductive activity, with no significant difference between them. Flow cytometry assay demonstrated that both DA and ASCs expressed HLA-ABC, CD29 and CD44, while didn't express CD45, CD34 and CD106. After two weeks of adipogenic differentiation, lipid droplets could be displayed by oil red O staining in both DA and ASCs. After two weeks of osteogenic differentiation, calcium salts mineralization in DA and ASCs could be detected by alizarin bordeaux staining. After two weeks of chondrogenic differentiation, matrix of cartilage cells in DA and ASCs could be detected by alcian blue staining.</p><p><b>CONCLUSIONS</b>Mature adipocytes can be dedifferentiated into DA in vitro. DA has strong reproductive activity, as well as osteogenic, chondrogenic ability and strong adipogenic ability. It expresses some of the stem cell-related cell surface proteins and is a promising seed cell for adipose tissue engineering.</p>

Epithelial-mesenchymal transition and human fetal prostate development / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the role and significance of epithelial-mesenchymal transition (EMT) and its transcriptional regulator Twist1 in the development of the human fetal prostate.</p><p><b>METHODS</b>Twenty-five human fetal prostate specimens at various developmental stages (16-39 weeks) were included in this study. EMT markers, such as E-Cadherin, N-Cadherin and Vimentin, and EMT transcriptional regulator Twist1 were determined by immunohistochemistry, and their relationship with the development of the human fetal prostate was analyzed.</p><p><b>RESULTS</b>E-Cadherin was expressed in the fetal prostate epithelium only, while Vimentin, N-Cadherin and Twist1 in both the epithelium and the stroma. The expression of E-Cadherin gradually increased, but those of Vimentin, N-Cadherin and Twist1 gradually decreased with the gestation stages. No significant changes were observed in the staining patterns of Vimentin, N-Cadherin and Twist1 in the stroma during the whole developmental process.</p><p><b>CONCLUSION</b>EMT is involved in the development of the human fetal prostate, which may promote epithelial cell motility to form prostatic bud tubules in early gestation stages and boost the differentiation of prostate epithelia in later stages.</p>

Characterization of prostate cancer cell lines and their epithelial-mesenchymal transition in subcutaneous tumors / 中华男科学杂志

<p><b>OBJECTIVE</b>To identify the differences in the expression of epithelial or mesenchymal standard proteins between prostate cancer cell lines and tumors, and to investigate the relationship between the process of the prostate cancer cell line forming subcutaneous tumors and epithelial-mesenchymal transition (EMT) by comparing the characteristics of different prostate cell lines forming subcutaneous tumors in SCID mice.</p><p><b>METHODS</b>We constructed prostate cancer models in male SCID mice by subcutaneous injection of 4 human prostate cancer cell lines DU145, Tsu, PC3 and LNCaP, and compared the characteristics of tumor formation. We used Western blot to detect the expressions of E-cadherin and Vimentin in the cancer cell lines and subcutaneous tumors, observed their differences before and after tumor formation, and explore the relationship between EMT and tumor formation.</p><p><b>RESULTS</b>The EMT positive cells DU145 and Tsu showed a higher rate and speed of tumor formation than the EMT negative ones PC3 and LNCaP. The expression of E-cadherin was down-regulated in DU145, up-regulated in Tsu, and absent in PC3 and LNCaP.</p><p><b>CONCLUSION</b>EMT positive cells have a stronger ability of forming tumors than EMT negative cells, and mesenchymal-epithelial transition does exist in subcutaneous tumor formation.</p>

Mesenchymal stem cells derived from rat epicardial versus epididymal adipose tissue

Some investigation has indicated that adipose-derived stem cells possess different surface epitopes and differentiation potential according to the localization of fat pad from which the cells were derived. In the present study proliferation capacity and aging of such cells were explored. Adherent cells were isolated from the collagenase digests of adipose tissues excised from rat epicardial and epididymal regions and propagated with several subcultures. The cells were then investigated whether or not they were able to differentiate into bone, cartilage and adipose cell lineages. Studied cells from two adipose tissues were also compared with respect to their in vitro proliferation capacity. The presence of senescent cells in the culture was determined and compared using senescence-associated [SA] beta-galactosidase staining method. Successful differentiations of the cells were indicative of their mesenchymal stem cells [MSCs] identity. Epicardial adipose-derived cells tended to have a short population doubling time [45 +/- 9.6 hr] than the epididymal adipose-derived stem cells [69 +/- 16 hr, P< 0.05]. Colonogenic activity and the growth curve characteristics were all better in the culture of stem cells derived from epicardial compared to epididymal adipose tissue. Comparatively more percentage of senescent cells was present at the cultures derived from epididymal adipose tissue [P< 0.05]. Our data emphasize on the differences existed between the stem cells derived from adipose depots of different anatomical sites in terms of their proliferative capacity and in vitro aging. Such data can help understand varying results reported by different laboratories involved in adipose stem cell investigations

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.