Eosinophilic globule cells in mouse MFH-like sarcomas: lectin histochemistry.

Lectin binding patterns in ten mouse malignant fibrous histiocytoma (MFH)-like sarcomas containing eosinophilic globule (EG) cells and in granular metrial gland (GMG) cells of mouse placenta were stained with nine lectins (Con A, LCA, WGA, DBA, SBA, e-PHA, PNA, RCA-I and UEA-I) by an avidin-biotin-peroxidase-complex method. EG cells stained strongly with DBA, SBA and PNA which are specific for N-acetyl-D-galactosamine and/or D-galactose. DBA and SBA bound throughout the cytoplasm including the globules; PNA reacted preferentially at the cell surface. There was no evidence that these three lectins were reactive for immature EG cells. WGA, RCA-I and e-PHA also gave a slightly to moderately positive reaction to globules of EG cells. The results indicate that the globules contain abundant O-linked sequences of sugars, but also a few N-linked residues. MFH tumor cells showed a variable degree of binding with Con A, RCA-I, and WGA, but did not react with DBA, SBA and PNA. On the other hand, GMG cells exhibited specific affinities for DBA, SBA and PNA with staining patterns similar to those of EG cells. These findings suggest that EG and GMG cells may be of the same cellular lineage.     

Eosinophilic globule cells in mouse MFH-like sarcomas. Light and electron microscopic studies

Light and electron microscopic observations were made on eosinophilic globule (EG) cells found in 27 subcutaneous malignant fibrous histiocytoma (MFH)-like sarcomas in aged ICR mice. These tumors, which were composed of fibroblast-like cells as the major component, with small numbers of histiocyte-like cells and undifferentiated cells, showed one or more of five growth patterns: storiform, pleomorphic, fascicular, myxoid, and hemangiopericytoma-like. EG cells were interspersed among the tumor cells and were also present in metastatic lesions. They were pleomorphic in shape and contained various numbers of cytoplasmic globules, which were positive with the periodic acid-Schiff reaction and resistant to diastase digestion. Electron microscopic observation revealed that these cells had small finger-like and pseudopodia-like projections, and contained varied numbers of characteristic osmiophilic globules and glycogen particles in the cytoplasm which seemed to become more abundant as the cells differentiated. The osmiophilic globules consisted of a dense homogeneous core and a marginal area rich in small vesicular membranous structures. A small population of EG cells exhibited features suggesting differentiation to fibroblasts; these were characterized by an increased amount of rough endoplasmic reticulum. It is suggested that the EG cell is a neoplastic cell, perhaps derived from a primitive mesenchymal cell, although an inflammatory cell origin is also possible.     

Proteomic signatures corresponding to histological classification and grading of soft-tissue sarcomas

We performed a global protein expression study on soft-tissue sarcoma in order to develop novel diagnostic biomarkers and allow molecular classification. 2-D difference gel electrophoresis was used to generate the global protein expression profiles of 80 soft-tissue sarcoma samples with seven different histological backgrounds. We found that 67 protein spots distinguished the subtypes of soft-tissue sarcoma. Hierarchical clustering with these 67 protein spots resulted in the grouping of all 80 sarcoma samples corresponding to the histological classification. We found that the expression pattern of tropomyosin isoforms was different in conventional and pleomorphic leiomyosarcomas. We also identified five proteins, including alpha-1-antitrypsin, alpha-actinin 1, HSP27, and elongation factor 2, that could differentiate between malignant fibrous histiocytomas and leiomyosarcomas in grade III into low-risk and high-risk groups, which differed significantly with respect to survival. These results establish proteomics as a powerful tool to develop novel biomarkers for diagnosis and molecular classification of soft-tissue sarcomas. Identification of proteins associated with survival in grade III sarcoma will allow delineation of a high-risk group that may benefit from adjuvant therapy and the exclusion of low-risk patients in whom additional therapies are unlikely to exhibit clinical benefit.     

Human mesenchymal stromal cells do not promote recurrence of soft tissue sarcomas in mouse xenografts after radiation and surgery

Background. Mesenchymal stromal cells (MSCs) promote wound healing, including after radiotherapy (RT) and surgery. The use of MSCs in regenerative medicine in the context of malignancy, such as to enhance wound healing post-RT/surgery in patients with soft tissue sarcomas (STSs), requires safety validation. The aim of this study was to determine the effects of human MSCs on STS growth in vitro and local recurrence and metastasis in vivo. Methods. Human primary STS and HT-1080 fibrosarcoma lines were transduced to express luciferase/eGFP (enhanced green fluorescent protein). Sarcoma cells were co-cultured or co-injected with bone marrow–derived MSCs for growth studies. Xenograft tumor models were established with STS lines in NOD/SCID/γ_c^null mice. To emulate a clinical scenario, subcutaneous tumors were treated with RT/surgery prior to MSC injection into the tumor bed. Local and distant tumor recurrence was studied using histology and bioluminescence imaging. Results. MSCs did not promote STS proliferation upon co-culture in vitro, which was consistent among MSCs from different donors. Co-injection of MSCs with sarcoma cells in mice exhibited no significant tumor-stimulating effect, compared with control mice injected with sarcoma cells alone. MSC administration after RT/surgery had no effect on local recurrence or metastasis of STS. Discussion. These studies are important for the establishment of a safety profile for MSC administration in patients with STS. Our data suggest that MSCs are safe in STS management after standard of care RT/surgery, which can be further investigated in early-phase clinical trials to also determine the efficacy of MSCs in reducing morbidity and to mitigate wound complications in these patients.     

Binucleate cells in mouse morulae

Summary Mouse morulae from two strains were examined in whole mounts after dissociation of embryos into single cells and were analysed in serial sections by light and electron microscopy. One or two binucleate cells per embryo were discovered in a statistically significant number of morulae. The frequency of morulae with binucleate cell(s) was higher in older morulae than in younger ones. Binucleate cells were always the outer cells of the embryo. Their ultrastructure did not differ from the ultrastructure of mononucleate cells. It is suggested that cell binuclearity at the morula stage is a possible way to polyploidization of nuclei, resulting in the formation of primary trophoblast giant cells.     

Nefarious NTRK oncogenic fusions in pediatric sarcomas: Too many to Trk

Neurotrophic Tyrosine Receptor Kinase (NTRK) genes undergo chromosomal translocations to create novel open reading frames coding for oncogenic fusion proteins; the N-terminal portion, donated by various partner genes, becomes fused to the tyrosine kinase domain of either NTRK1, NTRK2, or NTRK3. NTRK fusion proteins have been identified as driver oncogenes in a wide variety of tumors over the past three decades, including Pediatric Gliomas, Papillary Thyroid Carcinoma, Spitzoid Neoplasms, Glioblastoma, and additional tumors. Importantly, NTRK fusions function as drivers of pediatric sarcomas, accounting for approximately 15% of childhood cancers including Infantile Fibrosarcoma (IFS), a subset of pediatric soft tissue sarcoma (STS). While tyrosine kinase inhibitors (TKIs), such as larotrectinib and entrectinib, have demonstrated profound results against NTRK fusion-positive cancers, acquired resistance to these TKIs has resulted in the formation of gatekeeper, solvent-front, and compound mutations. We present a comprehensive compilation of oncogenic fusions involving NTRKs focusing specifically on pediatric STS, examining their biological signaling pathways and mechanisms of activation. The importance of an obligatory dimerization or multimerization domain, invariably donated by the N-terminal fusion partner, is discussed using characteristic fusions that occur in pediatric sarcomas. In addition, examples are presented of oncogenic fusion proteins in which the N-terminal partners may contribute additional biological activities beyond an oligomerization domain. Lastly, therapeutic approaches to the treatment of pediatric sarcoma will be presented, using first generation and second-generation agents such as selitrectinib and repotrectinib.     

A strategy for the suppression of tumorigenesis induced by biomaterials: Restoration of transformed phenotype of polyetherurethane-induced tumor cells by Cx43 transfection

Biomaterials such as polyetherurethans (PEUs) are the scaffolding, which is indispensable for the development of the bio-artificial organs. However, PEUs can induce tumors in subcutaneous implantation sites in rat. We have shown that the different inhibitory potential of gap junctional intercellular communication (GJIC) on the surface of the biomaterials, including PEUs, is a key step in determining the tumorigenic potential. Here we show that suppression of a gap junctional protein connexin 43 (Cx43) plays an important role in in vivo tumorigenesis induced by PEUs for the first time and that Cx43 transfection may be an effective strategy for preventing tumorigenesis induced by biomaterials. Rat tumor cell line U41 is derived from tumors in the subcutaneous implantation of PEU films. The GJIC and the expression of Cx43 were suppressed in U41. The restoration of normal phenotype, such as reduction of growth rate, recovery of contact inhibition and loss of colony formation ability in soft agar, was achieved by Cx43 transfection. These results strongly suggest that suppression of Cx43 expression plays an important role in the development of rat malignant fibrous histiocytoma (MFHC) caused by PEUs and that Cx43 transfection is effective for prevention of tumorigenesis induced by PEUs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Sertoli细胞饲养层对小鼠精原干细胞增殖的影响

摘要 目的 以小鼠睾丸支持细胞(Sertoli)为饲养层,小鼠胚胎成纤维细胞(STO) 饲养层做对照,研究它对小鼠精原干细胞增殖的影响。方法 用无血清StemPro-34 SFM培养基培养2～5日龄小鼠精原干细胞,分别用相差显微镜观察,免疫组化法研究Sertoli饲养层对精原干细胞生物学行为的影响。结果 发现精原干细胞在Sertoli及STO两种饲养层上的一周内的生物学行为非常相似,但培养1周后,Sertoli细胞作饲养层的培养体系中保留的精原干细胞要比对照组明显增多,约有30%的精原干细胞能存活下来并能维持存活到60d以上。结论 Sertoli细胞作饲养层明显促进精原干细胞的更新增殖。     

Centrifugal seeding of mammalian cells in nonwoven fibrous matrices

Three‐dimensional (3D) cell cultures have many advantages over two‐dimensional cultures. However, seeding cells in 3D scaffolds such as nonwoven fibrous polyethylene terephthalate (PET) matrices has been a challenge task in tissue engineering and cell culture bioprocessing. In this study, a centrifugal seeding method was investigated to improve the cell seeding efficiency in PET matrices with two different porosities (93% and 88%). Both the centrifugal force and centrifugation time were found to affect the seeding efficiency. With an appropriate centrifugation speed, a high 80?90% cell seeding efficiency was achieved and the time to reach this high seeding efficiency was less than 5 min. The seeding efficiency was similar for matrices with different porosities, although the optimal seeding time was significantly shorter for the low‐porosity scaffold. Post seeding cell viability was demonstrated by culturing colon cancer cells seeded in PET matrices for over 5 days. The centrifugal seeding method developed in this work can be used to efficiently and uniformly seed small fibrous scaffolds for applications in 3D cell‐based assays for high‐throughput screening. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Hyperplastic neuroretinopathy and disorder of pigment epithelial cells precede accelerated retinal degeneration in the SJL/N mouse

We have found a complex eye disease in the SJL/N mouse. This animal is closely related to the SJL/J mouse, which is homozygous for retinal degeneration (rd) and which also suffers from extraocular reticulum cell sarcomas at around 200 days of age. In the SJL/N animal, a high incidence of subretinal tumor is present at 9 days after birth. Furthermore, we have observed an extensive neuroretinal hyperplasia, a phenomenon that is termed hyperplastic neuroretinopathy, and that is probably the consequence of elevated levels of cytokines in the animals. In addition to these anomalies, the SJL/N mouse shows progressive dystrophy of the retinal pigment epithelium (RPE) from day 4 onwards, and accelerated photoreceptor cell degeneration is completed by day 16. The early RPE dystrophy appears to be a secondary autoimmune disease, since cells in this structure and in the choroid develop MHC class II antigens, whereas we suspect that the accelerated photoreceptor cell loss is induced by a soluble toxic agent. The F₁ progeny derived from cross-breeding the SJL/N and Balb/c +/+ strains also shows a high incidence of subretinal tumor and hyperplastic neuroretinopathy, but neither the RPE dystrophy nor retinal degeneration.     

探索Sertoli细胞对去除精原细胞的动态反应

目的探索Sertoli细胞对去除小鼠精原细胞后睾丸的动态反应。方法采用15、30和44 mg/kg的白消安腹腔注射法建立不同程度去除精原细胞的动物模型,处理后5 d和28 d时对睾丸进行组织学检测,评价精子发生状态,并运用实时定量荧光PCR技术检测这两个时期睾丸GDNF、PLZF、Nanog和GFRα1基因mRNA的表达量。结果在白消安处理后第5天,GDNF出现显著升高,且呈剂量依赖趋势,而PLZF与GFRɑ1并无显著变化,睾丸组织学观察亦无明显变化。在白消安处理后28 d时,GDNF、PLZF、Nanog、GFRɑ1基因mRNA相对表达量均出现大幅度的升高,睾丸组织学切片观察显示随着给药剂量的增加,精子发生受到的损伤愈加严重。结论 Sertoli细胞早在白消安处理后第5天就对精原细胞的变化发生了反应,Sertoli细胞分泌GDNF的能力发生代偿性增加,进而刺激精原干细胞自我更新速度加快,体现在Nanog和PLZF水平提高,从而实现精子发生的重建。     

Mouse embryonic stem cell-derived feeder cells support the growth of their own mouse embryonic stem cells

Feeder cells are usually used in culturing embryonic stem cells (ESCs) to maintain their undifferentiated and pluripotent status. To test whether mouse embryonic stem cells (mESCs) may be a source of feeder cells to support their own growth, 48 fibroblast-like cell lines were isolated from the same mouse embryoid bodies (mEBs) at three phases (10th day, 15th day, 20th day), and five of them, mostly derived from 15th day mEBs, were capable of maintaining mESCs in an undifferentiated and pluripotent state over 10 passages, even up to passage 20. mESCs cultured on the feeder system derived from these five cell lines expressed alkaline phosphatase and specific mESCs markers, including SSEA-1, Oct-4, Nanog, and formed mEBs in vitro and teratomas in vivo. These results suggest that mEB-derived fibroblasts (mEB-dFs) could serve as feeder cells that could sustain the undifferentiated growth and pluripotency of their own mESCs in culture. This study not only provides a novel feeder system for mESCs culture, avoiding a lot of disadvantages of commonly used mouse embryonic fibroblasts as feeder cells, but also indicates that fibroblast-like cells derived from mESCs take on different functions. Investigating the molecular mechanisms of these different functional fibroblast-like cells to act on mESCs will contribute to the understanding of the mechanisms of mESCs self-renewal.     

Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow

The future use of adult mesenchymal stem cells (MSCs) for human therapies depends on the establishment of preclinical studies with other mammals such as mouse. Surprisingly, purification and characterisation of murine MSCs were only poorly documented. The aim of this study was to purify mouse MSCs from adult bone marrow and to functionally characterise their abilities to differentiate along diverse lineages. Adherent cells from adult C57Bl/6J mouse bone marrow were depleted of granulo-monocytic cells and subsequently allowed to grow on fibronectin-coated dishes in presence of fetal bovine serum and growth factors. The growing fibroblastoid cell population primarily consisted of spindle- and star-shaped cells with significant renewal capacity as they were cultured until 30 passages (about 60 doubling population). We fully demonstrated the MSC phenotype of these cells by inducing them to differentiate along osteoblastic, adipocytic, and chondrocytic pathways. Mouse MSCs (mMSCs) sharing the same morphological and functional characteristics as human MSCs can be successfully isolated from adult bone marrow without previous mouse or bone marrow treatment. Therefore, mMSCs will be an important tool to study the in vivo behaviour and fate of this cell type after grafting in mouse pathology models.     

Differential expression of connexin 43 in mouse mammary cells

In this study we have employed suppressive subtractive hybridization (SSH) analysis to investigate differential gene expression in primary mouse mammary epithelial cells (PMMEC) cultured under mildly apoptotic/quiescent and differentiating conditions. Among a small group of genes whose expression was differentially regulated was connexin 43. In vitro, connexin 43 mRNA and protein were detectable in PMMEC cultured under proliferative or mildly apoptotic conditions. The level of connexin 43 mRNA expression in vivo was also investigated. High levels of expression were found to be associated with the periods of greatest glandular plasticity (pubertal expansion of the mammary tree, early pregnancy and during early involution). Thus, terminally differentiated cells in vivo and in vitro did not express connexin 43 mRNA suggesting that connexin 43 expression, and perhaps facilitated gap junction communication, is associated with undifferentiated progenitor cell populations.     

Expression profiles of mouse dendritic cell sarcoma are similar to those of hematopoietic stem cells or progenitors by clustering and principal component analyses

We isolated and screened two tumor cell clones DD1 and DG6 with different capacity of metastasis from the same parent cell line, a mouse dendritic cell (DC) sarcoma, using limited dilution method. The genome-wide expressions of DD1 and DG6 cells were detected by Affymetrix's MOE-430A microarray. The expression profiles related with mouse DC development were downloaded from GEO at NCBI and ArrayExpress at EBI database. In order to compare the expression of DC sarcoma and DC developmental arrays which was performed by MG-U74av2, we had screened the best matched probesets between MOE-430A and MG-U74av2 according to the probe identities from Affymetrix technical annotation. After the normalization of 11 housekeeping genes across the 34 arrays (2 DC sarcoma and 32 DC developmental arrays), all these expression profiles were analyzed by the methods of hierarchical clustering, principal component analysis, nearest-neighborhood, and self-organizing maps. The results indicate that expression profiles of DC sarcoma are closer to those of the DC progenitors and hematopoietic stem cells from bone marrow compared with the sorted DCs from spleen. The results support the hypothesis that cancers (tumors or sarcomas) arise from stem cells. It is suggested that the DC sarcomas are more similar to the DC progenitors and hematopoietic stem cells than the relative mature DCs in gene expressions on the large-scale.     

Ontogenesis of gastrin cells in the pyloric antrum and duodenum of the mouse

Summary Ontogenesis of gastrin cells was studied in the pyloroduodenal mucosa of the mouse using anti-human G17 serum, R-1301, and anti-human G34(1–15) serum, R-2703. R-1301-immunostained cells first appeared in the pyloric mucosa of 14-day-old fetuses. Cells stained with both R-1301 and R-2703 appeared immediately after birth, and gradually increased in number to the adult level. Most R-1301-reactive cells were also reactive to R-2703, whereas some cells that reacted with R-1301 exhibited very weak or no reaction with R-2703. The discrepancy between these two immunoreactivities is discussed.In the duodenum, a considerable number of R-1301-reactive cells were present from the perinatal stage and through out adult development. A few R-2703-reactive cells were seen in the duodenum of young mice but not of the adult.     

Eosinophilic granule cells in Carassius auratus scale epidermis

Eosinophilic granule cells (EGCs) were characterized in Carassius auratus scale epidermis in situ and in explants. Live EGCs were readily identified by the presence of numerous large cytoplasmic granules observed with DIC microscopy. Histochemical staining with toluidine blue and alcian blue yielded granule metachromasia and pale blue granules, respectively, both consistent with mammalian mast cell staining. However, EGCs also share some features with mammalian basophils as neutral red dye was selectively incorporated into EGC granules. EGCs within scale epidermis were actively motile, displaying average speeds of 16 μm/min and maximum speeds of greater than 40 μm/min and showing morphological plasticity during migration. The predominant motile phenotype was elongate with a well‐developed leading lamella, while a broader body motile morphology was observed to a lesser extent. A trailing, relatively unchanged uropod was associated with every motile EGC and invariably contained one or a few granules. A rounded EGC shape without a leading‐edge or trailing uropod was also observed and was generally associated with static cells. Individual cells readily switched between the three major shapes during motility; static cells could abruptly develop a polarized morphology, and actively motile cells switched between elongate and broad‐bodied shapes or the static, rounded shape.