Mast cells contribute to the stromal microenvironment in mammary gland branching morphogenesis

The stromal microenvironment regulates mammary gland branching morphogenesis. We have observed that mast cells are present in the mammary gland throughout its postnatal development and, in particular, are found around the terminal end buds and ductal epithelium of the pubertal gland. Mast cells contribute to allergy, inflammatory diseases, and cancer development but have not been implicated in normal development. Genetic and pharmacological disruption of mast cell function in the mammary gland revealed that mast cells are involved in rapid proliferation and normal duct branching during puberty, and this effect is independent of macrophage recruitment, which also regulates mammary gland development. For mast cells to exert their effects on normal morphogenesis required activation of their serine proteases and degranulation. Our observations reveal a novel role for mast cells during normal pubertal development in the mammary gland.     

Enhanced branching morphogenesis in mammary glands of mice lacking cell surface beta1,4-galactosyltransferase

Development of the mammary gland is influenced both by the systemic hormonal environment and locally through cell-cell and cell-extracellular matrix (ECM) interactions. We have previously demonstrated aberrant mammary gland morphogenesis in transgenic mice with elevated levels of the long isoform of beta1,4-galactosyltransferase 1 (GalT), a proportion of which is targeted to the plasma membrane, where it plays a role in cell-ECM interactions. Here, we show that mammary glands of mice lacking the long GalT isoform exhibit a complementary phenotype. Cell-surface GalT activity was reduced by over 60%, but because the short GalT isoform is intact, total GalT activity was reduced only slightly relative to wild type. Mammary glands from long GalT-null mice were characterized by excess branching, and this phenotype was accompanied by altered expression of laminin chains. Laminin alpha1 and alpha3 were reduced 2.4- and 3.0-fold, respectively, while expression of laminin gamma2 was elevated 2.3-fold. The expression and cleavage of laminin gamma2 have been correlated with branching and cell migration, and Western blotting revealed an altered pattern in gamma2 cleavage products in long GalT-null mammary glands. We then examined the expression of metalloproteases that cleave laminins or that have been shown to play a role in mammary gland morphogenesis. Expression of MT1-MMP, a membrane-bound protease that can cleave laminin gamma2, was elevated 5.5-fold in the long GalT-nulls. MMP 7 was also elevated 5.1-fold. Our results suggest that expression of surface GalT is important for the proper regulation of matrix expression and deposition, which in turn regulates the proper branching morphogenesis of the mammary epithelial ductal system.     

Mouse 3T3 fibroblasts under the influence of fibroblasts isolated from stroma of human basal cell carcinoma acquire properties of multipotent stem cells

Background information. Multipotent mesenchymal stem cells can participate in the formation of a microenvironment stimulating the aggressive behaviour of cancer cells. Moreover, cells exhibiting pluripotent ESC (embryonic stem cell) markers (Nanog and Oct4) have been observed in many tumours. Here, we investigate the role of cancer‐associated fibroblasts in the formation of stem cell supporting properties of tumour stroma. We test the influence of fibroblasts isolated from basal cell carcinoma on mouse 3T3 fibroblasts, focusing on the expression of stem cell markers and plasticity in vitro by means of microarrays, qRT‐PCR (quantitative real‐time PCR) and immunohistochemistry. Results. We demonstrate the biological activity of the cancer stromal fibroblasts by influencing the 3T3 fibroblasts to express markers such as Oct4, Nanog and Sox2 and to show differentiation potential similar to mesenchymal stem cells. The role of growth factors such as IGF2 (insulin‐like growth factor 2), FGF7 (fibroblast growth factor 7), LEP (leptin), NGF (nerve growth factor) and TGFβ (transforming growth factor β), produced by the stromal fibroblasts, is established to participate in their bioactivity. Uninduced 3T3 do not express the stem cell markers and show minimal differentiation potential. Conclusions. Our observations indicate the pro‐stem cell activity of cancer‐associated fibroblasts and underline the role of epithelial—mesenchymal interaction in tumour biology.     

A rat mammary gland cancer cell with stem cell properties of self-renewal and multi-lineage differentiation

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing cancer-initiating cells with stem cell properties at the single cell level has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell, derived from rat mammary adenocarcinoma has: the ability to serially re-generate mammospheres in long-term non-adherent cultures, the differentiation potential to generate all the cell lineages of the mammary gland and branched duct-like structures that recapitulate morphologically and functionally the ductal–alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multi-lineage differentiation and the tubular-like structure formation potential suggest that LA7 cells is a cancer stem model system to study the dynamics of tumor formation at the single cell level. Cinzia Cocola, Sveva Sanzone and Simonetta Astigiano have contributed equally to this work.     

Mammary gland morphogenesis in vitro: Extracellular requirements for the formation of tubules in collagen gels by a cloned rat mammary epithelial cell line

Summary The mechanism of induction of tubular outgrowths in vitro on floating collagen gels and the influence of extracellular factors on this process have been investigated using the clonal rat mammary epithelial cell line, Rama 25. Growth of Rama 25 on such floating gels causes their contraction. Contraction of the gel is accompanied by a 10-fold increase in the number of cells per unit area, a change in cell shape, and a convolution of the epithelial cell sheet. Gels folded over manually show an 11-times higher incidence of tubules along the folds than on the flat surface. Tubular formation begins when cords of cells develop from local proliferations of the cell sheet and become canalized. Tubules follow wrinkles in the gel and branch to yield monopodial, dichotomous, or lobular architecture. Hydrocortisone and insulin, in the presence of serum, stimulate both narrow and thick tubular structures on folded gels, whereas extra additions of 1 ng/ml cholera toxin or 100 ng/ml epidermal growth factor preferentially stimulate thick tubular structures. Floating glutaraldehyde-fixed gels, very thick collagen gels, and collagen gels prepared on the top of rigid steel grids fail to support the formation of tubules, suggesting that flexibility and access of the medium to basal surfaces are important to their genesis. Incorporation of hyaluronic acid into the gel matrix preferentially inhibits the thick tubular outgrowths. Thus, the branching tubular structures generated by Rama 25 can be influenced in different ways by various extracellular factors in the medium and in the gel. During the course of this work E. J. Ormerod was in receipt of a Ludwig Research Studentship.     

A Notch positive feedback in the intestinal stem cell niche is essential for stem cell self‐renewal

The intestinal epithelium is the fastest regenerative tissue in the body, fueled by fast‐cycling stem cells. The number and identity of these dividing and migrating stem cells are maintained by a mosaic pattern at the base of the crypt. How the underlying regulatory scheme manages this dynamic stem cell niche is not entirely clear. We stimulated intestinal organoids with Notch ligands and inhibitors and discovered that intestinal stem cells employ a positive feedback mechanism via direct Notch binding to the second intron of the Notch1 gene. Inactivation of the positive feedback by CRISPR/Cas9 mutation of the binding sequence alters the mosaic stem cell niche pattern and hinders regeneration in organoids. Dynamical system analysis and agent‐based multiscale stochastic modeling suggest that the positive feedback enhances the robustness of Notch‐mediated niche patterning. This study highlights the importance of feedback mechanisms in spatiotemporal control of the stem cell niche.     

聚N-异丙基丙烯酰胺细胞培养平台的研究进展

聚N-异丙基丙烯酰胺(poly(N-isopropylacrylamide),PNIPAAm),温敏性聚合物,可利用其温敏特性替代酶类物质或细胞刮刀用于贴壁细胞的收获,从而有效避免酶解和机械损伤,可为生物医药领域提供品质优良的种子细胞。重点阐述促进细胞有效粘附和快速脱附的温敏性PNIPAAm二维平面的研发情况,包括选取特殊基材、引入亲水基团、调节反应物比率、控制聚合物厚度/密度、提供适宜外力等方式,从而有效改善细胞对温敏性平面的适应性并降低染菌风险以及减少低温处理对细胞的影响。同时介绍PNIPAAm微载体、支架和凝胶等温敏性三维培养介质的研究进展,此方式不仅增加细胞生长面积,更可以模拟体内微环境,从而保持细胞原始生理特征,同时实现大规模扩增和非酶解收获细胞以及组织器官修复和重构的目标。最后简单说明PNIPAAm培养平台的应用,PNIPAAm的研发为再生医学的发展提供了崭新思路。     

MicroRNA: A new player in response to therapy for colorectal cancer

Colorectal cancer (CRC) is one of the important malignancies that result in cancer-related deaths worldwide. Multiple lines of evidence have indicated that different responses to therapy in CRC cells led to the failure of the current therapies. Hence, identification of the underlying cellular and molecular pathways involved in the emergence of different responses from CRC cells could contribute to finding and designing new therapeutic platforms to overcome the present limitations. Among the various targets involved in CRC pathogenesis, microRNAs (miRNAs) have key roles in many signaling pathways that are associated with the initiation and progression of CRC. Increasing evidence has confirmed that miRNAs as epigenetic regulators could play critical roles in the response (resistance or sensitivity) to therapy. Cancer stem cells are well-known players in resistance to therapy in CRC. They have been shown to play significant roles via inhibition and activation of many miRNA networks. Hence, miRNAs could be involved in the resistance and sensitivity of therapy in CRC cells via affecting different mechanisms, such as activation of cancer stem cells. Here, we summarized the role of various miRNAs in response to therapy of CRC cells. Moreover, we highlighted the roles of these molecules in the function of cancer stem cells, which are known as important players in the resistance to therapy in CRC.     

A respiratory syncytial virus persistent-infected cell line system reveals the involvement of SOCS1 in the innate antiviral response

HEp-2 cells persistently infected with respiratory syncytial virus(RSV) are a heterogeneous mixture of viral antigen-positive and-negative variants; however, the mechanism through which viral replication becomes latent remains unclear. In this study, we investigated the potential mechanism by which RSV escapes from innate immune surveillance. Persistent-infected RSV HEp-2 cells were isolated and cell clones were passaged. The RSV-persistent cells produced viruses at a lower titer, resisted wild-type RSV re-infection, and secreted high levels of interferon-β(IFN-β), macrophage inflammatory protein-1α(Mip-1α), interleukin-8(IL-8), and Rantes. Toll-like receptor 3(TLR3), retinoic acid inducible gene-I(RIG-I), and suppressor of cytokine signaling 1(SOCS1) levels were upregulated in these cells. The silencing of TLR3 m RNA decreased the expression of SOCS1 protein and the secretion of cytokines. RSV-persistent cells are in an inflammatory state; upregulation of SOCS1 is related to the TLR3 signaling pathway, which could be associated with the mechanism of viral persistence.     

A comparison of human mesenchymal stem cell osteogenesis in poly(ethylene glycol) hydrogels as a function of MMP-sensitive crosslinker and crosslink density in chemically defined medium

This study investigated osteogenesis of human mesenchymal stem cells encapsulated in matrix-metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG) hydrogels in chemically defined medium (10 ng/ml bone morphogenic factor-2). Thiol-norbornene photoclick hydrogels were formed with CRGDS and crosslinkers of PEG dithiol (nondegradable), CVPLS-LYSGC (P1) or CRGRIGF-LRTDC (P2; dash indicates cleavage site) at two crosslink densities. Exogenous MMP-2 degraded P1 and P2 hydrogels similarly. MMP-14 degraded P1 hydrogels more rapidly than P2 hydrogels. Cell spreading was greatest in P1 low crosslinked hydrogels and to a lesser degree in P2 low crosslinked hydrogels, but not evident in nondegradable and high crosslinked MMP-sensitive hydrogels. Early osteogenesis (Alkaline phosphatase [ALP] activity) was accelerated in hydrogels that facilitated cell spreading. Contrarily, late osteogenesis (mineralization) was independent of cell spreading. Mineralized matrix was present in P1 hydrogels, but only present in P2 high crosslinked hydrogels and not yet present in nondegradable hydrogels. Overall, the low crosslinked P1 hydrogels exhibited an accelerated early and late osteogenesis with the highest ALP activity (Day 7), greatest calcium content (Day 14), and greatest collagen content (Day 28), concomitant with increased compressive modulus over time. Collectively, this study demonstrates that in chemically defined medium, hydrogel degradability is critical to accelerating early osteogenesis, but other factors are important in late osteogenesis.     

The promise of stem cell markers in the diagnosis and therapy of epithelial dysplasia and oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer. Epithelial dysplasia is often initiated in the cells and cell nuclei adjacent to the epithelial cell membrane. Reduced cell–cell adhesions enable cancer cells to detach from the tumor and disseminate to other organs. The mutations in epithelial dysplasia markers such as E‐cadherin and epithelial cell adhesion molecules (CD326) can lead to proliferation, growth and survival of the tumor cells and persistence of numerous malignancies that play a key role in epithelial dysplasia of OSCC. Accordingly, these genes can be considered prognostic markers or potential therapeutic targets for the tailored management of patients with OSCC. The gene expression profile of OSCC stem cells indicates a differential pattern that facilitates establishing a cell signature. Owing to the highly tumorigenic behavior of cancer stem cells and the role of these cells in tumor differentiation, treatment resistance, relapse, and metastasis, we reviewed the role of stem cell markers in epithelial dysplasia and OSCC.     

A Pdgf‐cCreERT2 knock‐in mouse model for tracing PDGF‐C cell lineages during development

PDGF‐C, a member of the platelet‐derived growth factor (PDGF) family, plays important roles in the development of craniofacial structures, the neural system, the vascular system, and tumors. PDGF‐C could also be required for the regulation of certain types of stem or progenitor cells as suggested by its expression in the regions where these cells are located. To further characterize the role of PDGF‐C in development, we generated a Pdgf‐c^CreERT2 mouse strain, in which a tamoxifen‐inducible Cre (CreERT2) cDNA was specifically targeted into the Pdgf‐c genomic locus and controlled by the endogenous Pdgf‐c regulatory elements. We also showed that Cre activity in this mouse strain could be specifically induced by tamoxifen, which allowed the fate of PDGF‐C‐expressing cells to be traced at various stages of development. Using this model system, we demonstrated for the first time that PDGF‐C‐expressing cells could be multipotent, generating multiple cell lineages required for the formation of the cerebellum. Therefore, the Pdgf‐c^CreERT2 mouse strain generated in this study will be a valuable transgenic tool for exploring the function of PDGF‐C in development and stem cell biology.     

Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration

Three populations of myogenic cells were isolated from normal mouse skeletal muscle based on their adhesion characteristics and proliferation behaviors. Although two of these populations displayed satellite cell characteristics, a third population of long-time proliferating cells expressing hematopoietic stem cell markers was also identified. This third population comprises cells that retain their phenotype for more than 30 passages with normal karyotype and can differentiate into muscle, neural, and endothelial lineages both in vitro and in vivo. In contrast to the other two populations of myogenic cells, the transplantation of the long-time proliferating cells improved the efficiency of muscle regeneration and dystrophin delivery to dystrophic muscle. The long-time proliferating cells' ability to proliferate in vivo for an extended period of time, combined with their strong capacity for self-renewal, their multipotent differentiation, and their immune-privileged behavior, reveals, at least in part, the basis for the improvement of cell transplantation. Our results suggest that this novel population of muscle-derived stem cells will significantly improve muscle cell-mediated therapies.     

hiPSC-derived hepatocytes closely mimic the lipid profile of primary hepatocytes: A future personalised cell model for studying the lipid metabolism of the liver

Hepatocyte-like cells (HLCs) differentiated from human-induced pluripotent stem cells offer an alternative platform to primary human hepatocytes (PHHs) for studying the lipid metabolism of the liver. However, despite their great potential, the lipid profile of HLCs has not yet been characterized. Here, we comprehensively studied the lipid profile and fatty acid (FA) metabolism of HLCs and compared them with the current standard hepatocyte models: HepG2 cells and PHHs. We differentiated HLCs by five commonly used methods from three cell lines and thoroughly characterized them by gene and protein expression. HLCs generated by each method were assessed for their functionality and the ability to synthesize, elongate, and desaturate FAs. In addition, lipid and FA profiles of HLCs were investigated by both mass spectrometry and gas chromatography and then compared with the profiles of PHHs and HepG2 cells. HLCs resembled PHHs by expressing hepatic markers: secreting albumin, lipoprotein particles, and urea, and demonstrating similarities in their lipid and FA profile. Unlike HepG2 cells, HLCs contained low levels of lysophospholipids similar to the content of PHHs. Furthermore, HLCs were able to efficiently use the exogenous FAs available in their medium and simultaneously modify simple lipids into more complex ones to fulfill their needs. In addition, we propose that increasing the polyunsaturated FA supply of the culture medium may positively affect the lipid profile and functionality of HLCs. In conclusion, our data showed that HLCs provide a functional and relevant model to investigate human lipid homeostasis at both molecular and cellular levels.