Response of NIH 3T3 Fibroblast Cells on Laser-Induced Periodic Surface Structures on a 15×(Ti/Zr)/Si Multilayer System.

Ultrafast laser processing with the formation of periodic surface nanostructures on the 15×(Ti/Zr)/Si multilayers is studied in order to the improve cell response. A novel nanocomposite structure in the form of 15x(Ti/Zr)/Si multilayer thin films, with satisfying mechanical properties and moderate biocompatibility, was deposited by ion sputtering on an Si substrate. The multilayer 15×(Ti/Zr)/Si thin films were modified by femtosecond laser pulses in air to induce the following modifications: (i) mixing of components inside of the multilayer structures, (ii) the formation of an ultrathin oxide layer at the surfaces, and (iii) surface nano-texturing with the creation of laser-induced periodic surface structure (LIPSS). The focus of this study was an examination of the novel Ti/Zr multilayer thin films in order to create a surface texture with suitable composition and structure for cell integration. Using the SEM and confocal microscopies of the laser-modified Ti/Zr surfaces with seeded cell culture (NIH 3T3 fibroblasts), it was found that cell adhesion and growth depend on the surface composition and morphological patterns. These results indicated a good proliferation of cells after two and four days with some tendency of the cell orientation along the LIPSSs.

Improving the analytical toolbox to investigate copurifying host cell proteins presence: N-(4)-(ß-acetylglucosaminyl)- l-asparaginase case study.

Levels of host cell proteins (HCPs) in purification intermediates and drug substances (DS) of monoclonal antibodies (mAbs) must be carefully monitored for the production of safe and efficacious biotherapeutics. During the development of mAb1, an immunoglobulin G1 product, unexpected results generated with HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit triggered an investigation which led to the identification of a copurifying HCP called N-(4)-(ß-acetylglucosaminyl)-l-asparaginase (AGA, EC3.5.1.26) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The risk assessment performed indicated a low immunogenicity risk for the copurifying HCP and an ad hoc stability study demonstrated no mAb glycan cleavage and thus no impact on product quality. Fractionation studies performed on polishing steps revealed that AGA was coeluted with the mAb. Very interestingly, the native digestion protocol implemented to go deeper in the MS-HCP profiling was found to be incompatible with correct AGA detection in last purification intermediate and DS, further suggesting a hitchhiking behavior of AGA. In silico surface characterization of AGA also supports this hypothesis. Finally, the combined support of HCP ELISA results and MS allowed process optimization and removal of this copurifying HCP.

Unexpected features of acute T lymphoblastic lymphomas in Notch1IC transgenic rats.

Dysregulated Notch signaling accounts for the majority of acute T lymphoblastic leukemia/lymphoma (T-ALL) cases in humans. Here, we characterize lymphomas from Notch1IC transgenic rats, which develop T-ALL shortly after weaning, and show that they display a number of previously undocumented features. Starting from monoclonal thymic tumors, the CD4(+)CD8alphaalpha(+) lymphoma cells infiltrate the bone marrow and then spread to secondary lymphoid and non-lymphoid organs. However, major hallmarks of T-ALL cells in other murine models and human patients, such as constitutive NF-kappaB activity and increased levels of anti-apoptotic proteins, are remarkably absent in Notch1IC lymphomas. In contrast, CD30, a classic marker of Hodgkin lymphomas, is overexpressed in these tumors. Intriguingly, enforced Notch1 signaling up-regulates expression of Notch3, which has also been implicated in the pathogenesis of T-ALL. By blocking endogenous Notch signaling, we could demonstrate that Notch1IC is sufficient to induce sustained preTCR expression in transgenic thymocytes but not for their progression to the double-positive stage. This suggests that other Notch activities may also contribute to the phenotype of the transgenic rats. In summary, we anticipate this new animal model will help to further elucidate the role of Notch1 in the pathogenesis of T-ALL.

Differentiation potential of FDCPmix cells following injection into blastocysts.

Factor-dependent cell Paterson mixed potential (FDCPmix) cells are murine, multipotent, interleukin-3 (IL-3)-dependent progenitor cells, established from long-term bone marrow cultures. They show multilineage myeloid/erythroid and osteoclast differentiation capacity in vitro. FDCPmix cells are neither immortalised, leukaemic nor transformed and have no detectable karyotypic abnormalities. To investigate the broader differentiation potential of FDCPmix cells in vivo, they were injected into murine blastocysts, and the engraftment of donor cells in developing chimaeric embryos was analysed. FDCPmix cell progeny was detected predominantly in haematopoietic tissues, and immunohistochemical analysis revealed that the majority of donor-derived cells expressed the pan-haematopoietic marker CD45. Albumin expression indicative of hepatocytes was not detectable in FDCPmix-derived cells in chimaeric foetal livers. However, analysis of foetal brains of developing embryos revealed a low frequency of neural cell adhesion molecule-positive donor cells. These results suggest that the majority of FDCPmix cells injected into blastocysts follow haematopoietic differentiation programs, but that a small number of cells can assume the expression of neural markers.

Erythroid-like cells from neural stem cells injected into blastocysts.

OBJECTIVE: In contrast to embryonic stem (ES) cells, which are able to give rise to all cell types of the body, somatic stem cells have been thought to be more limited in their differentiation potential in that they are committed to generate only cells of their tissue of origin. Unexpectedly, some recent data suggest that somatic stem cells isolated from one tissue can also generate cells of heterologous tissues and organs, implying that somatic stem cells have a greater potential for differentiation. METHODS: To explore further the developmental potential of murine neural stem cells (NSCs) we injected cultured NSCs as neurospheres into preimplantation blastocysts and determined the seeding by donor cells in tissues of developing chimeric fetal and adult animals. RESULTS: We frequently detected progeny of injected NSCs both in embryos and in adult animals. In embryos we observed transient seeding of donor cells to hematopoietic tissues and generation of NSC-derived cells that express globin genes and an erythroid-specific cell-surface marker. In adults progeny of NSCs were mostly detected in neural tissues. The observed low level of chimerism of wild-type NSCs was increased if we injected stem cells expressing a bcl-2 transgene, without changing the seeding pattern. CONCLUSION: These results suggest that cultured NSCs, following their injection into blastocysts, generate at mid-gestation erythroid-like cells but later, in adult chimeric mice, engraftment mainly persisted in neural tissues.

Plasticity of hematopoietic stem cells and cellular memory.

Stem cell systems represent an effective and powerful approach for tissue development and regeneration of diverse tissue types. Common and defining features of these exceptional cells are the capacity for self-renewal and the potential for differentiation into multiple mature cell types. Recently, surprising new observations have indicated that stem cells isolated from one adult tissue can also give rise to mature cells of other cell lineages, irrespective of classical germ layer designations. This discovery has resulted in quantum leaps in both scientific knowledge and the potential applications of stem cells. The new findings contradict central dogmas of commitment and differentiation of stem and progenitor cells. However, the true potential of somatic stem cells is just emerging and the new findings have to be defined more fully and integrated into a unifying model of stem cell potential and behavior. Here we analyze the developmental potential of hematopoietic stem cells of mouse and man following their injection into the murine preimplantation blastocyst, an environment that allows the development of all cell lineages. In addition, we discuss the emerging lines of evidence of the developmental plasticity of hematopoietic and other somatic stem cells and consider how cellular memory of transcriptional states is established and may be potentially involved in this phenomenon.

Developmental potential of hematopoietic and neural stem cells: unique or all the same?

Like many other animals, mammals develop from fertilized oocytes - the ultimate stem cells. As embryogenesis proceeds, most cells lose developmental potential and eventually become restricted to a specific cell lineage. The result is the formation of a complete and structured mature organism with complex organs composed of a great variety of mature, mostly mitotically quiescent effector cells. However, along the way, some exceptional cells, known as somatic stem cells (SSCs) are set aside and maintain a high proliferation and tissue-specific differentiation potential. SSCs, in contrast to embryonic stem (ES) cells, which are able to give rise to all cell types of the body, have been regarded as being more limited in their differentiation potential in the sense that they were thought to be committed exclusively to their tissue of origin. However, recent studies have demonstrated that somatic stem cells from a given tissue can also contribute to heterologous tissues and thus show a broad nontissue restricted differentiation potential. The question arises: how plastic are somatic stem cells? To provide a tentative answer, we describe and review here recent investigations into the developmental potentials of two somatic stem cell types, namely hematopoietic and neural stem cells.