Improving the Study of Protein Glycosylation with New Tools for Glycopeptide Enrichment

High confidence methods are needed for determining the glycosylation profiles of complex biological samples as well as recombinant therapeutic proteins. A common glycan analysis workflow involves liberation of N-glycans from glycoproteins with PNGase F or O-glycans by hydrazinolysis prior to their analysis. This method is limited in that it does not permit determination of glycan attachment sites. Alternative proteomics-based workflows are emerging that utilize site-specific proteolysis to generate peptide mixtures followed by selective enrichment strategies to isolate glycopeptides. Methods designed for the analysis of complex samples can yield a comprehensive snapshot of individual glycans species, the site of attachment of each individual glycan and the identity of the respective protein in many cases. This chapter will highlight advancements in enzymes that digest glycoproteins into distinct fragments and new strategies to enrich specific glycopeptides

3D Engineering of Human Hematopoietic Niches in Perfusion Bioreactor

The hematopoietic microenvironment, also referred to as hematopoietic niche, is a functional three-dimensional (3D) unit of the bone marrow (BM) that planar culture systems cannot recapitulate. Existing limitations of 2D protocols are driving the development of advanced 3D methodologies, capable of superior modeling of the native organization and interactions between hematopoietic cells and their niche. Hereafter we describe the use of a 3D perfusion bioreactor for in vitro generation of human hematopoietic niches. The approach enables the recapitulation of the interactions between hematopoietic stem and progenitor cells (HSPCs), mesenchymal cells (MSCs), and their extracellular matrix in a 3D relevant setting. This was shown to support the functional maintenance of blood populations, self-distributing in the system compartments depending on their differentiation status. Such 3D niche modeling represents an advanced tool toward uncovering human hematopoiesis in relation to its host microenvironment, for both fundamental hematopoiesis and personalized medicine applications

Customizable 3D printed perfusion bioreactor for the engineering of stem cell microenvironments

Faithful modeling of tissues and organs requires the development of systems reflecting their dynamic 3D cellular architecture and organization. Current technologies suffer from a lack of design flexibility and complex prototyping, preventing their broad adoption by the scientific community. To make 3D cell culture more available and adaptable we here describe the use of the fused deposition modeling (FDM) technology to rapid-prototype 3D printed perfusion bioreactors. Our 3D printed bioreactors are made of polylactic acid resulting in reusable systems customizable in size and shape. Following design confirmation, our bioreactors were biologically validated for the culture of human mesenchymal stromal cells under perfusion for up to 2 weeks on collagen scaffolds. Microenvironments of various size/volume (6–12 mm in diameter) could be engineered, by modulating the 3D printed bioreactor design. Metabolic assay and confocal microscopy confirmed the homogenous mesenchymal cell distribution throughout the material pores. The resulting human microenvironments were further exploited for the maintenance of human hematopoietic stem cells. Following 1 week of stromal coculture, we report the recapitulation of 3D interactions between the mesenchymal and hematopoietic fractions, associated with a phenotypic expansion of the blood stem cell populations.Our data confirm that perfusion bioreactors fit for cell culture can be generated using a 3D printing technology and exploited for the 3D modeling of complex stem cell systems. Our approach opens the gates for a more faithful investigation of cellular processes in relation to a dynamic 3D microenvironment

Development of Humanized Ossicles : Bridging the Hematopoietic Gap

Ectopic 'humanized ossicles' (hOss) are miniaturized, engineered human bone organs in mice displaying a similar structure and function to native mouse bones. However, they are composed of human mesenchymal derived cells forming a humanized bone marrow niche. This in vivo reconstitution of human skeletal and hematopoietic compartments provides an opportunity to investigate the cellular and molecular processes involved in their establishment and functions in a human setting. However, current hOs strategies vary in their engineering methods and their downstream applications, undermining comprehensive exploitation of their potential. This review describes the specificities of the hOs models and highlights their potential and limits. Ultimately, we propose directions for the development of hOss as a technological platform for human hematopoietic studies

Comparative site-specific N-glycoproteome analysis reveals aberrant N-glycosylation and gives insights into mannose-6-phosphate pathway in cancer

Comparative site-specific N-glycoproteome analysis of tumorigenic and non-tumorigenic colon cancer cells provides a pool of potential N-glycosylation related cancer biomarkers and insights into the mannose-6-phosphate pathway in cancer

Manufacturing of Human Tissues as off-the-Shelf Grafts Programmed to Induce Regeneration

Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, a conceptual shift based on the manufacturing of devitalized human hypertrophic cartilage (HyC), as cell-free material inducing bone formation by recapitulating the developmental process of endochondral ossification, is reported. The strategy relies on a customized human mesenchymal line expressing bone morphogenetic protein-2 (BMP-2), critically required for robust chondrogenesis and concomitant extracellular matrix (ECM) enrichment. Following apoptosis-driven devitalization, lyophilization, and storage, the resulting off-the-shelf cartilage tissue exhibits unprecedented osteoinductive properties, unmatched by synthetic delivery of BMP-2 or by living engineered grafts. Scalability and pre-clinical efficacy are demonstrated by bioreactor-based production and subsequent orthotopic assessment. The findings exemplify the broader paradigm of programming human cell lines as biological factory units to engineer customized ECMs, designed to activate specific regenerative processes

Engineering human mini-bones for the standardized modeling of healthy hematopoiesis, leukemia, and solid tumor metastasis

The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Preclinical research relies on xenograft mouse models, but these models preclude the human-specific functional interactions of stem cells with their bone marrow microenvironment. Instead, human mesenchymal cells can be exploited for the in vivo engineering of humanized niches, which confer robust engraftment of human healthy and malignant blood samples. However, mesenchymal cells are associated with major reproducibility issues in tissue formation. Here, we report the fast and standardized generation of human mini-bones by a custom-designed human mesenchymal cell line. These resulting humanized ossicles (hOss) consist of fully mature bone and bone marrow structures hosting a human mesenchymal niche with retained stem cell properties. As compared to mouse bones, we demonstrate superior engraftment of human cord blood hematopoietic cells and primary acute myeloid leukemia samples and also validate hOss as a metastatic site for breast cancer cells. We further report the engraftment of neuroblastoma patient-derived xenograft cells in a humanized model, recapitulating clinically described osteolytic lesions. Collectively, our human mini-bones constitute a powerful preclinical platform to model bone-developing tumors using patient-derived materials