Culturing patient-derived malignant hematopoietic stem cells in engineered and fully humanized 3D niches.

Human malignant hematopoietic stem and progenitor cells (HSPCs) reside in bone marrow (BM) niches, which remain challenging to explore due to limited in vivo accessibility and constraints with humanized animal models. Several in vitro systems have been established to culture patient-derived HSPCs in specific microenvironments, but they do not fully recapitulate the complex features of native bone marrow. Our group previously reported that human osteoblastic BM niches (O-N), engineered by culturing mesenchymal stromal cells within three-dimensional (3D) porous scaffolds under perfusion flow in a bioreactor system, are capable of maintaining, expanding, and functionally regulating healthy human cord blood-derived HSPCs. Here, we first demonstrate that this 3D O-N can sustain malignant CD34+ cells from acute myeloid leukemia (AML) and myeloproliferative neoplasm patients for up to 3 wk. Human malignant cells distributed in the bioreactor system mimicking the spatial distribution found in native BM tissue, where most HSPCs remain linked to the niches and mature cells are released to the circulation. Using human adipose tissue-derived stromal vascular fraction cells, we then generated a stromal-vascular niche and demonstrated that O-N and stromal-vascular niche differentially regulate leukemic UCSD-AML1 cell expansion, immunophenotype, and response to chemotherapy. The developed system offers a unique platform to investigate human leukemogenesis and response to drugs in customized environments, mimicking defined features of native hematopoietic niches and compatible with the establishment of personalized settings.

Analysis of the subcapital two-part humerus fracture by fluoroscopy: objective criteria for classification and decision making.

BACKGROUND: Surgical decision making in the treatment of proximal humerus fractures (PHFx) is primarily based on fracture classification using standard radiographs. Due to the lack of objective criteria, this classification process is associated with high interobserver variation. In this study, we investigate the fluoroscopic analysis of humerus fractures through the surgical neck using a semi-quantitative determination of distinct angulation patterns of the proximal humerus as they appear in the image intensifier. METHODS: Using a saw bone model, defined subcapital 2-part fracture configurations were generated and assessed radiographically. Anatomical landmarks-including the greater and lesser tuberosity as well as anatomical neck-were identified using an image converter, and the exact degree of fracture displacement with 10° up to 70° (in 10° increments) of posterior, varus or combined posterior-varus angulation was compared to nondisplaced controls. From the resultant series of radiographs, the appearance of these angulations in anteroposterior (AP) and scapular Y-views were also visualized and defined. RESULTS: An angulation of 50° or more of any given 2-part fracture through the surgical neck is present when the greater tuberosity becomes the most proximal point in AP view (varus and combined posterior-varus angulation) or a bimodal form is found for the superior contour of the head with the lesser tuberosity being the most proximal point in the Y-view (posterior angulation). CONCLUSION: The radiological appearance of various PHFx constellations can be well visualized using the saw bone shoulder model. The presence of angulation in accordance with the Neer classification for group III fractures can be adequately determined by analyzing the relative position of the greater or lesser tuberosity to the humeral head calotte. This can assist the surgeon's decision on whether to operate or opt for a conservative approach. LEVEL OF EVIDENCE: Basic Science, Anatomy Study, Imaging.

In vitro biomimetic engineering of a human hematopoietic niche with functional properties.

In adults, human hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment. Our understanding of human hematopoiesis and the associated niche biology remains limited, due to human material accessibility and limits of existing in vitro culture models. The establishment of an in vitro BM system would offer an experimentally accessible and tunable platform to study human hematopoiesis. Here, we develop a 3D engineered human BM analog by recapitulating some of the hematopoietic niche elements. This includes a bone-like scaffold, functionalized by human stromal and osteoblastic cells and by the extracellular matrix they deposited during perfusion culture in bioreactors. The resulting tissue exhibited compositional and structural features of human BM while supporting the maintenance of HSPCs. This was associated with a compartmentalization of phenotypes in the bioreactor system, where committed blood cells are released into the liquid phase and HSPCs preferentially reside within the engineered BM tissue, establishing physical interactions with the stromal compartment. Finally, we demonstrate the possibility to perturb HSPCs' behavior within our 3D niches by molecular customization or injury simulation. The developed system enables the design of advanced, tunable in vitro BM proxies for the study of human hematopoiesis.

Harnessing human adipose-derived stromal cell chondrogenesis in vitro for enhanced endochondral ossification.

Endochondral ossification (ECO), the major ossification process during embryogenesis and bone repair, involves the formation of a cartilaginous template remodelled into a functional bone organ. Adipose-derived stromal cells (ASC), non-skeletal multipotent progenitors from the stromal vascular fraction (SVF) of human adipose tissue, were shown to recapitulate ECO and generate bone organs in vivo when primed into a hypertrophic cartilage tissue (HCT) in vitro. However, the reproducibility of ECO was limited and the major triggers remain unknown. We studied the effect of the expansion of cells and maturation of HCT on the induction of the ECO process. SVF cells or expanded ASC were seeded onto collagen sponges, cultured in chondrogenic medium for 3-6 weeks and implanted ectopically in nude mice to evaluate their bone-forming capacities. SVF cells from all tested donors formed mature HCT in 3 weeks whereas ASC needed 4-5 weeks. A longer induction increased the degree of maturation of the HCT, with a gradually denser cartilaginous matrix and increased mineralization. This degree of maturation was highly predictive of their bone-forming capacity in vivo, with ECO achieved only for an intermediate maturation degree. In parallel, expanding ASC also resulted in an enrichment of the stromal fraction characterized by a rapid change of their proteomic profile from a quiescent to a proliferative state. Inducing quiescence rescued their chondrogenic potential. Our findings emphasize the role of monolayer expansion and chondrogenic maturation degree of ASC on ECO and provides a simple, yet reproducible and effective approach for bone formation to be tested in specific clinical models.

Selective proapoptotic activity of polyphenols from red wine on teratocarcinoma cell, a model of cancer stem-like cell.

Cancer stem cells are expected to be responsible for tumor initiation and metastasis. These cells are therefore potential targets for innovative anticancer therapies. However, the absence of bona fide cancer stem cell lines is a real problem for the development of such approaches. Since teratocarcinoma cells are totipotent stem cells with a high degree of malignancy, we used them as a model of cancer stem cells in order to evaluate the anticancer chemopreventive activity of red wine polyphenols (RWPs) and to determine the underlying cellular and molecular mechanisms. We therefore investigated the effects of RWPs on the embryonal carcinoma (EC) cell line P19 which was grown in the same culture conditions as the most appropriate normal cell line counterpart, the pluripotent embryonic fibroblast cell line NIH/3T3. The present study indicates that RWPs selectively inhibited the proliferation of P19 EC cells and induced G1 cell cycle arrest in a dose-dependent manner. Moreover, RWPs treatment specifically triggered apoptosis of P19 EC cells in association with a dramatic upregulation of the tumor suppressor gene p53 and caspase-3 activation. Our findings suggest that the chemopreventive activity of RWPs on tumor initiation and development is related to a growth inhibition and a p53-dependent induction of apoptosis in teratocarcinoma cells. In addition, this study also shows that the EC cell line is a convenient source for studying the responses of cancer stem cells to new potential anticancer agents.

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.