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

Engineering human hematopoietic environments through ossicle and bioreactor technologies exploitation

The bone marrow microenvironment contains cellular niches that maintain the pool of hematopoietic stem and progenitor cells and support hematopoietic maturation. Malignant hematopoietic cells also co-opt normal cellular interactions to promote their own growth and evade therapy. In vivo systems used to study human hematopoiesis have been developed through transplantation into immunodeficient mouse models. However, incomplete cross-compatibility between the murine stroma and transplanted human hematopoietic cells limits the rate of engraftment and the study of relevant interactions. To supplement in vivo xenotransplantation models, complementary strategies have recently been developed, including the use of three-dimensional human bone marrow organoids in vivo, generated from bone marrow stromal cells seeded onto osteo-inductive scaffolds, as well as the use of ex vivo bioreactor models. These topics were the focus of the Spring 2020 International Society for Experimental Hematology New Investigator webinar. We review here the latest advances in generating humanized hematopoietic organoids and how they allow for the study of novel microenvironmental interactions

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

Osteoinductivity of engineered cartilaginous templates devitalized by inducible apoptosis

The role of cell-free extracellular matrix (ECM) in triggering tissue and organ regeneration has gained increased recognition, yet current approaches are predominantly based on the use of ECM from fully developed native tissues at nonhomologous sites. We describe a strategy to generate customized ECM, designed to activate endogenous regenerative programs by recapitulating tissue-specific developmental processes. The paradigm was exemplified in the context of the skeletal system by testing the osteoinductive capacity of engineered and devitalized hypertrophic cartilage, which is the primordial template for the development of most bones. ECM was engineered by inducing chondrogenesis of human mesenchymal stromal cells and devitalized by the implementation of a death-inducible genetic device, leading to cell apoptosis on activation and matrix protein preservation. The resulting hypertrophic cartilage ECM, tested in a stringent ectopic implantation model, efficiently remodeled to form de novo bone tissue of host origin, including mature vasculature and a hematopoietic compartment. Importantly, cartilage ECM could not generate frank bone tissue if devitalized by standard "freeze & thaw" (F&T) cycles, associated with a significant loss of glycosaminoglycans, mineral content, and ECM-bound cytokines critically involved in inflammatory, vascularization, and remodeling processes. These results support the utility of engineered ECM-based devices as off-the-shelf regenerative niches capable of recruiting and instructing resident cells toward the formation of a specific tissue

Osteoinductivity of engineered cartilaginous templates devitalized by inducible apoptosis

The role of cell-free extracellular matrix (ECM) in triggering tissue and organ regeneration has gained increased recognition, yet current approaches are predominantly based on the use of ECM from fully developed native tissues at nonhomologous sites. We describe a strategy to generate customized ECM, designed to activate endogenous regenerative programs by recapitulating tissue-specific developmental processes. The paradigm was exemplified in the context of the skeletal system by testing the osteoinductive capacity of engineered and devitalized hypertrophic cartilage, which is the primordial template for the development of most bones. ECM was engineered by inducing chondrogenesis of human mesenchymal stromal cells and devitalized by the implementation of a death-inducible genetic device, leading to cell apoptosis on activation and matrix protein preservation. The resulting hypertrophic cartilage ECM, tested in a stringent ectopic implantation model, efficiently remodeled to form de novo bone tissue of host origin, including mature vasculature and a hematopoietic compartment. Importantly, cartilage ECM could not generate frank bone tissue if devitalized by standard “freeze & thaw” (F&T) cycles, associated with a significant loss of glycosaminoglycans, mineral content, and ECM-bound cytokines critically involved in inflammatory, vascularization, and remodeling processes. These results support the utility of engineered ECM-based devices as off-the-shelf regenerative niches capable of recruiting and instructing resident cells toward the formation of a specific tissue

Engineered humanized bone organs maintain human hematopoiesis in vivo

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) environment, the so-called HSC niche, that provides pivotal factors for their maintenance. Although the cellular and molecular components of the mouse BM HSC niche have been extensively studied using genetically modified animals, relatively little is known about the counterpart human BM niche components. We previously illustrated, with a developmental tissue engineering approach, that human adult BM-derived mesenchymal stromal cells (MSCs) can develop into human bone organs (so-called ossicles) through endochondral ossification in vivo and that these human ossicles are able to maintain functional mouse HSCs. We here report that human ossicles in immunodeficient mice maintain human immature and mature hematopoiesis in vivo. Moreover, a higher percentage of human stem and progenitor cells are kept in quiescence in human ossicles as compared with mouse BM. These findings indicate that the human MSC-derived ossicles function as a hematopoietic niche and may potentially serve as a re-engineerable platform to study normal and diseased human hematopoiesis in a physiologically optimized environment

Fat-Derived Stromal Vascular Fraction Cells Enhance the Bone-Forming Capacity of Devitalized Engineered Hypertrophic Cartilage Matrix

Engineered and devitalized hypertrophic cartilage (HC) has been proposed as bone substitute material, potentially combining the features of osteoinductivity, resistance to hypoxia, capacity to attract blood vessels, and customization potential for specific indications. However, in comparison with vital tissues, devitalized HC grafts have reduced efficiency of bone formation and longer remodeling times. We tested the hypothesis that freshly harvested stromal vascular fraction (SVF) cells from human adipose tissue-which include mesenchymal, endothelial, and osteoclastic progenitors-enhance devitalized HC remodeling into bone tissue. Human SVF cells isolated from abdominal lipoaspirates were characterized cytofluorimetrically. HC pellets, previously generated by human bone marrow-derived stromal cells and devitalized by freeze/thaw, were embedded in fibrin gel with or without different amounts of SVF cells and implanted either ectopically in nude mice or in 4-mm-diameter calvarial defects in nude rats. In the ectopic model, SVF cells added to devitalized HC directly contributed to endothelial, osteoblastic, and osteoclastic populations. After 12 weeks, the extent of graft vascularization and amount of bone formation increased in a cell-number-dependent fashion (up to, respectively, 2.0-fold and 2.9-fold using 12 million cells per milliliter of gel). Mineralized tissue volume correlated with the number of implanted, SVF-derived endothelial cells (CD31+ CD34+ CD146+). In the calvarial model, SVF activation of HC using 12 million cells per milliliter of gel induced efficient merging among implanted pellets and strongly enhanced (7.3-fold) de novo bone tissue formation within the defects. Our findings outline a bone augmentation strategy based on off-the-shelf devitalized allogeneic HC, intraoperatively activated with autologous SVF cells.; This study validates an innovative bone substitute material based on allogeneic hypertrophic cartilage that is engineered, devitalized, stored, and clinically used, together with autologous cells, intraoperatively derived from a lipoaspirate. The strategy was tested using human cells in an ectopic model and an orthotopic implantation model, in immunocompromised animals