Due to spaceflight, astronauts experience serious, weightlessness-induced bone loss because of an unbalanced process of bone remodeling that involves bone marrow mes- enchymal stem cells (BMSCs), as well as osteoblasts, osteo- cytes, and osteoclasts. The effects of microgravity on osteo- cells have been extensively studied, but it is only recently that consideration has been given to the role of BMSCs. Pre- vious researches indicated that human BMSCs cultured in simulated microgravity (sim-μg) alter their proliferation and differentiation. The spaceflight opportunities for biomedical experiments are rare and suffer from a number of opera- tive constraints that could bias the validity of the experiment itself, but remain a unique opportunity to confirm and explain the effects due to microgravity, that are only par- tially activated/detectable in simulated conditions. For this reason, we carefully prepared the SCD – STEM CELLS DIFFERENTIATION experiment, selected by the European Space Agency (ESA) and now on the International Space Station (ISS). Here we present the preparatory studies per- formed on ground to adapt the project to the spaceflight constraints in terms of culture conditions, fixation and stor- age of human BMSCs in space aiming at satisfying the biological requirements mandatory to retrieve suitable sam- ples for post-flight analyses. We expect to understand better the molecular mechanisms governing human BMSC growth and differentiation hoping to outline new countermeasures against astronaut bone loss.
Research Containing: Differentiation
Mechanical unloading in microgravity is thought to induce tissue degeneration by various mechanisms, including inhibition of regenerative stem cell differentiation. To address this hypothesis, we investigated the effects of microgravity on early lineage commitment of mouse embryonic stem cells (mESCs) using the embryoid body (EB) model of tissue differentiation. We found that exposure to microgravity for 15 days inhibits mESC differentiation and expression of terminal germ layer lineage markers in EBs. Additionally, microgravity-unloaded EBs retained stem cell self-renewal markers, suggesting that mechanical loading at Earth’s gravity is required for normal differentiation of mESCs. Finally, cells recovered from microgravity-unloaded EBs and then cultured at Earth’s gravity showed greater stemness, differentiating more readily into contractile cardiomyocyte colonies. These results indicate that mechanical unloading of stem cells in microgravity inhibits their differentiation and preserves stemness, possibly providing a cellular mechanistic basis for the inhibition of tissue regeneration in space and in disuse conditions on earth.
Mechanical forces, including gravity, tension, compression, hydrostatic pressure, and fluid shear stress, play a vital role in human physiology and pathology. They particularly influence extracellular matrix (ECM) gene expression, ECM protein synthesis, and production of inflammatory mediators of many load-sensitive adult cells such as fibroblasts, chondrocytes, smooth muscle cells, and endothelial cells. Furthermore, the mechanical forces generated by cells themselves, known as cell traction forces (CTFs), also influence many biological processes such as wound healing, angiogenesis, and metastasis. Thus, the quantitative characterization of CTFs by qualities such as magnitude and distribution is useful for understanding physiological and pathological events at the tissue and organ levels. Recently, the effects of mechanical loads on embryonic and adult stem cells in terms of self-renewal, differentiation, and matrix protein expression have been investigated. While it seems certain that mechanical loads applied to stem cells regulate their self-renewal and induce controlled cell lineage differentiation, the detailed molecular signaling mechanisms responsible for these mechano-effects remain to be elucidated. Challenges in the fields of both adult- and stem-cell mechanobiology include devising novel experimental and theoretical methodologies to examine mechano-responses more closely to various forms of mechanical forces and mechanotransduction mechanisms of these cells in a more physiologically accurate setting. Such novel methodologies will lead to better understanding of various pathological diseases, their management, and translational applications in the ever expanding field of tissue engineering.
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Most cells in tissues contact an extracellular matrix on at least one surface. These complex mixtures of interacting proteins provide structural support and biological signals that regulate cell differentiation and may be important for stem cell differentiation. In this study, we have grown a rhesus monkey embryonic stem cell line in the presence of various extracellular matrix components in monolayer, in a NASA-developed rotating wall vessel bioreactor in vitro, and subcutaneously in vivo. We find that individual components of the extracellular matrix, such as laminin-1 or collagen 1, do not influence the growth or morphology of the cells. In contrast, a basement membrane extract, Matrigel, containing multiple extracellular matrix components, induces the cells within 4 days to form immature glandular- and tubular-like structures, many of which contain a lumen with polarized epithelium and microvilli. Such structures were seen in vitro when the cells were grown in the bioreactor and when the cells were injected into mice. These tubular- and glandular-like structures were polarized epithelia based on immunostaining for laminin and cytokeratin. The cell aggregates and tumors also contained additional mixed populations of cells, including mesenchymal cells and neuronal cells, based on immunostaining with vimentin and neuronal markers. An extract of cartilage, containing multiple cartilage matrix components, promoted chondrogenesis in vivo where alcian blue-stained cartilage nodules could be observed. Some of these nodules stained with von Kossa, indicating that they had formed calcified cartilage. We conclude that extracellular matrices can promote the differentiation of embryonic stem cells into differentiated cells and structures that are similar to the tissue from which the matrix is derived. Such preprogramming of cell differentiation with extracellular matrices may be useful in targeting stem cells to repair specific damaged organs.
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We differentiated mouse bone marrow cells in the presence of recombinant macrophage colony stimulating (rM-CSF) factor for 14 days during the flight of space shuttle Space Transportation System (STS)-126. We tested the hypothesis that the receptor expression for M-CSF, c-Fms was reduced. We used flow cytometry to assess molecules on cells that were preserved during flight to define the differentiation state of the developing bone marrow macrophages; including CD11b, CD31, CD44, Ly6C, Ly6G, F4/80, Mac2, c-Fos as well as c-Fms. In addition, RNA was preserved during the flight and was used to perform a gene microarray. We found that there were significant differences in the number of macrophages that developed in space compared to controls maintained on Earth. We found that there were significant changes in the distribution of cells that expressed CD11b, CD31, F4/80, Mac2, Ly6C and c-Fos. However, there were no changes in c-Fms expression and no consistent pattern of advanced or retarded differentiation during space flight. We also found a pattern of transcript levels that would be consistent with a relatively normal differentiation outcome but increased proliferation by the bone marrow macrophages that were assayed after 14 days of space flight. There also was a surprising pattern of space flight influence on genes of the coagulation pathway. These data confirm that a space flight can have an impact on the in vitro development of macrophages from mouse bone marrow cells.
Stem and progenitor cells isolated from the embryonic rat cerebral cortex were immobilized by matrix entrapment in three-dimensional (3D) Type I collagen gels, and cultured in serum-free medium containing basic fibroblast growth factor. The cells trapped within the collagen networks actively proliferated and formed clone-like aggregates. Neurons were the first differentiated cells to appear within the aggregates, followed by generation of astrocytes and oligodendrocytes. In addition, necrotic cores were developed as the aggregate diameter increased and cell viability declined significantly after 3 weeks in culture. To overcome these problems, the cell-collagen constructs were transferred to Rotary Wall Vessel bioreactors for up to 10 weeks. In the rotary culture, the collagen gels compacted 3-4 folds and a long-term growth and differentiation of neural stem and progenitor cells was dynamically maintained. Remarkably, the cell-collagen constructs formed a complex two-layered structure that superficially emulated to a certain extent the cerebral cortex of the embryonic brain in architecture and functionality. The engineered 3D tissue-like constructs displaying characteristic properties of neuronal circuits may have potential use in tissue replacement therapy for injured brain and spinal cord.
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From spindle to spherical: Is spherical shape a potential predictor of human mesenchymal stem cells with increased differentiation capability?
Because human mesenchymal stem cells (hMSCs) can proliferate indefinitely in an undifferentiated state and differentiate into various cell types, hMSCs are expected to be useful for cell replacement therapy. But the clinic application is limited by its differentiation efficiency of hMSCs. It has been proved that cells can be geometrically switched between gene programs for growth, apoptosis and differentiation. Previous studies showed that hMSCs started showing round when exposed to modeled microgravity (MMG), while their differentiation capability seemed enhanced simultaneously. Thus, this article briefly reviews such studies, and hypothesizes that “spherical shape” could be a potential predictor of hMSCs with potentiated differentiation capability.
Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to differentiate into osteoblasts, chondroblasts, myocytes, and adipocytes. They have potential for bone tissue engineering by the utilization of in vitro expanded cells with osteogenic capacity and their delivery to the appropriate sites via biomaterial scaffolds. The objective was to evaluate the potential of rat bone marrow MSCs to form 3D bone-like tissue by the use of mineralized poly(DL-lactic-co-glycolic acid) (PLGA) foam and osteoinductive medium under rotating culture conditions. PLGA foams were prepared by solvent casting and particulate leaching, then mineralized by incubating in simulated body fluid. MSCs isolated from the bone marrow of young Wistar rats were expanded and seeded on the mineralized scaffolds. The cell-polymer constructs were then cultured in a slow turning lateral vessel-type rotating bioreactor for 4 weeks under the effect of osteogenic inducers, b-glycerophosphate, ascorbic acid and dexamethasone. Mineralization was evaluated using FT-IR and increases in dry mass; morphology changes of the mineralized foams and cell adhesion was characterized by SEM; cell viability was monitored by MTT (3-(4,5-dimethylthia-zol-2-yl-2,5-diphenyl tetrazolium bromide). Osteogenic differentiation was determined by using immunohistochemistry (anti-Osteopontin). Results indicate the feasibility of bone tissue engineering with MSCs and mineralized PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures under appropriate bioreactor conditions.
Three-dimensional bioreactor cultures: A useful dynamic model for the study of cellular interactions
The ex vivo expansion of hematopoietic cells is a developing area with emphasis on bioreactor systems for amelioration of culture conditions. A rational design of bioreactors, especially those allowing microgravity, could permit the production of stem cells and will offer new approaches for studying the mechanisms of proliferation, differentiation, and signal transduction of cultured cells. The efficacy of two commercially available bioreactors (rotating-vessel miniPERM and static INTEGRA CL 350) to support long-term bone marrow cell cultures (LTBMCC) and three-dimensional growth of Hodgkin's lymphoma HD-MY-Z cells was investigated. In the miniPERM system, the growth of LTBMCC spheroids (containing 30-40 cells) was obtained. An essentially higher content of hematopoietic precursor cells (colony-forming units-granulocyte macrophage) was registered in the rotating-vessel system. In this bioreactor, a growth of large HD-MY-Z spheroids (containing 100-200 cells) was achieved. The composed mathematical models of the physicomechanical behavior of spheroids enabled the evaluation of the revolution frequency increase schedule. The differential equations took into account all inertial effects caused by the production module rotation movement as well as those caused by the relative movement of the spheroid in the fluid. The models aimed at the optimization of the rotation frequency increase schedule for different types of cells to reduce shear stress, augment productivity, and tolerate the growth of large spheroids. The models were numerically tested using MATLAB-SIMULINK software, and the trajectories of prestained HD-MY-Z spheroids were filmed. The coincidence of the theoretical and experimental trajectories was sufficient.
The use of murine embryonic stem cells, alginate encapsulation, and rotary microgravity bioreactor in bone tissue engineering
The application of embryonic stem cells (ESCs) in bone tissue engineering and regenerative medicine requires the development of suitable bioprocesses that facilitate the integrated, reproducible, automatable production of clinically-relevant, scaleable, and integrated bioprocesses that generate sufficient cell numbers resulting in the formation of three-dimensional (3D) mineralised, bone tissue-like constructs. Previously, we have reported the enhanced differentiation of undifferentiated mESCs toward the osteogenic lineage in the absence of embryoid body formation. Herein, we present an efficient and integrated 3D bioprocess based on the encapsulation of undifferentiated mESCs within alginate hydrogels and culture in a rotary cell culture microgravity bioreactor. Specifically, for the first 3 days, encapsulated mESCs were cultured in 50% (v/v) HepG2 conditioned medium to generate a cell population with enhanced mesodermal differentiation capability followed by osteogenic differentiation using osteogenic media containing ascorbic acid, β-glycerophosphate and dexamethasone. 3D mineralised constructs were generated that displayed the morphological, phenotypical, and molecular attributes of the osteogenic lineage, as well mechanical strength and mineralised calcium/phosphate deposition. Consequently, this bioprocess provides an efficient, automatable, scalable and functional culture system for application to bone tissue engineering in the context of macroscopic bone formation.