3D microtissue-derived human stem cells seeded on electrospun nanocomposites under shear stress: Modulation of gene expression.

OBJECTIVE: Different microenvironments trigger distinct differentiation of stem cells. Even without chemical supplementation, mechanical stimulation by shear stress may help to induce the desired differentiation. The cell format, such as three-dimensional (3D) microtissues (MTs), MT-derived cells or single cells (SCs), may have a pivotal impact as well. Here, we studied modulation of gene expression in human adipose-derived stem cells (ASCs) exposed to shear stress and/or after MT formation. MATERIALS AND METHODS: Electrospun meshes of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) at a weight ratio of 60:40 were seeded with human ASCs as MTs or as SCs and cultured in Dulbecco's modified Eagle's medium without chemical supplementation. After 2 weeks of static culture, the scaffolds were cultured statically for another 2 weeks or placed in a Bose® bioreactor with a flow rate per area of 0.16 mL cm-2 min-1. Stiffness of the scaffolds was assessed as a function of time. After 4 weeks, minimum stem cell criteria markers and selected markers of osteogenesis, endothelial cell differentiation, adipogenesis and chondrogenesis were analysed by quantitative real-time polymerase chain reaction. Additionally, cell distribution within the scaffolds and the allocation of the yes-associated protein (YAP) in the cells were assessed by immunohistochemistry. RESULTS: MTs decayed completely within 2 weeks after seeding on PLGA/aCaP. The osteogenic marker gene alkaline phosphatase and the endothelial cell marker gene CD31 were upregulated in MT-derived ASCs compared with SCs. Shear stress realised by fluid flow perfusion upregulated peroxisome proliferator-activated receptor gamma 2 expression in MT-derived ASCs and in SCs. The nuclear-to-cytoplasmic ratio of YAP expression was doubled under perfusion compared with that under static culture for MT-derived ASCs and SCs. CONCLUSIONS: Osteogenic and angiogenic commitments were more pronounced in MT-derived ASCs seeded on bone biomimetic electrospun nanocomposite PLGA/aCaP than in SCs seeded without induction medium. Furthermore, the static culture was superior to the perfusion regimen used here, as shear stress resulted in adipogenic commitment for MT-derived ASCs and SCs, although the YAP nuclear-to-cytoplasmic ratio indicated higher cell tensions under perfusion, usually associated with preferred osteogenic differentiation.

An Early Function of Polycystin-2 for Left-Right Organizer Induction in Xenopus.

Nodal signaling controls asymmetric organ placement during vertebrate embryogenesis. Nodal is induced by a leftward fluid flow at the ciliated left-right organizer (LRO). The mechanism of flow sensing, however, has remained elusive. pkd2 encodes the calcium channel Polycystin-2, which is required for kidney development and laterality, and may act in flow perception. Here, we have studied the role of Polycystin-2 in Xenopus and show that pkd2 is indispensable for left-right (LR) asymmetry. Knockdown of pkd2 prevented left-asymmetric nodal cascade induction in the lateral plate mesoderm. Defects were due to failure of LRO specification, morphogenesis, and, consequently, absence of leftward flow. Polycystin-2 synergizes with the unconventional nodal-type signaling molecule Xnr3 to induce the LRO precursor tissue before gastrulation, upstream of symmetry breakage. Our data uncover an unknown function of pkd2 in LR axis formation, which we propose represents an ancient role of Polycystin-2 during LRO induction in lower vertebrates.

Cyclic uniaxial compression of human stem cells seeded on a bone biomimetic nanocomposite decreases anti-osteogenic commitment evoked by shear stress.

OBJECTIVE: Chemical supplementation of culture media to induce differentiation of adult stem cells seeded on a scaffold may mask other differentiation triggers such as scaffold stiffness, chemical composition or mechanical stimulation. However, stem cells can be differentiated towards osteoblasts without any supplementation given an appropriate osteogenic scaffold and an adequate mechanical stimulation. MATERIALS AND METHODS: Electrospun meshes of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) in a weight ratio of 60:40 were seeded with human adipose-derived stem cells (ASCs) and cultured in DMEM. After two weeks of static cultivation, they were either further cultivated statically for another two weeks (group 1), or placed in a Bose® bioreactor with a flow rate per area of 0.16 mL cm-2 min1 (group 2). Furthermore, group 3 was also cultivated under perfusion, however, with an additional uniaxial cyclic compression. Stiffness of the scaffolds was assessed as a function of time. After a total of four weeks, minimum stem cell criteria markers as well as typical markers for osteogenesis, endothelial cell differentiation, adipogenesis and chondrogenesis were analyzed by quantitative real-time PCR, cell distribution within the scaffolds by histology and protein expression by immunohistochemistry. RESULTS: Dynamic conditions (perfusion ±â€¯uniaxial cyclic compression) significantly upregulated gene and protein expression of PPAR-γ-2 compared to static cultivation, while osteogenic markers were slightly downregulated. However, the compression in the perfusion bioreactor favored osteogenesis compared to mere perfusion as indicated by upregulation of ALP, Runx2 and collagen I. This behavior was not only attributed to the compressive load, but also to the significant increase in stiffness of the scaffold. Furthermore, CD105 was significantly upregulated under compression. CONCLUSIONS: Although an osteogenic electrospun composite material with an organic (PLGA) and an inorganic phase (aCaP nanoparticles) was used as scaffold, the dynamic cultivation as realized by either perfusion alone or an additional compression did not upregulate typical osteogenic genes when compared to static cultivation. In contrast, there was a significant upregulation of the adipogenic gene PPAR-γ-2. However, this anti-osteogenic starting point evoked by mere perfusion was partially reversed by an additional compression. Our findings exemplify that bone tissue engineering using adult stem cells should consider any other differentiations that may be triggered and overwhelm the desired differentiation, although experimental conditions theoretically provide cues to achieve it - like an osteogenic scaffold and mechanical stimulation.