Human adipose-derived mesenchymal stem cells: serial passaging,doubling time and cell senescence

Adult adipose-derived mesenchymal stem cells (AD-MSC) are very interesting to our research group because they are easy to harvest, they are abundant in humans, and they have potential clinical applications in autologous cell therapy for disc degeneration. We examined these cells through sequential serial passages to assess osteogenic and chondrogenic capabilities, mean doubling time and cell senescence. Osteogenic and chondrogenic potencies were maintained through 13 passages. Mean passage doubling time increased significantly with increasing passage number. When donor age was evaluated, passages 1-4 from older donors had significantly longer doubling times compared to cells from younger donors. Passages 5-11 showed similar findings when analyzed by donor age. The mean percent senescence increased significantly with cell passaging, rising from 0% at passage 1 to 3.4% at passage 13. These novel data suggest that caution should be exercised when using AD-MSC with long passage times.     

Human adipose-derived mesenchymal stem cells: serial passaging, doubling time and cell senescence

Adult adipose-derived mesenchymal stem cells (AD-MSC) are very interesting to our research group because they are easy to harvest, they are abundant in humans, and they have potential clinical applications in autologous cell therapy for disc degeneration. We examined these cells through sequential serial passages to assess osteogenic and chondrogenic capabilities, mean doubling time and cell senescence. Osteogenic and chondrogenic potencies were maintained through 13 passages. Mean passage doubling time increased significantly with increasing passage number. When donor age was evaluated, passages 1-4 from older donors had significantly longer doubling times compared to cells from younger donors. Passages 5-11 showed similar findings when analyzed by donor age. The mean percent senescence increased significantly with cell passaging, rising from 0% at passage 1 to 3.4% at passage 13. These novel data suggest that caution should be exercised when using AD-MSC with long passage times.     

Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of time of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can be reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the BV formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.     

Articular chondrocyte passage number: influence on adhesion, migration, cytoskeletal organisation and phenotype in response to nano- and micro-metric topography

The isolation and culture of articular chondrocytes is a prerequisite of their use in tissue engineering, but prolonged culture and passaging is associated with de-differentiation. In this paper we studied the influence of nanometric and micrometric grooves (85 nm to 8 microm in depth and 2 microm to 20 microm in width) on 1st and 2nd passage ovine chondrocytes since our earlier findings indicate that primary cells are not affected by such features. 1st and 2nd passage chondrocytes cultured on grooved substrata showed a polarisation of cell shape parallel to the groove long axis and F-actin condensations were evident at groove ridge boundaries. An increase in cell migration with increasing groove depth was observed. Both passages of chondrocytes maintained type II collagen expression, but to a lesser degree in 2nd. This study demonstrates that passage number alters the response of chondrocytes to micrometric and nanometric topography, and could be important in ex vivo cartilage engineering.     

Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring

Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle™ 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 μg mL⁻¹ 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide pools were measured during all cell cultivations and the nucleoside triphosphate/uridine ratios were calculated. These ‘nucleotide ratios’ changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.     

TIMP-2 promotes cell spreading and adhesion via upregulation of Rap1 signaling

We previously demonstrated that TIMP-2 treatment of human microvascular endothelial cells (hMVECs) activates Rap1 via the pathway of paxillin-Crk-C3G. Here, we show that TIMP-2 overexpression in hMVECs by adenoviral infection enhances Rap1 expression, leading to further increase in Rap1-GTP. TIMP-2 expression, previously reported to inhibit cell migration, also leads to cell spreading accompanied with increased cell adhesion. HMVECs stably expressing Rap1 display a similar phenotype as hMVECs-TIMP-2, whereas the expression of inactive Rap1 mutant, Rap1(38N), leads to elongated appearance with greatly reduced cell adhesion. Furthermore, the phenotype of hMVECs-Rap1(38N) was not reversed by TIMP-2 overexpression. TIMP-2 greatly promotes the association of Rap1 with actin. Therefore, these findings suggest that TIMP-2 mediated alteration in cell morphology requires Rap1, TIMP-2 may recruit Rap1 to sites of actin cytoskeleton remodeling necessary for cell spreading, and enhanced cell adhesion by TIMP-2 expression may hinder cell migration.     

Cytotoxicity and genotoxicity of zingiberene on different neuron cell lines in vitro

The main objective of this study is to investigate the cytotoxic, genotoxic and antioxidant properties of zingiberene (ZBN) in an in vitro rat brain cell culture study. The cytotoxic effect was determined against the rat neuron and N2a neuroblastoma (N2a-NB) cell lines using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while the antioxidant activity was assessed using the total antioxidant capacity (TAC) and total oxidative stress (TOS) assays. The effects on DNA damage were also evaluated in this study by the single cell gel electrophoresis assay. The results indicated that ZBN has an anti-proliferative activity suppressing the proliferation of N2a-NB cells at concentrations over 50 mg L⁻¹ and neuron cells at concentrations over 150 mg L⁻¹. In addition, ZBN treatments at higher doses (≤50 mg L⁻¹) led to increases of TOS levels in N2a-NB cell cultures. However 25 mg L⁻¹ of ZBN treatment caused increases of TAC levels in cultured neuron and N2a-NB cell cultures while ZBN at doses of 10–400 mg L⁻¹ did not increase the number of total damage score in both cell lines. This study clearly indicates that ZBN has a significant potential to be used as a natural anticancer agent in cultured N2a-NBs.     

Cdc42 and RhoA are differentially regulated during arachidonate-mediated HeLa cell adhesion

Cell adhesion to extracellular matrix requires stimulation of an eicosanoid signaling pathway through the metabolism of arachidonate by 5-lipoxygenase to leukotrienes and cyclooxygenase-1/2 to prostaglandins, as well as activation of the small GTPase signaling pathway involving Cdc42 and Rho. These signaling pathways direct remodeling of the actin cytoskeleton during the adhesion process, specifically the polymerization of actin during cell spreading and the bundling of actin filaments when cells migrate. However, few studies linking these signaling pathways have been described in the literature. We have previously shown that HeLa cell adhesion to collagen requires oxidation of arachidonic acid (AA) by lipoxygenase for actin polymerization and cell spreading, and cyclooxygenase for bundling actin filaments during cell migration. We demonstrate that small GTPase activity is required for HeLa cell spreading upon gelatin, and that Cdc42 is activated while Rho is downregulated during the spreading process. Using constitutively active and dominant negative expression studies, we show that Cdc42 is required for HeLa cell spreading and migration, while activated RhoA is antagonistic towards spreading. Constitutively active RhoA promotes cell migration and increases the degree of actin bundling in HeLa cells. Further, we demonstrate that activation of either the AA oxidation pathway or the small GTPase pathway cannot rescue inhibition of spreading when the alternate pathway is blocked. Our results suggest (1) both the eicosanoid signaling pathway and small GTPase activation are required during HeLa cell adhesion, and (2) these signaling pathways converge to properly direct remodeling of the actin cytoskeleton during HeLa cell spreading and migration upon collagen.     

Distinct roles of Cdc42 in thymopoiesis and effector and memory T cell differentiation

Cdc42 of the Rho GTPase family has been implicated in cell actin organization, proliferation, survival, and migration but its physiological role is likely cell-type specific. By a T cell-specific deletion of Cdc42 in mouse, we have recently shown that Cdc42 maintains naïve T cell homeostasis through promoting cell survival and suppressing T cell activation. Here we have further investigated the involvement of Cdc42 in multiple stages of T cell differentiation. We found that in Cdc42^−/− thymus, positive selection of CD4⁺CD8⁺ double-positive thymocytes was defective, CD4⁺ and CD8⁺ single-positive thymocytes were impaired in migration and showed an increase in cell apoptosis triggered by anti-CD3/-CD28 antibodies, and thymocytes were hyporesponsive to anti-CD3/-CD28-induced cell proliferation and hyperresponsive to anti-CD3/-CD28-stimulated MAP kinase activation. At the periphery, Cdc42-deficient naive T cells displayed an impaired actin polymerization and TCR clustering during the formation of mature immunological synapse, and showed an enhanced differentiation to Th1 and CD8⁺ effector and memory cells in vitro and in vivo. Finally, Cdc42^−/− mice exhibited exacerbated liver damage in an induced autoimmune disease model. Collectively, these data establish that Cdc42 is critically involved in thymopoiesis and plays a restrictive role in effector and memory T cell differentiation and autoimmunity.     

Establishment of a leukocyte cell line derived from peritoneal macrophages of fish,Labeo rohita (Hamilton, 1822)

A continuous leukocyte cell line with phagocytic activity was established from peritoneal macrophages of rohu, Labeo rohita (LRPM). LRPM was initiated from adherent mononuclear leukocytes isolated from peritoneal cavity of rohu, without use of any growth factors or feeder cells. These cells exhibited maximum growth at 30 °C in L-15 medium containing 20 % foetal bovine serum, and has been subcultured for more than 60 passages till date. The cells showed 85 % viability after 6 months of storage in liquid nitrogen. The species of origin of the LRPM was confirmed by the amplification and sequencing of 655 bp fragment of cytochrome oxidase subunit I of mitochondrial DNA. Functionally, LRPM showed phagocytic activity of yeast cells and fluorescent latex beads as evaluated by phase contrast and scanning electron microscopy, respectively. Immuno-modulators such as bacterial lipopolysaccharide and phorbol myristate acetate resulted in functional activation of LRPM; and enhanced their microbicidal activity through release of reactive oxygen species and nitric oxide. Culture supernatant from activated cells also revealed lysozyme activity. Cells of LRPM were positive for alpha-naphthyl acetate esterase enzyme indicating macrophage lineage. Our results indicate that this cell line can be a useful in vitro tool to study the role of macrophages in teleost immune system and to evaluate the effects of new aquaculture drugs. The LRPM cell line represents the first reported leukocyte cell line of peritoneal origin from any freshwater species of fish.     

Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV

Human dipeptidylpeptidase IV (hDPPIV) is an enzyme that is in hydrolase class and has various roles in different parts of human body. Its deficiency may cause some disorders in the gastrointestinal, neurologic, endocrinological and immunological systems of humans. In the present study, hDPPIV enzyme was expressed on Spodoptera frugiperda (Sf9) cell lines as a host cell, and the expression of hDPPIV was obtained by a baculoviral expression system. The enzyme production, optimum multiplicity of infection, optimum transfection time, infected and uninfected cell size and cell behavior during transfection were also determined. For maximum hDPPIV (269 mU mL⁻¹) enzyme, optimum multiplicity of infection (MOI) and time were 0.1 and 72 h, respectively. The size of infected cells increased significantly (P < 0.001) after 24 h post infection. The results indicated that Sf9 cell line was applicable to the large scale for hDPPIV expression by using optimized parameters (infection time and MOI) because of its high productivity (4.03 mU m L⁻¹ h⁻¹).     

Rac regulates integrin-mediated endothelial cell adhesion and migration on laminin-8

Blood vessel formation requires endothelial cell interactions with the extracellular matrix through cell surface receptors, and signaling events that control endothelial cell adhesion, migration, and lumen formation. Laminin-8 (alpha4beta1gamma1) is present in all basement membranes of blood vessels in fetal and adult tissues, but despite its importance in vessel formation, its role in endothelial cell adhesion and migration remains undefined. We examined adhesion and migration of HMEC-1 human microvascular endothelial cells on laminin-8 with an emphasis on the integrin-mediated signaling events, as compared with those on laminin-10/11 and fibronectin. We found that laminin-8 was less potent in HMEC-1 cell adhesion than laminin-1, laminin-10/11, and fibronectin, and mediated cell adhesion through alpha6beta1 integrin. Despite its weak cell-adhesive activity, laminin-8 was as potent as laminin-10/11 in promoting cell migration. Cells adhering to laminin-8 displayed streaks of thin actin filaments and formed lamellipodia at the leading edge of the cells, as observed with cells adhering to laminin-10/11, while cells on fibronectin showed thick actin stress fibers and large focal adhesions. Pull-down assays of GTP-loaded Rho, Rac, and Cdc42 demonstrated that Rac, but not Rho or Cdc42, was preferentially activated on laminin-8 and laminin-10/11, when compared with fibronectin. Furthermore, a dominant-negative mutant of Rac suppressed cell spreading, lamellipodial formation, and migration on laminin-8, but not on fibronectin. These results, taken together, indicate that Rac is activated during endothelial cell adhesion to laminin-8, and is pivotal for alpha6beta1 integrin-mediated cell spreading and migration on laminin-8.     

Embryonic stem cells conditioned medium enhances Wharton’s jelly-derived mesenchymal stem cells expansion under hypoxic condition

Mesenchymal stem cells (MSCs) are accepted as a promising tool for therapeutic purposes. However, low proliferation and early senescence are still main obstacles of MSCs expansion for using as cell-based therapy. Thus, clinical scale of cell expansion is needed to obtain a large number of cells serving for further applications. In this study, we investigated the value of embryonic stem cells conditioned medium (ESCM) for in vitro expansion of Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) as compared to typical culture medium for MSCs, Dulbecco’s modified Eagle’s medium with 1.0 g/l glucose (DMEM-LG) supplemented with 10 % FBS, under hypoxic condition. The expanded cells from ESCM (ESCM-MSCs) and DMEM-LG (DMEM-MSCs) were characterized for both phenotype and biological activities including proliferation rate, population doubling time, cell cycle distribution and MSCs characteristics. ESCM and DMEM-LG could enhance WJ-MSCs proliferation as 204.66 ± 10.39 and 113.77 ± 7.89 fold increase at day 12, respectively. ESCM-MSCs could express pluripotency genes including Oct-4, Oct-3/4, Nanog, Klf-4, C-Myc and Sox-2 both in early and late passages whereas the downregulations of Oct-4 and Nanog were detected in late passage cells of DMEM-MSCs. The 2 cell populations also showed common MSCs characteristics including normal cell cycle, fibroblastic morphology, cell surface markers expressions (CD29⁺, CD44⁺, CD90⁺, CD34⁻, CD45⁻) and differentiation capacities into adipogenic, chondrogenic and osteogenic lineages. Moreover, our results revealed that ESCM exhibited as a rich source of several factors which are required for supportive WJ-MSCs proliferation. In conclusion, ESCM under hypoxic condition could accelerate WJ-MSCs expansion while maintaining their pluripotency properties. Our knowledge provide short term and cost-saving in WJ-MSCs expansion which has benefit to overcome insufficient cell numbers for clinical applications by reusing the discarded cell culture supernates from human ES culture system. Moreover, these findings can also apply for stem cell banking, regenerative medicine and pharmacological applications.     

Acquisition of Sequences Homologous to Host DNA by Closed Circular Simian Virus 40 DNA II. Further Studies on the Serial Passage of Virus Clones

Three plaque isolates of SV40 strain 777 and 1 plaque isolate of strain 776 were grown to high-titer stocks and serially passaged, undiluted, in monkey BS-C-1 cells. In each case, the serial passaging procedure resulted in the accumulation of closed-circular SV40 DNA molecules containing covalently linked sequences homologous to reiterated host cell DNA (called substituted virus DNA). The relative yields, at a given passage level, of SV40 DNA with measurable homology to host DNA varied in different sets of serial passages, including passages of the same virus clone. More reproducible yields of substituted viral DNA progeny were obtained when the serial passaging procedure was initiated from earlier passages rather than from the original plaque-purified stock. Fractionation of closed-circular SV40 DNA molecules on alkaline sucrose gadients indicated that the majority of substituted virus DNA molecules are not plaque producers and are slightly smaller in size than plaque-forming DNA molecules which display no detectable homology to host DNA. Evidence that substituted SV40 DNA molecules replicate during serial undiluted passage was obtained from experiments which demonstrated (i) the presence of host sequences in replicative forms of the viral DNA and (ii) the incorporation of (3)H-thymidine into host sequences isolated from the mature substituted virus DNA molecule.     

Binding of human angiogenin inhibits actin polymerization

Angiogenin is a potent inducer of angiogenesis, a process of blood vessel formation. It interacts with endothelial and other cells and elicits a wide range of cellular responses including migration, proliferation, and tube formation. One important target of angiogenin is endothelial cell-surface actin and their interaction might be one of essential steps in angiogenin-induced neovascularization. Based on earlier indications that angiogenin promotes actin polymerization, we studied the binding interactions between angiogenin and actin in a wide range of conditions. We showed that at subphysiological KCl concentrations, angiogenin does not promote, but instead inhibits polymerization by sequestering G-actin. At low KCl concentrations angiogenin induces formation of unstructured aggregates, which, as shown by NMR, may be caused by angiogenin’s propensity to form oligomers. Binding of angiogenin to preformed F-actin does not cause depolymerization of actin filaments though it causes their stiffening. Binding of tropomyosin and angiogenin to F-actin is not competitive at concentrations sufficient for saturation of actin filaments. These observations suggest that angiogenin may cause changes in the cell cytoskeleton by inhibiting polymerization of G-actin and changing the physical properties of F-actin.     

A clonal analysis of anthocyanin accumulation by cell cultures of wild carrot

The accumulation of anthocyanin by clones and subclones from a cell suspension culture of wild carrot (Daucus carota L.) has been measured under standard conditions. Clones which accumulate low amounts of anthocyanin were shown, by recloning after maintenance by serial passage, to have become heterogenous and to contain cells with increased accumulation of anthocyanin. There appears to be a maximum amount of anthocyanin that clones can accumulate. Clones which accumulate the maximum amount of anthocyanin were shown by recloning after maintenance by serial passaging, to have become heterogenous and to contain many cells which accumulate less than the maximum possible amount of anthocyanin. When clones which accumulate the maximum amount of anthocyanin are maintained by serial passage, the decline in anthocyanin accumulation is different in different media. The results indicate that the changes in the ability of cells to accumulate anthocyanin involve no qualitative change in the genetic information of the cells, i.e., the changes are not the consequence of mutations.     

Schwann cells promote endothelial cell migration

Directed cell migration is a crucial orchestrated process in embryonic development, wound healing, and immune response. The underlying substrate can provide physical and/or chemical cues that promote directed cell migration. Here, using electrospinning we developed substrates of aligned poly(lactic-co-glycolic acid) nanofibres to study the influence of glial cells on endothelial cells (ECs) in a 3-dimensional (3D) co-culture model. ECs build blood vessels and regulate their plasticity in coordination with neurons. Likewise, neurons construct nerves and regulate their circuits in coordination with ECs. In our model, the neuro-vascular cross-talk was assessed using a direct co-culture model of human umbilical vein endothelial cells (HUVECs) and rat Schwann cells (rSCs). The effect of rSCs on ECs behavior was demonstrated by earlier and higher velocity values and genetic expression profiles different of those of HUVECs when seeded alone. We observed 2 different gene expression trends in the co-culture models: (i) a later gene expression of angiogenic factors, such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and (ii) an higher gene expression of genes involved in actin filaments rearrangement, such as focal adhesion kinase (FAK), Mitogen-activated protein kinase-activated protein kinase 13 (MAPKAPK13), Vinculin (VCL), and Profilin (PROF). These results suggested that the higher ECs migration is mainly due to proteins involved in the actin filaments rearrangement and in the directed cell migration rather than the effect of angiogenic factors. This co-culture model provides an approach to enlighten the neurovascular interactions, with particular focus on endothelial cell migration.     

Myosin Motors Drive Long Range Alignment of Actin Filaments

The bulk alignment of actin filament sliding movement, powered by randomly oriented myosin molecules, has been observed and studied using an in vitro motility assay. The well established, actin filament gliding assay is a minimal experimental system for studying actomyosin motility. Here, we show that when the assay is performed at densities of actin filaments approaching those found in living cells, filament gliding takes up a preferred orientation. The oriented patterns of movement that we have observed extend over a length scale of 10–100 μm, similar to the size of a mammalian cell. We studied the process of filament alignment and found that it depends critically upon filament length and density. We developed a simple quantitative measure of filament sliding orientation and this enabled us to follow the time course of alignment and the formation and disappearance of oriented domains. Domains of oriented filaments formed spontaneously and were separated by distinct boundaries. The pattern of the domain structures changed on the time scale of several seconds and the collision of neighboring domains led to emergence of new patterns. Our results indicate that actin filament crowding may play an important role in structuring the leading edge of migrating cells. Filament alignment due to near-neighbor mechanical interactions can propagate over a length scale of several microns; much greater than the size of individual filaments and analogous to a log drive. Self-alignment of actin filaments may make an important contribution to cell polarity and provide a mechanism by which cell migration direction responds to chemical cues.     

Filamin A mediates interactions between cytoskeletal proteins that control cell adhesion

Cell adhesion, spreading and migration on extracellular matrices are regulated by complex processes that involve the cytoskeleton and a large array of adhesion receptors, including the β1 integrin. Filamin A is a large, multi-domain, homodimeric actin binding protein that contributes to the mechanical stability of cells and interacts with several proteins that regulate cell adhesion including β1 integrin and several protein kinases. Here we review current data on the structure, mechanical properties and intracellular signaling functions of filamin that regulate cell adhesion. We also consider new data showing that interactions of filamin A with intermediate filaments and protein kinase C enable tight regulation of β1 integrin function and consequently early events in cell adhesion and migration on extracellular matrix proteins.     

Specificity of endothelial cell reorientation in response to cyclic mechanical stretching

Evidence suggests that cellular responses to mechanical stimuli depend specifically on the type of stimuli imposed. For example, when subjected to fluid shear stress, endothelial cells align along the flow direction. In contrast, in response to cyclic stretching, cells align away from the stretching direction. However, a few aspects of this cell alignment response remain to be clarified: (1) Is the cell alignment due to actual cell reorientation or selective cell detachment? (2) Does the resulting cell alignment represent a response of the cells to elongation or shortening, or both? (3) Does the cell alignment depend on the stretching magnitude or rate, or both? Finally, the role of the actin cytoskeleton and microtubules in the cell alignment response remains unclear. To address these questions, we grew human aortic endothelial cells on deformable silicone membranes and subjected them to three types of cyclic stretching: simple elongation, pure uniaxial stretching and equi-biaxial stretching. Examination of the same cells before and after stretching revealed that they reoriented. Cells subjected to either simple elongation or pure uniaxial stretching reoriented specifically toward the direction of minimal substrate deformation, even though the directions for the two types of stretching differed by only about 20°. At comparable stretching durations, the extent of cell reorientation was more closely related to the stretching magnitude than the stretching rate. The actin cytoskeleton of the endothelial cell subjected to either type of stretching was reorganized into parallel arrays of actin filaments (i.e., stress fibers) aligned in the direction of the minimal substrate deformation. Furthermore, in response to equi-biaxial stretching, the actin cytoskeleton was remodeled into a “tent-like” structure oriented out of the membrane plane—again towards the direction of the minimal substrate deformation. Finally, abolishing microtubules prevented neither the formation of stress fibers nor cell reorientation. Thus, endothelial cells respond very specifically to the type of deformation imposed upon them.