Agent-based approach for elucidating the release from collective arrest of cell motion in corneal epithelial cell sheet.

Changes in cell fluidity have been observed in various cellular tissues and are strongly linked to biological phenomena such as self-organization. Recent studies suggested variety of mechanisms and factors, which are still being investigated. This study aimed to investigate changes in cell fluidity in multi-layered cell sheets, by exploring the collective arrest of cell motion and its release in cultures of corneal epithelial cells. We constructed mathematical models to simulate the behaviors of individual cells, including cell differentiation and time-dependent changes in cell-cell connections, which are defined by stochastic or kinetic rules. Changes in cell fluidity and cell sheet structures were expressed by simulating autonomous cell behaviors and interactions in tissues using an agent-based model. A single-cell level spatiotemporal analysis of cell state transition between migratable and non-migratable states revealed that the release from collective arrest of cell motion was initially triggered by a decreased ability to form cell-cell connections in the suprabasal layers, and was propagated by chain migration. Notably, the disruption of cell-cell connections and stratification occurred in the region of migratable state cells. Hence, a modeling approach that considers time-dependent changes in cell properties and behavior, and spatiotemporal analysis at the single-cell level can effectively delineate emergent phenomena arising from the complex interplay of cells.

An in vitro culture platform to study the extracellular matrix remodeling potential of human mesenchymal stem cells.

The ability of mesenchymal stem cells (MSCs) to synthesize and degrade extracellular matrix (ECM) is important for MSC-based therapies. However, the therapeutic effects associated with ECM remodeling in cultured MSCs have been limited by the lack of a method to assess the ability of cultured cells to degrade ECM in vitro. Here, we describe a simple in vitro culture platform for studying the ECM remodeling potential of cultured MSCs using a high-density collagen (CL) surface. Cells on the CL surface have remarkable ability to degrade collagen fibrils by secreting matrix metalloproteinase (MMP); to study this, the marker collagen hybridizing peptide (CHP) was used. Confirming the ECM remodeling potential of MSCs with different population doublings (PDs), young and healthy γ-H2AX-negative cells, a marker of DNA damage and senescence, showed more extensive collagen degradation on the CL surface, whereas damaged cells of γ-H2AX-positive cells showed no collagen degradation. The frequency of γ-H2AX-/CHP + cells at PD = 0 was 49%, which was 4.9-fold higher than that at PD=13.07, whereas the frequency of γ-H2AX+/CHP- at PD=13.07 was 50%, which was 6.4-folds higher than that at PD=0. Further experimentation examining the in vitro priming effect of MSCs with the pro-inflammatory cytokine interferon-γ treatment showed increased frequency of cells with ECM remodeling potential with higher MMP secretion. Thus, this culture surface can be used for studying the ECM remodeling capacity of ex vivo-expanded MSCs in vitro and may serve as a platform for prediction in vivo ECM remodeling effect. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) remodeling potential of cultured mesenchymal stem cells (MSCs) is important for assessing the effectiveness of MSC-based therapy. However, methods to assess the ability of cultured cells to degrade ECM in vitro are still lacking. Here, we developed a simple in vitro culture platform to study the ECM remodeling potential of cultured MSCs using high-density collagen surfaces. This platform was used to evaluate the ECM remodeling potential of long-term ex vivo-expanded MSCs in vitro.

Study protocol for periodontal tissue regeneration with a mixture of autologous adipose-derived stem cells and platelet rich plasma: A multicenter, randomized, open-label clinical trial.

Introduction: Adipose-derived stem cells (ASCs) secrete various growth factors to promote wound healing and to regenerate various tissues, such as bone, cartilage, and fat tissue. Subcutaneous adipose tissue is a considerable cell source in clinical practice and can be collected relatively easily and safely under local anesthesia. Moreover, platelet-rich plasma (PRP), a plasma component containing many platelets purified by centrifuging the collected blood, also promotes wound healing. PRP can be easily gelled and is therefore attracting attention as a scaffolding material for transplanted cells. The usefulness of a mixture of ASCs and PRP for periodontal tissue regeneration has been in vitro demonstrated in our previous study. The aim of this study is to present the protocol of translation of tissue regeneration with ASCs and PRP into practical use, evaluating its efficacy. Methods: This study is a multicenter, randomized, open-label comparative clinical trial. Fifteen patients will be randomly assigned to the treatment with mixture of ASCs and PRP or enamel matrix derivate administration into periodontal tissue defects. Increase in height of new alveolar bone in the transplanted area will be evaluated. The evaluation will be performed using dental radiographs after 36 weeks of transplantation. Occurrence of adverse events will be evaluated as secondary outcome. Results: This clinical study was initiated after meeting the regulations to be complied with, including ethical review and regulatory notifications. Conclusions: If effective, this cell therapy using autologous mesenchymal stem cells can represent a useful medical technology for regeneration of periodontal defects.

Novel approach to enhance aggregate migration-driven epigenetic memory which induces cardiomyogenic differentiation on a dendrimer-immobilized surface.

The dynamic migratory behavior of human mesenchymal stem cells (hMSCs) has a significant impact on the epigenetic profiles that determine fate choice and lineage commitment during differentiation. Here we report a novel approach to enhance repeated migration-driven epigenetic memory which induces cardiomyogenic differentiation on a dendrimer surface with fifth generation (G5). Cells exhibited the formation of cell aggregates on the G5 surface through active migration with morphological changes, and these aggregates showed strong expression of the cardiac-specific marker cardiac troponin T (cTnT) at 10 days. When cell aggregates were passaged onto a fresh G5 surface over three passages of 40 days, the expression levels of the multiple cardiac-specific markers including GATA4, NKX2.5, MYH7, and TNNT2 were higher compared to those passaged as single cells. To investigate whether cardiomyogenic differentiation of hMSCs was enhanced by repeated aggregate migration-driven epigenetic memory, cells on the G5 surface were reseeded onto a fresh G5 surface during three passages using aggregate-based and single cell-based passage methods. Analyses of global changes in H3 histone modifications exhibited pattern of increased H3K9ac and H3K27me3, and decreased H3K9me3 in aggregate-based passage cultures during three passages. However, the pattern of their histone modification on the PS surface was repeated after the initialization and reformation during three passages in single cell-based passage cultures. Thus, repetitive aggregate migratory behavior during aggregate-based passage led to a greater degree of histone modification, as well as gene expression changes suggestive of cardiomyogenic differentiation.

Exogenous FGF-2 prolongs endothelial connection in multilayered human skeletal muscle cell sheet.

Angiogenesis is a pressing issue in tissue engineering associated with restoration of blood supply to ischemic tissues and promotion of rapid vascularization of tissue-engineered grafts. Fibroblast growth factor-2 (FGF-2) plays a vital role in processes such as angiogenesis and is an attractive candidate for tissue engineering. While skeletal muscle tissue engineering is established, the role of FGF-2 in endothelial function to promote angiogenesis after transplantation is unclear. Here, a culture system comprising a five-layered sheet of human skeletal muscle cells co-incubated on green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) mimicking in vivo angiogenesis was used to investigate the role of FGF-2 in vascularization of engineered tissues. The basal level of FGF-2 in cultured media of skeletal muscle cell sheets was undetectable. Therefore, cell sheets co-incubated with GFP-HUVECs were exogenously treated with 10 ng/mL FGF-2, and endothelial network formation was evaluated. After prolonged culture, the endothelial network length and connectivity increased following treatment with FGF-2 as compared with control treatment. The numbers of medium and long endothelial networks significantly increased inside the sheet longer than 0.2 and 0.4 cm, respectively, after FGF-2 treatment. Time-lapse microscopy monitoring dynamic endothelial behavior revealed that FGF-2-mediated maintenance of endothelial connection and retardation of endothelial network disconnection after 72 h. The present study suggests the precise role of FGF-2 in maintaining endothelial connection and the extent of the endothelial network in skeletal muscle cell sheets. This understanding can be applied to design in vitro pre-vascularized tissue and graft integration prospects.

Cell jamming, stratification and p63 expression in cultivated human corneal epithelial cell sheets.

Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Here we report an investigation into the quality of cultivated human corneal epithelial cell sheets using time-lapse imaging of the cell culture process every 20 minutes over 14 days to ascertain the level of cell jamming, a phenomenon in which cells become smaller, more rounded and less actively expansive. In parallel, we also assessed the expression of p63, an important corneal epithelial stem cell marker. The occurrence of cell jamming was variable and transient, but was invariably associated with a thickening and stratification of the cell sheet. p63 was present in all expanding cell sheets in the first 9 days of culture, but it's presence did not always correlate with stratification of the cell sheet. Nor did p63 expression necessarily persist in stratified cell sheets. An assessment of cell jamming, therefore, can shed significant light on the quality and regenerative potential of cultivated human corneal epithelial cell sheets.

Muscle lineage switching by migratory behaviour-driven epigenetic modifications of human mesenchymal stem cells on a dendrimer-immobilized surface.

Understanding of the fundamental mechanisms of epigenetic modification in the migration of human mesenchymal stem cells (hMSCs) provides surface design strategies for controlling self-renewal and lineage commitment. We investigated the mechanism underlying muscle lineage switching of hMSCs by cellular and nuclear deformation during cell migration on polyamidoamine dendrimer surfaces. With an increase in the dendrimer generation number, cells exhibited increased nuclear deformation and decreased lamin A/C and lamin B1 expression. Analysis of two repressive modifications (H3K9me3 and H3K27me3) and one activating modification (H3K9ac) revealed that H3K9me3 was suppressed, and H3K9ac and H3K27me3 were upregulated in the cultures on a higher-generation dendrimer surface. This induced significant hMSC lineage switching to smooth, skeletal, and cardiac muscle lineages. Thus, reorganizations of the nuclear lamina and cytoskeleton related to migration changes on dendrimer surfaces are responsible for the integrated regulation of histone modifications in hMSCs, thereby shifting the cells from the multipotent state to muscle lineages. These findings improve our understanding of the role of epigenetic modification in cell migration and provide new insights into how designed surfaces can be applied as cell-instructive materials in the field of biomaterial-guided differentiation of hMSCs to different cell types. STATEMENT OF SIGNIFICANCE: Stem cell engineering strategies currently applied the mechanical cues that emerge from cellular microenvironment to regulate stem cell behaviour. This study significantly improved our understanding of the mechanotransduction mechanism involving cell-ECM and cytoskeleton-nucleoskeleton interactions, and of nuclear genome regulation based on cellular responses to biomaterial modifications. The new insights into how the physical environment on a culture surface influences cell behaviour improve our understanding of mechanical control mechanisms of the interactions of cells with the extracellular environment. Our findings are also expected to contribute to and play an essential role in the development of future material strategies for creating artificial cell-instructive niches.

Maintenance of Neurogenic Differentiation Potential in Passaged Bone Marrow-Derived Human Mesenchymal Stem Cells Under Simulated Microgravity Conditions.

Human mesenchymal stem cells (hMSCs) are considered to be able to adapt to environmental changes induced by gravity during cell expansion. In this study, we investigated neurogenic differentiation potential of passaged hMSCs under conventional gravity and simulated microgravity conditions. Immunostaining, quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), and western blot analysis of neurogenic differentiation markers, neurofilament heavy (NF-H), and microtubule-associated protein 2 (MAP2) revealed that differentiated cells from the cells cultured under simulated microgravity conditions expressed higher neurogenic levels than those from conventional gravity conditions. The levels of NF-H and MAP2 in the cells from simulated microgravity conditions were consistent during passage culture, whereas cells from conventional gravity conditions exhibited a reduction of the neurogenic levels against an increase of their passage number. In growth culture, cells under simulated microgravity conditions showed less apical stress fibers over their nucleus with fewer cells having a polarization of lamin A/C than those under conventional gravity conditions. The ratio of lamin A/C to lamin B expression in the cells under simulated microgravity conditions was constant; however, cells cultured under conventional gravity conditions showed an increase in the lamin ratio during passages. Furthermore, analysis of activating H3K4me3 and repressive H3K27me3 modifications at promoters of neuronal lineage genes indicated that cells passaged under simulated microgravity conditions sustained the methylation during serial cultivation. Nevertheless, the enrichment of H3K27me3 significantly increased in the passaged cells cultured under conventional gravity conditions. These results demonstrated that simulated microgravity-coordinated cytoskeleton-lamin reorganization leads to suppression of histone modification associated with neurogenic differentiation capacity of passaged hMSCs.

Alterations in Nuclear Lamina and the Cytoskeleton of Bone Marrow-Derived Human Mesenchymal Stem Cells Cultured Under Simulated Microgravity Conditions.

Cells sense and respond to environmental changes induced by gravity. Although reactions to conventional culture have been intensively studied, little is known about the cellular reaction to simulated microgravity conditions. Thus, in this study, we investigated the effects of simulated microgravity on human mesenchymal stem cells using a three-dimensional clinostat (Gravite®), a recently developed device used to generate simulated microgravity condition in vitro. Our time-lapse analysis shows that cells cultured under conventional culture conditions have a stretched morphology and undergo unidirectional migration, whereas cells cultured under simulated microgravity conditions undergo multidirectional migration with directional changes of cell movement. Furthermore, cells cultured under conventional culture conditions maintained their spindle shape through fibronectin fibril formation in their bodies and focal adhesion stabilization with enriched stress fibers. However, cells cultured under simulated microgravity conditions were partially contracted and the fibril structures were degraded in the cell bodies. Additionally, paxillin phosphorylation in the cells cultured under simulated microgravity conditions was more intense at the cell periphery in regions near the leading and trailing edges, but was less expressed in the cell bodies compared with that observed in cells cultured under conventional culture conditions. Furthermore, lamin A/C, a major component of the nuclear lamina, was mainly located on the apical side in cells cultured under conventional culture conditions, indicating basal-to-apical polarization. However, cells cultured under simulated microgravity conditions showed lamin A/C localization on both the apical and basal sides. Taken together, these results demonstrate that simulated microgravity-driven fibronectin assembly affects nuclear lamina organization through the spatial reorganization of the cytoskeleton.

Large-scale culture of a megakaryocytic progenitor cell line with a single-use bioreactor system.

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single-use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large-scale production and feasibility of meeting clinical-grade standards. The current work evaluated the capacity of a single-use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (kL a) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h-1 , respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7-fold with a total cell number of 1.2 ± 0.2 × 109 cells L-1 . In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single-use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362-369, 2018.

Chondrogenesis and hypertrophy in response to aggregate behaviors of human mesenchymal stem cells on a dendrimer-immobilized surface.

OBJECTIVES: To investigate the behaviors of aggregates of human mesenchymal stem cells (hMSCs) on chondrogenesis and chondrocyte hypertrophy using spatiotemporal expression patterns of chondrogenic (type II collagen) and hypertrophic (type X collagen) markers during chondrogenesis. RESULTS: hMSCs were cultured on either a polystyrene surface or polyamidoamine dendrimer surface with a fifth generation (G5) dendron structure in chondrogenic medium and growth medium. At day 7, cell aggregates without stress fibers formed on the G5 surface and triggered differentiation of hMSCs toward the chondrogenic fate, as indicated by type II collagen being observed while type X collagen was undetectable. In contrast, immunostaining of hMSCs cultured on polystyrene, which exhibited abundant stress fibers and did not form aggregates, revealed no evidence of either type II and or type X collagen. At day 21, the morphological changes of the cell aggregates formed on the G5 surface were suppressed as a result of stress fiber formation. Type II collagen was observed throughout the aggregates whereas type X collagen was detected only at the basal side of the aggregates. Change of cell aggregate behaviors derived from G5 surface alone regulated chondrogenesis and hypotrophy, and this was enhanced by chondrogenic medium. CONCLUSIONS: Incubation of hMSCs affects the expression of type II and X collagens via effects on cell aggregate behavior and stress fiber formation.

Phenotypic heterogeneity of human retinal pigment epithelial cells in passaged cell populations.

Human retinal pigment epithelial (RPE) cells at different population doublings (PDs) were cultured for 28 days to examine their phenotypic heterogeneity in a confluent state. In an early population (PD = 2.8), cells showed a cobblestone-like appearance (type I), which gradually became small and tight, and eventually exhibited dark pigmentation. Some cells showed a dome-like structure (type II), which detached from the culture surface during culture. With increasing PD, the cells showed active migration that caused a shift in phenotype from a single layer of large, flattened cells (type III) to a multiple cell layers (stratified) with flattened, irregularly shaped cells (type IV). Immunostaining of specific RPE markers, ZO-1 and Na+/K+-ATPase revealed that cells have markedly decreased expressions in a late population (PD = 10.1). RPE phenotypes were classified into four types by measuring the nuclear size and local density. The frequencies of type I cells decreased with increasing PD value, while the frequencies of type III and IV cells increased along with the decrease in type I. The frequencies of type IV cells at PD = 10.1 had increased by 10.3-fold compared with PD = 2.8. From these results, the nuclear size and local density were proposed as indicators for understanding phenotypic heterogeneity of RPE cells in the passaged cell population during cell expansion. It is concluded that the population doubling level is an important factor to affect the transition of RPE phenotype and thereby to modulate the quality of cultured cells.

Analysis of gene expression profiles of Lactobacillus paracasei induced by direct contact with Saccharomyces cerevisiae through recognition of yeast mannan.

Co-culture of lactic acid bacteria (LAB) and yeast induces specific responses that are not observed in pure culture. Gene expression profiles of Lactobacillus paracasei ATCC 334 co-cultured with Saccharomyces cerevisiae IFO 0216 were analyzed by DNA microarray, and the responses induced by direct contact with the yeast cells were investigated. Coating the LAB cells with recombinant DnaK, which acts as an adhesive protein between LAB and yeast cells, enhanced the ratio of adhesion of the LAB cells to the yeast cells. The signals induced by direct contact were clarified by removal of the LAB cells unbound to the yeast cells. The genes induced by direct contact with heat-inactivated yeast cells were very similar to both those induced by the intact yeast cells and those induced by a soluble mannan. The top 20 genes upregulated by direct contact with the heat-inactivated yeast cells mainly encoded proteins related to exopolysaccharide synthesis, modification of surface proteins, and transport systems. In the case of the most upregulated gene, LSEI_0669, encoding a protein that has a region homologous to polyprenyl glycosylphosphotransferase, the expression level was upregulated 7.6-, 11.0-, and 8.8-fold by the heat-inactivated yeast cells, the intact yeast cells, and the soluble mannan, respectively, whereas it was only upregulated 1.8-fold when the non-adherent LAB cells were not removed before RNA extraction. Our results indicated that the LAB responded to direct contact with the yeast cells through recognition of mannan on the surface of the yeast.

Degradation of endothelial network in disordered tumor-containing cell sheet.

Tumor angiogenesis is an important event in tumor malignancy; and the vasculature formed in tumor region is typically dysfunctional. Multiple factors are associated with tumor vessel abnormalities, but the precise mechanism has not been fully understood. In the present study, a tumor-containing cell sheet was prepared by mixing a small population of human embryonal rhabdomyosarcoma (RMS) cells (RDs) with human skeletal muscle myoblasts (HSMMs) to mimic muscle tissue invaded by RMS cells. Sheet fluidity and the extracellular matrix (ECM) meshwork of the tumor-containing cell sheet were found to be elevated and disordered, demonstrating the disruptive effect of tumor cells on sheet structure. When green fluorescent protein expressing human umbilical vein endothelial cells (GFP-HUVECs) were co-cultured with the tumor-containing cell sheet, an endothelial network was formed, but degraded faster as a result of activated migration of endothelial cells in the tumor-containing cell sheet. This study suggested that disorganized tissue structure facilitate tumor angiogenesis by activation of endothelial cell migration.

Disruption of myoblast alignment by highly motile rhabdomyosarcoma cell in tissue structure.

Rhabdomyosarcoma (RMS) is a highly malignant tumor type of skeletal muscle origin, hallmarked by local invasion. Interaction between invasive tumor cells and normal cells plays a major role in tumor invasion and metastasis. Culturing tumor cells in a three-dimensional (3D) model can translate tumor malignancy relevant cell-cell interaction. To mimic tumor heterogeneity in vitro, a co-culture system consisting of a malignant embryonal rhabdomyosarcoma (ERMS) cell line RD and a normal human skeletal muscle myoblast (HSMM) cell line was established by cell sheet technology. Various ratios of RDs to HSMMs were employed to understand the quantitative effect on intercellular interactions. Disruption of sheet structure was observed in heterogeneous cell sheets having a low ratio of RDs to HSMMs, whereas homogeneous HSMM or RD sheets maintained intact structure. Deeper exploration of dynamic tumor cell behavior inside HSMM sheets revealed that HSMM cell alignment was disrupted by highly motile RDs. This study demonstrated that RMS cells are capable of compromising their surrounding environment through induced decay of HSMMs alignment in a cell-based 3D system. This suggests that muscle disruption might be a major consequence of RMS cell invasion into muscles, which could be a promising target to preventing tumor invasion.

Migration-driven aggregate behaviors of human mesenchymal stem cells on a dendrimer-immobilized surface direct differentiation toward a cardiomyogenic fate commitment.

Dynamic behaviors of cell aggregates on a dendrimer surface were investigated to drive the directed differentiation of human mesenchymal stem cells (hMSCs) toward a cardiomyogenic lineage. Cell aggregates on the polyamidoamine dendrimer surface with fifth-generation (G5) of dendron structure showed dynamic changes in morphology associated with repetitive stretching and contracting during migration. Spatial-temporal observations revealed cellular movement in single aggregates by their morphological change through stretching and contracting on the G5 surface, suggesting that the dynamic behavior of aggregate causes mixing of cells. However, aggregates without cell-substrate adhesions on the low-binding culture surface sustained their spherical morphology without cellular movement within a single aggregate. Furthermore, ß-catenin was observed at nuclei in aggregates on the G5 surface, and expression of the cardiomyocyte marker cardiac Troponin T (cTnT) was detected. However, ß-catenin localized to the nuclei only in the outer region of the aggregate on the low-binding culture surface, and cTnT expression was restricted at the exterior surface of the aggregates. These observations indicate that cell mixing within aggregates on the G5 surface induced the directed differentiation of hMSCs toward a cardiomyogenic lineage by nuclear translocation of ß-catenin through dissociation of cell-cell adhesions. These results suggest that migration-driven aggregate behaviors on the dendrimer surface caused repeated morphological changes of aggregate through stretching and contracting, leading to the directed differentiation of hMSCs toward a cardiomyogenic fate commitment.

Facilitation of uniform maturation of human retinal pigment epithelial cells through collective movement in culture.

Understanding of the fundamental mechanisms that govern tight junction formation of retinal pigment epithelial (RPE) cells provides surface design strategies for promoting their maturation in culture. RPE cells were cultured to investigate their migratory behavior and the expression of tight junction protein ZO-1 in the central and peripheral regions of a culture vessel. Regardless of locational differences in the culture vessel, the cells at day 1 were elongated in shape, did not form tight junctions, and migrated actively. As the culture progressed, the cells in the central region slowly moved with morphological change of a cobblestone-like shape via interaction between contact cells and exhibiting the shift from random migration to collective movement toward the center, accompanied by tight junction formation. On the other hand, the cells in the peripheral region maintained the random migration at day 5, meaning spatial heterogeneity in maturation in the vessel. At day 5, RPE cells were incubated in medium with Rac1 inhibitor and the exposure to the Rac1 inhibitor triggered the rapid conversion of migratory behavior from random migration to collective movement toward the center of the vessel, resulting in uniform maturation. These findings indicate that the change in migratory patterns is an important cues and the collective movement toward the center causes the facilitation of uniform maturation in the vessel.

Changes in human mesenchymal stem cell behaviors on dendrimer-immobilized surfaces due to mediation of fibronectin adsorption and assembly.

Dynamic changes of morphologies in human mesenchymal stem cells (hMSCs) were investigated on dendrimer surfaces with different capacities for fibronectin adsorption by changing the polymeric generation numbers of first (G1), third (G3), and fifth (G5) generations. The amount of adsorbed fibronectin on dendrimer surfaces increased with the generation number. Time-lapse observations revealed that cells on the G1 surface maintained their shape with formation of fibronectin fibrils in the bodies, introducing to the stabilization of focal adhesion with enriched stress fibers. Cells on the G3 surface showed partial contraction with degradation of fibril structures in the trailing edge. Cells on the G5 surface changed the shape by active extension and strong contracting without stabilization of focal adhesion through the formation of fibronectin aggregates and immature stress fibers. In addition, the paxillin which is a focal adhesion protein at lamellipodia was phosphorylated, leading to active lamellipodium protrusions. These results indicate that the amount and structure of fibronectin affects dynamic hMSC behaviors through the formation of cytoskeletons and focal adhesions.

Locational heterogeneity of maturation by changes in migratory behaviors of human retinal pigment epithelial cells in culture.

To better characterize human retinal pigment epithelial (RPE) cells, their maturation was studied by time-lapse observation and immunostaining of the tight junction protein ZO-1. During subconfluency with active migration, the cells had an elongated shape. During cell division to reach confluency, RPE cells became small and tight, exhibiting cobblestone-like morphology. In addition, RPE maturation at the peripheral region of the culture vessel was delayed when compared with the central region, demonstrating local heterogeneity during maturation. To correlate cellular migration and maturation, we compared frequencies of migration rate and number of ZO-1-positive cells at the central and peripheral regions. Cells having migration rates less than 5.0 µm/h in the central region were 1.4-fold higher than in the peripheral region at day 5. Regardless of locational differences in the culture vessel, the frequency of cells having migration rates less than 5.0 µm/h showed 90% agreement with the frequency of ZO-1-positive cells. To inhibit cell migration, RPE cells were exposed to medium containing 50 µg/ml Rac1 inhibitor at day 5. Frequencies of ZO-1-positive cells and cells having migration rates less than 5.0 µm/h at the peripheral region were similar to those at the central region. The results show that migration is an important factor affecting maturation, and demonstrate that location heterogeneity during maturation is caused by different migratory behaviors in the culture vessel.

Switching between self-renewal and lineage commitment of human induced pluripotent stem cells via cell-substrate and cell-cell interactions on a dendrimer-immobilized surface.

Understanding mechanisms that govern cell fate determination of human induced pluripotent stem cells (hiPSCs) could assist in maintenance of the undifferentiated state during cell expansion. We used polyamidoamine dendrimer surfaces with first-generation (G1), third-generation (G3) and fifth-generation (G5) of dendron structure in cultures of hiPSCs with SNL feeder cells. Cells on the G1 surface formed tightly packed colony with close cell-cell contacts during division and migration; those on the G3 surface exhibited loose or dispersed colony pattern by enhanced migration. On the G5 surface, formation of aggregated colony with ring-like structures occurred spontaneously. We found that the substrate-adsorbed fibronectin and feeder cell-secreted fibronectin appeared elevated levels with the varied generation numbers of dendrimer surfaces. This subsequently resulted in cell migration and in activation of paxillin of hiPSCs. Location-dependent expression of Rac1 induced rearrangement of E-cadherin-mediated cell-cell interactions on dendrimer surfaces, and was associated with alterations in the cell and colony morphology, and migratory behavior. Furthermore, caspase-3 occurred in apoptotic cells on dendrimer surfaces, concomitant with the loss of E-cadherin-mediated cell-cell interactions. Cells on the G1 surface were maintained in an undifferentiated state, while those on the G5 surface exhibited the early commitment to differentiation toward endodermal fates. We conclude that morphological changes associated with altered migration on the dendrimer surfaces were responsible for the coordinated regulation of balance between cell-cell and cell-substrate interactions, thereby switching their transition from self-renewal state to early endoderm differentiation in hiPSCs.