Mee-Hae Kim

Cardiomyogenic induction of human mesenchymal stem cells by altered Rho family GTPase expression on dendrimer-immobilized surface with d-glucose display

The commitment of stem cells to different lineages is regulated by many cues in the intercellular signals from the microenvironment system. In the present study, we found that alterations in Rho family GTPase activities derived from cytoskeletal formation can lead to guidance of cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs) during in vitro culture. To regulate the cytoskeletal formation of hMSCs, we employed a dendrimer-immobilized substrate that displayed D-glucose. With an increase in the dendrimer generation number, the cells exhibited active migration, accompanied by cell morphological changes of stretching and contracting. Fluorescence microscopy for F-actin, vinculin and glucose transporter1 (GLUT1) clarified the localization of integrin-mediated and GLUT-mediated anchoring, introducing the idea that the morphological changes of the cells were responsive to variations in the generation number of the dendrimer with d-glucose display. On the 5th-generation dendrimer surface, in particular, the cells exhibited RhoA down-regulation and Rac1 up-regulation during the culture, associated with alterations in the cellular morphology and migratory behaviors. It was found that cell aggregation was promoted on this surface, supporting the notion that an increase in N-cadherin-mediated cell-cell contacts and Wnt signaling regulate hMSC differentiation into cardiomyocyte-like cells.

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.

Enrichment of undifferentiated mouse embryonic stem cells on a culture surface with a glucose-displaying dendrimer

This article describes an in vitro culture system for embryonic stem (ES) cells, which are expected to serve as a cell source for transplantation because of their potential for indefinite expansion and pluripotency. We present a serial passaging protocol that permits the enrichment of undifferentiated ES cells by culturing them on a surface modified with a synthesized dendrimer having d-glucose as a functional ligand. The d-glucose-displaying dendrimer (GLU/D) surface caused mouse ES cells to form loosely attached spherical colonies, and the frequency of such colonies increased gradually with the number of passages. Analyses of alkaline phosphatase activity and the gene expression of pluripotency and early differentiation markers revealed that the spherical colony cells passaged four times (a total of 16days in culture) on the GLU/D surface acquired more of the characteristics of undifferentiated cells than the cells cultured on a conventional gelatin-coated surface. Moreover, the cells cultured on the GLU/D surface retained their germ-line transmission ability after four passages. These results indicate that this modified culture surface may be a useful tool for obtaining enriched preparations of undifferentiated ES cells.

Morphological regulation and aggregate formation of rabbit chondrocytes on dendrimer-immobilized surfaces with D-glucose display.

The present study describes dendrimer-immobilized surfaces with D-glucose display, used to regulate the morphology of rabbit chondrocytes by changing the generation number and density of the dendrimer, and to clarify the relation between cell morphology and chondrogenic expression on the prepared surfaces. When the generation number increased, the frequency of round-shaped cells increased, whereas, cell stretching was appreciably suppressed. Further suppression of stretching was significant on the G4-LD surface, on which the density of the immobilized dendrimer was lowered, and this phenomenon was thought to be responsible for the sparse display of D-glucose on the surface. The time-lapse observations revealed that the G4-LD surface caused repeated morphological changes of stretching and contracting, together with fluctuations in cell roundness. By the cytoskeletal staining of F-actin, the immature stress fibers were recognized in both round and stretched cells on the G4-LD surface. It was also found that N-cadherin expression was promoted on this surface, thus supporting the idea that an increase in cell-cell contacts may maintain chondrocytic phenotypes. These results suggest that a G4-LD surface can provide a culture environment to promote cell-cell interactions by N-cadherin-mediated adhesion during cell aggregation, thereby facilitating chondrocytic phenotypes of cells.

Comprehension of terminal differentiation and dedifferentiation of chondrocytes during passage cultures.

A high density collagen type I coated substrate (CL substrate) was used to evaluate the chondrocyte phenotypes in passaged cultures. With increasing age of cell population (population doubling (PD)=0-14.5), the frequency of non-dividing spindle shaped cells without ALP activity increased, accompanied with an increase in gene expression of collagen type I, meaning the senescence of dedifferentiated cells. At the middle age of cell population (PD=5.1 and 6.6), the high frequency of polygonal shaped cells with ALP activity existed on the CL substrate together with up-regulated expressions of collagen types II and X, indicating the terminal differentiation of chondrocytes. When the chondrocytes passaged up to the middle age were embedded in collagen gel, the high frequency of single hypertrophic cells with collagen type II formation was recognized, which supports the thought that the high gene expression of collagen type II was attributed to terminal differentiation rather than redifferentiation. These results show that the CL substrate can draw out the potential of terminal differentiation in chondrocytes, which is unattainable by a polystyrene surface, and that the CL substrate can be a tool to evaluate cell quality in three-dimensional culture with the collagen gel.

Kinetic analysis of deviation from the undifferentiated state in colonies of human induced pluripotent stem cells on feeder layers

Understanding of the fundamental mechanisms that govern unintentional differentiation of human induced pluripotent stem cells (hiPSCs) provides key strategies to maintain their undifferentiated state during cell expansion. This study focused on deviation from the undifferentiated state in hiPSC colonies during culture with feeder cells. Deviated cells from the undifferentiated state of hiPSCs in cultures with SNL and MEF feeder cells were observed at the center and periphery of the colonies, respectively, accompanied by dramatic changes in the cell morphology from small to large flattened shapes. It was found that the deviation of undifferentiated hiPSCs in culture with SNL feeder cells caused deviated cells in the center of the colony through spontaneous occurrence in a colony size‐dependent manner, whereas the deviation of undifferentiated hiPSCs in culture with MEF feeder cells caused deviated cells in the periphery of the colonies through accidental events during migration in a colony size‐independent manner. Based on a kinetic analysis of time‐lapse images of single hiPSC colonies, the specific growth rate for replication of deviated cells from the undifferentiated state in culture with SNL feeder cells was 1.83 and 3.57 times higher than those of undifferentiated cells and transformation, respectively, meaning that the deviation of undifferentiated hiPSCs dramatically expanded through replication of deviated cells from the undifferentiated state and transformation once deviation from the undifferentiated state had occurred. In the case of MEF feeder cells, the specific growth rates for replication of deviated cells from the undifferentiated state was 3.12 times higher than that of undifferentiated cells, whereas the rate by transformation exhibited a negligible level compared with the rates of replication for undifferentiated cells and deviated cells from undifferentiated state, meaning that deviation of undifferentiated hiPSCs dramatically expanded only through replication of deviated cells from the undifferentiated state. These results suggest that once deviation has occurred in a colony, the deviated cells from undifferentiated state undertake dramatic invasion to occupy the colony. Maintenance of the undifferentiated state in subcultures inevitably requires vigilant care to remove any colonies that include deviated cells from the undifferentiated state. Biotechnol. Bioeng. 2014;111: 1128–1138.

Glucose transporter mediation responsible for morphological changes of human epithelial cells on glucose-displayed surfaces

Cellular morphology is one of the important factors for coordinating cell signaling. In this study, the morphological variation via glucose transporter (GLUT)-mediated anchoring was investigated in the cultures of human mammary epithelial cells in the presence or absence of insulin on culture surfaces with the changed ratios of d- and l-glucose displayed. With increasing ratio of d-glucose displayed on the surfaces, the cells showed a stretched shape in the culture with 10 mug/cm(3) insulin, reaching the highest extent of cell stretching at 100%d-glucose display, whereas round cells were predominant at 0%d-glucose display. In the absence of insulin, on the other hand, the extent of cell stretching showed a concave profile in terms of the ratio of d-glucose displayed, the extent being highest at 50%d-glucose display. Blocking of integrin alpha(5)beta(1) or GLUTs1 and 4 on the cells using corresponding antibodies revealed that the primary mechanism for cell attachment was based on integrin-mediated binding, and that GLUTs1 and 4 contributed largely to morphological changes of cells. Confocal microscopy further revealed that GLUT4 localization occurred in response to d-glucose display as well as insulin addition. In the absence of insulin, GLUT4 spots were extensively observed in the cell body regardless of whether d-glucose was displayed or not. However, in the presence of insulin, the broad distribution of GLUT4 appeared on the basal and apical sides of cells at 100%d-glucose display, in contrast with its localization only on the apical side of cells at 0%d-glucose display. These results suggest that the quantitative balance between GLUTs on the cytoplasmic membrane and d-glucose displayed on a culture surface determines the cell morphology, as explained by the receptor saturation model.

Dendrimer-Immobilized Culture Surface as a Tool to Evaluate Formation of Cellular Cytoskeleton of Anchorage-Dependent Cells

Anchorage-dependent cultivation of human epithelial and keratinocyte cells was carried out on surfaces modified with synthesized dendrimers. Notable elongation of the epithelial cells was recognized on the culture surface immobilized with a dendrimer having D-glucose as a functional ligand, but not when a dendrimer having L-glucose was used or when the dendrimer was ligand-free. This morphological change was attributable to a temporary grasping of the cells at the D-glucose moiety via a glucose transporter-mediated mechanism present in the cell membrane. Following visualization of the actin filaments of the cells, it was considered that the cellular elongation on the D-glucose-bound dendrimer surface reflected the degree of formation of the cellular cytoskeleton. The cellular roundness was calculated by means of image analysis of the individual cells and employed as a parameter to evaluate the formation of the cellular cytoskeleton. In the culture of keratinocytes on the D-glucose-bound dendrimer surface, it was demonstrated that the decrease in the ratio of elongated cells (i.e., cells with a low roundness value) was correlated with the deterioration in the growth potential associated with cellular senescence.

Maintenance of an undifferentiated state of human induced pluripotent stem cells through migration-dependent regulation of the balance between cell-cell and cell-substrate interactions.

We present an outlook on the current strategies for maintaining and culturing human induced pluripotent stem cells (hiPSCs) in an undifferentiated state without affecting their pluripotency. First, cell structures and function are described in relation to interactions between hiPSCs and their surroundings. Second, the phenomenon of spontaneous deviation from undifferentiated hiPSCs in cultures with feeder cells is addressed, with a summary of current topics that are of particular interest to our studies. The key regulatory factors that can contribute to the growth and maintenance of undifferentiated hiPSCs are also discussed, with a summary of recent work toward a culture strategy to control hiPSC fate through balancing cell-cell and cell-substrate interactions. Finally, we discuss culture process design in our previous studies with respect to maintaining and expanding cultures of undifferentiated and pluripotent hiPSCs. We focus on the regulation of migration-dependent balance between cell-cell and cell-substrate interactions. This review offers novel insights into the regulation and processing of stem cells for research in regenerative medicine.

Maintenance of undifferentiated state of human induced pluripotent stem cells through cytoskeleton-driven force acting to secreted fibronectin on a dendrimer-immobilized surface

Understanding of the fundamental mechanisms that govern adhesive properties of human induced pluripotent stem cells (hiPSCs) to culture environments provides surface design strategies for maintaining their undifferentiated state during cell expansion. Polyamidoamine dendrimer surface with first-generation (G1) with dendron structure was used for co-cultures of hiPSCs and SNL feeder cells that formed tightly packed compact hiPSC colonies, similar to those on a conventional gelatin-coated surface. hiPSCs passaged up to 10 times on the G1 surface maintained their undifferentiated state. Immunostaining and reverse transcriptase PCR analysis of fibronectin showed that the secreted fibronectin matrix from feeder cells on the G1 surface contributed to hiPSC attachment. Compared with cells on the gelatin-coated surface, F-actin and paxillin immunostaining revealed a well-organized network of actin stress fibers and focal adhesion formation at cell-substrate sites in hiPSC colonies on the G1 surface. E-cadherin expression levels on these surfaces were almost same, but paxillin and Rac1 expression levels on the G1 surface were significantly higher than those on the gelatin-coated surface. Zyxin showed prominent expression on the G1 surface at sites of focal adhesion and cell-cell contact in colonies, whereas zyxin expression on the gelatin-coated surface was not observed in regions of cell-cell contact. These findings indicate that transduction of mechanical stimuli through actin polymerization at sites of focal adhesion and cell-cell contact results in maintenance of undifferentiated hiPSC colonies on G1 surface. The G1 surface enables a substrate design based on the mechanical cues in the microenvironment from feeder cells to expand undifferentiated hiPSCs in long-term culture.