Sari Vanhatupa

MAPK-induced Ser727 phosphorylation promotes SUMOylation of STAT1.

STAT1 (signal transducer and activator of transcription 1) is a critical mediator of IFN-gamma (interferon-gamma)-induced gene responses, and its function is regulated through phosphorylation of Tyr701 and Ser727. MAPK (mitogen-activated protein kinase) pathways mediate phosphorylation of Ser727 in response to microbial infections, stress stimuli and growth factors. Recently, STAT1 was found to become modified by PIAS (protein inhibitor of activated STAT)-mediated SUMO-1 (small ubiquitin-related modifier-1) conjugation at Lys703, but the regulation of this modification is largely unknown. Here, we have investigated the role of MAPK-induced Ser727 phosphorylation in regulation of STAT1 SUMOylation. Activation of the p38MAPK pathway by upstream activating kinase, MKK6 (MAPK kinase-6) or osmotic stress enhanced the SUMOylation of STAT1, which was counteracted by the p38MAPK inhibitor SB202190 or by dominant-negative p38MAPK. Activation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway by Raf-1 also enhanced Ser727 phosphorylation and SUMOylation of STAT1, and this induction was counteracted by PD98059 inhibitor. Mutation of Ser727 to alanine abolished the p38MAPK-induced SUMOylation. Furthermore, S727D and S727E mutations, which mimic the phosphorylation of Ser727, enhanced the basal SUMOylation of STAT1 and interaction between PIAS1 and STAT1. Taken together, these results identify Ser727 phosphorylation as a regulator of STAT1 SUMOylation and highlight the central role of Ser727 in co-ordination of STAT1 functions in cellular responses.

Enhancement of adhesion and promotion of osteogenic differentiation of human adipose stem cells by poled electroactive poly(vinylidene fluoride)

Poly(vinylidene fluoride) (PVDF) is a biocompatible material with excellent electroactive properties. Nonelectroactive α-PVDF and electroactive β-PVDF were used to investigate the substrate polarization and polarity influence on the focal adhesion (FA) size and number as well as on human adipose stem cells (hASCs) differentiation. hASCs were cultured on different PVDF surfaces adsorbed with fibronectin and FA size and number, total adhesion area, cell size, cell aspect ratio and FA density were estimated using cells expressing vinculin fused to enhanced green fluorescent protein. Osteogenic differentiation was also determined using a quantitative alkaline phosphatase assay. The surface charge of the poled PVDF films (positive or negative) influenced the hydrophobicity of the samples, leading to variations in the conformation of adsorbed extracellular matrix proteins, which ultimately modulated the stem cell adhesion on the films and induced their osteogenic differentiation.

Bioactive glass ions as strong enhancers of osteogenic differentiation in human adipose stem cells

Bioactive glasses are known for their ability to induce osteogenic differentiation of stem cells. To elucidate the mechanism of the osteoinductivity in more detail, we studied whether ionic extracts prepared from a commercial glass S53P4 and from three experimental glasses (2-06, 1-06 and 3-06) are alone sufficient to induce osteogenic differentiation of human adipose stem cells. Cells were cultured using basic medium or osteogenic medium as extract basis. Our results indicate that cells stay viable in all the glass extracts for the whole culturing period, 14 days. At 14 days the mineralization in osteogenic medium extracts was excessive compared to the control. Parallel to the increased mineralization we observed a decrease in the cell amount. Raman and Laser Induced Breakdown Spectroscopy analyses confirmed that the mineral consisted of calcium phosphates. Consistently, the osteogenic medium extracts also increased osteocalcin production and collagen Type-I accumulation in the extracellular matrix at 13 days. Of the four osteogenic medium extracts, 2-06 and 3-06 induced the best responses of osteogenesis. However, regardless of the enhanced mineral formation, alkaline phosphatase activity was not promoted by the extracts. The osteogenic medium extracts could potentially provide a fast and effective way to differentiate human adipose stem cells in vitro.

Direct laser writing and geometrical analysis of scaffolds with designed pore architecture for three-dimensional cell culturing

Traditional scaffold fabrication methods used in tissue engineering enable only limited control over essential parameters such as porosity, pore size and pore interconnectivity. In this study, we designed and fabricated five different types of three-dimensionally interconnected, highly porous scaffolds with precise control over the scaffold characteristics. We used two-photon polymerization (2PP) with a commercial polymer?ceramic material (Ormocomp?) for scaffold fabrication. Also for the first time, we analyzed the 2PP fabrication accuracy with respect to scaffold design parameters. Our results showed that the porosity values decreased up to 13% compared to the design specifications due to the fabrication process and the shrinkage of the material. Finally, we showed that our scaffolds supported human adipose stem cell adhesion and proliferation in a six day culture. By precise tuning of scaffold parameters, our design and fabrication method provides a novel approach for studying the effect of scaffold architecture on cell behavior in vitro.

Bone Morphogenetic Protein-2 Induces Donor-Dependent Osteogenic and Adipogenic Differentiation in Human Adipose Stem Cells

Bone morphogenetic protein‐2 (BMP‐2) is a growth factor used to stimulate bone regeneration in clinical applications. However, there are contradicting reports on the functionality of BMP‐2 in human adipose stem cells (hASCs), which are frequently used in tissue engineering. In this study, we analyzed the effects of BMP‐2 on SMAD1/5 signaling, proliferation, and differentiation in hASCs. Our results indicated that BMP‐2 induced dose‐dependent (25–100 ng/ml) activation of SMAD signaling. Furthermore, the cell proliferation analysis revealed that BMP‐2 (100 ng/ml) consistently decreased the proliferation in all the cell lines studied. However, the analysis of the differentiation potential revealed that BMP‐2 (100 ng/ml) exhibited a donor‐dependent dual role, inducing both osteogenic and adipogenic differentiation in hASCs. The quantitative alkaline phosphatase (qALP) activity and mineralization levels were clearly enhanced in particular donor cell lines by BMP‐2 stimulus. On the contrary, in other cell lines, qALP and mineralization levels were diminished and the lipid formation was enhanced. The current study also suggests that hASCs have accelerated biochemical responsiveness to BMP‐2 stimulus in human serum‐supplemented culture medium compared with fetal bovine serum. The production origin of the BMP‐2 growth factor is also important for its response: BMP‐2 produced in mammalian cells enhanced signaling and differentiation responses compared with BMP‐2 produced in Escherichia coli. These results explain the existing contradiction in the reported BMP‐2 studies and indicate the variability in the functional end mechanism of BMP‐2‐stimulated hASCs.

Sumoylation of Drosophila Transcription Factor STAT92E

STAT92E is an essential transcription factor in Drosophila melanogaster for the development of several organs and the immune system. The JAK/STAT pathway employs different evolutionary conserved regulatory mechanisms to control biological processes. Numerous transcription factors in both mammals and invertebrates have been shown to be either activated or inhibited by a covalent modification with a small ubiquitin-like modifier (Sumo). Here, we show that Drosophila STAT92E is modified by Sumo at a single lysine residue 187 in S2 cells. Mutation of Lys187 increases the transcriptional activity of STAT92E, thus suggesting that sumoylation of STAT92E has a repressive role in the regulation of the JAK/STAT pathway in Drosophila melanogaster.

Growth and Differentiation of Human Dental Pulp Stem Cells Maintained in Fetal Bovine Serum, Human Serum and Serum-free/Xeno-free Culture Media

Introduction: Dental pulp stem cells (DPSCs) are an accessible cell source with therapeutic applicability in regeneration of damaged tissues. Current techniques for expansion of DPSCs require the use of Fetal Bovine Serum (FBS). However, animal-derived reagents stage safety issues in clinical therapy. By expanding DPSCs in serumfree/ xenofree medium (SF/XF-M) or in medium containing human serum (HS-M), the problems can be eliminated. Therefore, the aim of our study was to identify suitable cell culture media alternatives for DPSCs. Methods: We studied the isolation, proliferation, morphology, cell surface markers (CD29, CD44, CD90, CD105, CD31, CD45 and CD146), stemness markers expression (Oct3/4, Sox2, Nanog and SSEA-4) and in vitro multilineage differentiation of DPSCs in HS-M or SF/XF-M in comparison to FBS-M. Results: DPSCs expressed the cell surface and stemness markers in all studied conditions. The proliferation analysis of cells cultured in different HS concentrations revealed that cells isolated in 20% HS-M and passaged in 10% or 15% HS-M supported the cell growth. Direct isolation of cells in SF/XF-M did not support cell proliferation. Therefore, cells cultured in 20% HS-M were used for further SF/XF-M studies. However, proliferation of DPSCs was significantly lower in SF/XF-M when compared with cells cultured in FBS-M and HS-M. In addition, proliferation of DPSCs in SF/XF-M could be enhanced by addition of 1% HS in cell culture medium. There were differences in osteogenic, chondrogenic and adipogenic differentiation efficacy between cells cultured in FBS, HS and SF/XF differentation media. More pronounced adipogenic and osteogenic differentiation was observed in HS differentiation medium, however, in FBS-M cultured cells more effective chondrogenic differentiation was detected. Conclusions: Our results indicate that HS is a suitable alternative to FBS for the expansion of DPSCs. The composition of SF/XF-M needs to be further optimized in terms of cell expandability and differentiation efficiency to reach clinical applicability.

Structure-function analysis indicates that sumoylation modulates DNA-binding activity of STAT1

BackgroundSTAT1 is an essential transcription factor for interferon-γ-mediated gene responses. A distinct sumoylation consensus site (ψKxE) 702IKTE705 is localized in the C-terminal region of STAT1, where Lys703 is a target for PIAS-induced SUMO modification. Several studies indicate that sumoylation has an inhibitory role on STAT1-mediated gene expression but the molecular mechanisms are not fully understood.ResultsHere, we have performed a structural and functional analysis of sumoylation in STAT1. We show that deconjugation of SUMO by SENP1 enhances the transcriptional activity of STAT1, confirming a negative regulatory effect of sumoylation on STAT1 activity. Inspection of molecular model indicated that consensus site is well exposed to SUMO-conjugation in STAT1 homodimer and that the conjugated SUMO moiety is directed towards DNA, thus able to form a sterical hindrance affecting promoter binding of dimeric STAT1. In addition, oligoprecipitation experiments indicated that sumoylation deficient STAT1 E705Q mutant has higher DNA-binding activity on STAT1 responsive gene promoters than wild-type STAT1. Furthermore, sumoylation deficient STAT1 E705Q mutant displayed enhanced histone H4 acetylation on interferon-γ-responsive promoter compared to wild-type STAT1.ConclusionsOur results suggest that sumoylation participates in regulation of STAT1 responses by modulating DNA-binding properties of STAT1.

A durable and biocompatible ascorbic acid-based covalent coating method of polydimethylsiloxane for dynamic cell culture

Polydimethylsiloxane (PDMS) is widely used in dynamic biological microfluidic applications. As a highly hydrophobic material, native PDMS does not support cell attachment and culture, especially in dynamic conditions. Previous covalent coating methods use glutaraldehyde (GA) which, however, is cytotoxic. This paper introduces a novel and simple method for binding collagen type I covalently on PDMS using ascorbic acid (AA) as a cross-linker instead of GA. We compare the novel method against physisorption and GA cross-linker-based methods. The coatings are characterized by immunostaining, contact angle measurement, atomic force microscopy and infrared spectroscopy, and evaluated in static and stretched human adipose stem cell (hASC) cultures up to 13 days. We found that AA can replace GA as a cross-linker in the covalent coating method and that the coating is durable after sonication and after 6 days of stretching. Furthermore, we show that hASCs attach and proliferate better on AA cross-linked samples compared with physisorbed or GA-based methods. Thus, in this paper, we provide a new PDMS coating method for studying cells, such as hASCs, in static and dynamic conditions. The proposed method is an important step in the development of PDMS-based devices in cell and tissue engineering applications.

The effect of equiaxial stretching on the osteogenic differentiation and mechanical properties of human adipose stem cells

Although mechanical cues are known to affect stem cell fate and mechanobiology, the significance of such stimuli on the osteogenic differentiation of human adipose stem cells (hASCs) remains unclear. In this study, we investigated the effect of long-term mechanical stimulation on the attachment, osteogenic differentiation and mechanical properties of hASCs. Tailor-made, pneumatic cell stretching devices were used to expose hASCs to cyclic equiaxial stretching in osteogenic medium. Cell attachment and focal adhesions were visualised using immunocytochemical vinculin staining on days 3 and 6, and the proliferation and alkaline phosphatase activity, as a sign of early osteogenic differentiation, were analysed on days 0, 6 and 10. Furthermore, the mechanical properties of hASCs, in terms of apparent Youngs modulus and normalised contractility, were obtained using a combination of atomic force microscopy based indentation and computational approaches. Our results indicated that cyclic equiaxial stretching delayed proliferation and promoted osteogenic differentiation of hASCs. Stretching also reduced cell size and intensified focal adhesions and actin cytoskeleton. Moreover, cell stiffening was observed during osteogenic differentiation and especially under mechanical stimulation. These results suggest that cyclic equiaxial stretching modifies cell morphology, focal adhesion formation and mechanical properties of hASCs. This could be exploited to enhance osteogenic differentiation.