Hydrodynamic modulation of embryonic stem cell differentiation by rotary orbital suspension culture

Embryonic stem cells (ESCs) can differentiate into all somatic cell types, but the development of effective strategies to direct ESC fate is dependent upon defining environmental parameters capable of influencing cell phenotype. ESCs are commonly differentiated via cell aggregates referred to as embryoid bodies (EBs), but current culture methods, such as hanging drop and static suspension, yield relatively few or heterogeneous populations of EBs. Alternatively, rotary orbital suspension culture enhances EB formation efficiency, cell yield, and homogeneity without adversely affecting differentiation. Thus, the objective of this study was to systematically examine the effects of hydrodynamic conditions created by rotary orbital shaking on EB formation, structure, and differentiation. Mouse ESCs introduced to suspension culture at a range of rotary orbital speeds (20–60 rpm) exhibited variable EB formation sizes and yields due to differences in the kinetics of cell aggregation. Computational fluid dynamic analyses indicated that rotary orbital shaking generated relatively uniform and mild shear stresses (≤2.5 dyn/cm²) within the regions EBs occupied in culture dishes, at each of the orbital speeds examined. The hydrodynamic conditions modulated EB structure, indicated by differences in the cellular organization and morphology of the spheroids. Compared to static culture, exposure to hydrodynamic conditions significantly altered the gene expression profile of EBs. Moreover, varying rotary orbital speeds differentially modulated the kinetic profile of gene expression and relative percentages of differentiated cell types. Overall, this study demonstrates that manipulation of hydrodynamic environments modulates ESC differentiation, thus providing a novel, scalable approach to integrate into the development of directed stem cell differentiation strategies. Biotechnol. Bioeng. 2010; 105: 611–626. © 2009 Wiley Periodicals, Inc.     

Remission in psoriatic arthritis: is it possible and how can it be predicted?

Introduction

Since remission is now possible in psoriatic arthritis (PsA) we wished to examine remission rates in PsA patients following anti tumour necrosis factor alpha (TNFα) therapy and to examine possible predictors of response.

Methods

Analysis of a prospective patient cohort attending a biologic clinic, between November 2004 and March 2008, was performed prior to commencing therapy and at regular intervals. Baseline clinical characteristics including demographics, previous disease-modifying antirheumatic drug (DMARD) response, tender and swollen joint counts, early morning stiffness, pain visual analogue score, patient global assessment, C reactive protein (CRP) and health assessment questionnaire (HAQ) were collected.

Results

A total of 473 patients (152 PsA; 321 rheumatoid arthritis (RA)) were analyzed. At 12 months remission, defined according to the disease activity score using 28 joint count and CRP (DAS28-CRP), was achieved in 58% of PsA patients compared to 44% of RA patients, significant improvement in outcome measures were noted in both groups (P < 0.05). Analysis of a subgroup of PsA and RA patients matched for DAS28-CRP at baseline also showed higher numbers of PsA patients achieving remission. Linear regression analysis identified the HAQ at baseline as the best predictor of remission in PsA patients (P < 0.001).

Conclusions

DAS28 remission is possible in PsA patients at one year following anti-TNF therapy, at higher rates than in RA patients and is predicted by baseline HAQ.     

Structural Sterols Are Involved in Both the Initiation and Tip Growth of Root Hairs in Arabidopsis thaliana

Structural sterols are abundant in the plasma membrane of root apex cells in Arabidopsis thaliana. They specifically accumulate in trichoblasts during the prebulging and bulge stages and show a polar accumulation in the tip during root hair elongation but are distributed evenly in mature root hairs. Thus, structural sterols may serve as a marker for root hair initiation and growth. In addition, they may predict branching events in mutants with branching root hairs. Structural sterols were detected using the sterol complexing fluorochrome filipin. Application of filipin caused a rapid, concentration-dependent decrease in tip growth. Filipin-complexed sterols accumulated in globular structures that fused to larger FM4-64–positive aggregates in the tip, so-called filipin-induced apical compartments, which were closely associated with the plasma membrane. The plasma membrane appeared malformed and the cytoarchitecture of the tip zone was affected. Trans-Golgi network/early endosomal compartments containing molecular markers, such as small Rab GTPase RabA1d and SNARE Wave line 13 (VTI12), locally accumulated in these filipin-induced apical compartments, while late endosomes, endoplasmic reticulum, mitochondria, plastids, and cytosol were excluded from them. These data suggest that the local distribution and apical accumulation of structural sterols may regulate vesicular trafficking and plasma membrane properties during both initiation and tip growth of root hairs in Arabidopsis.     

Production and characterization of a soluble, active form of Tva, the subgroup A avian sarcoma and leukosis virus receptor

The receptor for the subgroup A avian sarcoma and leukosis viruses [ASLV(A)] is the cellular glycoprotein Tva. A soluble form of Tva, sTva, was produced and purified with a baculovirus expression system. Using this system, 7 to 10 mg of purified sTva per liter of cultured Sf9 cells was obtained. Characterization of the carbohydrate modification of sTva revealed that the three N glycosylation sites in sTva were differentially utilized; however, the O glycosylation common to Tva produced in mammalian and avian cells was not observed. Purified sTva demonstrates significant biological activity, specifically blocking infection of avian cells by ASLV(A) with a 90% inhibitory concentration of approximately 25 pM. A quantitative enzyme-linked immunosorbent assay, developed to assess the binding of sTva to ASLV envelope glycoprotein, demonstrates that sTva has a high affinity for EnvA, with an apparent dissociation constant of approximately 0.3 nM. Once they are bound, a very stable complex is formed between EnvA and sTva, with an estimated complex half-life of 6 h. The soluble receptor protein described here represents a valuable tool for analysis of the receptor-envelope glycoprotein interaction and for structural analysis of Tva.     

Enhanced mixing and mass transfer in a recirculation loop results in high cell densities in a roller bottle reactor

A recirculation loop added to a large-scale roller bottle reactor resulted in high cell densities as compared to standard roller bottles. Four different mammalian cell lines reached an average maximum density equal to 5.4 x 10(6) cells /mL (sigma = 0.263), which was between 2.13 and 2.95 times greater than the densities in roller bottles without recirculation using the same cell lines. The high densities were maintained over long durations (>25 days) while the reactor operated with continuous perfusion. The increased densities are attributed to enhanced liquid mixing and oxygen transfer that occur as a result of the recirculation loop. Models were developed that describe axial liquid flow and oxygen transfer in both the sample loop and the reactor growth chamber. Axial dispersion and oxygen transfer coefficients are presented for a variety of operating conditions. The increased oxygen transfer characteristics of the reactor allow for easy scale-up of roller bottle cultures by operating at larger volumes with greater liquid depths than conventional roller bottles permit. The surface-area-to-volume ratio in the tests performed was 0.206 versus 1.16 cm(-1) in a standard roller bottle.     

Control of P2X(2) channel permeability by the cytosolic domain

ATP-gated P2X channels are the simplest of the three families of transmitter-gated ion channels. Some P2X channels display a time- and activation-dependent change in permeability as they undergo the transition from the relatively Na(+)-selective I(1) state to the I(2) state, which is also permeable to organic cations. We report that the previously reported permeability change of rat P2X(2) (rP2X(2)) channels does not occur at mouse P2X(2) (mP2X(2)) channels expressed in oocytes. Domain swaps, species chimeras, and point mutations were employed to determine that two specific amino acid residues in the cytosolic tail domain govern this difference in behavior between the two orthologous channels. The change in pore diameter was characterized using reversal potential measurements and excluded field theory for several organic ions; both rP2X(2) and mP2X(2) channels have a pore diameter of approximately 11 A in the I(1) state, but the transition to the I(2) state increases the rP2X(2) diameter by at least 3 A. The I(1) to I(2) transition occurs with a rate constant of approximately 0.5 s(-1). The data focus attention on specific residues of P2X(2) channel cytoplasmic domains as determinants of permeation in a state-specific manner.