Good manufacturing practice-compliant animal-free expansion of human bone marrow derived mesenchymal stroma cells in a closed hollow-fiber-based bioreactor

Mesenchymal stroma cells (MSC) are increasingly recognized for various applications of cell-based therapies such as regenerative medicine or immunomodulatory treatment strategies. Standardized large-scale expansions of MSC under good manufacturing practice (GMP)-compliant conditions avoiding animal derived components are mandatory for further evaluation of these novel therapeutic approaches in clinical trials.We applied a novel automated hollow fiber cell expansion system (CES) for in vitro expansion of human bone marrow derived MSC employing a GMP-compliant culture medium with human platelet lysate (HPL). Between 8 and 32 ml primary bone marrow aspirate were loaded into the hollow fiber CES and cultured for 15–27 days. 2–58 million MSC were harvested after primary culture. Further GMP-compliant cultivation of second passage MSC for 13 days led to further 10–20-fold enrichment. Viability, surface antigen expression, differentiation capacity and immunosuppressive function of MSC cultured in the hollow fiber CES were in line with standard criteria for MSC definition. We conclude that MSC can be enriched from primary bone marrow aspirate in a GMP-conform manner within a closed hollow fiber bioreactor and maintain their T lymphocyte inhibitory capacity. Standardized and reliable conditions for large scale MSC expansion pave the way for safe applications in humans in different therapeutic approaches.     

Short-Term Azithromycin Treatment Promotes Cornea Allograft Survival in the Rat

Background

Any inflammatory response following corneal transplantation may induce rejection and irreversible graft failure. The purpose of this study is to analyze the anti-inflammatory effect of azithromycin (AZM) following experimental keratoplasty in rats.

Methods

Corneal transplants were performed between Fisher-donor and Lewis-recipient rats. Recipients were postoperatively treated three times daily with AZM, miglyol, ofloxacin or dexamethasone eye drops. As an additional control, AZM was applied following syngeneic keratoplasty. Furthermore, short-term treatments with AZM for seven days perioperatively or with AZM only three days prior to the transplantation were compared to appropriate controls. All transplants were monitored clinically for opacity, edema, and vascularization. Infiltrating CD45⁺, CD4⁺, CD8⁺, CD25⁺, CD161⁺ and CD163⁺ cells were quantified via immunohistochemistry.

Results

AZM significantly promoted corneal graft survival compared with miglyol or ofloxacin treatment. This effect was comparable to topical dexamethasone. No adverse AZM effect was observed. Histology confirmed a significant reduction of infiltrating leukocytes. The short-term application of AZM for three days prior to transplantation or for seven days perioperatively reduced corneal graft rejection significantly compared with the controls.

Conclusions

Along with antibiotic properties, topical AZM has a strong anti-inflammatory effect. Following keratoplasty, this effect is comparable to topical dexamethasone without the risk of steroid-induced adverse effects. Short-term treatment with AZM three days prior to the transplantation was sufficient to promote graft survival in the rat keratoplasty model. We therefore suggest further assessing the anti-inflammatory function of topical AZM following keratoplasty in humans.     

The calcium-sensing receptor acts as a modulator of gastric acid secretion in freshly isolated human gastric glands

Gastric acid secretion is activated by two distinct pathways: a neuronal pathway via the vagus nerve and release of acetylcholine and an endocrine pathway involving gastrin and histamine. Recently, we demonstrated that activation of H(+)-K(+)-ATPase activity in parietal cells in freshly isolated rat gastric glands is modulated by the calcium-sensing receptor (CaSR). Here, we investigated if the CaSR is functionally expressed in freshly isolated gastric glands from human patients undergoing surgery and if the CaSR is influencing histamine-induced activation of H(+)-K(+)-ATPase activity. In tissue samples obtained from patients, immunohistochemistry demonstrated the expression in parietal cells of both subunits of gastric H(+)-K(+)-ATPase and the CaSR. Functional experiments using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein and measurement of intracellular pH changes allowed us to estimate the activity of H(+)-K(+)-ATPase in single freshly isolated human gastric glands. Under control conditions, H(+)-K(+)-ATPase activity was stimulated by histamine (100 microM) and inhibited by omeprazole (100 microM). Reduction of the extracellular divalent cation concentration (0 Mg(2+), 100 microM Ca(2+)) inactivated the CaSR and reduced histamine-induced activation of H(+)-K(+)-ATPase activity. In contrast, activation of the CaSR with the trivalent cation Gd(3+) caused activation of omeprazole-sensitive H(+)-K(+)-ATPase activity even in the absence of histamine and under conditions of low extracellular divalent cations. This stimulation was not due to release of histamine from neighbouring enterochromaffin-like cells as the stimulation persisted in the presence of the H(2) receptor antagonist cimetidine (100 microM). Furthermore, intracellular calcium measurements with fura-2 and fluo-4 showed that activation of the CaSR by Gd(3+) led to a sustained increase in intracellular Ca(2+) even under conditions of low extracellular divalent cations. These experiments demonstrate the presence of a functional CaSR in the human stomach and show that this receptor may modulate the activity of acid-secreting H(+)-K(+)-ATPase in parietal cells. Furthermore, our results show the viability of freshly isolated human gastric glands and may allow the use of this preparation for experiments investigating the physiological regulation and properties of human gastric glands in vitro.     

Cell Division, a new open access online forum for and from the cell cycle community

ABSTRACT : Cell Division is a new, open access, peer-reviewed online journal that publishes cutting-edge articles, commentaries and reviews on all exciting aspects of cell cycle control in eukaryotes. A major goal of this new journal is to publish timely and significant studies on the aberrations of the cell cycle network that occur in cancer and other diseases.     

Mesenchymal stem cell-based HLA-independent cell therapy for tissue engineering of bone and cartilage

Mesenchymal stem cells (MSC) can be obtained from human bone marrow aspirates and, thanks to their differentiation potential and excellent in vitro culture properties, represent an attractive cell line for the regeneration of mesenchymal tissue. Both in vitro and in vivo, they can differentiate into cartilage, bone, tendons and fat cells, and-in contrast to embryonic stem cells-they are not under ethical scrutiny. Cultured on three-dimensional scaffolds according to the tissue engineering concept, they have already been successfully employed for reconstruction of mesenchymal tissues in numerous studies involving both small and large animal models. Recently, immunological properties of MSC have been investigated by several groups. On the basis of the available literature, MSC have to be referred to as immune privileged, and they seem to be available for HLA-independent cell transplantation. While clinical MSC transplantation has also been successfully performed in pilot studies in humans, numerous points still remain to be clarified, underscoring the need for further intensive research before large-scale clinical application can be contemplated. Only then can it be shown whether the associated high expectations are justified.     

Alternative splicing switches the divalent cation selectivity of TRPM3 channels

TRPM3 is a poorly understood member of the large family of transient receptor potential (TRP) ion channels. Here we describe five novel splice variants of TRPM3, TRPM3alpha1-5. These variants are characterized by a previously unknown amino terminus of 61 residues. The differences between the five variants arise through splice events at three different sites. One of these splice sites might be located in the pore region of the channel as indicated by sequence alignment with other, better-characterized TRP channels. We selected two splice variants, TRPM3alpha1 and TRPM3alpha2, that differ only in this presumed pore region and analyzed their biophysical characteristics after heterologous expression in human embryonic kidney 293 cells. TRPM3alpha1 as well as TRPM3alpha2 induced a novel, outwardly rectifying cationic conductance that was tightly regulated by intracellular Mg(2+). However, these two variants are highly different in their ionic selectivity. Whereas TRPM3alpha1-encoded channels are poorly permeable for divalent cations, TRPM3alpha2-encoded channels are well permeated by Ca(2+) and Mg(2+). Additionally, we found that currents through TRPM3alpha2 are blocked by extracellular monovalent cations, whereas currents through TRPM3alpha1 are not. These differences unambiguously show that TRPM3 proteins constitute a pore-forming channel subunit and localize the position of the ion-conducting pore within the TRPM3 protein. Although the ionic selectivity of ion channels has traditionally been regarded as rather constant for a given channel-encoding gene, our results show that alternative splicing can be a mechanism to produce channels with very different selectivity profiles.     

Peptide blockers of PKG inhibit ROS generation by acetylcholine and bradykinin in cardiomyocytes but fail to block protection in the whole heart

Bradykinin and acetylcholine (ACh) trigger preconditioning by ATP-sensitive K(+) (K(ATP)) channel-dependent production of reactive oxygen species (ROS). Recent evidence suggests that ROS production may in turn be influenced by cGMP-dependent protein kinase (PKG). This study utilized DT-2 and DT-3 peptides, highly specific membrane-permeable blockers of PKG. Rabbit cardiomyocytes were incubated for 15 min in reduced MitoTracker red, which becomes fluorescent only after exposure to ROS. Bradykinin (400 nM) and ACh (250 microM) caused a 49.9 +/- 5.9% and 46.8 +/- 1.7% increase in ROS production, respectively (P < 0.005 vs. untreated cells). Coincubation with DT-3 (250 nM) abolished both the ACh- and bradykinin-induced ROS signal, whereas a nonpermeable form of the peptide (W45) had no effect on ACh-induced ROS production. DT-3 was unable to block ROS production from diazoxide (100 microM), a selective opener of mitochondrial K(ATP) channels, suggesting that these channels are downstream of PKG. DT-2 (125 nM) also prevented ACh from triggering ROS production. 8-(4-Chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (100 microM), a cGMP analog and potent direct activator of PKG, increased ROS production of cardiomyocytes by 44.7 +/- 7.1% (P < 0.001 vs. untreated cells). This increase was blocked by DT-2. Neither DT-2 nor DT-3 could block the anti-infarct effect of bradykinin in isolated rabbit hearts. Studies with fluorescent-tagged DT-3 revealed that it was confined to endothelial cells and never reached the myocytes. We conclude that both bradykinin and ACh trigger ROS generation by a pathway that includes PKG. Although the peptides may be inappropriate for a whole heart model, they are likely to become important tool drugs for elucidation of signal transduction pathways in cell preparations.     

Evolutionary Relationships Among Robust and Gracile Australopiths: An “Evo-devo” Perspective

Hominin fossils of gracile and robust australopith groups were found both in East and in South Africa. It is unclear, however, whether all robusts belong to a monophyletic Paranthropus clade, as the craniofacial resemblance among robust australopiths might only be a superficial correlate of similar masticatory adaptations and not evidence of shared ancestry. It has been suggested that the East African Australopithecus/Paranthropus boisei and the South African A./P. robustus might be convergent allometric variants of their gracile geographical neighbors A. afarensis and A. africanus. Here we approach the phylogenetic questions about robust and gracile australopiths from an ??evo-devo?? perspective, examining how simple alterations of development could contribute to the shape differences among hominin species. Using geometric morphometrics we compare gracile and robust australopith crania in the context of the allometric scaling patterns of Pan troglodytes, P. paniscus, and Gorilla gorilla. We examine support for two alternative evolutionary scenarios based on predictions derived from quantitative genetics models: either (1) A./P. robustus evolved in South Africa from the gracile A. africanus, or (2) A./P. robustus is a local variant of the eastern African A./P. boisei. We use developmental simulations to demonstrate that some robust characteristics (wide faces, anteriorly placed zygomatics, and facial dishing) can be predicted by allometric scaling along the ontogenetic trajectory of the gracile A. africanus. We find, however, that the facial differences between A. africanus specimens (Taung, Sts 5, Sts 71, and Stw 505) and A./P. robustus specimen SK 48 cannot be explained by allometric scaling alone. Facial shape differences between A./P. robustus SK 48 and A./P. boisei (KNM-ER 732, KNM-ER 406, OH 5) and the A./P. aethiopicus specimen KNM-WT 17000, on the other hand, can largely be explained by allometric scaling. This is consistent with a close evolutionary relationship of these robust taxa.     

Reproductive seasonality in captive wild ruminants: implications for biogeographical adaptation,photoperiodic control,and life history

Many ruminant species show seasonal patterns of reproduction. Causes for this are widely debated, and include adaptations to seasonal availability of resources (with cues either from body condition in more tropical, or from photoperiodism in higher latitude habitats) and/or defence strategies against predators. Conclusions so far are limited to datasets with less than 30 species. Here, we use a dataset on 110 wild ruminant species kept in captivity in temperate‐zone zoos to describe their reproductive patterns quantitatively [determining the birth peak breadth (BPB) as the number of days in which 80% of all births occur]; then we link this pattern to various biological characteristics [latitude of origin, mother‐young‐relationship (hider/follower), proportion of grass in the natural diet (grazer/browser), sexual size dimorphism/mating system], and compare it with reports for free‐ranging animals. When comparing taxonomic subgroups, variance in BPB is highly correlated to the minimum, but not the maximum BPB, suggesting that a high BPB (i.e. an aseasonal reproductive pattern) is the plesiomorphic character in ruminants. Globally, latitude of natural origin is highly correlated to the BPB observed in captivity, supporting an overruling impact of photoperiodism on ruminant reproduction. Feeding type has no additional influence; the hider/follower dichotomy, associated with the anti‐predator strategy of ‘swamping’, has additional influence in the subset of African species only. Sexual size dimorphism and mating system are marginally associated with the BPB, potentially indicating a facilitation of polygamy under seasonal conditions. The difference in the calculated Julian date of conception between captive populations and that reported for free‐ranging ones corresponds to the one expected if absolute day length was the main trigger in highly seasonal species: calculated day length at the time of conception between free‐ranging and captive populations followed a y = x relationship. Only 11 species (all originating from lower latitudes) were considered to change their reproductive pattern distinctively between the wild and captivity, with 10 becoming less seasonal (but not aseasonal) in human care, indicating that seasonality observed in the wild was partly resource‐associated. Only one species (Antidorcas marsupialis) became more seasonal in captivity, presumably because resource availability in the wild overrules the innate photoperiodic response. Reproductive seasonality explains additional variance in the body mass–gestation period relationship, with more seasonal species having shorter gestation periods for their body size. We conclude that photoperiodism, and in particular absolute day length, are genetically fixed triggers for reproduction that may be malleable to some extent by body condition, and that plasticity in gestation length is an important facilitator that may partly explain the success of ruminant radiation to high latitudes. Evidence for an anti‐predator strategy involving seasonal reproduction is limited to African species. Reproductive seasonality following rainfall patterns may not be an adaptation to give birth in periods of high resource availability but an adaptation to allow conception only at times of good body condition.     

Molecular breeding of polymerases for amplification of ancient DNA

In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. We describe a strategy for the recovery of genetic information from damaged DNA. By molecular breeding of polymerase genes from the genus Thermus (Taq (Thermus aquaticus), Tth (Thermus thermophilus) and Tfl (Thermus flavus)) and compartmentalized self-replication selection, we have evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000-60,000-year-old cave bear DNA, these outperformed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions at which Taq did not. Our results demonstrate that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archeology and forensic medicine.