Optimizing isolation culture and freezing methods to preserve Wharton's jelly's mesenchymal stem cell (MSC) properties: an MSC banking protocol validation for the Hellenic Cord Blood Bank.

BACKGROUND: Mesenchymal stem or stromal cells (MSCs) are a heterogeneous population that can be isolated from many tissues including umbilical cord Wharton's jelly (UC-WJ). Although initially limited in studies such as a hematopoietic stem cell transplantation adjuvant, an increasing number of clinical trials consider MSCs as a potential anti-inflammatory or a regenerative medicine agent. It has been proposed that creating a repository of MSCs would increase their availability for clinical applications. The aim of this study was to assess the optimal isolation and cryopreservation procedures to facilitate WJ MSC banking. STUDY DESIGN AND METHODS: Cells were isolated from UC-WJ using enzymatic digestion or plastic adhesion methods. Their isolation efficacy, growth kinetics, immunophenotype, and differentiation potential were studied, as well as the effects of freezing. Flow cytometry for common MSC markers was performed on all cases and differentiation was shown with histocytochemical staining. Finally, the isolation efficacy on cryopreserved WJ tissue fragments was tested. RESULTS: MSC isolation was successful using both isolation methods on fresh UC-WJ tissue. However, UC-WJ MSC isolation from frozen tissue fragments was impossible. Flow cytometry analysis revealed that only MSC markers were expressed on the surface of the isolated cells while differentiation assays showed that they were capable of trilinear differentiation. All the above characteristics were also preserved in isolated UC-WJ MSCs over the cryopreservation study period. CONCLUSION: These data showed that viable MSCs can only be isolated from fresh UC-WJ tissue, setting the foundation for clinical-grade banking.

Antagonists of retinoic acid and BMP4 affect fetal mouse osteogenesis and odontoblast differentiation.

Retinoic acid and bone morphogenetic protein (BMP4) are endogenous factors indispensable for the physiological development of vertebrates. The proximate aim of the present study was to investigate whether the natural compound citral (a retinoic acid synthesis inhibitor) and a monoclonal, anti-BMP4 antibody, administered to pregnant mice affect in the fetuses cranial osteogenesis and odontoblast differentiation. The present investigation was motivated by the fact that, retinoic acid inhibitors and BMP4 neutralizers may frequently contact human tissues (both intentional and unintentional, and/or unconsciously) inducing unanticipated effects. Our ultimate goal is the prevention of side effects and, future clinical implementation of the results. To this end, pregnant, white mice (balb-c Mus musculus) were intra-abdominally injected with either citral or anti-BMP4 antibody at the 9th gestational day. Newborns were processed within 5h, postnatal. Results were evaluated (a) macroscopically, (b) stereoscopically, following histochemical double staining of cartilage and osseous tissues and, (c) microscopically after (c(1)) histological staining of paraffin sections, and, (c(2)) immunohistochemical detection of apoptosis. Data indicate that in vivo administration of citral (biomimicking hypovitaminosis A) caused restriction/retardation of cranial chondrogenesis and osteogenesis. Apoptosis was not detected in teeth tissues. In vivo administration of anti-BMP4 antibody resulted in a transitory interference with the normal course of odontoblast differentiation and the production of pre-dentin, whereas, delay in the ossification also included the alveoli. Animals inspected in adulthood displayed a fairly normal phenotype. It is concluded that those two substances, under their concentrations experienced, are quite safe for the public.

Cranial and postcranial skeletal variations induced in mouse embryos by mobile phone radiation.

This study focuses on foetal development following mild daily exposure of pregnant mice to near field electromagnetic radiation emitted by a mobile phone. The investigation was motivated by the fact that the potentially hazardous electromagnetic radiation emitted by mobile phones is currently of tremendous public interest. Physically comparable pregnant mice were exposed to radiofrequency radiation GSM 900MHz emitted by a mobile phone. Within 5h after birth most cubs were fixed followed by double staining in toto, and conventional paraffin histology. Other cubs remained with their mothers until teeth eruption. Structural development was assessed by examining newborns for the presence of anomalies and/or variations in soft tissues and skeletal anatomy. Electromagnetic radiofrequency exposed newborns, externally examined, displayed a normal phenotype. Histochemical and histological studies, however, revealed variations in the exposed foetuses with respect to control ones concerning the ossification of cranial bones and thoracic cage ribs, as well as displacement of Meckelian cartilage. Littermates examined after teeth eruption displayed normal phenotypes. It is concluded that mild exposure to mobile phone radiation may affect, although transiently, mouse foetal development at the ossification level. The developmental variations observed could be explained by considering the different embryonic origin and mode of ossification of the affected skeletal elements.

Embryoid bodies from mouse stem cells express oxytocin receptor, Oct-4 and DAZL.

Oxytocin is a nine amino acid peptide involved in a wide spectrum of physiological functions; predominantly those concerning reproduction and differentiation are of interest. Oxytocin receptors are expressed at early developmental stages of mammals, suggesting that oxytocin might be involved in the determination of the germ stem cell line, at the very early stages of mammalian development. In this respect, the proximate aim of the present study was to confirm and further analyze the existence of oxytocin receptors at a very early level of cell commitment, that is, the determination of germ cells derived from embryoid bodies. To achieve our purpose we have cultured mouse embryonic stem cells under conditions inducing formation of embryoid bodies. In this work, ES cells were allowed to aggregate in a novel medium, "Stefanidis medium" from day 0 to day 14 until formed EBs. RNA was isolated from EBs and using RT-PCR we showed that EBs expressed Oct-4, OTR, OT, and DAZL. To demonstrate simultaneous expression immunocytochemistry was preformed, in which EBs showed strong immunoreactivity for both, OTR and DAZL molecular markers. We found that 35% of the cells displayed OTR, using flow cytometry. In addition, this novel medium showed to increase OTR mRNA. We propose, that at least in murine induced embryoid bodies there is simultaneous expression of oxytocin receptors and germ cell markers (DAZL) in many cells (expressing Oct-4). We thus conclude that, the oxytocin might indeed be a molecule playing a leading role in germ cell determination.

Botulinum neurotoxin: the ugly duckling.

This review presents a brief account of the most significant biological effects and clinical applications of botulinum neurotoxins, in a way comprehensive even for casual readers who are not familiar with the subject. The most toxic known substances in botulinum neurotoxins are polypeptides naturally synthesized by bacteria of the genus Clostridium. These polypeptides inhibit acetylcholine release at neuromuscular junctions, thus causing muscle paralysis involving both somatic and autonomic innervation. There is substantial evidence that this muscle-paralyzing feature of botulinum neurotoxins is useful for their beneficial influence on more than 50 pathological conditions such as spastic paralysis, cerebral palsy, focal dystonia, essential tremor, headache, incontinence and a variety of cosmetic interventions. Injection of adequate quantities of botulinum toxins in spastic muscles is considered as a highly hopeful procedure for the treatment of people who suffer from dystonia, cerebral palsy or have experienced a stroke. So far, numerous and reliable studies have established the safety and efficacy of botulinum neurotoxins and advocate wider clinical therapeutic and cosmetic applications.

A curriculum vitae of teeth: evolution, generation, regeneration.

The ancestor of recent vertebrate teeth was a tooth-like structure on the outer body surface of jawless fishes. Over the course of 500,000,000 years of evolution, many of those structures migrated into the mouth cavity. In addition, the total number of teeth per dentition generally decreased and teeth morphological complexity increased. Teeth form mainly on the jaws within the mouth cavity through mutual, delicate interactions between dental epithelium and oral ectomesenchyme. These interactions involve spatially restricted expression of several, teeth-related genes and the secretion of various transcription and signaling factors. Congenital disturbances in tooth formation, acquired dental diseases and odontogenic tumors affect millions of people and rank human oral pathology as the second most frequent clinical problem. On the basis of substantial experimental evidence and advances in bioengineering, many scientists strongly believe that a deep knowledge of the evolutionary relationships and the cellular and molecular mechanisms regulating the morphogenesis of a given tooth in its natural position, in vivo, will be useful in the near future to prevent and treat teeth pathologies and malformations and for in vitro and in vivo teeth tissue regeneration.

Oxytocin receptor is differentially expressed in mouse endometrium and embryo during blastocyst implantation.

The oxytocin (OT)-oxytocin receptor (OTR) system of the mammalian uterus has mainly been studied in relation to its involvement in the onset of labor. The aim of this study was to elucidate the in vivo expression and localization pattern of OTR in the mouse endometrium and embryo during implantation, as well as OTR mRNA expression in the in vitro developing mouse embryo. The expression of OTR or OT was detected immunohistochemically in uterine tissue sections of 5- to 8-week-old female mice between days 4 and 10 of an established pregnancy. In addition, the expression of OTR mRNA was detected by means of reverse transcription polymerase chain reaction (RT-PCR) in mouse oocytes and embryos up to the blastocyst stage. The mean ratios of normalized expression levels of OTR gene in all samples were also calculated. The recorded increase in OTR mRNA immediately after fertilization could mean a possible role of OT in this process, as OTR mRNA gradually decreased after the four-cell stage of pre-embryonic development. The differential expression of OTR during embryonic apposition and embryonic invasion/placentation in the mouse uterus suggests a potential role of OT in the implantation process of the mouse. It is possible that the interaction of OTR with the hormones included in the ovulation induction regiments utilized today in in vitro fertilization (IVF) could be affecting the receptivity/quality of the implanting endometrium.

Vertebrate limb development: from Harrison's limb disk transplantations to targeted disruption of Hox genes.

Various animal organs have long been used to investigate the cellular and molecular nature of embryonic growth and morphogenesis. Among those organs, the tetrapod limb has been preferentially used as a model system for elucidating general patterning mechanisms. At the appropriate time during the embryonic period, the limb territories are first determined at the right positions along the cephalocaudal axis of the animal body, and soon the limb buds grow out from the flanks as mesenchymal cell masses covered by simple ectoderm. The position, number, and identity of the limbs depend on the expression of specific Hox genes. Limb morphogenesis occurs along three axes, which become gradually fixed: first the anteroposterior axis, then the dorsoventral, and finally the proximodistal axis, along which the bulk of limb growth occurs. Growth of the limb in amniotes depends on the formation of the apical ectodermal ridge, which, by secreting many members of the fibroblast growth factors family, attracts lateral plate and somitic mesodermal cells, keeps these cells in the progress zone proliferating, and prevents their differentiation until an appropriate time period. Mutual interactions between mesoderm and ectoderm are important in the growth process, and signaling regions have been identified, such as the zone of polarizing activity, the dorsal limb ectoderm, and the apical ectodermal ridge. Several molecules have been found to play leading roles in various biological processes relevant to morphogenesis. Besides its intrinsic merit as a model for unraveling the mechanisms of development, the limb deserves considerable clinical interest because defects of limb development are the most common single category of congenital abnormalities.

Quantitative estimation of HRP-labeled sensory and motor neurons during nerve-dependent and nerve-independent periods of urodele limb regeneration.

The relationship between urodele regeneration and possible regeneration in mammalian prospects is hard to evidence, but the idea of possible regeneration of neural elements in people is an area of potential clinical importance that is under investigation. One of the great challenges of the future is to understand enough about the basic biology of animal regeneration and to use it for the betterment of the mankind. It is well established that the initial stages of urodele limb regeneration depend on the presence of intact nerve fibres connected to their cell bodies. The nerve fibres severed at the limb amputation level, regrow and invade the blastema, providing blastema cells with indispensable factors. These factors are elaborated within the neuron perikarya and transported via their axons to the blastema. Numerous studies have been so far performed and have elucidated the quantitative relationships between nerve fibres and limb regeneration. However, there are no reports dealing with the individual nerve cells at work. The aim of the present investigation was to analyse the quantitative participation and qualitative distinction of nerve cells innervating regenerating parts of the urodele limb and their possible interrelationship with the nerve-dependent and nerve-independent periods of regeneration. The cells under study are housed in the dorsal ganglia (sensory neurons) and in the ventral aspect of the spinal cord grey matter (motor neurons). As a means of visualizing the direct implication of these neurons during various regeneration periods, the enzyme horseradish peroxidase was chosen. A total of 34 animals were used, 21 experimental and 13 controls, in order to study labeled nerve cell fluctuations. The results are summarized as follows: (a) The first nerve cells incorporating HRP within 5 days post amputation are found in the dorsal ganglia. Motor neurons in the grey matter are labeled within 7 days. (b) The number of labeled perikarya increases during the nerve-dependent regeneration period (0-21 dpa). The percentage of implicated sensory neurons exceeds that found in the control series. (c) During the next, nerve-independent period, the number of participating labeled neurons decreases gradually. Such fluctuations in the number of labeled neurons might represent the metabolic status of these cells in their effort to provide the blastema cells with the factors needed at the appropriate time. The current findings support previous observations that the periods of dependence and independence of urodele limb regeneration from the integrated control of brachial nerves reflect changes in the metabolism of individual sensory and motor neurons.