Adipose tissue-derived mesenchymal stromal cells for clinical application: An efficient isolation approach.

PURPOSE OF THE STUDY: Mesenchymal stromal cells (MSCs) are considered a promising tool for cell therapy approaches. The translation of research-based cell culture protocols into procedures that comply with Good Manufacturing Practice (GMP) is critical. The aim of this study was to design a new method for the expansion of MSCs from Adipose Tissue (AT-MSCs) in compliance with GMP, without enzymatic tissue digestion and without the use of animal proteins as source of growth factors. PATIENTS AND METHODS: MSCs were expanded from 10 periumbilical biopsies. Our new isolation approach is based on: (1) disruption of AT with an automated, closed system; (2) use of GMP-grade medium without the addition of fetal bovine serum or platelet lysate; (3) use of human recombinant Trypsin. AT-MSCs cultured in α-MEM and minced by scalpel were used as control. RESULTS: It was possible to expand MSCs from all the AT-samples for at least eight passages. MSCs displayed the typical spindle-shape morphology, a high viability, multilineage differentiation potential and high expression levels of the typical MSC-specific surface antigens and genes. Compared to standard method, MSCs obtained with the new method showed higher yield, up to passage 6, and higher purity in terms of percentage of CD34 and CD45 markers. All AT-MSCs exhibit in vitro immunosuppressive capacity and possess a normal karyotype. CONCLUSIONS: Our data clearly demonstrate that our new approach permits to generate AT-MSCs fully compliant for therapeutic use and better at least in terms of quantity and purity than those obtained with the standard method.

Takotsubo Syndrome: Clinical Features, Pathogenesis, Treatment, and Relationship with Cerebrovascular Diseases.

PURPOSE OF REVIEW: This review paper aims to provide a complete and updated overview on the clinical and pathophysiological aspects of Takotsubo syndrome (TTS), including prognosis, therapy, and the association with cerebrovascular conditions. RECENT FINDINGS: TTS is an increasingly recognized non-ischemic cardiomyopathy characterized by sudden, temporary weakening of the myocardium, of which the pathogenesis is unknown. Although pathogenesis of TTS remains unclear, a complex interaction between catecholamine-mediated stimulation, myocardial stunning, and subsequent stress-related myocardial dysfunction seems to be the main pathophysiological mechanism. Stroke is linked to TTS by a dual relationship since it may induce TTS by catecholamine release even if TTS itself also may be complicated by left ventricular thrombi leading to stroke. Given its possible complications, including the association with neurological diseases, both cardiologist and neurologists should be aware about TTS in order to diagnose it promptly and to initiate appropriate therapeutic measures.

Prevalence and characteristics of right-to-left shunt in migraine with aura: a survey on 120 Italian patients.

Many lines of research have suggested a relationship between migraine with aura (MA) and patent foramen ovale. Right-to-left shunt (RLS) of blood might explain both the occurrence of MA attacks, as well as the increased risk of ischaemic stroke in these patients. We evaluated the prevalence and the characteristics of RLS in a series of 120 MA patients, who were studied with contrast-enhanced Transcranial Doppler examination. We found RLS in 61 of them. A latent RLS was found in 28%, a permanent RLS in 72%, a shower-curtain pattern was detected in 52% of the studied patients.

The postnatal rat aorta contains pericyte progenitor cells that form spheroidal colonies in suspension culture.

Pericytes play an important role in modulating angiogenesis, but the origin of these cells is poorly understood. To evaluate whether the mature vessel wall contains pericyte progenitor cells, nonendothelial mesenchymal cells isolated from the rat aorta were cultured in a serum-free medium optimized for stem cells. This method led to the isolation of anchorage-independent cells that proliferated slowly in suspension, forming spheroidal colonies. This process required basic fibroblast growth factor (bFGF) in the culture medium, because bFGF withdrawal caused the cells to attach to the culture dish and irreversibly lose their capacity to grow in suspension. Immunocytochemistry and RT-PCR analysis revealed the expression of the precursor cell markers CD34 and Tie-2 and the absence of endothelial cell markers (CD31 and endothelial nitric oxide synthase, eNOS) and smooth muscle cell markers (alpha-smooth muscle actin, alpha-SMA). In addition, spheroid-forming cells were positive for NG2, nestin, PDGF receptor (PDGFR)-alpha, and PDGFR-beta. Upon exposure to serum, these cells lost CD34 expression, acquired alpha-SMA, and attached to the culture dish. Returning these cells to serum-free medium failed to restore their original spheroid phenotype, suggesting terminal differentiation. When embedded in collagen gels, spheroid-forming cells rapidly migrated in response to PDGF-BB and became dendritic. Spheroid-forming cells cocultured in collagen with angiogenic outgrowths of rat aorta or isolated endothelial cells transformed into pericytes. These results demonstrate that the rat aorta contains primitive mesenchymal cells capable of pericyte differentiation. These immature cells may represent an important source of pericytes during angiogenesis in physiological and pathological processes. They may also provide a convenient supply of mural cells for vascular bioengineering applications.

Neural stem cells: an overview.

Multipotent stem cells are present in the majority of mammalian tissues where they are a renewable source of specialized cells. According to the several biological portions from which multipotent stem cells can be derived, they are characterized as a) embryonic stem cells (ESCs) isolated from the pluripotent inner-cell mass of the pre-implantation blastocyste-stage embryo; b) multipotent fetal stem cells (FSCs) from aborted fetuses; and c) adult stem cells (ASCs) localized in small zones of several organs known as "niche" where a subset of tissue cells and extracellular substrates can indefinitely house one or more stem cells and control their self-renewal and progeny production in vivo. ECSs have an high self-renewing capacity, plasticity and pluripotency over the years. Pluripotency is a property that makes a stem cell able to give rise to all cell type found in the embryo and adult animals.

Hyperhomocysteinemia and other thrombophilic risk factors in 26 patients with cerebral venous thrombosis.

Despite the continuous description of new conditions pre-disposing for cerebral venous thrombosis (CVT), no apparent cause is found in about 30% of cases. Hyperhomocysteinemia (hyper-Hcy) is an established risk factor for deep venous thrombosis and stroke but has not been clearly associated with increased risk of CVT. We assessed the prevalence of hyper-Hcy and other thrombophilic risk factors in a population of 26 consecutive patients with non-pyogenic CVT, by review of a prospectively maintained database. The prevalences of hyper-Hcy and prothrombin G20210A, factor V G1691A and methylenetetrahydrofolate reductase (MTHFR) C677T mutations in these patients were compared with those in 100 healthy controls and 100 patients with cerebroarterial disease. The prevalence of hyper-Hcy was greater in patients with CVT (10/26, 38.5%) than healthy controls (13/100; OR 4.18, 95% CI 1.58-11.16) and comparable with that in patients with cerebroarterial disease (42/100). No significant differences were found in the prevalences of prothrombin or MTHFR mutation. No factor V mutation was found. Our findings indicate that hyper-Hcy is associated with an increased risk of CVT. Additional prospective cohort studies on large series of patients are required to clarify the time relationship between hyper-Hcy and the thrombotic event.

BACE-2 is overexpressed in Down's syndrome.

Brain deposition of the amyloid-beta protein (Abeta) is a frequent complication of Down's syndrome (DS) patients. Abeta peptide is generated by endoproteolytic processing of Abeta precursor protein by gamma and beta secretases. Recently a transmembrane aspartyl protease, BACE, has been identified as the beta-secretase, and its homologous BACE-2 has also been described. BACE-2 gene resides on chromosome 21 in the obligate DS region. It cleaves Abeta precursor protein at its beta site and more efficiently at a different site within Abeta. In the present study we characterized the BACE-2 gene and protein expression in the DS patients and healthy control. We analyzed, by using a nonradioactive ribonuclease protection assay, the levels of BACE-2 mRNA expression in primary skin fibroblasts. The analysis revealed a 2.6-fold increase in BACE-2 mRNA levels in the DS group compared to the levels observed in the control group. Western blot analysis revealed no difference between DS and control in BACE-2 protein levels in the intracellular compartment. In the medium conditioned by fibroblast, we revealed an evident secretion of BACE-2 protein, represented by two different molecular weights, remarkably increased in DS fibroblasts. BACE-2 overexpression was also confirmed in the DS fetal brains and human neural embryonic DS stem cells in which conditioned media BACE-2 was secreted. These data highlight the importance of the extracellular compartment where BACE-2 overexpression could play a role in plaque formation in DS patients.

Neural stem cells. Biological features and therapeutic potential in Parkinson's disease.

AIM: Neural stem cells (NSC) are clonogenic cells, capable of self-renewal and multilineage differentiation, since, under the appropriated experimental conditions, they proliferate indefinitely as undifferentiated neurospheres or differentiate in neurons, astrocytes and oligodendrocytes. Parkinson's disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons. METHODS: Here we investigated the suitability of recently identified and characterized neuronal progenitor cells at eliciting functional recovery in unilateral 6HODA-lesioned mice. We describe herein that intrastriatal engraftment of stem cell-derived neurons isolated from the olfactory bulb to give rise dopaminergic-like neurons results in long lasting functional recovery in 6OHDA-injured mice. RESULTS: Unilateral injection of 6OHDA resulted in a progressive neurodegeneration of the nigro-striatal pathway. Likewise, the systemic administration of L-DOPA in these mice elicited a marked contralateral turning which was evident 1 week post, increased during the following week and than stabilize throughout the time of the experiment. Conversely, the intrastriatal implantation of partially differentiated stem cells at 14 days postlesion, resulted in a profound decrease in L-DOPA-induced circling behavior; interestingly, the effect was evident 1 week after the engraftment and was retained during the following 9 weeks. Detailed biochemical and immunohistochemical evaluation is currently under investigation in our laboratory. Conclusion. Our observation opens new perspectives for the treatment of neurodegeneration in Parkinson's disease.

Isolation and characterization of neural stem cells from the adult human olfactory bulb.

We have recently isolated stem cells deriving from the olfactory bulbs of adult patients undergoing particularly invasive neurosurgery. After improving our experimental conditions, we have now obtained neural stem cells according to clonal analysis. The cells can be expanded, established in continuous cell lines and differentiated into the three classical neuronal phenotypes (neurons, astrocytes, and oligodendrocytes). Also, after exposition to leukemia inhibitory factor, we are able to improve the number of neurons, an ideal biological source for transplantation in various neurodegenerative disorders.

Isolation and intracerebral grafting of nontransformed multipotential embryonic human CNS stem cells.

In this work, we show that the embryonic human brain contains multipotent central nervous system (CNS) stem cells, which may provide a continuous, standardized source of human neurons that could virtually eliminate the use of primary human fetal brain tissue for intracerebral transplantation. Multipotential stem cells can be isolated from the developing human CNS in a reproducible fashion and can be exponentially expanded for longer than 2 years. This allows for the establishment of continuous, nontransformed neural cell lines, which can be frozen and banked. By clonal analysis, reverse transcription polymerase chain reaction, and electrophysiological assay, we found that over such long-term culturing these cells retain both multipotentiality and an unchanged capacity for the generation of neuronal cells, and that they can be induced to differentiate into catechlaminergic neurons. Finally, when transplanted into the brain of adult rodents immunosuppressed by cyclosporin A, human CNS stem cells migrate away from the site of injection and differentiate into neurons and astrocytes. No tumor formation was ever observed. Aside from depending on scarce human neural fetal tissue, the use of human embryonic CNS stem cells for clinical neural transplantation should provide a reliable solution to some of the major problems that pertain to this field, and should allow determination of the safety characteristics of the donor cells in terms of tumorigenicity, viability, sterility, and antigenic compatibility far in advance of the scheduled day of surgery.

Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation.

Stem cells that can give rise to neurons, astroglia, and oligodendroglia have been found in the developing and adult central nervous system (CNS) of rodents. Yet, their existence within the human brain has not been documented, and the isolation and characterization of multipotent embryonic human neural stem cells have proven difficult to accomplish. We show that the developing human CNS embodies multipotent precursors that differ from their murine counterpart in that they require simultaneous, synergistic stimulation by both epidermal and fibroblast growth factor-2 to exhibit critical stem cell characteristics. Clonal analysis demonstrates that human C NS stem cells are multipotent and differentiate spontaneously into neurons, astrocytes, and oligodendrocytes when growth factors are removed. Subcloning and population analysis show their extensive self-renewal capacity and functional stability, their ability to maintain a steady growth profile, their multipotency, and a constant potential for neuronal differentiation for more than 2 years. The neurons generated by human stem cells over this period of time are electrophysiologically active. These cells are also cryopreservable. Finally, we demonstrate that the neuronal and glial progeny of long-term cultured human CNS stem cells can effectively survive transplantation into the lesioned striatum of adult rats. Tumor formation is not observed, even in immunodeficient hosts. Hence, as a consequence of their inherent biology, human CNS stem cells can establish stable, transplantable cell lines by epigenetic stimulation. These lines represent a renewable source of neurons and glia and may significantly facilitate research on human neurogenesis and the development of clinical neural transplantation.

Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain.

The subventricular zone (SVZ) of the adult mammalian forebrain contains kinetically distinct precursor populations that contribute new neurons to the olfactory bulb. Because among forebrain precursors there are stem-like cells that can be cultured in the presence of mitogens such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), we asked whether distinct subsets of stem-like cells coexist within the SVZ or whether the proliferation of a single type of SVZ stem-like cell is controlled by several GFs. We show that the latter is the case. Thus cells isolated from the SVZ coexpress the EGF and FGF receptors; by quantitative analysis, the number of stem-like cells isolated from the SVZ by either FGF2 or EGF is the same, whereas no additive effect occurs when these factors are used together. Furthermore, short-term administration of high-dose [3H]thymidine in vivo depletes both the EGF- and FGF2-responsive stem-like cell populations equally, showing they possess closely similar proliferation kinetics and likely belong to the constitutively proliferating SVZ compartment. By subcloning and population analysis, we demonstrate that responsiveness to more than one GF endows SVZ cells with an essential stem cell feature, the ability to vary self-renewal, that was until now undocumented in CNS stem-like cells. The multipotent stem cell-like population that expands slowly in the presence of FGF2 in culture switches to a faster growth mode when exposed to EGF alone and expands even faster when exposed to both GFs together. Analogous responses are observed when the GFs are used in the reverse order, and furthermore, these growth rate modifications are fully reversible.

Type II neurofibromatosis presenting as quadriceps atrophy.

A young woman aged 26 years presented with atrophy of the left quadriceps progressing over one year. Magnetic resonance imaging (MRI) showed a large lesion of the lumbar plexus compatible with neurinoma. Cerebral MRI revealed a lesion in the right eighth cranial nerve also compatible with neurinoma. On further questioning of the patient, it was learned that her mother had undergone surgery twice for acoustic neurinoma. Type II neurofibromatosis was diagnosed from the basis of clinical picture, neuroimaging findings and family history. This case is unusual for the lack of central nervous system (CNS) symptoms. Expansive radicular lesions compatible with neurinoma should prompt cerebral MRI. Regular examination of family members at risk and early diagnosis can decrease the high mortality associated with this condition.

Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor.

It has been established that the adult mouse forebrain contains multipotential (neuronal/glial) progenitor cells that can be induced to proliferate in vitro when epidermal growth factor is provided. These cells are found within the subventricular zone of the lateral ventricles, together with other progenitor cell populations, whose requirements for proliferation remain undefined. Using basic fibroblast growth factor (bFGF), we have isolated multipotential progenitors from adult mouse striatum. These progenitors proliferate and can differentiate into cells displaying the antigenic properties of astrocytes, oligodendrocytes, and neurons. The neuron-like cells possess neuronal features, exhibit neuronal electrophysiological properties, and are immunoreactive for GABA, substance P, choline acetyl-transferase, and glutamate. Clonal analysis confirmed the multipotency of these bFGF-dependent cells. Most significantly, subcloning experiments demonstrated that they were capable of self-renewal, which led to a progressive increase in population size over serial passaging. These results demonstrate that bFGF is mitogenic for multipotential cells from adult mammalian forebrain that possess stem cell properties.

Basic fibroblast growth factor supports the proliferation of epidermal growth factor-generated neuronal precursor cells of the adult mouse CNS.

Stem cells isolated from the CNS of both embryonic and adult mice undergo extensive proliferation in the presence of epidermal growth factor (EGF). Removal of EGF determines the differentiation of these cells into neurons and glia. We have recently demonstrated that basic fibroblast growth factor (bFGF) regulates the proliferation of EGF-generated progenitors of the embryonic mouse striatum. We report here that bFGF induces proliferation of some EGF-generated precursors of the adult mouse striatum which, in turn, differentiate in vitro into cells possessing neuron-like morphology and neuronal antigenic properties. These results demonstrate that EGF and bFGF can act sequentially to regulate the de novo generation of neurons from the adult mouse CNS in vitro and suggest the existence of a lineage relationship between EGF- and bFGF-responsive progenitor cells of the adult murine brain.

Response to L-DOPA in multiple system atrophy.

A poor response to L-DOPA in addition to parkinsonian, cerebellar, and autonomic signs is commonly regarded as indicative of clinical multiple system atrophy (MSA). We compared the motor response to a single oral administration of 250 mg L-DOPA/25 mg carbidopa in eight MSA patients and eight Parkinson's disease (PD) patients with the "on-off" phenomenon, evaluating L-DOPA peripheral pharmacokinetics. Motor response was consistently good in all PD patients, but only four MSA patients had a (moderate) response. Pharmacokinetic parameters did not differ between the groups. The varying extent of putaminal damage could be responsible for the differing motor response to L-DOPA in MSA patients.

Dexamethasone suppression test, melatonin and TRH-test in cluster headache.

The origin of Cluster Headache (CH) is still unknown. The periodicty and presence of symptoms due to both sympathetic and parasympathetic activity suggest the involvement of central nervous system structures, particularly the hypothalamus. To investigate hypothalamic involvement in CH, we employed a neuroendocrinological approach. We observed a normal dexamethasone suppression test (DST) in all patients, increased cortisol plasma levels in remission patients at 8.00am before and at the end of the DST, while only in CH patients during cluster period did we find a reduced TSH response to TRH and a reduced night-time melatonin peak. The neuroendocrinological derangements found in CH may be consistent with hypothalamic involvement.

Growth hormone responses to cholinergically active drugs in patients with dementia of the Alzheimer type.

Patients with dementia of the Alzheimer type (DAT) reportedly have reduced concentrations and function of some brain messengers, particularly acetylcholine and somatostatin, not only in the cerebral cortex, but also in subcortical structures, e.g., the hippocampus and the hypothalamus. We wished to determine the responsive pattern of DAT patients to neurohormonal and pharmacologic probes affecting growth hormone (GH) release through an interaction with hypothalamic cholinergic and somatostatinergic (SS) neurons. In 10 DAT patients, pyridostigmine (120 mg orally, p.o.), an inhibitor of acetylcholinesterase, induced an increase in GH levels similar to that elicited by the drug in age-matched controls. In 9 DAT patients, administration of GH-releasing hormone (GHRH, 1 microgram/kg body weight, intravenously, i.v.) induced an increase in plasma GH not different from that evidenced in control subjects. In DAT patients the GHRH-induced GH increase was completely inhibited by pretreatment with atropine (1 mg intramuscularly, i.m., 15 min before administration of GHRH). These findings are considered to indicate that in DAT patients, hypothalamic cholinergic and somatostatinergic neurons involved in control of somatotropic function are preserved.

Dopamine metabolism alterations in a manganese-treated pheochromocytoma cell line (PC12).

By monitoring dopamine metabolism in rat pheochromocytoma derived PC12 cell cultures during extended treatment with manganese chloride, we assessed the functional changes occurring in a dopaminergic system during the development of manganese-induced damage. Besides eliciting a specific toxic effect on PC12 cells, manganese induced the complete disappearance of extracellular (free) but not intracellular (vesicle stored) dopamine and its metabolite 3,4-dihydroxyphenylacetic acid. This effect was observed also using low manganese concentrations (1 microM) and mainly occurred by non-enzymatic catechol oxidation since it was still evident in a cell free medium and it was fully prevented by ascorbic acid but not by reduced glutathione. The possibility of a mere "non-biological" action was ruled out by the observation of an irreversible effect of manganese as manifested by the cells' apparent inability to release dopamine or 3,4-dihydroxyphenylacetic acid into the culture medium even after complete manganese removal (post-manganese incubation). That a free radical mechanism was not involved in the genesis of this irreversible effect was shown by the fact that neither ascorbic acid, catalase, superoxide dismutase nor glutathione-peroxidase were able to prevent the decrease in catecholamine levels in the "post-manganese" incubation medium when added at the same time as the manganese. The results establish this PC12 cell system as an in vitro model for studying interactions between manganese and catechols and provide a detailed description of the nature of the neurochemical alterations that this heavy metal can induce in a dopaminergic system.