Cancer stem cell immunophenotypes in oral squamous cell carcinoma

Background: The presence of cancer stem cell (CSC) antigens can be evidenced in some human tumors by phenotypic analysis through immunostaining. This study aims to identify a putative CSC immunophenotype in oral squamous cell carcinoma (OSCC) and determine its influence on prognosis. Methods: The following data were retrieved from 157 patents: age, gender, primary anatomic site, smoking and alcohol intake, recurrence, metastases, histologic classification, treatment, disease-free survival (DFS), and overall survival (OS). An immunohistochemical study for CD44 and CD24 was performed in a tissue microarray of 157 paraffin blocks of OSCCs. Results: In univariate analysis, the immunostaining pattern showed significant influences in relation to OS for alcohol intake and treatment, as well as for the CD44+ and CD44-/CD24- immunophenotypes. The multivariate test confirmed these associations. Conclusions: Based on our results, the CD44 immunostaining and the absence of immunoexpression of these two investigated markers can be used in combination with other clinicopathologic information to improve the assessment of prognosis in OSCC.

Stem cell proliferation under low intensity laser irradiation: A preliminary study

Background and Objectives: Phototherapy with low intensity laser irradiation has shown to be effective in promoting the proliferation of different cells. The aim of this in vitro study was to evaluate the potential effect of laser phototherapy (660 nm) on human dental pulp stem cell (hDPSC) proliferation. Study Design/Materials and Methods: The hDPSC cell strain was used. Cells cultured under nutritional deficit (10% FBS) were either irradiated or not (control) using two different power settings (20 mW/6 seconds to 40 mW/3 seconds), with an InGaAIP diode laser. The cell growth was indirectly assessed by measuring the cell mitochondrial activity through the MTT reduction-based cytotoxicity assay. Results: The group irradiated with the 20 mW setting presented significantly higher MTT activity at 72 hours than the other two groups (negative control-10% FBSand lased 40 mW with 3 seconds exposure time). After 24 hours of the first irradiation, cultures grown under nutritional deficit (10% FBS) and irradiated presented significantly higher viable cells than the non-irradiated cultures grown under the same nutritional conditions. Conclusions: Under the conditions of this study it was possible to conclude that the cell strain hDPSC responds positively to laser phototherapy by improving the cell growth when cultured under nutritional deficit conditions. Thus, the association of laser phototherapy and hDPSC cells could be of importance for future tissue engineering and regenerative medicine. Moreover, it opens the possibility of using laser phototherapy for improving the cell growth of other types of stem cells.

Tooth Tissue Engineering: Optimal Dental Stem Cell Harvest Based on Tooth Development

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications.

Stem cell Researches in Brazil: Present and Future Challenges

A bill allowing researches with human embryonic stem cells has been approved by the Brazilian Congress, originally in 2005 and definitively by the Supreme Court in 2008. However, several years before, investigations in Brazil with adult stem cells in vitro in animal models as well as clinical trials, were started and are currently underway. Here, we will summarize the main findings and the challenges of going from bench to bed, focusing on heart, diabetes, cancer, craniofacial, and neuromuscular disorders. We also call attention to the importance of publishing negative results on experimental trials in scientific journals and websites. They are of great value to investigators in the field and may avoid the repeating of unsuccessful experiments. In addition, they could be referred to patients seeking information, aiming to protect them against financial and psychological harm.

Human Stem Cell Cultures from Cleft Lip/Palate Patients Show Enrichment of Transcripts Involved in Extracellular Matrix Modeling By Comparison to Controls

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.

Maintenance and differentiation of neural stem cells

The adult mammalian brain contains self-renewable, multipotent neural stem cells (NSCs) that are responsible for neurogenesis and plasticity in specific regions of the adult brain. Extracellular matrix, vasculature, glial cells, and other neurons are components of the niche where NSCs are located. This surrounding environment is the source of extrinsic signals that instruct NSCs to either self-renew or differentiate. Additionally, factors such as the intracellular epigenetics state and retrotransposition events can influence the decision of NSC`s fate into neurons or glia. Extrinsic and intrinsic factors form an intricate signaling network, which is not completely understood. These factors altogether reflect a few of the key players characterized so far in the new field of NSC research and are covered in this review. (C) 2010 John Wiley & Sons, Inc. WIREs Syst Biol Med 2011 3 107-114 DOI:10.1002/wsbm:100

Long-Term Culture of Mouse Embryonic Stem Cell-Derived Adherent Neurospheres and Functional Neurons

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.

Early modulation of inflammation by mesenchymal stem cell after acute kidney injury

Therapy with stem cells has showed to be promising for acute kidney injury (AKI), although how it works is still controversial. Modulation of the inflammatory response is one possible mechanism. Most of published data relies on early time and whether the protection is still maintained after that is not known. Here, we analyzed whether immune modulation continues after 24 h of reperfusion. MSC were obtained from male Wistar rats. After 3-5 passages, cells were screened for CD73, CD90, CD44, CD45, CD29 and CD 31. In addition, MSC were submitted to differentiation in adipocyte and in osteocyte. AKI was induced by bilaterally clamping of renal pedicles for 60 min. Six hours after injury, MSC (2 x 105 cells) were administered intravenously. MSC-treated animals presented the lowest serum creatinine compared to non-treated animals (24 h: 1.3 +/- 0.21 vs. 3.23 +/- 0.89 mg/dl, p<0.05). The improvement in renal function was followed by a lower expression of IL-1b, IL-6 and TNF-alpha and higher expression of IL-4 and IL-10. However, 48 h after reperfusion, this cytokine profile has changed. The decrease in Th1 cytokines was less evident and IL-6 was markedly up regulated. PCNA analysis showed that regeneration occurs faster in kidney tissues of MSC-treated animals than in controls at 24 h. And also ratio of Bcl-2/Bad was higher at treated animals after 24 and 48 h. Our data demonstrated that the immunomodulatory effects of MSC occur at very early time point, changing the inflammation profile toward a Th2 profile. (C) 2009 Elsevier B.V. All rights reserved.

Administration of Neural Precursor Cells Ameliorates Renal Ischemia-Reperfusion Injury

In this study we evaluated whether administration of stem cells of neural origin (neural precursor cells, NPCs) could be protective against renal ischemia-reperfusion injury (IRI). We hypothesized that stem cell outcomes are not tissue-specific and that NPCs can improve tissue damage through paracrine mechanisms, especially due to immunomodulation. To this end, Wistar rats (200-250 g) were submitted to 1-hour ischemia and treated with NPCs (4 x 10(6) cells/animal) at 4 h of reperfusion. To serve as controls, ischemic animals were treated with cerebellum homogenate harvested from adult rat brain. All groups were sacrificed at 24 h of reperfusion. NPCs were isolated from rat fetus telencephalon and cultured until neurosphere formation (7 days). Before administration, NPCs were labeled with carboxyfluorescein diacetate succinimydylester (CFSE). Kidneys were harvested for analysis of cytokine profile and macrophage infiltration. At 24 h, NPC treatment resulted in a significant reduction in serum creatinine (IRI + NPC 1.21 + 0.18 vs. IRI 3.33 + 0.14 and IRI + cerebellum 2.95 + 0.78mg/dl, p < 0.05) and acute tubular necrosis (IRI + NPC 46.0 + 2.4% vs. IRI 79.7 + 14.2%, p < 0.05). NPC-CFSE and glial fibrillary acidic protein (GFAP)-positive cells (astrocyte marker) were found exclusively in renal parenchyma, which also presented GFAP and SOX-2 (an embryonic neural stem cell marker) mRNA expression. NPC treatment resulted in lower renal proinflammatory IL1-beta and TNF-alpha expression and higher anti-inflammatory IL-4 and IL-10 transcription. NPC-treated animals also had less macrophage infiltration and decreased serum proinflammatory cytokines (IL-1 beta, TNF-alpha and INF-gamma). Our data suggested that NPC therapy improved renal function by influencing immunological responses. Copyright (C) 2009 S. Karger AG, Basel

Involvement of Eukaryotic Translation Initiation Factor 5A (eIF5A) in Skeletal Muscle Stem Cell Differentiation

The eukaryotic translation initiation factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post-translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non-differentiated satellite cells and skeletal muscle. The treatment with NI-guanyl- 1,7-diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L-Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration.

ASPM gene expression in medulloblastoma

Medulloblastomas are the most common malignant tumors of the central nervous system in childhood. The incidence is about 19-20% between children younger than 16 years old with peak incidence between 4 and 7 years. Despite its sensibility to no specific therapeutic means like chemotherapy and radiotherapy, the treatment is very aggressive and frequently results in regression, growth deficit, and endocrine dysfunction. From this point of view, new treatment approaches are needed such as molecular targeted therapies. Studies in glioblastoma demonstrated that ASPM gene was overexpressed when compared to normal brain and ASPM inhibition by siRNA-mediated inhibits tumor cell proliferation and neural stem cell proliferation, supporting ASPM gene as a potential molecular target in glioblastoma. The aim of this work was to evaluate ASPM expression in medulloblastoma fragment samples, and to compare the results with the patient clinical features. Analysis of gene expression was performed by quantitative PCR real time using SYBR Green system in tumor samples from 37 children. The t test was used to analyze the gene expression, and Mann-Whitney test was performed to analyze the relationship between gene expressions and clinical characteristics. Kaplan-Meier test evaluated curve survival. All samples overexpressed ASPM gene more than 40-fold. However, we did not find any association between the overexpressed samples and the clinical parameters. ASPM overexpression may modify the ability of stem cells to differentiate during the development of the central nervous system, contributing to the development of medulloblastoma, a tumor of embryonic origin from cerebellar progenitor cells.

Limited Ca(2+) and PKA-pathway dependent neurogenic differentiation of human adult mesenchymal stem cells as compared to fetal neuronal stem cells

The ability of mesenchymal stem cells to generate functional neurons in culture is still a matter of controversy. In order to assess this issue, we performed a functional comparison between neuronal differentiation of human MSCs and fetal-derived neural stem cells (NSCs) based on morphological, immunocytochemical, and electrophysiological criteria. Furthermore, possible biochemical mechanisms involved in this process were presented. NF200 immunostaining was used to quantify the yield of differentiated cells after exposure to CAMP. The addition of a PKA inhibitor and Ca(2+) blockers to the differentiation medium significantly reduced the yield of differentiated cells. Activation of CREB was also observed on MSCs during maturation. Na(+)-, K(+)-, and Ca(2+)-voltage-dependent currents were recorded from MSCs-derived cells. In contrast, significantly larger Na(+) currents, firing activity, and spontaneous synaptic currents were recorded from NSCs. Our results indicate that the initial neuronal differentiation of MSCs is induced by CAMP and seems to be dependent upon Ca(2+) and the PKA pathway. However, compared to fetal neural stem cells, adult mesenchymal counterparts are limited in their neurogenic potential. Despite the similar yield of neuronal cells, NSCs achieved a more mature functional state. Description of the underlying mechanisms that govern MSCs` differentiation toward a stable neuronal phenotype and their limitations provides a unique opportunity to enhance our understanding of stem cell plasticity. (C) 2009 Elsevier Inc. All rights reserved.

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results

PURPOSE: Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.

Alveolar osseous defect in rat for cell therapy: preliminary report

PURPOSE: To study were to reproduce an alveolar bone defect model in Wistar rats to be used for testing the efficacy of stem cell therapies. Additionally, we also aimed to determine the osteogenesis process of this osseous defect in the 1 month period post-surgery. METHODS: The animals were randomly divided into two groups of 7 animals each. A gingivobuccal incision was made, and a bone defect of 28 mm² of area was performed in the alveolar region. Animals were killed at 2 weeks after surgery (n=7) and 4 weeks after surgery (n=7). RESULTS: The average area of the alveolar defect at time point of 2 weeks was 22.27 ± 1.31 mm² and the average area of alveolar defect at time point of 4 weeks was 9.03 ± 1.17 mm². The average amount of bone formation at time point of 2 weeks was 5.73 ± 1.31 mm² and the average amount of bone formation at time point of 4 weeks was 19 ± 1.17 mm². Statistically significant differences between the amount of bone formation at 2 weeks and 4 weeks after surgery were seen (p=0.003). CONCLUSION: The highest rate of ossification occurred mostly from 2 to 4 weeks after surgery. This observation suggests that 4 weeks after the bone defect creation should be a satisfactory timing to assess the potential of bone inductive stem cells to accelerate bone regeneration in Wistar rats.

Establishment of a protocol for obtention of neuronal stem cells lineages from the dog olfactory epithelium

A morphological and cell culture study from nasal mucosa of dogs was performed in order to establish a protocol to obtain a cell population committed to neuronal lineage, as a proposal for the treatment of traumatic and degenerative lesions in these animals, so that in the future these results could be applied to the human species. Twelve mongrel dogs of 60-day aged pregnancy were collected from urban pound dogs in São Paulo. Tissue from cribriform ethmoidal lamina of the fetuses was collected at necropsy under sterile conditions around 1h to 2h postmortem by uterine sections and sections from the fetal regions described above. Isolated cells of this tissue were added in DMEM/F-12 medium under standard conditions of incubation (5% CO², >37ºC). Cell culture based on isolated cells from biopsies of the olfactory epithelium showed rapid growth when cultured for 24 hours, showing phase-bright sphere cells found floating around the fragments, attached on culture flasks. After 20 days, a specific type of cells, predominantly ellipsoids or fusiform cells was characterized in vitro. The indirect immunofluorescence examination showed cells expressing markers of neuronal precursors (GFAP, neurofilament, oligodendrocyte, and III â-tubulin). The cell proliferation index showed Ki67 immunostaining with a trend to label cell groups throughout the apical region, while PCNA immunostaining label predominantly cell groups lying above the basal lamina. The transmission electron microscopy from the olfactory epithelium of dogs revealed cells with electron-dense cytoplasm and preserving the same distribution as those of positive cell staining for PCNA. Metabolic activity was confirmed by presence of euchromatin in the greatest part of cells. All these aspects give subsidies to support the hypothesis about resident progenitor cells among the basal cells of the olfactory epithelium, committed to renewal of these cell populations, especially neurons.