Cell cycle dynamics of mouse embryonic stem cells in the ground state and during transition to formative pluripotency.

Mouse embryonic stem cells (mESCs) can be maintained as homogeneous populations in the ground state of pluripotency. Release from this state in minimal conditions allows to obtain cells that resemble those of the early post-implantation epiblast, providing an important developmental model to study cell identity transitions. However, the cell cycle dynamics of mESCs in the ground state and during its dissolution have not been extensively studied. By performing live imaging experiments of mESCs bearing cell cycle reporters, we show here that cells in the pluripotent ground state display a cell cycle structure comparable to the reported for mESCs in serum-based media. Upon release from self-renewal, the cell cycle is rapidly accelerated by a reduction in the length of the G1 phase and of the S/G2/M phases, causing an increased proliferation rate. Analysis of cell lineages indicates that cell cycle variables of sister cells are highly correlated, suggesting the existence of inherited cell cycle regulators from the parental cell. Together with a major morphological reconfiguration upon differentiation, our findings support a correlation between this in vitro model and early embryonic events.

The Effects of Photobiomodulation Delivered by Light-Emitting Diode on Stem Cells from Human Exfoliated Deciduous Teeth: A Study on the Relevance to Pluripotent Stem Cell Viability and Proliferation.

OBJECTIVE: Photobiomodulation (PBM) can modulate the proliferation of some types of stem cells. However, few reports have addressed the effects of PBM delivered by light-emitting diode (LED) on stem cells obtained from the pulp tissue of deciduous teeth. The aim of the present study was to investigate the effect of PBM delivered by red LED (630 nm, 75 mW, 37 mW/cm2) with different radiant exposures on the cell cycle, mitochondrial membrane potential, and senescence of stem cells from human exfoliated deciduous teeth (SHED). MATERIALS AND METHODS: Cultures were irradiated with LED (2, 4, 8, 16, and 32 J/cm2). After 24 h, the cell cycle and mitochondrial membrane potential of the cultures were evaluated using flow cytometry. Nonirradiated cultures served as control. RESULTS: Cultures irradiated with 16 J/cm2 had higher percentages of cells in the synthesis phase than control cultures (p < 0.05), and no significant differences were found regarding the percentage of cells with viable mitochondria between irradiated and control cultures. No significant difference in cell senescence was found between control cultures and cultures irradiated with 2 or 16 J/cm2. CONCLUSIONS: LED irradiation at 630 nm (37 mW/cm2, 75 mW) with radiant exposure of 16 J/cm2 was capable of inducing a proliferative response in stem cells from the pulp tissue of deciduous teeth without affecting mitochondrial function or inducing senescence.

Injury-induced purinergic signalling molecules upregulate pluripotency gene expression and mitotic activity of progenitor cells in the zebrafish retina.

Damage in fish activates retina repair that restores sight. The purinergic signalling system serves multiple homeostatic functions and has been implicated in cell cycle control of progenitor cells in the developing retina. We examined whether changes in the expression of purinergic molecules were instrumental in the proliferative phase after injury of adult zebrafish retinas with ouabain. P2RY1 messenger RNA (mRNA) increased early after injury and showed maximal levels at the time of peak progenitor cell proliferation. Extracellular nucleotides, mainly ADP, regulate P2RY1 transcriptional and protein expression. The injury-induced upregulation of P2RY1 is mediated by an autoregulated mechanism. After injury, the transcriptional expression of ecto-nucleotidases and ecto-ATPases also increased and ecto-ATPase activity inhibitors decreased Müller glia-derived progenitor cell amplification. Inhibition of P2RY1 endogenous activation prevented progenitor cell proliferation at two intervals after injury: one in which progenitor Müller glia mitotically activates and the second one in which Müller glia-derived progenitor cells amplify. ADPßS induced the expression of lin28a and ascl1a genes in mature regions of uninjured retinas. The expression of these genes, which regulate multipotent Müller glia reprogramming, was significantly inhibited by blocking the endogenous activation of P2RY1 early after injury. We consistently observed that the number of glial fibrillary acidic protein-BrdU-positive Müller cells after injury was larger in the absence than in the presence of the P2RY1 antagonist. Ecto-ATPase activity inhibitors or P2RY1-specific antagonists did not modify apoptotic cell death at the time of peak progenitor cell proliferation. The results suggested that ouabain injury upregulates specific purinergic signals which stimulates multipotent progenitor cell response.

Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5-azacytidine and cultured in differentiation medium containing BMP2, BMP4 or follicular fluid.

This study aims to investigate the effect 5-azacytidine (5-Aza) during induction of pluripotency in bovine fibroblasts and to evaluate the effects of BMP2, BMP4 or follicular fluid in the differentiation of reprogrammed fibroblasts in primordial germ cells and oocytes. It also analysis the mRNA levels for OCT4, NANOG, REX, SOX2, VASA, DAZL, cKIT, SCP3, ZPA and GDF9 after culturing 5-Aza treated fibroblasts in the different tested medium. Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 µM of 5-Aza for 18 h, 36 h or 72 h. Then, the cells were cultured in DMEM/F12 supplemented with 10 ng/ml BMP2, 10 ng/ml BMP4 or 5% follicular fluid. After culture, morphological characteristics, viability and gene expression were evaluated by qPCR. Treatment of skin fibroblasts with 2.0 µM 5-Aza for 72 h significantly increased expression of mRNAs for SOX2, OCT4, NANOG and REX. The culture in medium supplemented with BMP2, BMP4 or follicular fluid for 7 or 14 days induced formation of oocyte-like cells, as well as the expression of markers for germ cells and oocyte. In conclusion, treatment of bovine skin-derived fibroblasts with 2.0 µM 5-Aza for 72 h induces the expression of pluripotency factors. Culturing these cells in differentiation medium supplemented with BMP2, BMP4 or follicular fluid induces morphological changes and promotes expression of markers for germ cells, meiosis and oocyte.

Morphological, molecular and FTIR spectroscopic analysis during the differentiation of kidney cells from pluripotent stem cells.

BACKGROUND: Kidney diseases are a global health problem. Currently, over 2 million people require dialysis or transplant which are associated with high morbidity and mortality; therefore, new researches focused on regenerative medicine have been developed, including the use of stem cells. RESULTS: In this research, we generate differentiated kidney cells (DKCs) from mouse pluripotent stem cells (mPSCs) analyzing their morphological, genetic, phenotypic, and spectroscopic characteristics along differentiation, highlighting that there are no reports of the use of Fourier transform infrared (FTIR) spectroscopy to characterize the directed differentiation of mPSCs to DKCs. The genetic and protein experiments proved the obtention of DKCs that passed through the chronological stages of embryonic kidney development. Regarding vibrational spectroscopy analysis by FTIR, bands related with biomolecules were shown on mPSCs and DKCs spectra, observing distinct differences between cell lineages and maturation stages. The second derivative of DKCs spectra showed changes in the protein bands compared to mPSCs. Finally, the principal components analysis obtained from FTIR spectra allowed to characterize chemical and structurally mPSCs and their differentiation process to DKCs in a rapid and non-invasive way. CONCLUSION: Our results indicated that we obtained DKCs from mPSCs, which passed through the chronological stages of embryonic kidney development. Moreover, FTIR spectroscopy resulted in a non-invasive, rapid and precise technic that together with principal component analysis allows to characterize chemical and structurally both kind of cells and also discriminate and determine different stages along the cell differentiation process.

Generation of LIF-independent induced pluripotent stem cells from canine fetal fibroblasts.

Takahashi and Yamanaka established the first technique in which transcription factors related to pluripotency are incorporated into the genome of somatic cells to enable reprogramming of these cells. The expression of these transcription factors enables a differentiated somatic cell to reverse its phenotype to an embryonic state, generating induced pluripotent stem cells (iPSCs). iPSCs from canine fetal fibroblasts were produced through lentiviral polycistronic human and mouse vectors (hOSKM/mOSKM), aiming to obtain pluripotent stem cells with similar features to embryonic stem cells (ESC) in this animal model. The cell lines obtained in this study were independent of LIF or any other supplemental inhibitors, resistant to enzymatic procedure (TrypLE Express Enzyme), and dependent on bFGF. Clonal lines were obtained from slightly different protocols with maximum reprogramming efficiency of 0.001%. All colonies were positive for alkaline phosphatase, embryoid body formation, and spontaneous differentiation and expressed high levels of endogenous OCT4 and SOX2. Canine iPSCs developed tumors at 120 days post-injection in vivo. Preliminary chromosomal evaluations were performed by FISH hybridization, revealing no chromosomal abnormality. To the best of our knowledge, this report is the first to describe the ability to reprogram canine somatic cells via lentiviral vectors without supplementation and with resistance to enzymatic action, thereby demonstrating the pluripotency of these cell lines.

Morphological, molecular and FTIR spectroscopic analysis during the differentiation of kidney cells from pluripotent stem cells

BACKGROUND: Kidney diseases are a global health problem. Currently, over 2 million people require dialysis or transplant which are associated with high morbidity and mortality; therefore, new researches focused on regenerative medicine have been developed, including the use of stem cells. RESULTS: In this research, we generate differentiated kidney cells (DKCs) from mouse pluripotent stem cells (mPSCs) analyzing their morphological, genetic, phenotypic, and spectroscopic characteristics along differentiation, highlighting that there are no reports of the use of Fourier transform infrared (FTIR) spectroscopy to characterize the directed differentiation of mPSCs to DKCs. The genetic and protein experiments proved the obtention of DKCs that passed through the chronological stages of embryonic kidney development. Regarding vibrational spectroscopy analysis by FTIR, bands related with biomolecules were shown on mPSCs and DKCs spectra, observing distinct differences between cell lineages and maturation stages. The second derivative of DKCs spectra showed changes in the protein bands compared to mPSCs. Finally, the principal components analysis obtained from FTIR spectra allowed to characterize chemical and structurally mPSCs and their differentiation process to DKCs in a rapid and non-invasive way. CONCLUSION: Our results indicated that we obtained DKCs from mPSCs, which passed through the chronological stages of embryonic kidney development. Moreover, FTIR spectroscopy resulted in a non-invasive, rapid and precise technic that together with principal component analysis allows to characterize chemical and structurally both kind of cells and also discriminate and determine different stages along the cell differentiation process.

AKT/GSK3ß signaling pathway is critically involved in human pluripotent stem cell survival.

Human embryonic and induced pluripotent stem cells are self-renewing pluripotent stem cells (PSC) that can differentiate into a wide range of specialized cells. Basic fibroblast growth factor is essential for PSC survival, stemness and self-renewal. PI3K/AKT pathway regulates cell viability and apoptosis in many cell types. Although it has been demonstrated that PI3K/AKT activation by bFGF is relevant for PSC stemness maintenance its role on PSC survival remains elusive. In this study we explored the molecular mechanisms involved in the regulation of PSC survival by AKT. We found that inhibition of AKT with three non-structurally related inhibitors (GSK690693, AKT inhibitor VIII and AKT inhibitor IV) decreased cell viability and induced apoptosis. We observed a rapid increase in phosphatidylserine translocation and in the extent of DNA fragmentation after inhibitors addition. Moreover, abrogation of AKT activity led to Caspase-9, Caspase-3, and PARP cleavage. Importantly, we demonstrated by pharmacological inhibition and siRNA knockdown that GSK3ß signaling is responsible, at least in part, of the apoptosis triggered by AKT inhibition. Moreover, GSK3ß inhibition decreases basal apoptosis rate and promotes PSC proliferation. In conclusion, we demonstrated that AKT activation prevents apoptosis, partly through inhibition of GSK3ß, and thus results relevant for PSC survival.

Cell therapy for Parkinson's disease: Functional role of the host immune response on survival and differentiation of dopaminergic neuroblasts.

Parkinson's disease (PD) is a neurodegenerative disorder, whose cardinal pathology is the loss of dopaminergic neurons in the substantia nigra. Current treatments for PD have side effects in the long term and do not halt disease progression or regenerate dopaminergic cell loss. Attempts to compensate neuronal cell loss by transplantation of dopamine-producing cells started more than 30 years ago, leading to several clinical trials. These trials showed safety and variable efficacy among patients. In addition to variability in efficacy, several patients developed graft-induced dyskinesia. Nevertheless, they have provided a proof of concept that motor symptoms could be improved by cell transplantation. Cell transplantation in the brain presents several immunological challenges. The adaptive immune response should be abolished to avoid graft rejection by the host. In addition, the innate immune response will always be present after transplanting cells into the brain. Remarkably, the innate immune response can have dramatic effects on the survival, differentiation and proliferation of the transplanted cells, but has been hardly investigated. In this review, we analyze data on the functional effects of signals from the innate immune system on dopaminergic differentiation, survival and proliferation. Then, we discussed efforts on cell transplantation in animal models and PD patients, highlighting the immune response and the immunomodulatory treatment strategies performed. The analysis of the available data lead us to conclude that the modulation of the innate immune response after transplantation can increase the success of future clinical trials in PD by enhancing cell differentiation and survival. This article is part of a Special Issue entitled SI: PSC and the brain.

Avances en la medicina reconstructiva: células madre y células madre inducidas / Advances in reconstructive medicine: stem cells and induced stem cells

La utilización de células indiferenciadas embrionarias y de células diferenciadas inducidas para que se comporten como las anteriores permite dar origen adiferentes tejidos que pueden ser usados en medicina reconstructiva en reemplazo de los deteriorados...

Therapeutic Options in Idiopathic Burning Mouth Syndrome: Literature Review

Introduction Burning mouth syndrome (BMS) is characterized by a burning sensation in the tongue, palate, lips, or gums of no well-defined etiology. The diagnosis and treatment for primary BMS are controversial. No specific laboratory tests or diagnostic criteria are well established, and the diagnosis is made by excluding all other possible disorders. Objective To review the literature on the main treatment options in idiopathic BMS and compare the best results of the main studies in 15 years. Data Synthesis We conducted a literature review on PubMed/MEDLINE, SciELO, and Cochrane-BIREME of work in the past 15 years, and only selected studies comparing different therapeutic options in idiopathic BMS, with preference for randomized and double-blind controlled studies. Final Comments Topical clonazepam showed good short-term results for the relief of pain, although this was not presented as a definitive cure. Similarly, α-lipoic acid showed good results, but there are few randomized controlled studies that showed the longterm results and complete remission of symptoms. On the other hand, cognitive therapy is reported as a good and lasting therapeutic option with the advantage of not having side effects, and it can be combined with pharmacologic therapy. .

The role of DNA repair in the pluripotency and differentiation of human stem cells.

All living cells utilize intricate DNA repair mechanisms to address numerous types of DNA lesions and to preserve genomic integrity, and pluripotent stem cells have specific needs due to their remarkable ability of self-renewal and differentiation into different functional cell types. Not surprisingly, human stem cells possess a highly efficient DNA repair network that becomes less efficient upon differentiation. Moreover, these cells also have an anaerobic metabolism, which reduces the mitochondria number and the likelihood of oxidative stress, which is highly related to genomic instability. If DNA lesions are not repaired, human stem cells easily undergo senescence, cell death or differentiation, as part of their DNA damage response, avoiding the propagation of stem cells carrying mutations and genomic alterations. Interestingly, cancer stem cells and typical stem cells share not only the differentiation potential but also their capacity to respond to DNA damage, with important implications for cancer therapy using genotoxic agents. On the other hand, the preservation of the adult stem cell pool, and the ability of cells to deal with DNA damage, is essential for normal development, reducing processes of neurodegeneration and premature aging, as one can observe on clinical phenotypes of many human genetic diseases with defects in DNA repair processes. Finally, several recent findings suggest that DNA repair also plays a fundamental role in maintaining the pluripotency and differentiation potential of embryonic stem cells, as well as that of induced pluripotent stem (iPS) cells. DNA repair processes also seem to be necessary for the reprogramming of human cells when iPS cells are produced. Thus, the understanding of how cultured pluripotent stem cells ensure the genetic stability are highly relevant for their safe therapeutic application, at the same time that cellular therapy is a hope for DNA repair deficient patients.

Matrix-bound heparan sulfate is essential for the growth and pluripotency of human embryonic stem cells.

Human embryonic stem (hES) cell production of heparan sulfate influences cell fate and pluripotency. Human ES cells remain pluripotent in vitro through the action of growth factors signaling, and the activity of these factors depends on interaction with specific receptors and also with heparan sulfate. Here, we tested the hypothesis that matrix-associated heparan sulfate is enough to maintain hES cells under low fibroblast growth factor-2 concentration in the absence of live feeder cells. To pursue this goal, we compared hES cells cultured either on coated plates containing live murine embryonic fibroblasts (MEFs) or on a matrix derived from ethanol-fixed MEFs. hES cells were analyzed for the expression of pluripotency markers and the ability to form embryoid bodies. hES cells cultured either on live mouse fibroblasts or onto a matrix derived from fixed fibroblasts expressed similar levels of Oct-4, SOX-2, Nanog, TRA-1-60 and SSEA-4, and they were also able to form cavitated embryoid bodies. Heparan sulfate-depleted matrix lost the ability to support the adherence and growth of hES cells, confirming that this glycosaminoglycan, bound to the extracellular matrix, is enough for the growth and attachment of hES cells. Finally, we observed that the ethanol-fixed matrix decreases by 30% the levels of Neu5Gc in hES cells, indicating that this procedure reduces xeno-contamination. Our data suggest that matrix-bound heparan sulfate is required for the growth and pluripotency of hES cells and that ethanol-fixed MEFs may be used as a "live cell"-free substrate for stem cells.

Potencialidad Celular Evolutiva y Medicina Regenerativa / Potential Evolutionary Cell and Regenerative Medicine

Las células derivadas del embrioblasto o macizo celular interno (MCI) del blastocisto corresponden a las células madres embrionarias, ellas son pluripotentes, debido a que tienen la capacidad de generar todas las estirpes celulares y al resto, se les conoce como células madres adultas porque tienen un potencial de diferenciación mucho más restringido. Dentro de las células adultas podemos reconocer las células madre hematopoyéticas de la médula ósea y las células madres mesenquimales presentes en casi todos los tejidos conectivos adultos y destinadas a regenerar y reparar tejidos. Se las ha descrito formando parte de la médula ósea, en el mesénquima del cordón umbilical, en el tejido adiposo y en el tejido nervioso. La obtención y utilización de células madres de tejidos embrionarios y adultos es el tema de interés actual, en una nueva área de la medicina que pretende regenerar órganos y funciones. En esta revisión se analizan los aspectos más interesantes de la obtención de células madres, con sus respectivas proyecciones en medicina humana y veterinaria.

Embryoblast derived cells or inner cell mass (ICM) of blastocysts correspond to embryonic stem cells, are pluripotent them, because they have the capacity to generate all cell lines and the remainder are known as stem cells adults because they have the potential for much more restricted differentiation. Within cells can recognize adult hematopoietic stem cells and bone marrow mesenchymal stem cells present in nearly all connective tissues and intended adult tissue repair and regeneration. They have been described as part of the bone marrow, umbilical cord mesenchyme in adipose tissue and nerve tissue. The collection and use of embryonic stem cells and adult tissues is the subject of current interest in a new area of medicine that aims to regenerate organs and functions. In this paper, we analyze the most interesting aspects of obtaining stem cells, with their projections in human and veterinary medicine.

Derivation and potential applications of pluripotent stem cells for regenerative medicine in horses

Background: The ability to create tissues using pluripotent stem cells to repair or replace tissue lost due to damage, i.e. regenerative medicine, is developing very rapidly in many fields of human medicine. For veterinarians, regenerative medicine has focused mainly in the use of stem cells for arthritis and tendon ligament repair, indicating a need for treating musculoskeletal injuries. Our objective is to review the available approaches being used to derive pluripotent stem cells and discuss their potential use for regenerative medicine in the horse. Review: Adult adipose- and bone marrow-derived mesenchymal stem cells (MSC) are being used in practice to treat injuries in horses. However, there is scarce scientific evidence of their effectiveness and little is known of the mechanisms by which such cell preparations improve the healing process. For instance, although early healing response of articular cartilage injury was improved by treatment with injection of MSC, they did not enhance the long-term tissue response, indicating that cell proliferation was attenuated. Better protocols for the isolation and clinical testing of equine MSC are required to confirm healing properties. In contrast to MSC, embryonic stem cells (ESC) derived from the inner-cell- mass (ICM) of blastocyst stage embryos carry the ability to proliferate indefinitely in vitro and, given appropriate and favorable conditions, can differentiate into any tissue in the body. Parthenogenesis (PG) and somatic cell nuclear transfer (SCNT) are used to obtain a genetic match to the host animal and, thereby, eliminate the risk of inducing immune rejection of the grafted tissue. However, apart from the typical markers of pluripotency, equine ESC also express markers of trophoblastic tissues, indicating that they are different and possibly less able to differentiate than the ESC lines obtained in other species. Consequently, further studies are underway to identify conditions to obtain fully pluripotent ESC lines from equine SCNT embryos. To overcome the limitations of ESC lines derived from equine embryos, induced pluripotent stem cells (iPSC) were derived using a piggyBac transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally controlled fashion. Our established fetal-derived iPSC lines express hallmark pluripotency markers, display a stable karyotype after prolonged culture, and are able to form teratomas in immunodeficient mice containing tissues from all three embryonic layers. By establishing a protocol for deriving stable iPSC lines in the horse, we expect that new opportunities will be shortly developed for regenerative therapies in this species. Conclusion: It is possible to derive autologous pluripotent stem cells in horses by using both ESC and iPSC-derived approaches. Although ESC lines are generally the gold standard of pluripotency, further research is required to improve the proliferative and pluripotency characteristics for clinical applications. On the other hand, equine iPSC show excellent stability during prolonged in vitro culture and have the capacity to differentiate into the three germ layers in vivo, suggesting that they could soon be used in pre-clinical trials. Therefore, further studies need to be performed to establish reliable protocols for assessing the regenerative properties of iPS and ESC for equine muscle-skeletal injuries.

Epiblast embryo stem cells give origin to adult pluripotent cell populations: primordial germ cell, pericytic and haematopoyetic stem cells: a review / Las células madre del epiblasto dan origen a poblaciones de células pluripotentes adultas: células germinales primordiales, células madre pericíticas y hematopoyéticas: revisión

Adult stem cells are great promise to the future of regenerative therapy, and understanding of its embryonic origin permit the discrimination of stem cell sources. Embryonic stem cells derived from inner cell mass of blastocyst originate the primordial germ cells, and pericyte stem cell associated to vessels endothelium in yolk sac. Currently, it is being proposed that embryonic primordial germ cell could originate hematopoietic stem cells based on the detection of germ cell markers (SSEA-1/TEC-1, Oct-4 and Nanog) in stem cell harvested from fetal liver and bone marrow. However, different experimental evidence points at two separate differentiation routes toward primordial germ cells, and hematopoietic stem cell with the same embryonic origin. The expression of undifferentiated stem cell markers in umbilical cord and placental vessels, such CD34, CXCR4, c-kit and OCT4 demonstrates the intimate relation between pericyte stem cells, endothelium, haematopoiesis, and primordial germ cells, which all originate from embryonic stem cell from the inner cell mass epiblast.

Las células madre adultas son una gran promesa para el futuro de la terapia regenerativa, y la comprensión de su origen embrionario permite la discriminación de las fuentes de células madre. Las células madre embrionarias derivadas del macizo celular interno del blastocisto originan las células germinales primordiales, y células madre pericíticas asociadas al endotelio de los vasos del saco vitelino. En la actualidad, se propone que las células germinales primordiales embrionarias podrían originar a las células madre hematopoyéticas sobre la base de la detección de marcadores de células germinales (SSEA-1/TEC-1 oct-4 y Nanog) en células madre extraídas de hígado fetal y médula ósea. Sin embargo, diferentes evidencias experimentales apuntan hacia dos vías separadas de diferenciación en células germinales primordiales, y en células madre hematopoyéticas con el mismo origen embrionario. La expresión de marcadores de células madre no diferenciadas en el cordón umbilical y los vasos de la placenta, como CD34, CXCR4, c-kit y OcT4 demuestra la íntima relación entre las células madre pericíticas, el endotelio y las células germinales primordiales, las que se originan en células madre embrionarias a partir del epiblasto del macizo celular interno.

Stem cells / Células-tronco

Stem cells can be classified as embryonic stem (ES) cells or adult stem cells considering their origin. If plasticity is considered, stem cells can be classified as totipotent, when stem cells retain the ability to give rise to an entire new organism. When stem cells lose this capacity, cells are named pluripotent stem cells, which can give rise to almost all mature cell types that compound an organism. Totipotent and pluripotent stem cells can be obtained from developing early-stage embryos. Multipotent is the group of adult stem cells with restricted plasticity. These cells can differentiate into a defined cell type related with a specific organ or tissue. ES cells can be propagated in vitro under undifferentiated system or with a series of protocols to induce cell differentiation. On the other hand, multipotent adult stem cells cannot be maintained in vitro in an undifferentiated form, except for a special class of adherent adult stem cells named mesenchimal stem cells, which can be expanded in vitro conserving their undifferentiated characteristics. Considering the ability to generate teratomas, ES cells were not used in experimental in vivo cell transplant. On the other hand, several experimental adult stem cells transplants have been performed with controversial results

Considerando a origem de obtenção, as células-tronco podem ser classificadas como células-tronco embrionárias (ES) ou como células-tronco adultas. Mas, se a plasticidade for considerada, as células-tronco podem ser classificadas como células totipotentes, quando as células-tronco preservam a capacidade de dar origem a um novo indivíduo completo. Quando as células-tronco perdem esta capacidade, passam a ser classificadas como células-tronco pluripotentes, que podem dar origem a praticamente todos os tipos celulares maduros que compõem um organismo. Células-tronco totipotentes e pluripotentes podem ser obtidas de estágios embrionários iniciais. O grupo de células-tronco que apresenta plasticidade restrita é denominado de multipotente. Estas células podem se diferenciar em determinado tipo celular comprometido com um órgão ou tecido específico. Células ES podem ser expandidas in vitro, mantendo sua forma indiferenciada, ou podem ser submetidas a uma série de protocolos, que irão induzir diferenciação in vitro. Por outro lado, as células-tronco adultas multipotentes não podem ser mantidas in vitro na forma indiferenciada, exceto uma subpopulação de célulastronco adultas aderentes, denominadas células-tronco mesenquimais, que podem ser mantidas in vitro na forma indiferenciada. Considerando a capacidade de gerar teratomas, as células ES não foram utilizadas para transplante celular experimental in vivo. Além disso, várias cirurgias de transplantes experimentais com células-tronco adultas têm sido realizadas, porém apresentando resultados controversos

Glial-guided neuronal migration in P19 embryonal carcinoma stem cell aggregates.

During development of the nervous system, neuronal precursors that originated in proliferative regions migrate along radial glial fibers to reach their final destination. P19 embryonal carcinoma (EC) stem cells exposed to retinoic acid (RA) differentiate into neurons, glia, and fibroblast-like cells. In this work, we induced P19 aggregates for 4 days with RA and plated them onto tissue culture dishes coated with poly-L-lysine. Several cells migrated out of and/or extended processes from the aggregates after 24 hr. Some cell processes were morphologically similar to radial glial fibers and stained for glial fibrillar acidic protein (GFAP) and nestin. Large numbers of migrating cells showed characteristics similar to those of bipolar migrating neurons and expressed the neuronal marker microtubule-associated protein 2. Furthermore, scanning electron microscopy analysis revealed an intimate association between the radial fibers and the migrating cells. Therefore, the migration of neuron-like cells on radial glia fibers in differentiated P19 aggregates resembled some of the migration models used thus far to study gliophilic neuronal migration. In addition, HPTLC analysis in this system showed the expression of 9-O-acetyl GD3, a ganglioside that has been associated with neuronal migration. Antibody perturbation assays showed that immunoblockage of 9-O-acetyl GD3 arrested neuronal migration in a reversible manner. In summary, we have characterized a new cell culture model for investigation of glial-guided neuronal migration and have shown that 9-O-acetyl GD3 ganglioside has an important role in this phenomenon.