An Update on Human Stem Cell-Based Therapy in Parkinson's Disease.

CDATA[Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease and it is characterized by the progressive loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc). Current pharmacological treatments for PD are only symptomatic and unfortunately there is still no cure for this disorder. Stem cell technology has become an attractive option to investigate and treat PD. Indeed, transplantation of fetal ventral mesencephalic cells into PD brains have provided proof of concept that cell replacement therapy can be beneficial for some patients, greatly improving their motor symptoms. However, ethical and practical aspects of tissue availability limit its widespread clinical use. Hence, the need of alternative cell sources are based on the use of different types of stem cells. Stem cell-based therapies can be beneficial by acting through several mechanisms such as cell replacement, trophic actions and modulation of inflammation. Here we review recent and current remarkable clinical studies involving stem cell-based therapy for PD and provide an overview of the different types of stem cells available nowadays, their main properties and how they are developing as a possible therapy for PD treatment.

Resistance of subventricular neural stem cells to chronic hypoxemia despite structural disorganization of the germinal center and impairment of neuronal and oligodendrocyte survival.

Chronic hypoxemia, as evidenced in de-acclimatized high-altitude residents or in patients with chronic obstructive respiratory disorders, is a common medical condition that can produce serious neurological alterations. However, the pathogenesis of this phenomenon is unknown. We have found that adult rodents exposed for several days/weeks to hypoxia, with an arterial oxygen tension similar to that of chronically hypoxemic patients, manifest a partially irreversible structural disarrangement of the subventricular neurogenic niche (subventricular zone) characterized by displacement of neurons and myelinated axons, flattening of the ependymal cell layer, and thinning of capillary walls. Despite these abnormalities, the number of neuronal and oligodendrocyte progenitors, neuroblasts, and neurosphere-forming cells as well as the proliferative activity in subventricular zone was unchanged. These results suggest that neural stem cells and their undifferentiated progeny are resistant to hypoxia. However, in vivo and in vitro experiments indicate that severe chronic hypoxia decreases the survival of newly generated neurons and oligodendrocytes, with damage of myelin sheaths. These findings help explain the effects of hypoxia on adult neurogenesis and provide new perspectives on brain responsiveness to persistent hypoxemia.

Cxcl12/Cxcr4 signaling controls the migration and process orientation of A9-A10 dopaminergic neurons.

CXCL12/CXCR4 signaling has been reported to regulate three essential processes for the establishment of neural networks in different neuronal systems: neuronal migration, cell positioning and axon wiring. However, it is not known whether it regulates the development of A9-A10 tyrosine hydroxylase positive (TH(+)) midbrain dopaminergic (mDA) neurons. We report here that Cxcl12 is expressed in the meninges surrounding the ventral midbrain (VM), whereas CXCR4 is present in NURR1(+) mDA precursors and mDA neurons from E10.5 to E14.5. CXCR4 is activated in NURR1(+) cells as they migrate towards the meninges. Accordingly, VM meninges and CXCL12 promoted migration and neuritogenesis of TH(+) cells in VM explants in a CXCR4-dependent manner. Moreover, in vivo electroporation of Cxcl12 at E12.5 in the basal plate resulted in lateral migration, whereas expression in the midline resulted in retention of TH(+) cells in the IZ close to the midline. Analysis of Cxcr4(-/-) mice revealed the presence of VM TH(+) cells with disoriented processes in the intermediate zone (IZ) at E11.5 and marginal zone (MZ) at E14. Consistently, pharmacological blockade of CXCR4 or genetic deletion of Cxcr4 resulted in an accumulation of TH(+) cells in the lateral aspect of the IZ at E14, indicating that CXCR4 is required for the radial migration of mDA neurons in vivo. Altogether, our findings demonstrate that CXCL12/CXCR4 regulates the migration and orientation of processes in A9-A10 mDA neurons.

SFRP1 and SFRP2 dose-dependently regulate midbrain dopamine neuron development in vivo and in embryonic stem cells.

Secreted Frizzled related proteins (sFRPs) are a family of proteins that modulate Wnt signaling, which in turn regulates multiple aspects of ventral midbrain (VM) and dopamine (DA) neuron development. However, it is not known which Wnt signaling branch and what aspects of midbrain DA neuron development are regulated by sFRPs. Here, we show that sFRP1 and sFRP2 activate the Wnt/planar-cell-polarity/Rac1 pathway in DA cells. In the developing VM, sFRP1 and sFRP2 are expressed at low levels, and sFRP1-/- or sFRP2-/- mice had no detectable phenotype. However, compound sFRP1-/-;sFRP2-/- mutants revealed a Wnt/PCP phenotype similar to that previously described for Wnt5a-/- mice. This included an anteroposterior shortening of the VM, a lateral expansion of the Shh domain and DA lineage markers (Lmx1a and Th), as well as an accumulation of Nurr1+ precursors in the VM. In vitro experiments showed that, while very high concentrations of SFRP1 had a negative effect on cell survival, low/medium concentrations of sFRP1 or sFRP2 promoted the DA differentiation of progenitors derived from primary VM cultures or mouse embryonic stem cells (ESCs), mimicking the effects of Wnt5a. We thus conclude that the main function of sFRP1 and sFRP2 is to enhance Wnt/PCP signaling in DA cells and to regulate Wnt/PCP-dependent functions in midbrain development. Moreover, we suggest that low-medium concentrations of sFRPs may be used to enhance the DA differentiation of ESCs and improve their therapeutic application.

GDNF is predominantly expressed in the PV+ neostriatal interneuronal ensemble in normal mouse and after injury of the nigrostriatal pathway.

Glial cell line-derived neurotrophic factor (GDNF) is absolutely required for survival of dopaminergic (DA) nigrostriatal neurons and protect them from toxic insults. Hence, it is a promising, albeit experimental, therapy for Parkinson's disease (PD). However, the source of striatal GDNF is not well known. GDNF seems to be normally synthesized in neurons, but numerous reports suggest GDNF production in glial cells, particularly in the injured brain. We have studied in detail striatal GDNF production in normal mouse and after damage of DA neurons with MPTP. Striatal GDNF mRNA was present in neonates but markedly increased during the first 2-3 postnatal weeks. Cellular identification of GDNF by unequivocal histochemical methods demonstrated that in normal or injured adult animals GDNF is expressed by striatal neurons and is not synthesized in significant amounts by astrocytes or microglial cells. GDNF mRNA expression was not higher in reactive astrocytes than in normal ones. Approximately 95% of identified neostriatal GDNF-expressing cells in normal and injured animals are parvalbumin-positive (PV+) interneurons, which only represent ~0.7% of all striatal neurons. The remaining 5% of GDNF+ cells are cholinergic and somatostatin+ interneurons. Surprisingly, medium spiny projection neurons (MSNs), the vast majority of striatal neurons that receive a strong DA innervation, do not express GDNF. PV+ interneurons constitute an oscillatory functional ensemble of electrically connected cells that control MSNs' firing. Production of GDNF in the PV+ neurons might be advantageous to supply synchronous activity-dependent release of GDNF in broad areas of the striatum. Stimulation of the GDNF-producing striatal PV+ ensemble in PD patients could have therapeutic effects.

Characterization of the functional properties of the neuroectoderm in mouse Cripto(-/-) embryos showing severe gastrulation defects.

During development of the mammalian embryo, there is a complex relation between formation of the mesoderm and the neuroectoderm. In mouse, for example, the role of the node and its mesendoderm derivatives in anterior neural specification is still debated. Mouse Cripto(-/-) embryos could potentially help settle this debate because they lack almost all embryonic endoderm and mesoderm, including the node and its derivatives. In the present paper, we show that Cripto(-/-) embryos can still form functional neural stem cells that are able to differentiate and maintain a neural phenotype both in vivo and in vitro. These data suggest that signals emanating from the mesoderm and endoderm might not be essential for the formation and differentiation of neural stem cells. However, we use grafting experiments to show that the Cripto(-/-) isthmus (the secondary organizer located at the midbrain-hindbrain boundary) loses its inductive ability. We further show that the Cripto(-/-)isthmus expresses lower amounts of the isthmic signalling molecule, Fgf8. Since nearby tissues remain competent to respond to exogenously added Fgf8, this reduction in Fgf8 levels in the Cripto(-/-) isthmus is the potential cause of the loss of patterning ability in graft experiments. Overall, we interpret our data to suggest that the mammalian node and primitive streak are essential for the development of the regional identities that control the specification and formation of the secondary organizers within the developing brain.

Identification of midbrain floor plate radial glia-like cells as dopaminergic progenitors.

The floor plate (FP), a signaling center and a structure rich in radial glia-like cells, has been traditionally thought to be devoid of neurons and neuronal progenitors. However, in the midbrain, the FP contains neurons of the dopaminergic (DA) lineage that require contact with radial glia-like cells for their induction. We, therefore, decided to explore the interaction relationship between radial glia and neurons during DA neurogenesis. Taking advantage of a novel FP radial glia-like cell culture system and retroviruses, DA neurons were lineage traced in vitro. In utero BrdU pulse-chases extensively labeled the midbrain FP and traced DA neurons both in vivo and in FP cultures. Moreover, from E9.5 to E13.5 the midbrain FP contained dividing cells only in the most apical part of the neuroepithelium, in cells identified as radial glia-like cells. We, therefore, hypothesized that midbrain FP radial glia-like cells could be DA progenitors and tested our hypothesis in vivo. Lineage tracing of DA progenitors with EGFP in Tis21-EGFP knock-in mice, and genetic fate mapping in GLAST::CreERT2/ZEG mice identified the neuroepithelium of the midbrain FP, and specifically, GLAST+ radial glia-like cells as DA progenitors. Combined, our experiments support the concept that the midbrain FP differs from other FP regions and demonstrate that FP radial glia-like cells in the midbrain are neurogenic and give rise to midbrain DA neurons.

Neuroprotective effect of adult hematopoietic stem cells in a mouse model of motoneuron degeneration.

Degenerative spinal motor diseases, like amyotrophic lateral sclerosis, are produced by progressive degeneration of motoneurons. Their clinical manifestations include a progressive muscular weakness and atrophy, which lead to paralysis and premature death. Current pharmacological therapies fail to stop the progression of motor deficits or to restore motor function. The purpose of our study was to explore the possible beneficial effect of mouse adult hematopoietic stem cells (hSCs) transplanted into the spinal cord of a mouse model of motoneuron degeneration. Our results show that grafted hSCs survive in the spinal cord. In addition, the number of motoneurons in the transplanted spinal cord is larger than in non-transplanted mdf mice at the same spinal cord segments and importantly, motor function significantly improves. These effects can be explained by the increased levels of glial cell line derived neurotrophic factor (GDNF) around host motoneurons produced by the grafted cells. Thus, these experiments demonstrate the neuroprotective effect of adult hSCs in the model employed and indicate that this cell type may contribute to ameliorating motor function in degenerative spinal motor diseases.

Adult stem cell therapy: dream or reality?

Adult stem cells may be an invaluable source of plastic cells for tissue regeneration. The bone marrow contains different subpopulations of adult stem cells easily accessible for transplantation. However the therapeutic value of adult stem cell is a question of debate in the scientific community. We have investigated the potential benefits of adult hematopoietic stem cell transplantation in animal models of demyelinating and motor neuron diseases. Our results suggest that transplantation of HSC have direct and indirect neuroregenerative and neuroprotective effects.

An efficient method for the derivation of mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) represent a unique tool for many researchers; however, the process of ESC derivation is often very inefficient and requires high specialization, training, and expertise. To circumvent these limitations, we aimed to develop a simple and efficient protocol based on the use of commercially available products. Here, we present an optimized protocol that we successfully applied to derive ESCs from several knockout mouse strains (Wnt-1, Wnt-5a, Lrp6, and parkin) with 50%-75% efficiency. The methodology is based on the use of mouse embryonic fibroblast feeders, knockout serum replacement (SR), and minimal handling of the blastocyst. In this protocol, all centrifugation steps (as well as the use of trypsin inhibitor) were avoided and replaced by an ESC medium containing fetal calf serum (FCS) after the trypsinizations. We define the potential advantages and disadvantages of using SR and FCS in individual steps of the protocol. We also characterize the ESCs for the expression of ESC markers by immunohistochemistry, Western blot, and a stem cell focused microarray. In summary, we provide a simplified and improved protocol to derive mESCs that can be useful for laboratories aiming to isolate transgenic mESCs for the first time.

Derivation of mouse embryonic stem cells.

Here we describe a simple and efficient protocol for derivation of germline chimera-competent mouse embryonic stem cells (mESCs) from embryonic day 3.5 (E3.5) blastocysts. The protocol involves the use of early-passage mouse embryonic fibroblast feeders (MEF) and the alternation of fetal bovine serum- and serum replacement (SR)-containing media. As compared to other available protocols for mESCs derivation, our protocol differs in the combination of commercial availability of all reagents, technical simplicity and high efficiency. mESC lines are derived with approximately 50% efficiency (50 independent mESC lines derived from 96 blastocysts). We believe that this protocol could be a good starting point for (i) setting up the derivation of mESC lines in a laboratory and (ii) incorporating further steps to improve efficiency or adapt the protocol to other applications. The whole process (from blastocyst extraction to the freezing of mESC line) usually takes between 15 and 20 d.

Presencia de microorganismos productores de b-lactamasa en dientes de pacientes con periodontitis apical crónica supurativa / Presence of microorganisms producing b-lactamases in teeth of patients with suppurative chronic apical periodontitis

Este artículo une los resultados de trabajos de grado realizados en el 2002 y en 2004 que tienen como propósito determinar la presencia de microorganismos productores de b-lactamasas en dientes con periodontitis apical crónica supurativa. La segunda parte se encaminó a obtener una ampliación de la muestra obtenida en la primera etapa del presente estudio y poder evaluar los resultados con un muestreo más representativo. Se tomaron 39 muestras de conductos con diangóstico de periodontitis apical crónica supurativa en el Hospital Rafael Uribe Uribe, que posteriormente fueron llevados al laboratorio de la Universidad El Bosque, en un medio de transporte VMGAIII. Se identificaron los bacilos gram negativos potencialmente productores de b-lactamasas mediante el sistema Rapid ID32A(R)(Biomerieux), a los que se les reálizó prueba de b-lactamasa Cefinase(R)(Becton Dickinson). De un total de 39 muestras, 14 no se tomaron en cuenta por resultar negativas (ausencia de microorganismos); en las 25 restantes se aisló: Prevotella denticola, Porphyromonas gingivalis, Porphyromonas asacharolitica, Actinomyces Spp., Prevotella intermedia/nigrecens, Propionibacterium acnes, A. meyeri, Fusobacterium Spp., A. odontoliticus, Prevotella melaninogenica, Peptostreptococcus magnus, Campylobacter Spp., Peptoestreptococcus micros, Peptoestreptococcus anaerobios, Serratia Marscenscens, Eubacterium Spp., y Staphilococcus Spp. Se encontró producción de b-lactamasa en Pophyromonas asacharolitica (1/1), Prevotella denticola (1/1), Prevotella intermedia (3/3), Prevotella melaninogenica (1/1), Prevotella intermedia/nigrecens (3/5), Serratia Marscenscens (1/1). Con los resultados obtenidos se sugiere determinar en una próxima investigación si estos microorganismos intervienen en los procesos de agudización de la periodontitis apical crónica supurativa y posteriormente determinar una adecuada terapia antibiótica

Haematopoietic progenitor cells from adult bone marrow differentiate into cells that express oligodendroglial antigens in the neonatal mouse brain.

Stem cells are self-renewable, pluripotent cells that, in adult life, proliferate by a characteristic asymmetric division in which one daughter cell is committed to differentiation whereas the other remains a stem cell. These cells are also characterized by their ability to differentiate into various cell types under heterotopic environmental influences. In the present study, we have explored the potential of adult haematopoietic bone marrow cells to differentiate into cells of oligodendroglial lineage under physiological, active myelinating conditions. We present evidence of generation of cells expressing oligodendroglial specific markers from a bone marrow subpopulation enriched on adult haematopoietic progenitor cells (CD117+) in vivo after intracerebral transplantation into the neonatal mouse brain. Our results suggest that adult bone marrow cells have the capacity to undergo differentiation from haematopoietic to oligodendroglial cells and add support the validity of bone marrow transplants as an alternative treatment for demyelinating diseases of the CNS including Multiple Sclerosis.