Human amniotic fluid cells form functional gap junctions with cortical cells.

The usage of stem cells is a promising strategy for the repair of damaged tissue in the injured brain. Recently, amniotic fluid (AF) cells have received a lot of attention as an alternative source of stem cells for cell-based therapies. However, the success of this approach relies significantly on proper interactions between graft and host tissue. In particular, the reestablishment of functional brain networks requires formation of gap junctions, as a key step to provide sufficient intercellular communication. In this study, we show that AF cells express high levels of CX43 (GJA1) and are able to establish functional gap junctions with cortical cultures. Furthermore, we report an induction of Cx43 expression in astrocytes following injury to the mouse motor cortex and demonstrate for the first time CX43 expression at the interface between implanted AF cells and host brain cells. These findings suggest that CX43-mediated intercellular communication between AF cells and cortical astrocytes may contribute to the reconstruction of damaged tissue by mediating modulatory, homeostatic, and protective factors in the injured brain and hence warrants further investigation.

Development of BMP7-producing human cells, using a third generation lentiviral gene delivery system.

Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor ß (TGF-ß) superfamily, plays important roles in the development of various tissues and organs in mouse and human. In particular, BMP7 is critical for the formation of the nervous system and it is considered to have therapeutic potential in brain injury and stroke. One approach to make BMP7 more suitable for therapeutic purposes is the development of efficient vectors that allow the consistent, reliable and cost-effective production of the BMP7 protein. In this study, we developed an efficient BMP7 delivery system, using a third generation lentiviral vector to produce functional BMP7 protein. The lentiviral transduction of several human cell types, including human embryonic kidney 293 (HEK293) cells, amniotic fluid cells, NTera2 neurons (NT2-N) and primary neuronal cultures resulted in BMP7 expression. The production of BMP7 protein was achieved for at least 4 weeks post-transduction, as determined by enzyme-linked immunosorbent assay (ELISA). SMAD phosphorylation and neuronal differentiation assays verified the bioactivity and functionality of the lentiviral-based BMP7 protein, respectively. In addition, the intracerebroventricular injection of the lentivirus resulted in exogenous BMP7 expression in both neurons and astrocytes in the mouse brain. Taken together, this gene delivery system provides a reliable source of functional BMP7 protein for future in vitro and in vivo studies.

Detection of acute brain injury by Raman spectral signature.

Brain injury can lead to irreversible tissue loss and functional deficit along with significant health care costs. Raman spectroscopy can be used as a non-invasive technique to provide detailed information on the molecular composition of diseased and damaged tissues. This technique was used to examine acute mouse brain injury, focusing on the motor cortex, a region directly involved in controlling execution of movement. The spectral profile obtained from the injured brain tissue revealed a markedly different signature, particularly in the amide I and amide III vibrational region when compared to that of healthy brain tissue. Most noticeably, there was a significant reduction of the amide I vibration at the acute injury site and the appearance of two distinct features at 1586 and 1618 cm(-1). Complementary immunohistochemical analysis of the injured brain tissue showed an abundant expression of Caspase 3 (a cysteine protease marker used for apoptosis), suggesting that the injury-induced specific Raman shifts may be correlated with cell death. Taken together, this study demonstrates that Raman spectroscopy can play an important role in detecting the changes that occur in the injured brain and provide a possible technology for monitoring the recovery process.

Two novel human NUMB isoforms provide a potential link between development and cancer.

We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

Novel RBPJ transcripts identified in human amniotic fluid cells.

The NOTCH signaling pathway plays important roles in stem cell maintenance, cell-fate determination and differentiation during development. Following ligand binding, the cleaved NOTCH intracellular domain (NICD) interacts directly with the recombinant signal binding protein for immunoglobulin kappa J region (RBPJ) transcription factor and the resulting complex targets gene expression in the nucleus. To date, four human RBPJ isoforms have been described in Entrez Gene, varying in the first 5'coding exons. Using an improved protocol, we were able to further identify all four known and five novel RBPJ transcript variants in human amniotic fluid (AF) cells, a cell type known for its stem cell characteristics. In addition, we used human embryonal carcinoma (EC) NTera2/D1 (NT2) cells and NT2-derived neuron and astrocytes to compare the expression pattern of RBPJ transcripts. Further examination of RBPJ transcripts showed that the novel splice variants contain open reading frames in-frame with the known isoforms, suggesting that they can putatively generate similar function proteins. All known and novel RBPJ transcripts contain the putative nuclear localization signal (NLS), an important component of RBPJ-mediated gene regulation.

Probing stemness and neural commitment in human amniotic fluid cells.

Recently, human amniotic fluid (AF) cells have attracted a great deal of attention as an alternative cell source for transplantation and tissue engineering. AF contains a variety of cell types derived from fetal tissues, of which a small percentage is believed to represent stem cell sub-population(s). In contrast to human embryonic stem (ES) cells, AF cells are not subject to extensive legal or ethical considerations; nor are they limited by lineage commitment characteristic of adult stem cells. However, to become therapeutically valuable, better protocols for the isolation of AF stem cell sub-populations need to be developed. This study was designed to examine the molecular components involved in self-renewal, neural commitment and differentiation of AF cells obtained at different gestational ages. Our results showed that, although morphologically heterogeneous, AF cells derived from early gestational periods ubiquitously expressed KERATIN 8 (K8), suggesting that the majority of these cells may have an epithelial origin. In addition, AF cells expressed various components of NOTCH signaling (ligands, receptors and target genes), a pathway involved in stem cell maintenance, determination and differentiation. A sub-population of K8 positive cells (<10%) co-expressed NESTIN, a marker detected in the neuroepithelium, neural stem cells and neural progenitors. Throughout the gestational periods, a much smaller AF cell sub-population (<1%) expressed pluripotency markers, OCT4a, NANOG and SOX2, from which SOX2 positive AF cells could be isolated through single cell cloning. The SOX2 expressing AF clones showed the capacity to give rise to a neuron-like phenotype in culture, expressing neuronal markers such as MAP2, NFL and NSE. Taken together, our findings demonstrated the presence of fetal cells with stem cell characteristics in the amniotic fluid, highlighting the need for further research on their biology and clinical applications.

Differentiation of mouse Neuro 2A cells into dopamine neurons.

Neuro 2A (N2a) is a mouse neural crest-derived cell line that has been extensively used to study neuronal differentiation, axonal growth and signaling pathways. A convenient characteristic of these cells is their ability to differentiate into neurons within a few days. However, most differentiation methods reported for N2a cells do not provide information about the neuronal types obtained after each treatment. In this study, we evaluated the generation of N2a dopamine neurons following treatment with a number of factors known to induce neuronal differentiation. Our results showed that N2a cells express Nurr-related factor 1 (Nurr1) and produce low levels of tyrosine hydroxylase (TH) and dopamine. Both TH and dopamine levels were significantly enhanced in the presence of dibutyryl cyclic adenosine monophosphate (dbcAMP), as evidenced by Western blot, immunocytochemistry and high performance liquid chromatography (HPLC). In contrast to dbcAMP, other factors such as transforming growth factor beta1 (TGF beta 1), bone morphogenetic protein 4 (BMP4), glial cell-derived neurotrophic factor (GDNF) and retinoic acid (RA) did not increase TH expression. Further investigation confirmed that the effect of dbcAMP on production of TH-positive neurons was mediated through cyclic AMP (cAMP) responsive element binding protein (CREB) and it was antagonized by RA. Thus, although various treatments can be used to generate N2a neurons, only dbcAMP significantly enhanced the formation of dopamine neurons. Taken together, this study provided a simple and reliable method to generate dopamine neurons for rapid and efficient physiological and pharmacological assays.

Astrocyte-secreted GDNF and glutathione antioxidant system protect neurons against 6OHDA cytotoxicity.

In recent years, GDNF has emerged as a protective and restorative agent in several models of neurodegeneration; however, the exact molecular mechanisms responsible for these effects are not yet fully understood. Here we examined the effects of astrocytes secreting GDNF on neurons subjected to 6OHDA toxicity using in vitro neuron-astroglia co-cultures. Astrocytes were transduced with lentiviral vectors carrying the GDNF gene under the control of either human glial fibrillary acidic protein or cytomegalovirus promoters. The overexpression of GDNF, regardless of the promoter employed, had no obvious adverse effects on astroglia and the engineered cells stably produced and secreted GDNF for extended periods of time (> or =3 weeks). These astrocytes very effectively protected neurons against 6OHDA, in both mouse and human co-culture systems. The neuroprotective effects were mediated not only by GDNF, but also by the antioxidant GSH since its depletion reduced the level of GDNF protection. Furthermore, neurons and astrocytes expressed different components of GDNF signaling complex, suggesting that they might utilize separate pathways to mediate autocrine and paracrine effects of GDNF.

Neuroregenerative strategies in the brain: emerging significance of bone morphogenetic protein 7 (BMP7).

Every year thousands of people suffer from brain injuries and stroke, and develop motor, sensory, and cognitive problems as a result of neuronal loss in the brain. Unfortunately, the damaged brain has a limited ability to enact repair and current modes of treatment are not sufficient to offset the damage. An extensive list of growth factors, neurotrophic factors, cytokines, and drugs has been explored as potential therapies. However, only a limited number of them may actually have the potential to effectively offset the brain injury or stroke-related problems. One of the treatments considered for future brain repair is bone morphogenetic protein 7 (BMP7), a factor currently used in patients to treat non-neurological diseases. The clinical application of BMP7 is based on its neuroprotective role in stroke animal models. This paper reviews the current approaches considered for brain repair and discusses the novel convergent strategies by which BMP7 potentially can induce neuroregeneration.

Role of Sox2 in the development of the mouse neocortex.

The mammalian neocortex is established from neural stem and progenitor cells that utilize specific transcriptional and environmental factors to create functional neurons and astrocytes. Here, we examined the mechanism of Sox2 action during neocortical neurogenesis and gliogenesis. We established a robust Sox2 expression in neural stem and progenitor cells within the ventricular zone, which persisted until the cells exited the cell cycle. Overexpression of constitutively active Sox2 in neural progenitors resulted in upregulation of Notch1, recombination signal-sequence binding protein-J (RBP-J) and hairy enhancer of split 5 (Hes5) transcripts and the Sox2 high mobility group (HMG) domain seemed sufficient to confer these effects. While Sox2 overexpression permitted the differentiation of progenitors into astroglia, it inhibited neurogenesis, unless the Notch pathway was blocked. Moreover, neuronal precursors engaged a serine protease(s) to eliminate the overexpressed Sox2 protein and relieve the repression of neurogenesis. Glial precursors and differentiated astrocytes, on the other hand, maintained Sox2 expression until they reached a quiescent state. Sox2 expression was re-activated by signals that triggered astrocytic proliferation (i.e., injury, mitogenic and gliogenic factors). Taken together, Sox2 appears to act upstream of the Notch signaling pathway to maintain the cell proliferative potential and to ensure the generation of sufficient cell numbers and phenotypes in the developing neocortex.

Neurogenesis and neuronal communication on micropatterned neurochips.

Neural networks are formed by accurate connectivity of neurons and glial cells in the brain. These networks employ a three-dimensional bio-surface that both assigns precise coordinates to cells during development and facilitates their connectivity and functionality throughout life. Using specific topographic and chemical features, we have taken steps towards the development of poly(dimethylsiloxane; PDMS) neurochips that can be used to generate and study synthetic neural networks. These neurochips have micropatterned structures that permit adequate cell positioning and support cell survival. Within days of plating, cells differentiate into neurons displaying excitability and communication, as evidenced by intracellular calcium oscillations and action potentials. The structural and functional capacities of such simple neural networks open up new opportunities to study synaptic communication and plasticity.