Retinoic acid regulates Sox2 expression during neuronal and glial differentiation in mouse P19 cells

Retinoic acid (RA) is a small lipophilic molecule that plays important roles in embryonic development by regulating cell proliferation, signaling and differentiation. There is compelling evidence that RA signaling controls the expression of a number of transcription factors in a spatio-temporal manner during these processes. In particular, Sox2, a transcription factor known for its involvement in stem cell fate commitment and neurogenesis may serve as a target of RA. In this study, we have used mouse embryonic carcinoma (EC) P19 cells to examine the effect of RA on Sox2 expression during neuronal and glial differentiation. Undifferentiated P19 cells express abundant levels of Sox2 during proliferation, a fact further supported by Sox2-DNA interaction throughout various stages of mitosis. In the presence of RA, P19 cells form embryonic bodies (EB) and differentiate into neurons and astrocytes in a chronological order similar to that of cell differentiation in the brain. RT-PCR, western blot and immunocytochemistry analyses show that the level of Sox2 is significantly reduced in P19 cells, as they differentiate into neurons, whereas P19 astrocytes maintain Sox2 expression. These results show that Sox2 is differentially expressed during neurogenesis and gliogenesis, suggesting its distinct functions throughout these processes.NRC publication: Ye

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 \u3b2 (TGF-\u3b2) 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 BMP7deliverysystem, using athirdgenerationlentiviral vector to produce functional BMP7 protein. The lentiviral transduction of several humancell 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 genedeliverysystem provides a reliable source of functional BMP7 protein for future in vitro and in vivo studies.Peer reviewed: YesNRC publication: Ye

Involvement of NOS3 in RA-induced neural differentiation of human NT2/D1 cells

Nitric oxide (NO) plays a key role in neurogenesis as a regulator of cell proliferation and differentiation. NO is synthesized from the amino acid L-arginine by nitric oxide synthases (NOS1, NOS2, and NOS3), which are encoded by separate genes and display different tissue distributions. We used an in vitro model of RA-induced neural differentiation of NT2 cells to examine which of the three NO-synthesizing enzymes is involved in this process. The results revealed a transient induction of NOS3 (known as the constitutively expressed endothelial nitric oxide synthase; eNOS) during the time course of the RA treatment. The peak of gene expression and the nuclear presence of NOS3 protein coincided with cell cycle exit of NT2-derived neuronal precursors. The subsequent analysis of cytosine methylation and histone H3 acetylation of the human NOS3 5\u2032 regulatory sequences indicated that epigenetic modifications, especially upstream of the proximal promoter ( 12734 to 12989, relative to exon 2 TSS at +1), were also taking place. NOS1 was expressed only in the differentiated neurons (NT2-N), whereas NOS2 was not expressed at all in this cellular model. Thus, a burst of NO production, possibly required to inhibit neural cell proliferation, was generated by the transient expression of NOS3. This pattern of gene expression, in turn, required epigenetic remodeling of its regulatory region.Peer reviewed: YesNRC publication: Ye

Traumatic brain injury: classification, models, and markers.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Due to its high incidence rate and often long-term sequelae, TBI contributes significantly to increasing costs of health care expenditures annually. Unfortunately, advances in the field have been stifled by patient and injury heterogeneity that pose a major challenge in TBI prevention, diagnosis, and treatment. In this review, we briefly discuss the causes of TBI, followed by its prevalence, classification, and pathophysiology. The current imaging detection methods and animal models used to study brain injury are examined. We discuss the potential use of molecular markers in detecting and monitoring the progression of TBI, with particular emphasis on microRNAs as a novel class of molecular modulators of injury and its repair in the neural tissue

A novel human induced pluripotent stem cell blood-brain barrier model: Applicability to study antibody-triggered receptor-mediated transcytosis

Abstract We have developed a renewable, scalable and transgene free human blood-brain barrier model, composed of brain endothelial cells (BECs), generated from human amniotic fluid derived induced pluripotent stem cells (AF-iPSC), which can also give rise to syngeneic neural cells of the neurovascular unit. These AF-iPSC-derived BECs (i-BEC) exhibited high transendothelial electrical resistance (up to 1500 Ω cm2) inducible by astrocyte-derived molecular cues and retinoic acid treatment, polarized expression of functional efflux transporters and receptor mediated transcytosis triggered by antibodies against specific receptors. In vitro human BBB models enable pre-clinical screening of central nervous system (CNS)-targeting drugs and are of particular importance for assessing species-specific/selective transport mechanisms. This i-BEC human BBB model discriminates species-selective antibody- mediated transcytosis mechanisms, is predictive of in vivo CNS exposure of rodent cross-reactive antibodies and can be implemented into pre-clinical CNS drug discovery and development processes