NGN2 mmRNA-Based Transcriptional Programming in Microfluidic Guides hiPSCs Toward Neural Fate With Multiple Identities

Recent advancements in cell engineering have succeeded in manipulating cell identity with the targeted overexpression of specific cell fate determining transcription factors in a process named transcriptional programming. Neurogenin2 (NGN2) is sufficient to instruct pluripotent stem cells (PSCs) to acquire a neuronal identity when delivered with an integrating system, which arises some safety concerns for clinical applications. A non-integrating system based on modified messenger RNA (mmRNA) delivery method, represents a valuable alternative to lentiviral-based approaches. The ability of NGN2 mmRNA to instruct PSC fate change has not been thoroughly investigated yet. Here we aimed at understanding whether the use of an NGN2 mmRNA-based approach combined with a miniaturized system, which allows a higher transfection efficiency in a cost-effective system, is able to drive human induced PSCs (hiPSCs) toward the neuronal lineage. We show that NGN2 mRNA alone is able to induce cell fate conversion. Surprisingly, the outcome cell population accounts for multiple phenotypes along the neural development trajectory. We found that this mixed population is mainly constituted by neural stem cells (45% \ub1 18 PAX6 positive cells) and neurons (38% \ub1 8 \u3b2IIITUBULIN positive cells) only when NGN2 is delivered as mmRNA. On the other hand, when the delivery system is lentiviral-based, both providing a constant expression of NGN2 or only a transient pulse, the outcome differentiated population is formed by a clear majority of neurons (88% \ub1 1 \u3b2IIITUBULIN positive cells). Altogether, our data confirm the ability of NGN2 to induce neuralization in hiPSCs and opens a new point of view in respect to the delivery system method when it comes to transcriptional programming applications

Hypoxia inducible factor‐2α importance for migration, proliferation, and self‐renewal of trunk neural crest cells

Background: The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)‐2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development. Results: Here, we show that HIF‐2α is expressed in trunk neural crest cells of human, murine, and avian embryos. Knockdown as well as overexpression of HIF‐2α in vivo causes developmental delays, induces proliferation, and self‐renewal capacity of neural crest cells while decreasing the proportion of neural crest cells that migrate ventrally to sympathoadrenal sites. Reflecting the in vivo phenotype, transcriptome changes after loss of HIF‐2α reveal enrichment of genes associated with cancer, invasion, epithelial‐to‐mesenchymal transition, and growth arrest. Conclusions: Taken together, these results suggest that expression levels of HIF‐2α must be strictly controlled during normal trunk neural crest development and that dysregulated levels affects several important features connected to stemness, migration, and development

A compact multifunctional microfluidic platform for exploring cellular dynamics in real-time using electrochemical detection

Downscaling of microfluidic cell culture and detection devices for electrochemical monitoring has mostly focused on miniaturization of the microfluidic chips which are often designed for specific applications and therefore lack functional flexibility. We present a compact microfluidic cell culture and electrochemical analysis platform with in-built fluid handling and detection, enabling complete cell based assays comprising on-line electrode cleaning, sterilization, surface functionalization, cell seeding, cultivation and electrochemical real-time monitoring of cellular dynamics. To demonstrate the versatility and multifunctionality of the platform, we explored amperometric monitoring of intracellular redox activity in yeast (Saccharomyces cerevisiae) and detection of exocytotically released dopamine from rat pheochromocytoma cells (PC12). Electrochemical impedance spectroscopy was used in both applications for monitoring cell sedimentation and adhesion as well as proliferation in the case of PC12 cells. The influence of flow rate on the signal amplitude in the detection of redox metabolism as well as the effect of mechanical stimulation on dopamine release were demonstrated using the programmable fluid handling capability. The here presented platform is aimed at applications utilizing cell based assays, ranging from e.g. monitoring of drug effects in pharmacological studies, characterization of neural stem cell differentiation, and screening of genetically modified microorganisms to environmental monitoring

Real-time monitoring of cellular dynamics using a microfluidic cell culture system with integrated electrode array and potentiostat

A versatile microfluidic, multichamber cell culture and analysis system with an integrated electrode array and potentiostat suitable for electrochemical detection and microscopic imaging is presented in this paper. The system, which allows on-line electrode cleaning and modification, was developed for real-time monitoring of cellular dynamics, exemplified in this work by monitoring of redox metabolism inside living yeast cells and dopamine release from PC12 cell

High serum levels of transforming growth factor β1 are associated with increased cortical thickness in cingulate and right frontal areas in healthy subjects

ABSTRACT

Semaphorin 6A Improves Functional Recovery in Conjunction with Motor Training after Cerebral Ischemia

Background: We have previously identified Semaphorin 6a (Sema6A) as an upregulated gene product in a gene expression screen in cortical ischemia [1]. Semaphorin 6a was regulated during the recovery phase following ischemia in the cortex. Semaphorin 6a is a member of the superfamily of semaphorins involved in axon guidance and other functions. We hypothesized that the upregulation indicates a crucial role of this molecule in post-stroke rewiring of the brain. Here we have tested this hypothesis by overexpressing semaphorin 6a in the cortex by microinjection of a modified AAV2-virus. A circumscribed cortical infarct was induced, and the recovery of rats monitored for up to 4 weeks using a well-established test battery (accelerated rotarod training paradigm, cylinder test, adhesive tape removal). We observed a significant improvement in post-ischemic recovery of animals injected with the semaphorin 6a virus versus animals treated with a control virus. We conclude that semaphorin 6a overexpressed in the cortex enhances recovery after cerebral ischemia

Short-Term Withdrawal of Mitogens Prior to Plating Increases Neuronal Differentiation of Human Neural Precursor Cells

Background: Human neural precursor cells (hNPC) are candidates for neural transplantation in a wide range of neurological disorders. Recently, much work has been done to determine how the environment for NPC culture in vitro may alter their plasticity. Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) are used to expand NPC; however, it is not clear if continuous exposure to mitogens may abrogate their subsequent differentiation. Here we evaluated if short-term removal of FGF-2 and EGF prior to plating may improve hNPC differentiation into neurons.Principal Findings: We demonstrate that culture of neurospheres in suspension for 2 weeks without EGF-FGF-2 significantly increases neuronal differentiation and neurite extension when compared to cells cultured using standard protocols. in this condition, neurons were preferentially located in the core of the neurospheres instead of the shell. Moreover, after plating, neurons presented radial rather than randomly oriented and longer processes than controls, comprised mostly by neurons with short processes. These changes were followed by alterations in the expression of genes related to cell survival.Conclusions: These results show that EGF and FGF-2 removal affects NPC fate and plasticity. Taking into account that a three dimensional structure is essential for NPC differentiation, here we evaluated, for the first time, the effects of growth factors removal in whole neurospheres rather than in plated cell culture.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Institutos do Milenio de Bioengenharia TecidualUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Ciencias Biomed, BR-21941 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilFAPESP: fellowCNPq: fellowWeb of Scienc