Damos las gracias a K. Deisseroth y S. Je para las construcciones lentiviral ChR2 y Synapsin1-tdTomato respectivamente, C. Chai para el intercambio de conocimientos y el protocolo en la generación de IPSC, WY Leong para el apoyo técnico, los miembros del laboratorio Goh para compartir reactivos y conocimientos. Este trabajo fue apoyado por Abbott Nutrición y el Centro Académico de Excelencia (ACE) premio de investigación de GlaxoSmithKline (GSK) para ELG, por la Fundación Nacional de Investigación de Singapur bajo su Programa de Investigación Competitiva (NRF 2008 NRF-CRP 002-082) a ELG y GJA, y por el (WCI) Programa del Instituto de clase mundial de la Fundación Nacional de Investigación de Corea (NRF), financiado por el Ministerio de Educación, Ciencia y Tecnología de Corea (MEST) (NRF número de concesión: WCI 2009-003) a GJA

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No conflicts of interest declared.

La optogenética proporciona precisión temporal y espacial para la activación de las poblaciones definidas de neuronas 13. En nuestros experimentos, todo el campo de el objetivo del microscopio se iluminó durante 30 segundos a sólo neuronas corticales foto-estimular expresan ChR2. Esto nos permitió determinar si las conexiones sinápticas se formaron entre diferentes poblaciones de neuronas dentro del co-cultivo. Nuestros resultados revelaron que la fotoestimulación aumenta la frecuencia PSC en neuronas derivadas de IPSC, lo que demuestra que estas neuronas reciben entrada sináptica de las neuronas corticales presináptica expresan ChR2. Esto, a su vez, estableció que las neuronas derivadas de IPSC incorporado con éxito en circuitos con las neuronas corticales. Hay varios pasos críticos para la generación de este sistema de co-cultivo. Es importante asegurarse de que los cultivos de astrocitos derivados de NPC ratón son saludables, con la muerte celular mínima, antes de proceder con el chapadode otros tipos de células. Las neuronas corticales y NPCs humanos atribuyen bien en astrocitos sanos y registros electrofisiológicos dependen en gran medida de las condiciones de cultivo. Los otros pasos clave en este protocolo son la electroporación y chapado de neuronas corticales en cultivos de astrocitos. Como un gran número de células mueren después de la electroporación, es importante asegurarse de que al menos 6 millones de neuronas corticales se electroporación para placas en cultivos de astrocitos en placas de 24 pocillos. Hemos observado que cuando se sometieron a electroporación menos de 6 millones de células, el número de neuronas que sobreviven no forman una red neuronal densa, que afectó registros electrofisiológicos. El número de neuronas corticales electroporación también debe ser coherente para cada experimento de co-cultivo para permitir la comparación entre los diferentes estudios. Para todas las etapas que implican la digestión enzimática, es esencial no dejar las células en solución digestivo durante demasiado tiempo ya que puede conducir a la muerte celular. Esteenfoque puede ser utilizado para detectar la capacidad de las neuronas derivadas de fibroblastos de pacientes específicos para formar contactos sinápticos con otras neuronas. Las neuronas derivadas de fibroblastos de pacientes que padecen el trastorno del desarrollo neurológico Síndrome de Rett (RTT) exhiben defectos de la transmisión sináptica: RTT neuronas muestran una disminución significativa en la frecuencia de PSC y la amplitud en comparación con las neuronas sanas, presumiblemente debido a la menor conectividad sináptica 14. Nuestro sistema de co-cultivo permite el examen de los efectos del fármaco sobre las neuronas que muestran defectos de desarrollo. Esto puede llevarse a cabo mediante la adición de compuestos de interés durante la siembra de NPC humanos enfermos, así como durante la exposición continua a los compuestos a lo largo del tiempo de la diferenciación neuronal. Si bien este sistema de co-cultivo también se puede utilizar como un modelo para las pruebas de drogas, una limitación de este enfoque es que el proceso de investigación de los efectos de cada compuesto candidato puede ser largo y tedioso comono es un método de cribado de alto rendimiento. Tras el tratamiento con los compuestos candidatos, neuronas derivadas de IPSC tienen que ser parcheado individualmente para determinar los efectos de cada compuesto sobre PSC. Por otra parte, en estos momentos no hay muchos disponibles fibroblastos y células iPS de pacientes. Por lo tanto, será de interés en el futuro próximo para tener más células del paciente y también para adaptar este protocolo para la selección de alto rendimiento. Por ejemplo, por marcación de neuronas derivadas de IPSC con un indicador de actividad, tales como sensores genéticamente codificados de iones o el potencial de membrana, sería posible aumentar la tasa de rendimiento sustancialmente por el procedimiento electrofisiológico tiempo-escalonamiento lado. Como todo el proceso de generación de este sistema de co-cultivo, desde la reprogramación de fibroblastos para la realización de registros electrofisiológicos, requiere más de 90 días, se recomienda que, o bien las células iPS o NPCs humanos ampliarse, después de la reprogramación y pasos de inducción neural, respectivamente,y criopreservados para reducir el tiempo necesario para futuros experimentos. En nuestros experimentos, expresamos ChR2 en neuronas corticales bajo el promotor synapsin1. Debido a que este promotor es pan-neuronal, que presumiblemente estaban photostimulating ambas neuronas corticales inhibidoras y excitadoras. Si el objetivo de los experimentos futuros es interrogar específicamente circuitos inhibitorios o excitatorios o bien, se podrían utilizar los promotores más específicos. Alternativamente, las neuronas corticales podrían obtenerse a partir de transgénicos (o virus inyectado ratones) donde la expresión ChR2 se dirige específicamente a las subpoblaciones genéticamente definidas de neuronas. Por ejemplo, los tejidos corticales de cosecha de un ratón que tiene la recombinasa Cre expresan en interneuronas que contiene parvalbúmina (interneuronas PV) que se cruza con un ratón con floxed ChR2 dará lugar a una situación en la que las únicas neuronas corticales que expresan ChR2 serán interneuronas PV 15. El uso de tales ChR2 interneuronas que expresan en nuestros co-culture sistema permitirá a la determinación de si una neurona parcheado derivados de IPSC es capaz de incorporar en un circuito con interneuronas PV. Basado en un estudio previo 9, las neuronas corticales son maduros con dendritas superposición después de 14 días in vitro. Por lo tanto, si la fotoestimulación no provoca una respuesta de un patched neurona derivada de IPSC, se debe indicar que la neurona no tiene la capacidad de hacer conexiones sinápticas con las neuronas corticales. Una neurona derivada de IPSC es capaz de recibir la entrada sináptica, tanto de otras neuronas derivadas de IPSC o neuronas corticales. Si se utilizaron grabaciones de patch clamp tradicional, no sería posible distinguir donde la entrada sináptica está viniendo. La ventaja de nuestro sistema es que las respuestas postsinápticas de entrada presináptica cortical pueden ser evocados y se identificaron selectivamente. Los procedimientos descritos aquí proporcionan un método sencillo para evaluar la capacidad de las neuronas derivadas de IPSC para integraren una red neuronal definido.

En los últimos años, nuestra comprensión de la neurona y la función del circuito neuronal han sido revolucionada por varias herramientas optogenética que controlan la excitabilidad neuronal. Una de estas herramientas es el canal catiónico activado por la luz, canalrodopsina-2 (ChR2): las neuronas que expresan ChR2 potenciales de acción del fuego en respuesta a la estimulación de la luz 1.3. Debido a que las neuronas no son normalmente sensibles a la luz, esta notable capacidad abre nuevas vías para el control temporal y espacial precisa de la actividad de las poblaciones genéticamente definidas de neuronas 4 y ha estudios de la función cerebral acelerado enormemente. ChR2 se ha aplicado a la investigación de células madre para diferentes propósitos, desde el seguimiento del desarrollo neuronal 5 a la actividad de las redes neuronales 6 regulación. Aquí hemos utilizado ChR2 para aumentar la utilidad de un sistema de co-cultivo neuronal. Sistemas de co-cultivo permiten la evaluación de las interacciones entre los diferentes tipos de células.Co-cultivos de neuronas y células gliales, los principales tipos de células del sistema nervioso, se utilizan comúnmente para investigar la señalización entre estos diferentes tipos de células, así como para evaluar la importancia de esta señalización para las respuestas fisiológicas, la propagación de los daños y para examinar la mecanismos moleculares que están potencialmente implicadas en esta señalización 7. Un estudio anterior reveló que iPS humanas se diferencian muy rápidamente en las neuronas de la presencia de la cultura que contiene neuronas de rata corticales primarias, los astrocitos, los oligodendrocitos y microglia 8. En este protocolo, se demuestra un método que utiliza ChR2 en un sistema de co-cultivo para interrogar a las conexiones sinápticas entre las neuronas corticales de rata y las neuronas derivadas de células madre pluripotentes inducidas humanas (iPSCs). Describimos pasos para diseccionar y tejidos cerebrales cosecha 9, así como a mantener y diferenciar ratón células progenitoras neurales (CPN) en astrocitos y NPCs humanos INTneuronas o para crear el sistema de co-cultivo. Para establecer este sistema de co-cultivo, se utilizó el método de reprogramación-integración libre descrito por Okita et al. 10 para generar células iPS humanas. Estos iPSCs fueron entonces diferenciarse eficientemente en NPC en condiciones definidas químicamente utilizando inhibidores de molécula pequeña tal como se describe por Li et al. 11 En los co-cultivos, las diferentes poblaciones de neuronas se pueden identificar a través de la detección de la expresión de ChR2 en neuronas corticales y etiquetado de neuronas derivadas de IPSC a través de la proteína fluorescente, en tándem de dímero de tomate (tdTomato). La combinación de registros electrofisiológicos de neuronas derivadas de IPSC con fotoestimulación optogenético de las neuronas corticales permite la evaluación de la capacidad de estos dos tipos de neuronas para formar circuitos entre sí. En concreto, la fotoestimulación de ChR2 que expresan las neuronas corticales evocan respuestas sinápticas en neuronas derivadas de IPSC que se han formado los circuitos sinápticoscon la red de neuronas corticales fotoestimulada. Se demuestra que el aumento de las corrientes postsinápticas están efectivamente detectados en neuronas derivadas de IPSC en tales condiciones. Este protocolo permite la evaluación de los circuitos sinápticos bajo una variedad de condiciones experimentales, incluyendo recapitulación de diferentes modelos de enfermedades neurológicas mediante el uso de células iPS derivadas de fibroblastos de pacientes con la enfermedad.

an optogenetic approach for assessing formation of neuronal connections in a co-culture system

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

Aquí, un protocolo que describe los varios pasos implicados en la generación de un co-cultivo que consiste en astrocitos, neuronas corticales y neuronas humanas se ilustra. CMPI humanas se obtuvieron mediante la reprogramación de fibroblastos usando vectores episomales de 10 y especificados en un linaje neuronal con un cóctel de inhibidores de molécula pequeña, por lo que NPC 11 que se mantuvieron a alta densidad (Figura 1A). Se requieren varios pasos para generar el co-cultivo: diferenciación de los NPCs de ratón en los astrocitos, chapado de neuronas corticales de rata que expresan ChR2, y la siembra de NPCs humanos etiquetados con tdTomato (Figura 1B). En el día 2 de diferenciación, glial fibrilar ácida de la proteína (GFAP) + astrocitos pueden ser fácilmente observados en nuestros cultivos (Figura 1C). Ratón NPC se les permitió diferenciarse por lo menos una semana antes de chapado neuronas corticales que expresan ChR2 sobre la monocapa de astrocitos (Figura 1D). Después de 34 semanas de diferenciación NPC humana, se detectaron las neuronas tdTomato + en las culturas (Figura 1E). Este sistema de co-cultivo permite la detección coherente de las corrientes postsinápticas de neuronas derivadas de IPSC individuales en respuesta a la estimulación de luz (Figura 2). Cuando son estimulados por la luz, los potenciales de acción se generaron en las neuronas corticales que expresan ChR2 (Figura 2B). IPSC derivado de las neuronas también son excitables (Figura 2C), que muestra el aumento de potencial de acción disparando como la amplitud de la despolarización de pulsos de corriente se incrementó (Figura 2D). Por lo tanto, estas células exhiben propiedades neuronales maduros. En la ausencia de luz, neuronas derivadas de IPSC recibieron entradas sinápticas espontáneas (Figura 2E). Estas corrientes hacia el interior postsinápticos se mediada principalmente por los receptores AMPA, porque las corrientes a través de los receptores GABA serían hacia fuera y los receptores NMDA no llevan una corrientet el potencial de mantenimiento de -70 mV. Al menos algunas de estas entradas eran de neuronas corticales presinápticos que expresan ChR2, debido a la estimulación de luz se incrementó en gran medida la frecuencia de las corrientes postsinápticas (Figura 2E). El curso temporal de este aumento de la entrada sináptica se muestra en la Figura 2F: fotoestimulación de las neuronas corticales se mantuvo a lo largo del 30 seg destello de luz larga. Si bien la frecuencia de PSC fue elevado cuando ChR2 que expresan las neuronas corticales fueron fotoestimuladas (Figura 2G), su amplitud no fue afectado (Figura 2H). En resumen, estos resultados indican que las neuronas derivadas de IPSC exhiben propiedades neuronales maduros y podrían formar conexiones sinápticas con las neuronas corticales presinápticas. <img alt="Figura 1" src="/files/ftp_upload/52408/52408fig1highres.jpg" /> Figura 1: Diagramas esquemáticos para la generación de co-cultivosistema. (A) Línea de tiempo para reprogramar fibroblastos humanos en células iPS y la inducción neural a los NPCs. (B) Plazo para la diferenciación terminal de NPCs humanos en neuronas maduras en cultivos de neuronas y astrocitos corticales. (C) Ratón NPCs diferenciar rápidamente en GFAP + astrocitos después de 2 días in vitro. (D) Después de una semana, las neuronas corticales que expresan ChR2 se sembraron en GFAP + astrocitos. (E) A las 4 a 5 semanas después de la diferenciación de los NPCs humanos, registros electrofisiológicos se realizaron en tdTomato + neuronas. Las barras de escala representan 100 micras. <img alt="Figura 2" src="/files/ftp_upload/52408/52408fig2highres.jpg" /> Figura 2: Análisis optogenética de conectividad sináptica funcional. derivados de IPSC (A) células etiquetadas con tdTomato (rojo) fueron co-cultivadas con neuronas corticales que expresa la luz-activadocanal, ChR2 (verde). Las grabaciones de neuronas derivadas de IPSC revelaron actuales (PSC) respuestas postsinápticas, que podrían provenir de cualquiera de las neuronas corticales u otras neuronas derivadas de IPSC. (B) Iluminación (barra azul) evoca una serie de potenciales de acción en las neuronas corticales que expresan ChR2. ( derivados de las células IPSC C) son evocados por inyección de corriente. (D) La cocción frente a la curva actual de las células derivadas de IPSC. (E) Fotoestimulación de las neuronas corticales ChR2 expresan (barra azul) aumentaron la frecuencia de las ventanillas únicas en las neuronas derivadas de IPSC . Por supuesto (F) Tiempo del aumento en la frecuencia PSC producido por fotoestimulación. Barra azul indica el tiempo de fotoestimulación. (G, H) mientras que la frecuencia PSC se incrementó por la fotoestimulación (G), la amplitud PSC no se vio afectada (H). * Indica p &lt;0,05 en comparación con el control con la prueba t de Student.

Watch this Scientific Journal Video about An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System at JoVE.com

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Un enfoque optogenética para Evaluar formación de conexiones neuronales en un sistema de co-cultura

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

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Developmental Biology

13.3K vistas.  Duke-NUS Graduate Medical School. A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2. 

Video: An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Cultura y Co-cultura de ratón ovarios y los folículos ováricos

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Investigación

Biología del Desarrollo

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

La encapsulación de alginato de células madre pluripotentes utilizando una boquilla-Co axial

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Enfoque preciso de la ablación celular para modelar lesiones renales agudas en el desarrollo de pez cebra

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Un enfoque estandarizado para la purificación multiespecífica de células germinales masculinas de mamíferos por disociación mecánica de tejidos y citometría de flujo

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

El ensayo de co-cultivo del anillo aórtico: Una herramienta conveniente para evaluar el potencial angiogénico de las células mesenquimales estromales In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Un modelo In Vitro de un sistema de placas paralelas de la perfusión para el estudio de adherencia bacteriana a los tejidos de injerto

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Sistema de ensayo de angiogénesis tridimensional utilizando esferoides de cocultura formados por células formadoras de colonias endoteliales y células madre mesenquimales

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Un método de cocultura para estudiar el crecimiento de Neurita en respuesta a las señales de paracrina de pulpa dental

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Un sistema optimizado de O9-1/hidrogel para estudiar señales mecánicas en células de cresta neural

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

Expresión de GFP inducida por la luz en embriones de pez cebra utilizando el sistema optogenético TAEL/C120

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...