eoe sub articles

EoE Sub Articles

1. Neural stem cell induction
NOTE: Depending on cell conditions, about 5 or 7 days after transfection, monolayer adherent cells will reach 30 - 50% confluence (Figure 1C).
<ol>
	<li>Remove the supernatants containing floating cells and spheres and then add 1 ml of neural progenitor medium (Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12), containing 1x N2 supplement, 0.1% (w/v) bovine serum albumin (BSA), 1% (v/v) antibiotics, 20 ng/ml of basic fibroblast growth factor (bFGF), and 20 ng/ml of epidermal growth factor (EGF)) into the adherent cells.</li>
	<li>Optionally, dissociate the cell spheres by gently pipetting and centrifuging the cells at 170 x g for 10 min. Remove the supernatant and resuspend the cells in 1 ml of neural progenitor medium in a new 24-well plate coated with matrigel per instruction as a backup.</li>
	<li>Change the medium every other day till the cells reach 60 - 80% confluent, usually after 1 week.</li>
	<li>Discard the supernatant and add in 1 ml of neural stem cell medium (serum-free medium, NSC SFM). Dissociate the cells using a cell scraper, followed by very gentle pipetting.</li>
	<li>Remove the cells and seed all of them into one well of a 6-well plate. Add another 1 ml of neural stem cell medium to make 2 ml media for each well. Incubate the cells at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Change the medium every other day.</li>
	<li>When cells reach 60% confluence, dissociate and replate the cells at a 1:3 ratio following the steps in 1.4 and 1.6.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell scraper</td>
			<td>Sarstedt</td>
			<td>83.183</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life technologies</td>
			<td>12400-024</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Life technologies</td>
			<td>17502-048</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A2934</td>
			<td>0.1% (w/v)</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life technologies</td>
			<td>17504-044</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>NSC serum free medium</td>
			<td>Life Technologies</td>
			<td>A1050901</td>
			<td>1X</td>
		</tr>
	</tbody>
</table>

direct reprogramming of hematopoietic progenitor cells into neural stem cells

Source: Wang, T., et al. <a href="https://app.jove.com/v/52298">Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells.</a> J. Vis. Exp, (2015)
This video demonstrates the direct reprogramming of genetically modified hematopoietic progenitor cells into neuronal stem cells. Transcription factors expressed by hematopoietic progenitor cells trigger a cascade of molecular events facilitating their transformation. Further specific media is used to select and grow induced neuronal stem cells.

<img alt="Figure 1" src="/files/ftp_upload/22403/22403_Figure1.jpg" /> 
Figure 1. Morphological changes of CD34 cells after Sendai virus transfection. (A) Significant number of CD34 cells can be observed at the time of transfection. (B) Sphere-like cell aggregates can be observed 24 hr after Sendai virus transfection, which expands during time. (C) Mostly, bipolar adherent cells appeared after 5 days of transfection....

Direct Reprogramming of Hematopoietic Progenitor Cells into Neural Stem Cells

1. Neural stem cell induction
NOTE: Depending on cell conditions, about 5 or 7 days after transfection, monolayer adherent cells will reach 30 - 50% ...

Begin with a cell suspension of genetically altered hematopoietic progenitor cells.
These cells express transcription factors required for their reprogramming into neural stem cells. This process is called direct reprogramming.
Transfer the cells to an extracellular matrix-coated multiwell plate with a neural progenitor medium, and incubate.
The extracellular matrix provides a three-dimensional scaffold for cell attachment and growth.
During incubation, transcription factors expressed by the hematopoietic progenitor cell initiate a sequence of molecular events that allow its transformation into a neural stem cell.
The neural progenitor medium contains factors that favor the survival of induced neural stem cells.
After incubation, replace this medium with a fresh neural progenitor medium to promote cell multiplication.
Detach the cells using a cell scraper.
Collect this suspension and transfer the cells to a well containing neural stem cell medium.
Incubate for maintenance of induced neural stem cells.

direct-reprogramming-hematopoietic-progenitor-cells-into-neural-stem

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

29 visualizações. Fonte: Wang, T., et al. Indução direta de células-tronco neurais humanas a partir de células progenitoras hematopoiéticas do sangue periférico. J. Vis. Exp, (2015) Este vídeo demonstra a reprogramação direta de células progenitoras hematopoiéticas geneticamente modificadas em células-tronco neuronais. Os fatores de transcrição expressos pelas células progenitoras hematopoiéticas desencadeiam uma cascata de eventos moleculares que facilitam sua transformação. Outros meios espec...

Vídeo: Reprogramação direta de células progenitoras hematopoiéticas em células-tronco neurais - Conceito

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.

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.

Uma Abordagem Optogenetic para Avaliar Formação das conexões neuronais em um 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 ...

JoVE Journal

Biologia do Desenvolvimento

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

Reprogramação Direta de Fibroblastos de Rato em Melanócitos

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Isolamento e Reprogramação Neuronal Direta de Astrócitos de Camundongos

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

Direct Reprogramming of Hematopoietic Progenitor Cells into Neural Stem Cells

Transcrição

Direct Reprogramming of Hematopoietic Progenitor Cells into Neural Stem Cells