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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Rat MSC Cultures
<ol>
	<li>Harvest of MSCs from the femur
	<ol>
		<li>Autoclave all dissection tools (i.e., fine dissecting scissors, blunt-tipped cutting scissors, and toothed forceps) at 180 &#176;C for at least 2 h before use.</li>
		<li>Prepare MSC growth medium comprising of minimal essential medium - alpha modification (&#945;MEM) supplemented with 15% fetal bovine serum (FBS) and penicillin/streptomycin (P/S, 1% v/v).</li>
		<li>Sacrifice young male Sprague Dawley rats (200-250 g body weight) by pentobarbitone overdose (240 mg/kg body weight, intraperitoneal). 
		NOTE: Marrow samples from different rats should be processed separately.</li>
		<li>Place the sacrificed animals in the supine position. Clean their abdomen and lower limbs thoroughly with 70% ethanol.</li>
		<li>Remove the skin and subcutaneous tissue over the medial thighs using fine dissecting scissors and forceps. Remove the thigh muscles circumferentially until the femur is exposed. Continue this proximally and distally until the knee and hip joints are seen. Disarticulate the femur through the hip and knee joint using blunt-tipped cutting scissors. 
		NOTE: Do not transect the femur to expose the marrow cavity at this stage. Transfer intact femurs to a laminar flow tissue culture hood for further processing.</li>
		<li>Use blunt-tipped cutting scissors to transect the distal and proximal ends of the femur through the metaphysis.</li>
		<li>Place a 70 &#956;m cell strainer over a 50 mL conical tube. Insert a 21 G, 10 mL syringe containing phosphate-buffered saline (PBS, 10 mM Na2HPO4, pH 7.4) into the exposed femoral canal and flush the marrow content into the conical tube by repeated flushing. 
		NOTE: Approximately 20 mL of PBS is used to flush each femur. If the color of the flushed content remains blood-stained and turbid, a larger volume can be used.</li>
		<li>Collect the cells by centrifugation at 480 x g for 5 min. Discard the supernatant. Resuspend the cell pellet in 10 mL of MSC growth medium. Plate the cells onto a 10 cm tissue culture dish. Place the tissue culture dishes into a cell incubator (37 &#176;C, 5% CO2). Record the initial day of plating as day 0. 
		NOTE: In all steps involving centrifugation, set the brake for maximal deceleration.</li>
	</ol>
	</li>
	<li>Establishment and expansion of MSC colonies 
	NOTE: This protocol relies on tissue culture plastic adherence to select MSCs from within the marrow cavity. The bone marrow content is allowed to adhere to the tissue culture plastic for 2 days.
	<ol>
		<li>On day 2, rinse the culture plates thrice with 10 mL of PBS to remove non-adherent cells. Replace the PBS with 10 mL of MSC growth medium after rinsing. Wash the cells with PBS and replenish them with MSC growth medium every 3 days. 
		NOTE: MSC colonies should be visible by day 6-7 (Figure 1A).</li>
		<li>Passage the cells by day 10 by removing the growth medium and rinsing the cells with PBS. Add 1.5 mL of recombinant enzymatic cell dissociation reagent and incubate at 37 &#176;C for 5 min. Add 3 mL of MSC growth medium to neutralize the reaction. Collect the detached cells by centrifugation at 250 x g for 5 min.</li>
		<li>Quantify the cells within the pellet using a hemocytometer after an appropriate dilution in PBS.</li>
		<li>Seed passaged cells at a density of 40,000 cells/cm2 into a 10-cm culture plate in an MSC growth medium. 
		NOTE: Rat MSCs should reach 80-90% confluence within 2 days of passaging (Figure 1B). Cells can be passaged as described in step 1.2.2 for up to 8 passages. MSCs can be characterized using immunocytochemistry and their capacity for trilineage differentiation (Figure 2). Only MSC cultures between passages 3 and 8 are subject to subsequent hypoxic preconditioning and neural progenitor enrichment. MSCs of greater passage number adopt a flattened morphology (Figure 1C) and do not produce sufficient neural progenitors. These cultures should be discarded.</li>
	</ol>
	</li>
</ol>
2. Preparation of Human BMSC Cultures
<ol>
	<li>Dilute 1 mL of human bone marrow aspirate with 9 mL of MSC growth medium and plate the cells on a 10-cm tissue culture dish. Maintain the cultures in a cell incubator (37 &#176;C, 5% CO2).</li>
	<li>Remove the medium after 2 days and gently rinse the cultures thrice with 10 mL of PBS to remove non-adherent cells. After the final rinse, remove the PBS and replace it with 10 mL of MSC growth medium. Replenish the growth medium after every 3 days of culture after the PBS rinse. 
	NOTE: MSC colonies should be visible by day 6-7. The number of colonies may vary between subjects.</li>
	<li>Passage the cells on day 10, as described in step 1.2.2. Quantify the cells within the pellet using a hemocytometer after an appropriate dilution in PBS. Seed the passaged cells at 40,000 cells/cm2 density onto a 10 cm culture plate in an MSC growth medium. 
	NOTE: Human MSCs (Figure 1D) demonstrate a similar morphology to rat MSCs and should reach 80-90% confluence within 2 days of passaging. They should be characterized using immunocytochemistry and their capacity for trilineage differentiation. As with rat MSCs, human MSCs between passages 3 and 8 are subject to subsequent hypoxic preconditioning and neural progenitor enrichment.</li>
</ol>
3. Hypoxic Preconditioning
<ol>
	<li>Disassemble the hypoxia chamber components (i.e., base, lid, trays) after releasing the ring clamp and clean the individual parts with 70% ethanol. Place the chamber components within a laminar flow tissue culture hood for sterilization under UV light for 15 min.</li>
	<li>Before hypoxic preconditioning, remove the medium and rinse the rat and human MSC cultures (sections 1 and 2) with 10 mL of PBS. Replace the PBS with 10 mL of MSC growth medium supplemented with 25 mM HEPES. 
	NOTE: The MSCs cultured upon 10 cm dishes should have reached 80-90% confluency before being subject to hypoxic preconditioning.</li>
	<li>Place the culture dishes within the hypoxia chamber. Reassemble the chamber components and tighten the ring clamp. Flush a gas mixture of 99% N2/1% O2 into the chamber at a flow rate of 10 L/min for 5 min.</li>
	<li>Seal the connecting ends of the hypoxia chamber to ensure no gas leakage. Place the chamber inside the cell incubator (37 &#176;C, 5% CO2) for 16 h.</li>
	<li>Upon completion of the hypoxic preconditioning, remove the cultures from the chamber in preparation for the subsequent neural progenitor enrichment culture.</li>
</ol>
4. Neural Progenitor Enrichment Culture
<ol>
	<li>Prepare neural progenitor medium comprised of Dulbecco&#39;s modified eagle medium/Ham&#39;s nutrient mixture F12 (DMEM/F12) supplemented with B27 (2% v/v), basic fibroblast growth factor (bFGF, 20 ng/mL), epidermal growth factor (EGF, 20 ng/mL), and P/S (1% v/v).</li>
	<li>Detach the hypoxic preconditioned rat/human MSCs, as described in step 1.2.2. Collect the detached cells by centrifugation at 250 x g for 5 min. Quantify the cells within the pellet using a hemocytometer after appropriate dilution in PBS.</li>
	<li>Resuspend the cells in neural progenitor medium and seed on low-attachment, 6-well plates at a density of 6,000 cells/cm2. Place the culture in a cell incubator (37 &#176;C, 5% CO2) for 12 days. Replenish 75% of the neural progenitor medium every 3 days. 
	NOTE: Sizeable non-adherent cell clusters should be observed by days 6-7. By day 10-12, neurospheres with a diameter &#8805; 100 &#956;m can be observed (Figure 3). Hypoxic preconditioned MSCs should yield more neurospheres than MSCs cultured under normoxic conditions.</li>
	<li>Collect neurospheres on day 12 by aspirating them into a 10-mL pipette and transferring them to a 15 mL conical tube. Centrifuge the neurospheres at 250 x g for 5 min. 
	NOTE: Neurospheres can be characterized on day 12 for neural progenitor markers, such as nestin and GFAP.</li>
</ol>
5. Generation of Schwann cell-like cells
<ol>
	<li>Prepare glial induction medium comprised of &#945;MEM supplemented with &#946;-Heregulin (100 ng/mL), bFGF (10 ng/ml), platelet-derived growth factor (PDGF-AA, 5 ng/mL), FBS (10%), and P/S (1% v/v).</li>
	<li>Plate the neurospheres prepared in section 4 in PDL/laminin-coated 6-well plates at a density of 5-10 spheres per cm2 in 1.5 mL of glial induction medium per well. Replace the glial induction medium every 2 days after rinsing the cells with PBS. 
	NOTE: Cells from seeded neurospheres are seen to migrate outwards by day 2. By day 7, migratory cells have a tapered appearance and should demonstrate immunopositivity for the Schwann cell markers p75 neurotrophin receptor (p75) and S100&#946;. These cells are referred to as SCLCs.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#945;MEM</td>
			<td>Sigmaaldrich</td>
			<td>&#160;M4526</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher scientific</td>
			<td>&#160;12400-024</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biosera</td>
			<td>&#160;FB-1280/500</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher scientific</td>
			<td>&#160;17504-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal growth factor (EGF)</td>
			<td>Thermofisher scientific</td>
			<td>&#160;PHG0313</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic fibroblast growth factor (bFGF)</td>
			<td>Peprotech</td>
			<td>&#160;100-18B/100UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Platelet-derived growth factor-AA (PDGF-AA)</td>
			<td>Peprotech</td>
			<td>&#160;100-13A</td>
			<td></td>
		</tr>
		<tr>
			<td>Heregulin beta-3, EGF domain (&#946;-Her)</td>
			<td>Millipore</td>
			<td>01-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine (PDL)</td>
			<td>Sigmaaldrich</td>
			<td>P7886-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermofisher scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / streptomycin (P/S)</td>
			<td>Thermofisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>&#160;Thermofisher Scientific</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm plate for adherent culture</td>
			<td>&#160;TPP</td>
			<td>93100</td>
			<td>&#160;Used for selection of MSCs</td>
		</tr>
		<tr>
			<td>6-well plate for adherent culture</td>
			<td>TPP</td>
			<td>92006</td>
			<td>&#160;Used for expansion of MSCs following passaging</td>
		</tr>
		<tr>
			<td>UltraLow 6-well plate for nonadherent culture</td>
			<td>Corning</td>
			<td>3471</td>
			<td>&#160;Used for neural progenitor enrichment</td>
		</tr>
		<tr>
			<td>anti-human CD90(Thy-1)</td>
			<td>BD Biosciences</td>
			<td>555593</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human CD73</td>
			<td>BD Biosciences</td>
			<td>550256</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human/rat STRO-1</td>
			<td>R&#38;D Systems</td>
			<td>MAB1038</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>555480</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxia chamber</td>
			<td>Billups-Rothenberg</td>
			<td>MIC-101</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigmaaldrich</td>
			<td>H4034-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human nestin</td>
			<td>R&#38;D Systems</td>
			<td>MAB1259</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat CD90(Thy-1)</td>
			<td>BD Biosciences</td>
			<td>554895</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat CD73</td>
			<td>&#160;BD Biosciences</td>
			<td>551123</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat nestin</td>
			<td>BD Biosciences</td>
			<td>MAB1259</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat CD45</td>
			<td>BD Biosciences</td>
			<td>554875</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-S100&#946;</td>
			<td>Dako</td>
			<td>Z031101</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p75</td>
			<td>Millipore</td>
			<td>MAB5386</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFAP</td>
			<td>Sigmaaldrich</td>
			<td>G3893</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Class III-beta tubulin (Tuj-1)</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human nuclei</td>
			<td>Millipore</td>
			<td>MAB1281</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating schwann cell-like cells from bone marrow stromal cells

Source: Tsui, Y., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55794">Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method for obtaining Schwann cell-like cells from multipotent bone marrow stromal cells (BMSCs). Initially, the BMSCs undergo hypoxia treatment, followed by directed differentiation into neuronal progenitor cells. Subsequently, specific media and culture conditions are employed to induce the transformation of these progenitor cells into Schwann cell-like cells.

<img alt="Figure 1" src="/files/ftp_upload/22393/22393_Figure1.jpg" /> 
Figure 1: Establishment of MSC colonies. Sizeable MSC colonies should be visible 6-7 days after the plating of bone marrow cells onto tissue culture plastic. A representative image of a rat MSC colony is shown (A), while human colonies demonstrate a similar appearance. Colonies can be passaged on day 10. Seen at higher magnification, both rat (B) and human (<stro...

Generating Schwann Cell-Like Cells from Bone Marrow Stromal Cells

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Rat MSC Cultures

	...

Begin with an adherent culture of bone marrow stromal cells.
Subject the cells to hypoxia, a condition characterized by reduced oxygen levels, for a required duration.
Isolate the hypoxia-preconditioned cells, then centrifuge and remove the supernatant. Resuspend the cells in neuronal progenitor media containing neuronal growth factors.&#160;
Transfer the cells to a low-attachment culture plate and incubate.&#160;
Hypoxia-preconditioning and neuronal growth factors induce stromal cell differentiation into neuronal progenitors.
The low-attachment plate surface maintains the progenitors in suspension, facilitating their proliferation and aggregation into three-dimensional neurospheres.
Harvest the neurospheres, centrifuge, and remove the supernatant. Resuspend the cells in glial induction media containing glia-specific growth factors.
Transfer the neurospheres to a culture plate coated with laminin, an extracellular matrix component, and poly-D-lysine, a positively-charged synthetic polymer.
The coated surface promotes cell adhesion, facilitating neurosphere attachment.
The glia-specific growth factors stimulate neuronal progenitor differentiation into Schwann cell-like cells that exhibit the characteristic tapered morphology of Schwann cells.

generating-schwann-cell-like-cells-from-bone-marrow-stromal-cells

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

34 조회수. 출처: Tsui, Y., et al. 성숙한 슈반 세포 생성을 위한 소스로서의 골수 유래 전구 세포의 저산소 전조건화.J. Vis. 특급 (2017년) 이 동영상은 다분화능 골수 기질 세포(BMSC)에서 슈반 세포 유사 세포를 얻는 방법을 보여줍니다. 초기에 BMSC는 저산소증(hypoxia) 치료를 받은 후 신경 전구 세포(neuronal progenitor cell)로 직접 분화합니다. 그 후, 특정 배지 및 배양 조건을 사용하여 이러한 전구 ?...

비디오: 골수 기질 세포에서 Schwann 세포 유사 세포 생성 - 개념

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

사람 골수에서 분리 Mesangiogenic 전구 세포 (MPC와)

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

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JoVE Journal

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Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

마우스 배아 줄기 세포에서 신경 전조및 뉴런의 분화 및 특성화

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Generating Schwann Cell-Like Cells from Bone Marrow Stromal Cells

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Generating Schwann Cell-Like Cells from Bone Marrow Stromal Cells