Name: visualization and quantification of mesenchymal cell adipogenic differentiation potential with a lineage specific marker
Uploaded: 2018-03-31T15:00:00.000+00:00
Duration: 13 min 26 s
Description: Watch this Scientific Journal Video about Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker at JoVE.com

We are grateful to the donors of the adipose tissues and the research professionals who collect and provide this resource to us for research use. We would like to acknowledge funding support by Allergan. We are also grateful to the University of Auckland, Faculty of Medical and Health Sciences Performance Based Research Fund for funding the cost of videoing and editing for submission of this article.

1. Preparing ASCs for Differentiation Assays
<ol>
	<li>Prepare tissue culture reagents and handle live cells in a sterile tissue culture hood.</li>
	<li>Prepare A0 medium: Dulbecco Modified Eagle Medium and Ham&#39;s F12 Medium (DMEM/F12), 1x glutamax, 1x penicillin/streptomycin.</li>
	<li>Prepare complete ASC medium: DMEM/F12, 1x glutamax, 1x penicillin/streptomycin, 10% fetal bovine serum.</li>
	<li>Use adherent purified ASCs, expanded in a T75 culture flask. Confirm the purity of ASCs using fluorescence-activated cell sorting (FACS)10.</li>
	<li>Detach cells by removing all medium from the T75 culture flask and adding 2 mL of cell dissociation enzyme.</li>
	<li>Leave the culture flask in the incubator (37 &#176;C, humidified, 5% CO2) for 5-10 min. Take the culture flask out of the incubator and firmly tap the side of flask. Check if the cells have detached from the culture flask using an inverted microscope.</li>
	<li>Add 13 mL of A0 medium to a T75 culture flask.</li>
	<li>Transfer 15 mL into a 15 mL centrifuge tube and centrifuge at 400 x g for 5 min.</li>
	<li>Discard the supernatant and re-suspend the cell pellet in 1 mL of complete ASC medium.</li>
	<li>Perform a cell count using hemocytometer.</li>
	<li>Dilute the cell suspension to a concentration of 25,000 cells mL-1 in complete ASC medium.</li>
	<li>Add 200 &#181;L of A0 medium to all periphery wells of the 96 well plate (flat bottom). This reduces the evaporation rate of the wells containing cells in the center.</li>
	<li>Transfer 200 &#181;L of cell suspension to a microwell plate to achieve a final cell density of 5 x 103 cells per well. Four wells are needed for each treatment group (triplicate technical repeats and a no primary antibody control for immunocytochemistry (ICC)).</li>
	<li>Leave microwell plate in the incubator (37 &#176;C, humidified, 5% CO2) until cells reach confluence (3-4 days).</li>
</ol>
2. Inducing Differentiation of ASCs
<ol>
	<li>Prepare adipogenic differentiation medium by supplementing complete ASC medium with 1 &#181;M dexamethasome, 10 &#181;M insulin and 200 &#181;M indomethacin. The complete differentiation medium is stable at 4 &#176;C for 1 month, so only make as required for 1 assay. Store at 4 &#176;C and when required heat an aliquot to 37 &#176;C in water bath.</li>
	<li>After 3-4 days, take microwell plate out of incubator and replace half of the culture medium with adipogenic differentiation medium.</li>
	<li>Perform a medium change every 2-3 days. 
	NOTE: Optimal adipogenic differentiation requires 14 days of culture in differentiation medium. Differentiated cells are analyzed using traditional dye based stains and gene-specific stains.</li>
</ol>
3. Staining for Differentiation - Traditional Dye Based Stains
<ol>
	<li>Prepare 4% formaldehyde solution by diluting 16% formaldehyde with PBS. Dilute only the amount needed for experiment and store tightly sealed in a centrifuge tube. 
	CAUTION: Formaldehyde is a potential carcinogen and can cause irritation to nose, throat and lungs upon inhalation. Perform all procedures relating to formaldehyde in a fume hood.</li>
	<li>Prepare Oil Red O stock solution by dissolving 300 mg in 100 mL of isopropanol.</li>
	<li>Take the microwell plate out of the incubator. The following steps can be performed in a non-sterile environment.</li>
	<li>Remove all medium from wells and fix cells with 4% formaldehyde for 30 min.</li>
	<li>During incubation time, prepare Oil Red O working solution. Working solution comprises 6 parts Oil Red O stock with 4 parts distilled water (dH2O). Mix well and let sit at room temperature for 20 min, before filtering using 0.2 &#181;m filter unit. Working solution is only stable for 2 h.</li>
	<li>Remove all formaldehyde from wells by pipetting.</li>
	<li>Wash cells once with 60% isopropanol and let the wells dry completely before proceeding.</li>
	<li>Add 50 &#181;L of Oil Red O working solution to each well and incubate at room temperature for 10 min.</li>
	<li>Remove Oil Red O solution and immediately add dH2O. Wash with dH2O 4 times before viewing under light microscope.</li>
</ol>
4. Staining for Differentiation - Gene Specific Antibodies
<ol>
	<li>Prepare stock solution of Tris buffered saline (TBS) by dissolving 80 g of NaCl, 2 g of KCl and 30 g of Tris Base to 1 L of dH2O (pH 8). Prepare working solution by diluting 1:10 using dH2O. Store at room temperature.</li>
	<li>Prepare immunobuffer (IB) (1% fetal bovine serum (FBS) in TBS). Label the 15 mL centrifuge tube with date and store at 4 &#176;C. IB can be used for 1 week from the date made.</li>
	<li>Prepare 0.25% casein blocker by dissolving 0.5 g of casein and 0.195 g of sodium azide in 200 mL of phosphate buffered saline (PBS). Completely dissolve the ingredients by stirring overnight. Aliquot into 15 mL centrifuge tubes and store in a -20 &#176;C freezer. Thawed casein solutions can be kept at 4 &#176;C for 1 month.</li>
	<li>Prepare DAPI solution by diluting stock DAPI 1:200 in TBS. Store at 4 &#176;C, protected from light.</li>
	<li>Prepare stock thimerosal solution (10 mg mL-1) by dissolving thimerosal in sterile PBS. Store at 4 &#176;C and dilute appropriate amounts to 0.4 mg mL-1 for use.</li>
	<li>Fix and permeabilize cells with ice-cold methanol (96 well plate) for 5 min. Wash cells once with 200 &#181;L of TBS.</li>
	<li>Block with 50 &#181;L of 0.25% casein at room temperature for 10 min and wash once with TBS.</li>
	<li>Add 50 &#181;L of primary antibodies diluted in IB (see Table 1 for antibody details) and incubate with lids closed for 1 h.</li>
	<li>Wash once with 200 &#181;L of TBS, and then 3 times with TBS for 5 min with gentle rocking.</li>
	<li>Add 50 &#181;L of secondary antibodies diluted in IB (see Table 2 for antibody details) with 1:2000 DAPI in each, incubate with lids closed for 1 h. Cover the plate with foil to prevent photo-bleaching.</li>
	<li>Wash once with TBS, and then twice with TBS for 15 min with gentle rocking.</li>
	<li>Remove all TBS and add 200 &#181;L of thimerosal solution.</li>
	<li>Store plate at 4 &#176;C, protected from light until imaging.</li>
</ol>
5. Imaging Cells using a High-Throughput Microscope
<ol>
	<li>Analyze cells stained with gene specific antibodies and fluorophore conjugated secondary antibodies (immunofluorescence) using a high content screening microscope controlled with accompanying software.</li>
	<li>Load the microwell plate into the microscope stage. On the plate acquisition control, select image at medium range magnification (10X objective lens). Make sure images are taken from the center of each well, with 50 &#181;m spacing in between each site to ensure that one cell does not appear in two adjacent fields.</li>
	<li>Select the wells to image.</li>
	<li>Select the appropriate channel combinations, exposure time and z-offset parameters. Refer to Table 3 for more detailed information of each filters (Supplementary Table 2).</li>
	<li>Acquire the assay using the acquisition wizard (images are automatically stored to Database server).</li>
	<li>Use alternative channel combinations to acquire plates stained with Oil Red O. (Supplementary Table 3).</li>
</ol>
6. Analyze Acquired Image
Note: Remote access to the database server enables quick and easy evaluation of the images acquired and use of the image analysis software.
<ol>
	<li>Open Cell Scoring module on the analysis software.</li>
	<li>Detect cell nuclei using DAPI channel (nuclear marker) and segment nuclei of interest with user defined parameters for size and signal intensity above background.</li>
	<li>Detect FABP4 signal using FITC channel. Segment differentiated cells with user defined parameters for size and signal intensity above background to select all FABP4 positive regions in each image.</li>
	<li>Open Transfluor module on the analysis software to analyze plates stained with Oil Red O.</li>
	<li>Specify the size and intensity of Oil Red O stained regions as &#39;pits&#39;.</li>
</ol>

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The authors declare no conflicts of interest.

This paper demonstrates the utility and advantages of FABP4 immunolabelling to accurately detect and quantify mature adipocytes derived from mesenchymal stromal cells. FABP4 is able to detect changes in expression of a lineage specific protein3,4,5 in mature adipocytes, contrary to other commonly used dyes which label macromolecular changes13,14 such as Oil Red O15, Nile Red16 and Sudan Black17. FABP4 immunolabelling allows for visualization and analysis of changes in cellular morphology. This is a distinctive advantage over previously described methods using quantitative reverse transcription PCR (RT-qPCR) that analyzes changes at the transcript level without any visualisation5,18,19,20, or flow cytometry that requires cells to be in suspension20, or Western blotting that requires cells to be lysed to isolate protein19,20. FABP4 immunolabelling is stable in fixed cells, does not leak out in solution and being a cytosolic protein when labelled in an adherent monolayer of cells, will overlap with the nucleus allowing for easy visualisation and added accuracy in the automated analysis.
High-content screening is advantageous because it is fast, accurate, reproducible and objective. It provides a platform for cost effective use of reagents and researcher time, since image acquisition and analysis require minimal manual manipulation and rely on the automatic processing of the instrument. Several factors were identified as critical to the accuracy and reproducibility of the high-content screening assays31. The quantification of antigen expression relies on image quality. For successful image analysis, images from each channel per well must be in focus and fall within an optimal dynamic range for gray values (Auto Expose settings are ideal). Out of focus or saturated images lead to erroneous results.
Some potential limitations of the methods described in this article include the following. The requirement for specialised imaging equipment and analysis software. While outside the scope of this article, alternative open source software options (for example, ImageJ32,33, Fiji34, CellProfiler32,35) can be used to perform similar image segmentation tasks; however they do require the skills to either download and tailor macros available online or write macros/commands for their specific analysis pipelines. Inherent to all automated imaging analysis pipelines is a level of background noise. This can largely be attributed to debris, poor image quality or analysis error, emphasizing the need for careful sample preparation and careful set-up of the imaging and analysis platform. The FABP4 label in mice has been found to detect undifferentiated progenitors30; while the controls in this study (which consist of undifferentiated cells) are devoid of specific FABP4 staining. This under-scores the necessity of checking FABP4 expression in undifferentiated progenitors in each biological context where the assay is employed.
In order to draw valid comparisons between the FABP4 labelling and Oil Red O staining, the output measurements from image analysis needed to be biologically relevant. Consequently, the measurement, &#39;% positive cells&#39; was used for FABP4, and &#39;area of staining per cell&#39; was used for Oil Red O. It is noteworthy that the measure &#39;% positive cells&#39; was not used for Oil Red O labelled cells in our study because it was not possible to attribute the labelled fat droplets accurately to a given nucleus; the fat droplets varied considerably in size, number and distribution across the cell body and rarely overlapped with the nucleus. Oil Red O staining variability exists between cells derived from different tissue sources; while the% positive cells measure was not appropriate for the current study, another group has used it successfully to quantify adipocytes derived from human gland stem cells22 where the Oil Red O staining appeared as one large fat droplet which spanned the cytoplasm and overlapped with nuclei and enabling data to be presented as% positive cells.
In contrast, the morphology of FABP4 immunolabelling is consistent in adipocytes derived from a range of different tissue sources23,24,25. The ability to quantify the proportion of cells within a culture capable of differentiation has a number of applications. Adult mesenchymal stromal cells hold significant promise for a diverse range of therapeutic uses. A significant issue that has arisen with clinical translation is the inherent variability in the capability of these cells between patients26, between sites of isolation27,28, and between methods for isolation12 and expansion of cell numbers12 for therapeutic use. It has been shown previously that cultures of adherent stromal cells are frequently heterogeneous, with some cells capable of differentiation and some not 12,17 as our FABP4 assay demonstrates for ASC cultures. Assays like this, combined with enrichment techniques, will help identify sub-populations within heterogeneous cultures capable of lineage-specific differentiation. Having a standardized, quantifiable assay such as this FABP4 assay provides a simple method for comparison between all of these variables, and the potential to evaluate therapeutic outcomes within and between clinical trials.
The quantification of FABP4 in a semi-automated fashion enables objectivity and reproducibility in analysis across many donor cases and samples. Furthermore as the label is cytoplasmic, it can potentially be used to analyze hypertrophy (enlargement of adipocyte size) in addition to hyperplasia (increase in adipocyte number), a distinction that is of considerable importance in obesity research, where hypertrophy is strongly associated with adipose dysfunction in obese individuals21. The obesity epidemic has spurred a significant amount of interest in identifying drugs capable of controlling lipid storage. This FABP4 assay also provides a simple readout for screening thousands of compounds for their ability to inhibit lipid accumulation.

One of the key requirements established by the International Society for Cellular Therapy (ISCT) to define a multipotent mesenchymal stromal cell is that the cells must have the ability to differentiate into the adipogenic, osteogenic and chondrogenic lineages1. Conventional methods of measuring differentiation into these three lineages rely on the detection of macromolecular products using chemical dyes1. Dyes such as Oil Red O (which stains fat droplets in cells that have undergone adipogenesis), are cheap and easy to use; however they fail to detect the specific changes in gene expression that occur when mesenchymal cells differentiate into each respective lineage2. Here, we have developed a differentiation assay which quantifies protein expression for a adipogenic lineage-specific marker, fatty acid binding protein-4 (FABP4)3,4,5. FABP4 was initially found in murine 3T3-L1 adipocytes3 and was later discovered to be expressed in human subcutaneous adipose tissue6. It is a cytosolic protein, which acts as a chaperone to guide fatty acid uptake by cells and is involved in the process of lipolysis4.
The precursor cells used for the differentiation assays were adipose derived mesenchymal stromal cells (ASCs)7,8. ASCs share many properties with bone marrow-derived mesenchymal stem cells (BM-MSCs), a major mesenchymal stem cell population in adults8,9. ASCs offer several advantages over BM-MSCs in a clinical application, as greater yield of cells can be isolated from more readily accessible tissue sources8,9. An isolated cell population needs to meet certain criteria to be defined as ASCs. First, they must show adherence to plastic tissue culture vessels in standard culture conditions1. They also must show specific surface antigen expression1. Uncultured ASCs are characterized by positive surface antigen expression of CD34, CD73, CD90, low expression of CD105 and negative expression of CD45 and HLA-DR10. ASCs purified by culturing on plastic for 28 days (adherent purified ASCs) show positive expression of CD73, CD90 and CD105 and negative expression of CD34, CD45 and HLA-DR10. Finally, cells must retain the ability to differentiate into various lineages1,7,8.
Adipogenic differentiation protocols induce striking upregulation of FABP4 expression amongst other adipocyte lineage genes, so we used immunochemistry to visualize FABP4 protein within cells, and then quantified FABP4 expression at the single cell level using an automated fluorescent high-content screening microscope. This method is advantageous over traditional dyes as it enables highly specific confirmation of adipocyte-lineage differentiation. Such gene specific lineage assays combined with high content screening methods also enable quantification of the proportion of cells within a heterogeneous cell preparation that are capable of differentiation down a particular lineage. In our studies we used the FABP4 assay to confirm loss of adipogenic differentiation potential of freshly isolated ASCs after cell culture.

<table><tbody><tr><td>Tris Base</td><td>Invitrogen</td><td>15504-020</td><td>Tris buffered saline</td></tr><tr><td>Sodium Chloride</td><td>Merck</td><td>1064041000</td><td>Tris buffered saline</td></tr><tr><td>Potassium Chloride</td><td>Merck</td><td>1049360500</td><td>Tris buffered saline</td></tr><tr><td>Sodium Azide</td><td>Scharlau</td><td>SO0091</td><td>Casein blocker solution</td></tr><tr><td>Casein</td><td>Sigma</td><td>C7078-500G</td><td>Casein blocker solution</td></tr><tr><td>PBS tablet</td><td>Sigma</td><td>P4417-100TAB</td><td>Phosphate buffered saline</td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11330-032</td><td>Cell culture</td></tr><tr><td>Penicillin/streptomycin</td><td>Invitrogen</td><td>15140-122</td><td>Cell culture</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td>Cell culture</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10091-148</td><td>Cell culture</td></tr><tr><td>TrypLE Select Enzyme (1X), no phenol red</td><td>Gibco</td><td>12563-029</td><td>Cell dissociation enzyme</td></tr><tr><td>96 well plate (flat bottom)</td><td>Falcon</td><td>FAL353072</td><td>Cell culture equipment</td></tr><tr><td>T75 culture flask, with filter cap</td><td>Greiner</td><td>658175</td><td>Cell culture equipment</td></tr><tr><td>Insulin</td><td>Sigma&amp;nbsp;</td><td>19278-5ML</td><td>Adipogenic differentiation media</td></tr><tr><td>dexamethasone</td><td>Sigma</td><td>D2915-100MG</td><td>Adipogenic differentiation media</td></tr><tr><td>indomethacin</td><td>sigma</td><td>I7378-5G</td><td>Adipogenic differentiation media</td></tr><tr><td>Oil-Red O</td><td>Sigma</td><td>O-0625</td><td>Classic stain for adipogenesis</td></tr><tr><td>Isopropanol</td><td>Merck</td><td>1.09634</td><td>Classic stain for adipogenesis</td></tr><tr><td>16% formaldehyde</td><td>Pierce</td><td>28908</td><td>Cell fixation</td></tr><tr><td>Acetone</td><td>Merck</td><td>100983</td><td>Cell fixation</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>Immunocytochemistry</td></tr><tr><td>Anti rabbit IgG alexa 488</td><td>Molecular Probes</td><td>A11008</td><td>Immunocytochemistry</td></tr><tr><td>Thimerosal</td><td>Sigma</td><td>T5125-10G</td><td>Immunocytochemistry</td></tr><tr><td>Rabbit anti-human fatty acid binding protein 4 (FABP4) antibody</td><td>Cayman Chemicals</td><td>10004944</td><td>Marker of adipogenesis</td></tr><tr><td>Centrifuge tubes (15mL)</td><td>Greiner</td><td>GR188271</td><td>General</td></tr><tr><td>Centrifuge tubes (50mL)</td><td>Greiner</td><td>GR227261-P</td><td>General</td></tr><tr><td>ImageXpress Micro XLS</td><td>Molecular Devices</td><td></td><td>high content screening machine</td></tr><tr><td>MetaXpress</td><td>Molecular Devices</td><td></td><td>high content screening software, version 5.4.01</td></tr></tbody></table>

visualization and quantification of mesenchymal cell adipogenic differentiation potential with a lineage specific marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

In this study, the adipogenic differentiation potential of ASCs with 3 different methods were measured. Immunofluorescent labelling of FABP4, showed that this marker localised to the cytoplasm of differentiated cells, and the labelling overlapped with DAPI-labelled nuclei (Figure 1A). Automated image analysis revealed a 24.6 fold increase in the % of FABP4 positive cells in the differentiated group in early passage cells compared to 6.9 fold increase in late passage cells (Figure 1B). Oil Red O staining was localized to fat droplets dispersed throughout the cytosol of differentiated cells, and the labelling rarely overlapped with DAPI-labelled nuclei; however from visual inspection, there are fewer cell nuclei associated with Oil Red O fat droplets in the late passage cells compared to early passage cells (Figure 2A). Automated image analysis revealed an 11.1 fold increase in the area of Oil Red O staining per cell in early passage cells and a 53.9 fold increase in staining area per cell in late passage cells (Figure 2B). Imaging of both stains was performed, and then quantified using Cell Scoring (FABP4) or Transfluor (Oil Red O) module in the software (Supplementary Figure 1, Supplementary Table 2-5). The upregulation of FABP4 expression was confirmed by Western blot analysis (Figure 3, Supplementary Figure 5). All 3 analysis methods revealed that early passage ASCs have higher adipogenic differentiation potential than late passage cells. The adipogenic differentiation did not affect total cell number per well (Supplementary Figure 2, 3). However, the nuclei size of FABP4-positive differentiated cells was significantly smaller than control cells for late passage ASCs only (Supplementary Figure 3).
We demonstrated that as cells were expanded in culture, or passaged, the percentage of cells within the culture capable of adipogenic differentiation decreased, consistent with previously published data11. It is important to note that factors such as age, body mass index or previous medical history12 could not be controlled for in this study and likely contributed to the variability observed between different donor samples (Supplementary Table 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57153/57153fig1.jpg" /> 
Figure 1: FABP4 immunolabelling shows that early passage cells have greater differentiation potential than later passage cells. A) Representative images of early and late passage, control and differentiated cells labelled with DAPI (nuclear stain) and FABP4 are shown alongside merge and segmentation images. The segmentation images display differentiated cells with a green overlay and undifferentiated cells with a red overlay. B) A significant increase in FABP4 expressing cells was observed with adipogenic differentiation in early and late passage cells, however, early passage cells demonstrated significantly greater increase in differentiation than late passage cells (Mann Whitney test * denotes P&#60;0.05 and *** denotes P&#60;0.001). The data shown is the average across early passage (p4; n=8) or late passage (p10; n=4) donor samples, &#177; SEM. <a href="/files/ftp_upload/57153/57153fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57153/57153fig2.jpg" /> 
Figure 2: Oil Red O staining shows that early passage cells have greater differentiation potential than later passage cells. A) The representative images of DAPI (nuclear stain) and Oil Red O (grey, lipid droplet stain) are shown alongside merge and segmentation images. The segmentation images depict all nuclei with green overlay and Oil Red O stained lipid droplets with a red overlay. B) A significant increase in Oil Red O staining area was observed with adipogenic differentiation. Early passage cells showed greater area of Oil Red O stain per cell compared to late passage cells. The area of Oil Red O staining per cell (&#956;m2) is used here as a measure of adipogenic differentiation potential. The data shown is the average across early passage (p4; n=8) or late passage (p10; n=4) donor samples, &#177; SEM (Mann Whitney test * denotes P=&#60;0.05 and *** denotes P=&#60;0.001). <a href="/files/ftp_upload/57153/57153fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57153/57153fig3.jpg" /> 
Figure 3: Western blot analysis of FABP4 protein expression level of differentiated early and late passage ASCs. Semi-quantitative analysis of the optical density of FABP4 bands as a ratio of total protein loading control, beta actin (ACTB), showed that FABP4 immunolabelling was significantly increased in early passage and to a lesser degree late passage differentiated cells. The data shown is the average across early passage (p4; n=8) or late passage (p10; n=4) donor samples, &#177; SEM (Mann Whitney test * denotes P=&#60;0.05 and *** denotes P=&#60;0.001). <a href="/files/ftp_upload/57153/57153fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Target antigen</td>
			<td>Isotype (clone)</td>
			<td>Species</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>FABP4</td>
			<td>Polyclonal</td>
			<td>Rabbit</td>
			<td>1:100</td>
		</tr>
	</tbody>
</table>
Table 1: Primary antibody
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Targeting antibody</td>
			<td>Fluorophore label</td>
			<td>Species</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Rabbit IgG</td>
			<td>Alexa fluoro 488</td>
			<td>Goat</td>
			<td>1:200</td>
		</tr>
	</tbody>
</table>
Table 2: Secondary antibody
Supplementary Figure 1: Overview of the image analysis virtual environment. The stained cells were imaged using an automated, high-throughput fluorescent microscope to avoid any source of bias. Images acquired using ImageXpress Micro XLS, were saved directly to the MDCStore Data Manager database, and made available for viewing through virtual desktop connections, which users use to optimize and test their specific analysis parameters. Images were analyzed with a dedicated &#39;autorun&#39; instance which enables multiple different users to send &#39;jobs&#39; to the autorun queue. Users can retrieve their data via the virtual instance once their &#39;jobs&#39; are completed. <a href="/files/ftp_upload/57153/Supplementary_Figure_1.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 2: Comparison of total cell count per well of control and differentiated wells for early and late passage ASCs, using FABP4 stained plate. The data shown is the average across early passage (p4; n=8) or late passage (p10; n=4) donor samples, &#177; SEM. There was no statistically significant difference between control and differentiated cells for both early and late passage ASCs. There were more cells per well for early passage cells compared to late passage cells. <a href="/files/ftp_upload/57153/Supplementary_Figure_2.tif" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 3: Comparison of total cell count per well of control and differentiated wells for early and late passage ASCs, using Oil Red O stained plate. The data shown is the average across early passage (p4; n=8) or late passage (p10; n=4) donor samples, &#177; SEM. There was no statistically significant difference between control and differentiated cells for both early and late passage ASCs. <a href="/files/ftp_upload/57153/Supplementary_Figure_3.tif" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 4: At later passages, differentiated cells that were FABP4 positive had significantly smaller nuclei area than cells in control wells. There was no statistically significant difference in nuclei area between control and differentiated cells in early passage. The data shown is the average across early passage (p4; n=8) or late passage (p10; n=4) donor samples, &#177; SEM. (Unpaired t test. *** denotes P=&#60;0.001). Mean &#177; SEM are shown.) <a href="/files/ftp_upload/57153/Supplementary_Figure_4.tif" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 5: Images of Western blot gels. Red channel depicts beta-actin. Green channel depicts FABP4 immunolabelling. <a href="/files/ftp_upload/57153/Supplementary_Figure_5.tiff" target="_blank">Please click here to download this figure.</a>
Supplementary Table 1: Clinicopathological information of donors <a href="/files/ftp_upload/57153/Supplementary_table_1.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 2: Imaging settings (FABP4) <a href="/files/ftp_upload/57153/Supplementary_table_2.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 3: Imaging settings (Oil Red O) <a href="/files/ftp_upload/57153/Supplementary_table_3.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 4: Table of analysis parameters used (FABP4). Cell Scoring module. <a href="/files/ftp_upload/57153/Supplementary_table_4.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 5: Table of analysis parameters used (Oil Red O). Trans Fluor module. <a href="/files/ftp_upload/57153/Supplementary_table_5.xlsx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker at JoVE.com

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

visualization-quantification-mesenchymal-cell-adipogenic

Research

JoVE Journal

Biology

9.8K Views.  The University of Auckland. Traditionele methoden voor de beoordeling van de differentiatie van de adipogenic zijn goedkoop en makkelijk te gebruiken, maar zijn niet specifiek voor wijzigingen in genexpressie. Hebben we een test om te kwantificeren mesenchymale celdifferentiatie in volwassen adipocytes met behulp van een specifieke marker van afkomst. Deze bepaling heeft diverse toepassingen in basisonderzoek en klinische geneeskunde.

Video: Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

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.

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

Developmental Biology

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications.
Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The protocol allows the generation of a pure adipocyte population from induced pluripotent stem cells (iPSCs). Retinoic acid is used to differentiate iPSCs into mesenchymal stem cells (MSCs) which are used for producing adipocytes. Then, a sorting approach based on Nile red staining is used to obtain pure adipocytes.

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the reliability and robustness of the source. We describe here a stepwise protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue (uMSCs), their immunophenotyping, and the propagation of such cultures over several passages. In this procedure, the viability of the uMSCs is high because there is no enzymatic digestion. Further, the removal of blood vessels, including the umbilical cord arteries and the vein, ensures that there is no contamination of cells of endothelial origin. Using flow cytometry, uMSCs upon isolation are CD45&#8722;CD34&#8722;, indicating an absence of cells from the hematopoietic lineage. Importantly, they express key surface markers, CD105, CD90, and CD73. Upon establishment of cultures, this paper describes an efficient method to induce differentiation in these uMSCs into the skeletal muscle lineage. A detailed analysis of myogenic progression in differentiated uMSCs reveals that uMSCs express Pax7, a marker for myogenic progenitors in the initial stages of differentiation, followed by the expression of MyoD and Myf5, and, finally, a terminal differentiation marker, myosin heavy chain (MyHC).

We describe a protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue and their differentiation into the skeletal muscle lineage.

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Immunology and Infection

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

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