This work was funded by a grant from Qatar National Research Fund (QNRF) (Grant No. NPRP10-1221-160041). Maryam Aghadi was supported by GSRA scholarship from Qatar National Research Fund (QNRF).

The study has been approved by the appropriate institutional research ethics committee and performed following the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The protocol was approved by the Institutional Review Board (IRB) of HMC (no. 16260/16) and QBRI (no. 2016-003). This work is also optimized for hESCs such as H1 and H9. Blood samples were obtained from healthy individuals with full informed consent. The iPSCs are generated from peripheral blood mononuclear cells (PBMCs) of healthy individuals.
1. Culturing and maintaining iPSCs
<ol>
	<li>Prepare basement membrane matrix-coated plates by reconstituting coating matrix in knockout-DMEM at a ratio of 1:80 and store at 4 &#176;C.</li>
	<li>Prepare iPSCs culture media by adding 50 mL of 10x stem cell supplement media to 500 mL of stem cell basal media, along with 5 mL of 100x penicillin-streptomycin (P/S) and store at 4 &#176;C for short term or at -20 &#176;C for long term use.</li>
	<li>Line the plates with coating matrix-1 mL for a 6-well plate, 500 &#181;L for a 12-well plate, 250 &#181;L for a 24-well plate-and incubate the plate at 37 &#176;C for 1-2 h.</li>
	<li>Remove an aliquot of iPSCs culture media and pre-warm at room temperature before use.</li>
	<li>Thaw a vial of iPSCs (ESCs or iPSCs) in a 37 &#176;C water bath and transfer to a 15 mL conical tube containing 2-3 mL of culture media.</li>
	<li>Centrifuge the tube at 120 x g for 4 min at room temperature (RT-23 &#176;C).</li>
	<li>Remove the supernatant and add 2 mL of fresh culture media supplemented with 10 &#181;M ROCK inhibitor (Y-27632). Plate the cells in one well of a matrix-coated 6-well plate and place the plate at 37 &#176;C.</li>
	<li>After 24 h, remove the media and replace it with fresh culture media.</li>
	<li>Change the media every day until the cells reach 80%-90% confluency.</li>
	<li>Upon reaching confluency, passage cells by following the steps outlined below.
	<ol>
		<li>Remove the media and wash the cells with Dulbecco&#39;s phosphate-buffered saline (DPBS).</li>
		<li>Add iPSCs dissociation reagent (see Table of Materials)-500 &#181;L for a well of a 6-well plate, 250 &#181;L for a well of a 24-well plate-and incubate for 1 min at 37 &#176;C.</li>
		<li>Remove the dissociation reagent and incubate the cells dry for 1 min at 37 &#176;C.</li>
		<li>Collect the cells using culture media-1 mL for a well of a 6-well plate and 250 &#181;L for a well of a 24-well plate-in a 15 mL conical tube and centrifuge at 120 x g for 4 min.</li>
		<li>Resuspend cells in culture media-2 mL for a well of a 6-well plate and 500 &#181;L for a well of a 24-well plate-supplemented with 10 &#181;M ROCK inhibitor and plate them on fresh matrix coated plates at 40% confluency.</li>
	</ol>
	</li>
</ol>
2. Differentiation of iPSC into MSCs
<ol>
	<li>Prepare MSC differentiation media by adding 15% fetal bovine serum (FBS) and 1% P/S to low glucose DMEM + pyruvate and store at 4 &#176;C.</li>
	<li>Upon reaching 80% confluency, use iPSCs for embryoid body (EB) formation following the steps outlined below.
	<ol>
		<li>Wash the cells with DPBS and incubate them with dissociation medium/EDTA-500 &#181;L for a well of a 6-well plate, 250 &#181;L for a well of a 24-well plate.</li>
		<li>Incubate at 37 &#176;C for 1 min, aspirate the dissociation reagent and keep the cells at 37 &#176;C for an additional 1 min. To start MSC differentiation, ~10-12 x 106 cells are required.</li>
		<li>Collect the cells in a 15 mL conical tube using culture media. Make sure to be very gentle while collecting to prevent cells from getting single and allow EB formation. Centrifuge the cells at 120 x g for 4 min.</li>
		<li>Resuspend the cells in 3 mL of MSC differentiation media containing 10 &#181;M ROCK inhibitor.</li>
		<li>Mix and distribute 0.5 mL/well in a 24-well ultra-low attachment plate. 
		NOTE: Usage of ultra-low attachment plate would encourage cell aggregation into EBs rather than their attachment on the surface.</li>
		<li>Place the plate in the incubator at 37 &#176;C.</li>
	</ol>
	</li>
	<li>After 24 h, induce the attained EBs with high retinoic acid (RA) treatment by following the steps outlined below.
	<ol>
		<li>Add 10 &#181;M RA to 3 mL of MSC differentiation media. Collect EBs in a 15 mL tube and allow them to settle down for 15 min.</li>
		<li>Remove the supernatant from EBs and add MSC differentiation media supplemented with 10 &#181;M RA.</li>
		<li>Resuspend gently and distribute 0.5 mL/well in the same 24-well ultra-low attachment plate.</li>
		<li>Place the plate in the incubator at 37 &#176;C. Do not disturb EBs for the next 48 h.</li>
		<li>After 48 h, collect EBs in a 15 mL tube and allow them to settle down for 15 min.</li>
		<li>Remove the supernatant from EBs and add MSC differentiation media supplemented with 0.1 &#181;M RA.</li>
		<li>Resuspend gently and distribute 0.5 mL/well in the same 24-well ultra-low attachment plate.</li>
		<li>Place the plate in the incubator at 37 &#176;C. Do not disturb EBs for the next 48 h.</li>
	</ol>
	</li>
	<li>Remove the RA added to the cells by following the steps outlined below.
	<ol>
		<li>After 48 h of the last RA treatment, collect the EBs and allow them to settle down for 15 min.</li>
		<li>Remove the supernatant and add DMEM low glucose media without cytokines.</li>
		<li>Resuspend gently and distribute 0.5 mL/well in a 24-well ultra-low attachment plate. Place the plate in the incubator at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Plate the iPSCs-derived EBs by following the steps outlined below.
	<ol>
		<li>After 48 h from the previous step (step 2.4), collect the EBs in a 15 mL tube and allow them to settle down for 15 min.</li>
		<li>Remove supernatant and resuspend in 2 mL of fresh MSC differentiation media.</li>
		<li>Transfer to two wells of a basement membrane matrix-coated 6-well plate.</li>
		<li>Change the media every other day for additional 5 days.</li>
		<li>After 5 days, remove the spent media and replace it with fresh MSC differentiation media containing 2.5 ng/mL of basic fibroblast growth factor (bFGF).</li>
	</ol>
	</li>
	<li>Passage the plated EBs when they reach 80%-90% confluency, by following the steps outlined below.
	<ol>
		<li>Wash the cells with DPBS, add trypsin-EDTA-500 &#181;L for a well of a 6-well plate-and incubate the cells at 37 &#176;C for 3 min.</li>
		<li>Collect the cells using MSC differentiation media in a 15 mL conical tube and spin at 750 x g for 4 min.</li>
		<li>Resuspend in MSC differentiation media with 2.5 ng/mL of bFGF and plate the cells on basement membrane matrix-coated plates at a ratio of 1:3.</li>
		<li>Repeat the passage when the cells reach 70%-80% confluency. It is expected to gain 3-6 million cells by 2-3 passages.</li>
	</ol>
	</li>
</ol>
3. Flow cytometry analysis of iPSCs-derived MSCs
NOTE: Upon undergoing 2-3 passages, the cells should be accessed for the efficiency of MSC differentiation. Differentiation will be considered successful if the cells express MSC differentiation markers-CD44, CD73, CD90, and CD105 at more than 90% efficiency, and do not express high levels of hematopoietic markers-CD14, CD19, CD34, and CD45. The efficiency of these markers can be accessed by following the steps below.
<ol>
	<li>Passage the cells using the steps outlined above (step 2.6) and attain 1 x 105 cells in one well of a v-bottom 96-well plate.</li>
	<li>Centrifuge the plate at 375&#160;x g for 4 min at 4 &#176;C.</li>
	<li>Resuspend 1 x 105 cells in 100 &#181;L of cold DPBS with 1 &#181;L of conjugated antibody (Ab) (see Table of Materials) and incubate at 4 &#176;C for 30-40 min preventing exposure to light.</li>
	<li>Resuspend another 1 x 105 cells in 100 &#181;L of cold DPBS with the respective isotype control of the conjugated Ab at a concentration of 1:100 ) and incubate at 4 &#176;C for 30-40 min preventing exposure to light.</li>
	<li>Following incubation, centrifuge the plate at 375&#160;x g for 4 min at 4 &#176;C. Discard the supernatant by shaking the plate over the sink.</li>
	<li>Resuspend the cells in 100 &#181;L of cold DPBS.</li>
	<li>Centrifuge the cells at 375&#160;x g for 4 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cells in 200 &#181;L of cold DPBS and collect in dark, cold 1.5 mL microcentrifuge tubes and keep them on ice until analyzed by fluorescence-activated cell sorting (FACS).</li>
	<li>For FACS analysis, distribute the cells using side scattered (SSC-A) versus forward scattered (FSC-A) to exclude the debris. Further, distribute the gated cells using forward scattered height (FSC-H) versus forward scattered area (FSC-A) to distinguish singlets from doublets from the live cell population. 
	NOTE: Cells were gated relative to the shift of isotype control for every marker, and a minimum of 10,000 gated events from every stained sample was used for analysis.</li>
</ol>
4. Differentiation of MSCs into adipocytes
<ol>
	<li>Prepare adipocyte differentiation basal media by adding 10% knockout serum replacement (KOSR), 1% Glutamine, 1% P/S, 4.5 ng/&#181;L of glucose to minimum essential media (MEM)-alpha and store at 4 &#176;C.</li>
	<li>Allow MSCs to reach above 90% confluency. Continue culturing them for another 48 h to allow them to undergo a period of growth arrest.</li>
	<li>Prepare complete adipocyte differentiation media by adding 100 &#181;g/mL of 3-Isobutyl-1-methylxanthine (IBMX), 1 &#181;M of dexamethasone, 0.2 U/mL of insulin, 100 &#181;M of indomethacin, and 10 &#181;M of rosiglitazone to the basal media.</li>
	<li>Remove MSC differentiation media and wash the cells using DPBS.</li>
	<li>Add complete adipocyte differentiation media-2 mL for a well of a 6-well plate and 1 mL for a well of a 12-well plate-and incubate the cells at 37 &#176;C. Change complete differentiation media every other day for 14 days.</li>
</ol>
5. Evaluation of the differentiation efficiency of adipocytes
<ol>
	<li>On day 14 of differentiation, check the efficiency of differentiation by staining cells for adipocyte maturation markers, FABP4, and adiponectin.</li>
	<li>Remove the media and wash the cells with DPBS.</li>
	<li>Fix the cells using 4% paraformaldehyde (PFA) - 200 &#181;L to a well of a 24-well plate -and incubate at room temperature for 15 min.</li>
	<li>Discard the PFA and wash using tris-buffered saline with 0.5% tween (TBST) and place it on a shaker at room temperature for 15 min. Repeat the process twice.</li>
	<li>Permeabilize the fixed cells with phosphate-buffered saline with 0.5% Triton X-100 (PBST) and place it on a shaker at room temperature for 15-20 min.</li>
	<li>Discard the PBST and add the blocking buffer (5%-6% bovine serum albumin (BSA) in PBST)-500 &#181;L for a well of a 6-well plate and 250 &#181;L for a well of a 12-well plate-and incubate at room temperature on the shaker for 40-60 min.</li>
	<li>Dilute the primary antibodies against FABP4, adiponectin in 2%-3% BSA, at a concentration of 1:500 (see Table of Materials). Add these antibodies together only if raised in different animals and place the plate on the shaker at 4 &#176;C, overnight.</li>
	<li>Remove the primary antibodies and wash the cells three times with TBST (15 min each) and place it on a shaker at room temperature.</li>
	<li>Prepare Alexa Fluor secondary antibodies in PBST (1:500). Incubate the cells in the secondary antibody combinations (as per the species in which the primary antibody is raised ) for 60 min at room temperature and cover the plate with aluminum foil to protect it from light.</li>
	<li>Discard the secondary antibodies, wash with TBST three times, and place the plate on the shaker.</li>
	<li>To stain the nuclei, add 1 &#181;g/mL of Hoechst 33342-200 &#181;L for a well of a 24-well plate-diluted in PBS and incubate for 5 min at room temperature.</li>
	<li>Discard the Hoechst solution and add PBS-500 &#181;L for a well of a 24-well plate-to the cells. Keep the plates covered from light until visualized using an inverted fluorescence microscope.</li>
</ol>
6. Sorting of adipocytes using Nile red
<ol>
	<li>Prepare Nile red working solution by adding 1 mg/mL Nile red stock solution in DMSO and store at -20 &#176;C. Right before use, thaw the Nile red stock and reconstitute in DPBS to attain 300 nM working solution concentration.</li>
	<li>On or after day 14 of adipocyte differentiation, discard the media from the cells and wash using DPBS.</li>
	<li>Add Nile red working solution -1 mL in a well of a 6-well plate- and incubate at 37 &#176;C for 15 min.</li>
	<li>Remove the Nile red solution and add trypsin-EDTA -500 &#181;L in a well of a 6-well plate- and incubate at 37 &#176;C for 4 min.</li>
	<li>Collect the cells using DMEM containing 5% FBS in a 15 mL conical tube. Centrifuge at 750 x g for 4 min.</li>
	<li>Remove the supernatant and resuspend in DPBS-1 mL for 1 x 106 cells. Centrifuge at 750 x g for 4 min.</li>
	<li>Remove the supernatant and resuspend in DPBS-1 mL for 1 x 106 cells. Use a FACS sorter to isolate the Nile red-positive cells using the FL1 channel.</li>
	<li>Re-culture the sorted cells in adipocyte differentiation media or collect the sorted cells for RNA and protein isolation.</li>
	<li>Extract RNA from the sorted cells and perform relative quantitative analysis of adipocyte differentiation markers, including FABP4, PPARG, and C/EBPA. The Nile red-positive cells show a significant upregulation in the gene expression of at least two folds compared to un-sorted cells.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neural+crest+cell+fate+by+the+retinoic+acid+and+Pparg+signalling+pathways.">Regulation of neural crest cell fate by the retinoic acid and Pparg signalling pathways.</a> Development. 137 (3), 389-394 (2010).</li><li>Ussar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ASC-1,+PAT2,+and+P2RX5+are+cell+surface+markers+for+white,+beige,+and+brown+adipocytes.">ASC-1, PAT2, and P2RX5 are cell surface markers for white, beige, and brown adipocytes.</a> Science Translational Medicine. 6 (247), (2014).</li><li>Festy, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+protein+expression+between+human+adipose+tissue-derived+stromal+cells+and+mature+adipocytes.">Surface protein expression between human adipose tissue-derived stromal cells and mature adipocytes.</a> Histochemistry and Cell Biology. 124 (2), 113-121 (2005).</li><li>Cai, L., Wang, Z., Ji, A., Meyer, J. M., vander Westhuyzen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scavenger+receptor+CD36+expression+contributes+to+adipose+tissue+inflammation+and+cell+death+in+diet-induced+obesity.">Scavenger receptor CD36 expression contributes to adipose tissue inflammation and cell death in diet-induced obesity.</a> PLoS One. 7 (5), 36785 (2012).</li><li>Mesuret, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neuronal+role+of+the+Alanine-Serine-Cysteine-1+transporter+(SLC7A10,+Asc-1)+for+glycine+inhibitory+transmission+and+respiratory+pattern.">A neuronal role of the Alanine-Serine-Cysteine-1 transporter (SLC7A10, Asc-1) for glycine inhibitory transmission and respiratory pattern.</a> Scientific Reports. 8 (1), 8536 (2018).</li><li>Silverstein, R. L., Febbraio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD36,+a+scavenger+receptor+involved+in+immunity,+metabolism,+angiogenesis,+and+behavior.">CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior.</a> Science Signaling. 2 (72), (2009).</li><li>Brooimans, R. A., van Wieringen, P. A., van Es, L. A., Daha, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+roles+of+decay-accelerating+factor,+membrane+cofactor+protein,+and+CD59+in+the+protection+of+human+endothelial+cells+against+complement-mediated+lysis.">Relative roles of decay-accelerating factor, membrane cofactor protein, and CD59 in the protection of human endothelial cells against complement-mediated lysis.</a> European Journal of Immunology. 22 (12), 3135-3140 (1992).</li><li>Davies, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD59,+an+LY-6-like+protein+expressed+in+human+lymphoid+cells,+regulates+the+action+of+the+complement+membrane+attack+complex+on+homologous+cells.">CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells.</a> The Journal of Experimental Medicine. 170 (3), 637-654 (1989).</li><li>Lapid, K., Graff, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form(ul)ation+of+adipocytes+by+lipids.">Form(ul)ation of adipocytes by lipids.</a> Adipocyte. 6 (3), 176-186 (2017).</li><li>Aldridge, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+validation+for+the+assessment+of+adipogenesis+of+multipotential+stromal+cells--a+direct+comparison+of+four+different+methods.">Assay validation for the assessment of adipogenesis of multipotential stromal cells--a direct comparison of four different methods.</a> Cytotherapy. 15 (1), 89-101 (2013).</li><li>Schaedlich, K., Knelangen, J. M., Navarrete Santos, A., Fischer, B., Navarrete Santos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+sort+ESC-derived+adipocytes.">A simple method to sort ESC-derived adipocytes.</a> Cytometry A. 77 (10), 990-995 (2010).</li><li>Costa, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+mesenchymal+stem+cells+from+natural+niches+to+culture+conditions:+implications+for+further+clinical+uses.">Functional heterogeneity of mesenchymal stem cells from natural niches to culture conditions: implications for further clinical uses.</a> Cellular and Molecular Life Sciences: CMLS. 78 (2), 447-467 (2021).</li></ol>

The authors declare that they have no competing interests.

This protocol holds paramount importance due to its ability to provide MSCs in high yield and efficiency. This mass-scale production of MSCs was made possible by transient incubation of iPSCs-derived EBs with 10 &#181;M of RA14,15. Transient treatment with 10 &#181;M of RA enhanced the MSC yield by 11.2 to 1542 folds14,15, with this protocol being applicable on both iPSCs and hPSCs. At this dose and durat...

Mesenchymal stem cells (MSCs) act as an effective transitory resource for producing cells of mesodermal origin like adipocytes, osteocytes, and chondrocytes, which could be further used for modeling their respective genetic disorders. However, previous approaches relied on attaining these MSCs from adult tissues1, which imposed the challenge of obtaining them in high numbers from the donors, and the limitation of keeping them functionally viable in suboptimal in vitro culture conditions1,2. These obstacles have produced a great demand of having a protocol for generating MSCs in vitro. Human induced pluripotent stem cells (iPSCs) can be used as a valuable source of MSCs, exhibiting MSC characteristics3,4,5. iPSCs-derived MSCs can be used as a therapeutic option in several diseases. Also, the ability of iPSCs-derived MSCs to generate adipocytes, makes them a valuable in vitro human model to study human adipogenesis, obesity, and adipocyte-associated disorders.
Current differentiation protocols of adipocytes can be classified into two groups, with one involving differentiation of adipocytes using chemical or protein-based cocktails giving a resultant yield of 30%-60%6,7,8,9, while the other involving genetic manipulation for robust induction of key transcription factors governing adipocytes development to give a yield of 80%-90%10,11. However, genetic manipulation doesn&#39;t recapitulate the natural process of adipocyte differentiation, and often masks the subtle paradigms arriving during adipogenesis, making it ineffective for disease modeling purposes12,13. Therefore, we present a way to sort chemically derived mature adipocytes from immature ones by fluorescently tagging lipid-bearing adipocytes using Nile red.
Here we present a protocol involving transient incubation of iPSCs derived embryoid bodies (EBs) with all-trans retinoic acid to produce a high number of rapidly proliferating MSCs, which could be further used for generating adipocytes14. We also present a way to sort chemically derived mature adipocytes from the heterogeneous differentiation pool by fluorescently tagging their lipid droplets using a lipophilic dye; Nile red. This would allow the generation of a pure population of mature adipocytes with enhanced functionality to accurately model adipocyte-associated metabolic disorders.

<table><tbody><tr><td>Adiponectin</td><td>Abcam</td><td>ab22554</td><td>Adipocyte maturation marker</td></tr><tr><td>anti-CD105</td><td>BD Pharmingen</td><td>560839</td><td>MSC differentiation marker</td></tr><tr><td>anti-CD14</td><td>BD Pharmingen</td><td>561712</td><td>MSC differentiation marker</td></tr><tr><td>anti-CD19</td><td>BD Pharmingen</td><td>555415</td><td>MSC differentiation marker</td></tr><tr><td>anti-CD34</td><td>BD Pharmingen</td><td>555824</td><td>MSC differentiation marker</td></tr><tr><td>anti-CD44</td><td>abcam</td><td>ab93758</td><td>MSC differentiation marker</td></tr><tr><td>anti-CD45</td><td>BD Pharmingen</td><td>&lt;br/&gt; 560975</td><td>MSC differentiation marker</td></tr><tr><td>anti-CD73</td><td>BD Pharmingen</td><td>550256</td><td>MSC differentiation marker</td></tr><tr><td>anti-CD90</td><td>BD Pharmingen</td><td>555596</td><td>MSC differentiation marker</td></tr><tr><td>bFGF</td><td>R&amp;amp;D</td><td>233-FP</td><td>MSC culture media supplement</td></tr><tr><td>C/EBPA</td><td>Abcam</td><td>ab40761</td><td>Adipocyte maturation marker</td></tr><tr><td>Dexamethasone</td><td>Torics</td><td>1126</td><td>Adipocyte differentiation media supplement</td></tr><tr><td>FABP4</td><td>Abcam</td><td>ab93945</td><td>Adipocyte maturation marker</td></tr><tr><td>Fetal bovine serum</td><td>ThermoFisher</td><td>10082147</td><td>MSC culture media supplement</td></tr><tr><td>Glutamax</td><td>ThermoFisher</td><td>35050-061</td><td>MSC culture media supplement</td></tr><tr><td>IBMX</td><td>Sigma Aldrich</td><td>I5879</td><td>Adipocyte differentiation media supplement</td></tr><tr><td>Indomethacin</td><td>Sigma Aldrich</td><td>I7378</td><td>Adipocyte differentiation media supplement</td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>91077C</td><td>Adipocyte differentiation media supplement</td></tr><tr><td>Knockout DMEM</td><td>ThermoFisher</td><td>12660012</td><td>Basal media for preparing matrigel</td></tr><tr><td>Low glucose DMEM</td><td>ThermoFisher</td><td>11885084</td><td>MSC culturing media</td></tr><tr><td>Matrigel</td><td>Corning</td><td>354230</td><td>Coating matrix</td></tr><tr><td>MEM-alpha</td><td>ThermoFisher</td><td>12561056</td><td>Adipocyte differentiation media</td></tr><tr><td>Nilered</td><td>Sigma Aldrich</td><td>19123</td><td>Sorting marker for adipocyte</td></tr><tr><td>Penicillin</td><td>ThermoFisher</td><td>15140122</td><td>MSC/Adipocyte media supplement</td></tr><tr><td>Phosphate-buffered saline</td><td>ThermoFisher</td><td>14190144</td><td>wash buffer</td></tr><tr><td>Pierce&amp;trade; 20X TBS Buffer</td><td>Thermo Fisher</td><td>28358</td><td>wash buffer</td></tr><tr><td>PPARG</td><td>Cell Signaling Technology</td><td>2443</td><td>Adipocyte maturation marker</td></tr><tr><td>ReLeSR</td><td>Stem Cell Technologies</td><td>5872</td><td>Dissociation reagent</td></tr><tr><td>Retinoic acid</td><td>Sigma Aldrich</td><td>R2625</td><td>MSC differentiation media supplement</td></tr><tr><td>Rock inhibitor</td><td>Tocris</td><td>1254/10</td><td>hPSC culture media supplement</td></tr><tr><td>Roziglitazone</td><td>Sigma Aldrich</td><td>R2408</td><td>Adipocyte differentiation media supplement</td></tr><tr><td>StemFlex</td><td>ThermoFisher</td><td>A334901</td><td>hPSC culture media</td></tr><tr><td>Triton</td><td>Thermo Fisher</td><td>28314</td><td>Permebealization reagent</td></tr><tr><td>Trypsin</td><td>ThermoFisher</td><td>25200072</td><td>Dissociation reagent</td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P7942</td><td>Wash buffer</td></tr></tbody></table>

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.

Schematic and morphology of cells during mesenchymal differentiation: Differentiation of iPSCs into MSCs involves various stages of development spanning across EB formation, MSC differentiation, and MSC expansion (Figure 1). During these stages of development, cells acquire various morphology owing to the different stimulatory chemicals they are subjected to. Upon initiating differentiation, cells are plated in suspension and are expected to be round, with defined cell borders, while being s...

Watch this Scientific Journal Video about Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders at JoVE.com

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

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 ...

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JoVE Journal

Developmental Biology

3.1K Views.  Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF). 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.

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

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

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

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Biology

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Characterizing Surface Antigens and Proliferation Ability in hiPSC-Driving MSCs

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

Author Spotlight: Advancements in iPSCs and Genetic Disease Research 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...