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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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> 
			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&#38;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&#8482; 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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Universidad de Cartagena UDEC

Research

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

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

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 ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

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

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Chapters in this video

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells