Name: robust differentiation of human ipscs into a pure population of adipocytes to study adipocyte-associated disorders
Uploaded: 2022-02-09T14:45:00
Description: 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

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 periph...

<ol>
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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...

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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 ...

Previous approaches relied on highly invasive methods for obtaining mesenchymal stem cells in vitro, but those mesenchymal stem cells had a limitation of producing heterogeneous pool of 30%with efficiency ranging from 30 to 60%Here, we present a protocol for producing high number of rapidly proliferating mesenchymal stem cells that can produce a pure population of mature adipocytes by sorting using Nile red. When the iPSCs have reached 80%confluence, wash the cells with DPBS and add dissociation medium containing EDTA. Incubate the cells at 37 degrees Celsius for one minute.After one minute, aspirate the dissociation reagent and place the cells at 37 degrees Celsius for an additional one minute. Next, remove the plate from the incubator. Add one milliliter of StemFlex media per well and carefully collect the cells in a 15-milliliter conical tube.Centrifuge the cells. Remove the supernatant, and gently resuspend them in three milliliters of MSC differentiation media containing 10 micromoles of a rock inhibitor. Mix the cell suspension and distribute 500 microliters per well in a 24-well ultra-low attachment plate.Then place the plate in an incubator at 37 degrees Celsius. After 24 hours, to induce the attained embryonic bodies, collect them in a 15-milliliter tube and allow them to settle down. After 15 minutes, remove the supernatant and add MSC differentiation medium supplemented with 10 micromolar retinoic acid.Resuspend the embryonic bodies gently and distribute 500 microliters per well in the same 24-well ultra-low attachment plate. Then place the plate in the incubator. After 48 hours, collect the embryonic bodies in a 15-milliliter tube and allow them to settle down for 15 minutes.Then remove the supernatant and add MSC differentiation medium supplemented with 0.1 micromolar retinoic acid. After resuspending the embryonic bodies, distribute 500 microliters per well in the same 24-well plate and place the plate in the incubator. Forty-eight hours after the last retinoic acid treatment, collect the embryonic bodies, remove the supernatant, and add DMEM low-glucose medium without cytokines.Gently resuspend and distribute 500 microliters of the cell suspension per well in the same 24-well ultra-low attachment plate, then incubate the plate at 37 degrees Celsius. After 48 hours, to plate the iPSC-derived embryonic bodies, collect the embryonic bodies in a 15-milliliter tube, allow them to settle, then remove the supernatant, and resuspend in two milliliters of fresh MSC differentiation medium. Next, transfer the suspension to two wells of a basement membrane matrix-coated six-well plate and incubate the cells at 37 degrees Celsius.After five days, replace the spent media with a fresh MSC differentiation medium containing 2.5 nanograms per milliliter of basic fibroblast growth factor and continue the MCS differentiation for up to 11 and 15 days. When the plated embryonic bodies have reached 80 to 90%confluence, passage them by washing with DPBS, adding Trypsin-EDTA, and incubating at 37 degrees Celsius for one minute. Remove the Trypsin-EDTA from the plate and incubate again at 37 degrees Celsius for one minute.Next, add one milliliter media per well. After three minutes, collect the cells using MSC differentiation media in a 15-milliliter conical tube and centrifuge the cells. Then resuspend the cells in an MSC differentiation medium containing basic fibroblast growth factor and plate the cells on basement membrane matrix-coated plates at a ratio of one to three.After the MSCs have reached 90%confluence, continue culturing them for another 48 hours to allow them to undergo a period of growth arrest. Then remove the medium and wash the cells with DBPS. After the wash, add complete adipocyte differentiation medium to the plate and incubate the cells at 37 degrees Celsius.Change the medium every other day for 14 days. Differentiate iPSC-derived MSCs into adipocytes. To sort the adipocytes using Nile red, prepare Nile red working solution in DMSO as described in the text manuscript.Before use, thaw the stock solution and reconstitute it in DPBS to attain a 300-nanomolar working solution concentration. On or after day 14 of adipocyte differentiation, discard the medium from the cells and wash using DPBS. Then add Nile red working solution.Cover the plate and incubate the cells at 37 degrees Celsius. After 15 minutes, replace the Nile red solution with Trypsin-EDTA and incubate the cells at 37 degrees Celsius for four minutes. Then collect the cells using DMEM containing 5%FBS in a 15-milliliter conical tube and centrifuge the cells.Remove the supernatant and resuspend the cells in DPBS at a density of 1 million cells per milliliter. Then using the FACS sorter, isolate the Nile red-positive cells using the FL-1 channel. Collect the sorted cells and reculture them in adipocyte differentiation media or proceed to RNA extraction followed by quantitative analysis of adipocyte differentiation markers.Upon initiating differentiation, cells plated in suspension are round with defined cell borders and are small to medium size in diameter. The viability of embryonic bodies is observed by the rapid proliferation behavior giving rise to more MSCs. This rapid proliferation behavior, along with their peculiar and elongated morphology, is retained even after passaging the MSCs onto fresh matrix-coated plates.Good differentiation produces reliable MSCs with greater than 90%expression efficiency of mesenchymal surface markers CD73, CD44, and CD90. In addition, assessment of cells for the absence of surface markers depicting the hematopoietic phenotype CD14, CD34, and CD19 showed less than 1%expression efficiency, High expression in cytoplasmic distribution of FABP4, a marker for terminally-differentiated adipocytes, indicates their developmental maturity. Additionally, high expression of adiponectin, another marker of adipocyte maturity, indicates that the adipocytes are functional enough to undergo lipid storage and adipogenesis in response to glucose signaling.Upon staining, Nile red exclusively binds to the lipid-bearing mature adipocytes, making it an effective tool for sorting mature adipocytes using fluorescent-activated flow cytometry. The Nile red-positive cells show a significant upregulation of maturation markers by at least two-folds compared to unsorted cells. You have to be very gentle while collecting cells during embryonic body formation and mesenchymal cell dissociation as that would highly impact the survival efficiency post-dissociation of mesenchymal stem cells.Pure population of adipocytes attained from mutation-carrying patients can be subjected to functional and the whole transcriptome sequencing to determine regulatory and signaling pathways affected by particular mutation without the data being affected by sample heterogeneity. This technique will allow scalable generation of mesenchymal stem cells in vitro, removing the need of harvesting them from human donors to readily differentiate them to adipocytes, chondrocytes, and osteocytes for understanding tissue-related disease pathogenesis.

Research

JoVE Journal

Developmental Biology

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Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

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Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

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Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

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