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

Maryam Aghadi; Manale Karam; Essam M. Abdelalim

doi:10.3791/63311

JoVE Journal > Developmental Biology

Biología del desarrollo

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

Published: February 09, 2022

doi:

10.3791/63311

Maryam Aghadi^1,2, Manale Karam¹, Essam M. Abdelalim^1,2

¹Diabetes Research Center, Qatar Biomedical Research Institute (QBRI),Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), ²College of Health and Life Sciences,Hamad Bin Khalifa University (HBKU), Qatar Foundation

Summary

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.

Abstract

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.

Introduction

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'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.

Protocol

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

Prepare basement membrane matrix-coated plates by reconstituting coating matrix in knockout-DMEM at a ratio of 1:80 and store at 4 °C.
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 °C for short term or at -20 °C for long term use.
Line the plates with coating matrix-1 mL for a 6-well plate, 500 µL for a 12-well plate, 250 µL for a 24-well plate-and incubate the plate at 37 °C for 1-2 h.
Remove an aliquot of iPSCs culture media and pre-warm at room temperature before use.
Thaw a vial of iPSCs (ESCs or iPSCs) in a 37 °C water bath and transfer to a 15 mL conical tube containing 2-3 mL of culture media.
Centrifuge the tube at 120 x g for 4 min at room temperature (RT-23 °C).
Remove the supernatant and add 2 mL of fresh culture media supplemented with 10 µM ROCK inhibitor (Y-27632). Plate the cells in one well of a matrix-coated 6-well plate and place the plate at 37 °C.
After 24 h, remove the media and replace it with fresh culture media.
Change the media every day until the cells reach 80%-90% confluency.
Upon reaching confluency, passage cells by following the steps outlined below.
1. Remove the media and wash the cells with Dulbecco's phosphate-buffered saline (DPBS).
2. Add iPSCs dissociation reagent (see Table of Materials)-500 µL for a well of a 6-well plate, 250 µL for a well of a 24-well plate-and incubate for 1 min at 37 °C.
3. Remove the dissociation reagent and incubate the cells dry for 1 min at 37 °C.
4. Collect the cells using culture media-1 mL for a well of a 6-well plate and 250 µL for a well of a 24-well plate-in a 15 mL conical tube and centrifuge at 120 x g for 4 min.
5. Resuspend cells in culture media-2 mL for a well of a 6-well plate and 500 µL for a well of a 24-well plate-supplemented with 10 µM ROCK inhibitor and plate them on fresh matrix coated plates at 40% confluency.

2. Differentiation of iPSC into MSCs

Prepare MSC differentiation media by adding 15% fetal bovine serum (FBS) and 1% P/S to low glucose DMEM + pyruvate and store at 4 °C.
Upon reaching 80% confluency, use iPSCs for embryoid body (EB) formation following the steps outlined below.
1. Wash the cells with DPBS and incubate them with dissociation medium/EDTA-500 µL for a well of a 6-well plate, 250 µL for a well of a 24-well plate.
2. Incubate at 37 °C for 1 min, aspirate the dissociation reagent and keep the cells at 37 °C for an additional 1 min. To start MSC differentiation, ~10-12 x 10⁶ cells are required.
3. 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.
4. Resuspend the cells in 3 mL of MSC differentiation media containing 10 µM ROCK inhibitor.
5. 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.
6. Place the plate in the incubator at 37 °C.
After 24 h, induce the attained EBs with high retinoic acid (RA) treatment by following the steps outlined below.
1. Add 10 µM RA to 3 mL of MSC differentiation media. Collect EBs in a 15 mL tube and allow them to settle down for 15 min.
2. Remove the supernatant from EBs and add MSC differentiation media supplemented with 10 µM RA.
3. Resuspend gently and distribute 0.5 mL/well in the same 24-well ultra-low attachment plate.
4. Place the plate in the incubator at 37 °C. Do not disturb EBs for the next 48 h.
5. After 48 h, collect EBs in a 15 mL tube and allow them to settle down for 15 min.
6. Remove the supernatant from EBs and add MSC differentiation media supplemented with 0.1 µM RA.
7. Resuspend gently and distribute 0.5 mL/well in the same 24-well ultra-low attachment plate.
8. Place the plate in the incubator at 37 °C. Do not disturb EBs for the next 48 h.
Remove the RA added to the cells by following the steps outlined below.
1. After 48 h of the last RA treatment, collect the EBs and allow them to settle down for 15 min.
2. Remove the supernatant and add DMEM low glucose media without cytokines.
3. Resuspend gently and distribute 0.5 mL/well in a 24-well ultra-low attachment plate. Place the plate in the incubator at 37 °C.
Plate the iPSCs-derived EBs by following the steps outlined below.
1. 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.
2. Remove supernatant and resuspend in 2 mL of fresh MSC differentiation media.
3. Transfer to two wells of a basement membrane matrix-coated 6-well plate.
4. Change the media every other day for additional 5 days.
5. 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).
Passage the plated EBs when they reach 80%-90% confluency, by following the steps outlined below.
1. Wash the cells with DPBS, add trypsin-EDTA-500 µL for a well of a 6-well plate-and incubate the cells at 37 °C for 3 min.
2. Collect the cells using MSC differentiation media in a 15 mL conical tube and spin at 750 x g for 4 min.
3. 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.
4. Repeat the passage when the cells reach 70%-80% confluency. It is expected to gain 3-6 million cells by 2-3 passages.

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.

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.
Centrifuge the plate at 375 x g for 4 min at 4 °C.
Resuspend 1 x 10⁵ cells in 100 µL of cold DPBS with 1 µL of conjugated antibody (Ab) (see Table of Materials) and incubate at 4 °C for 30-40 min preventing exposure to light.
Resuspend another 1 x 10⁵ cells in 100 µL of cold DPBS with the respective isotype control of the conjugated Ab at a concentration of 1:100 ) and incubate at 4 °C for 30-40 min preventing exposure to light.
Following incubation, centrifuge the plate at 375 x g for 4 min at 4 °C. Discard the supernatant by shaking the plate over the sink.
Resuspend the cells in 100 µL of cold DPBS.
Centrifuge the cells at 375 x g for 4 min at 4 °C. Discard the supernatant.
Resuspend the cells in 200 µL 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).
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.

4. Differentiation of MSCs into adipocytes

Prepare adipocyte differentiation basal media by adding 10% knockout serum replacement (KOSR), 1% Glutamine, 1% P/S, 4.5 ng/µL of glucose to minimum essential media (MEM)-alpha and store at 4 °C.
Allow MSCs to reach above 90% confluency. Continue culturing them for another 48 h to allow them to undergo a period of growth arrest.
Prepare complete adipocyte differentiation media by adding 100 µg/mL of 3-Isobutyl-1-methylxanthine (IBMX), 1 µM of dexamethasone, 0.2 U/mL of insulin, 100 µM of indomethacin, and 10 µM of rosiglitazone to the basal media.
Remove MSC differentiation media and wash the cells using DPBS.
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 °C. Change complete differentiation media every other day for 14 days.

5. Evaluation of the differentiation efficiency of adipocytes

On day 14 of differentiation, check the efficiency of differentiation by staining cells for adipocyte maturation markers, FABP4, and adiponectin.
Remove the media and wash the cells with DPBS.
Fix the cells using 4% paraformaldehyde (PFA) – 200 µL to a well of a 24-well plate -and incubate at room temperature for 15 min.
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.
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.
Discard the PBST and add the blocking buffer (5%-6% bovine serum albumin (BSA) in PBST)-500 µL for a well of a 6-well plate and 250 µL for a well of a 12-well plate-and incubate at room temperature on the shaker for 40-60 min.
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 °C, overnight.
Remove the primary antibodies and wash the cells three times with TBST (15 min each) and place it on a shaker at room temperature.
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.
Discard the secondary antibodies, wash with TBST three times, and place the plate on the shaker.
To stain the nuclei, add 1 µg/mL of Hoechst 33342-200 µL for a well of a 24-well plate-diluted in PBS and incubate for 5 min at room temperature.
Discard the Hoechst solution and add PBS-500 µL for a well of a 24-well plate-to the cells. Keep the plates covered from light until visualized using an inverted fluorescence microscope.

6. Sorting of adipocytes using Nile red

Prepare Nile red working solution by adding 1 mg/mL Nile red stock solution in DMSO and store at -20 °C. Right before use, thaw the Nile red stock and reconstitute in DPBS to attain 300 nM working solution concentration.
On or after day 14 of adipocyte differentiation, discard the media from the cells and wash using DPBS.
Add Nile red working solution -1 mL in a well of a 6-well plate- and incubate at 37 °C for 15 min.
Remove the Nile red solution and add trypsin-EDTA -500 µL in a well of a 6-well plate- and incubate at 37 °C for 4 min.
Collect the cells using DMEM containing 5% FBS in a 15 mL conical tube. Centrifuge at 750 x g for 4 min.
Remove the supernatant and resuspend in DPBS-1 mL for 1 x 10⁶ cells. Centrifuge at 750 x g for 4 min.
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.
Re-culture the sorted cells in adipocyte differentiation media or collect the sorted cells for RNA and protein isolation.
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.

Representative Results

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 small to medium size in diameter (Figure 2). Choice of culturing cells in suspension during the initial phase of differentiation allows it to closely resemble the process of natural embryonic development, making this phase highly crucial for successful differentiation. The phase of EB formation and RA treatment is followed by plating EBs on basement membrane matrix-coated plates. The viability of EBs upon plating can be accessed by observing their rapid proliferation behavior giving rise to more MSCs (Figure 2). This rapid proliferation behavior exhibited by MSCs is retained even after passaging them onto fresh matrix coated plates along with retaining peculiar, elongated morphology (Figure 2).

Quantitative assessment of MSC surface markers: Differentiation efficiency of MSCs is accessed by quantification of surface markers specific for MSC differentiation. Good differentiation producing reliable MSCs should show greater than 90% efficiency of mesenchymal surface markers CD73, CD44, and CD90 (Figure 3A). In addition to that, cells are also assessed for the absence of surface markers depicting hematopoietic phenotype, CD14, CD34, and CD19, and is therefore expected to show less than 1% expression efficiency for them (Figure 3B).

Differentiation of MSCs into adipocytes: Differentiation of MSCs into adipocytes can be accessed by staining for FABP4 and adiponectin. FABP4 is a cytoplasmic protein, and it is regarded as a marker for terminally differentiated adipocytes. Its high expression among adipocytes, with a cytoplasmic distribution, is a key sign of their developmental maturity (Figure 4A). In addition to FABP4, adiponectin is regarded as one of the important markers for adipocyte maturity. Its high expression indicates adipocytes are functional enough for undergoing lipid storage and adipogenesis in response to glucose signaling. Being a secretory protein, adiponectin exhibits globular morphology with every protein globule easily distinguishable within the cytoplasm (Figure 4B).

Staining and sorting of mature adipocytes using Nile red: Upon differentiation, mature adipocytes can be distinguished from their immature counterparts by staining for Nile red. Nile red binds to lipid-bearing adipocytes, a characteristic exclusive to mature adipocytes (Figure 5A). This along with the fluorescent bearing characteristic of Nile red makes it an effective tool for sorting mature adipocytes using fluorescent activated flow cytometry (Figure 5B). Effective sorting should result in the enhancement of maturation markers-PPARG, C/EBPA, and FABP4-by at least two folds, determined by quantitative real-time PCR (qRT-PCR) (Figure5C).

Figure 1: Schematic diagram showing the differentiation of iPSCs into MSCs and adipocytes. iPSCs are differentiated into MSCs using the embryoid body (EB) technique. The EBs are subjected to a short exposure of 10 µM of all-trans retinoic acid (RA). The generated MSCs are differentiated into 40%-77% adipocytes based on the iPSC line. The Nile red positive cells are sorted using FACS to obtain a purified population of mature adipocytes that can be used for studying adipocyte-associated disorders (disease modeling), identifying novel drugs, and eventually for personalized therapy. Please click here to view a larger version of this figure.

Figure 2: Differentiation of iPSCs into MSCs. Representative morphological images showing different stages of MSC differentiation at days 2 (D2), 11 (D11), 15 (D15), and 24 (D24). Embryoid bodies (EBs) generated in the presence of 10 µM of RA for 24 h were plated at day 8 of differentiation, followed by dissociation and passaging after 12-17 days of differentiation. The MSCs were passaged several times. Abbreviations: P2 = passage 2. Please click here to view a larger version of this figure.

Figure 3: Expression of MSC markers and hematopoietic markers in iPSC-derived MSCs. Representative flow cytometry histograms showing the expression of the MSC markers, CD73, CD44, and CD90, (A) and the hematopoietic markers, CD34, CD19, and CD14 (B) in the MSCs generated from iPSC-derived EBs treated with 10 µM of RA. The X-axis in the graph represents the fluorescent intensity. Please click here to view a larger version of this figure.

Figure 4: Differentiation of iPSC-derived MSCs into adipocytes. Immunostaining images showing the expression of FABP4 (A) and adiponectin (ADIPO) (B) in mature adipocytes derived from iPSCs. The nuclei were stained with Hoechst. Please click here to view a larger version of this figure.

Figure 5: Sorting of iPSC-derived adipocytes using Nile red. (A) Images showing bright field (BF) and Nile red-stained mature adipocytes. (B) Quantification of Nile red (PE-positive cells) in mature adipocytes using FACS. (C) Real-time PCR analysis showing the expression of C/EBPA, FABP4, and PPARG in sorted versus unsorted mature adipocytes. Data are represented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. Please click here to view a larger version of this figure.

Discussion

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 µM of RA¹⁴^,¹⁵. Transient treatment with 10 µM 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 duration of treatment, RA improves the proliferative and survival capacities of EB-forming cells by direct or indirect regulation of the expression of several genes involved in cell proliferation, apoptosis, and cell-cell and ECM-cell adhesions, which are critical for the survival and proliferation of iPSCs¹⁴^,¹⁶. The genes include, but are not limited to, transcription factors (such as EGFR4, SOX4), growth factors and growth factor receptors (such as IGF2, FGFR4), and adhesion molecules (such as FN1 and CAMs). However, in contrast to low doses (0.1-10 µM), at high doses (≥20 µM), RA negatively regulates proliferation and survival of EB-forming cells resulting in reduced PSC-derived EB number and size and thereby a decreased yield of MSCs¹⁴. RA is regarded as a proliferation inhibitor in several normal differentiated and cancerous cells¹⁷^,¹⁸^,¹⁹. In EBs, retinoid signaling is context (time, concentration, species, and cell line)-dependent; differentially affecting the self-renewal, survival, and differentiation of EB-forming cells by regulating distinct genes and signaling pathways²⁰^,²¹. Therefore, the usage of RA in an optimal time and concentration of RA-10 µM on day 3 of EB induction followed by dose reduction to 0.1 µM on day 5 for 2 days as described in the present protocol-is crucial to induce EB-forming cell survival and proliferation.

In addition to regulating growth and survival, RA does subject the treated EBs to differentiation delay as compared to cells non-treated with RA¹⁴. In fact, RA-treated EBs maintain their compact shape after plating and fail to differentiate into MSC-like cells, in contrast to RA-untreated EBs. This is consistent with previous studies reporting that short-term exposure to RA treatment inhibits cell differentiation through the suppression of WNT signaling²¹. Moreover, these RA-treated differentiation-delayed cells also showed enhanced expression of cadherin and extra-cellular matrix proteins¹⁴, which are known to play an important role in maintaining the pluripotent state of iPSCs¹⁶. To release the RA-mediated differentiation block, EBs should be dissociated, which results in disrupting cell adhesions and allows long-term MSC differentiation upon plating. Interestingly, RA treatment did hold a differentiation block over cells, but it did not maintain the cells in a pluripotent state. In fact, the EB-forming cells undergoing short-term exposure to 10 µM RA show significantly reduced expression of key pluripotency markers-OCT4, SOX2, and NANOG¹⁴.

The MSCs generated by short-term RA treatment of EBs have been shown to maintain their typical fibroblast-like morphology with abundant expression of MSC surface markers and their multipotency following cryopreservation, thus making these mass-produced MSCs storable for long-term expansion studies¹⁴. When subjecting them to adipogenic, chondrogenic, and osteogenic differentiation conditions, these MSCs could readily differentiate into the three mesodermal cell types thus making them an easily attainable source for modeling tissue-related diseases¹⁴. Thus, the stable and versatile in vitro behavior of the MSCs generated by the RA-mediated differentiation protocol provides them with paramount importance in research and application-based settings.

While the chondrogenic and osteogenic differentiation potentials of MSCs obtained from RA-treated EBs seem to be similar to those of the MSCs obtained from untreated EBs, the former was found to display an enhanced potential to differentiate into adipogenic lineage when subjected to adipogenic differentiation conditions¹⁴. This was evidenced by a 2- to 3-fold increase in intracellular lipid accumulation (Oil Red O staining) and adipocyte marker FABP4-positive cells in the differentiation pool of cells obtained after culturing the MSCs derived from RA-treated EBs with adipogenic differentiation media, as compared to MSCs derived from RA-untreated EBs. This could be the consequence of the regulation, by RA, of several signaling pathways governing adipocyte development such as Hippo, WNT, and ECM-cell interaction pathways, as revealed by RNA sequencing data from RA-treated and untreated EBs¹⁴^,²²^,²³^,²⁴^,²⁵. This enhanced ability of RA-derived MSCs to undergo adipogenic differentiation is valuable, as currently available protocols either lead to poor adipocyte yield or make use of genetic manipulation making the generated adipocytes invaluable for deriving natural-process recapitulated adipocytes. Adipocytes are classified into three types-white, brown, and beige. White adipocytes are classified by the presence of a single lipid droplet and play a role in energy storage. Whereas, brown adipocytes are involved in energy expenditure by substrate oxidation due to the very high abundance of mitochondria characterized by the expression of UCP1. Whereas the brown adipocytes that are found localized in white adipose tissue are known as beige-or brown-like-adipocytes. These MSC have the potential to give an abundant yield of white adipocytes given the pre-exposure of EBs to RA. Previous publications have stated selective induction of iPSC into cells expressing low UCP1 i.e., white adipocytes, rather than exposing cells with high UCP1 levels to RA²⁶. Previous publications have reported that RA produced from neural crest cells in mouse and zebrafish embryos plays an important role in white adipocyte formation²⁷^,²⁸.

Although the RA-based protocol allowed the generation of MSCs that provide increased yield of adipocytes reaching 48.5%-77.4% (vs. 22.5%-57.6% without RA treatment), not attaining >90% is still problematic when modeling multi-variant adipocyte-based genetic disorders in vitro. In fact, not reaching a pure adipocyte population could make results coming from multi-variant disease models ambivalent, as it would be hard to distinguish if the observed developmental differences are due to the different genetic makeups or due to inconsistent differentiation efficiencies. In order to circumvent this issue, it was important to sort the differentiated cells to obtain a pool of pure mature adipocytes, so that any differences in phenotypes could only be attributed to inherent genetic differences. Several studies have identified surface markers on adipocytes that could potentially be used for sorting. For example, work carried out by Ronald Kahn allowed the identification of the amino acid transporter ASC-1 as a novel surface marker on white adipocytes²⁹. In addition, studies extracting mature adipocytes from omental and subcutaneous regions have reported mature adipocytes to express CD34, CD36, and CD59 on their surfaces³⁰, where CD36 has been reported to function as a fatty acid transporter on the surface of mature adipocytes³¹. However, these studies have made use of heterogeneous populations of cells derived from the adipose tissue without specifying the expression of these markers to only mature adipocyte populations. Furthermore, these markers can be also expressed by other cell types and are not specific to adipocytes. For example, ASC-1 is present on both astrocytes and neurons³², CD34 is a marker of hematopoietic stem cells³³, CD36 is present on platelets, mononuclear phagocytes, hepatocytes, myocytes, and some epithelia³³, and CD59 is expressed on endothelial and lymphoid cells³⁴^,³⁵. Therefore, as an alternative solution, Nile red, the selective fluorescent stain for intracellular lipids, was used as a possible candidate for sorting adipocytes. Adipocytes store a significant bulk of lipids that can be released and used to produce energy, build membranes, or as signaling molecules that regulate metabolism³⁶. Nile red dye has previously been used in flow cytometry and microscopy to stain adipocytes derived from murine and human MSCs³⁷. Previous studies have reported usage of Nile red for ESC-derived adipocytes and enhancement of adipocyte markers post sorting³⁸. The adipocytes generated from the MSCs obtained by the present RA-based protocol were assessed for their ability to be stained by Nile red, indicating their maturity, and sorted to purify them. These Nile red-sorted cells exhibited a two to three-fold increase in the expression of the adipocyte maturation markers, including PPARG, C/EBPA, and FABP4 compared to unsorted cells, thus further increasing the yield of iPSCs-derived adipocytes. Although these markers are expressed before lipid accumulation, their expression tags a cell for terminal differentiation to lipid bearing adipocytes. Checking sorting efficiency by these markers allow us to identify a pool where all cells are express FABP4, CEBPa, and PPARg, indicating a pool, which was pre-destined for mature adipocyte formation. Cells are sorted based upon their staining potential to Nile red. Purification efficiency increased by two to three folds due to the high number of adipocytes in the unsorted fraction. The size of lipid bearing adipocytes vary largely during differentiation, where a pool of cells with identical size distribution are sorted. Unsorted fraction encompasses lipid-bearing adipocytes, but they are not fully mature and governed by dissimilar size proportions.

The heterogeneity of MSCs isolated from the human body has been previously reported³⁹. This heterogeneity depends on several factors, such as the MSC origin, donors, and conditions³⁹. This may lead to variations in their efficiency in treating different diseases. This study suggests that short RA treatment of hPSCs produced under good manufacturing practice (GMP)-compatible culture conditions would give a homogenous population of MSCs. This indicates that the current protocol is a promising approach for generating a large number of clinical-grade MSCs that can be used for MSC-based therapy.

The combination of the RA-based MSC differentiation protocol leading to adipocyte differentiation and Nile red-sorting protocol allowed us to obtain iPSCs-derived adipocytes with enhanced expression of functional markers and increased yield and purity. Thus, this combined protocol would allow the generation, in sufficient quantity and purity, of mature adipocytes from genetically distinct individuals and the potential uncovering of novel genetic variants behind adipocyte-related metabolic disorders.

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

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).

Materials

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

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Aghadi, M., Karam, M., Abdelalim, E. M. Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders. J. Vis. Exp. (180), e63311, doi:10.3791/63311 (2022).