Method Article

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

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DOI:

10.3791/63311

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February 9th, 2022

 ,  , 
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

In This Article

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.

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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

  1. Prepare basement membrane matrix-coated plates by reconstituting coating matrix in knockout-DMEM at a ratio of 1:80 and store at 4 °C.
  2. 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.
  3. 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.
  4. Remove an aliquot of iPSCs culture media and pre-warm at room temperature before use.
  5. 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.
  6. Centrifuge the tube at 120 x g for 4 min at room temperature (RT-23 °C).
  7. 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.
  8. After 24 h, remove the media and replace it with fresh culture media.
  9. Change the media every day until the cells reach 80%-90% confluency.
  10. 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

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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).
  6. 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.

  1. 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.
  2. Centrifuge the plate at 375 x g for 4 min at 4 °C.
  3. 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.
  4. 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.
  5. 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.
  6. Resuspend the cells in 100 µL of cold DPBS.
  7. Centrifuge the cells at 375 x g for 4 min at 4 °C. Discard the supernatant.
  8. 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).
  9. 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

  1. 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.
  2. Allow MSCs to reach above 90% confluency. Continue culturing them for another 48 h to allow them to undergo a period of growth arrest.
  3. 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.
  4. Remove MSC differentiation media and wash the cells using DPBS.
  5. 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

  1. On day 14 of differentiation, check the efficiency of differentiation by staining cells for adipocyte maturation markers, FABP4, and adiponectin.
  2. Remove the media and wash the cells with DPBS.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. Remove the primary antibodies and wash the cells three times with TBST (15 min each) and place it on a shaker at room temperature.
  9. 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.
  10. Discard the secondary antibodies, wash with TBST three times, and place the plate on the shaker.
  11. 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.
  12. 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

  1. 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.
  2. On or after day 14 of adipocyte differentiation, discard the media from the cells and wash using DPBS.
  3. Add Nile red working solution -1 mL in a well of a 6-well plate- and incubate at 37 °C for 15 min.
  4. 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.
  5. Collect the cells using DMEM containing 5% FBS in a 15 mL conical tube. Centrifuge at 750 x g for 4 min.
  6. Remove the supernatant and resuspend in DPBS-1 mL for 1 x 106 cells. Centrifuge at 750 x g for 4 min.
  7. 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.
  8. Re-culture the sorted cells in adipocyte differentiation media or collect the sorted cells for RNA and protein isolation.
  9. 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.

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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 s...

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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 durat...

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Disclosures

The authors declare that they have no competing interests.

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).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
AdiponectinAbcamab22554Adipocyte maturation marker
anti-CD105BD Pharmingen560839MSC differentiation marker
anti-CD14BD Pharmingen561712MSC differentiation marker
anti-CD19BD Pharmingen555415MSC differentiation marker
anti-CD34BD Pharmingen555824MSC differentiation marker
anti-CD44abcamab93758MSC differentiation marker
anti-CD45BD Pharmingen
560975		MSC differentiation marker
anti-CD73BD Pharmingen550256MSC differentiation marker
anti-CD90BD Pharmingen555596MSC differentiation marker
bFGFR&D233-FPMSC culture media supplement
C/EBPAAbcamab40761Adipocyte maturation marker
DexamethasoneTorics1126Adipocyte differentiation media supplement
FABP4Abcamab93945Adipocyte maturation marker
Fetal bovine serumThermoFisher10082147MSC culture media supplement
GlutamaxThermoFisher35050-061MSC culture media supplement
IBMXSigma AldrichI5879Adipocyte differentiation media supplement
IndomethacinSigma AldrichI7378Adipocyte differentiation media supplement
InsulinSigma Aldrich91077CAdipocyte differentiation media supplement
Knockout DMEMThermoFisher12660012Basal media for preparing matrigel
Low glucose DMEMThermoFisher11885084MSC culturing media
MatrigelCorning354230Coating matrix
MEM-alphaThermoFisher12561056Adipocyte differentiation media
NileredSigma Aldrich19123Sorting marker for adipocyte
PenicillinThermoFisher15140122MSC/Adipocyte media supplement
Phosphate-buffered salineThermoFisher14190144wash buffer
Pierce™ 20X TBS BufferThermo Fisher28358wash buffer
PPARGCell Signaling Technology2443Adipocyte maturation marker
ReLeSRStem Cell Technologies5872Dissociation reagent
Retinoic acidSigma AldrichR2625MSC differentiation media supplement
Rock inhibitorTocris1254/10hPSC culture media supplement
RoziglitazoneSigma AldrichR2408Adipocyte differentiation media supplement
StemFlexThermoFisherA334901hPSC culture media
TritonThermo Fisher28314Permebealization reagent
TrypsinThermoFisher25200072Dissociation reagent
Tween 20Sigma AldrichP7942Wash buffer

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Tags

Human IPSC DifferentiationMesenchymal Stem CellsAdipocyte DifferentiationNile Red SortingPure Adipocyte PopulationEmbryonic BodiesFACS SortingAdipocyte Maturation MarkersLipid Bearing Adipocytes

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