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

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 conditions^1,2. These obstacles have produced a great demand of having a protocol for generating MSCs

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

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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.......

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 RA^14,15. Transient treatment with 10 µM of RA enhanced the MSC yield by 11.2 to 1542 folds^14,15, with this protocol being applicable on both iPSCs and hPSCs. At this dose and durat.......

Materials

Name	Company	Catalog Number	Comments
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