Chondrogenic Differentiation of Human-Induced Pluripotent Stem Cells

Abstract

The generation of large quantities of genetically defined human chondrocytes remains a critical step for the development of tissue engineering strategies for cartilage regeneration and high-throughput drug screening. This protocol describes chondrogenic differentiation of human-induced pluripotent stem cells (hiPSCs), which can undergo genetic modification and the capacity for extensive cell expansion. The hiPSCs are differentiated in a stepwise manner in monolayer through the mesodermal lineage for 12 days using defined growth factors and small molecules. This is followed by 28 days of chondrogenic differentiation in a 3D pellet culture system using transforming growth factor beta 3 and specific compounds to inhibit off-target differentiation. The 6-week protocol results in hiPSC-derived cartilaginous tissue that can be characterized by histology, immunohistochemistry, and gene expression or enzymatically digested to isolate chondrocyte-like cells. Investigators can use this protocol for experiments including genetic engineering, in vitro disease modeling, or tissue engineering.

Keywords: Chondrogenesis; Human iPSCs; Stem cells; Tissue-engineered cartilage.

Fig. 1

Overview schematic of the protocol. hiPSCs undergo mesodermal differentiation in monolayer for 12 days. The cells go through the anterior primitive streak, paraxial mesoderm, early somite, sclerotome, and finally chondroprogenitor stage. Cells are then cultured in a 3D pellet culture to become chondrocytes and synthesize cartilaginous matrix. The protocol then has four options to either digest the tissue to isolate single cells or validate chondrogenesis with histology (Saf-O and IHC), biochemical assays (DMMB for sGAG and PicoGreen for dsDNA), and/or RT-qPCR

Fig. 2

Phase contrast images of cells throughout mesodermal differentiation. (a) Induce cells when they are 30–40% confluent. (b–f) As cells differentiate, from (b) anterior primitive streak, (c) paraxial mesoderm, (d) early somite, (e) sclerotome, to (f) chondroprogenitor, they spread and become more spindled. (g) If cells are induced at too high of a density, they may not fully differentiate and form nodules in the center of the colonies. Scale bar = 1 mm

Fig. 3

Anticipated results – gene expression and matrix quantification. (a–c) Chondrogenic transcription factor SOX9 gene expression should increase early, followed by matrix genes COL2A1 and ACAN. (d–e) Relative to the expression of COL2A1, lower gene expression of fibrocartilage and hypertrophic cartilage markers COL1A1 and COL10A1, respectively, was observed. (f) sGAG/DNA ratio should increase throughout chondrogenesis, reaching 20–30 ng/ng at day 28 and over 40 ng/ng at day 42. Mean ± SEM, n = 4. *p < 0.05 compared to previous time point

Fig. 4

Anticipated results – histology. (a–c) Robust, homogenous safranin-O staining for sGAGs in three different cell lines. (d–f) Pellet in panel a with IHC labeling of COL2A1 (d), COL1A1 (e), and COL10A1 (f). Scale bar = 500 μm