We show that the day 21 purified CPs (GFPhigh population) maintain improved chondrogenic capacity over the course of 5 passages compared to controls

We show that the day 21 purified CPs (GFPhigh population) maintain improved chondrogenic capacity over the course of 5 passages compared to controls. patient-specific or genetically-engineered cartilage for applications in drug screening for disease-modifying osteoarthritis drugs (DMOADs) [12]. Therefore, methods to rapidly and efficiently differentiate hiPSCs into chondrocytes in a reproducible and robust manner will be critical for their application in joint repair. An important goal of such protocols is to minimize variability in hiPSC differentiation potential, which may arise from characteristics of the donor and/or reprogramming method [13C15]. Although several approaches have been employed to generate articular chondrocytes from PSCs, most differentiation protocols have been based on trial-and-error delivery of growth factors without immediate consideration of the signaling pathways that direct and inhibit each stage of differentiation. Accordingly, chondrogenic differentiation is often dependent on the specific cell lines used, and broad application of iPSC chondrogenesis protocols has not been independently demonstrated with multiple cell lines [16]. Recently, critical insights from developmental biology have elucidated the sequence of inductive and repressive signaling pathways needed for PSC lineage specification to a number of cell fates [17C19]. By reproducing these reported signaling pathways locus, which encodes for type atorvastatin II collagen C an important structural constituent of articular cartilage [20C22]. However, this transgenic approach is not feasible for human iPSCs, which motivated our pursuit of gene editing methods to create a knock-in reporter of collagen II production at the human locus. In this study, we demonstrate the development and application of a step-wise differentiation protocol validated in three unique and well-characterized hiPSC lines. We examined gene expression profiles and cartilaginous matrix production during the course of differentiation. To further purify committed CPs, we then used CRISPR-Cas9 genome engineering technology to knock-in a GFP reporter at the collagen atorvastatin type II alpha 1 chain (chondrogenesis platforms for disease modeling and drug screening. Results Step-wise differentiation of hiPSCs into chondroprogenitor cells To establish a standardized protocol for hiPSC chondrogenesis, we optimized growth factor and small molecule concentrations using established principles of PSC differentiation along mesodermal lineages as described in Figure 1A [17]. To validate our differentiation approach, we measured expression of transcription factors representative of various stages of development with qRT-PCR and monitored cell morphology at multiple time points in three hiPSC lines (BJFF, ATCC, and RVR) (Figure 1B-G and S1). Over the course of differentiation, we observed a gradual decrease in expression of the pluripotency markers octamer-binding protein 4 (expression atorvastatin level at paraxial mesoderm stage, only the RVR-iPSC line is significantly different from its hiPSC stage. For expression level at chondroprogenitor stage, only RVR is not atorvastatin significantly different from its hiPSC stage. PS: primitive streak. Data points represent means and error bars signify SEM. Characterization of surface markers of atorvastatin hiPSCs and CP cells Upregulation of chondrogenic markers in CPs suggested that this stage may be appropriate for further chondrogenic differentiation. The RVR-iPSC line was assessed for surface marker expression levels at iPSC and CP cell stages (Figure 1H). The hiPSCs and CP cells exhibited distinct expression patterns of surface proteins (Table S1). hiPSCs exhibited a surface marker profile Mouse monoclonal to ApoE characteristic of primed hiPSCs (CD90+/CD24+/SSEA-4+/CD57+/CD45?). Pluripotency-specific markers SSEA-4/CD57/CD24 decreased in CP cells, and the CP cells displayed a moderate increase in the surface markers CD105, CD146, CD166, and CD271. Interestingly, we also observed that CP cells expressed surface proteins that are often absent on mesenchymal stem cells (MSCs) such as CD56, CD111, CD112, and CD117. Chondrogenic gene expression during chondrogenesis After mesodermal specification and pre-chondrogenesis of hiPSCs, we further differentiated cells in.