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1320 - Prospective Isolation of Chondroprogenitors from Human iPSCs Based on a Collagen II Reporter Allele Generated by CRISPR-Cas9 Genome Editing</b>

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Presented at

ORS 2019 Annual Meeting

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Abstract

INTRODUCTION: Human induced pluripotent stem cells (hiPSCs) offer a promising cell source for cartilage tissue engineering and in vitro disease modeling. However, generation of a homogenous chondrocyte population from hiPSCs remains a challenge. Recently, our lab established a step-wise differentiation protocol for hiPSC-derived chondrocytes through paraxial mesoderm specification [1]. To enhance homogeneity of the differentiated cell population, we also purified chondroprogenitor (CP) cells via a COL2-GFP reporter allele generated by CRISPR-Cas9 genome editing. While this method showed improved chondrogenesis and tissue homogeneity, the need for gene editing limits its translational applicability to patient-specific iPSC lines. The goal of this study was to characterize the surface markers of the COL2-GFP positive CPs and use these markers to prospectively isolate and purify CP cells from unedited hiPSC lines. Furthermore, the effect of in vitro expansion on chondrogenic potential of sorted and unsorted cells was also evaluated. METHODS: Human iPSCs (RVR cell line) containing a CRISPR-Cas9-edited COL2-GFP knock-in reporter were differentiated into the mesodermal lineage in a step-wise manner as previously described [1]. The cells were then stained for a variety of surface markers including CD45, CD73, CD105, CD146, CD166, CD271, BMPRI-β, and PDGFRβ. These markers were selected because they are either conventionally known to be present in mesenchymal stem cells (MSCs) [2] or highly expressed in CP cells measured via a cell-surface marker array in our recent study [1]. The COL2-GFP positive population was analyzed to identify the markers co-expressed with COL2 at the highest levels. An unmodified hiPSC line (BJFF, Washington University in Saint Louis) was also differentiated along the mesodermal lineage and then sorted for the identified marker set using a fluorescent activated cell sorter. Both sorted and unsorted cells from BJFF hiPSCs were passaged up to 4 times, and chondrogenic pellets were made with each passage. Pellets were harvested after 28 days for histological analysis. RESULTS: Surface marker staining identified a particular marker set (PDGFRβ+/CD146+/CD166+/CD45-) in COL2-GFP positive CP cells (Fig. 1A). Flow cytometry showed that the COL2-GFP+ population had very few cells expressing several putative stem cell surface markers such as CD73 (3%), CD271 (8%), BMPRI-β (0.04%), and CD105 (3.1%). However, a high proportion of GFP+ cells (51.7%) expressed PDGFRβ, CD146, and CD166. In the BJFF line, PDGFRβ+/CD146+/CD166+ triple positive CP cells averaged to be 17.4% of the total population while 8.5% were triple negative (Fig. 1B and C). Triple positive sorted cells showed an increase in homogeneity and robustness of cartilaginous matrix in both hiPSC lines when compared with unsorted cells, as shown by the Safranin-O staining for glycosaminoglycans (GAGs) (Fig. 2). In addition, sorting increased proliferation during expansion by approximately 10-fold after 4 passages (data not shown). Chondrogenic potential was present after one passage of CP cells but was reduced after two or more passages as exhibited by the histologic analysis for GAGs (Fig. 3). DISCUSSION: This current study identified a PDGFRβ+/CD146+/CD166+/CD45- cell population with a strong chondrogenic differentiation capacity from hiPSC-derived mesodermal cells. This population can homogenously differentiate into chondrocyte-like cells and produce hyaline cartilaginous matrix upon TGF-β3 induction. Our prospectively isolated cells appear to be similar to the PSC-derived mesenchyme (CD146+/CD166+/KDR-/CD326-/BMPRI-β-) defined by Evseenko’s group [3] as both cell populations were isolated from mesodermal cells and exhibited high chondrogenic potential. While MSCs are known to have relatively high expression of CD73, CD105, and CD271 [2], our triple positive sorted cells were low in these three markers, suggesting that these sorted cells are distinct from conventionally recognized MSCs. SIGNIFICANCE: This study identified a specific cell population that can generate homogenous chondrocyte-like cells from hiPSCs and thus provides a significant foundation for the applications of hiPSC-based cartilage repair and regeneration.

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All rights reserved.