Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing

Abstract

The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. Here we demonstrate cartilaginous matrix production in three unique hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified chondroprogenitors demonstrated an improved chondrogenic capacity compared to unselected populations. The ability to enrich for chrondroprogenitors and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications.

Prospective Isolation of Chondroprogenitors from Human iPSCs Based on Cell Surface Markers Identified using a CRISPR-Cas9-Generated Reporter

“…The results of scRNA-seq of sorted cells revealed that cells positive for PDGFRb, CD146, and CD166 exhibited enhanced cell homogeneity with decreased neurogenic subpopulations. These findings support the hypothesis that sorting of hiPSC-derived chondroprogenitor cells using surface markers can be used to purify progenitor cells with enhanced chondrogenic potential, without the need for genetic modification to improve hiPSC chondrogenesis (Adkar et al, 2018;Diekman et al, 2012).…”

“…We previously reported that chondroprogenitor cells at the end of mesodermal lineage differentiation had high expression of CD146 and CD166 (Adkar et al, 2018). In the present study, we observed that these markers were also highly co-expressed with COL2A1.…”

“…COL2A1-GFP reporter hiPSCs were differentiated into chondroprogenitor cells along the mesodermal lineage as previously described (Adkar et al, 2018). After differentiation, the cells were labeled for surface markers commonly associated with MSCs and/or chondroprogenitors in the developing limb bud (Chan et al, 2018;Lv et al, 2014;Wu et al, 2013).…”

“…Previously, our lab used green fluorescent protein (GFP) reporter systems to track the expression of collagen type II alpha 1 chain (COL2A1) in mouse (Diekman et al, 2012) and human (Adkar et al, 2018) iPSCs, allowing for the prospective isolation and purification of COL2A1-GFP+ chondrogenic cells during the differentiation process. Despite the fact that this approach significantly enhanced iPSC chondrogenesis (Adkar et al, 2018), genome editing is required to create a reporter line, hindering potential clinical translation. In this regard, the identification of cell surface markers that are directly representative of this COL2A1-positive population could greatly enhance the prospective isolation and purification of chondroprogenitors, without requiring genetic modifications to the cell line.…”

Designer Stem Cells: Genome Engineering and the Next Generation of Cell‐Based Therapies

“…With the advent of a multitude of new methods for cellular engineering, we propose that a new generation of stem cell therapies will emerge that can provide functional tissue replacements as well as exogenous or even self‐regulating capabilities for biologic drug delivery. In addition to such therapeutic applications of stem cells in vivo, genome edited stem cells may also allow for the development of in vitro models of disease or reporter systems for stem cell purification or for investigating the consequences of causative or associate genetic elements such as coding/noncoding single nucleotide polymorphisms, variable number tandem repeats, and so forth identified through genome‐wide association studies …”