Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have many promising applications, including the regeneration of injured heart muscles, cardiovascular disease modeling, and drug cardiotoxicity screening. Current differentiation protocols yield a heterogeneous cell population that includes pluripotent stem cells and different cardiac subtypes (pacemaking and contractile cells). The ability to purify these cells and obtain well-defined, controlled cell compositions is important for many downstream applications; however, there is currently no established and reliable method to identify hiPSC-derived cardiomyocytes and their subtypes. Here, we demonstrate that second harmonic generation (SHG) signals generated directly from the myosin rod bundles can be a label-free, intrinsic optical marker for identifying hiPSC-derived cardiomyocytes. A direct correlation between SHG signal intensity and cardiac subtype is observed, with pacemaker-like cells typically exhibiting ~70% less signal strength than atrial- and ventricular-like cardiomyocytes. These findings suggest that pacemaker-like cells can be separated from the heterogeneous population by choosing an SHG intensity threshold criteria. This work lays the foundation for developing an SHG-based high-throughput flow sorter for purifying hiPSC-derived cardiomyocytes and their subtypes.
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 (CPs) produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified CPs demonstrated an improved chondrogenic capacity compared with unselected populations. The ability to enrich for CPs and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications. Stem Cells 2019;37:65–76
- NSF-PAR ID:
- 10363227
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Stem Cells
- Volume:
- 37
- Issue:
- 1
- ISSN:
- 1066-5099
- Page Range / eLocation ID:
- p. 65-76
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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