skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Label-free imaging for quality control of cardiomyocyte differentiation
Abstract Human pluripotent stem cell (hPSC)-derived cardiomyocytes provide a promising regenerative cell therapy for cardiovascular patients and an important model system to accelerate drug discovery. However, cost-effective and time-efficient platforms must be developed to evaluate the quality of hPSC-derived cardiomyocytes during biomanufacturing. Here, we develop a non-invasive label-free live cell imaging platform to predict the efficiency of hPSC differentiation into cardiomyocytes. Autofluorescence imaging of metabolic co-enzymes is performed under varying differentiation conditions (cell density, concentration of Wnt signaling activator) across five hPSC lines. Live cell autofluorescence imaging and multivariate classification models provide high accuracy to separate low (< 50%) and high (≥ 50%) differentiation efficiency groups (quantified by cTnT expression on day 12) within 1 day after initiating differentiation (area under the receiver operating characteristic curve, 0.91). This non-invasive and label-free method could be used to avoid batch-to-batch and line-to-line variability in cell manufacturing from hPSCs.  more » « less
Award ID(s):
1648035
PAR ID:
10311299
Author(s) / Creator(s):
; ; ; ; ;
Date Published:
Journal Name:
Nature Communications
Volume:
12
Issue:
1
ISSN:
2041-1723
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. (, SPIE Photonics West)
    The efficient transition of hPSCs to definitive endoderm (DE) progeny is an essential step toward disease modeling and the manufacturing of a wide range of cellular therapeutics in medical relevant quantities. Two-photon excited fluorescence (TPEF) imaging, as a non-invasive, non-destructive, label-free modality for metabolic studies, reveals the distinct metabolic switches during DE differentiation in a real-time monitoring mode. Since metabolic pathways orchestrate important regulatory mechanisms that influence and determine cell fate decisions, TPEF imaging serves as an important enabling technology in hPSC-based tissue engineering applications affording non-invasive determination of metabolic biomarkers and informing optimizations of hPSCs differentiation processes. 
    more » « less
  2. ; ; ; ; ; ; ; ; ; ; et al (, Communications Biology)
    Current methods for producing cardiomyocytes from human induced pluripotent stem cells (hiPSCs) using 2D monolayer differentiation are often hampered by batch-to-batch variability and inef!cient puri!cation processes. Here, we introduce CM-AI, a novel arti!cial intelligence-guided laser cell processing platform designed for rapid, label-free puri!cation of hiPSC-derived cardiomyocytes (hiPSC-CMs). This approach signi!cantly reduces processing time without the need for chronic metabolic selection or antibody-based sorting. By integrating real-time cellular morphology analysis and targeted laser ablation, CM-AI selectively removes non-cardiomyocyte populations with high precision. This streamlined process preserves cardiomyocyte viability and function, offering a scalable and ef!cient solution for cardiac regenerative medicine, disease modeling, and drug disco 
    more » « less
  3. ; ; (, Biotechnology and Bioengineering)
    Abstract Human induced pluripotent stem cells (iPSCs) hold great promise for reducing the mortality of cardiovascular disease by cellular replacement of infarcted cardiomyocytes (CMs). CM differentiation via iPSCs is a lengthy multiweek process and is highly subject to batch‐to‐batch variability, presenting challenges in current cell manufacturing contexts. Real‐time, label‐free control quality attributes (CQAs) are required to ensure efficient iPSC‐derived CM manufacturing. In this work, we report that live oxygen consumption rate measurements are highly predictive CQAs of CM differentiation outcome as early as the first 72 h of the differentiation protocol with an accuracy of 93%. Oxygen probes are already incorporated in commercial bioreactors, thus methods presented in this work are easily translatable to the manufacturing setting. Detecting deviations in the CM differentiation trajectory early in the protocol will save time and money for both manufacturers and patients, bringing iPSC‐derived CM one step closer to clinical use. 
    more » « less
  4. ; (, Cells)
    Efforts to direct the specification of human pluripotent stem cells (hPSCs) to therapeutically important somatic cell types have focused on identifying proper combinations of soluble cues. Yet, whether exosomes, which mediate intercellular communication, play a role in the differentiation remains unexplored. We took a first step toward addressing this question by subjecting hPSCs to stage-wise specification toward cardiomyocytes (CMs) in scalable stirred-suspension cultures and collecting exosomes. Samples underwent liquid chromatography (LC)/mass spectrometry (MS) and subsequent proteomic analysis revealed over 300 unique proteins from four differentiation stages including proteins such as PPP2CA, AFM, MYH9, MYH10, TRA2B, CTNNA1, EHD1, ACTC1, LDHB, and GPC4, which are linked to cardiogenic commitment. There was a significant correlation of the protein composition of exosomes with the hPSC line and stage of commitment. Differentiating hPSCs treated with exosomes from hPSC-derived CMs displayed improved efficiency of CM formation compared to cells without exogenously added vesicles. Collectively, these results demonstrate that exosomes from hPSCs induced along the CM lineage contain proteins linked to the specification process with modulating effects and open avenues for enhancing the biomanufacturing of stem cell products for cardiac diseases. 
    more » « less
  5. ; ; ; ; ; ; (, Biotechnology Journal)
    Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are immature in their structure and function, limiting their potential in disease modeling, drug screening, and cardiac cellular therapies. Prior studies have demonstrated that coculture of hPSC‐derived CMs with other cardiac cell types, including endothelial cells (ECs), can accelerate CM maturation. To address whether the CM differentiation stage at which ECs are introduced affects CM maturation, the authors coculture hPSC‐derived ECs with hPSC‐derived cardiac progenitor cells (CPCs) and CMs and analyze the molecular and functional attributes of maturation. ECs have a more significant effect on acceleration of maturation when cocultured with CPCs than with CMs. EC coculture with CPCs increases CM size, expression of sarcomere, and ion channel genes and proteins, the presence of intracellular membranous extensions, and chronotropic response compared to monoculture. Maturation is accelerated with an increasing EC:CPC ratio. This study demonstrates that EC incorporation at the CPC stage of CM differentiation expedites CM maturation, leading to cells that may be better suited for in vitro and in vivo applications of hPSC‐derived CMs. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email