skip to main content


Title: 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 (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

</sec> </span> <a href='#' class='show open-abstract' style='margin-left:10px;'>more »</a> <a href='#' class='hide close-abstract' style='margin-left:10px;'>« less</a> <div style="clear:both;margin-bottom:20px;"></div> <dl class="dl-horizontal small"> <dt>NSF-PAR ID:</dt> <dd>10363227</dd> </dl> <dl class="dl-horizontal small"> <dt>Author(s) / Creator(s):</dt> <dd> <a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Adkar, Shaunak S.""><span class="author" itemprop="author">Adkar, Shaunak S.</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Wu, Chia-Lung""><span class="author" itemprop="author">Wu, Chia-Lung</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Willard, Vincent P.""><span class="author" itemprop="author">Willard, Vincent P.</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Dicks, Amanda""><span class="author" itemprop="author">Dicks, Amanda</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Ettyreddy, Adarsh""><span class="author" itemprop="author">Ettyreddy, Adarsh</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Steward, Nancy""><span class="author" itemprop="author">Steward, Nancy</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Bhutani, Nidhi""><span class="author" itemprop="author">Bhutani, Nidhi</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Gersbach, Charles A.""><span class="author" itemprop="author">Gersbach, Charles A.</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Guilak, Farshid""><span class="author" itemprop="author">Guilak, Farshid</span> <sup class="text-muted"></sup></a></dd> </dl> <dl class="dl-horizontal small"> <dt>Publisher / Repository:</dt> <dd itemprop="publisher">Oxford University Press</dd> </dl> <dl class="dl-horizontal small"> <dt>Date Published:</dt> <dd> <time itemprop="datePublished" datetime="2018-10-31">2018-10-31</time> </dd> </dl> <dl class="dl-horizontal small"> <dt>Journal Name:</dt> <dd>Stem Cells</dd> </dl> <dl class="dl-horizontal small"> <dt>Volume:</dt> <dd>37</dd> </dl> <dl class="dl-horizontal small"> <dt>Issue:</dt> <dd>1</dd> </dl> <dl class="dl-horizontal small"> <dt>ISSN:</dt> <dd>1066-5099</dd> </dl> <dl class="dl-horizontal small"> <dt>Page Range / eLocation ID:</dt> <dd>p. 65-76</dd> </dl> <dl class="dl-horizontal small"> <dt>Format(s):</dt> <dd>Medium: X</dd> </dl> <dl class="dl-horizontal small"> <dt>Sponsoring Org:</dt> <dd itemprop="sourceOrganization">National Science Foundation</dd> </dl> <div class="clearfix"></div> </div> </div> <div id="citation-addl" class="hidden-print"> <h5 id='mlt-header'>More Like this</h5> <ol class="item-list documents" id="citation-mlt" style="min-height: 80px;"> <li> <div class="article item document" itemscope itemtype="http://schema.org/TechArticle"> <div class="item-info"> <div class="title"> <a href="https://par.nsf.gov/biblio/10363223-intrinsic-label-free-signal-identifying-stem-cell-derived-cardiomyocyte-subtype" itemprop="url"> <span class='span-link' itemprop="name">An intrinsic, label-free signal for identifying stem cell-derived cardiomyocyte subtype</span> </a> </div> <div> <strong> <a class="misc external-link" href="https://doi.org/10.1002/stem.3127" target="_blank" title="Link to document DOI">https://doi.org/10.1002/stem.3127  <span class="fas fa-external-link-alt"></span></a> </strong> </div> <div class="metadata"> <span class="authors"> <span class="author" itemprop="author">Chang, Che-Wei</span> <span class="sep">; </span><span class="author" itemprop="author">Kao, Hillary K. J.</span> <span class="sep">; </span><span class="author" itemprop="author">Yechikov, Sergey</span> <span class="sep">; </span><span class="author" itemprop="author">Lieu, Deborah K.</span> <span class="sep">; </span><span class="author" itemprop="author">Chan, James W.</span> </span> <span class="year">( <time itemprop="datePublished" datetime="2019-12-09">December 2019</time> , Stem Cells) </span> </div> <div style="cursor: pointer;-webkit-line-clamp: 5;" class="abstract" itemprop="description"> <title>Abstract

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.

</sec> </div> <a href='#' class='show open-abstract' style='margin-left:10px;'>more »</a> <a href='#' class='hide close-abstract' style='margin-left:10px;'>« less</a> </div><div class="clearfix"></div> </div> </li> <li> <div class="article item document" itemscope itemtype="http://schema.org/TechArticle"> <div class="item-info"> <div class="title"> <a href="https://par.nsf.gov/biblio/10089550-serum-free-manufacturing-mesenchymal-stem-cell-tissue-rings-using-human-induced-pluripotent-stem-cells" itemprop="url"> <span class='span-link' itemprop="name">Serum-Free Manufacturing of Mesenchymal Stem Cell Tissue Rings Using Human-Induced Pluripotent Stem Cells</span> </a> </div> <div> <strong> <a class="misc external-link" href="https://doi.org/10.1155/2019/5654324" target="_blank" title="Link to document DOI">https://doi.org/10.1155/2019/5654324  <span class="fas fa-external-link-alt"></span></a> </strong> </div> <div class="metadata"> <span class="authors"> <span class="author" itemprop="author">Winston, Tackla S.</span> <span class="sep">; </span><span class="author" itemprop="author">Suddhapas, Kantaphon</span> <span class="sep">; </span><span class="author" itemprop="author">Wang, Chenyan</span> <span class="sep">; </span><span class="author" itemprop="author">Ramos, Rafael</span> <span class="sep">; </span><span class="author" itemprop="author">Soman, Pranav</span> <span class="sep">; </span><span class="author" itemprop="author">Ma, Zhen</span> </span> <span class="year">( <time itemprop="datePublished" datetime="2019-01-15">January 2019</time> , Stem Cells International) </span> </div> <div style="cursor: pointer;-webkit-line-clamp: 5;" class="abstract" itemprop="description"> Combination of stem cell technology and 3D biofabrication approaches provides physiological similarity to in vivo tissues and the capability of repairing and regenerating damaged human tissues. Mesenchymal stem cells (MSCs) have been widely used for regenerative medicine applications because of their immunosuppressive properties and multipotent potentials. To obtain large amount of high-quality MSCs without patient donation and invasive procedures, we differentiated MSCs from human-induced pluripotent stem cells (hiPSC-MSCs) using serum-free E6 media supplemented with only one growth factor (bFGF) and two small molecules (SB431542 and CHIR99021). The differentiated cells showed a high expression of common MSC-specific surface markers (CD90, CD73, CD105, CD106, CD146, and CD166) and a high potency for osteogenic and chondrogenic differentiation. With these cells, we have been able to manufacture MSC tissue rings with high consistency and robustness in pluronic-coated reusable PDMS devices. The MSC tissue rings were characterized based on inner diameter and outer ring diameter and observed cell-type-dependent tissue contraction induced by cell-matrix interaction. Our approach of simplified hiPSC-MSC differentiation, modular fabrication procedure, and serum-free culture conditions has a great potential for scalable manufacturing of MSC tissue rings for different regenerative medicine applications. </div> <a href='#' class='show open-abstract' style='margin-left:10px;'>more »</a> <a href='#' class='hide close-abstract' style='margin-left:10px;'>« less</a> </div><div class="clearfix"></div> </div> </li> <li> <div class="article item document" itemscope itemtype="http://schema.org/TechArticle"> <div class="item-info"> <div class="title"> <a href="https://par.nsf.gov/biblio/10424852-nanoengineering-gold-nanoribbon-embedded-isogenic-stem-cell-derived-cardiac-organoids" itemprop="url"> <span class='span-link' itemprop="name">Nanoengineering of gold nanoribbon-embedded isogenic stem cell-derived cardiac organoids</span> </a> </div> <div> <strong> <a class="misc external-link" href="https://doi.org/10.1039/D3RA01811C" target="_blank" title="Link to document DOI">https://doi.org/10.1039/D3RA01811C  <span class="fas fa-external-link-alt"></span></a> </strong> </div> <div class="metadata"> <span class="authors"> <span class="author" itemprop="author">Patino-Guerrero, Alejandra</span> <span class="sep">; </span><span class="author" itemprop="author">Esmaeili, Hamid</span> <span class="sep">; </span><span class="author" itemprop="author">Migrino, Raymond Q.</span> <span class="sep">; </span><span class="author" itemprop="author">Nikkhah, Mehdi</span> </span> <span class="year">( <time itemprop="datePublished" datetime="2023-06-05">June 2023</time> , RSC Advances) </span> </div> <div style="cursor: pointer;-webkit-line-clamp: 5;" class="abstract" itemprop="description"> Cardiac tissue engineering is an emerging field providing tools to treat and study cardiovascular diseases (CVDs). In the past years, the integration of stem cell technologies with micro- and nanoengineering techniques has enabled the creation of novel engineered cardiac tissues (ECTs) with potential applications in disease modeling, drug screening, and regenerative medicine. However, a major unaddressed limitation of stem cell-derived ECTs is their immature state, resembling a neonatal phenotype and genotype. The modulation of the cellular microenvironment within the ECTs has been proposed as an efficient mechanism to promote cellular maturation and improve features such as cellular coupling and synchronization. The integration of biological and nanoscale cues in the ECTs could serve as a tool for the modification and control of the engineered tissue microenvironment. Here we present a proof-of-concept study for the integration of biofunctionalized gold nanoribbons (AuNRs) with hiPSC-derived isogenic cardiac organoids to enhance tissue function and maturation. We first present extensive characterization of the synthesized AuNRs, their PEGylation and cytotoxicity evaluation. We then evaluated the functional contractility and transcriptomic profile of cardiac organoids fabricated with hiPSC-derived cardiomyocytes (mono-culture) as well as with hiPSC-derived cardiomyocytes and cardiac fibroblasts (co-culture). We demonstrated that PEGylated AuNRs are biocompatible and do not induce cell death in hiPSC-derived cardiac cells and organoids. We also found an improved transcriptomic profile of the co-cultured organoids indicating maturation of the hiPSC-derived cardiomyocytes in the presence of cardiac fibroblasts. Overall, we present for the first time the integration of AuNRs into cardiac organoids, showing promising results for improved tissue function. </div> <a href='#' class='show open-abstract' style='margin-left:10px;'>more »</a> <a href='#' class='hide close-abstract' style='margin-left:10px;'>« less</a> </div><div class="clearfix"></div> </div> </li> <li> <div class="article item document" itemscope itemtype="http://schema.org/TechArticle"> <div class="item-info"> <div class="title"> <a href="https://par.nsf.gov/biblio/10446232-viscoelasticity-adhesion-signaling-biomaterials-control-human-pluripotent-stem-cell-morphogenesis-culture" itemprop="url"> <span class='span-link' itemprop="name">Viscoelasticity and Adhesion Signaling in Biomaterials Control Human Pluripotent Stem Cell Morphogenesis in 3D Culture</span> </a> </div> <div> <strong> <a class="misc external-link" href="https://doi.org/10.1002/adma.202101966" target="_blank" title="Link to document DOI">https://doi.org/10.1002/adma.202101966  <span class="fas fa-external-link-alt"></span></a> </strong> </div> <div class="metadata"> <span class="authors"> <span class="author" itemprop="author">Indana, Dhiraj</span> <span class="sep">; </span><span class="author" itemprop="author">Agarwal, Pranay</span> <span class="sep">; </span><span class="author" itemprop="author">Bhutani, Nidhi</span> <span class="sep">; </span><span class="author" itemprop="author">Chaudhuri, Ovijit</span> </span> <span class="year">( <time itemprop="datePublished" datetime="2021-09-09">September 2021</time> , Advanced Materials) </span> </div> <div style="cursor: pointer;-webkit-line-clamp: 5;" class="abstract" itemprop="description"> <title>Abstract

Organoids are lumen‐containing multicellular structures that recapitulate key features of the organs, and are increasingly used in models of disease, drug testing, and regenerative medicine. Recent work has used 3D culture models to form organoids from human induced pluripotent stem cells (hiPSCs) in reconstituted basement membrane (rBM) matrices. However, rBM matrices offer little control over the microenvironment. More generally, the role of matrix viscoelasticity in directing lumen formation remains unknown. Here, viscoelastic alginate hydrogels with independently tunable stress relaxation (viscoelasticity), stiffness, and arginine–glycine–aspartate (RGD) ligand density are used to study hiPSC morphogenesis in 3D culture. A phase diagram that shows how these properties control hiPSC morphogenesis is reported. Higher RGD density and fast stress relaxation promote hiPSC viability, proliferation, apicobasal polarization, and lumen formation, while slow stress relaxation at low RGD densities triggers hiPSC apoptosis. Notably, hiPSCs maintain pluripotency in alginate hydrogels for much longer times than is reported in rBM matrices. Lumen formation is regulated by actomyosin contractility and is accompanied by translocation of Yes‐associated protein (YAP) from the nucleus to the cytoplasm. The results reveal matrix viscoelasticity as a potent factor regulating stem cell morphogenesis and provide new insights into how engineered biomaterials may be leveraged to build organoids.

 
more » « less
  • ABSTRACT  
    more » « less