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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> <div style="clear:both;margin-bottom:20px;"></div> <dl class="dl-horizontal small"> <dt>Authors:</dt> <dd> <a 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 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 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 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 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 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 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 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 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>Publication Date:</dt> <dd> <time itemprop="datePublished" datetime="2018-10-31">2018-10-31</time> </dd> </dl> <dl class="dl-horizontal small"> <dt>NSF-PAR ID:</dt> <dd>10363227</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>Page Range or eLocation-ID:</dt> <dd>p. 65-76</dd> </dl> <dl class="dl-horizontal small"> <dt>ISSN:</dt> <dd>1066-5099</dd> </dl> <dl class="dl-horizontal small"> <dt>Publisher:</dt> <dd itemprop="publisher">Oxford University Press</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 itemprop="name">An intrinsic, label-free signal for identifying stem cell-derived cardiomyocyte subtype</span> </a> </div> <div> <strong> <a class="misc doi-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 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> </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 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 doi-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 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> </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/10380081-synthetic-amyloid-beta-does-induce-robust-transcriptional-response-innate-immune-cell-culture-systems" itemprop="url"> <span itemprop="name">Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems</span> </a> </div> <div> <strong> <a class="misc doi-link" href="https://doi.org/10.1186/s12974-022-02459-1" target="_blank" title="Link to document DOI">https://doi.org/10.1186/s12974-022-02459-1  <span class="fas fa-external-link-alt"></span></a> </strong> </div> <div class="metadata"> <span class="authors"> <span class="author" itemprop="author">Quiroga, I. Y.</span> <span class="sep">; </span><span class="author" itemprop="author">Cruikshank, A. E.</span> <span class="sep">; </span><span class="author" itemprop="author">Bond, M. L.</span> <span class="sep">; </span><span class="author" itemprop="author">Reed, K. S. M.</span> <span class="sep">; </span><span class="author" itemprop="author">Evangelista, B. A.</span> <span class="sep">; </span><span class="author" itemprop="author">Tseng, J. H.</span> <span class="sep">; </span><span class="author" itemprop="author">Ragusa, J. V.</span> <span class="sep">; </span><span class="author" itemprop="author">Meeker, R. B.</span> <span class="sep">; </span><span class="author" itemprop="author">Won, H.</span> <span class="sep">; </span><span class="author" itemprop="author">Cohen, S.</span> <span class="sep">; </span><span class="author">et al</span></span> <span class="year">( <time itemprop="datePublished" datetime="2022-04-23">April 2022</time> , Journal of Neuroinflammation) </span> </div> <div class="abstract" itemprop="description"> <title>Abstract Background

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that impacts nearly 400 million people worldwide. The accumulation of amyloid beta (Aβ) in the brain has historically been associated with AD, and recent evidence suggests that neuroinflammation plays a central role in its origin and progression. These observations have given rise to the theory that Aβ is the primary trigger of AD, and induces proinflammatory activation of immune brain cells (i.e., microglia), which culminates in neuronal damage and cognitive decline. To test this hypothesis, many in vitro systems have been established to study Aβ-mediated activation of innate immune cells. Nevertheless, the transcriptional resemblance of these models to the microglia in the AD brain has never been comprehensively studied on a genome-wide scale.

Methods

We used bulk RNA-seq to assess the transcriptional differences between in vitro cell types used to model neuroinflammation in AD, including several established, primary and iPSC-derived immune cell lines (macrophages, microglia and astrocytes) and their similarities to primary cells in the AD brain. We then analyzed the transcriptional response of these innate immune cells to synthetic Aβ or LPS and INFγ.

Results

We found that human induced pluripotent stem cell (hIPSC)-derived microglia (IMGL) are the in vitro cell modelmore »that best resembles primary microglia. Surprisingly, synthetic Aβ does not trigger a robust transcriptional response in any of the cellular models analyzed, despite testing a wide variety of Aβ formulations, concentrations, and treatment conditions. Finally, we found that bacterial LPS and INFγ activate microglia and induce transcriptional changes that resemble many, but not all, aspects of the transcriptomic profiles of disease associated microglia (DAM) present in the AD brain.

Conclusions

These results suggest that synthetic Aβ treatment of innate immune cell cultures does not recapitulate transcriptional profiles observed in microglia from AD brains. In contrast, treating IMGL with LPS and INFγ induces transcriptional changes similar to those observed in microglia detected in AD brains.

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  • Abstract

    A major cause of chronic kidney disease (CKD) is glomerular disease, which can be attributed to a spectrum of podocyte disorders. Podocytes are non-proliferative, terminally differentiated cells. Thus, the limited supply of primary podocytes impedes CKD research. Differentiation of human pluripotent stem cells (hPSCs) into podocytes has the potential to produce podocytes for disease modeling, drug screening, and cell therapies. In the podocyte differentiation process described here, hPSCs are first induced to primitive streak-like cells by activating canonical Wnt signaling. Next, these cells progress to mesoderm precursors, proliferative nephron progenitors, and eventually become mature podocytes by culturing in a serum-free medium. Podocytes generated via this protocol adopt podocyte morphology, express canonical podocyte markers, and exhibit podocyte phenotypes, including albumin uptake and TGF-β1 triggered cell death. This study provides a simple, defined strategy to generate podocytes forin vitromodeling of podocyte development and disease or for cell therapies.

  • Abstract

    Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in termsmore »of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.

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"Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing". <em>Stem Cells</em> 37 (1). Country unknown/Code not available: Oxford University Press. <a href="https://doi.org/10.1002/stem.2931">https://doi.org/10.1002/stem.2931.</a> <a href="https://par.nsf.gov/biblio/10363227">https://par.nsf.gov/biblio/10363227</a>. </div> <div class="modal-footer"> <button class="btn btn-sm btn-default" data-dismiss="modal" aria-hidden="true">Close</button> </div> </div> </div> </div></li> <li class="links-format"><a href="#cite-bib" data-toggle="modal">BibTeX</a> <div id="cite-bib" class="modal" tabindex="-1" role="dialog" aria-labelledby="cite-bib_label" aria-hidden="true"> <div class="modal-dialog"> <div class="modal-content"> <div class="modal-header"> <button type="button" class="close" data-dismiss="modal" aria-hidden="true">×</button> <strong id="cite-bib_label">Cite: BibTeX Format</strong> </div> <div class="modal-body"> @article{osti_10363227,<br/> place = {Country unknown/Code not available}, title = {Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing}, url = {https://par.nsf.gov/biblio/10363227}, DOI = {10.1002/stem.2931}, abstractNote = {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}, journal = {Stem Cells}, volume = {37}, number = {1}, publisher = {Oxford University Press}, author = {Adkar, Shaunak S. and Wu, Chia-Lung and Willard, Vincent P. and Dicks, Amanda and Ettyreddy, Adarsh and Steward, Nancy and Bhutani, Nidhi and Gersbach, Charles A. and Guilak, Farshid}, }</div> <div class="modal-footer"> <button class="btn btn-sm btn-default" data-dismiss="modal" aria-hidden="true">Close</button> </div> </div> </div> </div></li> <li class="divider"></li> </ul> <ul class="nav nav-list" style="font-size: 14px; font-family: Arial Regular;"> <li class="nav-header header-format">Export Metadata</li> <li class="links-format"><a href="https://par.nsf.gov/endnote?osti_id=10363227">EndNote</a></li> <li class="links-format"><a href="https://par.nsf.gov/export/format:excel/osti-id:10363227">Excel</a></li> <li class="links-format"><a href="https://par.nsf.gov/export/format:csv/osti-id:10363227">CSV</a></li> <li class="links-format"><a href="https://par.nsf.gov/export/format:xml/osti-id:10363227">XML</a></li> <li class="divider"></li> </ul> <ul class="nav nav-list" style="font-size: 14px; 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