scholarly journals Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

2017 ◽  
Vol 28 (21) ◽  
pp. 2854-2874 ◽  
Author(s):  
Brock Roberts ◽  
Amanda Haupt ◽  
Andrew Tucker ◽  
Tanya Grancharova ◽  
Joy Arakaki ◽  
...  

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1–4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line–generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community.

2017 ◽  
Author(s):  
Brock Roberts ◽  
Amanda Haupt ◽  
Andrew Tucker ◽  
Tanya Grancharova ◽  
Joy Arakaki ◽  
...  

AbstractWe present a CRISPR/Cas9 genome editing strategy to systematically tag endogenous proteins with fluorescent tags in human inducible pluripotent stem cells. To date we have generated multiple human iPSC lines with GFP tags for 10 proteins representing key cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, lamin B1, non-muscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome editing methodology using Cas9 ribonuclear protein electroporation and fluorescence-based enrichment of edited cells resulted in <0.1-24% HDR across all experiments. Clones were generated from each edited population and screened for precise editing. ∼25% of the clones contained precise mono-allelic edits at the targeted locus. Furthermore, 92% (36/39) of expanded clonal lines satisfied key quality control criteria including genomic stability, appropriate expression and localization of the tagged protein, and pluripotency. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. The data described here, including our editing protocol, genetic screening, quality control assays, and imaging observations, can serve as an initial resource for genome editing in cell biology and stem cell research.


Author(s):  
Carol X.-Q. Chen ◽  
Narges Abdian ◽  
Gilles Maussion ◽  
Rhalena A. Thomas ◽  
Iveta Demirova ◽  
...  

AbstractInduced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a four-step workflow to assess our newly generated iPSCs for variations and reproducibility relative to each other and iPSCs obtained from external sources. Our benchmarks for evaluating iPSCs include examining iPSC morphology and proliferation in two different media conditions (mTeSR1 and Essential 8) and evaluating their ability to differentiate into each of the three germ layers, with a particular focus on neurons. Genomic stability in the human iPSCs was analyzed by G-band karyotyping and a qPCR-based stability test, and cell-line identity authenticated by Short Tandem Repeat (STR) analysis. Using standardized dual SMAD inhibition methods, all iPSC lines gave rise to neural progenitors that could subsequently be differentiated into cortical neurons. Neural differentiation was analyzed qualitatively by immunocytochemistry and quantitatively by q-PCR for progenitor, neuronal, cortical and glial markers. Taken together, we present a standardized quality control workflow to evaluate variability and reproducibility across and between iPSCs.HighlightsValidation of culture conditions is critical in the expansion and maintenance of an iPSC line.Characterization of pluripotency and genomic stability ensures each line is free of defects at the DNA level, while maintaining its ability to be directed into any of the three germ layers.Forebrain cortical neurons can be generated from all iPSC line tested; however, the morphology and expression pattern of these neurons can vary from line to line.


2019 ◽  
Vol 21 (1) ◽  
pp. 108 ◽  
Author(s):  
Mehdi Shafa ◽  
Tylor Walsh ◽  
Krishna Morgan Panchalingam ◽  
Thomas Richardson ◽  
Laura Menendez ◽  
...  

The clinical effectiveness of human induced pluripotent stem cells (iPSCs) is highly dependent on a few key quality characteristics including the generation of high quality cell bank, long-term genomic stability, post-thaw viability, plating efficiency, retention of pluripotency, directed differentiation, purity, potency, and sterility. We have already reported the establishment of iPSC master cell banks (MCBs) and working cell banks (WCBs) under current good manufacturing procedure (cGMP)-compliant conditions. In this study, we assessed the cellular and genomic stability of the iPSC lines generated and cryopreserved five years ago under cGMP-compliant conditions. iPSC lines were thawed, characterized, and directly differentiated into cells from three germ layers including cardiomyocytes (CMs), neural stem cells (NSCs), and definitive endoderm (DE). The cells were also expanded in 2D and 3D spinner flasks to evaluate their long-term expansion potential in matrix-dependent and feeder-free culture environment. All three lines successfully thawed and attached to the L7TM matrix, and formed typical iPSC colonies that expressed pluripotency markers over 15 passages. iPSCs maintained their differentiation potential as demonstrated with spontaneous and directed differentiation to the three germ layers and corresponding expression of specific markers, respectfully. Furthermore, post-thaw cells showed normal karyotype, negative mycoplasma, and sterility testing. These cells maintained both their 2D and 3D proliferation potential after five years of cryopreservation without acquiring karyotype abnormality, loss of pluripotency, and telomerase activity. These results illustrate the long-term stability of cGMP iPSC lines, which is an important step in establishing a reliable, long-term source of starting materials for clinical and commercial manufacturing of iPSC-derived cell therapy products.


2010 ◽  
Vol 22 (1) ◽  
pp. 192
Author(s):  
S. Li ◽  
T. Flisikowska ◽  
B. Kessler ◽  
T. Güngör ◽  
R. Kind ◽  
...  

Mesenchymal stem cells (MSC) are adult stem cells with fibroblast-like morphology, which can be easily isolated from bone marrow and expanded in culture. Mesenchymal stem cells are able to grow from a single cell into a cell clone, which makes them potentially useful for gene targeting. In our recent study we investigated the dynamics of epigenetic reprogramming following nuclear transfer (NT) with MSC and found that these cells can support development of cloned embryos as good as genetically identical fibroblasts (Brero et al. 2009 Cloning Stem Cells 11, 319-329). In the present study we tested whether live cloned rabbits can be produced from MSC. Nuclear donor cells were isolated from a 6-week-old transgenic Ali/Bas rabbit, expanded in culture, and assessed for their differentiation potential. Mesenchymal stem cells were transfected with a green fluorescent protein (GFP) reporter gene construct and stable cell clones were selected (GFP-MSC). The MSC and GFP-MSC were used for NT at passage 3 to 7 after serum starvation for 2 to 4 days. Nuclear transfer was performed essentially as described previously (Yang et al. 2007 Reproduction 133, 219-320). To assess the development to blastocyst, reconstructed embryos were cultured in B2 medium for 5 to 6 days, whereas for in vivo development embryos were cultured only overnight and then transferred into recipients at the 4- to 8-cell stage. In the MSC group, 844 oocytes were used, 793 (94%) of them fused, 698/786 (89%) cleaved, and 48/128 (38%) developed to blastocyst. After transfer of 483 cloned embryos into 13 recipients, 2 from 8 pregnant recipients gave birth to 10 (2.4%) rabbits, from which 2 and 1 survived for more than 7 days and 3 months, respectively. In the GFP-MSC group, 444 oocytes were used, 412 (93%) of them fused, 377/409 (92%) cleaved, and 97/178 (55%) developed to blastocyst. Transfer of 216 cloned embryos into 8 recipients resulted in 4 pregnancies. One recipient gave birth to 6 (3.7%) live and 2 stillborn rabbits, from which 2 and 1 rabbits survived for more than 3 days and 2 weeks, respectively. All cloned rabbits carried a GFP gene, and green fluorescence could be detected in the follicles of the skin under a fluorescence microscope (Zeiss Axiovert200, Carl Zeiss, Germany). Our study demonstrates that live cloned rabbits can be produced from genetically modified MSC, thus paving the way to generate gene targeted animals. This work is supported by Roche Diagnostic GmbH.


2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Lisi Deng ◽  
Guangze Liu ◽  
Xin Wu ◽  
Yaping Wang ◽  
Minghua Tong ◽  
...  

Background and Aim.Adipose derived mesenchymal stem cells (ADMSCs) may be an attractive source for acute and chronic liver injury because they are abundant and easy to obtain. We aim to investigate the efficacy of ADMSCs transplantation in the acute liver failure (ALF) caused by carbon tetrachloride (CCl4) in mice.Methods.ADMSCs were isolated from inguinal fat pads of enhanced green fluorescent protein (EGFP) transgenic mice and their surface markers and differentiation potential were analyzed. ALF models were established by infusion of CCl4and divided into two groups: control group; EGFP-ADMSCs transplantation group. The restoration of biological functions of the livers receiving transplantation was assessed via a variety of approaches such as survival rates, live function parameters, histological localization of EGFP-ADMSCs, and Immunofluorescence analysis.Results.ADMSCs were positive for CD105, CD44 but negative for CD45, CD34 and had adipogenic, osteogenic differentiation potential. The survival rate of transplantation group significantly increased compared to PBS group. Furthermore, the transplanted cells were well integrated into injured livers and produced albumin, cytokeratin-18.Conclusion.Direct transplantation of ADMSCs is an effective treatment for ALF. The transplanted ADMSCs exhibit the potential to differentiate into hepatocyte-like cells in the injured livers.


2015 ◽  
Vol 35 (6) ◽  
pp. 2299-2308 ◽  
Author(s):  
Jiong Yu ◽  
Xiaoru Su ◽  
Chengxing Zhu ◽  
Qiaoling Pan ◽  
Jinfeng Yang ◽  
...  

Background: Stem cell-based therapy in liver diseases has received increasing interest over the past decade, but direct evidence of the homing and implantation of transplanted cells is conflicting. Reliable labeling and tracking techniques are essential but lacking. The purpose of this study was to establish human placenta-derived mesenchymal stem cells (hPMSCs) expressing green fluorescent protein (GFP) and to assay their hepatic functional differentiation in vitro. Methods: The GFP gene was transduced into hPMSCs using a lentivirus to establish GFP+ hPMSCs. GFP+ hPMSCs were analyzed for their phenotypic profile, viability and adipogenic, osteogenic and hepatic differentiation. The derived GFP+ hepatocyte-like cells were evaluated for their metabolic, synthetic and secretory functions, respectively. Results: GFP+ hPMSCs expressed high levels of HLA I, CD13, CD105, CD73, CD90, CD44 and CD29, but were negative for HLA II, CD45, CD31, CD34, CD133, CD271 and CD79. They possessed adipogenic, osteogenic and hepatic differentiation potential. Hepatocyte-like cells derived from GFP+ hPMSCs showed typical hepatic phenotypes. Conclusions: GFP gene transduction has no adverse influences on the cellular or biochemical properties of hPMSCs or markers. GFP gene transduction using lentiviral vectors is a reliable labeling and tracking method. GFP+ hPMSCs can therefore serve as a tool to investigate the mechanisms of MSC-based therapy, including hepatic disease therapy.


2015 ◽  
Vol 27 (1) ◽  
pp. 254
Author(s):  
A. Congras ◽  
H. Barasc ◽  
C. Delcros ◽  
F. Vignoles ◽  
A. Pinton ◽  
...  

Chromosomal rearrangements have a crucial impact on the proper proceedings of meiosis and can lead by several mechanisms to the production of unbalanced gametes or to the complete arrest of gametes production. To assess the impact of these rearrangements in the early development of pig germ cells, we proposed to generate a library of stem cells from infertile boars that are carriers of chromosomal abnormalities as a new tool for the development of an in vitro differentiation system from pluripotent stem cells to germ cells. We report here the reprogramming of fibroblasts from an azoospermic boar carrying a reciprocal translocation t(Y:14) by integrative or nonintegrative viral overexpression of Oct4, Sox2, Klf4, and c-Myc. The iPS cell lines were characterised for pluripotency, cell cycle, and differentiation potential by conventional methods. Genomic stability was analysed by G-banding karyotype, comparative genomic hybridization, and FISH. The porcine iPS-like cell lines harbored characteristics of ground and naïve pluripotency when cultured in specific media. They expressed several pluripotency genes and harbored an ES-like cell cycle. Nevertheless, contrary to mouse and human iPS, they did not silence the integrated exogenes, leading to a poor differentiation potential. Moreover, cytogenetic analysis revealed a high genomic instability upon passaging, which suggests the development of a population with an increased selective advantage. We characterised the selected duplications and compared them to those previously described in other species. In contrast, the nonintegrative reprogrammation system gives us promising results regarding differentiation potential and genomic stability and will bring new insights into the molecular factors controlling and maintaining pluripotency in the pig species.


2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Intekhab Islam ◽  
Gopu Sriram ◽  
Mingming Li ◽  
Yu Zou ◽  
Lulu Li ◽  
...  

Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.


2021 ◽  
Author(s):  
Seon Hye Cheon ◽  
Foster D Ritchie ◽  
Janay M Vacharasin ◽  
Nicholas Marinelli ◽  
Collin Cheatle ◽  
...  

Genome editing and neuronal differentiation protocols have proliferated in the last decade. Mutations in genes that control pluripotency could lead to a potential obstacle with regards to the survival and differentiation potential of the genome-edited cell lines. Here we describe a protocol for the generation, and differentiation, of cell lines containing CRISPR/Cas9 induced mutations in the histone methyltransferase ASH1L. This chromatin modifier was previously implicated in hematopoietic stem cell pluripotency and is a major genetic risk factor for autism spectrum disorders (ASD). We find that haploinsufficiency of ASH1L leads to decreased NANOG gene expression leading to reduce cell survival and increased spontaneous differentiation. We report a method that provides improved single-cell survival with higher colony formation efficiency in ASH1L mutant stem cells. Additionally, we describe a modified dual-SMAD inhibition neuronal induction methodology that permits the successful generation of human neurons with mutations in ASH1L, in a smaller scale than previously reported methods. With our modified CRISPR-genome editing and neuronal differentiation protocols, it is possible to generate genome-edited stem cells containing mutations in genes that impact pluripotency and could affect subsequent cell lineage-specific differentiation. Our detailed technical report presents cost-effective strategies that will benefit researchers focusing on both translational and basic science using stem cell experimental systems.


2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Geraldine Xue En Tu ◽  
Yoon Khei Ho ◽  
Zhi Xu Ng ◽  
Ke Jia Teo ◽  
Tseng Tsai Yeo ◽  
...  

Abstract Background Mesenchymal stem cells (MSCs) serve as an attractive vehicle for cell-directed enzyme prodrug therapy (CDEPT) due to their unique tumour-nesting ability. Such approach holds high therapeutic potential for treating solid tumours including glioblastoma multiforme (GBM), a devastating disease with limited effective treatment options. Currently, it is a common practice in research and clinical manufacturing to use viruses to deliver therapeutic genes into MSCs. However, this is limited by the inherent issues of safety, high cost and demanding manufacturing processes. The aim of this study is to identify a facile, scalable in production and highly efficient non-viral method to transiently engineer MSCs for prolonged and exceptionally high expression of a fused transgene: yeast cytosine deaminase::uracil phosphoribosyl-transferase::green fluorescent protein (CD::UPRT::GFP). Methods MSCs were transfected with linear polyethylenimine using a cpg-free plasmid encoding the transgene in the presence of a combination of fusogenic lipids and β tubulin deacetylase inhibitor (Enhancer). Process scalability was evaluated in various planar vessels and microcarrier-based bioreactor. The transfection efficiency was determined with flow cytometry, and the therapeutic efficacy of CD::UPRT::GFP expressing MSCs was evaluated in cocultures with temozolomide (TMZ)-sensitive or TMZ-resistant human glioblastoma cell lines. In the presence of 5-fluorocytosine (5FC), the 5-fluorouracil-mediated cytotoxicity was determined by performing colometric MTS assay. In vivo antitumor effects were examined by local injection into subcutaneous TMZ-resistant tumors implanted in the athymic nude mice. Results At > 90% transfection efficiency, the phenotype, differentiation potential and tumour tropism of MSCs were unaltered. High reproducibility was observed in all scales of transfection. The therapeutically modified MSCs displayed strong cytotoxicity towards both TMZ-sensitive and TMZ-resistant U251-MG and U87-MG cell lines only in the presence of 5FC. The effectiveness of this approach was further validated with other well-characterized and clinically annotated patient-derived GBM cells. Additionally, a long-term suppression (> 30 days) of the growth of a subcutaneous TMZ-resistant U-251MG tumour was demonstrated. Conclusions Collectively, this highly efficient non-viral workflow could potentially enable the scalable translation of therapeutically engineered MSC for the treatment of TMZ-resistant GBM and other applications beyond the scope of this study.


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