Highly efficient scarless knock-in of reporter genes into human and mouse pluripotent stem cells via transient antibiotic selection

AbstractPluripotent stem cells (PSCs) edited with genetic reporters are useful tools for differentiation analysis and for isolation of specific cell populations for study. Reporter integration into the genome is now commonly achieved by targeted DNA nuclease-enhanced homology directed repair (HDR). However, human PSCs are known to have a low frequency of gene knock-in (KI) by HDR, making reporter line generation an arduous process. Here, we report a methodology for scarless KI of large fluorescent reporter genes into PSCs by transient selection with puromycin or zeocin. With this method, we can perform targeted KI of a single reporter gene with up to 65% efficiency, as well as simultaneous KI of two reporter genes into different loci with up to 11% efficiency. Additionally, we demonstrate that this method also works in mouse PSCs.

Download Full-text

Highly efficient scarless knock-in of reporter genes into human and mouse pluripotent stem cells via transient antibiotic selection

PLoS ONE ◽

10.1371/journal.pone.0201683 ◽

2018 ◽

Vol 13 (11) ◽

pp. e0201683 ◽

Cited By ~ 3

Author(s):

Valentin M. Sluch ◽

Xitiz Chamling ◽

Claire Wenger ◽

Yukan Duan ◽

Dennis S. Rice ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Reporter Genes ◽

Antibiotic Selection ◽

Highly Efficient ◽

Human And Mouse

Download Full-text

Physical cues of cell culture materials lead the direction of differentiation lineages of pluripotent stem cells

Journal of Materials Chemistry B ◽

10.1039/c5tb01276g ◽

2015 ◽

Vol 3 (41) ◽

pp. 8032-8058 ◽

Cited By ~ 43

Author(s):

Akon Higuchi ◽

Qing-Dong Ling ◽

S. Suresh Kumar ◽

Yung Chang ◽

Abdullah A. Alarfaj ◽

...

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Pluripotent Stem Cells ◽

Specific Cell ◽

Cell Lineages ◽

Physical Cues

Differentiation methods of hPSCs into specific cell lineages. Differentiation of hPSCsviaEB formation (types AB, A–D) or without EB formation (types E–H).

Download Full-text

Pluripotent stem cell SOX9 and INS reporters facilitate differentiation into insulin-producing cells

10.1101/2021.02.02.429390 ◽

2021 ◽

Author(s):

Rabea Dettmer ◽

Isabell Niwolik ◽

Ilir Mehmeti ◽

Anne Jörns ◽

Ortwin Naujok

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Beta Cells ◽

Pluripotent Stem Cells ◽

Reporter Genes ◽

Human Pluripotent Stem Cells ◽

Endogenous Gene ◽

Pancreatic Progenitors ◽

Insulin Producing Cells ◽

Endogenous Genes

AbstractDifferentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. The SOX9 gene plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9+ cells form the source for NEUROG3+ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2Kk-F2A-GFP2 reporter genes into the SOX9 locus and a P2A-mCherry reporter gene into the INS locus mediated by CRISPR/CAS9-technology. The knock-ins enable co-expression of the endogenous genes and reporter genes, report the endogenous gene expression and enable the purification of pancreatic progenitors and insulin-producing cells using FACS or MACS. Using these cell lines we established a new differentiation protocol geared towards SOX9+ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells.

Download Full-text

Abstract 65: Latrophilin-2 is a Specific Cell-surface Marker for Cardiac Progenitor Cells and Specifies Cardiac Lineage Commitment and Development

Circulation Research ◽

10.1161/res.119.suppl_1.65 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Hyun-Jai Cho ◽

Choon-Soo Lee ◽

Jin-Woo Lee ◽

Jung-Kyu Han ◽

Han-Mo Yang ◽

...

Keyword(s):

Stem Cells ◽

Cell Surface ◽

Progenitor Cells ◽

Pluripotent Stem Cells ◽

Cardiac Development ◽

Cardiac Differentiation ◽

Specific Cell ◽

Specific Marker ◽

Cardiac Progenitor Cells ◽

Cardiac Lineage

Backgrounds: The identification of a lineage-specific marker plays a pivotal role in understanding developmental process and is utilized to isolate a certain cell type with high purity for the therapeutic purpose. We here report a new cardiac-specific marker, and demonstrate its functional significance in the cardiac development. Methods and Results: When mouse pluripotent stem cells (ES and iPS cells) were stimulated with BMP4, Activin A, bFGF and VEGF, they differentiated into cardiac cells. To screen cell-surface expressing molecules on cardiac progenitor cells compared to undifferentiated mouse iPS and ES cells, we isolated Flk1+/PDGFRa+ cells at differentiation day 4 and performed microarray analysis. Among candidates, we identified a new G protein-coupled receptor, Latrophilin-2 (LPHN2) whose signaling pathway and its effect on cardiac differentiation is unknown. In sorting experiments under cardiac differentiation condition, LPHN2+ cells derived from pluripotent stem cells strongly expressed cardiac-related genes (Mesp1, Nkx2.5, aMHC and cTnT) and exclusively gave rise to beating cardiomyocytes, as compared with LPHN2- cells. LPHN2-/- mice revealed embryonically lethal and huge defects in cardiac development. Interestingly, LPHN2+/- heterozygotes were alive and fertile. For the purpose of cardiac regeneration, we transplanted iPS-derived LPHN2+ cells into the infarcted heart of adult mice. LPHN2+ cells differentiated into cardiomyocytes, and systolic function of left ventricle was improved and infarct size was reduced. We confirmed LPHN2 expression on human iPS and ES cell-derived cardiac progenitor cells and human heart. Conclusions: We demonstrate that LPHN2 is a functionally significant and cell-surface expressing marker for both mouse and human cardiac progenitor and cardiomyocytes. Our findings provide a valuable tool for isolating cardiac lineage cells from pluripotent stem cells and an insight into cardiac development and regeneration.

Download Full-text

Fluorescent Reporter Genes and the Analysis of Bacterial Regulatory Networks

Hybrid Systems Biology - Lecture Notes in Computer Science ◽

10.1007/978-3-319-27656-4_2 ◽

2015 ◽

pp. 27-50

Author(s):

Hidde de Jong ◽

Johannes Geiselmann

Keyword(s):

Regulatory Networks ◽

Reporter Genes ◽

Fluorescent Reporter ◽

Fluorescent Reporter Genes

Download Full-text

Human Pluripotent Stem Cell Culture: Current Status, Challenges, and Advancement

Stem Cells International ◽

10.1155/2018/7396905 ◽

2018 ◽

Vol 2018 ◽

pp. 1-17 ◽

Cited By ~ 30

Author(s):

Sushrut Dakhore ◽

Bhavana Nayer ◽

Kouichi Hasegawa

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Pluripotent Stem Cells ◽

Large Scale ◽

Embryonic Stem ◽

Current Status ◽

Specific Cell ◽

Therapeutic Applications ◽

Proliferative Ability

Over the past two decades, human embryonic stem cells (hESCs) have gained attention due to their pluripotent and proliferative ability which enables production of almost all cell types in the human body in vitro and makes them an excellent tool to study human embryogenesis and disease, as well as for drug discovery and cell transplantation therapies. Discovery of human-induced pluripotent stem cells (hiPSCs) further expanded therapeutic applications of human pluripotent stem cells (PSCs). hPSCs provide a stable and unlimited original cell source for producing suitable cells and tissues for downstream applications. Therefore, engineering the environment in which these cells are grown, for stable and quality-controlled hPSC maintenance and production, is one of the key factors governing the success of these applications. hPSCs are maintained in a particular niche using specific cell culture components. Ideally, the culture should be free of xenobiotic components to render hPSCs suitable for therapeutic applications. Substantial efforts have been put to identify effective components, and develop culture conditions and protocols, for their large-scale expansion without compromising on quality. In this review, we discuss different media, their components and functions, including specific requirements to maintain the pluripotent and proliferative ability of hPSCs. Understanding the role of culture components would enable the development of appropriate conditions to promote large-scale, quality-controlled expansion of hPSCs thereby increasing their potential applications.

Download Full-text

Derivation of induced pluripotent stem cells from ferret somatic cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00456.2019 ◽

2020 ◽

Vol 318 (4) ◽

pp. L671-L683

Author(s):

Jinghui Gao ◽

Sophia Petraki ◽

Xingshen Sun ◽

Leonard A. Brooks ◽

Thomas J. Lynch ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Patient Specific ◽

Preclinical Model ◽

Specific Cell ◽

Chronic Lung Diseases ◽

Human Ipscs ◽

Fetal Fibroblasts ◽

Induced Pluripotent

Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets.

Download Full-text

Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells

Development ◽

10.1242/dev.132209 ◽

2016 ◽

Vol 143 (9) ◽

pp. 1475-1481 ◽

Cited By ~ 26

Author(s):

Derek T. Peters ◽

Christopher A. Henderson ◽

Curtis R. Warren ◽

Max Friesen ◽

Fang Xia ◽

...

Keyword(s):

Stem Cells ◽

Cell Surface ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

Surface Marker ◽

Asialoglycoprotein Receptor ◽

Cell Surface Marker ◽

Specific Cell ◽

Specific Cell Surface

Download Full-text

Differential regulation of lineage commitment in human and mouse primed pluripotent stem cells by NuRD

10.1101/2020.02.05.935544 ◽

2020 ◽

Author(s):

Ramy Ragheb ◽

Sarah Gharbi ◽

Julie Cramard ◽

Oluwaseun Ogundele ◽

Susan Kloet ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Pluripotent Stem Cells ◽

Es Cells ◽

Pluripotent Cells ◽

Down Regulation ◽

Mouse Es Cells ◽

The Right ◽

Human And Mouse ◽

Pluripotency Genes

AbstractDifferentiation of mammalian pluripotent cells involves large-scale changes in transcription and, among the molecules that orchestrate these changes, chromatin remodellers are essential to initiate, establish and maintain a new gene regulatory network. The NuRD complex is a highly conserved chromatin remodeller which fine-tunes gene expression in embryonic stem cells. While the function of NuRD in mouse pluripotent cells has been well defined, no study yet has defined NuRD function in human pluripotent cells. We investigated the structure and function of NuRD in human induced pluripotent stem cells (hiPSCs). Using immunoprecipitation followed by mass-spectrometry in hiPSCs and in naive or primed mouse pluripotent stem cells, we find that NuRD structure and biochemical interactors are generally conserved. Using RNA sequencing, we find that, whereas in mouse primed stem cells and in mouse naïve ES cells, NuRD is required for an appropriate level of transcriptional response to differentiation signals, hiPSCs require NuRD to initiate these responses. This difference indicates that mouse and human cells interpret and respond to induction of differentiation differently.Graphical AbstractNuRD acts like a conductor in an orchestra.A. In the presence of NuRD (pink blob figure, centre) differentiation occurs in an ordered fashion in both mouse (left) and human (right) ES cells. Gene expression changes in both cell types are tightly controlled with down-regulation of pluripotency genes and up-regulation of lineage appropriate genes. This is akin to a group of musicians producing musical notes in the right order and at the right amplitude to create a coherent piece of music. B. Loss of “the conductor” NuRD results in increased transcriptional noise in both systems, indicated here as a low-level blanket of sound in both systems. Consequences of MBD3/NuRD loss differs between human and mouse ES cells. In mouse ES cells, differentiation cues lead to some down-regulation of pluripotency genes and incomplete progression along a lineage appropriate pathway. This is like musicians who know that they should be making music but who lose their way without a conductor’s influence. In human iPS cells the background level of noise without NuRD results in a lack of order to gene expression changes in response to differentiation. The noise from these “musicians” would be truly awful.

Download Full-text