iPSC CRISPR Facility

The iPSC CRISPR Facility is an expertise center intended to support, help, train and advise researchers with their respective iPSC and CRISPR-Cas9 experiments. The facility offers its services to and collaborates with researchers (both within the UMCG/RUG as well as external clients from academia and industry) that see possibilities for iPSC and / or CRISPR-Cas9 in their research. The facility offers to generate iPSC from human somatic cells derived from multiple sources and provides full characterization of iPSC clones with the option to introduce genetic changes using CRISPR-Cas9 technology.

ERIBA-Image-iPS-cells-color-2

Contact

If you are interested in making use of our expertise, please use the Request Form.

For questions, please contact:

Email:                    ips.crispr.facility@umcg.nl

Post address:       ERIBA, UMCG, Building 3226, PO Box 196, Internal Zip Code FA50, 9700 AD Groningen

 

[[Background iPSC and CRISPR-Cas9

Due to their potential to self-renew and their capacity to differentiate into any cell type, stem cells are of crucial interest both for basic research as well as for the development of regenerative therapies. While (embryonic) stem cells are difficult to acquire, the groundbreaking discovery that differentiated somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC) has generated an easy and virtually limitless source of stem cells.

Another great leap in biomedical science in recent years has been the discovery that the adaptive bacterial immune system CRISPR-Cas9 can be repurposed as a versatile tool for human genome editing. Many human diseases are caused by alterations in the DNA. Such genetic mutations can either be inherited or acquired during life. Over the past decade, many disease-causing mutations have been identified and techniques have been developed to quickly identify them in patients. Genome editing techniques including CRISPR-Cas9 have made it possible to revert disease mutations, thereby enabling the restoration of normal cell function. Performing genome editing on patient-derived iPSC offers an excellent tool for disease modelling and furthermore may allow to cure diseases in the future. While these techniques hold enormous therapeutic promise, for now it is unclear whether they are safe enough to be used on patients. The iPSC CRISPR facility set out to contribute to research that increases our understanding of iPSC and CRISPR-Cas9 and their combined therapeutic potential.

 

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[[Our Expertise & Services

The facility generates, characterizes and stores iPS cell lines derived from multiple cell types. Over the past 1.5 years we have set up differentiation protocols and we closely collaborate with a number of labs holding extensive experience with differentiating iPS cells into a wide variety of cell types. Moreover, the facility offers to perform several CRISPR genome editing strategies on both iPSC and other cell types. This combined expertise on iPSC and CRISPR allows us to support researchers during any stage of their iPSC and / or CRISPR project, be it either through advice, training or experimental help.

The facility’s services include:

 Generation of iPSC

  • Isolation of somatic cells from patient material (blood, urine, skin)
  • Reprogramming of somatic cells (lentiviral or episomal)
  • Generation of iPS cells

Characterization of iPSC

  • pluripotency marker gene expression (qPCR or NGS, immunofluorescence)
  • differentiation / lineage marker gene expression (qPCR or NGS, immunofluorescence)
  • teratoma formation
  • karyotyping (metaphase spreads)
  • single-cell DNA Seq

 

Differentiation of iPS and ES cells

  • The facility has protocols in place for the differentiation of iPS or ES cells into certain cell types (including neural progenitor cells, astrocytes, microglia and macrophages). We furthermore will test differentiation protocols for iPSC into other cell types upon request and hold collaborations with laboratories with ample experience in differentiating iPSC into a variety of different cell types.

CRISPR Cas9-mediated genome editing

  • Gene knock-out
  • Gene tagging
  • Gene alterations
  • Gene regulation (CRISPRi / CRISPRa)
  • Screens using human or mouse knock-out libraries
  • Visualization of genomic loci

CRISPR Cas9-mediated epigenome editing

  • Epigenetic writers and erasers for the regulation of gene expression (de Groote et al. NAR 2012, Cano-Rodriguez & Rots Curr Genet Med Rep 2016, Cano-Rodriguez et al. Nat Comm 2016)

Digital droplet PCR

  • Our platform is available for different applications (including CRISPR-Cas9, detection of rare mutations, copy-number variations, absolute quantification, etc.) for all researchers within the UMCG

Standard operating procedures

Our SOPs listed below will be made available upon request.

iPSC culture

  • Passaging of stem cell lines by cutting
  • Passaging of stem cell lines with ReLeSRTM
  • Passaging of stem cell lines with StemPro® Accutase® cell dissociation reagent
  • Freezing iPSC
  • Reviving iPSC
  • Preparation of Geltrex® coated plates
  • Preparation of Vitronectin coated plates

iPSC reprogramming

  • Isolation of PBMC from whole blood
  • Episomal reprogramming of PBMC
  • Isolation and expansion of epithelial cells from urine
  • Freezing urine-derived epithelial cells
  • Reviving of urine-derived epithelial cells
  • Episomal reprogramming of urine-derived cells using 4D nucleofection (OKSM)
  • Reprogramming of urine-derived cells using ReproRNATM-OKSGM
  • Lentiviral reprogramming of urine derived cells
  • Production of lentiviral reprogramming vector in HEK293 cells
  • Generation of fibroblast cultures from skin biopsies
  • Episomal reprogramming of fibroblasts using 4D nucleofection (OKSM)
  • iPSC expansion schedule (passage 0 to 10)

 iPSC characterization

  • Embryoid body (EB) formation
  • iPSC & EB culture on coverslips
  • Characterization of iPSC lines by immunofluorescence (IF)
  • Characterization of iPSC lines by qPCR
  • Karyotyping iPSC

CRISPR

  • Cloning of sgRNA protospacer into pX vectors (Zhang lab)
  • Plasmid preparation and test restriction
  • Recombinant Cas9 expression and purification
  • In vitro Cas9 cleavage assay
  • T7 endonuclease I assay
  • Digital droplet PCR (ddPCR)
  • Transfection of HEK293 cells using FuGene HD

Constructs and plasmids 190708 Plasmids and Constructs

CRISPR transgenic mouse facility

 

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[[The team

 

The iPSC CRISPR and the CRISPR transgenic mouse teams. From left to right and top to bottom: Jonas Seiler, Othman Alhazzaa, Sahil Gupta, Arun Thiruvalluvan, Niels Kloosterhuis, Eline Sportel, Bart van de Sluis, Floris Foijer, René Wardenaar, Nicolette Huijkman, Mathilde Broekhuis, and Eslie Rozema-Huizinga]]

[[iPSC CRISPR team

Floris Foijer

f.foijer@umcg.nl

Principal investigator and head of the iPSC CRISPR facility

 

Jonas Seiler

j.seiler@umcg.nl

Jonas received his PhD in 2016 at the University of Duisburg-Essen in the lab of Hemmo Meyer at the department of Molecular Biology. During his PhD he studied the role of protein phosphatese-1 (PP1) in mitotic chromosomal alignment and segregation in human cells. Following this, he investigated the role of the AAA ATPase VCP/p97 in PP1 biogenesis and could show through biochemical reconstitution that PP1 holoenzyme formation involves direct disassembly of an inactive intermediate complex by VCP/p97. He has experience with a broad spectrum of molecular biology and cell-based methods. Jonas joined the iPSC CRISPR facility in 2019 where he is involved in project management and the advancement of methods.

Arun Thiruvalluvan

a.thiruvalluvan@umcg.nl

During his PhD, He examined the potential use of human ES/iPSCs derived cells as a tool for stem cell-based therapy and disease modeling. He addressed myelin-restorative strategies by grafting human iPSC-derived oligodendrocytes in animal models for MS (cuprizone and EAE). Using a similar technology with forced transcription factor expression, he studied the transdifferentiation of astrocytes into oligodendrocytes as an alternative source for cell-based remyelination therapy. Moreover, he established MS patient-derived iPSC cell lines that may serve as disease modeling tools to study intrinsic mechanisms underlying MS pathogenesis. Besides that, he used iPS cells of patients with the genetic neurological disorders SCA-3 (Spinocerebellar ataxia) to obtained detailed insights into the pathogenic mechanisms underlying nerve cell degeneration in SCA3.

Sahil Gupta

s.gupta@umcg.nl

I procured my Masters in Biology, at Ludwig Maximillian University, Munich 2012-14. I performed my PhD at Goethe University Frankurt, Biochemistry I (2015-18) in the field of Cancer Immunology, focussing on mechanistic analysis of cytokine synergism in tumor associated macrophage of breast cancer microenvironment. I identified a cancer biomarker using state of art technologies such as RNA sequencing library preparation, CRISPR interference, CRISPR-Cas9 KOs in cells, confocal live cell imaging, in vitro functional assays, Chromatin Immunoprecipitation (ChIP). I joined the iPSC/CRISPR facility as postdoc in January 2018, establishing and optimizing new protocols for iPSC reprogramming of aneuploid cells and differentiation.

Mathilde Broekhuis

m.broekhuis@umcg.nl

Mathilde has been trained as a technician in medical biochemistry at the Saxion Hogeschool in Enschede. After her studies she moved to Rotterdam to work in the lab of Rob Pieters at the department of Pediatric Oncology. She performed cytotoxicity assays and mutation detection on material from leukemia patients. In 2009, Mathilde moved to the lab of Gerald de Haan at the UMCG/RUG. She assisted in fundamental research on the hematopoietic system, performing lineage tracing and in vitro analysis. Lab management also became one of her interests, especially when the lab moved to the new ERIBA institute. Mathilde was the first team member of the new iPSC CRISPR facility that was started up at the end of 2015. As a senior technician she is involved in setting up a the facility lab and performing iPSC and CRISPR-mediated genome engineering.

Eslie Rozema-Huizinga

g.c.huizinga@umcg.nl

Eslie graduated as a biomedical research technician at the Hanze Hogeschool in Groningen in 2016. She did her graduation project at the Pediatric Oncology department of Eveline de Bont in the UMCG. During this intership she assisted Walderik Zomerman with his work on the role of the CREB protein in medulloblastoma. After her studies she joined the iPSC CRISPR facility as a junior technician, to expand her lab skills in iPSC and CRISPR-CAS9 technology.

Othman Alhazzaa

o.a.a.alhazzaa@umcg.nl

 

René Wardenaar

r.wardenaar@umcg.nl

René Wardenaar obtained his PhD in 2016 from the University of Groningen. His thesis described the analysis of sequencing data (whole-genome and bisulfite sequencing) and tiling array data (MeDIP-chip) generated by several studies that are focused on quantifying the contribution of DNA methylation to heritable phenotypic variation in the model plant Arabidopsis thaliana. Part of his thesis got published  in Science in 2014, in which he, together with colleagues and collaborators, described how differentially methylated regions (DMRs) can contribute to phenotypic variation independently from sequence variation and act as bona fide epigenetic quantitative trait loci. Besides these plant-related studies he also participated in other projects that for example were focused on the role of DNA methylation in cervical cancer and the extent of structural variation in the Dutch population. He has a broad interest in biology and joined the iPSC – CRISPR facility in July 2018 where he is supporting the team with the analysis of various types of data.]]

[[CRISPR transgenic mouse team

Bart van de Sluis

a.j.a.van.de.sluis@umcg.nl

Principal investigator and head of the CRISPR transgenic mouse facility

 

Nicolette Huijkman

n.c.a.huijkman@umcg.nl

 

Marieke Smit

m.smit05@umcg.nl

 

Niels Kloosterhuis

n.j.kloosterhuis@umcg.nl

 

Eline Sportel

e.sportel@umcg.nl]]

 

 

 

 

 

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