Publications by authors named "Harald Mikkers"

31 Publications

Protocol for Isolation, Stimulation and Functional Profiling of Primary and iPSC-derived Human NK Cells.

Bio Protoc 2020 Dec 5;10(23):e3845. Epub 2020 Dec 5.

Department of Cell & Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands.

Natural killer (NK) cells are innate immune cells, characterized by their cytotoxic capacity, and chemokine and cytokine secretion upon activation. Human NK cells are identified by CD56 expression. Circulating NK cells can be further subdivided into the CD56 (~10%) and CD56 NK cell subsets (~90%). NK cell-like cells can also be derived from human induced pluripotent stem cells (iPSC). To study the chemokine and cytokine secretion profile of the distinct heterogenous NK cell subsets, intracellular flow cytometry staining can be performed. However, this assay is challenging when the starting material is limited. Alternatively, NK cell subsets can be enriched, sorted, stimulated, and functionally profiled by measuring secreted effector molecules in the supernatant by Luminex. Here, we provide a rapid and straightforward protocol for the isolation and stimulation of primary NK cells or iPSC-derived NK cell-like cells, and subsequent detection of secreted cytokines and chemokines, which is also applicable for a low number of cells.
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http://dx.doi.org/10.21769/BioProtoc.3845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842530PMC
December 2020

Reprogramming Urine-Derived Cells using Commercially Available Self-Replicative RNA and a Single Electroporation.

Curr Protoc Stem Cell Biol 2020 12;55(1):e124

LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands.

We describe a protocol for efficient generation of human-induced pluripotent stem cells (hiPSCs) from urine-derived cells (UDCs) obtained from adult donors using self-replicative RNA containing the reprogramming factors OCT3/4, SOX2, KLF4, GLIS1, and c-MYC (ReproRNA-OKSGM). After electroporation, transfection efficiency is quantified by measuring OCT3/4-expressing UDCs using flow cytometry and should be ≥0.1%. hiPSC colonies emerge within 3 weeks after transfection and express multiple pluripotency markers. Moreover, the UDC-derived hiPSCs are able to differentiate into cells of all three germ layers and display normal karyotypes. ReproRNA-OKSGM is available commercially and only requires a single transfection step so that the protocol is readily accessible, as well as straightforward. In addition to a detailed step-by-step description for generating clonal hiPSCs from UDCs using ReproRNA-OKSGM, we provide guidance for basic pluripotency characterization of the hiPSC lines. © 2020 The Authors. Basic Protocol: Reprogramming of urine-derived cells using ReproRNA-OKSGM Support Protocol 1: Determination of the pluripotency status of hiPSCs by flow cytometry Support Protocol 2: Characterization of functional pluripotency of hiPSCs.
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http://dx.doi.org/10.1002/cpsc.124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540473PMC
December 2020

Generation and genetic repair of 2 iPSC clones from a patient bearing a heterozygous c.1120del18 mutation in the ACVRL1 gene leading to Hereditary Hemorrhagic Telangiectasia (HHT) type 2.

Stem Cell Res 2020 07 28;46:101786. Epub 2020 May 28.

LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, 2333 ZC, The Netherlands; Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, 2333 ZC, The Netherlands.

Fibroblasts from a patient carrying a heterozygous 18bp deletion in exon 8 of the ACVRL1 gene (c.1120del18) were reprogrammed using episomal vectors. The in-frame deletion in ACVRL1 causes the loss of 6 amino acids of the protein, which is associated with Hereditary Hemorrhagic Telangiectasia (HHT) type 2 (Letteboer et al., 2005). CRISPR-Cas9 editing was used to genetically correct the mutation in the induced pluripotent stem cells (iPSCs). The top5-predicted off-target sites were not altered. Patient and isogenic iPSCs showed high pluripotent marker expression, in vitro differentiation capacity into all three germ layers and displayed a normal karyotype. The obtained isogenic pairs will enable proper in vitro disease modelling of HHT (Roman and Hinck, 2017).
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http://dx.doi.org/10.1016/j.scr.2020.101786DOI Listing
July 2020

In vitro modelling of alveolar repair at the air-liquid interface using alveolar epithelial cells derived from human induced pluripotent stem cells.

Sci Rep 2020 03 26;10(1):5499. Epub 2020 Mar 26.

Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands.

Research on acute and chronic lung diseases would greatly benefit from reproducible availability of alveolar epithelial cells (AEC). Primary alveolar epithelial cells can be derived from human lung tissue but the quality of these cells is highly donor dependent. Here, we demonstrated that culture of EpCAM cells derived from human induced pluripotent stem cells (hiPSC) at the physiological air-liquid interface (ALI) resulted in type 2 AEC-like cells (iAEC2) with alveolar characteristics. iAEC2 cells expressed native AEC2 markers (surfactant proteins and LPCAT-1) and contained lamellar bodies. ALI-iAEC2 were used to study alveolar repair over a period of 2 weeks following mechanical wounding of the cultures and the responses were compared with those obtained using primary AEC2 (pAEC2) isolated from resected lung tissue. Addition of the Wnt/β-catenin activator CHIR99021 reduced wound closure in the iAEC2 cultures but not pAEC2 cultures. This was accompanied by decreased surfactant protein expression and accumulation of podoplanin-positive cells at the wound edge. These results demonstrated the feasibility of studying alveolar repair using hiPSC-AEC2 cultured at the ALI and indicated that this model can be used in the future to study modulation of alveolar repair by (pharmaceutical) compounds.
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http://dx.doi.org/10.1038/s41598-020-62226-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099095PMC
March 2020

iPSC-Based Modeling of RAG2 Severe Combined Immunodeficiency Reveals Multiple T Cell Developmental Arrests.

Stem Cell Reports 2020 02 16;14(2):300-311. Epub 2020 Jan 16.

Department of Cell & Chemical Biology, Leiden University Medical Center, Leiden 2300 RC, The Netherlands; LUMC hiPSC Hotel, Leiden University Medical Center, Leiden 2333 ZC, The Netherlands. Electronic address:

RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7CD5 to CD4CD8. The impaired differentiation was accompanied by an increase in CD7CD56CD33 natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks.
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http://dx.doi.org/10.1016/j.stemcr.2019.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013232PMC
February 2020

De novo generation of a functional human thymus from induced pluripotent stem cells.

J Allergy Clin Immunol 2019 11 16;144(5):1416-1419.e7. Epub 2019 Jul 16.

Department of Immunohematology & Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2019.05.042DOI Listing
November 2019

Human iPSC-Derived Retinas Recapitulate the Fetal CRB1 CRB2 Complex Formation and Demonstrate that Photoreceptors and Müller Glia Are Targets of AAV5.

Stem Cell Reports 2019 05 4;12(5):906-919. Epub 2019 Apr 4.

Department of Ophthalmology, Leiden University Medical Center (LUMC), 2333 ZA Leiden, The Netherlands; Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences (KNAW), 1105 BA Amsterdam, The Netherlands. Electronic address:

Human retinal organoids from induced pluripotent stem cells (hiPSCs) can be used to confirm the localization of proteins in retinal cell types and to test transduction and expression patterns of gene therapy vectors. Here, we compared the onset of CRB protein expression in human fetal retina with human iPSC-derived retinal organoids. We show that CRB2 protein precedes the expression of CRB1 in the developing human retina. Our data suggest the presence of CRB1 and CRB2 in human photoreceptors and Müller glial cells. Thus the fetal CRB complex formation is replicated in hiPSC-derived retina. CRB1 patient iPSC retinal organoids showed disruptions at the outer limiting membrane as found in Crb1 mutant mice. Furthermore, AAV serotype 5 (AAV5) is potent in infecting human Müller glial cells and photoreceptors in hiPSC-derived retinas and retinal explants. Our data suggest that human photoreceptors can be efficiently transduced by AAVs in the presence of photoreceptor segments.
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http://dx.doi.org/10.1016/j.stemcr.2019.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522954PMC
May 2019

Generation of 3 human induced pluripotent stem cell lines LUMCi005-A, B and C from a Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type patient.

Stem Cell Res 2019 01 14;34:101359. Epub 2018 Dec 14.

Department of Human Genetics, LUMC, Leiden, The Netherlands.

Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type (HCHWA-D) is an autosomal dominant hereditary disease caused by a point mutation in exon 17 of the APP gene. We generated human induced pluripotent stem cells (hiPSCs) from a symptomatic HCHWA-D patient by using non-integrating Sendai virus (SeV). The newly generated hiPSCs express all pluripotency markers, have a normal karyotype, carry the Dutch mutation, can differentiate in the three germ layers in vitro and are SeV free.
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http://dx.doi.org/10.1016/j.scr.2018.101359DOI Listing
January 2019

Allele-specific repression of Sox2 through the long non-coding RNA Sox2ot.

Sci Rep 2018 01 10;8(1):386. Epub 2018 Jan 10.

Department of Molecular Cell Biology, Leiden University Medical Center, PO Box 9600, 2300RC, Leiden, The Netherlands.

The transcription factor Sox2 controls the fate of pluripotent stem cells and neural stem cells. This gatekeeper function requires well-regulated Sox2 levels. We postulated that Sox2 regulation is partially controlled by the Sox2 overlapping long non-coding RNA (lncRNA) gene Sox2ot. Here we show that the RNA levels of Sox2ot and Sox2 are inversely correlated during neural differentiation of mouse embryonic stem cells (ESCs). Through allele-specific enhanced transcription of Sox2ot in mouse Sox2eGFP knockin ESCs we demonstrate that increased Sox2ot transcriptional activity reduces Sox2 RNA levels in an allele-specific manner. Enhanced Sox2ot transcription, yielding lower Sox2 RNA levels, correlates with a decreased chromatin interaction of the upstream regulatory sequence of Sox2 and the ESC-specific Sox2 super enhancer. Our study indicates that, in addition to previously reported in trans mechanisms, Sox2ot can regulate Sox2 by an allele-specific mechanism, in particular during development.
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http://dx.doi.org/10.1038/s41598-017-18649-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762901PMC
January 2018

Production of iPS-Derived Human Retinal Organoids for Use in Transgene Expression Assays.

Methods Mol Biol 2018 ;1715:261-273

Department of Ophthalmology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.

In vitro retinal organoid modeling from human pluripotent stem cells is becoming more common place in many ophthalmic laboratories worldwide. These organoids mimic human retinogenesis through formation of organized layered retinal structures that display markers for typical retinal cell types. Pivotally these humanized retinal models provide a stepping stone to the clinic as therapeutic tools and are expected to provide a promising alternative to current animal models. Thus pluripotent stem cell based healthy as well as diseased human retinal organoids are attractive for use in drug potency assays and gene augmentation therapeutics. Here we outline an established protocol for generation of these retinal organoids and how they can be used in conjunction with adeno-associated virus vectors for transgene expression assays.
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http://dx.doi.org/10.1007/978-1-4939-7522-8_19DOI Listing
July 2018

Antisense Long Non-Coding RNAs Are Deregulated in Skin Tissue of Patients with Systemic Sclerosis.

J Invest Dermatol 2018 04 24;138(4):826-835. Epub 2017 Nov 24.

Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands.

Systemic sclerosis is an autoimmune disease characterized by fibrosis of skin and multiple organs of which the pathogenesis is poorly understood. We studied differentially expressed coding and non-coding genes in relation to systemic sclerosis pathogenesis with a specific focus on antisense non-coding RNAs. Skin biopsy-derived RNAs from 14 early systemic sclerosis patients and six healthy individuals were sequenced with ion-torrent and analyzed using DEseq2. Overall, 4,901 genes with a fold change >1.5 and a false discovery rate <5% were detected in patients versus controls. Upregulated genes clustered in immunologic, cell adhesion, and keratin-related processes. Interestingly, 676 deregulated non-coding genes were detected, 257 of which were classified as antisense genes. Sense genes expressed opposite of these antisense genes were also deregulated in 42% of the observed sense-antisense gene pairs. The majority of the antisense genes had a similar effect sizes in an independent North American dataset with three genes (CTBP1-AS2, OTUD6B-AS1, and AGAP2-AS1) exceeding the study-wide Bonferroni-corrected P-value (P < 0.0023, P = 1.1 × 10, 1.4 × 10, 1.7 × 10, respectively). In this study, we highlight that together with coding genes, (antisense) long non-coding RNAs are deregulated in skin tissue of systemic sclerosis patients suggesting a novel class of genes involved in pathogenesis of systemic sclerosis.
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http://dx.doi.org/10.1016/j.jid.2017.09.053DOI Listing
April 2018

In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting.

Nat Commun 2017 09 22;8(1):657. Epub 2017 Sep 22.

Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands.

Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.CRISPR-Cas9-based gene editing involves double-strand breaks at target sequences, which are often repaired by mutagenic non-homologous end-joining. Here the authors use Cas9 nickases to generate coordinated single-strand breaks in donor and target DNA for precise homology-directed gene editing.
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http://dx.doi.org/10.1038/s41467-017-00687-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5610252PMC
September 2017

Axin2-mTurquoise2: A novel reporter mouse model for the detection of canonical Wnt signalling.

Genesis 2017 10 19;55(10). Epub 2017 Sep 19.

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, The Netherlands.

The canonical Wnt signalling pathway has been implicated in organogenesis and self-renewal of essentially all stem cell systems. In vivo reporter systems are crucial to assess the role of Wnt signalling in the biology and pathology of stem cell systems. We set out to develop a Turquoise (TQ) fluorescent protein based Wnt reporter. We used a CRISPR-Cas9 approach to insert a TQ fluorescent protein encoding gene into the general Wnt target gene Axin2, thereby establishing a Wnt reporter mouse similar to previously generated Wnt reporter mice but with the mTurquoise2 gene instead of E. coli-β-galactosidase (LacZ). The use of mTurquoise2 is especially important in organ systems in which cells need to a be alive for further experimentation such as in vitro activation or transplantation studies. We here report successful generation of Axin2-TQ mice and show that cells from these mice faithfully respond to Wnt signals. High Wnt signals were detected in the intestinal crypts, a classical Wnt signalling site in vivo, and by flow cytometry in the thymus. These mice are an improved tool to further elucidate the role of Wnt signalling in vivo.
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http://dx.doi.org/10.1002/dvg.23068DOI Listing
October 2017

Caught in a Wnt storm: Complexities of Wnt signaling in hematopoiesis.

Exp Hematol 2016 06 23;44(6):451-7. Epub 2016 Mar 23.

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands; Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.

The Wnt signaling pathway is an evolutionary conserved pathway that is involved in the development of almost every organ system in the body and provides self-renewal signals for most, if not all, adult stem cell systems. In recent years, this pathway has been studied by various research groups working on hematopoietic stem cells, resulting in contradicting conclusions. Here, we discuss and interpret the results of these studies and propose that Wnt dosage, the source of hematopoietic stem cells, and interactions with other pathways explain these disparate results.
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http://dx.doi.org/10.1016/j.exphem.2016.03.004DOI Listing
June 2016

Comment on "Functional Analysis of a Complement Polymorphism (rs17611) Associated with Rheumatoid Arthritis".

J Immunol 2015 Jul;195(1):3-4

Department of Rheumatology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands; Department of Human Genetics, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.

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http://dx.doi.org/10.4049/jimmunol.1500822DOI Listing
July 2015

Cell replacement therapies: is it time to reprogram?

Hum Gene Ther 2014 Oct 17;25(10):866-74. Epub 2014 Sep 17.

1 Department of Molecular Cell Biology, Leiden University Medical Center , 2300RC Leiden, The Netherlands .

Hematopoietic stem cell transplantations have become a very successful therapeutic approach to treat otherwise life-threatening blood disorders. It is thought that stem cell transplantation may also become a feasible treatment option for many non-blood-related diseases. So far, however, the limited availability of human leukocyte antigen-matched donors has hindered development of some cell replacement therapies. The Nobel-prize rewarded finding that pluripotency can be induced in somatic cells via expression of a few transcription factors has led to a revolution in stem cell biology. The possibility to change the fate of somatic cells by expressing key transcription factors has been used not only to generate pluripotent stem cells, but also for directly converting somatic cells into fully differentiated cells of another lineage or more committed progenitor cells. These approaches offer the prospect of generating cell types with a specific genotype de novo, which would circumvent the problems associated with allogeneic cell transplantations. This technology has generated a plethora of new disease-specific research efforts, from studying disease pathogenesis to therapeutic interventions. Here we will discuss the opportunities in this booming field of cell biology and summarize how the scientists in the Netherlands have joined efforts in one area to exploit the new technology.
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http://dx.doi.org/10.1089/hum.2014.097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4180125PMC
October 2014

Reconsidering pluripotency tests: do we still need teratoma assays?

Stem Cell Res 2013 Jul 26;11(1):552-62. Epub 2013 Mar 26.

SET Foundation, Frankfurt am Main, Germany.

The induction of teratoma in mice by the transplantation of stem cells into extra-uterine sites has been used as a read-out for cellular pluripotency since the initial description of this phenomenon in 1954. Since then, the teratoma assay has remained the assay of choice to demonstrate pluripotency, gaining prominence during the recent hype surrounding human stem cell research. However, the scientific significance of the teratoma assay has been debated due to the fact that transplanted cells are exposed to a non-physiological environment. Since many mice are used for a result that is heavily questioned, it is time to reconsider the teratoma assay from an ethical point of view. Candidate alternatives to the teratoma assay comprise the directed differentiation of pluripotent stem cells into organotypic cells, differentiation of cells in embryoid bodies, the analysis of pluripotency-associated biomarkers with high correlation to the teratoma forming potential of stem cells, predictive epigenetic footprints, or a combination of these technologies. Each of these assays is capable of addressing one or more aspects of pluripotency, however it is essential that these assays are validated to provide an accepted robust, reproducible alternative. In particular, the rapidly expanding number of human induced pluripotent stem cell lines, requires the development of simple, affordable standardized in vitro and in silico assays to reduce the number of animal experiments performed.
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http://dx.doi.org/10.1016/j.scr.2013.03.001DOI Listing
July 2013

Prospects and challenges of induced pluripotent stem cells as a source of hematopoietic stem cells.

Ann N Y Acad Sci 2012 Aug;1266:179-88

Department of Molecular Cell Biology, Regenerative Medicine Program, Leiden University Medical Center, Leiden, the Netherlands.

Many life-threatening hematological diseases are now treated by bone marrow transplantations, i.e., infusion of hematopoietic stem cells (HSCs). HSC transplantations are a valid option for the treatment of a variety of metabolic disorders, and even for solid tumors and some refractory severe autoimmune diseases. Unfortunately, the frequency and outcome of HSC transplantations are limited by a shortage of suitable donors. Induced pluripotent stem cells (iPSCs)--somatic cells that have acquired pluripotent stem cell characteristics by the ectopic expression of pluripotency-inducing factors--have been proposed as an alternative source of HSCs. Possible applications include cells of autologous, of autologous and genetically modified, or of allogeneic origin. Here, we provide a perspective on the distinct opportunities of iPSCs and discuss the challenges that lie ahead.
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http://dx.doi.org/10.1111/j.1749-6632.2012.06629.xDOI Listing
August 2012

Induced pluripotent stem cells and severe combined immunodeficiency: merely disease modeling or potentially a novel cure?

Pediatr Res 2012 Apr 8;71(4 Pt 2):427-32. Epub 2012 Feb 8.

Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.

For most, but not all, types of severe combined immunodeficiency (SCID) the underlying molecular defects are known, in principle allowing the cure of affected children via gene therapy. Typically such approaches have used autologous hematopoietic stem cells modified to express a therapeutic gene via γ-retroviral vectors. Insertional mutagenesis has emerged as a significant risk for successful application of this type of gene therapy. Therefore, lentiviral vectors with a self-inactivating design have been developed. Recent advances in stem cell technology using induced pluripotent stem cells (iPSCs) allow an entire different approach to gene therapy for SCID and other genetic disorders, namely by correction of the affected gene in patient-specific iPSCs followed by hematopoietic differentiation. Here, we review these recent advances in the field from an efficacy and safety point of view.
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http://dx.doi.org/10.1038/pr.2011.65DOI Listing
April 2012

[Stem cells: therapeutic applications and experimental techniques].

Ned Tijdschr Geneeskd 2011 ;155(47):A3565

Leids Universitair Medisch Centrum, afd. Moleculaire Celbiologie, Leiden, the Netherlands.

Unlabelled: Stem cells are being used more frequently for research and experimental therapy, but as yet the clinical applications of stem cells are limited. Pluripotent stem cells, with embryonic stem cells as the most well know example, can differentiate into each cell type; in contrast, tissue specific stem cells can only form one or more cell types within one type of tissue. It has been possible for some time to reprogram different types of somatic cells into pluripotent stem cells. Such stem cells are termed induced pluripotent stem cells (iPS cells). iPS cells can also be created from cells of patients with genetic conditions. Research into mechanisms of pathology and new medicines can be carried out with these against a specific genetic background. Clinical application of such iPS cells is not to be expected in the short term. Facilities are being established in different Dutch academic centres to create iPS cells for scientific research.

Conflict Of Interest: none declared.
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July 2013

Differentiation of induced pluripotent stem cells into functional oligodendrocytes.

Glia 2011 Jun 24;59(6):882-92. Epub 2011 Mar 24.

Department of Neuroscience, University Medical Centre Groningen, A.Deusinglaan 1, 9713AV Groningen, The Netherlands.

The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell-based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in-vitro (co-culture with DRG neurons) as well as in-vivo (after implantation in the demyelinated corpus callosum of cuprizone-treated mice). We report the importance of complete purification of the iPS-derived OPC suspension to prevent the contamination with teratoma-forming iPS cells.
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http://dx.doi.org/10.1002/glia.21159DOI Listing
June 2011

Long non-coding RNAs: Guardians of development.

Differentiation 2010 Nov-Dec;80(4-5):175-83. Epub 2010 Aug 11.

Department of Molecular Cell Biology, Regenerative Medicine Program, Leiden University Medical Center, Postal zone S1P, P.O. Box 9600, 2300RC Leiden, The Netherlands.

Two decades ago, the existence of long non-coding RNAs (lncRNAs) was discovered. In the following genomics era more transcribed non-coding genomic regions were identified. These were initially regarded as transcriptional noise and did not receive a lot of attention. Emerging data on several of these long non-coding transcripts have refuted this hypothesis by demonstrating that non-coding RNAs (ncRNAs) are important for regulating transcription and cell signaling. A special subset of the lncRNAs affecting gene transcription appears to orchestrate major developmental programs. Here, we discuss the mechanisms by which lncRNAs regulate transcription, and review the evidence that links this class of lncRNAs to a role in development.
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http://dx.doi.org/10.1016/j.diff.2010.07.003DOI Listing
February 2011

Tumors originating from induced pluripotent stem cells and methods for their prevention.

Ann N Y Acad Sci 2009 Sep;1176:197-204

Department of Molecular Cell Biology, Regenerative Medicine Program, Leiden University Medical Center, Leiden, the Netherlands.

Pluripotent stem cells represent an almost unlimited source of most somatic cell types, providing them with great potential for cell-based therapies. The earliest methods used for generating human pluripotent stem cells as embryonic stem cells from human embryos suffered from ethical and technical drawbacks. These problems have been solved in part through the efficient induction of pluripotency in somatic cells using forced expression of a tetrad of factors. Here, we describe the formation of rhabdomyosarcomas originating from factor-induced pluripotent stem (iPS) cells derived from mouse neural stem cells. This underscores the commonly accepted notion that the use of retroviral delivery methods for inducing pluripotency will not be suited for clinical applications. However, the iPS cell field is developing rapidly. Safer protocols are now available for producing pluripotent stem cells. Here the current state-of-the-art in this field will be discussed.
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http://dx.doi.org/10.1111/j.1749-6632.2009.04563.xDOI Listing
September 2009

A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.

Nat Protoc 2009 30;4(5):789-98. Epub 2009 Apr 30.

Division of Molecular Genetics, Cancer Genomics Centre, Netherlands Cancer Institute, Plesmanlaan, Amsterdam, The Netherlands.

Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA isolation, DNA digestion, linker or splinkerette ligation, primary and secondary PCR amplification, and sequencing. This method, which takes about 1 week to perform, has allowed us to isolate hundreds of thousands of insertion sites from mouse tumors and, unlike other methods, has been specifically optimized for the murine leukemia virus (MuLV), and can easily be performed in a 96-well plate format for the efficient multiplex isolation of insertion sites.
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http://dx.doi.org/10.1038/nprot.2009.64DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3627465PMC
September 2009

Induced pluripotency with endogenous and inducible genes.

Exp Cell Res 2008 Oct 9;314(17):3255-63. Epub 2008 Jul 9.

Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.

The recent discovery that two partly overlapping sets of four genes induce nuclear reprogramming of mouse and even human cells has opened up new possibilities for cell replacement therapies. Although the combination of genes that induce pluripotency differs to some extent, Oct4 and Sox2 appear to be a prerequisite. The introduction of four genes, several of which been linked with cancer, using retroviral approaches is however unlikely to be suitable for future clinical applications. Towards developing a safer reprogramming protocol, we investigated whether cell types that express one of the most critical reprogramming genes endogenously are predisposed to reprogramming. We show here that three of the original four pluripotency transcription factors (Oct4, Klf4 and c-Myc or MYCER(TAM)) induced reprogramming of mouse neural stem (NS) cells exploiting endogenous SoxB1 protein levels in these cells. The reprogrammed neural stem cells differentiated into cells of each germ layer in vitro and in vivo, and contributed to mouse development in vivo. Thus a combinatorial approach taking advantage of endogenously expressed genes and inducible transgenes may contribute to the development of improved reprogramming protocols.
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http://dx.doi.org/10.1016/j.yexcr.2008.06.024DOI Listing
October 2008

Genetic manipulation of adult mouse neurogenic niches by in vivo electroporation.

Nat Methods 2008 Feb 20;5(2):189-96. Epub 2008 Jan 20.

Department of Cell and Molecular Biology, Medical Nobel Institute, SE-171 77 Stockholm, Sweden.

Targeted ectopic expression of genes in the adult brain is an invaluable approach for studying many biological processes. This can be accomplished by generating transgenic mice or by virally mediated gene transfer, but these methods are costly and labor intensive. We devised a rapid strategy that allows localized in vivo transfection of plasmid DNA within the adult neurogenic niches without detectable brain damage. Injection of plasmid DNA into the ventricular system or directly into the hippocampus of adult mice, followed by application of electrical current via external electrodes, resulted in transfection of neural stem or progenitor cells and mature neurons. We showed that this strategy can be used for both fate mapping and gain- or loss-of-function experiments. Using this approach, we identified an essential role for cadherins in maintaining the integrity of the lateral ventricle wall. Thus, in vivo electroporation provides a new approach to study the adult brain.
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http://dx.doi.org/10.1038/nmeth.1174DOI Listing
February 2008

Deconstructing stemness.

EMBO J 2005 Aug 21;24(15):2715-9. Epub 2005 Jul 21.

Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden.

Stem cells are unique in their capacity to self-renew and generate differentiated progeny to maintain tissues throughout life. A common molecular program for stem cells has remained elusive. We discuss what the molecular logic of stemness may be. We suggest that it may not be coupled to distinct cellular properties such as self-renewal or multipotency, but rather to the stable suspension at a specific developmental stage. In this view, the stem cell niche allows a cell to maintain a transcriptional accessibility enabling the generation of specific differentiated progeny.
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http://dx.doi.org/10.1038/sj.emboj.7600749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1182244PMC
August 2005

Mice deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth factors.

Mol Cell Biol 2004 Jul;24(13):6104-15

Division of Molecular Genetics and Centre of Biomedical Genetics, Netherlands Cancer Institute, Amsterdam, The Netherlands.

The Pim family of proto-oncogenes encodes a distinct class of serine/threonine kinases consisting of PIM1, PIM2, and PIM3. Although the Pim genes are evolutionarily highly conserved, the contribution of PIM proteins to mammalian development is unclear. PIM1-deficient mice were previously described but showed only minor phenotypic aberrations. To assess the role of PIM proteins in mammalian physiology, compound Pim knockout mice were generated. Mice lacking expression of Pim1, Pim2, and Pim3 are viable and fertile. However, PIM-deficient mice show a profound reduction in body size at birth and throughout postnatal life. In addition, the in vitro response of distinct hematopoietic cell populations to growth factors is severely impaired. In particular, PIM proteins are required for the efficient proliferation of peripheral T lymphocytes mediated by synergistic T-cell receptor and interleukin-2 signaling. These results indicate that members of the PIM family of proteins are important but dispensable factors for growth factor signaling.
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http://dx.doi.org/10.1128/MCB.24.13.6104-6115.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC480904PMC
July 2004

Retroviral insertional mutagenesis: tagging cancer pathways.

Adv Cancer Res 2003 ;88:53-99

Division of Molecular Genetics and Centre of Biomedical Genetics, Netherlands Cancer Institute 1066 CX, Amsterdam, The Netherlands.

Slow transforming retroviruses, such as the Moloney murine leukemia virus (M-MuLV), induce tumors upon infection of a host after a relatively long latency period. The underlying mechanism leading to cell transformation is the activation of proto-oncogenes or inactivation of tumor suppressor genes as a consequence of proviral insertions into the host genome. Cells carrying proviral insertions that confer a selective advantage will preferentially grow out. This means that proviral insertions mark genes contributing to tumorigenesis, as was demonstrated by the identification of numerous proto-oncogenes in retrovirally induced tumors in the past. Since cancer is a complex multistep process, the proviral insertions in one clone of tumor cells also represent oncogenic events that cooperate in tumorigenesis. Novel advances, such as the launch of the complete mouse genome, high-throughput isolation of proviral flanking sequences, and genetically modified animals have revolutionized proviral tagging into an elegant and efficient approach to identify signaling pathways that collaborate in cancer.
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http://dx.doi.org/10.1016/s0065-230x(03)88304-5DOI Listing
November 2003

Proviral activation of the tumor suppressor E2a contributes to T cell lymphomagenesis in EmuMyc transgenic mice.

Oncogene 2002 Sep;21(43):6559-66

Division of Molecular Genetics and Centre of Biomedical Genetics, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

The basic helix-loop-helix factor E2A plays an important role in the development of B and T lymphocytes. In addition, E2a has been implicated as a gene with tumor suppressor activity, since mice deficient for E2a succumb to T cell lymphomas. We have performed retroviral tagging in EmuMyc transgenic mice to identify genes that contribute to lymphomagenesis. The EmuMyc transgenic mouse is a well-established model of a common translocation in human B cell lymphomas. Analyses of the proviral insertion sites in the MuLV-induced lymphomas revealed that a number of T cell lymphomas carried proviral insertions in the promoter region of E2a. These proviral insertions yield hybrid viral-E2a mRNAs resulting in a marked rise in E2A protein levels. The proviral insertions in E2a were predominantly of clonal origin indicating that E2a insertions are early events in these T cell lymphomas. The primary oncogenic effect of E2A is likely to be associated with enhancement of transcription of the c-Myc transgene via binding to the regulatory immunoglobulin enhancers. The results herein thus provide the first evidence that in a specific setting E2A overexpression can contribute to T-lymphomagenesis. This implies that E2a contains oncogenic features in addition to the previously described tumor suppressive properties.
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http://dx.doi.org/10.1038/sj.onc.1205930DOI Listing
September 2002