Lars Bolund

Aarhus University, Aarhus, Central Jutland, Denmark

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Publications (329)1702.49 Total impact

  • Lin Lin · Lars Bolund · Yonglun Luo
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    ABSTRACT: Mesenchymal stem cells (MSCs) are adult stem cells with the capacity of self-renewal and multilineage differentiation, and can be isolated from several adult tissues. However, isolating MSCs from adult tissues for cell therapy is hampered by the invasive procedure, the rarity of the cells and their attenuated proliferation capacity when cultivated and expanded in vitro. Human MSCs derived from induced pluripotent stem cells (iPSC-MSCs) have now evolved as a promising alternative cell source for MSCs and regenerative medicine. Several groups, including ours, have reported successful derivation of functional iPSC-MSCs and applied these cells in MSC-based therapeutic testing. Still, the current experience and understanding of iPSC-MSCs with respect to production methods, safety and efficacy are primitive. In this review, we highlight the methodological progress in iPSC-MSC research, describing the importance of choosing the right sources of iPSCs, iPSC reprogramming methods, iPSC culture systems, embryoid body intermediates, pathway inhibitors, basal medium, serum, growth factors and culture surface coating. We also highlight some progress in the application of iPSC-MSCs in direct cell therapy, tissue engineering and gene therapy.
    Current Stem Cell Research & Therapy 07/2015; · 2.86 Impact Factor
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    ABSTRACT: DNA methylation has important roles in the regulation of gene expression and cellular specification. Reduced representation bisulfite sequencing (RRBS) has prevailed in methylation studies due to its cost-effectiveness and single-base resolution. The rapid accumulation of RRBS data demands well designed analytical tools. To streamline the data processing of DNA methylation from multiple RRBS samples, we present a flexible pipeline named SMAP, whose features include: (i) handling of single-and/or paired-end diverse bisulfite sequencing data with reduced false-positive rates in differentially methylated regions; (ii) detection of allele-specific methylation events with improved algorithms; (iii) a built-in pipeline for detection of novel single nucleotide polymorphisms (SNPs); (iv) support of multiple user-defined restriction enzymes; (v) conduction of all methylation analyses in a single-step operation when well configured. Simulation and experimental data validated the high accuracy of SMAP for SNP detection and methylation identification. Most analyses required in methylation studies (such as estimation of methylation levels, differentially methylated cytosine groups, and allele-specific methylation regions) can be executed readily with SMAP. All raw data from diverse samples could be processed in parallel and 'packetized' streams. A simple user guide to the methylation applications is also provided.
    07/2015; 4(1):29. DOI:10.1186/s13742-015-0070-9
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    Dataset: nbt.3200-S1
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    ABSTRACT: The human genome is diploid, and knowledge of the variants on each chromosome is important for the interpretation of genomic information. Here we report the assembly of a haplotype-resolved diploid genome without using a reference genome. Our pipeline relies on fosmid pooling together with whole-genome shotgun strategies, based solely on next-generation sequencing and hierarchical assembly methods. We applied our sequencing method to the genome of an Asian individual and generated a 5.15-Gb assembled genome with a haplotype N50 of 484 kb. Our analysis identified previously undetected indels and 7.49 Mb of novel coding sequences that could not be aligned to the human reference genome, which include at least six predicted genes. This haplotype-resolved genome represents the most complete de novo human genome assembly to date. Application of our approach to identify individual haplotype differences should aid in translating genotypes to phenotypes for the development of personalized medicine.
    Nature Biotechnology 05/2015; 33(6). DOI:10.1038/nbt.3200 · 39.08 Impact Factor
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    ABSTRACT: Irinotecan (SN38) and oxaliplatin are chemotherapeutic agents used in the treatment of colorectal cancer. However, the frequent development of resistance to these drugs represents a considerable challenge in the clinic. Alus as retrotransposons comprise 11% of the human genome. Genomic toxicity induced by carcinogens or drugs can reactivate Alus by altering DNA methylation. Whether or not reactivation of Alus occurs in SN38 and oxaliplatin resistance remains unknown. We applied reduced representation bisulfite sequencing (RRBS) to investigate the DNA methylome in SN38 or oxaliplatin resistant colorectal cancer cell line models. Moreover, we extended the RRBS analysis to tumor tissue from 14 patients with colorectal cancer who either did or did not benefit from capecitabine + oxaliplatin treatment. For the clinical samples, we applied a concept of 'DNA methylation entropy' to estimate the diversity of DNA methylation states of the identified resistance phenotype-associated methylation loci observed in the cell line models. We identified different loci being characteristic for the different resistant cell lines. Interestingly, 53% of the identified loci were Alu sequences- especially the Alu Y subfamily. Furthermore, we identified an enrichment of Alu Y sequences that likely results from increased integration of new copies of Alu Y sequence in the drug-resistant cell lines. In the clinical samples, SOX1 and other SOX gene family members were shown to display variable DNA methylation states in their gene regions. The Alu Y sequences showed remarkable variation in DNA methylation states across the clinical samples. Our findings imply a crucial role of Alu Y in colorectal cancer drug resistance. Our study underscores the complexity of colorectal cancer aggravated by mobility of Alu elements and stresses the importance of personalized strategies, using a systematic and dynamic view, for effective cancer therapy.
    BMC Genomics 05/2015; 16(1). DOI:10.1186/s12864-015-1552-y · 4.04 Impact Factor
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    ABSTRACT: Hydatidiform moles are associated with an excess of paternal genome set(s) or abnormal imprinting of multiple imprinted genes. However, it appears unlikely that all imprinted genes are relevant for the pathogenesis of a hydatidiform mole. We studied two rare cases of triploid, diandric moles that were tetrasomic for chromosome 6. DNA-marker analysis showed that in one mole the fourth chromosome 6 originated in the father, and in the other mole the fourth chromosome 6 originated in the mother. Histopathologic revision disclosed that both moles were partial moles with no significant difference in the phenotypes. It is unlikely that a major gene involved in the pathogenesis of hydatidiform mole, or a major gene involved in determining the severity of the molar phenotype, is located on chromosome 6.
    The Journal of reproductive medicine 05/2015; 60(5-6):261-4. · 0.58 Impact Factor
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    ABSTRACT: It has previously been suggested that embryos developing from intracytoplasmic sperm-injected (ICSI) zygotes with three pronuclei (3PN) are endowed with a mechanism for self-correction of triploidy to diploidy. 3PN are also observed in zygotes after conventional in vitro fertilization (IVF). The parental origin, however, differs between the two fertilization methods. Whereas the vast majority of 3PN IVF zygotes are of dispermic origin and thus more likely to have two centrioles, the 3PN ICSI zygotes are digynic in origin and therefore, more likely to have one centriole. In the present study, we examine whether the parental origin of 3PN embryos correlates with the karyotype. The karyotype of each nucleus was estimated using four sequential fluorescence in situ hybridizations-each with two probes-resulting in quantitative information of 8 different chromosomes. The karyotypes were then compared and correlated to the parental origin. 3PN ICSI embryos displayed a significantly larger and more coordinated reduction from the assumed initial 3 sets of chromosomes than 3PN IVF embryos. The differences in the parental origin-and hence the number of centrioles-between the 3PN IVF and the 3PN ICSI zygotes are likely to be the cause of the differences in karyotypes.
    03/2015; 42(1):14-21. DOI:10.5653/cerm.2015.42.1.14
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    ABSTRACT: Building a population-specific catalogue of single nucleotide variants (SNVs), indels and structural variants (SVs) with frequencies, termed a national pan-genome, is critical for further advancing clinical and public health genetics in large cohorts. Here we report a Danish pan-genome obtained from sequencing 10 trios to high depth (50 × ). We report 536k novel SNVs and 283k novel short indels from mapping approaches and develop a population-wide de novo assembly approach to identify 132k novel indels larger than 10 nucleotides with low false discovery rates. We identify a higher proportion of indels and SVs than previous efforts showing the merits of high coverage and de novo assembly approaches. In addition, we use trio information to identify de novo mutations and use a probabilistic method to provide direct estimates of 1.27e-8 and 1.5e-9 per nucleotide per generation for SNVs and indels, respectively.
    Nature Communications 01/2015; 6:5969. DOI:10.1038/ncomms6969 · 10.74 Impact Factor
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    ABSTRACT: Previously, we established a simple method for deriving mesenchymal stem cells (MSCs) from human induced pluripotent stem cells (iPSC-MSCs). These iPSC-MSCs were capable of forming osteogenic structures in scaffolds and nanofibers. The objective of this study is to systematically characterize the mesenchymal characteristics of the iPSC-MSCs by comparing them to bone marrow-derived MSCs (BM-MSCs). Two iPSC-MSC lines (named as mRNA-iPSC-MSC-YL001 and lenti-iPSC-MSC-A001) and one BM-MSC line were used for the study. Cell proliferation, presence of mesenchymal surface markers, tri-lineage differentiation capability (osteogenesis, chondrogenesis, adipogenesis), and expression of "stemness" genes were analyzed in these MSC lines. The iPSC-MSCs were similar to BM-MSCs in terms of cell morphology (fibroblast-like) and surface antigen profile: CD29+, CD44+, CD73+, CD90+, CD105+, CD11b-, CD14-, CD31-, CD34-, CD45- and HLA-DR-. A faster proliferative capability was seen in both iPSC-MSCs lines compared to the BM-MSCs. The iPSC-MSCs showed adequate capacity of osteogenesis and chondrogenesis compared to the BM-MSCs, while less adipogenic potential was found in the iPSC-MSCs. The iPSC-MSCs and the tri-lineage differentiated cells (osteoblasts, chondrocytes, adipocytes) all lack expression of "stemness" genes: OCT4, SOX2, GDF3, CRIPTO, UTF1, DPPA4, DNMT3B, LIN28a, and SAL4. The MSCs derived from human iPSCs with our method have advanced proliferation capability and adequate osteogenic and chondrogenic properties compared to BM-MSCs. However, the iPSC-MSCs were less efficient in their adipogenicity, suggesting that further modifications should be applied to our method to derive iPSC-MSCs more closely resembling the naïve BM-MSCs if necessary.
    Stem Cell Research & Therapy 01/2015; 6(1-1):144. DOI:10.1186/s13287-015-0137-7 · 4.63 Impact Factor
  • Somatic Genome Manipulation Advances, Methods, and Applications, 1 edited by Xiu-Qing Li • Danielle J. Donnelly Thomas G. Jensen, 01/2015: chapter 2; Spriner.
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    ABSTRACT: Next generation sequencing (NGS) is now being used for detecting chromosomal abnormalities in blastocyst trophectoderm (TE) cells from in vitro fertilized embryos. However, few data are available regarding the clinical outcome, which provides vital reference for further application of the methodology. Here, we present a clinical evaluation of NGS-based preimplantation genetic diagnosis/screening (PGD/PGS) compared with single nucleotide polymorphism (SNP) array-based PGD/PGS as a control. A total of 395 couples participated. They were carriers of either translocation or inversion mutations, or were patients with recurrent miscarriage and/or advanced maternal age. A total of 1,512 blastocysts were biopsied on D5 after fertilization, with 1,058 blastocysts set aside for SNP array testing and 454 blastocysts for NGS testing. In the NGS cycles group, the implantation, clinical pregnancy and miscarriage rates were 52.6% (60/114), 61.3% (49/80) and 14.3% (7/49), respectively. In the SNP array cycles group, the implantation, clinical pregnancy and miscarriage rates were 47.6% (139/292), 56.7% (115/203) and 14.8% (17/115), respectively. The outcome measures of both the NGS and SNP array cycles were the same with insignificant differences. There were 150 blastocysts that underwent both NGS and SNP array analysis, of which seven blastocysts were found with inconsistent signals. All other signals obtained from NGS analysis were confirmed to be accurate by validation with qPCR. The relative copy number of mitochondrial DNA (mtDNA) for each blastocyst that underwent NGS testing was evaluated, and a significant difference was found between the copy number of mtDNA for the euploid and the chromosomally abnormal blastocysts. So far, out of 42 ongoing pregnancies, 24 babies were born in NGS cycles; all of these babies are healthy and free of any developmental problems. This study provides the first evaluation of the clinical outcomes of NGS-based pre-implantation genetic diagnosis/screening, and shows the reliability of this method in a clinical and array-based laboratory setting. NGS provides an accurate approach to detect embryonic imbalanced segmental rearrangements, to avoid the potential risks of false signals from SNP array in this study.
    12/2014; 3(1):30. DOI:10.1186/2047-217X-3-30
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    ABSTRACT: Logistic regression analysis based on data from 822 Han Chinese oldest old aged 92+ demonstrated that interactions between carrying FOXO1A-266 or FOXO3-310 or FOXO3-292 and tea drinking at around age 60 or at present time were significantly associated with lower risk of cognitive disability at advanced ages. Associations between tea drinking and reduced cognitive disability were much stronger among carriers of the genotypes of FOXO1A-266 or FOXO3-310 or FOXO3-292 compared with noncarriers, and it was reconfirmed by analysis of three-way interactions across FOXO genotypes, tea drinking at around age 60, and at present time. Based on prior findings from animal and human cell models, we postulate that intake of tea compounds may activate FOXO gene expression, which in turn may positively affect cognitive function in the oldest old population. Our empirical findings imply that the health benefits of particular nutritional interventions, including tea drinking, may, in part, depend upon individual genetic profiles.
    The Journals of Gerontology Series A Biological Sciences and Medical Sciences 06/2014; 70(4). DOI:10.1093/gerona/glu060 · 4.98 Impact Factor
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    ABSTRACT: The recombinant adeno-associated virus (rAAV) has proven to be an efficient and attractive tool for targeted genome engineering. Here we present a novel method employing the Golden Gate cloning strategy for fast and efficient construction of rAAV-based gene knockout or single-nucleotide knockin vectors. Two vectors, pGolden-Neo and pGolden-Hyg, were generated as common assembling modules to confer antibiotic resistance to the targeting vector. To validate the method, we then generated two rAAV-based targeting vectors: pAAV-pTP53-KO and pAAV-hTauP301L-KI. Furthermore, we generated a pGolden-AAV plasmid that allows one-step generation of an rAAV-based targeting vector. Our new methodology for rAAV targeting vector assembly is efficient, accurate, time-saving, and cost-effective.
    BioTechniques 05/2014; 56(5):263-8. DOI:10.2144/000114169 · 2.75 Impact Factor
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    ABSTRACT: To analyze the correlation between the genetic constitution and the phenotype in triploid pregnancies. 158 triploid pregnancies identified in hospitals in Western Denmark from April 1986 to April 2010. Clinical data and karyotypes were collected retrospectively and archived samples were retrieved. The parental origin of the genome, either double paternal contribution (PPM) or double maternal contribution (MMP) was determined by analyzing methylation levels at imprinted sites. There were significantly more PPM than MMP cases (p<0.01). In MMP cases the possible karyotypes had similar frequencies, whereas in PPM cases 43% had the karyotype 69,XXX, 51% the karyotype 69,XXY, and 6% the karyotype 69,XYY. Molar phenotype was only seen in PPM cases. However, PPM cases with a non-molar phenotype were also seen. For both parental genotypes, various fetal phenotypes were seen at autopsy. Levels of human chorionic gonadotropin in maternal serum (MS-hCG) were low in MMP cases and varying in PPM cases, some being as low as in the MMP cases. In a triploid pregnancy, suspicion of hydatidiform mole at US, by macroscopic inspection of the evacuated tissue, at histology, or due to a high MS-hCG level each predict the parental type PPM with a very high specificity. In contrast the sensitivity of these observations is less than 100%.
    American journal of obstetrics and gynecology 03/2014; 211(4). DOI:10.1016/j.ajog.2014.03.039 · 3.97 Impact Factor
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    ABSTRACT: Handmade cloning (HMC) has been used to generate transgenic pigs for biomedical research. Recently, we found that parthenogenetic activation (PA) of porcine oocytes and improved HMC efficiency could be achieved by treatment with sublethal high hydrostatic pressure (HHP). However, the molecular mechanism underlying the effects of HHP treatment on embryonic development is poorly understood and so was investigated in the present study. Thus, in the present study, we undertook genome-wide gene expression analysis in HHP-treated and untreated oocytes, as well as in 4-cell and blastocyst stage embryos derived by PA or HMC. Hierarchical clustering depicted stage-specific genomic expression profiling. At the 4-cell and blastocyst stages, 103 and 163 transcripts were differentially expressed between the HMC and PA embryos, respectively (P<0.05). These transcripts are predominantly involved in regulating cellular differentiation, gene expression and cell-to-cell signalling. We found that 44 transcripts were altered by HHP treatment, with most exhibiting lower expression in HHP-treated oocytes. Genes involved in embryonic development were prominent among the transcripts affected by HHP. Two of these genes (INHBB and ME3) were further validated by quantitative reverse transcription-polymerase chain reaction. We also observed that HHP treatment activated expression of the imprinting gene DLX5 in 4-cell PA embryos. In conclusion, our genomic expression profiling data suggest that HHP alters the RNA constitution in porcine oocytes and affects the expression of imprinting genes during embryonic development.
    Reproduction Fertility and Development 03/2014; 26(3):469-84. DOI:10.1071/RD13037 · 2.58 Impact Factor
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    ABSTRACT: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. Time-lapse analysis. Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p < 0.001), displayed longer duration of the 3-cell stage (p < 0.001), and arrested development from the compaction stage and onwards (p < 0.001). For the IVF embryos, the 2nd and 3rd cleavage cycles were completed within the expected time frame. However, timing of the cell divisions within the cleavage cycles differed between the two groups. In contrast, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0.001). More 3PN IVF than ICSI embryos displayed early cleavage into 3 cells (p < 0.001). This study reports differences in cleavage patterns between 2PN and 3PN embryos and for the first time demonstrates differences in the cleavage pattern between 3PN IVF and ICSI embryos.
    Journal of Assisted Reproduction and Genetics 01/2014; 31(4). DOI:10.1007/s10815-014-0178-3 · 1.77 Impact Factor
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    ABSTRACT: In this paper, we mine full mtDNA sequences from an exome capture data set of 2000 Danes, showing that it is possible to get high-quality full-genome sequences of the mitochondrion from this resource. The sample includes 1000 individuals with type 2 diabetes and 1000 controls. We characterise the variation found in the mtDNA sequence in Danes and relate the variation to diabetes risk as well as to several blood phenotypes of the controls but find no significant associations. We report 2025 polymorphisms, of which 393 have not been reported previously. These 393 mutations are both very rare and estimated to be caused by very recent mutations but individuals with type 2 diabetes do not possess more of these variants. Population genetics analysis using Bayesian skyline plot shows a recent history of rapid population growth in the Danish population in accordance with the fact that >40% of variable sites are observed as singletons.European Journal of Human Genetics advance online publication, 22 January 2014; doi:10.1038/ejhg.2013.282.
    European journal of human genetics: EJHG 01/2014; 22(8). DOI:10.1038/ejhg.2013.282 · 4.23 Impact Factor
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    ABSTRACT: Biomimetic nanofibrous scaffolds combined with stem cells are promising for bone tissue engineering. In the present study, we have employed nano-hydroxyapatite (nHAp) contained polycaprolactone (PCL) nanofibers as a biomimetic nanofibrous scaffold, and mesenchymal stem cells derived from human induced pluripotent stem cells (hiPS-MSCs) as the novel stem cells sources. The response of hiPS-MSCs on the nanofibrous scaffolds in terms of cell proliferation and differentiation into the osteoblastic phenotype was investigated by XTT assay, scanning electron microscopy (SEM), osteogenic genes expression (runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), collagen I (COL1A1), and osteocalcin (OC)), ALP activity, and calcium deposition. It is clearly shown that the hiPS-MSCs attached, and proliferated on the nanofibrous scaffolds. Compared with PCL nanofibers without nHAp, the cells on the nHAp contained nanofibers demonstrated superior capabilites to differentiate to form calcified extrac
    RSC Advances 01/2014; 4(11-11):5734-5739. DOI:10.1039/C3RA44181D · 3.84 Impact Factor
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    ABSTRACT: Development of resistance to tamoxifen is an important clinical issue in the treatment of breast cancer. Tamoxifen resistance may be the result of acquisition of epigenetic regulation within breast cancer cells, such as DNA methylation, resulting in changed mRNA expression of genes pivotal for estrogen-dependent growth. Alternatively, tamoxifen resistance may be due to selection of pre-existing resistant cells, or a combination of the two mechanisms. To evaluate the contribution of these possible tamoxifen resistance mechanisms, we applied modified DNA methylation-specific digital karyotyping (MMSDK) and digital gene expression (DGE) in combination with massive parallel sequencing to analyze a well-established tamoxifen-resistant cell line model (TAMR), consisting of 4 resistant and one parental cell line. Another tamoxifen-resistant cell line model system (LCC1/LCC2) was used to validate the DNA methylation and gene expression results. Significant differences were observed in global gene expression and DNA methylation profiles between the parental tamoxifen-sensitive cell line and the 4 tamoxifen-resistant TAMR sublines. The 4 TAMR cell lines exhibited higher methylation levels as well as an inverse relationship between gene expression and DNA methylation in the promoter regions. A panel of genes, including NRIP1, HECA and FIS1, exhibited lower gene expression in resistant vs. parental cells and concurrent increased promoter CGI methylation in resistant vs. parental cell lines. A major part of the methylation, gene expression, and pathway alterations observed in the TAMR model were also present in the LCC1/LCC2 cell line model. More importantly, high expression of SOX2 and alterations of other SOX and E2F gene family members, as well as RB-related pocket protein genes in TAMR highlighted stem cell-associated pathways as being central in the resistant cells and imply that cancer-initiating cells/cancer stem-like cells may be involved in tamoxifen resistance in this model. Our data highlight the likelihood that resistant cells emerge from cancer-initiating cells/cancer stem-like cells and imply that these cells may gain further advantage in growth via epigenetic mechanisms. Illuminating the expression and DNA methylation features of putative cancer-initiating cells/cancer stem cells may suggest novel strategies to overcome tamoxifen resistance.
    Breast cancer research: BCR 12/2013; 15(6):R119. DOI:10.1186/bcr3588 · 5.88 Impact Factor

Publication Stats

11k Citations
1,702.49 Total Impact Points

Institutions

  • 1979–2015
    • Aarhus University
      • Institute of Human Genetics
      Aarhus, Central Jutland, Denmark
  • 2003–2011
    • Beijing Genomics Institute
      Bao'an, Guangdong, China
  • 1997
    • IT University of Copenhagen
      København, Capital Region, Denmark
  • 1996
    • Swedish University of Agricultural Sciences
      • Institutionen för husdjursgenetik
      Uppsala, Uppsala, Sweden
  • 1992–1995
    • Aarhus University Hospital
      Aarhus, Central Jutland, Denmark
  • 1993
    • The John F. Kennedy Institute, Denmark
      Glostrup, Capital Region, Denmark
  • 1990
    • Duke University
      Durham, North Carolina, United States
  • 1980
    • University of Oslo
      Kristiania (historical), Oslo, Norway