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Blixt, M., Hellsand, M., Konjusha, D., Zhang, H., Stenfelt, S., Åkesson, M., . . . Hallböök, F. (2022). MYCN induces cell-specific tumorigenic growth in RB1-proficient human retinal organoid and chicken retina models of retinoblastoma. Oncogenesis, 11(1)
Open this publication in new window or tab >>MYCN induces cell-specific tumorigenic growth in RB1-proficient human retinal organoid and chicken retina models of retinoblastoma
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2022 (English)In: Oncogenesis, E-ISSN 2157-9024, Vol. 11, no 1Article in journal (Refereed) Published
Abstract [en]

Retinoblastoma is a rare, intraocular paediatric cancer that originates in the neural retina and is most frequently caused by bi-allelic loss of RB1 gene function. Other oncogenic mutations, such as amplification and increased expression of the MYCN gene, have been found even with proficient RB1 function. In this study, we investigated whether MYCN over-expression can drive carcinogenesis independently of RB1 loss-of-function mutations. The aim was to elucidate the events that result in carcinogenesis and identify the cancer cell-of-origin. We studied the chicken retina, a well-established model for studying retinal neurogenesis, and generated over-expression of MYCN by in ovo electroporation. In parallel, we established an equivalent human stem cell-derived retinal organoid (retinoid) model system. We found that over-expression of MYCN induced tumorigenic growth with high frequency in RB1-proficient chicken retinas and human retinoids. In both systems, the tumorigenic cells expressed markers for undifferentiated cone photoreceptor/horizontal cell progenitors. The over-expression resulted in metastatic retinoblastoma within 7-9 weeks in chicken. MYCN cells could be grown in vitro and, when orthotopically injected, formed tumours that infiltrated the sclera and optic nerve and expressed markers for undifferentiated cones. Investigation of the tumour cell phenotype determined that the potential for neoplastic growth was embryonic stage-dependent and featured a cell-specific resistance to apoptosis in the cone/horizontal cell lineage, but not in ganglion or amacrine cells. We conclude that MYCN over-expression is sufficient to drive tumorigenesis and that a cell-specific resistance to apoptosis in the cone/horizontal cell lineage mediates the cancer phenotype.

Place, publisher, year, edition, pages
Springer NatureSpringer Nature, 2022
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-315611 (URN)10.1038/s41389-022-00409-3 (DOI)000814269100001 ()35729105 (PubMedID)
Available from: 2017-02-16 Created: 2017-02-16 Last updated: 2024-01-15Bibliographically approved
Ghaderi Berntsson, S., Kristoffersson, A., Daniilidou, M., Dahl, N., Ekström, C., Semnic, R., . . . Landtblom, A.-M. (2020). Aniridia with PAX6 mutations and narcolepsy. Journal of Sleep Research, 29(6), Article ID e12982.
Open this publication in new window or tab >>Aniridia with PAX6 mutations and narcolepsy
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2020 (English)In: Journal of Sleep Research, ISSN 0962-1105, E-ISSN 1365-2869, Vol. 29, no 6, article id e12982Article in journal (Refereed) Published
Abstract [en]

PAX6 gene mutations cause a variety of eye and central nervous system (CNS) abnormalities. Aniridia is often accompanied by CNS abnormalities such as pineal gland atrophy or hypoplasia, leading to disturbed circadian rhythm and sleep disorders. Less is known on the coincidence of narcolepsy in this patient group. We aimed to find out whether the circadian rhythm or sleep-wake structure was affected in patients with aniridia. Four members of a family segregating with congenital aniridia in two generations were included in the study. The patients were subjected to genetic testing for a PAX6 mutation, multiple sleep latency test, whole-brain magnetic resonance imaging (MRI), hypocretin-1 in cerebrospinal fluid, and Human Leukocyte Antigen DQ beta1*06:02. All four members were heterozygous for the pathogenic c.959-1G>A mutation in the PAX6 gene. Sleep disturbance was observed in all family members. The index patient was diagnosed with narcolepsy. MRI showed a hypoplastic pineal gland in all members. We describe the first case of a patient with PAX6 haploinsufficiency, aniridia and pineal gland hypoplasia diagnosed with narcolepsy type-1, suggesting a complex sleep disorder pathogenesis.

Keywords
absence of iris, narcolepsy, PAX6 haploinsufficiency, pineal gland atrophy
National Category
Neurology Ophthalmology
Research subject
Neurology; Ophtalmology
Identifiers
urn:nbn:se:uu:diva-402602 (URN)10.1111/jsr.12982 (DOI)000507560500001 ()31943460 (PubMedID)
Funder
Swedish Research Council, 2015-4870Swedish Research Council, 2015-02424The Swedish Brain Foundation, FO2019-0210
Available from: 2020-01-17 Created: 2020-01-17 Last updated: 2023-11-21Bibliographically approved
Sato, D. X., Rafati, N., Ring, H., Younis, S., Feng, C., Blanco-Aguiar, J. A., . . . Andersson, L. (2020). Brain Transcriptomics of Wild and Domestic Rabbits Suggests That Changes in Dopamine Signaling and Ciliary Function Contributed to Evolution of Tameness. Genome Biology and Evolution, 12(10), 1918-1928
Open this publication in new window or tab >>Brain Transcriptomics of Wild and Domestic Rabbits Suggests That Changes in Dopamine Signaling and Ciliary Function Contributed to Evolution of Tameness
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2020 (English)In: Genome Biology and Evolution, ISSN 1759-6653, E-ISSN 1759-6653, Vol. 12, no 10, p. 1918-1928Article in journal (Refereed) Published
Abstract [en]

Domestication has resulted in immense phenotypic changes in animals despite their relatively short evolutionary history. The European rabbit is one of the most recently domesticated animals, but exhibits distinct morphological, physiological, and behavioral differences from their wild conspecifics. A previous study revealed that sequence variants with striking allele frequency differences between wild and domestic rabbits were enriched in conserved noncoding regions, in the vicinity of genes involved in nervous system development. This suggests that a large proportion of the genetic changes targeted by selection during domestication might affect gene regulation. Here, we generated RNA-sequencing data for four brain regions (amygdala, hypothalamus, hippocampus, and parietal/temporal cortex) sampled at birth and revealed hundreds of differentially expressed genes (DEGs) between wild and domestic rabbits. DEGs in amygdala were significantly enriched for genes associated with dopaminergic function and all 12 DEGs in this category showed higher expression in domestic rabbits. DEGs in hippocampus were enriched for genes associated with ciliary function, all 21 genes in this category showed lower expression in domestic rabbits. These results indicate an important role of dopamine signaling and ciliary function in the evolution of tameness during rabbit domestication. Our study shows that gene expression in specific pathways has been profoundly altered during domestication, but that the majority of genes showing differential expression in this study have not been the direct targets of selection.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2020
Keywords
transcriptome, domestication, the European rabbit, evolution of tameness, dopamine, amygdala
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-430581 (URN)10.1093/gbe/evaa158 (DOI)000593024100020 ()32835359 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2021-01-14 Created: 2021-01-14 Last updated: 2021-01-14Bibliographically approved
Mäkeläinen, S., Hellsand, M., van Der Heiden, A. D., Andersson, E., Thorsson, E., Hoist, B. S., . . . Bergström, T. F. (2020). Deletion in the Bardet-Biedl Syndrome Gene TTC8 Results in a Syndromic Retinal Degeneration in Dogs. Genes, 11(9), Article ID 1090.
Open this publication in new window or tab >>Deletion in the Bardet-Biedl Syndrome Gene TTC8 Results in a Syndromic Retinal Degeneration in Dogs
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2020 (English)In: Genes, ISSN 2073-4425, E-ISSN 2073-4425, Vol. 11, no 9, article id 1090Article in journal (Refereed) Published
Abstract [en]

In golden retriever dogs, a 1 bp deletion in the canineTTC8gene has been shown to cause progressive retinal atrophy (PRA), the canine equivalent of retinitis pigmentosa. In humans,TTC8is also implicated in Bardet-Biedl syndrome (BBS). To investigate if the affected dogs only exhibit a non-syndromic PRA or develop a syndromic ciliopathy similar to human BBS, we recruited 10 affected dogs to the study. The progression of PRA for two of the dogs was followed for 2 years, and a rigorous clinical characterization allowed a careful comparison with primary and secondary characteristics of human BBS. In addition to PRA, the dogs showed a spectrum of clinical and morphological signs similar to primary and secondary characteristics of human BBS patients, such as obesity, renal anomalies, sperm defects, and anosmia. We used Oxford Nanopore long-read cDNA sequencing to characterize retinal full-lengthTTC8transcripts in affected and non-affected dogs, the results of which suggest that three isoforms are transcribed in the retina, and the 1 bp deletion is a loss-of-function mutation, resulting in a canine form of Bardet-Biedl syndrome with heterogeneous clinical signs.

Place, publisher, year, edition, pages
MDPI, 2020
Keywords
Bardet-Biedl syndrome (BBS), primary cilia, ciliopathy, BBS8, progressive retinal atrophy (PRA), retinitis pigmentosa
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-424628 (URN)10.3390/genes11091090 (DOI)000580752000001 ()32962042 (PubMedID)
Funder
Swedish Research Council Formas, 221-2014-1005
Available from: 2020-11-09 Created: 2020-11-09 Last updated: 2022-08-24Bibliographically approved
Edwards, S. J., Carannante, V., Kuhnigk, K., Ring, H., Tararuk, T., Hallböök, F., . . . Brismar, H. (2020). High-Resolution Imaging of Tumor Spheroids and Organoids Enabled by Expansion Microscopy. Frontiers in Molecular Biosciences, 7, Article ID 208.
Open this publication in new window or tab >>High-Resolution Imaging of Tumor Spheroids and Organoids Enabled by Expansion Microscopy
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2020 (English)In: Frontiers in Molecular Biosciences, E-ISSN 2296-889X, Vol. 7, article id 208Article in journal (Refereed) Published
Abstract [en]

Three-dimensional cell cultures are able to better mimic the physiology and cellular environments found in tissuesin vivocompared to cells grown in two dimensions. In order to study the structure and function of cells in 3-D cultures, light microscopy is frequently used. The preparation of 3-D cell cultures for light microscopy is often destructive, including physical sectioning of the samples, which can result in the loss of 3-D information. In order to probe the structure of 3-D cell cultures at high resolution, we have explored the use of expansion microscopy and compared it to a simple immersion clearing protocol. We provide a practical method for the study of spheroids, organoids and tumor-infiltrating immune cells at high resolution without the loss of spatial organization. Expanded samples are highly transparent, enabling high-resolution imaging over extended volumes by significantly reducing light scatter and absorption. In addition, the hydrogel-like nature of expanded samples enables homogenous antibody labeling of dense epitopes throughout the sample volume. The improved labeling and image quality achieved in expanded samples revealed details in the center of the organoid which were previously only observable following serial sectioning. In comparison to chemically cleared spheroids, the improved signal-to-background ratio of expanded samples greatly improved subsequent methods for image segmentation and analysis.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2020
Keywords
expansion, microscopy, spheroid, organoid, lightsheet, imaging
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-423588 (URN)10.3389/fmolb.2020.00208 (DOI)000576034000001 ()
Available from: 2020-10-29 Created: 2020-10-29 Last updated: 2021-04-21Bibliographically approved
Harun-Or-Rashid, M. & Hallböök, F. (2019). Alpha 2-Adrenergic Receptor Agonist Brimonidine Stimulates ERK1/2 and AKT Signaling via Transactivation of EGF Receptors in the Human MIO-M1 Müller Cell Line. Current Eye Research, 44(1), 34-45
Open this publication in new window or tab >>Alpha 2-Adrenergic Receptor Agonist Brimonidine Stimulates ERK1/2 and AKT Signaling via Transactivation of EGF Receptors in the Human MIO-M1 Müller Cell Line
2019 (English)In: Current Eye Research, ISSN 0271-3683, E-ISSN 1460-2202, Vol. 44, no 1, p. 34-45Article in journal (Refereed) Published
Abstract [en]

Purpose: Alpha 2-adrenergic receptor (α2-ADR) agonists are used clinically for a range of indications including reducing elevated intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Animal experiments show that α2-ADR agonists attenuate the injury-induced Müller cell dedifferentiation by a mechanism that involves activation and regulation of extracellular signal-regulated kinase (ERK) 1/2 leading to transactivation of epidermal growth factor receptors (EGFRs). The purpose of this study was to study and corroborate the activation of this system in human cells.

Material and Methods: The human Müller cell line MIO-M1 was treated with the α2A-ADR agonist brimonidine in combination with inhibitors for Src-kinase, EGFR-kinase, matrix metalloproteinase (MMP) as well as small interfering RNAs (siRNAs) for the EGFR. The cells were analyzed using immunocytochemistry, quantitative PCR and western blot techniques.

Results: Our results show that human MIO-M1 cells express α2A-ADRs and that stimulation of these receptors caused a robust increase of ERK1/2 and protein kinase B (PKB/AKT) (Thr-308) phosphorylation in MIO-M1 cells. P-ERK1/2 and P-AKT (Thr-308) signaling was mediated by Src-kinase and associated with phosphorylation of tyrosine residue of epidermal growth factor receptor (P-EGFR Y1173). In addition, the agonist caused activation of MMPs. These effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-kinase inhibitor (AG1478), EGFR-siRNA and a MMP inhibitor (GM6001).

Conclusion: The results confirm that this human Müller cell line responds to ADR stimulation with phosphorylation of ERK and AKT, which suggests that it is possible to pharmacologically target ADR to modulate the early events in human Müller cell dedifferentiation in a similar fashion as been shown for chicken Müller cells.

Abbreviations: CRALBP: cellular retinaldehyde binding protein; EGFR: epidermal growth factor receptor; ERK1/2: extracellular signal-regulated kinase 1/2; GS: glutamine synthetase; GPCR: G protein-coupled receptor; IR: immunoreactivity; MAPK: mitogen-activated protein kinase; MMP: matrix metalloproteinase; P-ERK1/2: phospho-ERK1/2; qRT-PCR: quantitative reverse transcriptase PCR

Keywords
AKT pathway, Alpha 2-adrenergic receptors, Brimonidine, EGF receptor, ERK1/2, Matrix metalloproteinases, MIO-M1 human Müller cell, and Src-kinase
National Category
Neurosciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-281576 (URN)10.1080/02713683.2018.1516783 (DOI)000454952100006 ()30198788 (PubMedID)
Funder
Swedish Research Council, 2016-01641
Note

Title in thesis list of papers: Alpha2-Adrenergic Agonist Brimonidine Stimulates ERK1/2 and AKT Signaling Via Transactivation of EGF Receptors in MIO-M1 Human Müller Cells

Available from: 2016-03-24 Created: 2016-03-24 Last updated: 2019-01-25Bibliographically approved
Mäkeläinen, S., Gòdia, M., Hellsand, M., Viluma, A., Hahn, D., Makdoumi, K., . . . Bergström, T. F. (2019). An ABCA4 loss-of-function mutation causes a canine form of Stargardt disease. PLOS Genetics, 15(3), Article ID e1007873.
Open this publication in new window or tab >>An ABCA4 loss-of-function mutation causes a canine form of Stargardt disease
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2019 (English)In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 15, no 3, article id e1007873Article in journal (Refereed) Published
Abstract [en]

Autosomal recessive retinal degenerative diseases cause visual impairment and blindness in both humans and dogs. Currently, no standard treatment is available, but pioneering gene therapy-based canine models have been instrumental for clinical trials in humans. To study a novel form of retinal degeneration in Labrador retriever dogs with clinical signs indicating cone and rod degeneration, we used whole-genome sequencing of an affected sib-pair and their unaffected parents. A frameshift insertion in the ATP binding cassette subfamily A member 4 (ABCA4) gene (c.4176insC), leading to a premature stop codon in exon 28 (p.F1393Lfs*1395), was identified. In contrast to unaffected dogs, no full-length ABCA4 protein was detected in the retina of an affected dog. The ABCA4 gene encodes a membrane transporter protein localized in the outer segments of rod and cone photoreceptors. In humans, the ABCA4 gene is associated with Stargardt disease (STGD), an autosomal recessive retinal degeneration leading to central visual impairment. A hallmark of STGD is the accumulation of lipofuscin deposits in the retinal pigment epithelium (RPE). The discovery of a canine homozygous ABCA4 loss-of-function mutation may advance the development of dog as a large animal model for human STGD.

Place, publisher, year, edition, pages
Public Library of Science (PLoS), 2019
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-382473 (URN)10.1371/journal.pgen.1007873 (DOI)000462994900009 ()30889179 (PubMedID)
Funder
Swedish Research Council Formas, 221-2014-1005Swedish Kennel Club, P2012-0015Swedish Kennel Club, N2013-0020Swedish Kennel Club, P2014-0018Swedish Kennel Club, P2015-0012
Available from: 2019-05-02 Created: 2019-05-02 Last updated: 2023-06-30Bibliographically approved
Brusini, I., Carneiro, M., Wang, C., Rubin, C.-J., Ring, H., Afonso, S., . . . Andersson, L. (2018). Changes in brain architecture are consistent with altered fear processing in domestic rabbits. Proceedings of the National Academy of Sciences of the United States of America, 115(28), 7380-7385
Open this publication in new window or tab >>Changes in brain architecture are consistent with altered fear processing in domestic rabbits
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2018 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 28, p. 7380-7385Article in journal (Refereed) Published
Abstract [en]

The most characteristic feature of domestic animals is their change in behavior associated with selection for tameness. Here we show, using high-resolution brain magnetic resonance imaging in wild and domestic rabbits, that domestication reduced amygdala volume and enlarged medial prefrontal cortex volume, supporting that areas driving fear have lost volume while areas modulating negative affect have gained volume during domestication. In contrast to the localized gray matter alterations, white matter anisotropy was reduced in the corona radiata, corpus callosum, and the subcortical white matter. This suggests a compromised white matter structural integrity in projection and association fibers affecting both afferent and efferent neural flow, consistent with reduced neural processing. We propose that compared with their wild ancestors, domestic rabbits are less fearful and have an attenuated flight response because of these changes in brain architecture.

National Category
Psychology Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-355452 (URN)10.1073/pnas.1801024115 (DOI)000438050900076 ()29941556 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilThe Swedish Brain FoundationEuropean Social Fund (ESF)
Available from: 2018-06-29 Created: 2018-06-29 Last updated: 2018-09-25Bibliographically approved
Blixt, M. K. E., Konjusha, D., Ring, H. & Hallböök, F. (2018). Zinc finger gene nolz1 regulates the formation of retinal progenitor cells and suppresses the Lim3/Lhx3 phenotype of retinal bipolar cells in chicken retina. Developmental Dynamics, 247(4), 630-641
Open this publication in new window or tab >>Zinc finger gene nolz1 regulates the formation of retinal progenitor cells and suppresses the Lim3/Lhx3 phenotype of retinal bipolar cells in chicken retina
2018 (English)In: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 247, no 4, p. 630-641Article in journal (Refereed) Published
Abstract [en]

Background: The zinc-finger transcription factor Nolz1 regulates spinal cord neuron development by interacting with the transcription factors Isl1, Lim1, and Lim3, which are also important for photoreceptors, horizontal and bipolar cells during retinal development. We, therefore, studied Nolz1 during retinal development.

Results: Nolz1 expression was seen in two waves during development: one early (peak at embryonic day 3-4.5) in retinal progenitors and one late (embryonic day 8) in newly differentiated cells in the inner nuclear layer. Overexpression and knockdown showed that Nolz1 decreases proliferation and stimulates cell cycle withdrawal in retinal progenitors with effects on the generation of retinal ganglion cells, photoreceptors, and horizontal cells without triggering apoptosis. Overexpression of Nolz1 gave more p27 positive cells. Sustained overexpression of Nolz1 in the retina gave fewer Lim3/Lhx3 bipolar cells.

Conclusions: We conclude that Nolz1 has multiple functions during development and suggest a mechanism in which Nolz1 initially regulates the proliferation state of the retinal progenitor cells and then acts as a repressor that suppresses the Lim3/Lhx3 bipolar cell phenotype at the time of bipolar cell differentiation.

Place, publisher, year, edition, pages
WILEY, 2018
Keywords
chicken embryo, differentiation, horizontal cells, in ovo electroporation, Isl1, Lim1, morpholino, p27, piggyback, photoreceptors, repressor
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-350726 (URN)10.1002/dvdy.24607 (DOI)000427563200005 ()29139167 (PubMedID)
Funder
Swedish Research Council, MH521.2013.3346]Swedish Childhood Cancer Foundation, PR20150122]
Available from: 2018-05-17 Created: 2018-05-17 Last updated: 2022-04-06Bibliographically approved
Stenfelt, S., Blixt, M. K. E., All-Ericsson, C., Hallböök, F. & Boije, H. (2017). Heterogeneity in retinoblastoma: a tale of molecules and models. CLINICAL AND TRANSLATIONAL MEDICINE, 6, Article ID 42.
Open this publication in new window or tab >>Heterogeneity in retinoblastoma: a tale of molecules and models
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2017 (English)In: CLINICAL AND TRANSLATIONAL MEDICINE, ISSN 2001-1326, Vol. 6, article id 42Article, review/survey (Refereed) Published
Abstract [en]

Retinoblastoma, an intraocular pediatric cancer, develops in the embryonic retina following biallelic loss of RB1. However, there is a wide range of genetic and epigenetic changes that can affect RB1 resulting in different clinical outcomes. In addition, other transformations, such as MYCN amplification, generate particularly aggressive tumors, which may or may not be RB1 independent. Recognizing the cellular characteristics required for tumor development, by identifying the elusive cell-of-origin for retinoblastoma, would help us understand the development of these tumors. In this review we summarize the heterogeneity reported in retinoblastoma on a molecular, cellular and tissue level. We also discuss the challenging heterogeneity in current retinoblastoma models and suggest future platforms that could contribute to improved understanding of tumor initiation, progression and metastasis in retinoblastoma, which may ultimately lead to more patient-specific treatments.

Place, publisher, year, edition, pages
BIOMED CENTRAL LTD, 2017
Keywords
Cancer, Cell-of-origin, Genetics, Horizontal cells, MYCN, Photoreceptors, OTX2, RB1
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-346610 (URN)10.1186/s40169-017-0173-2 (DOI)000414747600001 ()
Available from: 2018-03-22 Created: 2018-03-22 Last updated: 2019-01-22Bibliographically approved
Projects
Generation of neurons from retinal progenitors: To divide or not to divide? [2010-02542_VR]; Uppsala UniversityExploring and regulating the neurogenic potential of retinal progenitor cells [2013-03346_VR]; Uppsala UniversityRetinal stem and progenitor cells in regeneration, normal and neoplastic growth [2016-01641_VR]; Uppsala University
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-7552-187x

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