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Orzechowski Westholm, JakubORCID iD iconorcid.org/0000-0002-6849-6220
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Publications (10 of 13) Show all publications
von Berlin, L., Orzechowski Westholm, J., Ratz, M. & Frisén, J. (2023). Early fate bias in neuroepithelial progenitors of hippocampal neurogenesis. Hippocampus, 33(4), 391-401
Open this publication in new window or tab >>Early fate bias in neuroepithelial progenitors of hippocampal neurogenesis
2023 (English)In: Hippocampus, ISSN 1050-9631, E-ISSN 1098-1063, Vol. 33, no 4, p. 391-401Article in journal (Refereed) Published
Abstract [en]

Hippocampal adult neural stem cells emerge from progeny of the neuroepithelial lineage during murine brain development. Hippocampus development is increasingly well understood. However, the clonal relationships between early neuroepithelial stem cells and postnatal neurogenic cells remain unclear, especially at the single-cell level. Here we report fate bias and gene expression programs in thousands of clonally related cells in the juvenile hippocampus based on single-cell RNA-seq of barcoded clones. We find evidence for early fate restriction of neuroepithelial stem cells to either neurogenic progenitor cells of the dentate gyrus region or oligodendrogenic, non-neurogenic fate supplying cells for other hippocampal regions including gray matter areas and the Cornu ammonis region 1/3. Our study provides new insights into the phenomenon of early fate restriction guiding the development of postnatal hippocampal neurogenesis.

Keywords
clonal relationships, fate choice, fate specification, neuroepithelial precursors, neuroepithelial stem cells
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:su:diva-213903 (URN)10.1002/hipo.23482 (DOI)000896659400001 ()36468233 (PubMedID)2-s2.0-85143893599 (Scopus ID)
Available from: 2023-02-06 Created: 2023-02-06 Last updated: 2023-04-24Bibliographically approved
Vromman, M., Anckaert, J., Bortoluzzi, S., Buratin, A., Chen, C.-Y., Chu, Q., . . . Volders, P.-J. (2023). Large-scale benchmarking of circRNA detection tools reveals large differences in sensitivity but not in precision. Nature Methods, 20(8), 1159-1169
Open this publication in new window or tab >>Large-scale benchmarking of circRNA detection tools reveals large differences in sensitivity but not in precision
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2023 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 20, no 8, p. 1159-1169Article in journal (Refereed) Published
Abstract [en]

The detection of circular RNA molecules (circRNAs) is typically based on short-read RNA sequencing data processed using computational tools. Numerous such tools have been developed, but a systematic comparison with orthogonal validation is missing. Here, we set up a circRNA detection tool benchmarking study, in which 16 tools detected more than 315,000 unique circRNAs in three deeply sequenced human cell types. Next, 1,516 predicted circRNAs were validated using three orthogonal methods. Generally, tool-specific precision is high and similar (median of 98.8%, 96.3% and 95.5% for qPCR, RNase R and amplicon sequencing, respectively) whereas the sensitivity and number of predicted circRNAs (ranging from 1,372 to 58,032) are the most significant differentiators. Of note, precision values are lower when evaluating low-abundance circRNAs. We also show that the tools can be used complementarily to increase detection sensitivity. Finally, we offer recommendations for future circRNA detection and validation. This study describes benchmarking and validation of computational tools for detecting circRNAs, finding most to be highly precise with variations in sensitivity and total detection. The study also finds over 315,000 putative human circRNAs.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-221121 (URN)10.1038/s41592-023-01944-6 (DOI)001029639500002 ()37443337 (PubMedID)2-s2.0-85164517473 (Scopus ID)
Available from: 2023-09-19 Created: 2023-09-19 Last updated: 2023-09-19Bibliographically approved
Brandão, L. E., Espes, D., Orzechowski Westholm, J., Martikainen, T., Westerlund, N., Lampola, L., . . . Cedernaes, J. (2022). Acute sleep loss alters circulating fibroblast growth factor 21 levels in humans: A randomised crossover trial. Journal of Sleep Research, 31(2), Article ID e13472.
Open this publication in new window or tab >>Acute sleep loss alters circulating fibroblast growth factor 21 levels in humans: A randomised crossover trial
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2022 (English)In: Journal of Sleep Research, ISSN 0962-1105, E-ISSN 1365-2869, Vol. 31, no 2, article id e13472Article in journal (Refereed) Published
Abstract [en]

The hormone fibroblast growth factor 21 (FGF21) modulates tissue metabolism and circulates at higher levels in metabolic conditions associated with chronic sleep–wake disruption, such as type 2 diabetes and obesity. In the present study, we investigated whether acute sleep loss impacts circulating levels of FGF21 and tissue-specific production, and response pathways linked to FGF21. A total of 15 healthy normal-weight young men participated in a randomised crossover study with two conditions, sleep loss versus an 8.5-hr sleep window. The evening before each intervention, fasting blood was collected. Fasting, post-intervention morning skeletal muscle and adipose tissue samples underwent quantitative polymerase chain reaction and DNA methylation analyses, and serum FGF21 levels were measured before and after an oral glucose tolerance test. Serum levels of FGF21 were higher after sleep loss compared with sleep, both under fasting conditions and following glucose intake (~27%–30%, p = 0.023). Fasting circulating levels of fibroblast activation protein, a protein which can degrade circulating FGF21, were not altered by sleep loss, whereas DNA methylation in the FGF21 promoter region increased only in adipose tissue. However, even though specifically the muscle exhibited transcriptional changes indicating adverse alterations to redox and metabolic homeostasis, no tissue-based changes were observed in expression of FGF21, its receptors, or selected signalling targets, in response to sleep loss. In summary, we found that acute sleep loss resulted in increased circulating levels of FGF21 in healthy young men, which may occur independent of a tissue-based stress response in metabolic peripheral tissues. Further studies may decipher whether changes in FGF21 signalling after sleep loss modulate metabolic outcomes associated with sleep or circadian disruption.

Keywords
adipose tissue, circadian misalignment, DNA methylation, insulin resistance, skeletal muscle, tissue-specific
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:su:diva-197306 (URN)10.1111/jsr.13472 (DOI)000692037200001 ()34476847 (PubMedID)2-s2.0-85114115540 (Scopus ID)
Available from: 2021-09-29 Created: 2021-09-29 Last updated: 2022-03-28Bibliographically approved
Ratz, M., von Berlin, L., Larsson, L., Martin, M., Orzechowski Westholm, J., La Manno, G., . . . Frisén, J. (2022). Clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics. Nature Neuroscience, 25(3), 285-294
Open this publication in new window or tab >>Clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics
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2022 (English)In: Nature Neuroscience, ISSN 1097-6256, E-ISSN 1546-1726, Vol. 25, no 3, p. 285-294Article in journal (Refereed) Published
Abstract [en]

Ratz et al. present an easy-to-use method to barcode progenitor cells, enabling profiling of cell phenotypes and clonal relations using single-cell and spatial transcriptomics, providing an integrated approach for understanding brain architecture. The mammalian brain contains many specialized cells that develop from a thin sheet of neuroepithelial progenitor cells. Single-cell transcriptomics revealed hundreds of molecularly diverse cell types in the nervous system, but the lineage relationships between mature cell types and progenitor cells are not well understood. Here we show in vivo barcoding of early progenitors to simultaneously profile cell phenotypes and clonal relations in the mouse brain using single-cell and spatial transcriptomics. By reconstructing thousands of clones, we discovered fate-restricted progenitor cells in the mouse hippocampal neuroepithelium and show that microglia are derived from few primitive myeloid precursors that massively expand to generate widely dispersed progeny. We combined spatial transcriptomics with clonal barcoding and disentangled migration patterns of clonally related cells in densely labeled tissue sections. Our approach enables high-throughput dense reconstruction of cell phenotypes and clonal relations at the single-cell and tissue level in individual animals and provides an integrated approach for understanding tissue architecture.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:su:diva-203213 (URN)10.1038/s41593-022-01011-x (DOI)000761885700001 ()35210624 (PubMedID)2-s2.0-85125392438 (Scopus ID)
Available from: 2022-03-28 Created: 2022-03-28 Last updated: 2022-03-28Bibliographically approved
Bruhn-Olszewska, B., Davies, H., Sarkisyan, D., Juhas, U., Rychlicka-Buniowska, E., Wójcik, M., . . . Dumanski, J. P. (2022). Loss of Y in leukocytes as a risk factor for critical COVID-19 in men. Genome Medicine, 14(1), Article ID 139.
Open this publication in new window or tab >>Loss of Y in leukocytes as a risk factor for critical COVID-19 in men
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2022 (English)In: Genome Medicine, E-ISSN 1756-994X, Vol. 14, no 1, article id 139Article in journal (Refereed) Published
Abstract [en]

Background: The COVID-19 pandemic, which has a prominent social and economic impact worldwide, shows a largely unexplained male bias for the severity and mortality of the disease. Loss of chromosome Y (LOY) is a risk factor candidate in COVID-19 due to its prior association with many chronic age-related diseases, and its impact on immune gene transcription.

Methods: Publicly available scRNA-seq data of PBMC samples derived from male patients critically ill with COVID-19 were reanalyzed, and LOY status was added to the annotated cells. We further studied LOY in whole blood for 211 COVID-19 patients treated at intensive care units (ICU) from the first and second waves of the pandemic. Of these, 139 patients were subject to cell sorting for LOY analysis in granulocytes, low-density neutrophils (LDNs), monocytes, and PBMCs.

Results: Reanalysis of available scRNA-seq data revealed LDNs and monocytes as the cell types most affected by LOY. Subsequently, DNA analysis indicated that 46%, 32%, and 29% of critically ill patients showed LOY above 5% cut-off in LDNs, granulocytes, and monocytes, respectively. Hence, the myeloid lineage that is crucial for the development of severe COVID-19 phenotype is affected by LOY. Moreover, LOY correlated with increasing WHO score (median difference 1.59%, 95% HDI 0.46% to 2.71%, p=0.025), death during ICU treatment (median difference 1.46%, 95% HDI 0.47% to 2.43%, p=0.0036), and history of vessel disease (median difference 2.16%, 95% HDI 0.74% to 3.7%, p=0.004), among other variables. In 16 recovered patients, sampled during ICU stay and 93–143 days later, LOY decreased significantly in whole blood and PBMCs. Furthermore, the number of LDNs at the recovery stage decreased dramatically (median difference 76.4 per 10,000 cell sorting events, 95% HDI 55.5 to 104, p=6e−11).

Conclusions: We present a link between LOY and an acute, life-threatening infectious disease. Furthermore, this study highlights LOY as the most prominent clonal mutation affecting the myeloid cell lineage during emergency myelopoiesis. The correlation between LOY level and COVID-19 severity might suggest that this mutation affects the functions of monocytes and neutrophils, which could have consequences for male innate immunity.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-213908 (URN)10.1186/s13073-022-01144-5 (DOI)000898816800001 ()36514076 (PubMedID)2-s2.0-85143837302 (Scopus ID)
Available from: 2023-02-06 Created: 2023-02-06 Last updated: 2024-07-04Bibliographically approved
Zaghlool, A., Niazi, A., Björklund, Å. K., Orzechowski Westholm, J., Ameur, A. & Feuk, L. (2021). Characterization of the nuclear and cytosolic transcriptomes in human brain tissue reveals new insights into the subcellular distribution of RNA transcripts. Scientific Reports, 11(1), Article ID 4076.
Open this publication in new window or tab >>Characterization of the nuclear and cytosolic transcriptomes in human brain tissue reveals new insights into the subcellular distribution of RNA transcripts
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2021 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 11, no 1, article id 4076Article in journal (Refereed) Published
Abstract [en]

Transcriptome analysis has mainly relied on analyzing RNA sequencing data from whole cells, overlooking the impact of subcellular RNA localization and its influence on our understanding of gene function, and interpretation of gene expression signatures in cells. Here, we separated cytosolic and nuclear RNA from human fetal and adult brain samples and performed a comprehensive analysis of cytosolic and nuclear transcriptomes. There are significant differences in RNA expression for protein-coding and lncRNA genes between cytosol and nucleus. We show that transcripts encoding the nuclear-encoded mitochondrial proteins are significantly enriched in the cytosol compared to the rest of protein-coding genes. Differential expression analysis between fetal and adult frontal cortex show that results obtained from the cytosolic RNA differ from results using nuclear RNA both at the level of transcript types and the number of differentially expressed genes. Our data provide a resource for the subcellular localization of thousands of RNA transcripts in the human brain and highlight differences in using the cytosolic or the nuclear transcriptomes for expression analysis.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-192470 (URN)10.1038/s41598-021-83541-1 (DOI)000621412400031 ()33603054 (PubMedID)
Available from: 2021-04-21 Created: 2021-04-21 Last updated: 2022-09-15Bibliographically approved
Sork, H., Conceicao, M., Corso, G., Nordin, J., Lee, Y. X., Krjutskov, K., . . . Mager, I. (2021). Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion. Cells, 10(6), Article ID 1543.
Open this publication in new window or tab >>Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion
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2021 (English)In: Cells, E-ISSN 2073-4409, Vol. 10, no 6, article id 1543Article in journal (Refereed) Published
Abstract [en]

The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R-2 = 0.69-0.87), quantitatively an outstanding 96.2-99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 degrees C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.

Keywords
extracellular vesicles, small RNA, SEC, extracellular RNA, miRNA
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-196280 (URN)10.3390/cells10061543 (DOI)000665466500001 ()34207405 (PubMedID)
Available from: 2021-09-06 Created: 2021-09-06 Last updated: 2022-02-25Bibliographically approved
Cedernaes, J., Schonke, M., Orzechowski Westholm, J., Mi, J., Chibalin, A., Voisin, S., . . . Benedict, C. (2018). Acute sleep loss results in tissue-specific alterations in genome-wide DNA methylation state and metabolic fuel utilization in humans. Science Advances, 4(8), Article ID eaar8590.
Open this publication in new window or tab >>Acute sleep loss results in tissue-specific alterations in genome-wide DNA methylation state and metabolic fuel utilization in humans
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2018 (English)In: Science Advances, E-ISSN 2375-2548, Vol. 4, no 8, article id eaar8590Article in journal (Refereed) Published
Abstract [en]

Curtailed sleep promotes weight gain and loss of lean mass in humans, although the underlying molecular mechanisms are poorly understood. We investigated the genomic and physiological impact of acute sleep loss in peripheral tissues by obtaining adipose tissue and skeletal muscle after one night of sleep loss and after one full night of sleep. We find that acute sleep loss alters genome-wide DNA methylation in adipose tissue, and unbiased transcriptome-, protein-, and metabolite-level analyses also reveal highly tissue-specific changes that are partially reflected by altered metabolite levels in blood. We observe transcriptomic signatures of inflammation in both tissues following acute sleep loss, but changes involving the circadian clock are evident only in skeletal muscle, and we uncover molecular signatures suggestive of muscle breakdown that contrast with an anabolic adipose tissue signature. Our findings provide insight into how disruption of sleep and circadian rhythms may promote weight gain and sarcopenia.

National Category
Biological Sciences Cell and Molecular Biology
Identifiers
urn:nbn:se:su:diva-160135 (URN)10.1126/sciadv.aar8590 (DOI)000443498100025 ()30140739 (PubMedID)
Available from: 2018-09-17 Created: 2018-09-17 Last updated: 2022-03-23Bibliographically approved
Coenen-Stass, A. M. L., Sork, H., Gatto, S., Godfrey, C., Bhomra, A., Krjutskov, K., . . . Roberts, T. C. (2018). Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD. Molecular Therapy Nucleic Acids, 13, 1-15
Open this publication in new window or tab >>Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD
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2018 (English)In: Molecular Therapy Nucleic Acids, E-ISSN 2162-2531, Vol. 13, p. 1-15Article in journal (Refereed) Published
Abstract [en]

Extracellular small RNAs (sRNAs), including microRNAs (miRNAs), are promising biomarkers for diseases such as Duchenne muscular dystrophy (DMD), although their biological relevance is largely unknown. To investigate the relationship between intracellular and extracellular sRNA levels on a global scale, we performed sRNA sequencing in four muscle types and serum from wild-type, dystrophic mdx, and mdx mice in which dystrophin protein expression was restored by exon skipping. Differentially abundant sRNAs were identified in serum (mapping to miRNA, small nuclear RNA [snRNA], and PIWI-interacting RNA [piRNA] loci). One novel candidate biomarker, miR-483, was increased in both mdx serum and muscle, and also elevated in DMD patient sera. Dystrophin restoration induced global shifts in miRNA (including miR-483) and snRNA-fragment abundance toward wild-type levels. Specific serum piRNA-like sRNAs also responded to exon skipping therapy. Absolute miRNA expression in muscle was positively correlated with abundance in the circulation, although multiple highly expressed miRNAs in muscle were not elevated in mdx serum, suggesting that both passive and selective release mechanisms contribute to serum miRNA levels. In conclusion, this study has revealed new insights into the sRNA biology of dystrophin deficiency and identified novel DMD biomarkers.

National Category
Biological Sciences Cell and Molecular Biology
Identifiers
urn:nbn:se:su:diva-163702 (URN)10.1016/j.omtn.2018.08.005 (DOI)000452325300001 ()30219269 (PubMedID)
Available from: 2019-01-18 Created: 2019-01-18 Last updated: 2023-11-03Bibliographically approved
Zaghlool, A., Ameur, A., Wu, C., Orzechowski Westholm, J., Niazi, A., Manivannan, M., . . . Feuk, L. (2018). Expression profiling and in situ screening of circular RNAs in human tissues. Scientific Reports, 8, Article ID 16953.
Open this publication in new window or tab >>Expression profiling and in situ screening of circular RNAs in human tissues
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2018 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 8, article id 16953Article in journal (Refereed) Published
Abstract [en]

Circular RNAs (circRNAs) were recently discovered as a class of widely expressed noncoding RNA and have been implicated in regulation of gene expression. However, the function of the majority of circRNAs remains unknown. Studies of circRNAs have been hampered by a lack of essential approaches for detection, quantification and visualization. We therefore developed a target-enrichment sequencing method suitable for screening of circRNAs and their linear counterparts in large number of samples. We also applied padlock probes and in situ sequencing to visualize and determine circRNA localization in human brain tissue at subcellular levels. We measured circRNA abundance across different human samples and tissues. Our results highlight the potential of this RNA class to act as a specific diagnostic marker in blood and serum, by detection of circRNAs from genes exclusively expressed in the brain. The powerful and scalable tools we present will enable studies of circRNA function and facilitate screening of circRNA as diagnostic biomarkers.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-162782 (URN)10.1038/s41598-018-35001-6 (DOI)000450280500013 ()30446675 (PubMedID)
Available from: 2018-12-28 Created: 2018-12-28 Last updated: 2022-09-15Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0002-6849-6220

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