Ronghui Li

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Targeted capture sequencing identifies genetic variations of GRK4 and RDH8 in Han Chinese with essential hypertension in Xinjiang

PLOS ONE, 2021

Essential hypertension is a common cardiovascular disease with complex etiology, closely related ... more Essential hypertension is a common cardiovascular disease with complex etiology, closely related to genetic and environmental factors. The pathogenesis of hypertension involves alteration in vascular resistance caused by sympathetic nervous system (SNS) and renin angiotensin system (RAS). Susceptibility factors of hypertension vary with regions and ethnicities. In this study, we conducted target capture sequencing on 54 genes related to SNS and RAS derived from a collection of Han nationality, consisting of 151 hypertension patients and 65 normal subjects in Xinjiang, China. Six non-synonymous mutations related to hypertension were identified, including GRK4 rs1644731 and RDH8 rs1801058, Mutations are predicted to affect 3D conformation, force field, transmembrane domain and RNA secondary structure of corresponding genes. Based on protein interaction network and pathway enrichment, GRK4 is predicted to participate in hypertension by acting on dopaminergic synapse, together with inte...

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Mitochondrial DNA Mutations Associated with Essential Hypertension in Xin Jiang Han Chinese Population

Background: Genetic factors contribute to essential hypertension (EH) etiology, however, causal g... more Background: Genetic factors contribute to essential hypertension (EH) etiology, however, causal genes haven't been identi ed. Mitochondrial dysfunction is common in EH. However, evidence supporting mitochondrial DNA (mtDNA) mutation involved in EH in Chinese Han is lacking. We aimed to characterize relationship between mtDNA mutation and EH in China. Methods: Totally 216 individuals including 151 EH patients and 65 controls from Chinese Han population in Xinjiang were sequenced for whole mtDNA genome. Novel variations and haplogroups were identi ed for each mtDNA sequence. Frequencies of gene mutations were compared between cases and controls, and functional changes of mtDNA genes associated with EH were predicted. Results: Haplogroups of Chinese Han population in Xinjiang were consistent with those in northern China. No association of mitochondrial haplogroup with EH was observed. Nine novel variations and three EHassociated mutations were identi ed. Variants in mutation m.12361A>G, m15662A>G and m.1598G>A were predicted to affect functions of ND5, CYTB and 12S rRNA, respectively. Conclusions: Our results have provided a new clue for mitochondrial genetic characteristics in etiology of EH in Chinese Han population in Xinjiang.

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Mitochondrial DNA Mutations Associated with Type 2 Diabetes Mellitus in Chinese Uyghur Population

Scientific reports, Jan 5, 2017

A hospital-based case-control study was conducted to investigate potential association between mi... more A hospital-based case-control study was conducted to investigate potential association between mitochondrial DNA and Type 2 diabetes mellitus (T2DM) in Chinese Uyghur population. We sequenced mitochondrial DNA from 210 Uyghur individuals including 88 T2DM patients and 122 controls. Using haplogroup classification and association test, we found that haplogroup H (odds ratio [OR] = 1.40; 95% confidence interval [CI]: 1.20-1.64; P = 0.0005138) and D4 (odds ratio = 1.47; 95% CI: 1.22-1.77; P = 0.001064) were associated with an increased risk of T2DM in Chinese Uyghur population. Two markers of haplogroup D4 and H, MT-ATP8 m.8414 T > G (p.Leu17Phe) and m.2706 G > A encoding 16S rRNA in mitochondria, were predicted to affect the structure of MT-ATP8 and 16S RNA, respectively, and may be involved in the pathogenesis of T2DM. Our study provides a new clue for mitochondrial DNA in the etiology of T2DM in Chinese Uyghur population.

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Detection of cell markers from single cell RNA-seq with sc2marker

BMC Bioinformatics, Jul 12, 2022

Background Advances in single-cell RNA sequencing (scRNA-seq) methods have revolutionized biomedi... more Background Advances in single-cell RNA sequencing (scRNA-seq) methods have revolutionized biomedical research by allowing the transcriptomes of millions of cells to be studied at the same time. This has helped to uncover molecular processes that drive cell differentiation and complex diseases [1, 2]. Many computational methods have been proposed for scRNA-seq data analysis [1, 3], including unsupervised analysis to characterize novel and disease-specific cell sub-populations such as rare stromal and immune cells [4-6] that cannot be detected by other methods. The delineation of small panels with marker genes that characterize such sub-populations is of particular importance for further molecular characterization and validation of the detected cells. For example, flow cytometry can be used to physically isolate cells and quantify cell populations or the expression of markers for both research and clinical applications [7]. However, flow cytometry requires a small panel of antibodies (< 50) that target previously characterized cell surface proteins that can be used as markers for cell types of interest. Multiplex immunohistochemistry (IHC) imaging allows protein abundance to be measured at a cellular level in tissue cross-sections, which allows

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Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis

Cell Stem Cell, Apr 1, 2021

Highlights d Mesenchymal progenitor cells are fibrosis-driving cells in the bone marrow d Inflamm... more Highlights d Mesenchymal progenitor cells are fibrosis-driving cells in the bone marrow d Inflammation in the stroma characterizes pre-fibrosis and TGF-b signaling fibrosis d Stromal S100A8/S100A9 marks disease progression in MPN patients d Tasquinimod, inhibiting S100A8/S100A9 signaling, ameliorates the MPN phenotype

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M cell maturation and cDC activation determine the onset of adaptive immune priming in the neonatal Peyer’s patch

Immunity, Jun 1, 2023

Endothelium-derived stromal cells contribute to hematopoietic bone marrow niche formation

Cell Stem Cell, Apr 1, 2021

Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Thei... more Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR+ cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration.

Transcriptional Landscape of the Microenvironment in Bone Marrow Fibrosis at Single Cell Level

Blood, Nov 13, 2019

Although the molecular alterations in hematopoietic cells which drive the development of myelopro... more Although the molecular alterations in hematopoietic cells which drive the development of myeloproliferative neoplasms (MPN) have been largely defined, reactive cellular alterations in the non-hematopoietic compartment remain rather obscure and have not been studied at single cell level. We therefore profiled enriched non-hematopoietic bone marrow cells by scRNAseq in bone marrow (BM) fibrosis compared to healthy marrow. BM fibrosis was induced by transplantation of hematopoietic stem and progenitor cells (HSPCs) with overexpression of Thrombopoietin (ThPO) into lethally irradiated mice. As ThPO-overexpression robustly leads to reticulin fibrosis in all mice (100%), we were able to study 1) pre-fibrosis (5 weeks after transplantation; reticulin fibrosis grade 0) and 2) manifest bone marrow fibrosis (10 weeks after transplantation, reticulin grade 2-3). The analysis revealed a total of 8 distinct clusters: 1-4) subpopulations of mesenchymal stromal cells (MSC-1: adipogenic, MSC-2: osteogenic, MSC-3: transition, MSC-4: interferonhigh), 5) osteoblastic lineage cells (OLCs), 6) arterial endothelial cells (ECs) and 7-8) Schwann cell precursors (SCP-1: non-myelinating SCPs; SCP-2: myelinating SCPs). Exposure to ThPO overexpressing HSPCs resulted in an overrepresentation of adipogenic MSCs at the expense of all other MSC subclusters. Differential gene expression analysis revealed a functional reprogramming of the "adipogenic" expanding MSCs with down-regulation of hematopoiesis-support and induction of a secretory phenotype including upregulation of various extracellular matrix (ECM) proteins driving fibrosis. Interestingly, only two MSC subclusters gained significant ECM expression indicating myofibroblast differentiation. Expansion of OLCs in BM fibrosis suggested a differentiation of the underrepresented MSC subpopulations into osteolineage cells which was confirmed by pseudotime analysis. Myelinating SCPs, highly expressing interleukin-33 (IL-33), showed the largest expansion in fibrosis. IL-33 is described to play a significant role in solid organ fibrosis by having both pro- and anti-fibrotic effects. Nerve injury triggers the expansion of myelinating and non-myelinating Schwann cells to promote repair, suggesting that mSCPs increase as compensatory and regenerative mechanism for the previously described MPN-induced sympathetic neuropathy. Dissection of cellular and molecular alterations in pre-fibrosis and manifest fibrosis demonstrated that only one MSC subpopulation was already significantly expanded in the pre-fibrotic phase, but only showed minor transcriptional changes. The upregulation of ECM proteins, osteogenesis as well as proinflammatory genes were hallmark features of manifest fibrosis. Interestingly, the overrepresentation of IL-33 expressing mSCPs was more pronounced in the pre-fibrotic phase, indicating that the expansion is a regenerative phenomenon failing in the stages of manifest fibrosis. Our findings were validated in the clinically relevant JAK2(V617F)-induced model of myelofibrosis. In conclusion, we here identified two distinct MSC subsets that are pro-fibrotic and contribute to osteosclerosis in PMF. The functional reprogramming of these MSCs in the bone marrow niche was accompanied by expansion of mSCPs with regenerative capacities, most likely caused by neural damage and Schwann cell death triggered by mutant HSCs. Disclosures Crysandt: Amgem: Other: travel grant; Pfizer: Other: travel grant; Gilead: Other: travel grant; Incyte: Membership on an entity's Board of Directors or advisory committees; celgene: Other: travel grant. Koschmieder:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers-Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AOP Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis Foundation: Research Funding.

Dissecting the Functional Reprogramming of the Microenvironment in Bone Marrow Fibrosis at the Single Cell Level

Social Science Research Network, 2019

Functional contributions of individual members of the bone marrow microenvironment to myelofibros... more Functional contributions of individual members of the bone marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPN) are incompletely understood. Consequently, we aimed to generate a comprehensive map of the stroma in MPN/MF on a single cell level. Our analysis revealed two distinct mesenchymal stromal cells (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic stage and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. In parallel, IL-33-expressing myelinating Schwann cell progenitors expanded, likely as a repair mechanism for the previously described neuropathy in MPN. IL-33 had anti-fibrotic effects on MSCs but induced expression of the alarmin S100A8 leading to loss-of-hematopoiesis support. Importantly, we demonstrate that S100A8 expression in stroma and patient plasma marks fibrosis progression and might even serve as a biomarker for disease progression.

Mapping the cardiac vascular niche in heart failure

Nature Communications, May 31, 2022

The cardiac vascular and perivascular niche are of major importance in homeostasis and during dis... more The cardiac vascular and perivascular niche are of major importance in homeostasis and during disease, but we lack a complete understanding of its cellular heterogeneity and alteration in response to injury as a major driver of heart failure. Using combined genetic fate tracing with confocal imaging and single-cell RNA sequencing of this niche in homeostasis and during heart failure, we unravel cell type specific transcriptomic changes in fibroblast, endothelial, pericyte and vascular smooth muscle cell subtypes. We characterize a specific fibroblast subpopulation that exists during homeostasis, acquires Thbs4 expression and expands after injury driving cardiac fibrosis, and identify the transcription factor TEAD1 as a regulator of fibroblast activation. Endothelial cells display a proliferative response after injury, which is not sustained in later remodeling, together with transcriptional changes related to hypoxia, angiogenesis, and migration. Collectively, our data provides an extensive resource of transcriptomic changes in the vascular niche in hypertrophic cardiac remodeling.

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Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

Nucleic Acids Research, Jan 3, 2019

RNA can directly bind to purine-rich DNA via Hoogsteen base pairing, forming a DNA:RNA triple hel... more RNA can directly bind to purine-rich DNA via Hoogsteen base pairing, forming a DNA:RNA triple helical structure that anchors the RNA to specific sequences and allows guiding of transcription regulators to distinct genomic loci. To unravel the prevalence of DNA:RNA triplexes in living cells, we have established a fast and cost-effective method that allows genome-wide mapping of DNA:RNA triplex interactions. In contrast to previous approaches applied for the identification of chromatin-associated RNAs, this method uses protein-free nucleic acids isolated from chromatin. High-throughput sequencing and computational analysis of DNA-associated RNA revealed a large set of RNAs which originate from non-coding and coding loci, including superenhancers and repeat elements. Combined analysis of DNA-associated RNA and RNA-associated DNA identified genomic DNA:RNA triplex structures. The results suggest that triplex formation is a general mechanism of RNA-mediated target-site recognition, which has major impact on biological functions.

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Neonatal Peyer’s patch cDC activation as a pacemaker of postnatal immune maturation

bioRxiv (Cold Spring Harbor Laboratory), Mar 4, 2022

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Genetic barcoding systematically compares genes in del(5q) MDS and reveals a central role for CSNK1A1 in clonal expansion

Blood Advances, 2022

How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (H... more How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSCs) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, we performed a systematic comparison on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single-clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1−/+ multipotent progenitors showed a proproliferative gene signature and HSCs showed a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1−/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. We therefore propose a crucial role for Csnk1a1 haploinsufficiency in the selective advantage of 5q-HSCs, implemented by creation of a unique competitive advantage thro...

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The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates

SummaryColonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization eve... more SummaryColonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 µm from the outer rim of iPSC colonies. Single- cell RNA-sequencing demonstrated thatOCT4highsubsets have pronounced up-regulation of the TGF-β pathway, particularly of NODAL and its inhibitor LEFTY, at the rim of the colonies. Furthermore, calcium-dependent cell-cell interactions were found to be relevant for the self-organization. Interestingly, after 5 to 7 days, the iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size, without any enzymatic or mechanical treatment. Withi...

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Deconstructing the Clonal Advantage and Clonal Stability of 5q- Candidate Genes in Del(5q) MDS on a Single Cell Level

Blood, 2019

Hematopoietic Stem/Progenitor cells (HSPCs) with 5q haploinsufficiency in del(5q) myelodysplastic... more Hematopoietic Stem/Progenitor cells (HSPCs) with 5q haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) acquire a clonal advantage in the bone marrow and out-compete normal hematopoiesis. A critical, yet unsolved question remains: how does genetic haploinsufficiency in del(5q) cells contribute to the clonal advantage of HSPCs? We investigated the role of haploinsufficiency for three candidate genes in the common deleted region on chromosome 5 (Csnk1a1, Egr1 and Apc) in direct competition with each other and wild-type (wt) cells on a single cell level by employing a novel lentiviral genetic barcoding strategy. We introduced genotype and cell-specific barcodes into HSPCs from murine models haploinsufficient for Csnk1a1, Egr1 or Apc. Barcoded HSPCs were sort-purified, genotypes mixed and subsequently competitively transplanted into lethally irradiated mice and re-transplanted after 16 weeks in a secondary transplant. The barcoded progeny was reliably recovered from peripheral ...

Transcriptional Landscape of the Microenvironment in Bone Marrow Fibrosis at Single Cell Level

Blood, 2019

Detection of cell markers from single cell RNA-seq with sc2marker

Zenodo (CERN European Organization for Nuclear Research), Nov 15, 2021

Single cell sequencing allow the detection of rare cell types in complex tissues. The detection o... more Single cell sequencing allow the detection of rare cell types in complex tissues. The detection of markers for these cell types are useful for further biological analysis as flow cytometry and imaging for either physical isolation or spatial characterization of these cells. Nevertheless, only few computational approaches consider the problem of selection of small marker genes from single cell sequencing so far. We propose here sc2marker, which is based on a weighted residual measure and database of proteins with antibodies, to select markers for imaging or flow cytometry. We evaluate the performance of sc2marker on single cell data of immune and stromal cells, which shows its superior performance in both time and accuracy in ranking known markers in contrast to competing methods.

Detection of cell markers from single cell RNA-seq with sc2marker

BMC Bioinformatics

Background Single-cell RNA sequencing (scRNA-seq) allows the detection of rare cell types in comp... more Background Single-cell RNA sequencing (scRNA-seq) allows the detection of rare cell types in complex tissues. The detection of markers for rare cell types is useful for further biological analysis of, for example, flow cytometry and imaging data sets for either physical isolation or spatial characterization of these cells. However, only a few computational approaches consider the problem of selecting specific marker genes from scRNA-seq data. Results Here, we propose sc2marker, which is based on the maximum margin index and a database of proteins with antibodies, to select markers for flow cytometry or imaging. We evaluated the performances of sc2marker and competing methods in ranking known markers in scRNA-seq data of immune and stromal cells. The results showed that sc2marker performed better than the competing methods in accuracy, while having a competitive running time.

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A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts

Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate det... more Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-b1 and TGF-b receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu. RESULTS The Reprogramming of Fibroblasts Starts with a MET Morphological differences between elongated fibroblasts and mouse ESCs are profound (Figure 1A). During reprogramming,

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