Lei Tian

All Publications

• S100A10 is a critical mediator of GAS6/AXL-induced angiogenesis in renal cell carcinoma. Cancer research Xiao, Y., Zhao, H., Tian, L., Nolley, R., Diep, A. N., Ernst, A., Fuh, K. C., Miao, Y. R., von Eyben, R., Leppert, J. T., Brooks, J. D., Peehl, D. M., Giaccia, A. J., Rankin, E. B. 2019

  Abstract

  Angiogenesis is a hallmark of cancer that promotes tumor progression and metastasis. However, antiangiogenic agents have limited efficacy in cancer therapy due to the development of resistance. In clear cell renal cell carcinoma (ccRCC), AXL expression is associated with antiangiogenic resistance and poor survival. Here, we establish a role for GAS6/AXL signaling in promoting the angiogenic potential of ccRCC cells through the regulation of the plasminogen receptor S100A10. Genetic and therapeutic inhibition of AXL signaling in ccRCC tumor xenografts reduced tumor vessel density and growth under the renal capsule. GAS6/AXL signaling activated the expression of S100A10 through SRC to promote plasmin production, endothelial cell invasion and angiogenesis. Importantly, treatment with the small molecule AXL inhibitor cabozantinib or an ultra-high affinity soluble AXL Fc fusion decoy receptor (sAXL) reduced the growth of a pazopanib-resistant ccRCC patient-derived xenograft. Moreover, the combination of sAXL synergized with pazopanib and axitinib to reduce ccRCC patient-derived xenograft growth and vessel density. These findings highlight a role for AXL/S100A10 signaling in mediating the angiogenic potential of ccRCC cells and support the combination of AXL inhibitors with antiangiogenic agents for advanced ccRCC.

  View details for DOI 10.1158/0008-5472.CAN-19-1366

  View details for PubMedID 31585940

• Generation of Quiescent Cardiac Fibroblasts from Human Induced Pluripotent Stem Cells for In Vitro Modeling of Cardiac Fibrosis. Circulation research Zhang, H., Tian, L., Shen, M., Wu, H., Gu, M., Tu, C., Paik, D. T., Wu, J. C. 2019

  Abstract

  RATIONALE: Activated fibroblasts are the major cell type that secrete excessive extracellular matrix in response to injury, contributing to pathological fibrosis and leading to organ failure. Effective anti-fibrotic therapeutic solutions, however, are not available due to the poorly defined characteristics and unavailability of tissue-specific fibroblasts. Recent advances in single-cell RNA-sequencing (scRNA-seq) fill such gaps of knowledge by enabling delineation of the developmental trajectories and identification of regulatory pathways of tissue-specific fibroblasts among different organs.OBJECTIVE: This study aims to define the transcriptome profiles of tissue-specific fibroblasts using recently reported mouse scRNA-seq atlas, and to develop a robust chemically defined protocol to derive cardiac fibroblasts (CFs) from human induced pluripotent stem cells (iPSCs) for in vitro modeling of cardiac fibrosis and drug screening.METHODS AND RESULTS: By analyzing the single-cell transcriptome profiles of fibroblasts from 10 selected mouse tissues, we identified distinct tissue-specific signature genes, including transcription factors that define the identities of fibroblasts in the heart, lungs, trachea, and bladder. We also determined that CFs in large are of the epicardial lineage. We thus developed a robust chemically-defined protocol that generates CFs from human iPSCs. Functional studies confirmed that iPSC-derived CFs preserved a quiescent phenotype and highly resembled primary CFs at the transcriptional, cellular, and functional levels. We demonstrated that this cell-based platform is sensitive to both pro- and anti-fibrosis drugs. Finally, we showed that crosstalk between cardiomyocytes and CFs via the atrial/brain natriuretic peptide-natriuretic peptide receptor 1 pathway is implicated in suppressing fibrogenesis.CONCLUSIONS: This study uncovers unique gene signatures that define tissue-specific identities of fibroblasts. The bona fide quiescent CFs derived from human iPSCs can serve as a faithful in vitro platform to better understand the underlying mechanisms of cardiac fibrosis and to screen anti-fibrotic drugs.

  View details for DOI 10.1161/CIRCRESAHA.119.315491

  View details for PubMedID 31288631

• Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution. Circulation research Goodyer, W. R., Beyersdorf, B., Paik, D. T., Tian, L., Li, G., Buikema, J. W., Chirikian, O., Choi, S., Venkatraman, S., Adams, E. L., Tessier-Lavigne, M., Wu, J. C., Wu, S. M. 2019

  Abstract

  RATIONALE: The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node (SAN), atrioventricular node (AVN), His bundle, bundle branches (BB) and Purkinje fibers (PF). Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate due to inherent obstacles including small cell numbers, large cell type heterogeneity, complex anatomy and difficulty in isolation. Single-cell RNA-sequencing (scRNA-seq) allows for genome-wide analysis of gene expression at single-cell resolution.OBJECTIVE: Assess the transcriptional landscape of the entire CCS at single-cell resolution by scRNA-seq within the developing mouse heart.METHODS AND RESULTS: Wild-type, embryonic day 16.5 mouse hearts (n=6 per zone) were harvested and three zones of microdissection were isolated, including: Zone I - SAN region; Zone II - AVN/His region; and Zone III - BB/PF region. Tissue was digested into single cell suspensions, isolated, reverse transcribed and barcoded prior to high-throughput sequencing and bioinformatics analyses. scRNA-seq was performed on over 22,000 cells and all major cell types of the murine heart were successfully captured including bona fide clusters of cells consistent with each major component of the CCS. Unsupervised weighted gene co-expression network analysis led to the discovery of a host of novel CCS genes, a subset of which were validated using fluorescent in situ hybridization as well as whole mount immunolabelling with volume imaging (iDISCO+) in three-dimensions on intact mouse hearts. Further, subcluster analysis unveiled isolation of distinct CCS cell subtypes, including the clinically-relevant but poorly characterized "transitional cells" that bridge the CCS and surrounding myocardium.CONCLUSIONS: Our study represents the first comprehensive assessment of the transcriptional profiles from the entire CCS at single-cell resolution and provides a gene atlas for facilitating future efforts in conduction cell identification, isolation and characterization in the context of development and disease.

  View details for DOI 10.1161/CIRCRESAHA.118.314578

  View details for PubMedID 31284824

• Identifying the Transcriptome Signatures of Calcium Channel Blockers in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes CIRCULATION RESEARCH Lam, C., Tian, L., Belbachir, N., Wnorowski, A., Shrestha, R., Ma, N., Kitani, T., Rhee, J., Wu, J. C. 2019; 125 (2): 212–22

  View details for DOI 10.1161/CIRCRESAHA.118.314202

  View details for Web of Science ID 000474146200010

• Single cell expression analysis reveals anatomical and cell cycle-dependent transcriptional shifts during heart development. Development (Cambridge, England) Li, G., Tian, L., Goodyer, W., Kort, E. J., Buikema, J. W., Xu, A., Wu, J., Jovinge, S., Wu, S. M. 2019

  Abstract

  The heart is a complex organ composed of multiple cell and tissue types. Cardiac cells from different regions of the growing embryonic heart exhibit distinct patterns of gene expression, which are thought to contribute to heart development and morphogenesis. Single cell RNA sequencing allows genome-wide analysis of gene expression at the single cell level. Here, we analyzed cardiac cells derived from early stage developing hearts by single cell RNA-seq and identified cell cycle gene expression as a major determinant of transcriptional variation. Within cell cycle stage-matched CMs from a given heart chamber, we found that CMs in the G2/M phase downregulated sarcomeric and cytoskeletal markers. We also identified cell location-specific signaling molecules that may influence the proliferation of other nearby cell types. Our data highlight how variations in cell cycle activity selectively promote cardiac chamber growth during development, reveal profound chamber-specific cell cycle-linked transcriptional shifts, and open the way to deeper understanding of pathogenesis of congenital heart disease.

  View details for DOI 10.1242/dev.173476

  View details for PubMedID 31142541

• Human-Induced Pluripotent Stem Cell Model of Trastuzumab-Induced Cardiac Dysfunction in Patients With Breast Cancer CIRCULATION Kitani, T., Ong, S., Lam, C., Rhee, J., Zhang, J. Z., Oikonomopoulos, A., Ma, N., Tian, L., Lee, J., Telli, M. L., Witteles, R. M., Sharma, A., Sayed, N., Wu, J. C. 2019; 139 (21): 2451–65

  View details for DOI 10.1161/CIRCULATIONAHA.118.037357

  View details for Web of Science ID 000469018300014

• A Human iPSC Double-Reporter System Enables Purification of Cardiac Lineage Subpopulations with Distinct Function and Drug Response Profiles CELL STEM CELL Zhang, J. Z., Termglinchan, V., Shao, N., Itzhaki, I., Liu, C., Ma, N., Tian, L., Wang, V. Y., Chang, A. Y., Guo, H., Kitani, T., Wu, H., Lam, C., Kodo, K., Sayed, N., Blau, H. M., Wu, J. C. 2019; 24 (5): 802-+

  View details for DOI 10.1016/j.stem.2019.02.015

  View details for Web of Science ID 000466726500017

• Single-Cell RNA Sequencing of Human Embryonic Stem Cell Differentiation Delineates Adverse Effects of Nicotine on Embryonic Development STEM CELL REPORTS Guo, H., Tian, L., Zhang, J. Z., Kitani, T., Paik, D. T., Lee, W., Wu, J. C. 2019; 12 (4): 772–86

  View details for DOI 10.1016/j.stemcr.2019.01.022

  View details for Web of Science ID 000463364400011

• A Human iPSC Double-Reporter System Enables Purification of Cardiac Lineage Subpopulations with Distinct Function and Drug Response Profiles. Cell stem cell Zhang, J. Z., Termglinchan, V., Shao, N., Itzhaki, I., Liu, C., Ma, N., Tian, L., Wang, V. Y., Chang, A. C., Guo, H., Kitani, T., Wu, H., Lam, C. K., Kodo, K., Sayed, N., Blau, H. M., Wu, J. C. 2019

  Abstract

  The diversity of cardiac lineages contributes to the heterogeneity of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs). Here, we report the generation of a hiPSC TBX5Clover2 and NKX2-5TagRFP double reporter to delineate cardiaclineages and isolate lineage-specific subpopulations. Molecular analyses reveal that four different subpopulations can be isolated based on the differential expression of TBX5 and NKX2-5, TBX5+NKX2-5+, TBX5+NKX2-5-, TBX5-NKX2-5+, and TBX5-NKX2-5-, mimicking the first heart field, epicardial, second heart field, and endothelial lineages, respectively. Genetic and functional characterization indicates that each subpopulation differentiates into specific cardiac cells. We further identify CORIN as a cell-surface marker for isolating the TBX5+NKX2-5+ subpopulation and demonstrate the use of lineage-specific CMs for precise drug testing. We anticipate that this tool will facilitate theinvestigation of cardiac lineage specification and isolation of specific cardiac subpopulations for drug screening, tissue engineering, and disease modeling.

  View details for PubMedID 30880024

• Human Induced Pluripotent Stem Cell Model of Trastuzumab-Induced Cardiac Dysfunction in Breast Cancer Patients. Circulation Kitani, T., Ong, S. G., Lam, C. K., Rhee, J. W., Zhang, J. Z., Oikonomopoulos, A., Ma, N., Tian, L., Lee, J., Telli, M. L., Witteles, R. M., Sharma, A., Sayed, N., Wu, J. C. 2019

  Abstract

  Molecular targeted chemotherapies have been shown to significantly improve cancer patient outcomes, but often cause cardiovascular side effects that limit their use and impair patients' quality of life. Cardiac dysfunction induced by these therapies, especially trastuzumab, shows a distinct cardiotoxic clinical phenotype compared to cardiotoxicity induced by conventional chemotherapies.We employed the human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) platform to determine the underlying cellular mechanisms in trastuzumab-induced cardiac dysfunction. We assessed the effects of trastuzumab on structural and functional properties in iPSC-CMs from healthy individuals and performed RNA-sequencing (RNA-seq) to further examine the effect of trastuzumab on iPSC-CMs. We also generated iPSCs from patients receiving trastuzumab and examined whether patients' phenotype could be recapitulated in vitro using patient-specific iPSC-CMs.We found that clinically relevant doses of trastuzumab significantly impaired the contractile and calcium handling properties of iPSC-CMs without inducing cardiomyocyte death or sarcomeric disorganization. RNA-seq and subsequent functional analysis revealed mitochondrial dysfunction and altered cardiac energy metabolism pathway as primary causes of trastuzumab-induced cardiotoxic phenotype. Human iPSC-CMs generated from patients who received trastuzumab and experienced severe cardiac dysfunction were more vulnerable to trastuzumab treatment, compared to iPSC-CMs generated from patients who did not experience cardiac dysfunction following trastuzumab therapy. Importantly, metabolic modulation with AMPK activators could avert the adverse effects induced by trastuzumab.Our results indicate that alterations in cellular metabolic pathways in cardiomyocytes could be a key mechanism underlying the development of cardiac dysfunction following trastuzumab therapy; therefore, targeting the altered metabolism may be a promising therapeutic approach for trastuzumab-induced cardiac dysfunction.

  View details for PubMedID 30866650

• Modeling Cardiovascular Risks of E-Cigarettes With Human-Induced Pluripotent Stem Cell-Derived Endothelial Cells. Journal of the American College of Cardiology Lee, W. H., Ong, S. G., Zhou, Y., Tian, L., Bae, H. R., Baker, N., Whitlatch, A., Mohammadi, L., Guo, H., Nadeau, K. C., Springer, M. L., Schick, S. F., Bhatnagar, A., Wu, J. C. 2019; 73 (21): 2722–37

  Abstract

  Electronic cigarettes (e-cigarettes) have experienced a tremendous increase in use. Unlike cigarette smoking, the effects of e-cigarettes and their constituents on mediating vascular health remain understudied. However, given their increasing popularity, it is imperative to evaluate the health risks of e-cigarettes, including the effects of their ingredients, especially nicotine and flavorings.The purpose of this study was to investigate the effects of flavored e-cigarette liquids (e-liquids) and serum isolated from e-cigarette users on endothelial health and endothelial cell-dependent macrophage activation.Human-induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and a high-throughput screening approach were used to assess endothelial integrity following exposure to 6 different e-liquids with varying nicotine concentrations and to serum from e-cigarette users.The cytotoxicity of the e-liquids varied considerably, with the cinnamon-flavored product being most potent and leading to significantly decreased cell viability, increased reactive oxygen species (ROS) levels, caspase 3/7 activity, and low-density lipoprotein uptake, activation of oxidative stress-related pathway, and impaired tube formation and migration, confirming endothelial dysfunction. Upon exposure of ECs to e-liquid, conditioned media induced macrophage polarization into a pro-inflammatory state, eliciting the production of interleukin-1β and -6, leading to increased ROS. After exposure of human iPSC-ECs to serum of e-cigarette users, increased ROS linked to endothelial dysfunction was observed, as indicated by impaired pro-angiogenic properties. There was also an observed increase in inflammatory cytokine expression in the serum of e-cigarette users.Acute exposure to flavored e-liquids or e-cigarette use exacerbates endothelial dysfunction, which often precedes cardiovascular diseases.

  View details for DOI 10.1016/j.jacc.2019.03.476

  View details for PubMedID 31146818

• Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells. Circulation research Paik, D. T., Tian, L., Lee, J., Sayed, N., Chen, I. Y., Rhee, S., Rhee, J., Kim, Y., Wirka, R. C., Buikema, J. W., Wu, S. M., Red-Horse, K., Quertermous, T., Wu, J. C. 2018

  Abstract

  Rationale: Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology. Objective: Here we performed droplet-based single-cell RNA-sequencing (scRNA-seq) of the human iPSCs following iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. Methods and Results: Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody-conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of nitric oxide. Non-endothelial cell populations resulting from the differentiation protocol were identified, which included immature and atrial-like cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with four major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes respectively. Conclusions: Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of non-endothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity.

  View details for PubMedID 29986945

• Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells. Circulation Ma, N., Zhang, J., Itzhaki, I., Zhang, S. L., Chen, H., Haddad, F., Kitani, T., Wilson, K. D., Tian, L., Shrestha, R., Wu, H., Lam, C. K., Sayed, N., Wu, J. C. 2018

  Abstract

  Background -The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic "healthy" individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity. Methods -To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a "healthy" asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3, reported by the ClinVar database to be "likely pathogenic." Humaninduced pluripotent stem cells (iPSCs) were derived from the heterozygous VUSMYL3(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected "healthy" control; (2) homozygous VUSMYL3(170C>A); (3) heterozygous frameshift mutation MYL3(170C>A/fs); and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUSMYL3(170C>A), lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs). Results -The heterozygous VUSMYL3(170C>A)-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUSMYL3(170C>A)- and frameshift mutation MYL3(170C>A/fs)-iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3(170C>G), and a carrier-specific iPSC-CMs line, carrying a MYBPC3(961G>A) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments. Conclusions -Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrierspecific manner in a dish. As such, this platform represents a promising VUS riskassessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.

  View details for PubMedID 29914921