GeneSTAR Research Center - Genetic Studies of Atherosclerosis Risk

Induced Pluripotent Stem Cells, 
 Megakaryocytes, and Platelets
 

This is a new grant commencing in 2011. Below we outline the Phases. GeneSTAR's NextGen is a collaborative project with the Divisions of General Internal Medicine, Hematology, and Cardiology, The Institute for Cell Engineering, and the Johns Hopkins Genetics Core.  The project has multiple Principal Investigators. Under the leadership of Drs.  Lewis Becker from GeneSTAR and Linzhao Cheng from hematology, the study will begin with a 2 year laboratory phase, a portion of which will be guided by the University of Tokyo (Dr. Hiro Nakauchi) and University of Kyoto (Dr. Koji Eto).
 
Platelets in the circulating blood mediate normal hemostasis, but may also initiate pathological arterial thrombosis that produce heart attacks and strokes.  In our GeneSTAR GWAS study of native platelet and post-aspirin platelet function, we found many signals of genome-wide significance. The mechanism has remained largely undefined because most signals have occurred in introns or intergenic regions rather than in protein coding regions of known genes.
 
In addition, platelets are derived from megakaryocytes in the bone marrow, but themselves are anucleate with little residual mRNA. In this 3 phase study, we will examine the functional genomics of these associations in order to define novel risk assessment paradigms and identify new therapeutic targets for cardiovascular and thrombotic disorders.
 
In Phase I, under the direction of Dr. Linzhao Cheng with assistance from Drs. Nakauchi and Eto, 
  • developed an efficient method to generate human pluripotent stem cells (iPS) from peripheral blood mononuclear cells,
  • developed methods to generate differentiated megakaryocytes (Mks) from these human iPS,
  • determined that these differentiated Mks look like normal Mks and possess the cell markers of naturally occurring Mks, and that the whole-genome genotype of these differentiated Mks remains “true” to the original genotype.
 
 Dr. Cheng describes the iPSC process.
 
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Creating iPS from Whole Blood
Linzhao Cheng,PhD
 In Phase II, also under Dr. Cheng's direction, we :
 
  • developed an efficient method to generate iPS cells in batches from at least 10 individuals at a time using PBMCs from 20 GeneSTAR subjects
  • developed methods to generate Mks from these batches and performed RNAseq.  
 
In Phase III  under the direction of the GeneSTAR team, Dr. Lewis Becker has
 
  • generatde iPS cells from 258 selected study subjects who have had whole genome sequencing, a GWAS and extensive platelet phenotyping, chosen by phenotype and/or genotype for SNP variants with genome-wide significant associations in white and African American subjects.
  • Mks were differentiated from iPS cells for each subject selected. We examined gene expression profiling from the differentiated Mks from each subject using the Human Exon 1.0 ST Array from Affymetrix (containing all known gene transcripts and expressed sequence tags (ESTs) in humans), and confirmed the expression.
  • The expression of selected proteins corresponding to the expressed mRNAs is being examined by mass spectrometry at the laboratory of Dr. Jenny Van Eyk, Cedars Sinai Research Institute, Los Angeles CA.
  • For the significant genotype/phenotype SNP associations we have foundboth for native and post-ASA platelet function, we are comparing gene expression profiles from Mks by genotype.. 
  • We are determining whether elevated transcripts are associated with expression of the corresponding proteins.
  • We are determining the relationship between genetic variants across the genome and gene transcript levels (eQTLs) using multi-dimensional analysis to help understand how genetic variants, particularly in intergenic regions, may produce functional genomic effects.  This will provide an eQTL database for megakaryocytes that isshared with the scientific community.
  • We are also comparing gene expression profiles for Mks from subjects with high vs. low platelet aggregation response to different agonists (collagen, ADP, epinephrine, and arachidonic acid), at baseline and after aspirin.
  • We are determining whether some expressed transcripts code for proteins in known functional pathways.

In Phase III work includes collaborations of GeneSTAR's Dr. Rasika Mathias, an expert in  genetic analyses, with Drs, Jeff Leek, Kai Kammers, and Margaret Taub in the Department of Biostatistics in the Bloomberg School of Public Health. We are also studying mutation rates in iPSC and copy number variation with the expertise of Dr. Ingo Ruczinski, Department of Biostatistics.

Cell Repository:
We maintain an iPSC banking repository for all 257 cell lines at Johns Hopkins in the Becker Laboratory in Cardiology. 

The cells are also available  at WiCell, Inc. the home of the entire NHLBI NextGEN Project. WiCell can be searched using the following link to the Becker cells from GeneSTAR.. 


WiCell Repository
Platelet Aggregation (Dr. Lewis Becker, The Johns Hopkins University)

 Becker Lab’s Next Gen Cell LinesThis collection, from Dr. Lewis Becker (The Johns Hopkins University), was generated to enable the study of the genetic basis of human variation in native platelet function and platelet responsiveness to aspirin. The iPS cell lines from this study are included within this collection. 

This collection contains 198 human induced pluripotent stem cell lines derived under highly efficient clinically compliant conditions. The cell lines are comprised of healthy brothers, sisters, and offspring of index cases and siblings of persons of early onset coronary disease (< 60 years of age). None of the subjects were affected with clinical coronary disease, stroke, or other overt vascular disease phenotypes at the time of the study. Pedigrees are detailed in the “Genetically Related Cell Lines” field of each cell line’s web page.  The ages of donors range from 28 to 86 and ethnicities include European American and African American. 

About the Next Generation Genetic Association Studies (Next Gen) Program         

These cell lines were created as Next Generation Genetic Association Studies (Next Gen) Program, which was a five-year, $80 million program to investigate functional genetic variation in humans by assessing cellular profiles that are surrogates for disease phenotypes. To achieve this, researchers from multiple institutions across the U.S. were awarded grants to derive iPS cell lines from more than 1,500 individuals representing various conditions as well as healthy controls for use in functional genomic (‘disease in a dish’) research. This extensive panel includes a diverse set of age, gender and ethnic backgrounds, and therefore will be an invaluable tool for evaluations across demographics. Further enhancing the utility of these cell lines are data sets such as phenotyping, GWAS, genome sequencing, gene expression and -omics analyses (e.g., lipidomic, proteomic, methylomic) that will be made available with the cell lines.
 
 

 iPS Megakaryocytes, and Platelets in GeneSTAR
 
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Please do not copy, reproduce or print without explicit permission.(Linzhao Cheng, PhD)
 


Lewis Becker, MD
Robert L. Levy Professor, Cardiology
Principal Investigator