GeneSTAR Research Program - Study of Atherosclerosis Risk in Families
NHLBI NextGen
 
Induced Pluripotent Stem Cells, Megakaryocytes, and Platelets
 
Background
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, we will:
  • develop an efficient method to generate human pluripotent stem cells (iPS) from peripheral blood mononuclear cells,
  • develop methods to generate differentiated megakaryocytes (Mks) from these human iPS,
  • determine 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.
 
Below, Dr. Cheng describes this process.
 
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Creating iPS from Whole Blood
Linzhao Cheng,PhD
 In Phase II, also under Dr. Cheng's direction, we will:
 
  • develop an efficient method to generate iPS cells in batches from at least 10 individuals at a time using PBMCs from 20 GeneSTAR subjects
  • develop methods to generate Mks from these batches and perform mRNA microarrays of iPS-derived Mks from these 20 individuals.  Further characterization of these Mks will be done as in Phase I, Aim 2.
 Megakaryocyte
 
In Phase III  under the direction of the GeneSTAR team, Dr. Lewis Becker will work with Dr. Cheng to :
 
  • generate iPS cells from at least 400 selected study subjects who have had a GWAS and platelet phenotyping, chosen by phenotype and/or genotype for SNP variants with genome-wide significant associations in white and African American subjects.
  • Mks will be differentiated from iPS cells for each subject selected. We will examine 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 confirm the expression of selected mRNA transcripts by quantitative RT-PCR. 
  • The expression of selected proteins corresponding to the expressed mRNAs will be examined by mass spectrometry.
  • For the significant genotype/phenotype SNP associations we have found in the GWAS, both for native and post-ASA platelet function, we will compare gene expression profiles from Mks by genotype at each SNP location (both for signals in genes and intergenic regions). 
  • We will determine whether elevated transcripts are associated with expression of the corresponding proteins.
  • We will determine the relationship between genetic variants across the genome (2.5 million genotyped plus imputed SNPs) 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 can be shared with the scientific community.
  • We will compare 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 will determine whether some expressed transcripts code for proteins in known functional pathways.
  • For 5 of our most interesting significant results involving genotypes, phenotypes, gene expression profiles, and/or protein expression, we will produce platelets from the relevant differentiated megakaryocytes and perform functional characterization using aggregometry and flow cytometry. 
 
In Phase III work will include the collaborations of Dr. Diane Becker in analysis and clinical management, Dr. Rasika Mathias, an expert in  genetic analyses, and Dr. Jennifer Van Eyck in proteomics, in addition to Dr. Alan Scott of the Johns Hopkins Genomics Core. Dr. Rehan Qayyum will work with Dr. Nauder Faraday on the functional studies and platelet biology.
 
 Figure: Production of iPS Megakaryocytes, and Platelets
 
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Please do not copy, reproduce or print without explicit permission.(Linzhao Cheng, PhD)