iPSC Hematologic and Vascular Therapies

Basic and Translational Research of iPSC-Based Hematologic and Vascular Therapies

This Hub is comprised of investigators from Stanford University (PI: John P. Cooke, M.D., Ph.D.) and Johns Hopkins University (PI: Alan Friedman, M.D.) and will focus on the safe reprogramming and differentiation of adult cells to blood-forming cell lines for eventual application to blood or vascular disorders.

We intend to define early genetic and epigenetic mechanisms in induced pluripotent stem cell (iPSC) reprogramming; to generate safe iPSC lacking malignant potential; improve methods for generating endothelial cells (EC) and engraftable adult hematopoietic stem cells (HSC) from iPSC; and assess the clinical utility of iPSCs for vascular and blood diseases in preclinical models.

The component Projects of our Hub are:

Stanford University

Project 1: Novel Regulators to Enhance iPSC Derivation and Differentiation to EC (Helen Blau, Wing Wong).

Project 2: iPSC Engineering and Characterization (Renee Reijo Pera, James Swartz, Marius Wernig).

Project 3: iPSC-ECs for Therapeutic Angiogenesis: Determinants of Differentiation and Function (John Cooke).

Johns Hopkins University

Project 1: Epigenetic Mechanisms Underlying Tumorigenic Potential of iPSC (Stephen Baylin).

Project 2: Generation of Hemangioblasts and HSC from Human iPSC (Elias Zambidis).

Project 3: Role of Runx1 and Cooperating Factors in Specifying Adult HSC from the Hemangioblast (Friedman).

SCIENTIFIC MISSION AND GOALS

Our mission. The Stanford-Johns Hopkins Research Hub is a collaborative and tightly knit group of investigators who intend to develop novel technologies toward nuclear reprogramming and to generate new insights into directed differentiation, with the ultimate goal of developing cellular therapies for endothelial and hematologic diseases. Our group brings expertise in stem cell biology (Renee Reijo Pera and Marius Wernig); nuclear reprogramming and differentiation (Helen Blau, Stephen Baylin, and Alan Friedman); bioinformatics of transcriptional and epigenetic profiles (Wing Wong and Leslie Cope); protein bioengineering of reprogramming factors (James Swartz); and translational application of cell therapies for blood and vascular disease (Elias Zambidis and John Cooke). Nobel laureate Roger Kornberg, an expert in epigenetic regulation, chairs the scientific oversight committee for the Hub, and Vittorio Sebastiano will direct a vigorous Skills Development program in stem cell biology. We intend to gain a deeper understanding of the molecular pathways underlying nuclear reprogramming, and to provide the mechanistic clarity required for directed differentiation to hematopoietic stem cells (HSC) and endothelial cells (EC). The principles that emerge will be useful to the entire Consortium.

Our ongoing collaborations. Our work together, catalyzed by the RFA, has already begun. From our scientific exchanges and preliminary collaborations, the following 3 affinity groups have evolved:

1) Early events in iPSC Derivation
Rational generation of iPSC, and the directed differentiation of these cells, suffer from a paucity of information regarding the early genetic and epigenetic events that occur during reprogramming. Helen Blau (Stanford) first documented the plasticity of the differentiated state in mammalian cells 20 years ago, when she induced nuclear reprogramming by cell fusion.[1,2] More recently she has used cell fusion strategies and contemporary molecular and bioinformatic methods to gain insights into the early events of reprogramming to pluripotentiality. She works hand-in-glove with Wing Wong (Stanford) who has created much of the bioinformatics infrastructure (RNA-Seq, ChIP-chip and ChIP-Seq) that broadly serves the scientific community in this arena.[3-5] Stephen Baylin (Hopkins) is a world-renowned expert in basic epigenetic mechanisms that lead to the malignant phenotype [6,7] and his expertise in DNA-methyltransferases (DNMT) will be applied toward defining the mechanisms underlying formation of key epigenetic complexes that mediate iPSC generation in response to reprogramming factors. Blau and Baylin will work with Renee Reijo Pera, Marius Wernig (Stanford) and Elias Zambidis (Hopkins) to bring novel insights gained into early reprogramming to bear on the problem of optimal iPSC generation. Thus, Blau, Baylin, Wong, Reijo Pera, Wernig, and Zambidis represent our first affinity group, focused on defining the early events and mechanisms underlying reprogramming and translating these advances. The expertise of Alan Friedman in elucidating the details of transcriptional control of differentiation programs will also be valuable as these efforts proceed. The work of these investigators will inform the nuclear reprogramming efforts of our second affinity group, and will help to refine the methods of differentiation used by the third affinity group to derive therapeutic lineages from iPSC.

2) Engineering the iPSC
For clinical application of iPSCs, safety and function of the cells must be unambiguous. As the director of the Stanford stem cell bank, Renee Reijo Pera has uses a wide array of established and novel approaches to characterize iPSCs. She has defined genetic abnormalities within human embryonic stem cells (hESC) and iPSC lines via comparative genome hybridization (CGH) and other means.[8] Her work is complemented by that of Steve Baylin who has recently found striking similarities in epigenetic gene silencing between some iPSC lines and human cancer cells, highlighting the importance of developing methods to generate safer iPSC that lack cancer phenotypes. Marius Wernig (Stanford) contributes his extensive experience with direct reprogramming and integrative genomic analysis [9,10] together with his transgenic dox-inducible mice and reporter cell lines for screening novel reprogramming factors or small molecules.[11] Elias Zambidis is optimizing the use of non-integrating adenoviral or EBV vectors and is reprogramming adult marrow CD34+ hematopoietic stem cells (HSC) to iPSC as a means to obtain iPSC that more effectively generate hemangioblasts, HSC, and endothelium.[12] A key issue in generating safe iPSC is the need to avoid inadvertent integration of foreign DNA, which remains a hurdle for DNA-based strategies. Jim Swartz (Stanford) is a protein engineer, who developed a cell-free protein synthesis system for the efficient generation of cell permeable reprogramming factors [13,14], and is collaborating with John Cooke, Renee Reijo Pera and Sheng Ding to refine this reprogramming approach. In addition to ensuring the safety and function of iPSCs, Dr. Reijo Pera will apply the same rigorous genetic, epigenetic and mitochondrial analyses to compare iPSC-derived HSC and ECs to their normal adult counterparts.

3) iPSC-derived cells for blood and vascular disease
This affinity group will gain new insights and refine methods to generate iPSC-derived endothelial cells (iPSC-ECs) and iPSC-HSCs. Elias Zambidis (Hopkins) has contributed to the accumulating evidence for the existence of a human hemangioblast, and has defined the role of angiotensin signaling in hemangioblast differentiation to hematopoietic or endothelial lineage.[15] Zambidis has also uncovered hemangioblast-specific miRNAs, a subset of which target DNMTs, and will investigate their biology and translational utility with the assistance of Joshua Mendell (Hopkins), an expert in miRNA biology. Alan Friedman (Hopkins) has discovered that Runx1, a master transcription factor for specifying adult HSC, couples cell proliferation and differentiation due to its interaction with histone deacetylases (HDACs) regulated by cyclin-dependent kinase (cdk) phosphorylation.[16,17] He is poised to define how Runx1 gene regulation and protein interactions direct hemangioblasts to the hematopoietic rather than the endothelial lineage. In addition, Friedman will globally characterize RNAs and miRNAs expressed in hemangioblasts, HSC, and endothelial progenitors and will utilize ChIP:Seq to discover novel factors that cooperate with Runx1 to specify HSC or which help specify the endothelial fate. The insights gained by Drs. Zambidis and Friedman are leveraged by John Cooke and Helen Blau (Stanford) to enhance the generation of iPSC-EC. The Cooke lab has successfully generated EC from murine embryonic stem cells (ESC-EC) [18] and employed molecular imaging and perfusion technologies to document the incorporation of ESC-EC into the vasculature, and is now poised to generate and characterize human iPSC-EC in this model. Dr. Cooke will in addition seek to improve EC generation from iPSC using novel factors discovered by Friedman or Blau and using ACE effector pathways uncovered by Zambidis. Dr. Blau is further defining early events in EC differentiation using her heterokaryon model, and Dr. Zambidis proposes to collaborate with her to define novel determinants of hemangioblast and HSC development. Drs. Zambidis, Cooke, Blau, and Friedman are a 3rd nexus of interaction within our Research Hub, differentiating and applying iPSC-derived hematopoietic and endothelial cells.