Transplantation of donor hematopoietic (i.e. blood) stem cells (HSCs) into a patient’s bone marrow is the standard of care for a variety of inherited blood deficiencies and malignancies. However, this procedure relies on finding a genetically matched donor and can lead to problems if the match is not perfect. Thus, hematological disorders that require bone marrow transplants from a donor are accompanied by the risk of graft vs. host disease (GVHD), which can have fatal consequences. With the advent of induced pluripotent stem cell (iPSC) technologies (i.e. the ability to convert normal cells in our body to cells that have the potential to become any cell type) we are now at the forefront of unlimited potential to generate patient-specific therapies. The ability to obtain pluripotent cells from patients suffering from blood disorders that have the potential to be corrected (e.g. using gene therapy), and culturing them into HSCs that could then be transplanted back into the same patient, could provide opportunities to treat a variety of hematological diseases. We have shown the proof-of-principle of this possibility by deriving iPSCs from Fanconi anemia patients, and correcting them to generate disease-free blood cells. Yet despite these many advances, the problem remains that human HSCs derived from pluripotent cells in vitro are not able to fully reconstitute the blood system. In this project, we propose a method of comparing human HSCs of both in vitro and in vivo origin, to understand why this barrier exists. Using Next Generation Sequencing technology, we will determine gene network and genomic architecture differences between these two types of HSCs. We will test the ability of gene products identified as having a likely causative role in the functional differences between in vivo and in vitro HSCs to drive iPSCs into fully functional HSCs. Prominent researchers throughout the world have been working on ways to develop HSCs in vitro that could be used in the clinic. Despite creative and tireless efforts, this challenge remains. It is our goal that the results obtained from the proposed research provide a foundation by which researchers could more strategically address this obstacle, and enable this critical step towards the treatment of numerous hematological disorders.
Statement of Benefit to California:
The principle objective of this proposal is to develop methods to derive functional hematopoietic stem cells (HSCs) in vitro for use in regenerative medicine. This would potentially lead to improved treatment strategies for individuals with diseases affecting blood cells, such as sickle cell disease and Fanconi anemia, and reduced medical costs for the care of these patients in the State of California. Currently transplantation of donor HSCs into a patient’s bone marrow is standard of care for a variety of inherited blood deficiencies and malignancies. However, this procedure relies on finding a genetically matched donor and can lead to problems if the match is not perfect. The methods we propose to develop will enable the derivation of patient specific HSCs that are fully functional and capable of reconstituting the hematopoietic system. This will lay the ground work for future studies using gene therapy to correct inherited mutations in HSCs derived from somatic cells of a patient and the transplantation of these genetically corrected HSCs back into the bone marrow of the individual. The end goal of this work is to be able to provide a permanent cure for inherited diseases affecting blood cells which are often debilitating and common illnesses. Our project will also pilot new DNA sequencing methods that will have a substantial impact on stem cell biology research. These methods are likely to be used in the future to systematically validate the origin, quality and safety of stem cell derivatives, enabling the clinical implementation of stem cell based therapies. Our research will allow the State of California to continue to be at the forefront of stem cell research and regenerative medicine enabling our hopes for rapid translation of basic science to new therapies for disease to be fulfilled. All intellectual property generated by our research will be developed under CIRM guidelines for the maximum benefit of the State of California.
The ultimate goal of this proposal is to derive hematopoietic stem cells (HSCs) in vitro that are fully functional and capable of reconstituting the hematopoietic system. The production of true HSCs from human pluripotent stem cells (hPSCs) continues to be a significant roadblock for investigators in this field. The applicant proposes to study this problem and hopes to identify key molecular differences between in vivo isolated HSCs and induced pluripotent stem cells (iPSC)-derived HSCs that will inform a differentiation protocol for generating reconstitution-capable HSCs in vitro. The applicants propose 3 aims to accomplish this goal. First, the investigators will generate genetically identical sets of in vivo and in vitro HSCs by collecting HSC samples and reprogramming a portion of each sample to iPSCs. The second aim is to then examine the molecular differences between in vivo and in vitro HSCs. The investigators will use Next Generation Sequencing techniques and other genomic and informatics approaches. In the third aim, the investigators hope to differentiate iPSCs into fully functional HSCs. They will test the ability of gene products identified as having a likely causative role in the functional differences between in vivo and in vitro HSCs to drive iPSCs into fully functional HSCs.
Significance and Innovation:
- The inability to produce functional HSC has been a major barrier in developing relevant therapies based on hematopoietic stem cell transplantation/replacement; therefore, the significance of the proposal is high.
- Although the methods used throughout the proposal are not necessarily novel, they are state of the art for the most part and the approach is modestly innovative.
- The applicant takes a novel approach to compare global molecular profiles of in vivo obtained HSCs with genetically identical HSCs derived in vitro from iPSCs.
Feasibility and Experimental Design:
- Reviewers generally agreed that Aim 3 is poorly developed. Several concerns arise for Aim 3 since it is unclear how the gene products identified in Aims 1 and 2 will be prioritized, that combinations of products are likely needed, and that a precise level of expression and coordinated sequence of factors may be required which cannot be accomplished using the lentiviral system proposed.
- A reviewer noted the studies in Aim 3 are technically feasible, but doubt arises that fully functional HSC can be generated and no substantive alternative strategy is proposed.
- Reviewers noted that it is unlikely the applicants will identify one factor or one signature that would enable the transition from iPSC to HSC. It is more likely a combination of contributors. Therefore, feasibility to the end goal is an issue since it will be very difficult to find something through sequencing and genomic approaches with no plan for how to sift through the data.
- The reviewers were also concerned whether in vivo HSCs (CD34+/CD45+ cells) from the bone marrow would be too far downstream of the differentiation pathway to identify the critical factors for in vitro differentiation of HSCs, which may reflect an "upstream" profile.
- A reviewer noted that the proposal relies on the fact that different differentiation conditions would cause different patterns of gene expression and/or biological potential of resulting blood cells. The reviewer felt that the applicants should provide evidence that this is the case because, despite the lack of citations for groups that have already performed similar analysis, this is not the outcome others have described.
- One reviewer noted that the proposal presents strong supporting preliminary data and the use of state of the art techniques. The experiments proposed are feasible within the timelines proposed.
Principal Investigator (PI) and Research Team:
- The principal investigator (PI) and research team are appropriately qualified to carry out the proposed studies with greatest strengths in informatics and genomics.
- The PI is a world leader in genomic research and has made major contributions to the field. The PI has an impressive publication record and is well funded.
- The PI has chosen a strong group of collaborators who will bring their own specialist strengths to this study.
- Reviewers expressed concern that an insufficient number of full-time researchers are proposed to actually do the work. Only one full-time researcher is proposed.
Responsiveness to RFA:
- Reviewers agreed that the proposal fully meets the RFA objectives as it is exploring basic mechanisms. The study aims to define the global molecular profiles of in vivo obtained HSCs with genetically identical HSCs derived in vitro from iPSCs with a view to using this information to overcome the inability of human HSCs derived in vitro from pluripotent cells to reconstitute the hematopoietic system.