The overall goal of Project 3 is to develop a method to efficiently and reproducibly differentiate induced pluripotent stem (IPS) cells into hematopoietic stem cells (HSCs).
Aim 1 of this project will determine the best conditions for the mesodermal differentiation of iPS cells and the generation of HSCs.
This aim will test the hypothesis that an engineered human stromal cell line offers the best method to reliably differentiate IPS cells into hematopoietic precursors. Experiments will compare methods of differentiating IPS cells using embryoid bodies (EB) cultures and stromal cell lines to support the differentiation of IPS cells into HSCs. Various elements of these two differentiation methods will be studied to determine the best method to promote mesodermal differentiation and HSC creation.
The second aim i s to determine the optimal cytokine conditions for the survival, growth and expansion of HSCs generated from iPS cells.
This aim will test the hypothesis that HSCs generated from IPS are more similar in their cytokine responses to fetal HSCs than adult HSCs. Accumulation of HSCs in culture requires conditions that favor their survival and minimize their differentiation into committed progenitors. Various cytokines known to play a role in the early stages of hematopoiesis will be tested in combination to compare the similarity of HSCs generated from iPS cells to those isolated from fetal tissues, umbilical cord blood and adult peripheral blood. These experiments will optimize culture conditions for the production of HSCs.
The third aim will determine the variability in the capacity of different IPS cell lines to differentiate into HSCs.
This aim will test the hypothesis that different iPS cells lines are similarly capable to form HSCs that can generate long-term multilineage reconstitution in immunodeficient mice.
This aim will test isolated HSCs derived from multiple IPS cell lines for their capacity to provide long-term reconstitution without teratoma or leukemia formation, chromosomal abnormalities or other obvious functional deficiencies. The outcome of the experiments will be development of technology to efficiently generate transplantable HSCs from genetically corrected IPS cells.

Public Health Relevance

Project 3 develops methods of generating transplantable hematopoietic stem cells from pluripotent stem cells. This supports the overall program project goals to develop new methods of treating hemoglobinopathies using genetically correcting patient cells to generate blood stem cells for autologous transplantation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Program Projects (P01)
Project #
5P01DK088760-03
Application #
8532890
Study Section
Special Emphasis Panel (ZDK1-GRB-6)
Project Start
Project End
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
3
Fiscal Year
2013
Total Cost
$253,997
Indirect Cost
Name
University of California San Francisco
Department
Type
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Suzuki, Shingo; Sargent, R Geoffrey; Illek, Beate et al. (2016) TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs. Mol Ther Nucleic Acids 5:e273
Beyer, Ashley I; Muench, Marcus O (2016) Comparison of human hematopoietic reconstitution in different strains of immunodeficient mice. Stem Cells Dev :
Ye, Lin; Wang, Jiaming; Tan, Yuting et al. (2016) Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia. Proc Natl Acad Sci U S A 113:10661-5
Baimukanova, Gyulnar; Miyazawa, Byron; Potter, Daniel R et al. (2016) Platelets regulate vascular endothelial stability: assessing the storage lesion and donor variability of apheresis platelets. Transfusion 56 Suppl 1:S65-75
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Mahajan, Maya M; Cheng, Betty; Beyer, Ashley I et al. (2015) A quantitative assessment of the content of hematopoietic stem cells in mouse and human endosteal-bone marrow: a simple and rapid method for the isolation of mouse central bone marrow. BMC Hematol 15:9
Muench, Marcus O; Beyer, Ashley I; Fomin, Marina E et al. (2014) The adult livers of immunodeficient mice support human hematopoiesis: evidence for a hepatic mast cell population that develops early in human ontogeny. PLoS One 9:e97312
Xie, Fei; Ye, Lin; Chang, Judy C et al. (2014) Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Res 24:1526-33
Ye, Lin; Wang, Jiaming; Beyer, Ashley I et al. (2014) Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection. Proc Natl Acad Sci U S A 111:9591-6
Fomin, M E; Togarrati, P P; Muench, M O (2014) Progress and challenges in the development of a cell-based therapy for hemophilia A. J Thromb Haemost 12:1954-65

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