Inability to generate self-renewing hematopoietic stem cells from pluripotent stem cells (PSC) or via reprogramming has proven to be a major bottleneck that inhibits the use of in vitro engineered hematopoietic cells for therapeutic purposes. We hypothesize that defective induction of the correct transcriptional networks governing HSC self-renewal during the critical stages of hematopoietic development prevents the emergence of fully functional HSC in an in vitro setting. Guided by the transcriptional profile of highly purified human fetal liver HSC, we aim to identify key transcriptional regulators that govern self-renewal in developing human HSC and use this knowledge to develop new strategies to improve the function of in vitro derived hematopoietic cells. To identify the best candidates for self-renewal regulators, we took advantage of a novel human HSC surface marker, GPI80, to purify a population that is highly enriched for the true self-renewing HSC during human development. This strategy facilitated the identification of a set of transcriptional regulators that are unique to the self-renewing GPI80 HSC as compared to their downstream progeny, and are also suppressed in PSC-HPC. Utilizing our newly defined HSC culture system, which allows the study of human multilineage HSPC (hematopoietic stem/progenitor cell) hierarchy in vitro, we will first perform lentiviral knockdown of these candidate HSC regulators in primary human fetal liver GPI80+ HSC to test their importance for protecting HSC self-renewal and multipotency. In parallel, we will also investigate the degree to which the induction of these candidate self-renewal regulators extends HSC activity in cultured HSC, and/or even converts the transcriptional program in non-self-renewing GPI80-HPC, bringing them functionally and molecularly closer to fetal liver HSC. Finally, we will define the hierarchy of transcriptional networks associated with HSC self-renewal by analyzing the genomic binding sites and downstream gene expression networks for the individual transcriptional regulators that are involved in maintaining HSC self-renewal. Identification of the key upstream regulators that can confer self-renewal in developing HSC will both increase our knowledge of the fundamental regulatory mechanisms governing human HSC as well as pave the way for developing novel approaches for the in vitro generation of HSC for therapeutic use.

Public Health Relevance

Defining the mechanisms that establish and maintain self-renewing hematopoietic stem cells (HSC) will be critical for generating transplantable HSC from pluripotent stem cells or other cellular sources for therapeutic applications. We will take advantage of a novel human fetal HSC surface marker, GPI80 that has allowed us to define the unique nuclear regulatory program that distinguishes highly self-renewing HSC from downstream progenitors during human development. Defining the hierarchy and function of these novel HSC regulators will create a unique toolbox for generating human HSC in culture to treat blood disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK100959-01A1
Application #
8825314
Study Section
Molecular and Cellular Hematology (MCH)
Program Officer
Bishop, Terry Rogers
Project Start
2014-09-20
Project End
2017-07-31
Budget Start
2014-09-20
Budget End
2015-07-31
Support Year
1
Fiscal Year
2014
Total Cost
$277,200
Indirect Cost
$97,200
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095