Defining Regulators of Human Hematopoietic Stem Cells
Författare
Summary, in English
With the aim to discover new growth factors supporting stem cells in culture, we have assessed 276 extrinsic signaling molecules for their effect on CB-derived HSPCs. We identified the immunoregulatory chemokine (C-C motif) ligand 28 (CCL28) as a novel growth- and survival factor for primitive hematopoietic cells. CCL28 strongly supported the proliferation and clonogenic potential of hematopoietic progenitors from different ontogenetic origins, and significantly enhanced the ability of cultured putative HSCs to long-term reconstitute immunodeficient mice. Thus, CCL28 represents one of the few cytokines that can maintain the primitive properties and functional integrity of cultured human HSPCs. Furthermore, we identified myostatin propeptide, a naturally occurring inhibitor of the transforming growth factor-ß (TGF-ß) family member myostatin, as a novel promoter of HSPC proliferation during ex vivo culture.
Based on the limited self-renewal capacity of HSCs in vitro, we have developed a forward RNA interference (RNAi)-based screening method that allows the discovery of novel genes implicated in stem- and progenitor cell proliferation. Using pooled lentiviral short hairpin RNA (shRNA) libraries transduced into CB cells, we have identified short hairpins designed against exostoses 1 (shExt1), phospholipase C zeta 1 (shPLCZ1) and serine threonine kinase 38 (shSTK38) as new fate determinants for HSPC differentiation, proliferation, and self-renewal, respectively. However, first-generation RNAi screening in primary cells yielded a considerable amount of target gene-unrelated, yet shRNA-specific events (so-called ‘off-target effects’), exemplified by shSTK38. Tracking of library-transduced HSPCs by next-generation sequencing significantly improved the resolution and feasibility of the screening approach and identified inhibition of MAPK14/p38α as means to promote expansion of undifferentiated cells, as shown by both RNAi and pharmacological modification of p38 using small molecule inhibitors.
Taken together, in this thesis we employed different screening approaches to identify novel regulators of primitive human hematopoietic cells. We conclude that systematic growth factor screenings as well as forward RNAi-based technologies are powerful tools to detect and subsequently define novel mediators of HSPC fate decisions.
Avdelning/ar
Publiceringsår
2013
Språk
Engelska
Publikation/Tidskrift/Serie
Lund University Faculty of Medicine Doctoral Dissertation Series
Volym
2013:71
Fulltext
Dokumenttyp
Doktorsavhandling
Förlag
Division of Molecular Medicine and Gene Therapy
Ämne
- Hematology
Nyckelord
- High-throughput screening
- Expansion
- Hematopoietic stem cell
- Cord blood
- RNA interference
- extrinsic Signaling molecules
Status
Published
Handledare
ISBN/ISSN/Övrigt
- ISSN: 1652-8220
- ISBN: 978-91-87449-41-3
Försvarsdatum
15 juni 2013
Försvarstid
09:00
Försvarsplats
Segerfalksalen, BMC A10, Sölvegatan 17, Lund
Opponent
- Hal E. Broxmeyer (Professor)