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Defining Regulators of Human Hematopoietic Stem Cells

Karlsson, Christine LU (2013) In Lund University Faculty of Medicine Doctoral Dissertation Series 2013:71.
Abstract
Bone marrow transplantation (BMT) is a conceptual and elegant example of stem cell therapy, rendered possible by the dual capacity of hematopoietic stem cells (HSCs) to self-renew and differentiate. These defining features ultimately ensure the production of all blood cell lineages and simultaneous maintenance of the stem cell pool, thereby maintaining lifelong homeostasis. Banked umbilical cord blood (CB) is an abundant and readily available stem cell resource. However, the relatively low numbers of hematopoietic stem- and progenitor cells (HSPCs) present in a typical single CB unit and the associated delay in engraftment restrict its routine applicability to primarily children. Despite intense global efforts to find strategies that would... (More)
Bone marrow transplantation (BMT) is a conceptual and elegant example of stem cell therapy, rendered possible by the dual capacity of hematopoietic stem cells (HSCs) to self-renew and differentiate. These defining features ultimately ensure the production of all blood cell lineages and simultaneous maintenance of the stem cell pool, thereby maintaining lifelong homeostasis. Banked umbilical cord blood (CB) is an abundant and readily available stem cell resource. However, the relatively low numbers of hematopoietic stem- and progenitor cells (HSPCs) present in a typical single CB unit and the associated delay in engraftment restrict its routine applicability to primarily children. Despite intense global efforts to find strategies that would enable the ex vivo amplification of transplantable stem cells, the culture of HSCs outside their natural environment has proven difficult. It is therefore important to understand how HSC self-renewal, proliferation, and differentiation are integrated, which molecules participate in their regulation and how these could be modified for clinical benefit.



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. (Less)
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author
supervisor
opponent
  • Professor Broxmeyer, Hal E., Indiana School of Medicine, Indiana, USA
organization
publishing date
type
Thesis
publication status
published
subject
keywords
High-throughput screening, Expansion, Hematopoietic stem cell, Cord blood, RNA interference, extrinsic Signaling molecules
in
Lund University Faculty of Medicine Doctoral Dissertation Series
volume
2013:71
pages
98 pages
publisher
Division of Molecular Medicine and Gene Therapy
defense location
Segerfalksalen, BMC A10, Sölvegatan 17, Lund
defense date
2013-06-15 09:00:00
ISSN
1652-8220
ISBN
978-91-87449-41-3
language
English
LU publication?
yes
id
55fc028b-77c8-480a-a613-3ad4825ae9b2 (old id 3799435)
date added to LUP
2016-04-01 13:58:13
date last changed
2019-05-22 04:31:46
@phdthesis{55fc028b-77c8-480a-a613-3ad4825ae9b2,
  abstract     = {{Bone marrow transplantation (BMT) is a conceptual and elegant example of stem cell therapy, rendered possible by the dual capacity of hematopoietic stem cells (HSCs) to self-renew and differentiate. These defining features ultimately ensure the production of all blood cell lineages and simultaneous maintenance of the stem cell pool, thereby maintaining lifelong homeostasis. Banked umbilical cord blood (CB) is an abundant and readily available stem cell resource. However, the relatively low numbers of hematopoietic stem- and progenitor cells (HSPCs) present in a typical single CB unit and the associated delay in engraftment restrict its routine applicability to primarily children. Despite intense global efforts to find strategies that would enable the ex vivo amplification of transplantable stem cells, the culture of HSCs outside their natural environment has proven difficult. It is therefore important to understand how HSC self-renewal, proliferation, and differentiation are integrated, which molecules participate in their regulation and how these could be modified for clinical benefit.<br/><br>
<br/><br>
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. <br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.}},
  author       = {{Karlsson, Christine}},
  isbn         = {{978-91-87449-41-3}},
  issn         = {{1652-8220}},
  keywords     = {{High-throughput screening; Expansion; Hematopoietic stem cell; Cord blood; RNA interference; extrinsic Signaling molecules}},
  language     = {{eng}},
  publisher    = {{Division of Molecular Medicine and Gene Therapy}},
  school       = {{Lund University}},
  series       = {{Lund University Faculty of Medicine Doctoral Dissertation Series}},
  title        = {{Defining Regulators of Human Hematopoietic Stem Cells}},
  url          = {{https://lup.lub.lu.se/search/files/3697930/3799444.pdf}},
  volume       = {{2013:71}},
  year         = {{2013}},
}