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Molecular and cellular analysis of Lhx2 function in hematopoietic stem cells
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The formation of blood, hematopoiesis, is a dynamic process originating from a small number of hematopoietic stem cells (HSCs). To sustain hematopoiesis throughout life HSCs have the unique capacity to differentiate into all mature hematopoietic lineages as well as generating more HSCs by a mechanism referred to as self-renewal. However, the regulation of these processes is largely unknown. During embryonic development HSCs expand in the fetal liver, indicating that this environment supports HSC self-renewal. The LIM-homeobox gene Lhx2 is expressed in the fetal liver during this period and Lhx2 null mutant mice die in utero due to severe anemia caused by an environmental defect in the fetal liver. Embryonic stem cells differentiate in vitro, forming embryoid bodies (EBs) containing various tissues including hematopoietic progenitor cells. Introduction of Lhx2 into this system by retroviral transfer led to the generation of cytokine dependent HSC-like cell lines that were multipotent and expressed surface markers similar to embryonic HSCs. However, the specificity and efficiency of this event could not be elucidated.

To further evaluate the function of Lhx2 expression during hematopoietic development, Lhx2 was introduced into an ES cell system where expression could be efficiently turned on. This approach revealed that Lhx2 induce self-renewal of distinct multipotent hematopoietic progenitor/stem cells present in the EB, with the ability to form HSC-like cell lines. The Lhx2 induced self-renewal is growth factor specific since stem cell factor and interleukin-6 are necessary and sufficient for this process. However, Lhx2 expression blocked erythroid differentiation and interfered with early ES cell commitment, indicating that the effect of Lhx2 is cell type specific.

Since HSCs of early embryonic origin are inefficient in engrafting adult recipients upon transplantation, we wanted to address whether we could generate cell lines retaining this capacity by expression of Lhx2 in hematopoietic cells from adult bone marrow. This led to the generation of clonal and cytokine dependent HSC-like cell lines capable of generating erythroid, myeloid and lymphoid cells upon transplantation into lethally irradiated recipients. When transplanted into stem cell-deficient mice, they contributed to circulating erythrocytes for at least 18 months, revealing a remarkable potential for self-renewal and differentiation in vivo. However, expression of Lhx2 was maintained in vivo and most engrafted mice developed a transplantable myeloproliferative disorder resembling human chronic myeloid leukemia. Thus, elucidation of the mechanism for Lhx2 function in HSC-like cell lines would give insights into both normal and pathological regulation of HSCs.

Down-regulation of Lhx2 expression in HSC-like cell lines with inducible Lhx2 expression led to rapid loss of stem cell characteristics and differentiation into various hematopoietic cell types. Thus, global gene expression analysis comparing Lhx2+ HSC-like cell lines to their Lhx2- progeny would give insights into the molecular basis for Lhx2 function in stem cells. A number of differentially expressed genes overlapped with previously reported HSC enriched genes, further emphasizing the resemblance between HSCs and the HSC-like cell lines also at the molecular level. Moreover, a number of genes were identified with functions or expression patterns related to Lhx2 in other organs. Collectively, these data suggest that these HSC-like cell lines represent a relevant model system for normal HSCs on the molecular and the functional level as well as for evaluating Lhx2 function in the development of various tissues in the embryo as well as in disease.

Place, publisher, year, edition, pages
Umeå: Umeå centrum för molekylär medicin (UCMM) , 2007. , 81 p.
Keyword [en]
Hematopoietic stem cells, Lhx2, cell lines, self renewal, SCF, IL-6, chronic myeloproliferative disorder, global gene expression analysis
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1389ISBN: 978-91-7264-395-6 (print)OAI: oai:DiVA.org:umu-1389DiVA: diva2:140848
Public defence
2007-10-13, Betula, 6M, Norrlands universitetssjukhus, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2007-10-10 Created: 2007-10-10 Last updated: 2009-09-24Bibliographically approved
List of papers
1. Lhx2 promotes self-renewal of a distinct multipotent hematopoietic progenitor cell in embryoid bodies
Open this publication in new window or tab >>Lhx2 promotes self-renewal of a distinct multipotent hematopoietic progenitor cell in embryoid bodies
2008 (English)In: PLoS one, ISSN 1932-6203, Vol. 3, no 4, e2025- p.Article in journal (Refereed) Published
Abstract [en]

The molecular mechanisms regulating the expansion of the hematopoietic system including hematopoietic stem cells (HSCs) in the fetal liver during embryonic development are largely unknown. The LIM-homeobox gene Lhx2 is a candidate regulator of fetal hematopoiesis since it is expressed in the fetal liver and Lhx2−/− mice die in utero due to severe anemia. Moreover, expression of Lhx2 in embryonic stem (ES) cell-derived embryoid bodies (EBs) can lead to the generation of HSC-like cell lines. To further define the role of this transcription factor in hematopoietic regulation, we generated ES cell lines that enabled tet-inducible expression of Lhx2. Using this approach we observed that Lhx2 expression synergises with specific signalling pathways, resulting in increased frequency of colony forming cells in developing EB cells. The increase in growth factor-responsive progenitor cells directly correlates to the efficiency in generating HSC-like cell lines, suggesting that Lhx2 expression induce self-renewal of a distinct multipotential hematopoietic progenitor cell in EBs. Signalling via the c-kit tyrosine kinase receptor and the gp130 signal transducer by IL-6 is necessary and sufficient for the Lhx2 induced self-renewal. While inducing self-renewal of multipotential progenitor cells, expression of Lhx2 inhibited proliferation of primitive erythroid precursor cells and interfered with early ES cell commitment, indicating striking lineage specificity of this effect.

National Category
Cell and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:umu:diva-2625 (URN)10.1371/journal.pone.0002025 (DOI)18431502 (PubMedID)
Available from: 2007-10-10 Created: 2007-10-10 Last updated: 2010-09-14Bibliographically approved
2. Hematopoietic progenitor/stem cells immortalized by Lhx2 generate functional hematopoietic cells in vivo.
Open this publication in new window or tab >>Hematopoietic progenitor/stem cells immortalized by Lhx2 generate functional hematopoietic cells in vivo.
2002 (English)In: Blood, ISSN 0006-4971, Vol. 99, no 11, 3939-3946 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-2626 (URN)12010792 (PubMedID)
Available from: 2007-10-10 Created: 2007-10-10Bibliographically approved
3. Lhx2 expression in hematopoietic progenitor/stem cells in vivo causes a chronic myeloproliferative disorder and altered globin expression.
Open this publication in new window or tab >>Lhx2 expression in hematopoietic progenitor/stem cells in vivo causes a chronic myeloproliferative disorder and altered globin expression.
Show others...
2003 (English)In: Haematologica, ISSN 0390-6078, Vol. 88, no 12, 1336-1347 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-2627 (URN)14687986 (PubMedID)
Available from: 2007-10-10 Created: 2007-10-10Bibliographically approved
4. Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression
Open this publication in new window or tab >>Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression
Show others...
2006 (English)In: BMC Genomics, ISSN 1471-2164, Vol. 7, 75- p.Article in journal (Refereed) Published
Abstract [en]

Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types.

Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development.

Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin.

Keyword
Animals, Cell Differentiation, Down-Regulation, Doxycycline/metabolism/pharmacology, Embryo/cytology/metabolism, Gene Expression Regulation, Hematopoietic Stem Cells/drug effects/*metabolism, Homeodomain Proteins/genetics/*metabolism, In Situ Hybridization, Mice, Models; Biological, Oligonucleotide Array Sequence Analysis, Tetracycline/metabolism/pharmacology, Transcription Factors/genetics/*metabolism
National Category
Cell and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:umu:diva-5917 (URN)10.1186/1471-2164-7-75 (DOI)16600034 (PubMedID)
Available from: 2007-12-03 Created: 2007-12-03 Last updated: 2010-09-14Bibliographically approved

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