University of Groningen Regulatory role of fibroblast growth factors on hematopoietic stem cells Yeoh, Joyce Siew Gaik IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2007 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Yeoh, J. S. G. (2007). Regulatory role of fibroblast growth factors on hematopoietic stem cells. s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 09-04-2021
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University of Groningen
Regulatory role of fibroblast growth factors on hematopoietic stem cellsYeoh, Joyce Siew Gaik
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.
Document VersionPublisher's PDF, also known as Version of record
Publication date:2007
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):Yeoh, J. S. G. (2007). Regulatory role of fibroblast growth factors on hematopoietic stem cells. s.n.
CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.
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39
CHAPTER 2
Fibroblast growth factor-1 and 2 preserve long-term
repopulating ability of hematopoietic stem cells in
serum-free cultures
Joyce S. G. Yeoh1, Ronald van Os1, Ellen Weersing1,
Albertina Ausema1, Bert Dontje1, Edo Vellenga2, Gerald
de Haan1
1 Department of Cell Biology, Section Stem Cell Biology, University
Medical Centre Groningen, The Netherlands 2 Department of Hematology, University Medical Centre Groningen, The
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42. Austin TW, Solar GP, Ziegler FC, Liem L, Matthews W. A role for the Wnt gene family in hematopoiesis: expansion of multilineage progenitor cells. Blood 1997;89:3624-3635.
43. Sperger JM, Chen X, Draper JS et al. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc.Natl.Acad.Sci.U.S.A 2003;100:13350-13355.
44. Dailey L, Ambrosetti D, Mansukhani A, Basilico C. Mechanisms underlying differential responses to FGF signaling. Cytokine & Growth Factor Reviews 2005;16:233-247.
45. Small D, Kovalenko D, Soldi R et al. Notch activation suppresses fibroblast growth factor-dependent cellular transformation. J.Biol.Chem. 2003;278:16405-16413.
46. Moon RT, Brown JD, Torres M. WNTs modulate cell fate and behavior during vertebrate development. Trends Genet. 1997;13:157-162.
47. Israsena N, Hu M, Fu W, Kan L, Kessler JA. The presence of FGF2 signaling determines whether beta-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells. Dev.Biol. 2004;268:220-231.
48. Zhang CC, Lodish HF. Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood 2005;105:4314-4320.
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51. Wolf NS. The haemopoietic microenvironment. Clin.Haematol. 1979;8:469-500.
52. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978;4:7-25.
53. Arai F, Hirao A, Ohmura M et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004;118:149-161.
54. Calvi LM, Adams GB, Weibrecht KW et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425:841-846.
55. Dorshkind K. Regulation of hemopoiesis by bone marrow stromal cells and their products. Annu.Rev.Immunol. 1990;8:111-137.
56. Stier S, Ko Y, Forkert R et al. Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size. Journal of Experimental.Medicine 2005;201:1781-1791.
66
67
CHAPTER 3
Effects of fibroblast growth factor overexpression and
cellular localization on hematopoietic stem cell
function
Joyce S. G. Yeoh, Ellen Weersing, Bert Dontje, Leonid
Bystrykh, Ronald van Os, Gerald de Haan
Department of Cell Biology, Section Stem Cell Biology, University
Medical Centre Groningen, The Netherlands
In preparation
Fibroblast growth factor overexpression
68
Abstract Exogenous addition of Fibroblast Growth Factor (FGF)-1 and FGF-2 maintains and
expands long-term repopulating hematopoietic stem cells (HSCs) in vitro. These
proteins are also highly expressed by Lin-Sca-1+c-Kit+ (LSK) cells. In this study we
retrovirally transduced post 5-fluorouracil (5-FU) bone marrow (BM) cells with
retroviral vectors which express wild-type (WT) FGF-1 and WT FGF-2 to examine
their cell intrinsic role in hematopoietic cell expansion. In addition, we examine the
role of nucleocytoplasmic trafficking of FGFs in hematopoietic cells by
overexpressing two mutant isoforms of FGF-1 in which a serine residue at the
phosphorylation site is exchanged for glutamic acid (S130E) or alanine (S130A).
S130E mimics the phosphorylated state of FGF-1 and should hypothetically be
constitutively exported to the cytoplasm whilst S130A should remain in the nucleus.
A third mutant was created by inserting an artificial nuclear localization signal (NLS)
upstream of FGF-2. Using fluorescence microscopy, we demonstrate that FGF-1,
FGF-2 and S130E predominantly localizes to the cytoplasm whilst 2% of S130A and
11% of NLS/FGF-2 overexpressing cells shown nuclear localization of FGF-1 and
FGF-2 respectively. We present evidence demonstrating that in an in vivo competitive
repopulation assay, stem cells expressing S130A engrafted into secondary recipients
with superior kinetics compared to WT cells, suggesting that nuclear localization of
FGF-1 may improve hematopoietic stem cell functioning.
Fibroblast growth factor overexpression
69
Introduction A small population of HSCs plays a pivotal role in the lifelong maintenance of
hematopoiesis. A critical property of HSCs is their ability to undergo self-renewal.
The relative inability to expand HSCs ex vivo imposes substantial limitations on the
current use of HSC transplantation. While studies have shown that some self renewal
is clearly possible in vitro1-3, the magnitude of expansion obtained for human and
murine HSCs is modest4-7.
In studies aimed to maintain HSCs in vitro, recent attention has been focused on the
large family of FGFs which are involved in embryonic development and adult tissue
homeostasis. To date, there are 22 members of the FGF family and only four distinct
FGF receptors (FGFRs) (reviewed by Ornitz and Itoh 2001)8. There is a growing body
of evidence demonstrating the role of FGFs in hematopoiesis in general and HSCs in
particular. For example, previous in vitro studies showed impaired hematopoietic
development in FGFR-/- embryoid bodies (EB), such that the number of blast colonies,
primitive erythroid and myeloid progenitors were greatly reduced9. FGFs, in
particular FGF-2, have been shown to sustain the proliferation of hematopoietic
progenitor cells, maintaining their primitive phenotype10;11. FGF-1 induces
granulopoiesis12 and megakaryocytopoiesis13;14. Recently, our group showed that
Fgfr-1, -3 and -4 are expressed by mouse primitive hematopoietic cell subsets and that
FGF-1 was involved in the expansion of multi-lineage (lymphoid and myeloid), long-
term (LT) repopulating HSCs 15. Additionally, we recently reported that
unfractionated bone marrow cells could be cultured in the combination of FGF-1 and
FGF-2 for up to five weeks without loss of stem cell repopulation activity. We
showed that this originated from FGF cultured HSCs16.
Several lines of evidence exist indicating that nuclear localization of FGFs may be
required for the mitogenic effect in certain conditions, in different cells types. For
example, radiolabeled exogenous FGF-1 localized to the nuclear fraction and was
shown to stimulate DNA synthesis and cell proliferation in cells containing receptors
for FGF-117. In glioma cells and in primary cultures of human astrocytes, cell
proliferation rate and nuclear association of FGF-2 was reported to change in
parallel18. These observations support the notion that nuclear translocation of FGFs
could be related to mitogenesis in different cells.
Fibroblast growth factor overexpression
70
Studies have shown that the translocation of FGF-1 from the cytosol to the nucleus
requires tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) activity and that
phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC) at the only
functional phosphorylation site (Serine 130) 19-22. In contrast to FGF-1, FGF-2
contains both autocrine and intracrine effects resulting from the existence of different
isoforms. For example, human FGF-2 contains five different forms; a low molecular
mass form (18kDa), which acts as an autocrine/paracrine factor and four high
molecular mass forms (21-22, 22.5, 24 and 34kDa) which are intracrine effectors.
These four high molecular mass forms are generated by differential initiation of
translation sites from an upstream CUG codon. They contain at least two short N-
terminal extensions in which the NLS is located23;24. Only N-terminally extended
forms initiated at upstream CUG codons are translocated to the nucleus while the
normal 18 kDa AUG-initiated form is confined to the cytoplasm25-28. When the
intracrine FGF-2 forms were expressed in NIH 3T3 cells, high proliferation rates and
growth in soft agar were observed29 and stimulated cell growth under low-serum
conditions was evident30;31
In previous studies, our goal was to maintain and expand HSCs in vitro by
exogenously adding FGF-1 and FGF-2 to serum-free media15;16. In the current study
we focused on retroviral overexpression of FGF-1 and FGF-2 on HSCs to promote
expansion and maintenance of hematopoietic cells in both in vitro and in vivo studies.
Parallel to this, we examined the effects of nuclear localized FGFs and their ability to
provide long-term (LT) repopulation. To clarify the role of differential localization of
FGFs in maintaining hematopoietic cells, we created two FGF-1 mutants. Firstly, a
serine residue at the phosphorylation site (amino acid 130) was exchanged with
glutamic acid to mimic phosphorylated FGF-1, which is expected to be constitutively
transported to the cytosol (S130E)22. In the second mutant the same serine residue was
exchanged for alanine, which is expected to remain in the nucleus (S130A)22. A third
mutant for FGF-2 was also created whereby an upstream nuclear localization signal
(NLS) was inserted into the FGF-2 coding sequence.
Using retroviral overexpression, long-term in vivo repopulation and principles of
limiting dilution assays to quantitatively determine hematopoietic cell expansion for
both WT FGF-1 and FGF-2 and their mutant forms, we demonstrate that in in vitro
assay, endogenous FGFs confer increased mitogenic activity to BM cells. However, it
does not provide enhanced long-term repopulation in primary and secondary in vivo
Fibroblast growth factor overexpression
71
repopulating assays. Fluorescence microscopy images indicated that FGF-1, FGF-2
and S130E were preferentially located in the cytoplasm. In S130A and NLS/FGF-2
overexpressing cells, only 2% of FGF-1 and 11% of FGF-2 positive cells showed
3. Glimm H, Eaves CJ. Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture. Blood 1999;94:2161-2168.
4. Miller CL, Eaves CJ. Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proc Natl Acad Sci U S A 1997;94:13648-13653.
5. Conneally E, Cashman J, Petzer A, Eaves C. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc.Natl.Acad.Sci.U.S.A 1997;94:9836-9841.
6. Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J.Exp.Med. 1997;186:619-624.
7. Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. The Journal of Clinical Investigation 2000;105:1013-1021.
9. Faloon P, Arentson E, Kazarov A et al. Basic fibroblast growth factor positively regulates hematopoietic development. Development 2000;127:1931-1941.
10. Allouche M, Bikfalvi A. The role of fibroblast growth factor-2 (FGF-2) in hematopoiesis. Prog.Growth Factor Res. 1995;6:35-48.
11. Allouche M, Bayard F, Clamens S et al. Expression of basic fibroblast growth factor (bFGF) and FGF-receptors in human leukemic cells. Leukemia 1995;9:77-86.
12. Yang M, Li K, Lam AC et al. Platelet-derived growth factor enhances granulopoiesis via bone marrow stromal cells. Int.J.Hematol. 2001;73:327-334.
13. Bikfalvi A, Han ZC, Fuhrmann G. Interaction of fibroblast growth factor (FGF) with megakaryocytopoiesis and demonstration of FGF receptor expression in megakaryocytes and megakaryocytic-like cells. Blood 1992;80:1905-1913.
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14. Chen QS, Wang ZY, Han ZC. Enhanced growth of megakaryocyte colonies in culture in the presence of heparin and fibroblast growth factor. Int.J.Hematol. 1999;70:155-162.
15. de Haan G, Weersing E, Dontje B et al. In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1. Developmental Cell 2003;4:241-251.
16. Yeoh JS, van Os R, Weersing E et al. Fibroblast growth factor-1 and 2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures. Stem Cells 2006;24:1564-1572.
17. Wiedlocha A, Falnes PO, Rapak A et al. Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization. Mol.Cell Biol. 1996;16:270-280.
18. Joy A, Moffett J, Neary K et al. Nuclear accumulation of FGF-2 is associated with proliferation of human astrocytes and glioma cells. Oncogene 1997;14:171-183.
19. Wiedlocha A, Nilsen T, Wesche J et al. Phosphorylation-regulated nucleocytoplasmic trafficking of internalized fibroblast growth factor-1. Mol.Biol.Cell 2005;16:794-810.
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21. Klingenberg O, Wiedlocha A, Olsnes S. Effects of mutations of a phosphorylation site in an exposed loop in acidic fibroblast growth factor. J.Biol.Chem. 1999;274:18081-18086.
22. Klingenberg O, Wiedlocha A, Rapak A et al. Inability of the acidic fibroblast growth factor mutant K132E to stimulate DNA synthesis after translocation into cells. J.Biol.Chem. 1998;273:11164-11172.
23. Arnaud E, Touriol C, Boutonnet C et al. A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. Mol.Cell Biol. 1999;19:505-514.
24. Dono R, James D, Zeller R. A GR-motif functions in nuclear accumulation of the large FGF-2 isoforms and interferes with mitogenic signalling. Oncogene 1998;16:2151-2158.
25. Bugler B, Amalric F, Prats H. Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor. Mol.Cell Biol. 1991;11:573-577.
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27. Davis MG, Zhou M, Ali S et al. Intracrine and autocrine effects of basic fibroblast growth factor in vascular smooth muscle cells. J.Mol.Cell Cardiol. 1997;29:1061-1072.
28. Quarto N, Finger FP, Rifkin DB. The NH2-terminal extension of high molecular weight bFGF is a nuclear targeting signal. J.Cell Physiol 1991;147:311-318.
29. Bikfalvi A, Klein S, Pintucci G et al. Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms. J Cell Biol. 1995;129:233-243.
30. Arese M, Chen Y, Florkiewicz RZ et al. Nuclear activities of basic fibroblast growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals. Mol.Biol.Cell 1999;10:1429-1444.
31. Vagner S, Touriol C, Galy B et al. Translation of CUG- but not AUG-initiated forms of human fibroblast growth factor 2 is activated in transformed and stressed cells. J Cell Biol. 1996;135:1391-1402.
32. de Haan G, Van Zant G. Intrinsic and extrinsic control of hemopoietic stem cell numbers: mapping of a stem cell gene. J.Exp.Med. 1997;186:529-536.
33. de Haan G, Szilvassy SJ, Meyerrose TE et al. Distinct functional properties of highly purified hematopoietic stem cells from mouse strains differing in stem cell numbers. Blood 2000;96:1374-1379.
34. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-408.
35. Ploemacher RE, van der Sluijs JP, van Beurden CA, Baert MR, Chan PL. Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse. Blood 1991;78:2527-2533.
36. Morrison SJ, Uchida N, Weissman IL. The biology of hematopoietic stem cells. Annu.Rev.Cell Dev.Biol. 1995;11:35-71.
37. Okada S, Nakauchi H, Nagayoshi K et al. Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule. Blood 1991;78:1706-1712.
38. Alessi DR, Andjelkovic M, Caudwell B et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 1996;15:6541-6551.
39. Tagawa T, Kuroki T, Vogt PK, Chida K. The cell cycle-dependent nuclear import of v-Jun is regulated by phosphorylation of a serine adjacent to the nuclear localization signal. J.Cell Biol. 1995;130:255-263.
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40. Engel K, Kotlyarov A, Gaestel M. Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation. EMBO J. 1998;17:3363-3371.
41. Prudovsky I, Bagala C, Tarantini F et al. The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export. J.Cell Biol. 2002;158:201-208.
42. Crcareva A, Saito T, Kunisato A et al. Hematopoietic stem cells expanded by fibroblast growth factor-1 are excellent targets for retrovirus-mediated gene delivery. Exp.Hematol. 2005;33:1459-1469.
43. Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr.Relat Cancer 2000;7:165-197.
44. Olsnes S, Klingenberg O, Wiedlocha A. Transport of exogenous growth factors and cytokines to the cytosol and to the nucleus. Physiol Rev. 2003;83:163-182.
45. Wiedlocha A, Sorensen V. Signaling, internalization, and intracellular activity of fibroblast growth factor. Curr.Top.Microbiol.Immunol. 2004;286:45-79.
46. Baldin V, Roman AM, Bosc-Bierne I, Amalric F, Bouche G. Translocation of bFGF to the nucleus is G1 phase cell cycle specific in bovine aortic endothelial cells. EMBO J 1990;9:1511-1517.
47. Imamura T, Engleka K, Zhan X et al. Recovery of mitogenic activity of a growth factor mutant with a nuclear translocation sequence. Science 1990;249:1567-1570.
48. Wiedlocha A, Falnes PO, Madshus IH, Sandvig K, Olsnes S. Dual mode of signal transduction by externally added acidic fibroblast growth factor. Cell 1994;76:1039-1051.
49. Bouche G, Gas N, Prats H et al. Basic fibroblast growth factor enters the nucleolus and stimulates the transcription of ribosomal genes in ABAE cells undergoing G0----G1 transition. Proc.Natl.Acad.Sci.U.S.A 1987;84:6770-6774.
50. Renko M, Quarto N, Morimoto T, Rifkin DB. Nuclear and cytoplasmic localization of different basic fibroblast growth factor species. J.Cell Physiol 1990;144:108-114.
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52. Calvi LM, Adams GB, Weibrecht KW et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425:841-846.
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103
104
105
Chapter 4
Mobilized peripheral blood stem cells provide rapid
reconstitution but impaired long-term engraftment
Joyce S. G. Yeoh, Albertina Ausema, Piet Wierenga,
Gerald de Haan, Ronald van Os
Department of Cell Biology, Section Stem Cell Biology, University
Medical Centre Groningen, The Netherlands
Submitted
Mobilized peripheral blood stem cells have impaired long-term engraftment
106
Abstract In this study, we use competitive repopulation assay to compare the quality and
frequency of stem cells isolated from mobilized blood with stem cells isolated from
bone marrow (BM). Lin-Sca-1+c-Kit+ (LSK) cells were harvested from control BM
and peripheral blood of mice following Granulocyte Colony Stimulating Factor (G-
CSF) administration. LSK cells were used because of their resemblance with human
CD34+ cells. We confirmed that transplantation of phenotypically defined mobilized
peripheral blood (MPB) stem cells results in rapid recovery of blood counts.
However, in vitro results indicated that LSK cells purified from MPB had lower
cobblestone area forming cell (CAFC) day 35 activity compared to BM. Additionally,
evaluation of chimerism after co-transplantation of LSK cells purified from blood and
BM revealed that MPB stem cells contained 25-fold less repopulation potential
compared to BM stem cells. Competitive repopulating unit (CRU) frequency analysis
showed that freshly isolated MPB LSK cells have 8.8-fold fewer cells with long-term
repopulating ability compared to BM LSK cells. Secondary transplantation showed no
further decline in contribution of hematopoiesis relative to BM. We conclude that the
reduced frequency of stem cells within the LSK population of MPB, rather than
poorer quality, causes a reduced repopulation potential of MPB.
Mobilized peripheral blood stem cells have impaired long-term engraftment
107
Introduction The initial source of hematopoietic cells used for transplantations was bone marrow1.
However, due to the faster regeneration of both circulating neutrophils (9-11 days) 2;3
and platelets, peripheral blood stem cells have become the primary source of
hematopoietic stem cells for clinical transplantation over the past 10 years4.
Although multiple hematopoietic growth factors are capable of inducing mobilization
of hematopoietic progenitors, G-CSF is at present one of the most used mobilizing
molecule in clinical protocols5. The efficiency of G-CSF to mobilize bone marrow
precursors and long-term repopulating cells was initially shown in preclinical studies.
Molineux et al., observed a marked increase of the colony-forming unit spleen (CFU-
S) pool in the peripheral blood of mice treated with repeated doses of G-CSF6. In
addition, it was observed that G-CSF alone and in combination with SCF or IL-7
mobilizes hematopoietic precursors capable of both radioprotection and generating
sustained lympho-haematopoiesis in transplanted recipients7. In vitro data from
human patients appear consistent with the concept that the quality of human
mobilized peripheral blood progenitor cells is at least equivalent to that corresponding
to bone marrow grafts8;9.
Surprisingly, despite the prevalent use of hematopoietic stem cell mobilization in
clinical transplantation, few reports exist describing the competitive repopulating
quality of mobilized stem cells compared to bone marrow stem cells following G-CSF
treatment. Most available reports only outlined the differences in the kinetics and
efficiency of engraftment, in homing properties and in cell cycle profiles between
mobilized blood stem cells and those isolated from resting bone marrow5;10-13.
In view of the increased use of peripheral blood stem cells in clinical transplant
settings, it is of relevance to investigate long term functioning of stem cells isolated
from different sources. In this study, we directly assessed the function of mobilized
peripheral blood stem cells compared to control bone marrow stem cells when co-
transplanted in a single recipient mouse in an in vivo competitive repopulation assay.
We show a reduced frequency of repopulating stem cells in purified G-CSF-mobilized
peripheral blood LSK stem cells, which caused a 25-fold reduction in repopulation
potential compared with BM LSK cells.
Mobilized peripheral blood stem cells have impaired long-term engraftment
108
Materials and Methods Mice
Female C57BL/6 (B6), C57BL/6.SJL (CD45.1), (C57BL/6 x C57BL6.SJL) F1
(CD45.1/2) or C57BL/6-Tg(ACTB-EGFP)10sb/J transgenic GFP (GFP) mice were
used as donors, competitors or recipients of blood and marrow stem cells depending
on the experimental model. CD45.1 and transgenic GFP mice were originally
obtained from the Jackson Laboratory (Bar Harbor, Maine) and bred in our local
animal facility. Wild type female B6 mice were purchased from Harlan (Horst, The
Netherlands) and maintained under clean conventional conditions in the animal
facilities of University Medical Centre Groningen (The Netherlands). Mice were fed
ad libitum with food pellets and acidified tap water (pH = 2.8). All animal procedures
were approved by the local animal ethics committee of the University Medical Centre
Groningen.
Mobilization and harvesting of stem cells
Bone marrow cells were harvested by flushing the femoral content with α-MEM
(GibcoBRL, Invitrogen, CA) supplemented with 2% fetal calf serum (FCS;
Netherlands). The collected blood cell suspension (5 ml) was centrifuged over an
equal volume of Lympholyte-M (Cedarlane Laboratories Ltd, Hornby, Canada) at 400
x g for 30 minutes at room temperature. After centrifugation, the mononuclear cells
within the opaque interface layer were isolated and washed in IMDM/5% FCS for 5
minutes at 2,000 rpm at 4oC. Alternatively, red blood cells were lysed using
ammonium chloride (NH4Cl) without prior density separation. Nucleated cells were
measured on a Coulter Counter Model Z2 (Coulter Electronics, Hialeah, FL).
Mobilized peripheral blood stem cells have impaired long-term engraftment
109
Isolation of Lin-Sca-1+c-Kit+ cells
Bone marrow and mobilized peripheral blood cells were stained as previously
described15 with biotinylated lineage-specific antibodies Mouse Lineage Panel,
containing anti-CD45R, anti-CD11b, anti-TER119, anti-Gr-1 and anti-CD3e (BD
Pharmingen, San Diego, CA), FITC-anti-Sca-1 and APC-anti-c-kit (BD Pharmingen,
San Diego, CA). Biotinylated antibodies were visualized with streptavidin-PE
(Pharmingen, San Diego, CA). After antibody staining, cells were sorted by a
MoFlow cell sorter (DakoCytomation, Fort Collins, CO). Lin-Sca-1+c-Kit+ (LSK) and
Lin- non-Sca-1+c-Kit+ cells were sorted and used in transplantation assays or in in
vitro CAFC assays.
Long term competitive repopulation ability
Female B6, CD45.1 or transgenic GFP mice were used as donors for competitor cells.
Female B6 mice were used as recipients in all experiments. Recipient mice were
irradiated with 9.5 Gy γ-rays (0.7026 Gy/min) in a CIS Biointernational IBL 637 137Cs-source, 20-24 hours prior to transplantation. For competitive repopulation
determination, unfractionated cells or mobilized peripheral blood LSK cells, were
mixed with competitor cells (unfractionated or LSK bone marrow cells) and
intravenously transplanted into recipient mice. Each transplant group consisted of 8-
10 recipients. Following transplantation, blood samples (60μl) were taken monthly to
determine donor chimerism. Levels of chimerism were determined by detecting the
presence of GFP+ or CD45.1+ and CD45.2+ cells in transplanted mice. To detect
CD45.1+ and CD45.2+ cells, cells were stained with anti-CD45.2 (FITC) and anti-
CD45.1 (PE) antibodies (BD Pharmingen, San Diego, CA) for 30 minutes and
analyzed on a flow cytometer (FACS Calibur; Becton Dickinson Biosciences, San
Jose, CA). In addition, the competitive repopulating index (CRI) was determined. CRI
is a relative measure of the competitive ability of test cells to that of fresh bone
marrow cells. The CRI was calculated by taking the ratio of WBC derived from
mobilized blood cells to bone marrow cells in the circulation and dividing it by the
ratio of mobilized blood cells to bone marrow cells transplanted. A CRI value of one
indicates by definition that mobilized peripheral blood cells and bone marrow cells
have equal competitive ability.
Mobilized peripheral blood stem cells have impaired long-term engraftment
110
Competitive Repopulation Unit assay
B6 recipient mice were transplanted with a series of diluted CD45.1 LSK cells (1,200,
600 and 300) from mobilized blood and control bone marrow and with a fixed number
of B6 competitor cells (5 x 105). Twelve weeks after transplantation, donor cell
contribution in the peripheral blood was determined. Recipients with a contribution of
≥ 5% in both myeloid and lymphoid lineages were considered to be positive.
To evaluate and quantify the repopulating potential of mobilized blood LSK cells and
control BM LSK cells, the frequency of competitive repopulation units (CRU) was
calculated. CRU frequencies per 1,000 LSK were calculated from the resultant
percentage positive recipients by limiting dilution analysis procedures which uses
Poisson statistics16.
Cobblestone area forming cell assays
Cobblestone area forming cell assays (CAFC) were performed as described17-19 to
assess the number of hematopoietic progenitor cells (CAFC day 7) or more primitive
stem cells (CAFC day 35) in mobilized peripheral blood stem cells.
Secondary Transplantations
In one of the competitive repopulation experiments, in which recipients were
transplanted in different ratios with CD45.1 mobilized peripheral blood LSK cells and
CD45.2 bone marrow LSK competitor cells, mice were sacrificed for secondary
transplantations. Bone marrow cells from B6 primary chimeric recipients were
isolated on basis of CD45 isoform. CD45.1 bone marrow cells represent cells derived
from mobilized peripheral blood LSK population (CD45.1 MPB LSK derived bone
marrow) and CD45.2 (B6) bone marrow cells represent cells derived from bone
34. Kaufman CL, Colson YL, Wren SM et al. Phenotypic characterization of a novel bone marrow-derived cell that facilitates engraftment of allogeneic bone marrow stem cells. Blood 1994;84:2436-2446.
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CHAPTER 5
Summarizing Discussion and Future Perspectives
Summarizing Discussion and Future Perspectives
134
Summarizing Discussion Expansion and maintenance of self renewing primitive hematopoietic stem cells
(HSCs) would have major implications in the areas of stem cell transplantation and
gene therapy. The ultimate goal of many scientists is to successfully expand and
maintain stem cells in their primary functional characteristic, namely their ability to
engraft and sustain long-term hematopoiesis. Establishing the ideal in vitro growth
conditions to expand and maintain HSCs has proven to be difficult. Recently, strong
evidence is emerging indicating that the large family of fibroblast growth factors
(FGFs) and FGF receptors (FGFRs) may play a key role in stem cell maintenance.
Chapter 1 provides a conceptual overview on FGFs and their receptors in
maintaining tissue homeostasis in HSCs, neural stem cells (NSCs) and embryonic
stem (ES) cells, in order to retain the self renewal capacity of stem cells.
Commonalities do exist between these three distinct stem cell systems. We summarize
evidence that FGFs are growth factors crucial for the regulation and culturing of all
three of these stem cell systems.
In Chapter 2 we studied the role of FGFs to maintain HSC function by culturing
unfractionated bone marrow (BM) cells in serum-free media supplemented only with
FGF-1, FGF-2 or the combination of both (FGF-1 + 2). Bone marrow cells were
cultured for a total of five weeks and then competitively transplanted into lethally
irradiated hosts. Cells cultured in FGF-1 + 2 showed a 5-fold and 1.5-fold increase in
repopulating units after culturing for one and five weeks respectively. In addition, we
co-cultured Lin-Sca-1+c-Kit+ (LSK) with unfractionated BM cells for a total of five
weeks in FGF-1 + 2 and transplanted without competitors into lethally irradiated
recipients. Overall, the data demonstrated that FGF cultured BM cells can be
maintained for up to five weeks while retaining long-term repopulating ability
(LTRA) and that all FGF-induced stem cell activity was derived from the LSK
population. Furthermore, the data signifies the importance of the stem cell niche, a
specialized microenvironment that houses, regulates and protects stem cells1. We
could not culture purified LSK cells in serum-free medium supplemented only with
FGF-1 and/or FGF-2. However in the presence of unfractionated BM cells, LSK cells
proliferated, suggesting that FGFs may be acting on other cell types to induce stem
cell activity in vitro. Receptors for FGF-1 have been shown to be present on primitive
hematopoietic cell subsets2. Therefore, it is also highly probable that FGFs maintain
Summarizing Discussion and Future Perspectives
135
stem cells by acting on FGFRs expressed on the stem cells. Additionally, non-LSK
cells present in the stem cell niche may carry FGFRs and therefore be responsive to
FGFs, playing an important role in the maintenance of stem cells. We speculate that
BM elements in the co-culture act as a pseudo niche facilitating the proliferation of
stem cells in vitro. These results demonstrate that we can maintain stem cell in culture
for up to five weeks with LTRA.
Quantitative PCR (QPCR) analysis in Chapter 3 demonstrated that FGF-1 and FGF-2
were predominantly expressed in LSK cells. The presence of both FGFRs2 and high
expression levels of FGFs on LSK cells strongly implies that FGFs may regulate
HSCs by autocrine signaling. To further examine the role of FGFs on HSCs, in
Chapter 3 we retrovirally overexpressed FGF-1 and FGF-2 in 5-Fluorouracil (5-FU)
treated BM cells. In addition, to assess the role of nucleocytoplasmic trafficking of
FGFs in hematopoietic cells, two mutant isoforms which altered the phosphorylation
status of FGF-1 were created and overexpressed. In the first mutant, the serine residue
at phosphorylation site 130 was exchanged for glutamic acid (S130E). This mutant
was expected to mimic the phosphorylated state of FGF-1, constitutively exporting
FGF-1 to the cytoplasm3. For the second mutant, the serine was exchanged for alanine
(S130A) which was hypothesized to prevent FGF-1 phosphorylation and remain
localized in the nucleus3. Higher molecular weight isoforms of human FGF-2 (21-22,
22.5, 24 and 34kDa) contain and upstream nuclear localization signal (NLS) which
translocates FGF-2 into the nucleus4;5. The smaller 18kDa isoform is confined to the
cytoplasm6-8. To create a nuclear localized isoform of mouse FGF-2, an artificial NLS
was inserted upstream of FGF-2 (NLS/FGF-2).
Fluorescent images of hematopoietic cells overexpressing wild-type (WT) FGF-1,
S130E or WT FGF-2 retroviral vectors showed that both FGF-1 and FGF-2 were
expressed predominantly in the cytoplasm. In contrast, overlay images of
hematopoietic cells overexpressing S130A and NLS/FGF-2 revealed that 2% of FGF-
1 positive cells and 11% of FGF-2 positive cells were localized in the nucleus
respectively. Although FGF-1 and FGF-2 mutant proteins were not predominantly
localized in the nucleus, compared to WT FGF-1, S130E and WT FGF-2 nuclear
localization did increase. More sensitive methods such as cell fractionation studies
should be performed to clarify these results. Recently, using cell fractionation studies,
Wiedlocha et al. showed that WT FGF-1 was found in all fractions (membrane,
cytoplasmic and nucleus) of the cell, S130A was found in the nuclear fraction
Summarizing Discussion and Future Perspectives
136
whereas S130E was mainly in the cytosolic fraction9. These data indicated that the
translocation of FGFs appears to be a dynamic process and each FGF can be regulated
differently.
Unfortunately, transplantation studies with WT FGF-1, WT FGF-2, S130E and
NLS/FGF-2 overexpressing cells resulted in low donor chimerism levels in primary
recipients and little to no reconstitution in secondary recipients. This was unexpected
as previous studies from our group (as described in Chapter 2) had shown that the
unfractionated BM cells treated with exogenously added FGF-1 and/or FGF-2 were
capable of long-term repopulation2;10. These results suggest that the constitutive
overexpression of FGF-1 and FGF-2 does not increase the repopulating potential of
hematopoietic cells. Interestingly, secondary recipients transplanted with S130A
overexpressing cells showed a delayed but marked increase in donor chimerism levels
and a significant increase in GFP+ cells. These results strongly suggest that nuclear
localized FGF-1 may play an important role in maintaining stem cell quality.
In vivo competitive transplantation assay is the ‘gold standard’ to test whether BM
derived cells are indeed HSCs with the potential for reconstituting all hematopoietic
lineages. The competitive repopulation assay11 has two key features. The first is that it
enables the detection of a very primitive class of hematopoietic stem cells and the
survival of lethally irradiated mice transplanted with very low numbers of such cells.
The second is the use of a limiting dilution experimental design to allow stem cell
quantitation.
To highlight the effectiveness of this assay, in Chapter 4 we use the competitive
transplantation assay to compare the functional qualities of mobilized peripheral
blood (MPB) stem cells to normal BM stem cells. Mobilized peripheral blood has
been used for the past 10 years in place of BM as a source of stem cells for
transplants. Their ease of collection and ability to promote faster regeneration of
neutrophils12;13 and platelets makes them a primary source for HSC transplantation.
Transplantation of MPB stem cells in competition with BM stem cells demonstrated
that MPB stem cells have a reduced long term repopulation potential. This impairment
in repopulation potential was due to the presence of fewer stem cells rather than a
decrease in stem cell quality. In actual fact, secondary transplantation of MPB stem
cells indicated that the quality of stem cells from MPB did not decrease after
transplantation and that exhaustion of initially engrafted stem cells is similar for both
Summarizing Discussion and Future Perspectives
137
BM and MPB stem cells. Clearly, in a clinical setting, more blood stem cells must be
transplanted to compensate for the decrease frequency of stem cells.
Future Perspectives In this thesis the exogenous and endogenous effects of FGF-1 and FGF-2 were
examined. We have shown that FGFs, in particular FGF-1 and FGF-2 play an
important role in regulating and maintaining stem cells.
In total, 22 FGFs exists (not including spliced forms) and we have shown that two out
of 22 FGFs are able to maintain HSCs. Most stem cell studies carried out are
restricted to FGF-1 or FGF-2. From mouse knock-out studies it has appeared that
other FGFs are more potent. For example, deletion of FGF-414, FGF-815-18, FGF-919
and FGF-1020 result in embryonic lethality whereas FGF-1 and FGF-2 knockout
mice21 are viable and fertile. Given their pleiotropic effects, similar receptor binding
properties, overlapping patterns of expression and sequence similarities, functionally
redundancy is likely to occur. It will be interesting to assess the effects of other FGFs,
in particular FGF-4, FGF-8, FGF-9 and FGF-10 on stem cells. Such studies would
increase our understanding on the biological role of FGFs on HSC maintenance and
regulation.
The mechanistic action of FGFs on stem cells remains unknown. Our results suggest
that the intracellular function of nuclear localized FGF-1 is biologically significant.
This indicates that the nuclear import/export trafficking pathway of FGFs may be key
to understanding the mechanistic action of FGFs. Future studies should be aimed at
assessing the trafficking pathway of FGFs in stem cells. Firstly, it will be interesting
to determine which FGFs bind to which FGFR and to what affinity. It should be noted
that a large number of splice variants of FGFR genes exist and must be taken into
account. Secondly, it would be appealing to determine the stage of the cell cycle
which enables the precise cueing of the nuclear localization of FGFs. This may
provide valuable information as to how nuclear localized FGF-1 (S130A) maintained
stem cell quality. Thirdly, it would be interesting to determine whether all FGFs,
which are highly homologous, have the same trafficking pathway.
The competitive transplantation assay serves as the only tool to detect long-term
repopulating stem cells with the potential for reconstituting all hematopoietic lineages.
We highlighted the effectiveness of this assay to study the qualities of MPB stem cells
Summarizing Discussion and Future Perspectives
138
compared to normal BM stem cells. Blood cells mobilized with Granulocyte-Colony
Stimulating Factor (G-CSF) have reduced repopulation ability due to a lower
frequency of stem cells. Many growth factors capable of migrating stem cells from the
BM to the blood exist. Each mobilizing agent, alone or in combination with
chemotherapeutic agents affects the stem cell differently. It will therefore be
interesting to examine the effects of different mobilization regimes and whether this
will improve the stem cell frequency and repopulating ability of blood stem cells. This
knowledge may be relevant and change techniques used in established clinical
application.
Summarizing Discussion and Future Perspectives
139
References 1. Schofield R. The relationship between the spleen colony-forming cell and the
haemopoietic stem cell. Blood Cells 1978;4:7-25.
2. de Haan G, Weersing E, Dontje B et al. In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1. Developmental Cell 2003;4:241-251.
3. Klingenberg O, Widlocha A, Rapak A et al. Inability of the acidic fibroblast growth factor mutant K132E to stimulate DNA synthesis after translocation into cells. J.Biol.Chem. 1998;273:11164-11172.
4. Arnaud E, Touriol C, Boutonnet C et al. A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. Mol.Cell Biol. 1999;19:505-514.
5. Dono R, James D, Zeller R. A GR-motif functions in nuclear accumulation of the large FGF-2 isoforms and interferes with mitogenic signalling. Oncogene 1998;16:2151-2158.
6. Bugler B, Amalric F, Prats H. Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor. Mol.Cell Biol. 1991;11:573-577.
7. Florkiewicz RZ, Baird A, Gonzalez AM. Multiple forms of bFGF: differential nuclear and cell surface localization. Growth Factors 1991;4:265-275.
8. Quarto N, Finger FP, Rifkin DB. The NH2-terminal extension of high molecular weight bFGF is a nuclear targeting signal. J.Cell Physiol 1991;147:311-318.
9. Wiedlocha A, Nilsen T, Wesche J et al. Phosphorylation-regulated nucleocytoplasmic trafficking of internalized fibroblast growth factor-1. Mol.Biol.Cell 2005;16:794-810.
10. Yeoh JS, van Os R, Weersing E et al. Fibroblast growth factor-1 and 2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures. Stem Cells 2006;24:1564-1572.
11. Harrison DE. Competitive repopulation: a new assay for long-term stem cell functional capacity. Blood 1980;55:77-81.
12. Goldman J. Peripheral blood stem cells for allografting. Blood 1995;85:1413-1415.
13. Korbling M, Champlin R. Peripheral blood progenitor cell transplantation: a replacement for marrow auto- or allografts. Stem Cells 1996;14:185-195.
14. Feldman B, Poueymirou W, Papaioannou VE, DeChiara TM, Goldfarb M. Requirement of FGF-4 for postimplantation mouse development. Science 1995;267:246-249.
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15. Meyers EN, Lewandoski M, Martin GR. An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat.Genet. 1998;18:136-141.
16. Reifers F, Bohli H, Walsh EC et al. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 1998;125:2381-2395.
17. Shanmugalingam S, Houart C, Picker A et al. Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon. Development 2000;127:2549-2561.
18. Sun X, Meyers EN, Lewandoski M, Martin GR. Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 1999;13:1834-1846.
19. Colvin JS, Green RP, Schmahl J, Capel B, Ornitz DM. Male-to-female sex reversal in mice lacking fibroblast growth factor 9. Cell 2001;104:875-889.
20. Sekine K, Ohuchi H, Fujiwara M et al. Fgf10 is essential for limb and lung formation. Nature Genetics 1999;21:138-141.
21. Miller DL, Ortega S, Bashayan O, Basch R, Basilico C. Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice. Mol.Cell Biol. 2000;20:2260-2268.
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Nederlandse samenvatting Stamcellen zijn primitieve cellen met het vermogen zichzelf te vernieuwen en te
differentiëren in andere celtypen. Ze hebben het unieke vermogen schade in weefsels
te herstellen en/of andere cellen te activeren voor dit herstelproces. Stamcellen
worden onderverdeeld in embryonale of volwassen stamcellen. De studie beschreven
in dit proefschrift is gefocust op stamcellen uit het beenmerg (BM), die bloedcellen
produceren. Deze cellen worden hematopoietische stamcellen genoemd (HSCs).
HSC’s zijn in staat zich te vernieuwen en te differentiëren in een verscheidenheid van
gespecialiseerde bloedcellen zoals rode- (erytrocyten) en witte bloedcellen
(leukocyten). HSCs zijn verantwoordelijk voor het dagelijks aanmaken van miljarden
bloedcellen. Het is vanwege deze eigenschappen dat HSCs in de kliniek gebruikt
worden bij de behandeling van kanker, voornamelijk leukemie en lymfoma, kankers
onstaan uit bloedcellen. Ze worden na zware chemotherapie en/of radiotherapie
getransplanteerd om vernieuwe bloedcelaanmaak te garanderen.
HSCs kunnen geïsoleerd worden uit het BM en de bloedcirculatie. Het aantal
stamcellen in beenmerg en de bloedcirculatie is echter erg klein, daar er in BM slechts
minder dan 1 stamcel per 105 BM cellen aanwezig is. Het gevolg hiervan is dat
onderzoekers zijn gaan zoeken naar kweekmethodes met als doel meer primitieve
HSCs te genereren voor transplantatie. Om dit te verwezenlijken hebben onderzoekers
muizen-HSCs blootgesteld aan groeifactoren in een in vitro milieu.
Groeifactoren zijn kleine eiwitten die op cellulair niveau verschillende effecten
teweeg kunnen brengen. Er bestaan vele soorten groeifactoren. In dit proefschrift zijn
voornamelijk proeven beschreven die zich richten op de effecten van fibroblast
groeifactoren (FGFs) op de HSCs. FGFs zijn groeifactoren die specifiek reageren op
fibroblasten door het hele lichaam. Fibroblasten produceren de bouwstoffen van
fibreus weefsel, dat overal in het lichaam te vinden is. Tot op heden zijn 22 FGFs van
mens en muis bekend. FGFs zijn vooral bekend van wege hun functie tijdens
embryogenese wanneer de FGFs de ontwikkeling van organen zoals de longen en
ledematen besturen. Bij volwassenen spelen FGFs een belangrijke rol bij herstel van
weefsels, regeneratie, metabolisme en angiogenesis. FGFs verzorgen een biologische
respons door zich te binden aan gespecialiseerde eiwitten die receptoren genoemd
worden. Tot op heden zijn er in gewervelde dieren vier FGF-receptoren (FGFR)
geïdentificeerd, namelijk Fgfr1- Fgfr4.
Nederlandse Samenvatting
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Dit proefschrift richt zich op handhaving en groei van jonge HSCs onder invloed van
FGFs in zowel in vitro als in vivo studies. Hoofdstuk 1 geeft een algemeen overzicht
van de regulerende rol van FGFs bij de handhaving van zich zelf vernieuwende
stamcellen om veroudering van HSCs, neurale stamcellen (NSCs) en embryonale
stamcellen (ES) tegen te gaan. Veroudering gaat gepaard met een langzaam
achteruitgaan van weefselfunctie, inclusief het afnemen van de groei van nieuwe
cellen en een afname van het herstelvermogen. Veroudering wordt ook geassocieerd
met de toename van het ontstaan van kanker in alle weefsels waarin stamcellen
aanwezig zijn. Dit geeft aan dat de balans tussen proliferatie, overleving en
differentiatie van stamcellen streng gereguleerd moet zijn. Deze waarnemingen
suggereren dat er een link bestaat tussen verouderingsprocessen en de rol van
stamcellen in het vernieuwingproces van stamcellen. We veronderstellen dat FGFs
met hun receptoren weefsel-homeostase tijdens veroudering ondersteunen door
vernieuwing, handhaving en proliferatie van HSCs, NSCs en ES te reguleren.
In hoofdstuk 2 bestudeerden we de effecten van de groeifactoren FGF1, FGF2, alleen
en in combinatie, op normale muizen BM cellen in kweken met serum-vrij medium.
De BM cellen die op deze manier, gedurende 1,3 en 5 weken met FGFs gekweekt zijn
werden onderzocht op hun vermogen gedurende lange tijd nieuwe bloedcellen te
vormen na transplantatie. Daartoe werden ze gemengd met vers geïsoleerde BM
cellen. Dit celmengsel werd geïnjecteerd in muizen die vooraf een hoge
bestralingsdosis toegediend kregen om er voor te zorgen dat de endogene cellen van
de muis niet meer functioneel zouden zijn. De gekweekte cellen en de vers
geisoleerde BM cellen verschillen op het Ly5 (CD45) locus, wat ervoor zorgt dat de
cellen aan de hand van hun fenotype nog te onderscheiden zijn na transplantatie. Deze
manier van transplantatie staat bekend als de competitieve transplantatie assay, en
stelt ons in staat te bewijzen dat met FGF gekweekte BM cellen inderdaad HSCs zijn.
Na transplantatie werd het bloed van de getransplanteerde muis geanalyseerd.
Wanneer getransplanteerde cellen, die behandeld waren met FGF-groeifactoren, na 24
weken terug gevonden worden in het bloed kan aangenomen worden dat er stamcellen
aanwezig waren. Er wordt in dat geval aangenomen dat deze stamcellen zorgen voor
kunnen lange termijn repopulatie, dus permanente bloedeelvorming. In hoofdstuk 2
laten we zien dat de stamcelactiviteit van BM cellen gekweekt met FGFs tot 5 weken
in stand gehouden kan worden, doordat deze cellen in de ontvanger muizen een lange
termijn repopulatie teweeg brengen. Vervolgens is gekeken of deze stamcelactiviteit
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146
afkomstig was van al bestaande HSCs of dat ze ontstaan zijn uit een andere populatie
cellen. Stamcellen uit BM kunnen gezuiverd worden door selectie op basis van hun
primitieve HSC-fenotypering. Stamcellen hebben geen lineage marker, maar wel Sca-
1 and c-Kit stamcel-markers (LSK). Deze cellen worden LSK-cellen genoemd. De
LSK cellen werden gezuiverd en gedurende 5 weken verder gekweekt en in
combinatie met normale BM cellen met alleen FGF-1+2 in serum vrij medium. Na 5
weken werden alle cellen getransplanteerd in muizen die een dodelijke
bestralingsdosis toegediend hadden gekregen. Analyse van het bloed liet zien dat de
geïsoleerde en gezuiverde stamcellen bijdragen aan de bloedcelvorming na
transplantatie. Dus de stamcelactiviteit in FGF gestimuleerde BM kweken is
afkomstig van LSK stamcellen.
In hoofdstuk 3 onderzoeken we de rol van FGFs bij de intrinsieke regulatie en
handhaving van HSCs. De hypothese was dat FGFs van belang zijn voor het
handhaven van de stamcelactiviteit van HSC. Als het niveau van FGFs in stamcellen
verhoogd zou worden door retrovirale overexpressie, zou de stamcelactiviteit
verhoogd worden of langer gewaarborgd kunnen zijn. Om dit te onderzoeken zijn BM
cellen van met 5-fluorouracil (5-FU) behandelde muizen getransduceerd met FGF-1
of FGF-2. 5-Fluorouracil is een middel dat dient om beenmergcellen te verrijken voor
primitieve cellen en deze te activeren. Dit verbetert de transductie van stamcellen.
Het doel van de transductie was om in stamcellen een extra FGF-1 of FGF-2 gen in te
brengen. FGFs komen dan tot overexpressie hetgeen een hogere niveau van deze
eiwitten in de cel oplevert. De met het FGF-1 of FGF-2 gen getransduceerde cellen
werden getest in het in vivo competitieve transplantatiemodel. De cellen die extra
FGF-1 of FGF-2 produceren, hadden echter geen voordeel boven cellen die geen extra
FGFs produceerden. Ook op de lange termijn, na doortransplantatie van
getransduceerde cellen in secundaire ontvanger muizen, leverde een extra FGF-1 of
FGF-2 geen voordeel op voor hematopoietische stamcellen.
Er wordt gesuggereerd dat de locatie van FGFs in de cel belangrijk is voor zijn
functie. FGFs aanwezig binnen in de cellen zouden verantwoordelijk zijn voor de
mitogene effecten. Om dit te onderzoeken hebben we verscheidene mutanten van FGF
gecreëerd om de invloed van localisatie van FGF op zijn functie in hematopoietische
stamcellen te onderzoeken. Twee mutaties werden aangebracht in het FGF-1 gen.
Voor de eerste mutant werd het eiwit S130E ontwikkeld, dat de gefosforyleerde fase
van FGF-1 zou moeten nabootsen, waardoor deze naar het cytoplasma van de nucleus
Nederlandse Samenvatting
147
getransporteerd kon worden. De tweede mutant, S130A, bootst de niet
gefosforyleerde fase van FGF-1 na, en veronderstelt dat FGF-1 in de kern gehouden
wordt. Om de FGF-2 van het cytoplasma over te brengen naar de kern werd een
kunstmatig nucleair localisatiesignaal (NLS) ingebracht vóór het FGF-2 gen