- Email: [email protected]
Low c-Kit Expression Level Induced by Stem Cell Factor Does Not Compromise Transplantation of Hematopoietic Stem Cells
Accepted Manuscript Low c-Kit expression level induced by stem cell factor does not compromise transplantation of hematopoietic stem cells Chia-Ling Chen, MSc, Katerina Faltusova, MSc, Martin Molik, BSc, Filipp Savvulidi, BSc, Ko-Tung Chang, Ph.D., Emanuel Necas, M.D., Ph.D. PII:
S1083-8791(16)00166-X
DOI:
10.1016/j.bbmt.2016.03.017
Reference:
YBBMT 54230
To appear in:
Biology of Blood and Marrow Transplantation
Received Date: 20 November 2015 Accepted Date: 11 March 2016
Please cite this article as: Chen CL, Faltusova K, Molik M, Savvulidi F, Chang KT, Necas E, Low c-Kit expression level induced by stem cell factor does not compromise transplantation of hematopoietic stem cells, Biology of Blood and Marrow Transplantation (2016), doi: 10.1016/j.bbmt.2016.03.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Low c-Kit expression level induced by stem cell factor does not compromise transplantation of hematopoietic stem cells
Institute of Pathological Physiology, First Faculty of Medicine, Charles University in Prague,
Czech Republic 2
M AN U
1
SC
BSc1, Ko-Tung Chang, Ph.D.2#, Emanuel Necas, M.D., Ph.D.1#
RI PT
Chia-Ling Chen, MSc1,2*, Katerina Faltusova, MSc1*, Martin Molik, BSc1, Filipp Savvulidi,
Department of Biological Science and Technology, National Pingtung University of Science
TE D
and Technology, Pingtung, Taiwan
*C-L.Chen and K.Faltusova contributed equally to the paper
E.Necas ([email protected]) and K-T.Chang ([email protected]): corresponding
AC C
authors
EP
#
Short title: c-Kit and Bone Marrow Transplantation
The author for communication Emanuel Necas, M.D., Ph.D. Institute of Pathological Physiology 1st Faculty of Medicine
ACCEPTED MANUSCRIPT Charles University in Prague, U Nemocnice 5, 128 53 Prague Czech Republic Telephone:+420-224965901, Fax:+420-224965916
RI PT
e-mail: [email protected]
SC
ABSTRACT
M AN U
The c-Kit expression level is decreased in regenerating bone marrow and such bone marrow performs poorly when co-transplanted with normal bone marrow. We asked whether diminished numbers of c-Kit receptors on HSPCs following their internalization induced by the binding of the cytokine stem cell factor (SCF) would jeopardize transplantability of
TE D
HSPCs. We used a battery of functional assays to evaluate the capacity of HSPCs with a markedly different c-Kit expression levels to be transplanted. Surprisingly, our experiments testing the homing of transplanted HSPCs to bone marrow of recipient mice and their short-
EP
term and long-term engraftment did not reveal any defects in HSPCs with severely reduced numbers of c-Kit receptor molecules. This unexpected result can be ascribed to the fact that
AC C
HSPCs exposed to SCF replace the consumed c-Kit receptors rapidly. The paper demonstrates that exposure of HSPCs to SCF and diminished number of c-Kit receptors in their cell membranes do not compromise the capacity of HSPCs to reconstitute damaged hematopoietic tissue.
Key words: c-Kit receptor, hematopoietic stem cell, bone marrow transplantation, stem cell factor, irradiation
ACCEPTED MANUSCRIPT Introduction In their seminal paper Harrison and Astle 1 reported that bone marrow from sublethally irradiated donors performed very poorly in competitive repopulation assays. This phenomenon has not been fully deciphered to this day. Interestingly, Simonnet et al. 2
RI PT
reported significantly decreased expression level of the c-Kit receptor in hematopoietic stem and progenitor cells (HSPCs) of mice recovering from sublethal irradiation. The c-Kit
tyrosine kinase receptor is a fundamental marker of mouse hematopoietic stem and progenitor
. c-Kit is activated after binding its ligand, the stem cell factor (SCF; also known as c-Kit
M AN U
5
SC
cells (HSPCs) 3,4 important for HSPCs survival, proliferation or quiescence and self-renewal
ligand, steel factor or mast cell growth factor). c-Kit receptors form homodimers after binding SCF and are internalized and degraded 6. The SCF gene is significantly up-regulated in bone marrow damaged by irradiation 7,8 which could cause the low c-Kit expression level
TE D
in HSPCs of sublethally irradiated mice due to enhanced c-Kit consumption. During studies into the bone marrow regeneration from a small number of repopulating cells, we were confronted with poor performance of regenerating bone marrow when competing
EP
with normal bone marrow, and with low c-Kit expression level in HSPCs. We hypothesized that a particular density of c-Kit receptors on transplanted HSPCs could determine which type
AC C
of the supporting niche the cell would engraft into. The high density of c-Kit on HSPCs from normal bone marrow would allow to the cells to engraft into highly supportive niches. On the other hand, the low density of c-Kit on HSPCs from regenerating bone marrow would direct the cells towards less supportive niches. The notion was indirectly supported by the role of c-Kit/membrane-bound SCF interactions in the initial lodgement of transplanted stem cells 9 and in the functional positioning of stem cells to their niches 10. We decided to test the hypothesis by downregulation of c-Kit with exogenous SCF, and by testing the impact of markedly decreased c-Kit receptor density on the outcome of HSPC
ACCEPTED MANUSCRIPT transplantation. Results of this study convincingly demonstrated that an exposure of HSPCs to high SCF concentrations causing significant downregulation of c-Kit receptors in HSPCs, is not harmful in regard to their capacity to be transplanted. However, the hypothesis assuming that the density of c-Kit receptors predisposes transplanted HSPCs to engraft the niches with
RI PT
different supportive capacity could not be conclusively refuted because the HSPCs exposed to SCF replaced the internalized and consumed c-Kit receptors rapidly.
SC
Materials and Methods
M AN U
Mice
C57BL/6NCrl (CD45.2), B6.SJL-Ptprca Pepcb/BoyJ (CD45.1), dual CD45.2/CD45.1 F1 hybrid mice (F1) and C57Bl/6-Tg(UBC-GFP)30Scha/J mice were bred in the institutional animal facility and kept in a clean conventional part of the facility during the experiments.
TE D
Three-month to six-month-old adult mice were used in the experiments. The experiments were approved by the Laboratory Animal Care and Use Committee of the First Faculty of
Republic.
AC C
Cytokines
EP
Medicine, Charles University, and the Ministry of Education, Youth and Sports of the Czech
Recombinant mouse Stem Cell Factor (SCF) was bought from PeproTech (USA), catalog numbers # 250-03 and AF-450-33, respectively. SCF was dissolved in deionized water at a concentration of 10 µg/mL and stored at -80 °C in an ultra-low temperature freezer.
Exposure of bone marrow to SCF in vitro
Bone marrow cells (BMCs) obtained from normal mice were incubated in the Dulbecco´s Minimum Essential Medium (DMEM) for 60 minutes in presence/absence of SCF. Cell were
ACCEPTED MANUSCRIPT then stained for flow cytometry analysis to determine the median fluorescence intensity (MFI) of the anti-c-Kit antibody in immature cells negative for lineage markers (Lin-).
Administration of SCF in vivo
RI PT
SCF was injected intraperitoneally (i.p.) or intravenously (i.v.) to normal adult mice
C57BL/6NCrl, males or females, in the amount 1000 ng per mouse. Mice were sacrificed from 1 hour to 46 hours afterwards. Bone marrow was collected from one femur and analyzed
SC
for c-Kit median fluorescence intensity (MFI) on immature hematopoietic cells. The MFI values were compared to the corresponding MFI values of bone marrow from an untreated
M AN U
mouse which were taken to be 100% in each experiment.
Antibodies
An A700- or FITC-conjugated anti-Lineage cocktail of antibodies (B220, CD3, Gr-1, Mac-1,
TE D
and Ter-119), PE/CY7-conjugated anti-Sca-1, BV421-conjugated anti-c-Kit, APC-conjugated
USA.
Flow cytometry
EP
anti-CD150, and FITC-conjugated anti-CD48 antibodies were used, all from BioLegend, CA,
AC C
BMCs were analyzed using a digital FACS Canto II flow cytometer, equipped with 405 nm (60 mW), 488 nm (20 mW) and 633 nm (15 mW) laser lines (BD Biosciences, USA). Briefly, after multicolor staining bone marrow cells were filtered using 70µm Nylon Cell Strainers (BD Falcon, USA) and were analyzed by the flow cytometer. BD FACS Diva software version 6.1.3 was used for data acquisition. CS&T beads (BD Biosciences, USA) were used for the automated cytometer setup and the performance tracking procedure before measurements. The proper compensation matrix was created by running single-stained control samples (automatic compensation). The compensation matrix was then controlled and
ACCEPTED MANUSCRIPT manually adjusted (if necessary) for each measurement. The generated flow cytometry data were analyzed using FlowJo vX software (Treestar, USA). To properly interpret flow cytometry data, Fluorescence-Minus-One (FMO) controls were used for gating.
RI PT
c-Kit fluorescence intensity (MFI) Anti-c-Kit antibody minus one samples (FMO) were used to distinguish between c-Kit+ and c-Kit- BMCs. c-Kit+ cells lacking lineage markers, Lin-c-Kit+ cells, were divided into Sca-1
SC
negative (LS-K) and Sca-1 positive (LSK) cells (Fig. 1). LSK cells were further classified into four types characterized by the specific patterns of CD150 and CD48 markers according to
M AN U
Kiel et al. 11. The median fluorescence intensity (MFI) thus indicates the fluorescence intensity of the anti-c-Kit antibody of the particular cell type, recorded over the entire range of c-Kit intensities. An example of normal and depressed c-Kit fluorescence intensity in LSK
TE D
cells is shown in Fig. 3.
Colony-forming units-spleen (CFU-S) assay
Male C57BL/6 mice were irradiated with 9 Gy and injected intravenously with 6 x 104 bone
EP
marrow cells incubated for 60 minutes with/without SCF. Mice were sacrificed 8 or 10 days after the injection, spleens were fixed in Tellesniczsky´s solution (1:1:20 ratio of
AC C
formalin/acetic acid/70% alcohol) and colonies were counted on the convex surface of the spleens.
Homing of transplanted Lin-c-Kit+ cells
Recipient female CD45.1 mice were irradiated with a sublethal dose of 4 Gy three days before the transplantation of congenic BMCs originating either from CD45.2 mice or from UBCGFP transgenic mice. Before transplantation, BMCs were split into two equal parts and
ACCEPTED MANUSCRIPT incubated for 60 min at 37oC with/without 500 or 1000 ng of SCF per mL in a DMEM medium with 1% bovine serum albumin. The cells were then washed with PBS, a sample was withdrawn to be stained with anti-lineage cocktail, anti-Sca-1 antigen and anti-c-Kit antibodies for analysis of the c-Kit expression level. The remaining cells were transplanted to
RI PT
recipient mice through the retro-orbital venous plexus. Seventeen or 20 hours later,
transplanted mice were sacrificed, the femoral bone marrow was collected, stained with antilineage cocktail, anti-Sca-1 and anti-c-Kit antibodies, or screened for the CD45.1 and CD45.2
SC
allotype or GFP+ and GFP- BMCs. Flow cytometry was used to determine the percentage of
M AN U
the donor LS-K and LSK cells in the bone marrow of recipient mice.
Competitive bone marrow cell transplantation
Three variants of competitive transplantation assays were employed: (1) BMCs incubated with/without SCF were transplanted into sublethally (6 or 6.5 Gy) irradiated congenic
TE D
recipients to compete with repopulating cells surviving in irradiated mice; (2) tested BMCs, or Lin-c-Kit+ separated cells, from two congenic mice strains were incubated with/without SCF, the samples exposed to SCF were mixed 1:1 with the congenic control samples incubated
EP
without SCF, and the mixtures were transplanted into irradiated F1 hybrid recipients; (3) BMCs from CD45.1 mice were incubated with/without SCF and transplanted into non-
AC C
irradiated or mildly (1 Gy) irradiated transgenic UBC-GFP mice.
Analysis of chimeric hematopoiesis in transplanted mice
At various times after transplantation, a sample of peripheral blood was obtained from the retro-orbital sinus with 75 mm/60 µL heparinized capillary tubes. Samples were washed and stained with FITC-conjugated anti-Ly5.1 and A700-conjugated anti-Ly5.2 antibodies (BioLegend, CA). The percentage of donor-derived nucleated blood cells was determined
ACCEPTED MANUSCRIPT after 4 weeks, primarily reflecting the hematopoietic activity of the transplanted short-term repopulating cells (STRCs), and then up to 9 months, reflecting the hematopoietic activity of the long-term repopulating cells (LTRCs).
RI PT
Statistical analysis
Statistical analysis was performed with Graph Pad Prism version 5 (GraphPad Software, CA). Unpaired two-tailed t-test was used to compare results from corresponding experimental
SC
groups. P values < 0.05 were considered statistically significant. * P<0.05; ** P<0.01; ***
M AN U
P<0.001.
Results
Regenerating bone marrow performs poorly in transplantation assays Fig. 2A shows spleen colonies generated by injecting 105 normal BMCs and 5x105 bone
TE D
marrow cells collected 16 days after irradiation of mice with a sublethal dose of 6 Gy. The percentage of Lin-c-Kit+ cells in compared bone marrow was 2.95% in the normal bone marrow and 0.75% in the regenerating bone marrow. The regenerating bone marrow was ~ 6
EP
times less efficient in generating day-12 spleen colonies compared to normal bone marrow.
AC C
The low transplantation potential of regenerating bone marrow was highly expressed when an equal number of normal and regenerating Lin-c-Kit+ cells collected 14 days after irradiation were co-transplanted. There were 83 times more nucleated blood cells derived from normal bone marrow after 1 month and 480 times more after 6 months (Fig. 2B).
SCF downregulates c-Kit expression level in a dose-dependent manner Figure 3 shows that an exposure of bone marrow to SCF downregulates c-Kit in Lin-c-Kit+ cells proportionately to the SCF concentration.
ACCEPTED MANUSCRIPT c-Kit receptors consumed after binding SCF are replaced by new c-Kit receptors within 12-14 hours
For a proper interpretation of the results from competitive transplantation experiments, it was
RI PT
important to determine how rapidly the internalized and degraded c-Kit receptors can be replaced. As no data had been available on the ability of HSPCs to replace downregulated and consumed c-Kit receptors, we determined the c-Kit kinetics after their downregulation
SC
induced by SCF in several in vivo experiments. Mice were injected with SCF either
intraperitoneally or intravenously and their bone marrow was collected 1-46 hours later. c-Kit
M AN U
receptors were suppressed for 3 hours after SCF injection, but completely recovered by 12-14 hours after SCF administration (Fig. 4A,B).
The downregulation of c-Kit by SCF has no impact on the capacity of repopulating cells
TE D
to be transplanted
We tested possible deleterious effects of a severely downregulated c-Kit receptors on the homing of transplanted HSPCs to the bone marrow, their capacities to generate spleen
AC C
hematopoiesis.
EP
colonies (CFU-S), and to compete with normal bone marrow in the reconstitution of damaged
The homing of transplanted HSPCs to the bone marrow of sublethally irradiated
recipient mice is not affected by downregulated c-Kit
We downregulated c-Kit in the bone marrow collected from CD45.2 and UBC-GFP mice and transplanted it to congenic sublethally irradiated CD45.1 mice. Seventeen and 20 hours after the transplantation, the percentage of donor LS-K and LSK cells in all LS-K and LSK cells of recipient mice was determined. No significant differences were present between results from
ACCEPTED MANUSCRIPT transplantation of aliquots of the SCF-treated and control BMCs although they markedly differed in the c-Kit expression level (Fig. 5). Engraftment of transplanted bone marrow or sorted Lin-c-Kit+ cells is not affected
RI PT
by downregulated c-Kit
We downregulated c-Kit receptors by exposing BMCs to SCF in vitro and determined the capacity of the cells to establish blood cell production after transplantation. The colony-
SC
forming spleen assay (CFU-S) was not significantly affected by SCF (Fig. 6A). The next assay used a transplantation of the control and SCF-exposed bone marrow to sublethally
M AN U
irradiated mice with a different CD45 allotype and the detection of nucleated donor cells in the blood. This test is quantitative 12,13 and it should sensitively detect any diminished capacity of the SCF-exposed bone marrow to compete with the repopulating hematopoietic cells that survived in recipient mice 14. No significant difference in the engraftment of cells
TE D
with highly different densities of c-Kit receptors (c-Kit fluorescence profiles not shown) were found in two independent experiments (Fig. 6B, C). A competitive transplantation of normal and SCF-exposed samples of congenic (CD45.1 and CD45.2) bone marrow cells, which is the
EP
most frequently used assay to compare the capacity of HSPCs to reconstitute damaged hematopoiesis, did not demonstrate a significant effect of c-Kit downregulation on the ability
AC C
of BMCs to be transplanted either. (Fig. 6D). Due to reciprocal controls between CD45.1 and CD45.2 cells, the significant differences observed in the experiment could be ascribed by a higher content of long-term repopulating cells in the CD45.2 bone marrow donors, a phenomenon that had previously been reported 15. Next we sorted Lin-c-Kit+ cells from bone marrows collected from CD45.1 and CD45.2 mice. The cells were incubated with/without SCF and a 1:1 mixture of the congenic cells, mixing cells incubated with/without SCF reciprocally, were transplanted to irradiated dual CD45.2/CD45.1 F1 hybrid recipients. The
ACCEPTED MANUSCRIPT c-Kit expression level of CD45.1 and CD45.2 cells in transplanted cell mixtures differed markedly according to whether they were exposed or were not to SCF (not shown). No significant effect of the different c-Kit expression level was demonstrated in this experiment (Fig. 6E). Independently, we had noted that transgenic UBC-GFP mice 16 engraft congenic
RI PT
bone marrow without any conditioning or intensively after very low doses of irradiation. We used this feature of UBC-GFP mice to test whether a markedly different density of c-Kit
receptors would affect engraftment of HSPCs in the environment of completely undamaged or
SC
mildly damaged hematopoiesis of recipient mice. As in all previously described experiments, the capacity of repopulating donor cells to produce blood cells were not significantly altered,
M AN U
despite significant differences in the level of c-Kit expression (Fig. 6F, G). Collectively, all of the various transplantation assays failed to demonstrate importance of the c-Kit expression level (manipulated by SCF) on the ability of bone marrow cells to home and engraft into the
Discussion
TE D
hematopoietic tissue of recipient mice.
EP
Stem cell factor (SCF) is a component of the niche that supports and controls HSPCs in the
AC C
bone marrow 9,17,18. Transplanted HSPCs have to re-establish the physiological interactions between c-Kit receptors and SCF present in their niches. Therefore, the intensity of c-Kit expression level in transplanted HSPCs might play a crucial role in the biology of HSPCs transplantation.
We speculated that the HSPCs with a high density of c-Kit receptors might engraft into more supportive niches after transplantation than the HSPCs expressing c-Kit at a low level. It has previously been shown that blocking c-Kit receptors with the anti-c-Kit antibody ACK2 significantly reduced their transplantation 19. Driessen et al.9 and Kimura et al. 17 then
ACCEPTED MANUSCRIPT demonstrated the significance of c-Kit for the initial lodgement of transplanted hematopoietic stem cells within the endosteal bone marrow region, and to the functional positioning of hematopoietic stem cells to the niche. Grinenko et al.20 and Shin et al.21 demonstrated that inherent small differences in the c-Kit expression level in hematopoietic stem cells influence
RI PT
their engraftment after transplantation. In present experiments, we utilized the ability of SCF to downregulate c-Kit receptors by inducing their internalization and degradation. However, in a striking contrast to our expectations, grossly diminished c-Kit expression level in HSPCs
SC
did not affect the homing/lodgement and engraftment of transplanted HSPCs in a battery of
M AN U
transplantation-based assays.
These results prompted us to establish whether HSPCs can replace the consumed c-Kit receptors and how rapidly this can be achieved. All types of HSPCs reconstituted normal c-Kit density within 12-14 hours after SCF administration to mice. SCF is rapidly cleared from the circulation of mice with a half-life of ̴2 hours after intravenous application
22
, and
TE D
our results thus show that HSPCs exposed to SCF can rapidly reconstitute the density of c-Kit receptors to their inherently programmed level. The rapid dynamics of c-Kit receptors is therefore important for a proper interpretation of our transplantation tests that did not
EP
demonstrate any significant effect of a widely varying c-Kit density in transplanted HSPCs
AC C
upon their transplantation.
Previous experiments which demonstrated significance of c-Kit in the homing and lodgement of transplanted HSPCs 9,19 and in the positioning of HSPCs in their niches 17 utilized either the blockade of c-Kit receptors by the ACK2 antibody 9,19 or HSPCs with inherently diminished c-Kit function 17. Broudy et al.19 used functional colony-forming assays to measure transplanted HSPCs in bone marrow of lethally irradiated recipients after blocking c-Kit for 4 hours prior to the transplantation. Driessen 9 and co-workers localized the transplanted LinSca-1+c-Kit+ cells in the endosteal bone marrow region directly. In contrast, the experimental
ACCEPTED MANUSCRIPT approaches that we utilized to manipulate the c-Kit expression level in HSPCs were more physiological and fully reversible. We also used a direct determination of transplanted cells by flow cytometry in the bone marrow of recipients in our homing/lodgement experiments. Driessen et al.9 demonstrated a decreased proportion of transplanted cells with blocked c-Kit
RI PT
receptors within the endosteal region 15 hours post-transplant but the number of transplanted cells detected per recipient femoral section was not, in agreement with results, altered. Importantly, our experimental setting corresponded to conditions to which HSPCs are
SC
exposed during their generation or expansion ex vivo. It is thus significant that we
M AN U
demonstrated the ability of HSPCs to rapidly replace c-Kit receptors consumed after their activation by SCF, and that even markedly reduced numbers of c-Kit receptors do not hamper the ability of HSPCs to be transplanted.
The tested hypothesis assuming that depressed c-Kit expression in HSPCs in regenerating
TE D
bone marrow significantly contributes to their inferiority in transplantation assays cannot be ultimately rejected on basis of our negative results regarding the impact of c-Kit downregulation on HSPCs transplantation. This is because the SCF induced downregulation
EP
of c-Kit receptor was a transient phenomenon lasting less than 12-14 hours, while the whole process of engraftment of transplanted HSPCs may require more time.
AC C
In summary, the paper demonstrates that HSPCs exposed to SCF do not show decreased transplantability in the aftermath of c-Kit receptor internalization and degradation. It also demonstrates that HSPCs efficiently replace the consumed c-Kit receptors.
ACKNOWLEDGMENTS
The study was supported by the Grant Agency of the Czech Republic: GACR 14-25515S. It also received institutional support from projects PRVOUK-P24/LF1, SVV 2014–260 033,
ACCEPTED MANUSCRIPT UNCE 204021 and BIOCEV CZ.1.05/1.1.00/02.0109. In part, the study was supported by the Ministry of Science and Technology in Taiwan (NSC 99-2314-B-020-001-MY3).
The authors indicate no potential conflicts of interest.
Authorship statement:
RI PT
Conflict of interest statement:
SC
C-L.C.: collection and analysis of data, manuscript writing; K.F.: collection and analysis of data, manuscript writing; M.M.: collection of data; F.S.: flow cytometry analyses supervision
M AN U
and cell sorting; K-T.C.: conception and design, final approval; E.N.: conception and design, data interpretation, manuscript writing, final approval.
1.
TE D
References
Harrison DE, Astle CM. Loss of stem cell repopulating ability upon transplantation. Effects of donor age, cell number, and transplantation procedure. J Exp Med.
EP
1982;156(6):1767-79. Available at:
AC C
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2186863&tool=pmcentrez &rendertype=abstract. Accessed February 3, 2016.
2.
Simonnet AJ, Nehmé J, Vaigot P, Barroca V, Leboulch P, Tronik-Le Roux D. Phenotypic and functional changes induced in hematopoietic stem/progenitor cells after gamma-ray radiation exposure. Stem Cells. 2009;27(6):1400-9. doi:10.1002/stem.66.
3.
Ogawa M, Matsuzaki Y, Nishikawa S, et al. Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med. 1991;174(1):63-71. Available at:
ACCEPTED MANUSCRIPT http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2118893&tool=pmcentrez &rendertype=abstract. Accessed April 24, 2015.
4.
Okada S, Nakauchi H, Nagayoshi K, Nishikawa S, Miura Y, Suda T. In vivo and in
RI PT
vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells. Blood. 1992;80(12):3044-50. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1281687. Accessed April 24, 2015.
Kent D, Copley M, Benz C, Dykstra B, Bowie M, Eaves C. Regulation of
SC
5.
hematopoietic stem cells by the steel factor/KIT signaling pathway. Clin Cancer Res.
6.
M AN U
2008;14(7):1926-30. doi:10.1158/1078-0432.CCR-07-5134.
Lennartsson J, Rönnstrand L. Stem cell factor receptor/c-Kit: from basic science to clinical implications. Physiol Rev. 2012;92(4):1619-49.
7.
TE D
doi:10.1152/physrev.00046.2011.
Geissler EN, Ryan MA, Housman DE. The dominant-white spotting (W) locus of the mouse encodes the c-kit proto-oncogene. Cell. 1988;55(1):185-92. Available at:
Limanni A, Baker WH, Chang CM, Seemann R, Williams DE, Patchen ML. c-kit
AC C
8.
EP
http://www.ncbi.nlm.nih.gov/pubmed/2458842. Accessed April 21, 2015.
ligand gene expression in normal and sublethally irradiated mice. Blood.
1995;85(9):2377-84. Available at: http://www.ncbi.nlm.nih.gov/pubmed/7537111. Accessed February 3, 2016.
9.
Driessen RL, Johnston HM, Nilsson SK. Membrane-bound stem cell factor is a key regulator in the initial lodgment of stem cells within the endosteal marrow region. Exp Hematol. 2003;31(12):1284-1291. doi:10.1016/j.exphem.2003.08.015.
ACCEPTED MANUSCRIPT 10.
Kimura Y, Ding B, Imai N, Nolan DJ, Butler JM, Rafii S. c-Kit-mediated functional positioning of stem cells to their niches is essential for maintenance and regeneration of adult hematopoiesis. PLoS One. 2011;6(10):e26918.
11.
RI PT
doi:10.1371/journal.pone.0026918.
Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial
12.
SC
niches for stem cells. Cell. 2005;121(7):1109-21. doi:10.1016/j.cell.2005.05.026.
Down JD, Tarbell NJ, Thames HD, Mauch PM. Syngeneic and allogeneic bone
M AN U
marrow engraftment after total body irradiation: dependence on dose, dose rate, and fractionation. Blood. 1991;77(3):661-9. Available at:
http://www.ncbi.nlm.nih.gov/pubmed/1991176. Accessed October 30, 2015.
Forgacova K, Savvulidi F, Sefc L, Linhartova J, Necas E. All hematopoietic stem cells
TE D
13.
engraft in submyeloablatively irradiated mice. Biol Blood Marrow Transplant. 2013;19(5):713-9. doi:10.1016/j.bbmt.2013.02.012.
Michalova J, Savvulidi F, Sefc L, Faltusova K, Forgacova K, Necas E. Hematopoietic
EP
14.
stem cells survive circulation arrest and reconstitute hematopoiesis in myeloablated
AC C
mice. Biol Blood Marrow Transplant. 2011;17(9):1273-81.
doi:10.1016/j.bbmt.2011.07.007.
15.
Waterstrat A, Liang Y, Swiderski CF, Shelton BJ, Van Zant G. Congenic interval of CD45/Ly-5 congenic mice contains multiple genes that may influence hematopoietic stem cell engraftment. Blood. 2010;115(2):408-17. doi:10.1182/blood-2008-03-
143370.
ACCEPTED MANUSCRIPT 16.
Schaefer BC, Schaefer ML, Kappler JW, Marrack P, Kedl RM. Observation of antigendependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell Immunol. 2001;214(2):110-22. doi:10.1006/cimm.2001.1895.
Kimura Y, Ding B, Imai N, Nolan DJ, Butler JM, Rafii S. c-Kit-Mediated Functional
RI PT
17.
Positioning of Stem Cells to Their Niches Is Essential for Maintenance and
Regeneration of Adult Hematopoiesis. Rota M, ed. PLoS One. 2011;6(10):e26918.
18.
SC
doi:10.1371/journal.pone.0026918.
Oguro H, Ding L, Morrison SJ. SLAM family markers resolve functionally distinct
M AN U
subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell. 2013;13(1):102-16. doi:10.1016/j.stem.2013.05.014.
19.
Broudy VC, Lin NL, Priestley G V, Nocka K, Wolf NS. Interaction of stem cell factor
TE D
and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen. Blood. 1996;88(1):75-81. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8704204. Accessed April 24, 2015.
Grinenko T, Arndt K, Portz M, et al. Clonal expansion capacity defines two
EP
20.
consecutive developmental stages of long-term hematopoietic stem cells. J Exp Med.
21.
AC C
2014;211(2):209-215. doi:10.1084/jem.20131115.
Shin JY, Hu W, Naramura M, Park CY. High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias. J Exp Med.
2014;211(2):217-31. doi:10.1084/jem.20131128.
22.
Lynch DH, Jacobs C, DuPont D, et al. Pharmacokinetic parameters of recombinant mast cell growth factor (rMGF). Lymphokine Cytokine Res. 1992;11(5):233-243.
ACCEPTED MANUSCRIPT Legends to figures
FIGURE 1 Gating strategy to analyze immature hematopoietic cells in the bone marrow with
RI PT
depressed c-Kit expression level. c-Kit FMO samples were used to delimit c-Kit+ cells. c-Kit+ cells lacking lineage markers (Lin-) were further characterized according to the expression of Sca-1 antigen and CD150 and
SC
CD48 markers. The mean values ± SD of the percentage of CD150/CD48 LSK subtypes are
M AN U
from 10 independent determinations.
FIGURE 2 Spleen colonies and blood cell production derived from transplanted normal and regenerating bone marrow.
(A) Lethally irradiated mice were transplanted with 105 normal bone marrow cells or with
TE D
5x105 bone marrow cells obtained 16 days after sublethal irradiation with 6 Gy. c-Kit expression profiles of Lin-c-Kit+ cells in the donor bone marrows was determined and the results are presented. Spleens were collected 12 days after transplantation. (B) Equal numbers
EP
(20000 per mouse) of Lin-c-Kit+ cells from normal bone marrow (CD45.1) and from bone
AC C
marrow collected 14 days after irradiation of mice (CD45.2) with 6 Gy were co-transplanted 1:1 to lethally irradiated CD45.1/CD45.2 F1 hybrid mice. The ratio of CD45.1 to CD45.2 nucleated blood cells was determined in peripheral blood 1 month and 6 months after transplantation. c-Kit expression profiles of Lin-c-Kit+ cells in the donor bone marrows was determined and the results are presented. FIGURE 3 Exposure to SCF in vitro decreased c-Kit receptors in Lin-c-Kit+ cells proportionally to the SCF concentration.
ACCEPTED MANUSCRIPT Aliquots of normal bone marrow cells were incubated for 60 minutes in DMEM without (Control) or with various concentrations of SCF. After incubation the cells were washed and stained with lineage cocktail antibodies and anti-Sca-1 and anti c-Kit antibodies. In experiments which tested the transplantability of HSPCs with downregulated c-Kit receptors
RI PT
(Figs. 5 and 6) the concentrations of SCF concentrations 100-1000 ng/mL were used.
FIGURE 4 c-Kit receptors are fully reconstituted on HSPCs within 12-14 hours after their
SC
consumption induced by SCF.
Bone marrow was collected 1-14 hours after 1000 ng of SCF given intraperitoneally (A) or
M AN U
after 1-46 hours after 1000 ng of SCF given intravenously (B). Control mice were given PBS and were included in each experiment. The median fluorescence intensity index (MFI) was related to the values in control bone marrow in various cell types in each experiment. Data pooled from six independent experiments are presented and each time point shows the results
TE D
from single mice.
FIGURE 5 Downregulation of c-Kit by SCF did not influence the homing of transplanted LS-
EP
K and LSK cells in the bone marrow of sublethally irradiated mice.
Bone marrow was split into two aliquots, incubated with/without SCF and transplanted, 11
AC C
million CD45.2 (A) or 40 million UBC-GFP cells (B), to congenic CD45.1 mice irradiated 3 days earlier with a dose of 4 Gy. After seventeen hours in the CD45.2 experiment (A) and 20 hours in the UBC-GFP experiment (B) the bone marrow of recipient mice was analyzed for the proportion of donor LS-K or LSK cells in all cells of the particular phenotype.
FIGURE 6 The downregulation of c-Kit by SCF did not influence the capacity of bone marrow cells or separated HSPCs to be transplanted.
ACCEPTED MANUSCRIPT (A) CFU-S assay: BMCs incubated with/without SCF were transplanted (60,000 cells per mouse) to 9-Gy-irradiated mice. Spleen colonies were counted after 8 or 10 days. (B-C) 10 million (B) or 8 million (C) BMCs incubated with/without SCF were transplanted to congenic sublethally irradiated (6 Gy or 6.5 Gy) mice. The downregulation of c-Kit (not shown) did not
RI PT
decrease the engraftment of transplanted cells. (D) BMCs from CD45.1 and CD45.2 mice were split into two aliquots and incubated with/without SCF. After washing, congenic
samples were mutually mixed in a 1:1 ratio, analyzed for c-Kit expression level, and co-
SC
transplanted to irradiated (6.5 Gy) dual CD45.2/CD45.1 F1 mice. CD45.2 cells were more represented independently whether they were or were not exposed to SCF. Therefore, no
M AN U
effect of the markedly different density of c-Kit receptors on the engraftment of transplanted HSPCs was demonstrated. (E) A similar experiment used double sorted Lin-c-Kit+ CD45.1 and CD45.2 cells (96% and 98% purity). The cells were split into two aliquots and incubated with/without SCF, washed, mixed in a 1:1 ratio and co-transplanted to irradiated (6.5 Gy) dual
TE D
CD45.2/CD45.1 F1 mice. Markedly different c-Kit expression level (not shown) had no significant effect on competing of the repopulating cells. (F,G) BMCs of CD45.1 mice were incubated with/without SCF and transplanted, 26 million cells to each normal (F) or 15
EP
million cells to each 1 Gy irradiated (G) UBC-GFP mouse. No effect of exposure to SCF was
AC C
demonstrated. Data from individual mice and their means are presented throughout the figure.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights 1. Regenerating HSPCs suffer from transplantation defect and have a low c-Kit 2. SCF efficiently downregulate c-Kit in HSPCs 3. HSPCs with different density of c-Kit engraft equally
AC C
EP
TE D
M AN U
SC
RI PT
4. c-Kit down-regulated by SCF is replaced within 12 hours
S1083-8791(16)00166-X
DOI:
10.1016/j.bbmt.2016.03.017
Reference:
YBBMT 54230
To appear in:
Biology of Blood and Marrow Transplantation
Received Date: 20 November 2015 Accepted Date: 11 March 2016
Please cite this article as: Chen CL, Faltusova K, Molik M, Savvulidi F, Chang KT, Necas E, Low c-Kit expression level induced by stem cell factor does not compromise transplantation of hematopoietic stem cells, Biology of Blood and Marrow Transplantation (2016), doi: 10.1016/j.bbmt.2016.03.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Low c-Kit expression level induced by stem cell factor does not compromise transplantation of hematopoietic stem cells
Institute of Pathological Physiology, First Faculty of Medicine, Charles University in Prague,
Czech Republic 2
M AN U
1
SC
BSc1, Ko-Tung Chang, Ph.D.2#, Emanuel Necas, M.D., Ph.D.1#
RI PT
Chia-Ling Chen, MSc1,2*, Katerina Faltusova, MSc1*, Martin Molik, BSc1, Filipp Savvulidi,
Department of Biological Science and Technology, National Pingtung University of Science
TE D
and Technology, Pingtung, Taiwan
*C-L.Chen and K.Faltusova contributed equally to the paper
E.Necas ([email protected]) and K-T.Chang ([email protected]): corresponding
AC C
authors
EP
#
Short title: c-Kit and Bone Marrow Transplantation
The author for communication Emanuel Necas, M.D., Ph.D. Institute of Pathological Physiology 1st Faculty of Medicine
ACCEPTED MANUSCRIPT Charles University in Prague, U Nemocnice 5, 128 53 Prague Czech Republic Telephone:+420-224965901, Fax:+420-224965916
RI PT
e-mail: [email protected]
SC
ABSTRACT
M AN U
The c-Kit expression level is decreased in regenerating bone marrow and such bone marrow performs poorly when co-transplanted with normal bone marrow. We asked whether diminished numbers of c-Kit receptors on HSPCs following their internalization induced by the binding of the cytokine stem cell factor (SCF) would jeopardize transplantability of
TE D
HSPCs. We used a battery of functional assays to evaluate the capacity of HSPCs with a markedly different c-Kit expression levels to be transplanted. Surprisingly, our experiments testing the homing of transplanted HSPCs to bone marrow of recipient mice and their short-
EP
term and long-term engraftment did not reveal any defects in HSPCs with severely reduced numbers of c-Kit receptor molecules. This unexpected result can be ascribed to the fact that
AC C
HSPCs exposed to SCF replace the consumed c-Kit receptors rapidly. The paper demonstrates that exposure of HSPCs to SCF and diminished number of c-Kit receptors in their cell membranes do not compromise the capacity of HSPCs to reconstitute damaged hematopoietic tissue.
Key words: c-Kit receptor, hematopoietic stem cell, bone marrow transplantation, stem cell factor, irradiation
ACCEPTED MANUSCRIPT Introduction In their seminal paper Harrison and Astle 1 reported that bone marrow from sublethally irradiated donors performed very poorly in competitive repopulation assays. This phenomenon has not been fully deciphered to this day. Interestingly, Simonnet et al. 2
RI PT
reported significantly decreased expression level of the c-Kit receptor in hematopoietic stem and progenitor cells (HSPCs) of mice recovering from sublethal irradiation. The c-Kit
tyrosine kinase receptor is a fundamental marker of mouse hematopoietic stem and progenitor
. c-Kit is activated after binding its ligand, the stem cell factor (SCF; also known as c-Kit
M AN U
5
SC
cells (HSPCs) 3,4 important for HSPCs survival, proliferation or quiescence and self-renewal
ligand, steel factor or mast cell growth factor). c-Kit receptors form homodimers after binding SCF and are internalized and degraded 6. The SCF gene is significantly up-regulated in bone marrow damaged by irradiation 7,8 which could cause the low c-Kit expression level
TE D
in HSPCs of sublethally irradiated mice due to enhanced c-Kit consumption. During studies into the bone marrow regeneration from a small number of repopulating cells, we were confronted with poor performance of regenerating bone marrow when competing
EP
with normal bone marrow, and with low c-Kit expression level in HSPCs. We hypothesized that a particular density of c-Kit receptors on transplanted HSPCs could determine which type
AC C
of the supporting niche the cell would engraft into. The high density of c-Kit on HSPCs from normal bone marrow would allow to the cells to engraft into highly supportive niches. On the other hand, the low density of c-Kit on HSPCs from regenerating bone marrow would direct the cells towards less supportive niches. The notion was indirectly supported by the role of c-Kit/membrane-bound SCF interactions in the initial lodgement of transplanted stem cells 9 and in the functional positioning of stem cells to their niches 10. We decided to test the hypothesis by downregulation of c-Kit with exogenous SCF, and by testing the impact of markedly decreased c-Kit receptor density on the outcome of HSPC
ACCEPTED MANUSCRIPT transplantation. Results of this study convincingly demonstrated that an exposure of HSPCs to high SCF concentrations causing significant downregulation of c-Kit receptors in HSPCs, is not harmful in regard to their capacity to be transplanted. However, the hypothesis assuming that the density of c-Kit receptors predisposes transplanted HSPCs to engraft the niches with
RI PT
different supportive capacity could not be conclusively refuted because the HSPCs exposed to SCF replaced the internalized and consumed c-Kit receptors rapidly.
SC
Materials and Methods
M AN U
Mice
C57BL/6NCrl (CD45.2), B6.SJL-Ptprca Pepcb/BoyJ (CD45.1), dual CD45.2/CD45.1 F1 hybrid mice (F1) and C57Bl/6-Tg(UBC-GFP)30Scha/J mice were bred in the institutional animal facility and kept in a clean conventional part of the facility during the experiments.
TE D
Three-month to six-month-old adult mice were used in the experiments. The experiments were approved by the Laboratory Animal Care and Use Committee of the First Faculty of
Republic.
AC C
Cytokines
EP
Medicine, Charles University, and the Ministry of Education, Youth and Sports of the Czech
Recombinant mouse Stem Cell Factor (SCF) was bought from PeproTech (USA), catalog numbers # 250-03 and AF-450-33, respectively. SCF was dissolved in deionized water at a concentration of 10 µg/mL and stored at -80 °C in an ultra-low temperature freezer.
Exposure of bone marrow to SCF in vitro
Bone marrow cells (BMCs) obtained from normal mice were incubated in the Dulbecco´s Minimum Essential Medium (DMEM) for 60 minutes in presence/absence of SCF. Cell were
ACCEPTED MANUSCRIPT then stained for flow cytometry analysis to determine the median fluorescence intensity (MFI) of the anti-c-Kit antibody in immature cells negative for lineage markers (Lin-).
Administration of SCF in vivo
RI PT
SCF was injected intraperitoneally (i.p.) or intravenously (i.v.) to normal adult mice
C57BL/6NCrl, males or females, in the amount 1000 ng per mouse. Mice were sacrificed from 1 hour to 46 hours afterwards. Bone marrow was collected from one femur and analyzed
SC
for c-Kit median fluorescence intensity (MFI) on immature hematopoietic cells. The MFI values were compared to the corresponding MFI values of bone marrow from an untreated
M AN U
mouse which were taken to be 100% in each experiment.
Antibodies
An A700- or FITC-conjugated anti-Lineage cocktail of antibodies (B220, CD3, Gr-1, Mac-1,
TE D
and Ter-119), PE/CY7-conjugated anti-Sca-1, BV421-conjugated anti-c-Kit, APC-conjugated
USA.
Flow cytometry
EP
anti-CD150, and FITC-conjugated anti-CD48 antibodies were used, all from BioLegend, CA,
AC C
BMCs were analyzed using a digital FACS Canto II flow cytometer, equipped with 405 nm (60 mW), 488 nm (20 mW) and 633 nm (15 mW) laser lines (BD Biosciences, USA). Briefly, after multicolor staining bone marrow cells were filtered using 70µm Nylon Cell Strainers (BD Falcon, USA) and were analyzed by the flow cytometer. BD FACS Diva software version 6.1.3 was used for data acquisition. CS&T beads (BD Biosciences, USA) were used for the automated cytometer setup and the performance tracking procedure before measurements. The proper compensation matrix was created by running single-stained control samples (automatic compensation). The compensation matrix was then controlled and
ACCEPTED MANUSCRIPT manually adjusted (if necessary) for each measurement. The generated flow cytometry data were analyzed using FlowJo vX software (Treestar, USA). To properly interpret flow cytometry data, Fluorescence-Minus-One (FMO) controls were used for gating.
RI PT
c-Kit fluorescence intensity (MFI) Anti-c-Kit antibody minus one samples (FMO) were used to distinguish between c-Kit+ and c-Kit- BMCs. c-Kit+ cells lacking lineage markers, Lin-c-Kit+ cells, were divided into Sca-1
SC
negative (LS-K) and Sca-1 positive (LSK) cells (Fig. 1). LSK cells were further classified into four types characterized by the specific patterns of CD150 and CD48 markers according to
M AN U
Kiel et al. 11. The median fluorescence intensity (MFI) thus indicates the fluorescence intensity of the anti-c-Kit antibody of the particular cell type, recorded over the entire range of c-Kit intensities. An example of normal and depressed c-Kit fluorescence intensity in LSK
TE D
cells is shown in Fig. 3.
Colony-forming units-spleen (CFU-S) assay
Male C57BL/6 mice were irradiated with 9 Gy and injected intravenously with 6 x 104 bone
EP
marrow cells incubated for 60 minutes with/without SCF. Mice were sacrificed 8 or 10 days after the injection, spleens were fixed in Tellesniczsky´s solution (1:1:20 ratio of
AC C
formalin/acetic acid/70% alcohol) and colonies were counted on the convex surface of the spleens.
Homing of transplanted Lin-c-Kit+ cells
Recipient female CD45.1 mice were irradiated with a sublethal dose of 4 Gy three days before the transplantation of congenic BMCs originating either from CD45.2 mice or from UBCGFP transgenic mice. Before transplantation, BMCs were split into two equal parts and
ACCEPTED MANUSCRIPT incubated for 60 min at 37oC with/without 500 or 1000 ng of SCF per mL in a DMEM medium with 1% bovine serum albumin. The cells were then washed with PBS, a sample was withdrawn to be stained with anti-lineage cocktail, anti-Sca-1 antigen and anti-c-Kit antibodies for analysis of the c-Kit expression level. The remaining cells were transplanted to
RI PT
recipient mice through the retro-orbital venous plexus. Seventeen or 20 hours later,
transplanted mice were sacrificed, the femoral bone marrow was collected, stained with antilineage cocktail, anti-Sca-1 and anti-c-Kit antibodies, or screened for the CD45.1 and CD45.2
SC
allotype or GFP+ and GFP- BMCs. Flow cytometry was used to determine the percentage of
M AN U
the donor LS-K and LSK cells in the bone marrow of recipient mice.
Competitive bone marrow cell transplantation
Three variants of competitive transplantation assays were employed: (1) BMCs incubated with/without SCF were transplanted into sublethally (6 or 6.5 Gy) irradiated congenic
TE D
recipients to compete with repopulating cells surviving in irradiated mice; (2) tested BMCs, or Lin-c-Kit+ separated cells, from two congenic mice strains were incubated with/without SCF, the samples exposed to SCF were mixed 1:1 with the congenic control samples incubated
EP
without SCF, and the mixtures were transplanted into irradiated F1 hybrid recipients; (3) BMCs from CD45.1 mice were incubated with/without SCF and transplanted into non-
AC C
irradiated or mildly (1 Gy) irradiated transgenic UBC-GFP mice.
Analysis of chimeric hematopoiesis in transplanted mice
At various times after transplantation, a sample of peripheral blood was obtained from the retro-orbital sinus with 75 mm/60 µL heparinized capillary tubes. Samples were washed and stained with FITC-conjugated anti-Ly5.1 and A700-conjugated anti-Ly5.2 antibodies (BioLegend, CA). The percentage of donor-derived nucleated blood cells was determined
ACCEPTED MANUSCRIPT after 4 weeks, primarily reflecting the hematopoietic activity of the transplanted short-term repopulating cells (STRCs), and then up to 9 months, reflecting the hematopoietic activity of the long-term repopulating cells (LTRCs).
RI PT
Statistical analysis
Statistical analysis was performed with Graph Pad Prism version 5 (GraphPad Software, CA). Unpaired two-tailed t-test was used to compare results from corresponding experimental
SC
groups. P values < 0.05 were considered statistically significant. * P<0.05; ** P<0.01; ***
M AN U
P<0.001.
Results
Regenerating bone marrow performs poorly in transplantation assays Fig. 2A shows spleen colonies generated by injecting 105 normal BMCs and 5x105 bone
TE D
marrow cells collected 16 days after irradiation of mice with a sublethal dose of 6 Gy. The percentage of Lin-c-Kit+ cells in compared bone marrow was 2.95% in the normal bone marrow and 0.75% in the regenerating bone marrow. The regenerating bone marrow was ~ 6
EP
times less efficient in generating day-12 spleen colonies compared to normal bone marrow.
AC C
The low transplantation potential of regenerating bone marrow was highly expressed when an equal number of normal and regenerating Lin-c-Kit+ cells collected 14 days after irradiation were co-transplanted. There were 83 times more nucleated blood cells derived from normal bone marrow after 1 month and 480 times more after 6 months (Fig. 2B).
SCF downregulates c-Kit expression level in a dose-dependent manner Figure 3 shows that an exposure of bone marrow to SCF downregulates c-Kit in Lin-c-Kit+ cells proportionately to the SCF concentration.
ACCEPTED MANUSCRIPT c-Kit receptors consumed after binding SCF are replaced by new c-Kit receptors within 12-14 hours
For a proper interpretation of the results from competitive transplantation experiments, it was
RI PT
important to determine how rapidly the internalized and degraded c-Kit receptors can be replaced. As no data had been available on the ability of HSPCs to replace downregulated and consumed c-Kit receptors, we determined the c-Kit kinetics after their downregulation
SC
induced by SCF in several in vivo experiments. Mice were injected with SCF either
intraperitoneally or intravenously and their bone marrow was collected 1-46 hours later. c-Kit
M AN U
receptors were suppressed for 3 hours after SCF injection, but completely recovered by 12-14 hours after SCF administration (Fig. 4A,B).
The downregulation of c-Kit by SCF has no impact on the capacity of repopulating cells
TE D
to be transplanted
We tested possible deleterious effects of a severely downregulated c-Kit receptors on the homing of transplanted HSPCs to the bone marrow, their capacities to generate spleen
AC C
hematopoiesis.
EP
colonies (CFU-S), and to compete with normal bone marrow in the reconstitution of damaged
The homing of transplanted HSPCs to the bone marrow of sublethally irradiated
recipient mice is not affected by downregulated c-Kit
We downregulated c-Kit in the bone marrow collected from CD45.2 and UBC-GFP mice and transplanted it to congenic sublethally irradiated CD45.1 mice. Seventeen and 20 hours after the transplantation, the percentage of donor LS-K and LSK cells in all LS-K and LSK cells of recipient mice was determined. No significant differences were present between results from
ACCEPTED MANUSCRIPT transplantation of aliquots of the SCF-treated and control BMCs although they markedly differed in the c-Kit expression level (Fig. 5). Engraftment of transplanted bone marrow or sorted Lin-c-Kit+ cells is not affected
RI PT
by downregulated c-Kit
We downregulated c-Kit receptors by exposing BMCs to SCF in vitro and determined the capacity of the cells to establish blood cell production after transplantation. The colony-
SC
forming spleen assay (CFU-S) was not significantly affected by SCF (Fig. 6A). The next assay used a transplantation of the control and SCF-exposed bone marrow to sublethally
M AN U
irradiated mice with a different CD45 allotype and the detection of nucleated donor cells in the blood. This test is quantitative 12,13 and it should sensitively detect any diminished capacity of the SCF-exposed bone marrow to compete with the repopulating hematopoietic cells that survived in recipient mice 14. No significant difference in the engraftment of cells
TE D
with highly different densities of c-Kit receptors (c-Kit fluorescence profiles not shown) were found in two independent experiments (Fig. 6B, C). A competitive transplantation of normal and SCF-exposed samples of congenic (CD45.1 and CD45.2) bone marrow cells, which is the
EP
most frequently used assay to compare the capacity of HSPCs to reconstitute damaged hematopoiesis, did not demonstrate a significant effect of c-Kit downregulation on the ability
AC C
of BMCs to be transplanted either. (Fig. 6D). Due to reciprocal controls between CD45.1 and CD45.2 cells, the significant differences observed in the experiment could be ascribed by a higher content of long-term repopulating cells in the CD45.2 bone marrow donors, a phenomenon that had previously been reported 15. Next we sorted Lin-c-Kit+ cells from bone marrows collected from CD45.1 and CD45.2 mice. The cells were incubated with/without SCF and a 1:1 mixture of the congenic cells, mixing cells incubated with/without SCF reciprocally, were transplanted to irradiated dual CD45.2/CD45.1 F1 hybrid recipients. The
ACCEPTED MANUSCRIPT c-Kit expression level of CD45.1 and CD45.2 cells in transplanted cell mixtures differed markedly according to whether they were exposed or were not to SCF (not shown). No significant effect of the different c-Kit expression level was demonstrated in this experiment (Fig. 6E). Independently, we had noted that transgenic UBC-GFP mice 16 engraft congenic
RI PT
bone marrow without any conditioning or intensively after very low doses of irradiation. We used this feature of UBC-GFP mice to test whether a markedly different density of c-Kit
receptors would affect engraftment of HSPCs in the environment of completely undamaged or
SC
mildly damaged hematopoiesis of recipient mice. As in all previously described experiments, the capacity of repopulating donor cells to produce blood cells were not significantly altered,
M AN U
despite significant differences in the level of c-Kit expression (Fig. 6F, G). Collectively, all of the various transplantation assays failed to demonstrate importance of the c-Kit expression level (manipulated by SCF) on the ability of bone marrow cells to home and engraft into the
Discussion
TE D
hematopoietic tissue of recipient mice.
EP
Stem cell factor (SCF) is a component of the niche that supports and controls HSPCs in the
AC C
bone marrow 9,17,18. Transplanted HSPCs have to re-establish the physiological interactions between c-Kit receptors and SCF present in their niches. Therefore, the intensity of c-Kit expression level in transplanted HSPCs might play a crucial role in the biology of HSPCs transplantation.
We speculated that the HSPCs with a high density of c-Kit receptors might engraft into more supportive niches after transplantation than the HSPCs expressing c-Kit at a low level. It has previously been shown that blocking c-Kit receptors with the anti-c-Kit antibody ACK2 significantly reduced their transplantation 19. Driessen et al.9 and Kimura et al. 17 then
ACCEPTED MANUSCRIPT demonstrated the significance of c-Kit for the initial lodgement of transplanted hematopoietic stem cells within the endosteal bone marrow region, and to the functional positioning of hematopoietic stem cells to the niche. Grinenko et al.20 and Shin et al.21 demonstrated that inherent small differences in the c-Kit expression level in hematopoietic stem cells influence
RI PT
their engraftment after transplantation. In present experiments, we utilized the ability of SCF to downregulate c-Kit receptors by inducing their internalization and degradation. However, in a striking contrast to our expectations, grossly diminished c-Kit expression level in HSPCs
SC
did not affect the homing/lodgement and engraftment of transplanted HSPCs in a battery of
M AN U
transplantation-based assays.
These results prompted us to establish whether HSPCs can replace the consumed c-Kit receptors and how rapidly this can be achieved. All types of HSPCs reconstituted normal c-Kit density within 12-14 hours after SCF administration to mice. SCF is rapidly cleared from the circulation of mice with a half-life of ̴2 hours after intravenous application
22
, and
TE D
our results thus show that HSPCs exposed to SCF can rapidly reconstitute the density of c-Kit receptors to their inherently programmed level. The rapid dynamics of c-Kit receptors is therefore important for a proper interpretation of our transplantation tests that did not
EP
demonstrate any significant effect of a widely varying c-Kit density in transplanted HSPCs
AC C
upon their transplantation.
Previous experiments which demonstrated significance of c-Kit in the homing and lodgement of transplanted HSPCs 9,19 and in the positioning of HSPCs in their niches 17 utilized either the blockade of c-Kit receptors by the ACK2 antibody 9,19 or HSPCs with inherently diminished c-Kit function 17. Broudy et al.19 used functional colony-forming assays to measure transplanted HSPCs in bone marrow of lethally irradiated recipients after blocking c-Kit for 4 hours prior to the transplantation. Driessen 9 and co-workers localized the transplanted LinSca-1+c-Kit+ cells in the endosteal bone marrow region directly. In contrast, the experimental
ACCEPTED MANUSCRIPT approaches that we utilized to manipulate the c-Kit expression level in HSPCs were more physiological and fully reversible. We also used a direct determination of transplanted cells by flow cytometry in the bone marrow of recipients in our homing/lodgement experiments. Driessen et al.9 demonstrated a decreased proportion of transplanted cells with blocked c-Kit
RI PT
receptors within the endosteal region 15 hours post-transplant but the number of transplanted cells detected per recipient femoral section was not, in agreement with results, altered. Importantly, our experimental setting corresponded to conditions to which HSPCs are
SC
exposed during their generation or expansion ex vivo. It is thus significant that we
M AN U
demonstrated the ability of HSPCs to rapidly replace c-Kit receptors consumed after their activation by SCF, and that even markedly reduced numbers of c-Kit receptors do not hamper the ability of HSPCs to be transplanted.
The tested hypothesis assuming that depressed c-Kit expression in HSPCs in regenerating
TE D
bone marrow significantly contributes to their inferiority in transplantation assays cannot be ultimately rejected on basis of our negative results regarding the impact of c-Kit downregulation on HSPCs transplantation. This is because the SCF induced downregulation
EP
of c-Kit receptor was a transient phenomenon lasting less than 12-14 hours, while the whole process of engraftment of transplanted HSPCs may require more time.
AC C
In summary, the paper demonstrates that HSPCs exposed to SCF do not show decreased transplantability in the aftermath of c-Kit receptor internalization and degradation. It also demonstrates that HSPCs efficiently replace the consumed c-Kit receptors.
ACKNOWLEDGMENTS
The study was supported by the Grant Agency of the Czech Republic: GACR 14-25515S. It also received institutional support from projects PRVOUK-P24/LF1, SVV 2014–260 033,
ACCEPTED MANUSCRIPT UNCE 204021 and BIOCEV CZ.1.05/1.1.00/02.0109. In part, the study was supported by the Ministry of Science and Technology in Taiwan (NSC 99-2314-B-020-001-MY3).
The authors indicate no potential conflicts of interest.
Authorship statement:
RI PT
Conflict of interest statement:
SC
C-L.C.: collection and analysis of data, manuscript writing; K.F.: collection and analysis of data, manuscript writing; M.M.: collection of data; F.S.: flow cytometry analyses supervision
M AN U
and cell sorting; K-T.C.: conception and design, final approval; E.N.: conception and design, data interpretation, manuscript writing, final approval.
1.
TE D
References
Harrison DE, Astle CM. Loss of stem cell repopulating ability upon transplantation. Effects of donor age, cell number, and transplantation procedure. J Exp Med.
EP
1982;156(6):1767-79. Available at:
AC C
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2186863&tool=pmcentrez &rendertype=abstract. Accessed February 3, 2016.
2.
Simonnet AJ, Nehmé J, Vaigot P, Barroca V, Leboulch P, Tronik-Le Roux D. Phenotypic and functional changes induced in hematopoietic stem/progenitor cells after gamma-ray radiation exposure. Stem Cells. 2009;27(6):1400-9. doi:10.1002/stem.66.
3.
Ogawa M, Matsuzaki Y, Nishikawa S, et al. Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med. 1991;174(1):63-71. Available at:
ACCEPTED MANUSCRIPT http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2118893&tool=pmcentrez &rendertype=abstract. Accessed April 24, 2015.
4.
Okada S, Nakauchi H, Nagayoshi K, Nishikawa S, Miura Y, Suda T. In vivo and in
RI PT
vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells. Blood. 1992;80(12):3044-50. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1281687. Accessed April 24, 2015.
Kent D, Copley M, Benz C, Dykstra B, Bowie M, Eaves C. Regulation of
SC
5.
hematopoietic stem cells by the steel factor/KIT signaling pathway. Clin Cancer Res.
6.
M AN U
2008;14(7):1926-30. doi:10.1158/1078-0432.CCR-07-5134.
Lennartsson J, Rönnstrand L. Stem cell factor receptor/c-Kit: from basic science to clinical implications. Physiol Rev. 2012;92(4):1619-49.
7.
TE D
doi:10.1152/physrev.00046.2011.
Geissler EN, Ryan MA, Housman DE. The dominant-white spotting (W) locus of the mouse encodes the c-kit proto-oncogene. Cell. 1988;55(1):185-92. Available at:
Limanni A, Baker WH, Chang CM, Seemann R, Williams DE, Patchen ML. c-kit
AC C
8.
EP
http://www.ncbi.nlm.nih.gov/pubmed/2458842. Accessed April 21, 2015.
ligand gene expression in normal and sublethally irradiated mice. Blood.
1995;85(9):2377-84. Available at: http://www.ncbi.nlm.nih.gov/pubmed/7537111. Accessed February 3, 2016.
9.
Driessen RL, Johnston HM, Nilsson SK. Membrane-bound stem cell factor is a key regulator in the initial lodgment of stem cells within the endosteal marrow region. Exp Hematol. 2003;31(12):1284-1291. doi:10.1016/j.exphem.2003.08.015.
ACCEPTED MANUSCRIPT 10.
Kimura Y, Ding B, Imai N, Nolan DJ, Butler JM, Rafii S. c-Kit-mediated functional positioning of stem cells to their niches is essential for maintenance and regeneration of adult hematopoiesis. PLoS One. 2011;6(10):e26918.
11.
RI PT
doi:10.1371/journal.pone.0026918.
Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial
12.
SC
niches for stem cells. Cell. 2005;121(7):1109-21. doi:10.1016/j.cell.2005.05.026.
Down JD, Tarbell NJ, Thames HD, Mauch PM. Syngeneic and allogeneic bone
M AN U
marrow engraftment after total body irradiation: dependence on dose, dose rate, and fractionation. Blood. 1991;77(3):661-9. Available at:
http://www.ncbi.nlm.nih.gov/pubmed/1991176. Accessed October 30, 2015.
Forgacova K, Savvulidi F, Sefc L, Linhartova J, Necas E. All hematopoietic stem cells
TE D
13.
engraft in submyeloablatively irradiated mice. Biol Blood Marrow Transplant. 2013;19(5):713-9. doi:10.1016/j.bbmt.2013.02.012.
Michalova J, Savvulidi F, Sefc L, Faltusova K, Forgacova K, Necas E. Hematopoietic
EP
14.
stem cells survive circulation arrest and reconstitute hematopoiesis in myeloablated
AC C
mice. Biol Blood Marrow Transplant. 2011;17(9):1273-81.
doi:10.1016/j.bbmt.2011.07.007.
15.
Waterstrat A, Liang Y, Swiderski CF, Shelton BJ, Van Zant G. Congenic interval of CD45/Ly-5 congenic mice contains multiple genes that may influence hematopoietic stem cell engraftment. Blood. 2010;115(2):408-17. doi:10.1182/blood-2008-03-
143370.
ACCEPTED MANUSCRIPT 16.
Schaefer BC, Schaefer ML, Kappler JW, Marrack P, Kedl RM. Observation of antigendependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell Immunol. 2001;214(2):110-22. doi:10.1006/cimm.2001.1895.
Kimura Y, Ding B, Imai N, Nolan DJ, Butler JM, Rafii S. c-Kit-Mediated Functional
RI PT
17.
Positioning of Stem Cells to Their Niches Is Essential for Maintenance and
Regeneration of Adult Hematopoiesis. Rota M, ed. PLoS One. 2011;6(10):e26918.
18.
SC
doi:10.1371/journal.pone.0026918.
Oguro H, Ding L, Morrison SJ. SLAM family markers resolve functionally distinct
M AN U
subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell. 2013;13(1):102-16. doi:10.1016/j.stem.2013.05.014.
19.
Broudy VC, Lin NL, Priestley G V, Nocka K, Wolf NS. Interaction of stem cell factor
TE D
and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen. Blood. 1996;88(1):75-81. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8704204. Accessed April 24, 2015.
Grinenko T, Arndt K, Portz M, et al. Clonal expansion capacity defines two
EP
20.
consecutive developmental stages of long-term hematopoietic stem cells. J Exp Med.
21.
AC C
2014;211(2):209-215. doi:10.1084/jem.20131115.
Shin JY, Hu W, Naramura M, Park CY. High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias. J Exp Med.
2014;211(2):217-31. doi:10.1084/jem.20131128.
22.
Lynch DH, Jacobs C, DuPont D, et al. Pharmacokinetic parameters of recombinant mast cell growth factor (rMGF). Lymphokine Cytokine Res. 1992;11(5):233-243.
ACCEPTED MANUSCRIPT Legends to figures
FIGURE 1 Gating strategy to analyze immature hematopoietic cells in the bone marrow with
RI PT
depressed c-Kit expression level. c-Kit FMO samples were used to delimit c-Kit+ cells. c-Kit+ cells lacking lineage markers (Lin-) were further characterized according to the expression of Sca-1 antigen and CD150 and
SC
CD48 markers. The mean values ± SD of the percentage of CD150/CD48 LSK subtypes are
M AN U
from 10 independent determinations.
FIGURE 2 Spleen colonies and blood cell production derived from transplanted normal and regenerating bone marrow.
(A) Lethally irradiated mice were transplanted with 105 normal bone marrow cells or with
TE D
5x105 bone marrow cells obtained 16 days after sublethal irradiation with 6 Gy. c-Kit expression profiles of Lin-c-Kit+ cells in the donor bone marrows was determined and the results are presented. Spleens were collected 12 days after transplantation. (B) Equal numbers
EP
(20000 per mouse) of Lin-c-Kit+ cells from normal bone marrow (CD45.1) and from bone
AC C
marrow collected 14 days after irradiation of mice (CD45.2) with 6 Gy were co-transplanted 1:1 to lethally irradiated CD45.1/CD45.2 F1 hybrid mice. The ratio of CD45.1 to CD45.2 nucleated blood cells was determined in peripheral blood 1 month and 6 months after transplantation. c-Kit expression profiles of Lin-c-Kit+ cells in the donor bone marrows was determined and the results are presented. FIGURE 3 Exposure to SCF in vitro decreased c-Kit receptors in Lin-c-Kit+ cells proportionally to the SCF concentration.
ACCEPTED MANUSCRIPT Aliquots of normal bone marrow cells were incubated for 60 minutes in DMEM without (Control) or with various concentrations of SCF. After incubation the cells were washed and stained with lineage cocktail antibodies and anti-Sca-1 and anti c-Kit antibodies. In experiments which tested the transplantability of HSPCs with downregulated c-Kit receptors
RI PT
(Figs. 5 and 6) the concentrations of SCF concentrations 100-1000 ng/mL were used.
FIGURE 4 c-Kit receptors are fully reconstituted on HSPCs within 12-14 hours after their
SC
consumption induced by SCF.
Bone marrow was collected 1-14 hours after 1000 ng of SCF given intraperitoneally (A) or
M AN U
after 1-46 hours after 1000 ng of SCF given intravenously (B). Control mice were given PBS and were included in each experiment. The median fluorescence intensity index (MFI) was related to the values in control bone marrow in various cell types in each experiment. Data pooled from six independent experiments are presented and each time point shows the results
TE D
from single mice.
FIGURE 5 Downregulation of c-Kit by SCF did not influence the homing of transplanted LS-
EP
K and LSK cells in the bone marrow of sublethally irradiated mice.
Bone marrow was split into two aliquots, incubated with/without SCF and transplanted, 11
AC C
million CD45.2 (A) or 40 million UBC-GFP cells (B), to congenic CD45.1 mice irradiated 3 days earlier with a dose of 4 Gy. After seventeen hours in the CD45.2 experiment (A) and 20 hours in the UBC-GFP experiment (B) the bone marrow of recipient mice was analyzed for the proportion of donor LS-K or LSK cells in all cells of the particular phenotype.
FIGURE 6 The downregulation of c-Kit by SCF did not influence the capacity of bone marrow cells or separated HSPCs to be transplanted.
ACCEPTED MANUSCRIPT (A) CFU-S assay: BMCs incubated with/without SCF were transplanted (60,000 cells per mouse) to 9-Gy-irradiated mice. Spleen colonies were counted after 8 or 10 days. (B-C) 10 million (B) or 8 million (C) BMCs incubated with/without SCF were transplanted to congenic sublethally irradiated (6 Gy or 6.5 Gy) mice. The downregulation of c-Kit (not shown) did not
RI PT
decrease the engraftment of transplanted cells. (D) BMCs from CD45.1 and CD45.2 mice were split into two aliquots and incubated with/without SCF. After washing, congenic
samples were mutually mixed in a 1:1 ratio, analyzed for c-Kit expression level, and co-
SC
transplanted to irradiated (6.5 Gy) dual CD45.2/CD45.1 F1 mice. CD45.2 cells were more represented independently whether they were or were not exposed to SCF. Therefore, no
M AN U
effect of the markedly different density of c-Kit receptors on the engraftment of transplanted HSPCs was demonstrated. (E) A similar experiment used double sorted Lin-c-Kit+ CD45.1 and CD45.2 cells (96% and 98% purity). The cells were split into two aliquots and incubated with/without SCF, washed, mixed in a 1:1 ratio and co-transplanted to irradiated (6.5 Gy) dual
TE D
CD45.2/CD45.1 F1 mice. Markedly different c-Kit expression level (not shown) had no significant effect on competing of the repopulating cells. (F,G) BMCs of CD45.1 mice were incubated with/without SCF and transplanted, 26 million cells to each normal (F) or 15
EP
million cells to each 1 Gy irradiated (G) UBC-GFP mouse. No effect of exposure to SCF was
AC C
demonstrated. Data from individual mice and their means are presented throughout the figure.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights 1. Regenerating HSPCs suffer from transplantation defect and have a low c-Kit 2. SCF efficiently downregulate c-Kit in HSPCs 3. HSPCs with different density of c-Kit engraft equally
AC C
EP
TE D
M AN U
SC
RI PT
4. c-Kit down-regulated by SCF is replaced within 12 hours