Promotive effect of macrophage colony-stimulating factor on long-term engraftment of murine hematopoietic stem cells

www.elsevier.com/locate/issn/10434666 Cytokine 31 (2005) 447e453

Promotive effect of macrophage colony-stimulating factor on long-term engraftment of murine hematopoietic stem cells Chiharu Imada 1, Mai Hasumura 1, Katsuhiko Nawa* Discovery Research Laboratory, Daiichi Pharmaceutical Company Limited, 1-16-13, Kita-Kasai, Edogawa, Tokyo 134-8630, Japan Received 7 October 2004; received in revised form 1 June 2005; accepted 6 July 2005

Abstract Large ex vivo expansion of hematopoietic stem cells (HSCs) sufficient for use in clinical applications has not been achieved, although the influence of some cytokines including SCF, IL-11, Flt3-L, and TPO for this purpose has been reported. We present evidence for an indirect effect of macrophage colony-stimulating factor (M-CSF) on expansion of murine HSCs. Fresh Linÿ/low cells were isolated from Ly5.1 mouse bone marrow and cultured with or without M-CSF in the presence of SCF C IL-11 C Flt3-L or SCF C IL-11 C TPO for 6 days. The expanded cells were harvested and transplanted into lethally irradiated Ly5.2 recipients with competitor cells. Culture of Linÿ/low cells with M-CSF significantly enhanced long-term engraftment. When the more enriched HSC populations of Linÿ/low c-KitC Sca-1C cells were used as a source of HSCs, such a promotive effect was not observed, in agreement with negative expression of the M-CSF receptor (c-Fms). However, co-culture with Linÿ/low c-FmsC resulted in a significant increase of long-term engraftment. These results suggested that M-CSF is an indirect stimulator for ex vivo expansion of HSCs in the presence of SCF, IL-11, Flt3-L, and TPO. These observations provide new directions for ex vivo expansion and insight into new engraftment regulation through M-CSF signaling. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Hematopoietic stem cells; Long-term repopulation; M-CSF; c-Fms; Ex vivo expansion

1. Introduction Hematopoietic stem cells (HSCs) are defined as cells with extensive proliferative potential and capability to differentiate into all hematopoietic lineages. Specific cellsurface marker(s) identifying HSCs are not available and HSCs are evaluated through their unique ability for long-term repopulation (LTR) of all hematopoietic

Abbreviations: HSCs, Hematopoietic stem cells; SCF, Stem cell factor; IL-11, Interleukin-11; Flt3-L, Fms-like tyrosine kinase 3-ligand; TPO, Thrombopoietin; M-CSF, Macrophage colony-stimulating factor. * Corresponding author. Tel.: C81 3 5696 4672; fax: C81 3 5696 8196. E-mail address: [email protected] (K. Nawa). 1 These authors contributed equally to this study. 1043-4666/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2005.07.001

lineages in transplant models [1]. By using this assay system, others and we have shown that the LTR of HSCs is regulated positively by cytokines such as stem cell factor (SCF), interleukin-11 (IL-11), fms-like tyrosine kinase 3-ligand (Flt3-L), and thrombopoietin (TPO) [2e7]. Macrophage colony-stimulating factor (M-CSF) is a lineage-specific hematopoietic regulator that stimulates the survival, proliferation, and differentiation of monocytes/macrophages [8]. The effects of M-CSF are mediated by c-Fms, a high-affinity receptor tyrosine kinase encoded by the c-fms proto-oncogene [9]. While previous studies showed that HSCs contain low levels or no mRNA for c-Fms [10e12], we evaluated the influence of M-CSF on their long-term repopulating activity (LTR-activity) in Linÿ/low cells. LTR-activity was promoted in an indirect fashion and the presence of

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Linÿ/low c-FmsC cells that can respond to M-CSF was found to be important.

2. Results 2.1. Characterization of long-term repopulating assay A quantitative analysis of HSCs is required for expansion studies and we normalized the LTR-assay system for linearity (Fig. 1). Increasing numbers of Ly5.1-donor bone marrow cells were transplanted into lethally irradiated Ly5.2 recipients with 1 ! 106 Ly5.2 competitor cells. After 24 weeks, the donor contribution (ratio of %Ly5.1 to %Ly5.2) was proportional to the number of donor cells with a high correlation coefficient (R2 Z 0.996). The linear portion of the curve was used for quantitative analyses. 2.2. Promotive effect of M-CSF on the long-term repopulating activity of HSCs Since HSCs in bone marrow are enriched in Linÿ/low cells [13,14], we used this population as a source of HSCs for the primary expansion study. Several cytokines including SCF, IL-11, Flt3-L, and TPO have been shown to be efficient to some extent for promoting the ex vivo expansion of HSCs [2e7]. To confirm these cytokine effects, we cultured Linÿ/low cells with SCF, IL-11, and Flt3-L or TPO for 6 days and examined changes 1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

0

2

4

6

8

10

Ly5.1 BM cells injected, x 10-5 cells Fig. 1. Linearity of long-term repopulating assay. Bone marrow cells were freshly isolated from Ly5.1 mice and the indicated number transplanted into lethally irradiated mice (Ly5.2) with one million competitor cells. After 24 weeks, peripheral blood was analyzed for the donor contribution by flow cytometry. Data represent mean G SEM (n Z 4).

in their LTR-activities (Table 1). Fresh Linÿ/low cells (40,000) showed 19.1 G 1.9% and 18.5 G 1.3% of average engraftment level at 12 and 24 weeks posttransplantation, respectively, suggesting that their bone marrow attained steady state production by 12 weeks after transplantation. Linÿ/low cells cultured with the indicated cytokines required longer periods (R24 weeks) to reconstitute steady state conditions. Therefore, we compared long-term engraftment levels at 24 weeks after transplantation. LTR-activities of Linÿ/low increased slightly in cultures containing SCF C IL-11 C Flt3-L and SCF C IL-11 C TPO, but these increases were not significant. The further addition of M-CSF to the cultures resulted in a significant increase of LTR-activity compared with fresh control group. These high engraftment levels were observed even after 32 weeks posttransplantation (data not shown). Addition of 10 ng/mL M-CSF was near optimal since the promotive effect was inhibited slightly at 100 ng/mL M-CSF (data not shown). All mice showed multilineage reconstitution at 24 weeks post-transplantation. An example of a reconstitution with cells cultured in the presence of SCF C IL11 C Flt3-L C M-CSF is shown in Fig. 2. These results show that M-CSF promotes the LTR-activities of HSCs in the presence of SCF, IL-11, Flt3-L, and TPO. 2.3. Expression analysis of M-CSF receptor (c-Fms) on HSCs HSCs are reportedly negative for c-Fms mRNA expression [10e12]. To confirm that HSCs do not express c-Fms protein on their cell surface, Linÿ/low cells were isolated from fresh bone marrow cells and sorted into c-Fmsÿ and c-FmsC sub-populations. Cells positive for the c-Fms were found at low frequency (z5%) (Fig. 3A). Because the ratio of Linÿ/low c-Fmsÿ to Linÿ/low c-FmsC cells was approximately 20:1, 100,000 Linÿ/low c-Fmsÿ or 5000 Linÿ/low c-FmsC cells were transplanted into lethally irradiated mice. Donor contributions were observed only in mice transplanted with the c-Fmsÿ cells, whereas c-FmsC cells had no LTR-activity (Fig. 3B). This result from the phenotypic analysis confirmed that HSCs are negative for the expression of c-Fms in agreement with the previous studies on mRNA levels [10e12]. Taken together, MCSF appears to be an indirect stimulator for the regulation of HSCs. 2.4. Effect of Linÿ/low c-FmsC cells on the expansion of Linÿ/low c-KitC Sca-1C HSCs When Linÿ/low cells were used for the expansion study, the promotive effects of M-CSF were observed as described above. We assessed the effect of M-CSF on the more enriched HSC populations, the Linÿ/low c-KitC Sca-1C cells (Fig. 4A). Fresh Linÿ/low c-KitC

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C. Imada et al. / Cytokine 31 (2005) 447e453 Table 1 Effect of M-CSF on the ex vivo expansion of Linÿ/low hematopoietic stem cells Cytokines

Total cells (!104)

12 Weeks %Ly5.1

RU

%Ly5.1

RU (-fold)

Fresh SCF C IL-11 C Flt3-L SCF C IL-11 C Flt3-L C M-CSF SCF C IL-11 C TPO SCF C IL-11 C TPO C M-CSF

4 174 G 9 238 G 15 116 G 14 125 G 26

19.1 G 1.9 36.2 G 1.9 48.5 G 1.6 33.8 G 2.5 52.1 G 4.3

0.24 G 0.03 0.57 G 0.04 0.95 G 0.06 0.52 G 0.06 1.14 G 0.18

18.5 G 1.3 24.3 G 2.3a 46.1 G 2.1* 31.1 G 4.4a 47.4 G 4.1*

0.23 G 0.02 0.33 G 0.04 0.87 G 0.07 0.47 G 0.10 0.94 G 0.16

24 Weeks

(1.0) (1.4) (3.8) (2.0) (4.1)

Ly5.1 Linÿ/low cells (40,000 cells) were cultured for 6 days in the presence of the indicated cytokines (100 ng/mL) with or without 10 ng/mL M-CSF. The expanded cells were harvested, counted with a hemocytometer, and transplanted together with one million competitor cells. After 12 and 24 weeks, peripheral blood was collected and analyzed for donor cell contribution by flow cytometry. Data represent mean G SEM from three independent experiments (n Z 9). *p ! .01. a Not significantly compared with fresh control.

Sca-1C cells (1250 cells) displayed 14.4 G 1.6% of the average engraftment level at 24 weeks post-transplantation. When 10,000 Linÿ/low c-KitC Sca-1C cells were cultured for 5 days with SCF C IL-11 C Flt3-L and transplanted into 8 recipient mice (1250 fresh control cells equivalent per mouse), the average engraftment level was 18.3 G 3.1% with no significant change. Further addition of M-CSF to the culture had no effect on the donor cell engraftment (15.3 G 2.2%). These results suggested that the M-CSF requires Linÿ/low c-FmsC cells as a mediator to increase HSC LTR-activity. Thus, we examined the effect of Linÿ/low c-FmsC cells on the expansion of Linÿ/low c-KitC Sca-1C cells in the presence of M-CSF (Fig. 4B). Linÿ/low c-FmsC cells had no LTR-activity (Fig. 3), and the engraftment level in the two populations was evaluated. Analysis to discriminate between the long-term engraftment levels of Linÿ/low c-FmsC and Linÿ/low c-KitC Sca-1C cells was facilitated by the use of Ly5.1/Ly5.2 mice whose peripheral nucleated cells were double positive for the expression of Ly5.1 and Ly5.2. We isolated Linÿ/low c-FmsC and Linÿ/low c-KitC Sca-1C cells from Ly5.1/Ly5.2 and Ly 5.1 mice, respectively. Fresh Linÿ/low c-KitC Sca-1C cells (1250) showed 12.0 G 3.0% of the average engraftment level at 24 weeks post-transplantation. When 10,000 Linÿ/low c-KitC Sca-1C cells were co-cultivated with 4700 Linÿ/low c-FmsC cells for 5 days in the presence of

3. Discussion Our results that HSCs are negative for c-Fms expression are in agreement with earlier studies showing undetectable c-Fms mRNA in HSCs [10e12]. Although c-Fms is expressed primarily in bone marrow macrophages 103

103

18

18

6

11

102

6

100

101

Ly5.1

102

101 34

100

42

100

10

CD3ε

101

103

14

2

102

B220

Mac-1/Gr-1

103

SCF C IL-11 C Flt3-L and transplanted into 8 recipient mice (1250 fresh control cells equivalent per mouse), the average engraftment level of Linÿ/low c-KitC Sca-1C cells was slightly increased to 20.4 G 2.9% without significant change. Further addition of M-CSF resulted in a significant increase of the engraftment level (33.8 G 3.2%). Ly5.1C Ly5.2C cells constituted less than 2% of peripheral nucleated cells in each recipient, suggesting that Linÿ/low c-FmsC cells have little LTR-activities in agreement with the result of Fig. 3. These results suggested that Linÿ/low c-FmsC cells mediate the influence of M-CSF promotion for long-term engraftment of Linÿ/low c-KitC Sca-1C cells. In Linÿ/low cell cultures, the use of 40,000e80,000 cells per well of a 6-well plate was sufficient for detecting the promotive effect of M-CSF. c-FmsC cells are approximately 5% of Linÿ/low (Fig. 3), so that 2000e4000 c-FmsC cells were used in the culture systems. Approximately 4700 Linÿ/low c-FmsC cells were co-cultured with Linÿ/low c-KitC Sca-1C cells.

100

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Ly5.1 ÿ/low

102

101 100

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40

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101

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103

Ly5.1

Fig. 2. Multilineage hematopoietic reconstitution by M-CSF-stimulated Lin cells. After 24 weeks post-transplantation, peripheral blood samples from the recipient mice were stained with donor- and lineage-specific antibodies, and analyzed by flow cytometry. The data were derived from an experiment with SCF C IL-11 C Flt3-L C M-CSF (10 ng/mL).

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A)

B) 60 (+)

50

Ly5.1( )

Relative cell count

(-)

40 30 20 10

100

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0

c-Fms

c-Fms(-)

ÿ/low

c-Fms(+)

ÿ/low

Fig. 3. Expression analysis of c-Fms on Lin hematopoietic stem cells. Lin cells were stained with anti-c-Fms (solid line) or isotype control (gray filled), followed by FITC-conjugated secondary antibody and sorted based on c-Fms expression (A). Either 100,000 Linÿ/low c-Fmsÿ cells or 5000 Linÿ/low c-FmsC cells were transplanted together with 5 ! 105 competitor cells (n Z 3 or 5). After 24 weeks, peripheral blood was collected for flow cytometric analysis (B).

[15], co-culture of Linÿ/low c-KitC Sca-1C cells with M-CSF-stimulated macrophages had no significant effect on increasing long-term engraftment of donor HSCs (C.I., M.H. and K.N., unpublished data). In contrast, Linÿ/low c-FmsC cells enhanced LTR-activity of Linÿ/low c-KitC Sca-1C cells in the presence of M-CSF. The targets of M-CSF for the regulation of stem cells may be endothelial cells and/or stromal cells associated with bone marrow Linÿ/low c-FmsC cells. While a study to identify the regulatory cells is ongoing, it has been shown that 10 ng/mL M-CSF accelerates the differentiation of endothelial precursor cells [16] and the formation of stromal cells, which have a stimulating activity for high proliferative potential colony-forming cells [17,18]. There are several potential mechanisms by which MCSF/Linÿ/low c-FmsC cells may increase the LTR-

A

activity of HSCs. First, a humoral factor secreted from Linÿ c-FmsC cell progeny may have increased their LTR-activity in the presence of SCF, IL-11 and Flt3-L. Alternatively, contact with progeny of Linÿ c-FmsC cells may up-regulate the long-term engraftment of HSCs. Cultivation of Linÿ/low or Linÿ/low c-KitC Sca-1C cells with SCF, IL-11 and Flt3-L in our system had little ability to increase the LTR of HSCs. These results are inconsistent with a prior study that observed a 3- to 4-fold amplification of HSCs using these cytokine combinations in a serum-free culture [2]. While this discrepancy is not clear, the presence of serum seems to promote HSC differentiation and/or apoptosis and the influence of M-CSF under serum-free condition needs to be evaluated.

B 50 P<0.05

Ly5.1 (

)

40

NS

30

20

10

0 Fresh ÿ/low

- M-CSF ÿ/low

+ M-CSF

Fresh ÿ/low

- M-CSF

+ M-CSF

Fig. 4. Co-culture of Lin c-Kit Sca-1 cells with Lin c-Fms cells. (A) Lin c-Kit Sca-1 cells (10,000 cells) were isolated from Ly5.1 mice bone marrow and cultured in a 6-well plate with or without 10 ng/mL M-CSF in the presence of SCF C IL-11 C Flt3-L. Lethally irradiated Ly5.2 mice (n Z 8) received 5 ! 105 competitor cells (Ly5.2) together with 1250 fresh Ly5.1 Linÿ/low c-kitC Sca-1C cells or with progeny that grew from these cells after 5 days in culture. The donor cell contribution (Ly5.1) was analyzed at 24 weeks post-transplantation. (B) Purified Linÿ/low c-KitC Sca-1C cells (10,000 cells) were cultured for 5 days with 4700 Linÿ/low c-FmsC cells with or without M-CSF in the presence of SCF C IL-11 C Flt3-L. The expanded cells were harvested by gentle pipetting and transplanted into lethally irradiated mice (n Z 8) in the same manner. After 24 weeks, peripheral blood was analyzed for the donor (Ly5.1C Ly5.2ÿ) contribution by flow cytometry. Data represent mean G SEM (n Z 8). Data are representative of two separate experiments. C

C

C

C

C

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Mice homozygous for the osteopetrotic mutation (op/op) possess an inactivating mutation in the coding region of the M-CSF gene [19,20]. Recently, mice homozygous for a targeted c-Fms-null mutation were also studied and compared with op/op mice [21]. In the bone marrow of both mice, there were no significant differences in the frequency of hematopoietic progenitor cells compared with their frequency in wild mice, although the level of HSCs was not investigated. Although M-CSF apparently is not critical for the development and growth of HSCs, it will be interesting to examine the levels of in vivo-expanded stem cells after the transplantation of wild-type stem cells into lethally irradiated op/op mice. The administration of M-CSF after bone marrow transplantation resulted in an earlier recovery of neutrophils and platelets [22,23]. These effects may be partly due to more inducible self-renewal and/or differentiation of HSCs. In this study, we showed that M-CSF has a stimulatory effect on LTR-activity of HSCs. It is unclear whether M-CSF enhances selfrenewal of HSCs or modulates their homing capability to recipient bone marrow. 4. Materials and methods 4.1. Animals C57BL/6J-Ly5.1 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and used as the donor

Lin-

source of bone marrow cells. C57BL/6J-Ly5.2 mice were purchased from Clea Japan (Tokyo, Japan) and used as recipients. Ly5.1/Ly5.2 (C57BL/6J-Ly5.1 ! C57BL/6JLy5.2) mice were bred from the above breeding pairs and maintained in a specific pathogen-free room at our animal facility. Donors of purified HSCs and irradiated recipients were 8e12 weeks of age. Irradiated mice were provided with sterilized food and acidified water. All animal experiments were approved by the institutional ethics committee. 4.2. Cell preparations Isolation of Linÿ/low and Linÿ/low c-KitC Sca-1C cells was performed as described previously [7]. Briefly, Ly5.1 bone marrow cells were harvested by flushing the femurs and tibiae with Hanks’ balanced salt solution containing 5% fetal bovine serum (HBSSeFBS) and incubated at 4  C with a cocktail of biotinylated lineage-specific monoclonal antibodies (anti-B220, anti-Gr-1, antiMac-1, anti-Ly-1, and anti-Ter-119; all from Stemcell Technologies, Vancouver, Canada). Linÿ/low cells were enriched with anti-biotin tetrameric antibody complexes and magnetic dextran beads (Stemcell Technologies) according to the manufacturer’s instruction manual. Phenotypic analysis showed that the majority of Linhigh cells could be depleted from bone marrow cells (Fig. 5). The purity of Linÿ/low cells was O96% throughout the experiments.

Linhigh

Relative cell count

Linlow

100

101

102

103

Lineage marker Fig. 5. Preparation and characterization of Linÿ/low cells from adult bone marrow. Linÿ/low cells were isolated from Ly5.1 mice bone marrow by magnetic cell sorting and characterized for their expression of lineage markers, B220, Ly-1, Ter-119, Mac-1 and Gr-1. Cell samples before and after column purification were incubated with streptavidinePE and analyzed by flow cytometry. Black filled, purified Linÿ/low cells; gray filled, unfractionated bone marrow cells; dotted, negative control.

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To analyze c-Fms expression on hematopoietic stem cells, Linÿ/low cells were labeled with anti-murine-c-Fms (Upstate, Lake Placid, NY), followed by FITC-conjugated secondary antibody. Linÿ/low Fmsÿ and Linÿ/low FmsC cells were sorted on an Epics Elite cell sorter (Beckman Coulter, Miami, FL) at a flow rate of 1000 cells/s. For preparation of Linÿ/low c-KitC Sca-1C cells, the purified Linÿ/low cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-cKit (BD Pharmingen, San Jose, CA) and phycoerythrin (PE)-conjugated anti-Sca-1 (BD Pharmingen) and sorted into FITC- and PE-positive cells in the same manner [7]. The purity of the sorted populations was O98% throughout the cell-sorting experiments. 4.3. Suspension culture

were analyzed by staining with biotinylated anti-CD3e, -B220, -Gr-1, and -CD11b (Mac-1), followed by development with streptavidinePE. In some experiments, the relative repopulating activities of HSCs were compared by the use of repopulation units (RU), whose value was defined by the following equation [7,24,25]: RU Z %Ly5.1/(100 ÿ %Ly5.1). 4.6. Statistical analyses The significance of each set of values was assessed by the unpaired 2-tailed Student’s t test. Statistical significance was defined as p ! .05.

Acknowledgements

Purified cells were cultured in a 6-well plate with RPMI 1640 containing 5% fetal bovine serum at 37  C in 5% CO2 in air as previously reported [7]. All cytokines were from PeproTech (Rocky Hill, NJ). SCF, IL-11, Flt3-L and TPO were each used at 100 ng/mL. After incubation for 5e6 days without medium replacement, cells were harvested with HBSSe FBS and used for further analysis.

We thank Drs. M. Obinata (University of Tohoku) and A. Miyajima (University of Tokyo) for helpful discussion, and Dr. K. Ishibashi and his staff for the breeding and maintenance of mice. We are also grateful to Drs. M. Furusawa and T. Horiuchi for their helpful support and encouragement.

4.4. Co-cultivation of Linÿ/low c-KitC Sca-1C cells with Linÿ/low c-FmsC cells

References

ÿ/low

ÿ/low

Lin c-Fms and Lin c-Kit Sca-1 cells were isolated from Ly5.1/Ly5.2 and Ly 5.1 mice, respectively, and these two populations were co-cultured together in a 6-well plate with or without 10 ng/mL human M-CSF in the presence of SCF C IL-11 C Flt3-L. After 5 days, non-adherent and weakly adherent cells were harvested by gentle pipetting and analyzed for the long-term engraftment of the donor stem cells. C

C

C

4.5. Transplantation procedures and long-term reconstitution Competitive repopulation was examined over a period of 24 weeks to detect HSCs with long-term engraftment capabilities [7,24]. Ly5.2-recipient mice were irradiated with a single lethal dose of 9.5 Gy from an X-ray source and Ly5.1 test-cell samples were transplanted together with freshly isolated Ly5.2-unfractionated bone marrow cells as competitor cells. To assess reconstitution of the bone marrow, peripheral blood was drawn from the retro-orbital sinus with heparin-coated micropipettes and analyzed for the percent of Ly5.1 (test cell-derived)lymphoid and -myeloid cells by using FITC-conjugated anti-Ly5.1 on an Epics XL flow cytometer. Cells were also stained with 7-amino actinomycin D (SigmaAldrich, St. Louis, MO) to exclude dead cells. Donor contributions in the T cell, B cell, and myeloid lineages

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