Fibroblast Growth Factor-1 and -2 Preserve Long-Term Repopulating Ability of Hem - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:05 |只看该作者 |倒序浏览 |打印

作者：Joyce S.G. Yeoha, Ronald van Osa, Ellen Weersinga, Albertina Ausemaa, Bert Dontjea, Edo Vellengab, Gerald de Haana作者单位：a Department of Cell Biology, Section Stem Cell Biology, University Medical Centre Groningen, Groningen, The Netherlands;b Department of Hematology, University Medical Centre Groningen, Groningen, The Netherlands

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      【摘要】
   In this study, we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells, in serum-free medium, supplemented only with fibroblast growth factor (FGF)-1, FGF-2, or FGF-1  2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assays, high levels of stem cell activity were detectable at 1, 3, and 5 weeks after initiation of culture. FGFs as single growth factors failed to support cultures of highly purified Lin¨CSca-1 c-Kit (LSK) cells. However, cocultures of purified CD45.1 LSK cells with whole BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant, showing that HSC activity originated from LSK cells. Subsequently, we tested the reconstituting potential of cells cultured in FGF-1 2 with the addition of early acting stimulatory molecules, stem cell factor  interleukin-11 Flt3 ligand. The addition of these growth factors resulted in a strong mitogenic response, inducing rapid differentiation and thereby completely overriding FGF-dependent stem cell conservation. Importantly, although HSC activity is typically rapidly lost after short-term culture in vitro, our current protocol allows us to sustain stem cell repopulation potential for periods up to 5 weeks.
      【关键词】 Hematopoietic stem cells Serum-free culture Preservation Fibroblast growth factors; E" O, H/ s' O$ N+ O
   INTRODUCTION& w+ W' R) f% E

Hematopoietic stem cells (HSCs) play a vital role in establishing and maintaining hematopoiesis throughout life. Key to all stem cell transplantation therapies is the unique property of HSCs to undergo self-renewal and to functionally repopulate the tissue of origin when transplanted into a myeloablated recipient.( `4 }, k& M( W; y: V  {

It has been shown that HSCs can undergo a large number of self-renewing divisions in vivo, where the actual number of HSCs can increase. For example, in mice, it has been shown that a single stem cell can regenerate and maintain the entire lymphohematopoietic system after transplantation into an irradiated or immunocompromised host .( {1 _& b3 f2 Y2 N. K9 Q2 i3 [4 o& b, K
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Recent evidence suggests that signaling molecules involved in embryonic development, when the hematopoietic system is first formed, play an important role in regulating stem cell self-renewal. These include Wnt, Bmp, and Shh family members . In addition, we showed that all long-term repopulating HSC activity is contained in the lineage-depleted, FGF receptor (FGFR)-positive cell population in mouse bone marrow (BM).$ g' a, f  f* ?: ~) w' }

FGF-1 belongs to the family of FGFs of which, to date, 22 FGFs and four FGF receptors have been identified in vertebrate genomes ./ o6 N6 q  l& t, Q2 s
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The role of FGFs for in vitro maintenance of HSCs has remained largely unexplored. In the present study, we compared the growth of HSCs in serum-free medium supplemented with FGF-1 and/or FGF-2. We show that long-term repopulating stem cells can be conserved in vitro for periods up to 5 weeks.
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MATERIALS AND METHODS2 j9 Y! ^8 ^; k, U1 y8 l& K

Mice9 f$ ^/ f  m/ m( k3 s

Female C57BL/6 SJL CD45.1 mice, originally obtained from The Jackson Laboratory (Bar Harbor, ME, http://www.jax.org) and bred in our local animal facility, were used as a donor source of HSCs. C57BL/6-Tg(ACTB-EGFP)10sb/J transgenic green fluorescent protein (GFP) mice originally purchased from The Jackson Laboratory were bred in our local animal facility and also used in certain experiments. Wild-type female C57BL/6 mice were purchased from Harlan (Horst, The Netherlands, http://www.harlaneurope.com) and maintained under clean conventional conditions in the animal facilities of the Central Animal Facilities, University of Groningen (The Netherlands). Mice were fed ad libitum with food pellets and acidified tap water (pH 2.8). All animal procedures were approved by the local animal ethics committee of the University of Groningen.

Hematopoietic Cells, W5 w. r9 X! E+ ?! w- C# i7 s
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Mice were sacrificed by cervical dislocation, and BM cells were obtained by crushing both femora. Marrow cells were resus-pended in -minimum essential medium (µ-MEM; Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) supplemented with 2% fetal calf serum (FCS; Gibco-BRL). The cell suspensions were filtered through a 100-µM cell strainer (BD Falcon, Two Oak Park, MA, http://www.bdbiosciences.com) to remove debris. Cells were counted on a Coulter Counter Model Z2 (Coulter Electronics, Hialeah, FL, http://www.beckmancoulter.com).

Stem Cell Expansion Culture System: c* p/ _1 y2 O; w

Unfractionated C57BL/6.SJL CD45.1 BM cells were cultured at 5 x 106 cells per well in a six-well plate (Corning Incorporated, Corning, NY, http://www.corning.com) in StemSpan serum-free medium (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) in the presence of 10 ng/ml recombinant human FGF-1 (Gibco, Grand Island, NY, http://www.invitrogen.com), or with 10 ng/ml FGF-2 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), or a combination of both cytokines at 10 ng/ml each. Culture media were also supplemented with 10 µg/ml heparin (H3149; Sigma-Aldrich). In some experiments, unfractionated C57BL/6.SJL 5.1 cells isolated and cultured with StemSpan serum-free medium, 10 µg/ml heparin, and FGF-1 2 were treated with a cocktail of hematopoietic growth factors (GFs). A cocktail of stem cell factor (SCF) (300 ng/ml) (Amgen, Thousand Oaks, CA, http://www.amgen.com), interleukin (IL)-11 (20 ng/ml) (R&D Systems, Minneapolis, http://www.rndsystems.com), and Flt3 ligand (Flt3L) (1 ng/ml) (Immunex, Seattle, http://immunex.com) was added to the cultures for 1, 3, and 5 weeks. Nonadherent cells were harvested weekly, counted to determine growth kinetics, and reintroduced into the expansion culture, and fresh GFs were added to the culture. At 1, 3, and 5 weeks of culture, nonadherent and adherent cells were harvested and counted in preparation for cell analysis and in vivo transplantation assay into lethally irradiated C57BL/6 mice.. }0 ^  E2 V7 D5 a. C% ]9 B; V- g
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Isolation of Lin¨CSca-1  c-Kit  Cells
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Freshly isolated C57BL/6 BM cells were stained with biotinylated lineage-specific antibodies (Mouse Lineage Panel, containing anti-CD45R, anti-CD11b, anti-TER119, anti-Gr-1, and anti-CD3e (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), fluorescein isothiocyanate (FITC)-anti-Sca-1, and allophycocyanin (APC)-anti-c-kit (BD Pharmingen). Lin¨CSca-1 c-Kit  (LSK) cells were stained as described . Cells were either analyzed on the FACS Calibur (Becton, Dickinson and Company, San Jose, CA, http://www.bd.com) or sorted by a MoFlow cell sorter (DakoCytomation, Fort Collins, CO, http://www.dako.com).  Z, r3 |2 d' ~
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Cell Analysis
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FGF-expanded cells were spun for cytospin preparation. Cytospin preparations were stained with May-Gr¨¹nwald-Giemsa. Cytospots were washed with distilled water and allowed to air-dry before analysis under a microscope.2 `$ I8 ~2 k: q; }8 Z( J

Levels of chimerism were determined by detecting the presence of GFP or CD45.1- and CD45.2-positive cells in transplanted mice. To detect CD45.1- and CD45.2-positive cells, cells were stained with anti-CD45.2 (FITC) and CD45.1 (phycoerythrin) antibodies (BD Pharmingen) for 30 minutes and analyzed on a flow cytometer (FACS Calibur; Becton, Dickinson and Company).9 V4 k) b4 p0 o7 M: G% w2 Y7 O) {0 m

Cobblestone Area-Forming Cell Assays+ _& \! l3 y6 Z
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Cobblestone area-forming cell (CAFC) assays were performed as described .

In Vivo Transplantation Assays
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Female C57BL/6 mice were used to provide competitor cells and as recipient mice. BM cells were obtained by flushing the femoral content three times with -MEM supplemented with 2% FCS. Recipient mice were irradiated with 9.5-Gy -rays (0.7026 Gy/minute) in an IBL 637 cesium 137 source (CIS bio-international, Gifsur-Yvette, France, http://www.cisbiointernational.fr), 24 hours prior to transplantation. For competitive repopulation determination, varying doses of cultured BM cells were mixed with a constant number of BM competitor cells. Thus, recipient mice were intravenously transplanted with different dilutions of expanded stem cells, with or without 2 x 105 life-sparing C57BL/6 BM competitor cells. Each transplant group consisted of six recipients. After transplantation, blood samples (60 µl) were taken monthly from the retro-orbital sinus for flow cytometer analysis. At the time of sacrifice, chimerism in BM samples was analyzed by fluorescence-activated cell sorting (FACS) in the same manner. For each recipient, the competitive repopulating index (CRI) was determined. CRI is a relative measure of the competitive ability of cultured cells in comparison with that of fresh BM cells. The CRI was calculated by using the following formula:
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A CRI value of 1 indicates by definition that cultured cells and competitor cells have equal competitive ability. The repopulation ability of our cells can also be measured in repopulation units (RUs). The RU takes into account the total number of cells generated. Each RU is equivalent to the repopulation function of 100,000 competitor BM cells. The RU was calculated using the following formula:# \$ U7 ?8 U. }  X2 [* t* F' h% i

RESULTS1 }# n; ^4 Z0 x& l

Cell Growth Kinetics and Cell Morphology0 s6 T7 S% H) ?  G' @
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We used FGF-1 and FGF-2 separately and the combination of both FGFs as the only stimulus in serum-free media to culture CD45.1 BM cells for a period up to 5 weeks. One and 3 weeks after initiation of culture, a decrease in the number of cells was observed. The number of cells per well had dropped dramatically from 5 x 106 to an average of approximately 5 x 105 cells for all FGF conditions (Fig. 1A). Five weeks after the initiation of culture, cells treated with FGF-1 and/or FGF-2 had increased close to the input cell number, whereas cells cultured only in serum-free media remained low throughout the 5-week culture period at 1 x 105 (Fig. 1A). As a first screen test, prior to in vivo assays, in vitro CAFC assays were set up. CAFC subsets were quantified in cells harvested from the FGF-1 2 expansion cultures at 3 and 5 weeks. The absolute number of day 7 CAFCs that were harvested from 3-week cultures had increased 1.5-fold (Fig. 1B). Interestingly, a substantial 26-fold expansion of day 7 CAFCs was observed at the 5-week culture time point. In contrast, day 35 CAFCs at week-3 cultures were slightly lower than input, whereas a 3.5-fold expansion was apparent after 5 weeks of culture (Fig. 1B)./ w9 P4 a1 L1 ~1 ?% y

Figure 1. Cell growth, morphology, and phenotype of cells. (A): Growth kinetics of unfractionated bone marrow (BM) cells in culture for 5 weeks. These are four representative cultures out of 30 cultures. Some were performed in 25-cm2 flasks; the remaining were performed in six-well plates. Cells were cultured in serum-free medium supplemented with FGF-1, FGF-2, or FGF-1 2 in the presence of 10 µg/ml heparin. (B): Unfractionated BM cells and cells cultured for 3 and 5 weeks with FGF-1 2 were placed in limiting dilutions in a 96-well plate, and absolute numbers of day 7 and 35 CAFCs were compared with input cells. The input values refer to freshly isolated untreated BM cells. Day 7 and 35 CAFC activity was higher for cells after a culturing period of 5 weeks with FGF-1 2; p values were calculated using Student¡¯s t test, and Poisson-based limiting dilution analysis was used to determine the CAFC frequency. (C): May-Gr¨¹nwald-Giemsa staining was performed on BM cells cultured in the presence of FGF-1 2 at day 0 of initiation of culture. (D): May-Gr¨¹nwald-Giemsa staining was performed on BM cells cultured in the presence of FGF-1 2 at 3 weeks after initiation of culture. (E): May-Gr¨¹nwald-Giemsa staining was performed on BM cells cultured in the presence of FGF-1 2 at 5 weeks after initiation of culture. Abbreviations: CAFC, cobblestone area-forming cell; FGF, fibroblast growth factor; N.S., not significant., i! V2 v8 x; Z, }
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May-Gr¨¹nwald-Giemsa staining of the starting cell population (Fig. 1C) and of FGF-1 2-treated cells showed an accumulation of macrophages and blast-like cells after 3 and 5 weeks of culture (Fig. 1D, 1E). The presence of blast-like cells and extensive CAFC activity indicated the possible existence of immature cells with stem cell properties in FGF-stimulated cultures.
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Long-Term Competitive In Vivo Reconstitution of FGF-Expanded Cells

To assess whether cells cultured with FGF-1, FGF-2, or FGF-1 2 contained stem cell activity, we competitively transplanted congenic B6.CD45.1 or transgenic B6.GFP  FGF-expanded cells with CD45.2 BM cells at 1, 3, and 5 weeks after the initiation of culture. In each group of transplants, six recipient mice were transplanted. Animals receiving 1-week cultured cells were transplanted with 2.5 x 105 cultured cells and 2.5 x 105 B6 CD45.2 BM cells. As shown in Figure 2A and Table 1, after 1 week of culturing, the CRI of FGF-1 2-cultured cells was approximately 50, 18 weeks after transplant. Interestingly, CRI levels of FGF-1 2-cultured cells increased with time, suggesting engraftment of relatively more cells with long-term repopulation ability. Average chimerism levels after 18 weeks were 96% (Table 1). After 3 weeks of culturing, recipient mice received 1.8 x 105, 2.2 x 105, and 3.5 x 105 FGF-1, FGF-2, and FGF-1 2-cultured cells, respectively, together with 2 x 105 B6 CD45.2 competitor cells. Chimerism levels of approximately 80% were achieved, corresponding to a CRI level of approximately 5, 16 weeks after transplant. No significant differences were observed between the FGF groups (Fig. 2B; Table 1). As expected, cells cultured for 3 and 5 weeks in serum-free medium without any supplements had no long-term reconstituting activity. Remarkably, 5 weeks after culturing, 1.2 x 106 FGF-2 and 1.1 x 106 FGF-1 x 2 cells still outcompeted 2 x 105 freshly isolated BM cells, although CRI values dropped significantly compared with cells cultured for 1 or 3 weeks (Fig. 2C; Table 1). Low CRI values of 0.5 ¡À 0.2 for FGF-1-cultured cells were obtained, suggesting that these cells provide little competitive repopulation ability (Fig. 2C) even though 1.62 x 106 cells were transplanted with 2 x 105 B6 CD45.2 BM cells. The reconstitution activity of FGF-cultured cells, as indicated by the total number of RUs per well, correlates with the CRI values (Fig. 2D; Table 1). A fivefold increase in RU was observed with cells treated with FGF-1 2 for 1 week. After 5 weeks of culturing, both FGF-2 and FGF-1 2-cultured cells had a 1.5-fold increase in RU compared with input cells (Fig. 2D; Table 1).! s% b# ^$ N9 s- @7 }$ ?8 A; T
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Figure 2. In vivo competitive transplantation assay of FGF-cultured cells. (A): B6 CD45.1 cells cultured in serum-free medium alone or in the presence of FGF-1, FGF-2, or both FGF-1 2 were transplanted into lethally irradiated B6 CD45.2 recipient mice after 1 week of culture. (B): B6 CD45.1 cells cultured in serum-free medium alone or in the presence of FGF-1, FGF-2, or both FGF-1 2 were transplanted into lethally irradiated B6 CD45.2 recipient mice after 3 weeks of culture. (C): B6 CD45.1 cells cultured in serum-free medium alone or in the presence of FGF-1, FGF-2, or both FGF-1 2 were transplanted into lethally irradiated B6 CD45.2 recipient mice after 5 weeks of culture. For 1-week culture, mice were transplanted with 2.5 x 105 cultured cells and 2.5 x 105 B6 CD45.2 bone marrow (BM) cells. Recipients receiving 3-week cultured cells were transplanted with 1.8 x 105 FGF-1, 2.2 x 105 FGF-2, 3.5 x 105 FGF-1 2, and 7.5 x 104 no-FGF-treated cells with 2 x 105 B6 CD45.2 BM cells. Five weeks after culturing, 1.6 x 106 FGF-1, 1.2 x 106 FGF-2, 1.1 x 106 FGF-1 2, and 4.8 x 105 no-FGF-treated cells were transplanted into recipient mice with 2 x 105 B6 CD45.2 BM cells. Average CRI ¡À SD was calculated in each group consisting of six mice. (D): The absolute number of repopulating units of cultured cells was compared with input cells. Each RU is equivalent to the repopulation function of 100,000 competitor BM cells. Therefore, at initiation of culture, 5 x 106 whole BM cells contain 50 RUs. After 1 week of culturing in FGF-1 2, 253 RUs were generated. Both FGF-2 and FGF-1 2-cultured cells after 5 weeks produced 75 RUs. Abbreviations: CRI, competitive repopulating index; FGF, fibroblast growth factor; RU, repopulation unit.
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Table 1. In vivo reconstitution of FGF-expanded cells8 a! \3 V7 B, w6 L2 m$ C; C
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Radioprotection and Long-Term In Vivo Reconstitution of FGF-Expanded Cells0 B. K; j+ O  g( `" _6 l6 G" S
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To test the ability of expanded cells in a more clinically relevant model, 1 x 104 and 5 x 104 FGF-1 2 expanded cells were transplanted in lethally irradiated C57BL/6 mice without competitor cells. After 3 weeks of culture, 1 x 104 and 5 x 104 cells were able to stably engraft into most recipient mice, providing long-term repopulation. Transplantation of 1 x 104 cells provided radioprotection to 60% of animals (Fig. 3A). This value increased to 80% when 5 x 104 FGF-1 2-expanded cells were transplanted (Fig. 3A). The survival rate was higher when culture time was extended to 5 weeks, with 1 x 104 expanded cells providing radioprotection to 80% of animals (Fig. 3A). A further increase in survival was observed with 5 x 104 expanded cells providing radioprotection to 100% of the mice (Fig. 3A). In all cases, a radioprotection endpoint of 4 weeks after transplant was used. Animals that died of hematopoietic failure died within 14 days after transplant. Transplantation of 1 x 104 cells cultured for 3 weeks resulted in an average chimerism of 50%, whereas 5 x 104 cells engrafted with an average chimerism of 75%, 28 weeks after transplant (Fig. 3B). Chimerism results from transplants carried out with cells from 5-week cultures are shown in Figure 3C. With 1 x 104 FGF-1 2-expanded cells, engraftment levels steadily increased, and stabilized after 8 weeks, and 26 weeks after transplant donor contribution was more than 90%. Mice transplanted with 5 x 104 cells showed an average level of chimerism of more than 95%.

Figure 3. Radioprotective potential of fibroblast growth factor (FGF)-expanded cells. (A): Survival rate of mice transplanted only with expanded cells. (B): Unfractionated B6 CD45.1 bone marrow (BM) cells were cultured in FGF-1 2 for 3 weeks. (C): Unfractionated B6 CD45.1 BM cells were cultured in FGF-1 2 for 5 weeks. Recipient CD45.2 C57BL/6 mice were lethally irradiated and transplanted with 1 x 104 or 5 x 104 FGF-1 2-cultured cells without life-sparing competitor cells. Analysis of chimerism was performed, and average results are shown.

Growing of LSK Cells in Cocultures

In our 3- and 5-week FGF cultures described above, the percentage of LSK cells was analyzed prior to transplantation. After 3 weeks of culturing, LSK frequencies of FGF-1-cultured cells was 0.7% whereas those of FGF-2 and FGF-1 2 cells were 1.3%. LSK frequencies increased after 5 weeks of culturing to 0.9% for FGF-1 cells and 2.8% for FGF-2 and FGF-1 2 cells (data not shown). Despite the high percentage of LSK cells in these cultures compared with normal BM cells, which have an LSK frequency of approximately 0.2%, we were not successful at culturing purified LSK HSCs or bulk Lin and Lin cells in serum-free medium supplemented with FGF-1 2 (data not shown). Thus, we speculated that either the stem cell growth in unfractionated BM cultures did not originate from LSK cells, or alternatively, that an accessory population of cells contained within the BM was required. To test this hypothesis, we sorted LSK cells from B6 CD45.1 mice and cocultured 7 x 103 and 5 x 104 of these cells in the presence of 5 x 106 CD45.2 unfractionated BM cells. FACS analysis showed that 5 x 106 unfractionated CD45.2 BM cells contained approximately 5,500 LSK cells. Purified stem cells and whole BM were cocultured for 5 weeks. After culture, all cells were harvested and 2 x 105 cells were transplanted into lethally irradiated recipients without competitors. The percentage of white blood cells originating from the purified LSK CD45.1 fraction or from the CD45.2  unfractionated cells was assessed in the recipients. If only LSK cells were responsible for the FGF-stimulated stem cell activity in unfractionated BM, we would expect chimerism levels of the sorted 7,000 CD45.1 LSK cells to reach or come close to 60% (7,000 CD45.1 LSK cells 5,500 LSK CD45.2 cells: 7,000/ = 56%). Strikingly, 16 weeks after transplant, chimerism levels had increased to 51%, 52%, and 64%, implying that indeed all FGF-induced stem cell activity is derived from the LSK population (Fig. 4A). Chimerism levels in recipients transplanted with 5 x 104 CD45.1 LSK cells cultured in 5 x 106 CD45.2 unfractionated BM cells ranged from 70% to 99% 16 weeks after transplant (Fig. 4B). Engraftment was seen in all mice transplanted. The estimated expected level of chimerism in these animals was 50,000/(50,000 5,500) = 90%, clearly well in range with the experimental findings. These results suggest that FGFs may be acting both on LSK cells or on other cell types, indirectly affecting LSK cells to induce stem cell activity in vitro.' m4 |4 o/ e4 U1 F: ~

Figure 4. Highly purified CD45.1 Lin¨CSca-1 c-Kit  (LSK) cells cocultured with unfractionated CD45.2 bone marrow (BM) cells. (A): Seven thousand B6 CD45.1 LSK cells were cocultured with 5 x 106 B6 CD45.2 whole BM cells (estimated to contain 5,500 CD45.2 LSK cells) for 5 weeks in serum-free medium supplemented with fibroblast growth factor (FGF)-1 x 2. Subsequently, 2 x 105 cultured cells were transplanted into B6 CD45.2 mice without additional competitor cells. At the start of the culture, the percentage of CD45.1 LSK cells in the coculture was 7,000/(7,000 5,500) = 56%. Twelve weeks after transplant, chimerism levels of all mice steadily increased to an average of 57%. (B): Five-week coculture of 50,000 B6 CD45.1 LSK cells and 5 x 106 B6 CD45.2 whole BM cells was set up and similarly transplanted. The percentage of CD45.1 LSK cells in this culture was 50,000/(50,000 5,500) = 90%. Chimerism levels in transplanted recipients rapidly increased, reaching 83%. Chimerism levels for each transplanted recipient are denoted by different symbols.. K, c$ G) p- J4 r' c

Effect of SCF, IL-11, and Flt3L on FGF-1 2-Induced Clonogenic Activity In Vitro

Although single LSK cells cultured in FGF-1 2 did not divide, they did remain visible for 7 days (data not shown). Thus, it appeared that the addition of FGF-1 2 to cell cultures prolonged the lifespan of the cells but did not induce a strong enough mitogenic signal. More classic hematopoietic GFs such as SCF, IL-11, and Flt3L have a much stronger proliferating effect and are shown to maintain stem cells in short-term cultures . Therefore, we cultured cells with a cocktail of SCF, IL-11, and Flt3L with or without the addition of FGF-1 2. After 5 weeks of culturing, cell numbers of GF-treated cells had exponentially increased from 5 x 106 to 7 x 108, similar to GF FGF-1 2-cultured cells (Fig. 5A; Table 2). We next tested whether FGFs would be able to maintain stemness of cells when used in combination with SCF, IL-11, and Flt3L, which provides stronger mitogenic signals.
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Figure 5. Effects of GF treatment on FGF-cultured cells. (A): Growth kinetics of unfractionated bone marrow (BM) cells cultured with a cocktail of GFs (SCF, IL-11, and Flt3L) in the presence or absence of FGF-1 2. Cell growth exponentially increased from 5 x 106 to 7 x 108. (B): Day 7 and 35 CAFC content of unfractionated BM cells and cells cultured for 3 weeks with FGF-1 2, GFs (SCF, IL-11, and Flt3L) alone, and GFs FGF-1 2. No day 35 CAFC activity was observed for cells cultured with GFs alone or GFs FGF-1 2; p values were calculated using Student¡¯s t test, and Poisson-based limiting dilution analysis was used to determine the CAFC frequency. (C): CRI ¡À SD values (shown in Table 2) for mice transplanted with varying cell doses of FGF-1 2, GF, and GF FGF-1 2-cultured cells in competition with 2 x 105 B6.CD45.2 cells. Data are the same as in Figure 2 for FGF-1 2 and are included only for comparison. Abbreviations: CAFC, cobblestone area-forming cell; CRI, competitive repopulating index; FGF, fibroblast growth factor; Flt3L, Flt3 ligand; GF, growth factor; IL, interleukin; N.D., not detected; N.S., not significant; SCF, stem cell factor.
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Table 2. Effect of the addition of stem cell factor, interleukin-11, and flt3 ligand on FGF-1 2-induced stem cell activity  c4 J# W6 Y3 X0 H+ C% O

CAFC assays were carried out to determine clonogenic activity of cells treated with a cocktail of GFs. We observed a significant (p
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Effect of GFs on FGF-1 2-Induced Stem Cell Activity In Vivo, ]# B0 Q0 M) Q1 `& y$ U7 z2 K2 ~

Finally, we determined whether cells cultured in FGF-1 2 with or without SCF, IL-11, and Flt3L provided engraftment in vivo. To this end, varying cell doses ranging from 1.8 x 105 to 2 x 106 B6 CD45.1 cultured cells were transplanted in competition with 2 x 105 freshly isolated B6 CD45.2 BM cells and compared with results for transplanted FGF-1 2 cells (Fig. 2). Although highly elevated CRI levels were observed 16 weeks after transplant, when FGF-1 2 treated cells were cultured for 1 week, the CRI had dropped to 1 for cells cultured with GFs alone or GF FGF-1 2-treated cells (Fig. 5C; Table 2). Continued culturing of cells for 3 and 5 weeks decreased CRI values for all conditions. All competitive repopulation ability was lost after 3 weeks of culturing with GFs alone or with the addition of GF FGF-1 2 (Fig. 5C; Table 2).
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DISCUSSION

In the present study, we tested the potential of different FGFs to support HSC growth in serum-free medium. In all three FGF conditions, FGF-1, FGF-2, and FGF-1 2, similar trends in overall cell growth and cell morphology were observed. Although not evident from the growth and morphology of the cultured cells, the addition of FGF-1 and/or -2 to serum-free medium proved to be an effective culture condition to support primitive HSCs. Our in vitro CAFC data and in vivo reconstitution results clearly document that bona fide long-term repopulating stem cells can be preserved in vitro for up to 5 weeks when FGFs are added to the medium (Figs. 1, 2).

To test the repopulating potential of FGF-treated stem cells in a clinically relevant model, cultured cells were transplanted into lethally irradiated mice without competitor cells. Importantly, BM cells cultured for 3 and 5 weeks in the presence of only FGF-1 2 were able to provide radioprotection and reconstitution.1 d4 }% v5 U  _* l+ M

Thus, our data clearly document that FGFs (of which we tested two out of a family of 22) can be added to a growing list of signaling proteins that act on primitive stem cells. Interestingly, whereas "classic" hematopoietic GFs turn out to have limited potential in sustaining and expanding HSCs .7 Z- M' z+ q1 ?4 S7 h- M3 n

We were unable to culture purified LSK cells with only FGFs, whereas recently Zhang and Lodish were able to culture purified BM side population cells with a greater than eightfold increase in repopulating HSCs when grown in low levels of SCF, thrombopoietin (TPO), IGF-2, and FGF-1 in serum-free medium for 10 days . In our purified cell culture system, such crosstalk with other signaling networks was not possible, suggesting that the combination of SCF, TPO, IGF-2, and FGF-1 is better suited for the expansion of a purified population of HSCs. The results of Zhang and Lodish and our results highlight the importance of FGF in an in vitro culture system to maintain HSCs; however, our findings also suggest that the effect of FGFs on stem cells requires other stimuli. The addition of SCF, IL-11, and Flt3L increased the proliferation of stem cells. We tested whether FGFs were able to maintain the primitiveness of stem cells, thereby negating the potential differentiation effect of these GFs when placed in combination. Thus, GF FGF-1 2-cultured cells would have been expected to provide competitive reconstitution whereas GF-only cultures would have been expected to provide no or very little reconstitution. Unfortunately, this was evidently not the case (Fig. 5). The optimal combination of GFs required for maintaining primitive HSC activity remains to be discovered.

Our studies were not aimed at delineating the molecular consequences of incubating BM cells with FGFs. Consequently, we can only speculate on how FGFs maintain stem cells in whole BM cultures. It has been shown that receptors for FGF-1 are present on primitive hematopoietic cell subsets . We speculate that for HSCs to be properly maintained and amplified in vitro, the whole BM in coculture must act as a niche, facilitating stem cells to expand.8 K5 p+ R4 s5 y8 p( h

It is tempting to postulate that many stem cell expansion studies have not been so successful, because cultures almost invariably were initiated with purified cells. As shown in our purification studies, disruption of HSCs from their niche is likely to have detrimental effects on their subsequent developmental potential. Our study represents a clear example of an in vitro system using FGFs, capable of supporting primitive HSCs for an extended period of time. Future studies will be aimed at creating a niche for stem cells in vitro, while stimulating their proliferation.
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DISCLOSURES4 f0 x4 W8 n% f5 P& G! G
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS3 t" L# n: x6 A1 K" J2 d3 n. C
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We thank Geert Mesander and Henk Moes for assistance with cell sorting and the animal facility staff for taking care of the mice. This work was supported by grants from the Dutch Cancer Society (RUG 2000-2182 and RUG 2000-2183), National Institutes of Health (R01-HL073710), European Union (EU-LSHC-CT-2004-503436), and the Ubbo Emmius Foundation.
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