<title>1. INTRODUCTION</title> <p>Stem cells
are unique cells that retain the ability to divide and proliferate throughout
postnatal life to provide progenitor cells that can progressively become
committed to produce specialized cells. Many cytokines have been reported as
inducers of stem cell proliferation and differentiation, one of them is FL
(Flt3 ligand) which can prolong maintenance of primitive human cord blood cells
in stromal-free suspension cultures [##REF##8145851##1##–##REF##10828034##4##]. However, FL-containing
cultures still cause the loss of repopulating capacity of primitive cells [##REF##8618433##5##–##REF##14614158##10##]. </p> <p>We have extracted
and identified a new legume lectin from Hyactinth bean [##UREF##0##11##]. It is a ligand of
receptor tyrosine kinase Flt3 which is strictly expressed in hematopoietic and
neural cell lines [##REF##11640895##12##–##REF##7622010##14##]. Gabriella has reported a new flt3 ligand which has the ability to
preserve hematopoietic progenitor cells in vitro for 4 weeks [##REF##9892687##15##]. The gene and
protein sequences of our protein are quite similar to Gabriella’s protein [##UREF##0##11##],
besides our protein is also capable of long time in vitro preservation of
hematopoietic progenitor cells. According to previous reports [##REF##9892687##15##–##REF##10843844##19##], we named our protein FRIL too and found that FRIL could
maintain hematopoietic stem cell in <italic>G</italic>
<sub>0</sub>/<italic>G</italic>
<sub>1</sub> phase for at
least 4 weeks. Low activation of FRIL’s receptor Flt3 is considered the key
mechanism through which FRIL preserves cells. Further experiments showed that
FRIL inhibited hematopoietic stem/progenitor cell (HSPC) apoptosis by
upregulating p53, reduced activation of MAPK and phosphorylation of STAT5,
disturbed formation of AP-1 complex, and in the end suppressed the
expression/activation of cell cycle proteins [##UREF##2##20##–##UREF##3##22##].</p> <p>Over the last
decade, neural stem cell research provided penetrating insights into plasticity
and regenerative medicine. Stem cells have been isolated from both embryonic
and adult nervous system [##REF##17474295##23##, ##REF##17465732##24##]. Although with the help of extrinsic
signaling factors progress has been made in understanding fundamental stem cell
properties, processes that determine cell fate or that maintain a cell’s
primitive state are still unknown. As a following experiment to find out
whether FRIL can preserve neural progenitor cells as it does on HSPCs, we first
identified the isolated neural progenitor cells (NPCs) and their flt3
expressing levels, then we supplemented FRIL into different NPC suspension
culture media, observed cell morphological changes, analyzed cell growth and
cell surface marker expression, finally we used semiquantitive RT-PCR to verify
the changes in gene expression.</p>
<title>2. MATERIALS AND METHODS</title> <title>2.1. NPCs isolation and cell culture</title> <p>E13.5 SD rats (experimental animals department, Peking University, Beijing, China) were
sacrificed, then fetal embryonic telencephalon and mesencephalon were dissected
under the dissecting microscope, after digestion in the sterilized tube, cells
were seeded in 25 cm<sup>2</sup> flask for general culture and passage. Meanwhile statistically supplemented
EGF(20 ng/mL), bFGF(20 ng/mL), and FRIL(300 <italic>μ</italic>g/mL, 1 : 1000) into basic culture
media(98% DMEM/F12 (1 : 1) and 2% B27/N2) to form eight groups of culture media
(control, EGF, bFGF, FRIL, EGF&amp;bFGF, EGF&amp;FRIL, bFGF&amp;FRIL,
EGF&amp;bFGF&amp;FRIL). After second passage, equal numbers of NPCs were seeded
with these different culture media. Furthermore, some NPCs were seeded in
differentiation media which contains 5% fetal bovine serum. Culture media comprised
a low percent of glucose and did not contain mannose which may exerted negative
effect on FRIL’s function [##REF##9892687##15##].</p> <title>2.2. Identification of NPCs and expression of flt3 on NPCs</title> <p>P2 neurospheres were transferred into poly-l-lysine (pll) precoated 24-well plate,
incubated for 8 hours at 37°C, and then stained by nestin for immunofluorescence
for the purpose of NPCs identification. Meanwhile, some other neurospheres were
treated with HRP conjugated FRIL in order to find out the proportion of flt3
expressed on NPCs.</p> <title>2.3. Cell counting assay</title> <p>Cells were harvested at day 7
following seeding, resuspended with PBS after digestion and centrifuge, and
then seeded on 96-well plate together with 100 <italic>μ</italic>L solution and 10 <italic>μ</italic>L CCK-8(Cat. no.
CK04, Dojindo Laboratories Co, Japan.) per well. Plate was incubated at 37°C, for 4 hours, then the absorbance at
450 nm was measured using microplate reader with a reference wavelength at 650 nm(Bio-Rad Model 550). Data was analyzed by SAS 8.2 software.</p> <title>2.4. Morphological observation</title> <p>P3 NPCs were harvested and digested
into single cell, cells were counted and seeded into 25 cm<sup>2</sup> flasks with a 
desity of 5 × 10<sup>4</sup> per mL. Each flask was supplemented with only one media described above, and NPC
morphology was observed everyday.</p> <title>2.5. Immunofluorescence assay</title> <p>NPCs were seeded into pll precoated
24-well plate and incubated at 37°C for 8 hours, fixed with 4%
paraformaldehyde for 20 minutes, washed with PBS 5 minutes three times, 0.3%
Triton X-100 20 minutes, PBS 5 minutes three times, then were incubated with
primary antibody for 2 hours at 37°C, washed, and incubated with
secondary antibody for 30 minutes at 37°C. Primary antibodies that were used
include nestin (Mouse Anti-Nestin Monoclonal Antibody, Cat. no. MAB353, Chemicon, Inc, USA.), GFAP (Cat. no. ZA-0117, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.), NSE
(RABBIT ANTI-NSE, Cat. no. BA0535, Boster Biotechnology Co. Ltd.), Tubulin
(Monoclonal Anti-<italic>β</italic>-Tubulin III antibody produced in mouse Clone
SDL.3D10, ascites fluid, Cat. no. T 8660, Sigma-Aldrich Co, USA.), O4 (Mouse Anti-Oligodendrocyte
Marker O4 Monoclonal Antibody, Cat. no. MAB345, Chemicon, Inc. USA.). All primary
antibodies were diluted with PBS and 10% normal goat serum. Secondary
antibodies included FITC (Fluorescein-Conjugated) AffiniPure Goat Anti-Mouse
IgG (H+L) (Cat. no. ZF-0312, Beijing Zhongshan Golden Bridge Biotechnology Co.,
Ltd.), Rhodamine (TRITC)-Conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (Cat.
no. ZF-0313, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.),
Rhodamine (TRITC)-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Cat. no.
ZF-0316, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). When cells
were stained for nestin together with GFAP, Tubulin or NSE, primary and
secondary antibodies for each were added simultaneously. 4′,6′-Diamidino-2-phenylindole
dihydrochloride (DAPI; 1 : 10000) was used in all fluorescence facilitating cell
counts. Images for cell counts were captured from six equally spaced,
predetermined areas of each well under fluorescence microscope. NPCs, neurons,
glials, and other cells on the images were counted and analyzed.</p> <title>2.6. Semiquantitive RT-PCR assay</title> <p>mRNA was isolated using trizol kit
according to manufacture protocols, then mRNA was reverse transcripted in a
final volume of 100 <italic>μ</italic>L, 42°C 5 minutes, 42°C 1 hour, 94°C 5 minutes, −20°C. Then, <italic>ACTIN</italic> F 5′TACCACTGGCATCG
RGATGGACT
3′ R 5′TCCTTCTGCATCCTGTCGGCAAT 3′ 94°C 30 seconds, 59°C 30 seconds, 72°C 30 seconds, 25 cycles, 72°C 5 minutes, 4°C. <italic>GFAP</italic> F 5′GAAGAAAACCGCATCACCAT3′ R 5′GCACACCTCACATCACATCC3′ 94°C 30 seconds, 57°C 30 seconds, 72°C 30 seconds, 25 cycles, 72°C 5 minutes, 4°C. <italic>Nestin</italic> F 5′GGAGCCATTGTGGTCTACTGA3′ R 5′TCCCACCGCTGTTGATTT3′ 94°C 30sec, 56°C 30 seconds, 72°C 30 seconds, 28 cycles, 72°C 5 minutes, 4°C. <italic>NeuroD1</italic> F 5′ACCTGCTGCCCAGAGTTTTA3′ R 5′CAGAGGCTACCGAGGACTTG3′
94°C 30 seconds, 59°C 30 seconds, 72°C 30 seconds, 35
cycles, 72°C 5 minutes, 4°C. <italic>NeuroD2</italic> F 5′GGGACTCGCCTTCTCTCTCT3′R 5′CTATCCCCGAAACTCAGCAG3′ 94°C 30 seconds, 59°C 30 seconds, 72°C 30 seconds, 40
cycles, 72°C 5 minutes, 4°C. <italic>NeuroD3</italic> F 5′CAGTAGTCCCTCGGCTTCAG3′R 5′TAGACTGGGGCAGGAAAGAA3′
94°C 30 seconds, 59°C 30 seconds, 72°C 30 seconds, 40
cycles, 72°C 5 minutes, 4°C. <italic>Ngn3</italic> F 5′AGGGGACACACGATTAGCAG3′R 5′GGTCTCTTGGGACACTTGGA3′ 94°C 30 seconds, 59°C 30 seconds, 72°C 30 seconds, 38
cycles, 72°C 5 minutes, 4°C. <italic>Notch</italic> F 5′GGTGGACATTGACGAGTGTG3′ R 5′CCCTTGAGGCATAAGCAGAG3′ 94°C 30 seconds, 59°C 30 seconds, 72°C 30 seconds, 27
cycles, 72°C 5 minutes, 4°C. 20 <italic>μ</italic>L of each
samples of PCRs were run on 10% TAE PAGE stained with ethidium bromide. Gel
image were saved as TIFF files using AlphaImager3300 (Serial no. 981878, Alpha Inc, China.). Gel band
quantitation analysis was performed via analysis tools of AlphaImager3300.</p>
<title>3. RESULTS</title> <title>3.1. NPCs identification, flt3 expression, and neural progenitor cell growth assay</title> <p>Primer (P0) cells
began to form regular spheres on day 2 after dissection (##FIG##0##Figure 1(a)##), although
some cell death was evident by the presence of cell fragments. More than 98% of
the P2 cells were nestin-positive, indicating they were NPCs (Figures ##FIG##0##1(c)##, ##FIG##0##1(d)##).
While more than 95% of cells were positive for FRIL-HRP stain, indicating that
most NPCs expressed flt3 protein (##FIG##0##Figure 1(b)##).</p> <p>Then it came to
the NPCs in vitro growth assay. FRIL can promote NPCs growth (<italic>P</italic> &lt; .0001).
However, FRIL is less effective than bFGF (<italic>P</italic> &lt; .0001) and EGF (<italic>P</italic> &lt; .0001)
(##FIG##1##Figure 2##).</p> <title>3.2. FRIL delayed formation of neurospheres and induced cell adhesion</title> <p>When seeded,
NPCs formed spheres later in media with FRIL than those without FRIL. Although
there were neurospheres in FRIL containing media, these spheres were smaller
than those formed in media without FRIL (Figures ##FIG##2##3(b)##, ##FIG##2##3(d)##, ##FIG##2##3(f)##, ##FIG##2##3(h)##).
Especially in FRIL medium, there were many single/two cells and some small
neurospheres which mostly had 3 or 4 cells (##FIG##2##Figure 3(b)##).</p> <p>After P3 NPCs
were seeded, there was no difference on cell growth in media with/without FRIL
from day 1 to day 4 after seeding. However, after day 4, cells in FRIL containing
media adhered to the flask surface (##FIG##2##Figure 3##). And this phenomenon took place
again when NPCs were cultured after passage.</p> <p>NPCs cultured
in differentiation media showed difference from day 3 to day 7 after seeding. Cells
in serum media seemed to begin to grow on day 3 and covered the bottom of well/flask
on day 6/day 7, while cells in FRIL and serum containing media still spoted on
the bottom or just formed some colonies. However, from day 7 on cells in FRIL and
serum-containing media reached confluency. Most cells showed glial-like
morphology. 1∼3 neuron-like cells per well existed in serum-containing media,
while 13∼21 cells were present in FRIL and serum-containing media (data not
shown).</p> <title>3.3. FRIL reduced GFAP expression while maintained NSE expression</title> <p>We immunostained
cells, nestin for NPCs, GFAP for astrocytes, NSE/tubulin for neurons, and O4
for oligodendrocytes. Fewer FRIL treated NPCs differentiated into astrocytes than
those not treated by FRIL, despite the fact that they had adhered to the plates
(##FIG##3##Figure 4##). Oligodendrocytes were not found. NSE positive cells were not found
in culture media containing EGF and EGF&amp;FRIL too. The number of NSE
positive cells in FRIL containing media was slightly higher than that without
FRIL, although NSE positive rates were small. In addition, tubulin positive
cells were not found in any media.</p> <title>3.4. Semiquantitive RT-PCR analysis affirmed FRIL inhibition in glial differentiation</title> <p>On the basis
of previous findings [##REF##11239394##25##–##REF##11239431##27##], we chosed 5 genes, namely, <italic>GFAP</italic> and <italic>Notch</italic> (an indicator of GFAP), <italic>NeuroD1</italic>, <italic>NeuroD2</italic>, <italic>NeuroD3</italic>, and <italic>Ngn3</italic> (which
serves as upstream regulator of NeuroD) to examine gene expression of cells in
eight different cell culture media. <italic>GFAP</italic> and <italic>nestin</italic> were expressed in each
group, <italic>nestin</italic> expression was similar,
while <italic>GFAP</italic> expression in FRIL
containing groups was lower than those without FRIL (##FIG##4##Figure 5##). <italic>Notch</italic> was not found in control and serum-treated
groups, and its expression was quite opposite to that of <italic>GFAP</italic>. Neuronal gene expression was weak, there was no <italic>NeuroD3</italic> expression, <italic>NeuroD2</italic> was only expressed in 3 media,
although expression in <italic>NeuroD3</italic> and <italic>Ngn3</italic> was much higher in FRIL and serum media
than in serum media. Although FRIL might has no function on neuronal differentiation,
these RT-PCR results suggested that FRIL inhibited neural progenitor cells from
autodifferentiating into glials.</p>
<title>4. DISCUSSION</title> <p>98.6% nestin positive cells showed that to some extent all cells at P2 were NPCs. 95.7% flt3
expression confirmed the similarities between NPCs and HSCs, although this contradicted
to Brazel’s findings in which flt3 was expressed only in NPCs in dorsal root ganglions
[##REF##11640895##12##]. The similarities between NPCs and HSCs [##REF##12702659##28##, ##REF##11438738##29##], especially flt3
expression, gave us hints on FRIL-NPCs study.</p> <p>Previous studies showed that the plant lectin FRIL was successfully extracted from <italic>Dolichos lablab</italic> using a mannose affinity
column [##UREF##0##11##, ##REF##9892687##15##]. Although mannose or <italic>α</italic>-methyl <italic>α</italic>-D-mannoside were reported exerting a negative effects, FRIL
had the ability of preserving HSCs in vitro for a long time [##REF##15224142##21##, ##UREF##3##22##]. In this
study, we reported that FRIL could also preserve neural progenitor cells as it
does on hematopoietic stem/progenitor cells, it slowed NPC aggregation and
growth, on the other hand it inhibited NPC glial autodifferentiation, although
it might not affect NPC neuronal autodifferentiation. At present, the common
widely studied neural differentiation pathways are Notch, Shh, and so forth
[##REF##11438738##29##, ##REF##9915700##30##]. Based on these pathways, both neuronal and glial RT-PCR results are
in coincident with the results of immunofluorescence. No matter what kinds of
development in which FRIL is involved, the mechanism is still unclear and
further researches in signal pathways should be done. Depending on our previous
studies that FRIL can affect activities of MAPK, STAT5, and p53, FRIL neural
mechanical researches that may be more complicated could be performed.</p> <p>There have
been reports that stem cells can be induced differentiating into neural cells,
however cytokines that effectively control these cells are still inadequate.
FRIL’s ability to functionally preserve hematopoietic and neural cells may
significantly extend the range and time for manipulating these cells. Experiments
are underway to find out whether FRIL can improve applications in neural stem
cell transplantation, treatment of neural degenerative diseases, and cell
therapy, and FRIL’s properties of fuctionally select, preserve, and synchronize
hematopoietic cell population could also give some hints.</p>
<p>Recommended by Yang Liu</p> <p>In vitro maintenance of stem cells is crucial for many clinical applications. Stem cell preservation factor FRIL (Flt3 receptor-interacting lectin) is a plant lectin extracted from <italic>Dolichos Lablab</italic> and has been found preserve hematopoietic stem cells in vitro for a month in our previous studies. To investigate whether FRIL can preserve neural progenitor cells (NPCs), it was supplemented into serum-free suspension culture media. FRIL made NPC grow slowly, induced cell adhesion, and delayed neurospheres formation. However, FRIL did not initiate NPC differentiation according to immunofluorescence and semiquantitive RT-PCR results. In conclusion, FRIL could also preserve neural progenitor cells in vitro by inhibiting both cell proliferation and differentiation.</p>
<title>ACKNOWLEDGMENTS</title> <p>The authors would like to thank Professor Janet Benson (Lovelace Respiratory
Research Institute, NM, USA) for her advice on this manuscript revision. X. Pei
was supported by National High Technology Research and Development Program of China (no. 2006AA02A107), the
Major State Basic Research Program of China (no. 2005CB522702).</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>Neural progenitor cell identification and Flt3
expression. (a) Neurospheres under bright field. (b) Flt3-HRP staining on
neural spheres. (c) nestin immunofluorescence on neurospheres, RED nestin, BLUE
DAPI. (d) Nestin immunofluorescence on single NPC, GREEN nestin, BLUE DAPI.</p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>Cell counting assay. Cells were cultured in eight
media with different growth factors combination represented in figure by
control, FRIL, bFGF, bFGF&amp;FRIL, EGF, EGF&amp;FRIL, bFGF&amp;EGF,
bFGF&amp;EGF&amp;FRIL. Cell numbers were transferred into absorption value
obtained from microplate reader Bio-Rad Model 550.</p></caption></fig> <fig id="fig3" position="float"><label>Figure 3</label><caption><p>morphological observation of NPCs cultured in
different media. (a) NPCs in control medium. (b) NPCs in FRIL medium. (c) NPCs
in bFGF medium. (d) NPCs in bFGF&amp; FRIL medium. (e) NPCs in EGF medium. (f)
NPCs in EGF&amp;FRIL medium. (g) NPCs in bFGF &amp;EGF medium. (h) NPCs in
bFGF&amp;EGF&amp;FRIL medium.</p></caption></fig> <fig id="fig4" position="float"><label>Figure 4</label><caption><p>GFAP and nestin immunofluorescence on NPCs cultured
in different media. (a) NPCs in control medium. (b) NPCs in FRIL medium. (c)
NPCs in bFGF medium. (d) NPCs in bFGF&amp;FRIL medium. (e) NPCs in EGF medium. (f)
NPCs in EGF&amp;FRIL medium. (g) NPCs in bFGF&amp;EGF medium. (h) NPCs in
bFGF&amp;EGF&amp;FRIL medium. GREEN nestin, RED GFAP, BLUE DAPI.</p></caption></fig> <fig id="fig5" position="float"><label>Figure 5</label><caption><p>semiquantitive RT-PCR of different genes in NPCs
cultured in different media. Lane: (C) NPCs in control medium. (F) NPCs in FRIL
medium. (b) NPCs in bFGF medium. (E) NPCs in EGF medium. (bF) NPCs in
bFGF&amp;FRIL medium. (bE) NPCs in bFGF&amp;EGF medium. (EF) NPCs in
EGF&amp;FRIL medium. (bEF) NPCs in bFGF&amp;EGF&amp;FRIL medium. (S) NPCs in
serum medium. (FS) NPCs in serum&amp;FRIL medium. Row: ngn3, neurogenin3.
NOTCH, notch. NeuroD3, NeuroD3. NeuroD2, NeuroD2. NeuroD1, NeuroD1. Nestin,
nestin. GFAP, GFAP. ACTIN, actin.</p></caption></fig>
<graphic xlink:href="CDI2008-531317.001"/> <graphic xlink:href="CDI2008-531317.002"/> <graphic xlink:href="CDI2008-531317.003"/> <graphic xlink:href="CDI2008-531317.004"/> <graphic xlink:href="CDI2008-531317.005"/>
[{"label": ["11"], "surname": ["Xie", "Xie", "Li", "Bai", "Pei"], "given-names": ["X", "C", "J", "C-X", "X"], "article-title": ["Extraction, purification and function analysis of a new lectin which can maintain the multipotential of hematopoietic stem/progenitor cells in vitro"], "italic": ["Sheng Wu Hua Xue Yu Sheng Wu Wu Li Jin Zhan"], "year": ["2004"], "volume": ["31"], "issue": ["2"], "fpage": ["134"], "lpage": ["140"]}, {"label": ["17"], "surname": ["Kari", "Scott", "Yung"], "given-names": ["LK", "B", "C"], "article-title": ["Characterization of FRIL, a mannose binding lectin of "], "italic": ["Dolicos lablab"]}, {"label": ["20"], "surname": ["Xie", "Pei"], "given-names": ["C", "X"], "article-title": ["The mechanisms of a new plant lectin FRIL from "], "italic": ["Dolichos lablab", "Sheng li ke xue jin zhan [Progress in physiology]"], "year": ["2003"], "volume": ["34"], "issue": ["2"], "fpage": ["127"], "lpage": ["131"]}, {"label": ["22"], "surname": ["Xie", "Xie", "Li"], "given-names": ["X", "C", "J"], "article-title": ["Studies on the mechanism that how could lectin FRIL preserve hematopoietic stem/progenitor cells in vitro"], "italic": ["Chinese Journal of Hematology"], "year": ["2004"], "volume": ["25"], "fpage": ["501"], "lpage": ["504"]}]
Clin Dev Immunol. 2008 Aug 5; 2008:531317