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Original Article

Cell Research (2006) 16: 909=96915.=20 doi:10.1038/sj.cr.7310104; published online 7 November 2006

Differentiation profile of brain tumor stem cells: a = comparative=20 study with neural stem cells

Quan Bin Zhang¹,=20 Xiao Yan Ji¹,=20 Qiang Huang¹,=20 Jun Dong¹,=20 Yu De Zhu¹=20 and Qing Lan¹

¹Neurosurgical Department and = Brain Tumor=20 Research Laboratory, Second Affiliated Hospital of Suzhou University, = Suzhou=20 215004, China

Correspondence: Qiang Huang, Tel: +86-512-67783937; = E-mail: szqhdnah@pub.sz.jsinfo.net=

Received 18 May 2006; Revised=20 23 August 2006; Accepted 19 September 2006. =

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Abstract

Understanding of the differentiation profile of = brain tumor=20 stem cells (BTSCs), the key ones among tumor cell population, through = comparison=20 with neural stem cells (NSCs) would lend insight into the origin of = glioma and=20 ultimately yield new approaches to fight this intractable disease. Here, = we=20 cultured and purified BTSCs from surgical glioma specimens and NSCs from = human=20 fetal brain tissue, and further analyzed their cellular biological = behaviors,=20 especially their differentiation property. As expected, NSCs = differentiated into=20 mature neural phenotypes. In the same differentiation condition, = however, BTSCs=20 exhibited distinguished differences. Morphologically, cells grew = flattened and=20 attached for the first week, but gradually aggregated and reformed = floating=20 tumor sphere thereafter. During the corresponding period, the expression = rate of=20 undifferentiated cell marker CD133 and nestin in BTSCs kept decreasing, = but 1=20 week later, they regained ascending tendency. Interestingly, the = differentiated=20 cell markers GFAP and -tubu= linIII=20 showed an expression change inverse to that of undifferentiated cell = markers.=20 Taken together, BTSCs were revealed to possess a capacity to resist=20 differentiation, which actually represents the malignant behaviors of=20 glioma.

Keywords:

brain tumor stem cell, neural stem cell,=20 differentiation

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Introduction

Glioma is the most common neoplasm of central nervous = system in=20 human and remains difficult to cure despite rapid advances in diagnostic = imaging=20 techniques and multimodality therapy. For the past decades, huge = endeavors have=20 been dedicated to elucidate the cellular and molecular mechanisms of = this=20 intractable disease; however, no substantial breakthrough has been = achieved due=20 to the fact that brain tumor mass is composed of heterogeneous = combination of=20 cells. According to Reya et al. ¹,=20 among tumor cell population, there always exist stem cells that are rare = but=20 critical in tumorigenesis. Following the identification of tumor stem = cells from=20 leukemia and breast cancer ^2,=20
3,=20 brain tumor stem cells (BTSCs), as recently reported, were cultured from = both=20 surgical specimens and cultured glioma cell lines, according to the = following=20 three lines of evidence: (1) expressing known neural stem cell (NSC) = surface=20 marker CD133 and nestin, and generating spheres that are morphologically = indistinguishable from neurospheres; (2) self-renewing and = proliferating; and=20 (3) producing differentiated progeny in vitro or recapitulating = the=20 parental tumor mass when implanted into the immunodeficient animals = ^{4,=20
5,=20
6,=20
7,=20
8,=20
9}.=20 Singh et al. ⁴=20 observed that, under differentiation condition, all tumor spheres grew = attached=20 as monolayer, lacking expression of undifferentiated cell marker CD133 = and=20 nestin. Tumor stem cells have the characteristic of forming tumor = sphere;=20 however, the fact that only a small proportion of cells within tumor = spheres are=20 true stem cells makes it necessary to isolate and focus on tumor stem = cells if=20 the exact biological behaviors of this special cell fraction are to be = fully=20 understood. In this study, we purified BTSCs from brain tumor spheres, = and=20 isolated human NSCs, the mostly likely glioma cell-of-origin ^{10,=20
11,=20
12},=20 from neurospheres, and made a comparative study to specify the = differentiation=20 profile of BTSCs.

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Material and Methods

Tumor samples and human fetal brain tissue

Tumor tissues were obtained from a 52-year-old = female=20 patient undergoing twice operations within 2 months for rapid relapse. = Both=20 primary and recurrent lesions were pathologically diagnosed as mixed = glioma=20 consisting of anaplastic ependymoma and astrocytoma. Human fetal brain = tissue=20 was obtained from the consent-informed patient receiving induced = abortion in=20 accordance with protocols approved by the Research Ethics Board at the = Second=20 Affiliated Hospital of Suzhou University.

Tumor sphere and neurosphere culture

Briefly, the fresh tumor specimens were washed, = deprived of=20 vessels, acutely dissociated in PBS and subjected to enzymatic = dissociation as=20 described previously ².=20 Cells were suspended in defined stem cell medium consisting of = Dulbecco's=20 modified Eagle's medium/F-12 (Gibco), N₂ supplement (Gibco), = EGF (20=20 ng/ml, Invitrogen) and bFGF (20 ng/ml, Gibco), and plated at a density = of 510⁶=20 live cells per 75 cm² flask. Cells were fed by changing half = of the=20 medium every 3 days. Brain tissue from a 1-month-old embryo was = subjected to the=20 same procedure as that mentioned above to get neurospheres. Both tumor = spheres=20 and neurospheres were enzymatically dissociated into single cells for = expanded=20 culture when they were large in size and high in density.

Purification of BTSCs and NSCs

Neurospheres, tumor spheres as well as cells = differentiated=20 from tumor spheres were harvested, subjected to enzymatic dissociation = and=20 resuspended in 1PBS=20 containing BSA and 2 mM EDTA. Magnetic labeling with 1 ml CD133/1 = Microbead/1=20 million cells was performed using the CD133 Cell isolation kit (Miltenyi = Biotec). Cell sorting was carried out on the auto-MACS machine (Miltenyi = Biotec), with collected positive and negative fractions. The efficiency = of cell=20 sorting was further evaluated by flow cytometry (BD FACS Vantage).

Subsphere forming assay

Firstly, both tumor spheres and neurospheres were=20 dissociated into single cells and transferred to 96-well plates at = density=20 ranging from 10 cells to 200 cells per well. Cultures were fed by = changing 50 ml=20 of defined stem cell medium every 3 days till day 10. Secondly, both = tumor=20 spheres and neurospheres were subject to differentiation condition (in = medium=20 containing 10% FBS) for 7 days, then transferred to and cultured in = defined stem=20 cell medium. Thirdly, sorted CD133⁺ tumor cells and = CD133^-=20 tumor cells were plated to 96-well plates containing defined stem cell = medium.=20 Formation of subspheres was examined with phase-contrast microscopy.

Differentiation assay of BTSCs and NSCs

Immunofluorescent staining of BTSCs and = NSCs Purified=20 BTSCs and NSCs were plated onto glass coverslips coated in = poly-L-ornithine=20 (Sigma) in a 24-well plate. Differentiation was induced with culture = medium=20 added with 10% FBS. Coverslips were taken out on days 0 (4 h later), 3, = 7 and=20 10, fixed with 4% paraformaldehyde and immunostained with antibodies = against=20 CD133 (Miltenyi Biotec), nestin (BDbioscience Co.), GFAP (Santa Cruz = Co.) and=20 MAP2. Laser scanning confocal microscope (Carl Zess Co.) was employed to = examine=20 the costaining of the above cell markers during differentiation of BTSCs = and=20 NSCs.

Detection of the expressing rates of cell surface = markers=20 BTSCs and NSCs were transferred to the differentiation condition = mentioned=20 above, and morphological changes of cells were observed under = phase-contrast=20 microscopy. On days 0, 3, 7, 10 and 21, cells were collected and checked = with=20 flow cytometry to determine the expressing rates of cell surface = markers,=20 including CD133, nestin, GFAP and -tubu= linIII=20 (BDbioscience Co.). Each test was repeated three times, and the results = were=20 processed with SSPS software.

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Results

Tumor spheres and subspheres were successfully cultured = and=20 confirmed to be exclusively generated by CD133⁺ glioma = cell

Within 24-48 h of culturing of single-cell = suspensions made=20 from both primary and recurrent tumor tissues, neurosphere-like = clusters,=20 usually termed tumor spheres, emerged and increased in diameter and = density=20 steadily (Figure=20 1A=961C). Neurospheres were also observed after the same duration, = but grew=20 slowly when compared with tumor spheres (data not shown). After tumor = spheres=20 were harvested and subjected to subsphere forming assay by limited = dilution,=20 subspheres appeared as expected. After being in differentiation = condition for 7=20 days, both tumor sphere and neurosphere cells were observed to migrate = from=20 spheres and become flat and adherent. However, when they were = re-switched to=20 defined stem cell medium, a difference occurred: monolayer adherent = cells from=20 tumor spheres gradually retracted their processes, floated up and formed = rolling=20 spheres again, whereas cells from neurospheres kept their morphology = unchanged.=20 This interesting phenomenon was also described by Yuan et al. = ¹³.=20 Since CD133 is regarded as the putative surface marker for BTSCs ^{4,=20
9,=20
13},=20 single CD133⁺ glioma cell should be able to form tumor = sphere. To=20 prove this, we plated CD133⁺ cells and CD133^- = glioma cells=20 into 96 wells containing defined stem cell medium. As a result,=20 CD133⁺ glioma cells proliferated in culture as floating = spheres,=20 whereas CD133^-glioma cells got attached to the culture = dishes, and=20 unlike CD133⁺ glioma cells, they did not form spheres (Figure=20 2).

Figure=20 1.

<= /A>

Both primary and recurrent gliomas from the same patient formed=20 neurosphere-like colonies, termed tumor spheres. (A) Small tumor = spheres=20 emerged within 48 h of culturing of single-cell suspension = (magnification 40).=20 (B and C) Tumor spheres are shown from primary and = recurrent=20 gliomas, respectively (magnification 10).=20 (D) Tumor spheres maintained for long term in vitro seem = to=20 morphologically vary from primary tumor spheres (magnification 10).

Full=20 figure and legend (29K)

Figure=20 2.

<= /A>

Comparison of clonogenicity between CD133⁺ and CD133- = glioma=20 cells. (A) Single CD133⁺ cells purified with magnetic = cell=20 sorting system produced small tumor spheres within 48 h of culturing in = defined=20 stem cell medium (magnification 10).=20 (B) One tumor sphere is shown formed by one CD133⁺ = glioma cell=20 (magnification 40).=20 (C and D) CD133^- glioma cell could not generate = tumor=20 sphere but grew flattened and attached in the same culture condition=20 (magnification 10).

Full=20 figure and legend (56K)

BTSCs were kept for long term

At the closure of this experiment, tumor spheres = from both=20 primary and recurrent tumor tissues have been kept for more than 18 = months and=20 expanded for more than 150 passages. Impressively, about 4 months after = the=20 primary sphere culture, not only did the morphology of the tumor spheres = alter=20 but also the proliferating rate of tumor sphere cells was obviously = increased,=20 with the doubling time shortened from 1 week to 3 or 4 days (Figure=20 1D and data not shown). Nevertheless, NSCs could only be maintained = for no=20 more than 3 months and no growth acceleration was observed.

BTSCs and NSCs were purified effectively with magnetic = bead cell=20 sorting system

CD133, a putative cell surface marker of human = NSCs, was=20 recently found to identify an exclusive subpopulation of glioma cells = that=20 possess stem-like activity ⁴.=20 We made an attempt to isolate BTSCs by magnetic cell sorting with = antibody=20 against CD133. As a result, CD133⁺ cells sorted from tumor = spheres=20 accounted for a minor fraction of tumor cell population, ranging from = 3.8% to=20 4.0%; however, when tumor spheres differentiated, the frequency of=20 CD133⁺ cells decreased sharply to 1.8=962.0%. The efficiency = of=20 magnetic cell sorting was evaluated by flow cytometry, showing that the = purity=20 exceeded 80% in CD133⁺ cell fraction and 95% in = CD133^-=20 cell fraction. Isolation of NSCs from neurospheres with magnetic cell = sorting=20 was also performed, with satisfactory outcomes (detailed in Figure=20 3).

Figure=20 3.

<= /A>

The efficiency of magnetic bead cell sorting was evaluated by flow = cytometry,=20 with the first peak (gate M1) representing cells negative for CD133-=20 phycoerythrin expression, and the second peak (gate M2) representing=20 CD133⁺ cells. (A and B) Purity was 83.02% in=20 CD133⁺glioma cell fraction and 97.7% in CD133^- = glioma cell=20 fraction. (C and D) Purity of sorted CD133⁺ and = CD133-=20 neural cells reached 85.06% and 96.68%, respectively.

Full=20 figure and legend (37K)

BTSCs demonstrated intrinsic potential to resist=20 differentiation

For the first week in differentiation condition, = both NSCs=20 and BTSCs gradually adhered to the bottom of flasks and morphologically = became=20 flat and fusiform. With maintenance of differentiation condition, NSCs = still=20 grew attached, but BTSCs gradually retracted their processes, floated up = and=20 reformed suspending spheres (Figure=20 4). At the beginning of differentiation, immunofluorescence detected = by=20 laser scanning confocal microscope demonstrated costaining of = undifferentiated=20 cell surface markers (CD133 and nestin), showing lack of differentiated = cell=20 markers (GFAP and MAP2) on BTSCs. However, on day 10 of differentiation, = BTSCs=20 still exhibited positive costaining of CD133 and nestin, although = apparent=20 staining of GFAP emerged (Figure=20 5). In NSCs, the staining of CD133 and nestin gradually faded with=20 differentiation, and almost disappeared by day 10. The differentiation = assay=20 with flow cytometry confirmed the immunofluorescent results. In the = first week=20 of differentiation, the expression rates (number of marker positive = cells/total=20 number of examined cells) of undifferentiated cell markers CD133 and = nestin in=20 BTSCs kept decreasing as expected, but increased from day 10 (Figure=20 6A). During the corresponding period, differentiated markers GFAP = and -tubu= lin=20 experienced an "up-down" tendency, which was inverse to that of = undifferentiated=20 markers (Figure=20 6A). The same experiment was also performed in NSCs; however, in = this case,=20 expression rates of CD133 and nestin kept decreasing, while those of = GFAP and=20 -tubu= lin kept=20 increasing, and no turning point emerged (Table=20 1).

Figure=20 4.

<= /A>

In differentiation condition, BTSCs and NSCs exhibited apparent = difference in=20 morphological changes (magnification 10).=20 (A=96F) Reforming of tumor spheres by BTSCs in = differentiation=20 condition: tumor spheres in defined stem cell medium (A); sorted = single=20 BTSC suspension was transferred to differentiation condition (B); = cells=20 became fusiform and adherent on day 4 of differentiation (C); = cells were=20 mostly fusiform on day 7 (D); cells began to retract processes = and float=20 up by day 10 (E); and tumor sphere was reformed 21 days later = (F).=20 (G=96I) Morphological changes of NSCs in the same = differentiation=20 condition: neurosphere in defined stem cell medium (G); on day 7 = of=20 differentiation, NSCs varied in morphology, but were predominantly = fusiform=20 (H); and neurosphere did not recur like tumor sphere by day 21=20 (I).

Full=20 figure and legend (74K)

Figure=20 5.

<= /A>

Immunofluorescence performed on BTSCs during differentiation was = checked with=20 laser scanning confocal microscope (magnification 800),=20 with nuclei of BTSCs stained by DAPI (in blue, shown in the C = picture of=20 all the three groups of photos". (1) At the beginning of = differentiation,=20 cells were stained for undifferentiated marker CD133 (in red, A) = and=20 nestin (in green, B). (D) costaining of CD133 and nestin = when=20 pictures were merged. (2) Nestin (in green, A) and CD133 = (in red,=20 B) staining was still positive after differentiation for 10 days. = (D) Costaining of nestin and CD133was showed, but nestin staining = was=20 predominant. (3) Ten days after differentiation, nestin staining = (in=20 green), GFAP staining (in red) and costaining of nestin and GFAP were=20 demonstrated in (A), (B) and (D) = respectively.

Full=20 figure and legend (56K)

Figure=20 6.

<= /A>

In differentiation condition, the expression rates of differentiated = cell=20 markers GFAP and -tubu= linIII in=20 BTSCs were found to keep rising for the first week, but declined = thereafter (in=20 the left). During the corresponding period, the expressing tendency of=20 undifferentiated cell markers CD133 and nestin was perfectly inverse to = that of=20 differentiated cell markers (in the right). The expression rate refers = to the=20 number of marker positive cells divided by total number of examined = cells.

Full=20 figure and legend (25K)

Table=20 1 - Expression rates of differentiated cell surface markers.

Full=20 table

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Discussion

It is traditionally believed that glioma may develop = from NSCs=20 that undergo abnormal differentiation or from differentiated cell types = that=20 acquire malignance by de-differentiating in response to oncogenic = mutation=20 ^{14,=20
15,=20
16,=20
17}.=20 Most recently, the study on tumorigenesis of glioma was further = complicated by=20 cell-cell fusion theory, in which mature cells could gain = de-differentiation=20 through fusion with stem cells ^{18,=20
19,=20
20}.=20 Although the target cell of transformation mutation is still under=20 investigation, there is considerable evidence that glioma is most likely = derived=20 from NSCs: (1) fewer mutations may be required to maintain self-renewal = than to=20 activate self-renewal ectopically, that is, NSCs is more sensitive to = oncogenic=20 stimuli; (2) NSCs often persist for relatively long term, which means = there is a=20 much greater opportunity for mutation to accumulate in NSCs ^{10,=20
11,=20
21,=20
22}.=20 The successful identification of BTSCs from solid glioma with the = technique used=20 to identify NSCs supports the linkage between glioma and NSCs, as both = of them=20 (1) express CD133; (2) form rolling spheres; (3) possess the capacity = for=20 self-renewal, proliferation and multi-differentiation; and (4) share = similar=20 signaling pathway in cell cycle progression ^{23,=20
24,=20
25,=20
26}.=20 The similarity shared by BTSCs and NSCs evidently suggests the = derivation or=20 origination of BTSCs from NSCs. It is thus particularly interesting to = study the=20 biological behaviors of BTSCs through comparison with NSCs.

Yuan et al. ¹³=20 reported that tumor sphere could re-form after induction of = differentiation if=20 re-subjected to defined stem medium. In this study, we found BTSCs could = re-form=20 tumor spheres even in the persistent differentiation condition. The = expression=20 of differentiated and undifferentiated markers seemed to coincide with = the=20 morphological changes of BTSCs in the differentiation assay. To sum up, = the=20 differentiation process of BTSCs seemed unstable (floating down and up). = Actually, both the morphological changes and the shifting of cell marker = expression denoted BTSCs' intrinsic capacity to maintain = undifferentiated state=20 or resist differentiation. Both positive and negative regulators, = including=20 internal and external signals, control the cell fate of stem cells. = Negative=20 regulators keep the stem cells undifferentiated, while positive = regulators=20 instruct the commitment and differentiation of stem cells ^27,=20
28.=20 In the same differentiation condition, NSCs differentiated to the mature = neural=20 cell types, while BTSCs rebounded to keep undifferentiated status. This=20 difference indicates internal signaling pathway regulating = differentiation has=20 been severely shifted by accumulated gene mutation(s) in BTSCs; in other = words,=20 genes giving rise to differentiation have been suppressed by genes = keeping stem=20 cell undifferentiated. To determine the genes responsible for the=20 dedifferentiation through arrayed comparative genome hybridization and = other=20 methods is of great significance in probing the molecular mechanism of = brain=20 tumor initiation. Abnormal differentiation is characteristic of and = responsible=20 for the malignance of glioma. The strategy aimed to induce glioma cells = into=20 terminal differentiation has been carried out in our center in the past = years;=20 however, only temporary and incomplete differentiation was achieved = ^{29,=20
30,=20
31}.=20 The differentiation characters that BTSCs display make it reasonable to=20 conjecture that BTSCs are responsible for the unsatisfactory outcome of=20 differentiation-inducing treatment. How to induce BTSCs to mature status = is=20 critical in this therapy modality.

The identification of BTSC is due to, at least in part, = the=20 forming of tumor sphere by BTSCs. However, BTSCs were also identified = from=20 adherent and monolayer tumor cell population ⁶.=20 Then, what makes the tumor sphere? In BTSCs' differentiation assay, = growth into=20 adherent status was accompanied by the decrease of CD133 and nestin, = while=20 reforming of tumor sphere concurred with the increase of CD133 and = nestin. We=20 also separated BTSCs from tumor spheres and monolayer tumor cell = population, and=20 found that the frequency of BTSCs in tumor sphere was almost twice as = much as=20 that in monolayer cell population (4% versus 2%). Taken together, the = proportion=20 of BTSCs in tumor cell population may account for the formation of tumor = sphere;=20 however, more studies are necessary to get a clear answer.

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Acknowledgements

The current research was supported by National Natural = Science=20 Foundation of China (No. 30371457 and 30400457). We would express our = thanks to=20 the staff of Nerve Regeneration Key Laboratory of Jiangsu Province, = Nantong=20 University, who kindly helped to perform the Immunofluorescent analysis = with=20 laser scanning confocal microscope.

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Original Article

Differentiation profile of brain tumor stem cells: a = comparative=20 study with neural stem cells

Abstract

Keywords:

Introduction

Material and Methods

Tumor samples and human fetal brain tissue

Tumor sphere and neurosphere culture

Purification of BTSCs and NSCs

Subsphere forming assay

Differentiation assay of BTSCs and NSCs

Results

Tumor spheres and subspheres were successfully cultured = and=20 confirmed to be exclusively generated by CD133+ glioma = cell

Figure=20 1.

Figure=20 2.

BTSCs were kept for long term

BTSCs and NSCs were purified effectively with magnetic = bead cell=20 sorting system

Figure=20 3.

BTSCs demonstrated intrinsic potential to resist=20 differentiation

Figure=20 4.

Figure=20 5.

Figure=20 6.

Table=20 1 - Expression rates of differentiated cell surface markers.

Discussion

References

Acknowledgements

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Tumor spheres and subspheres were successfully cultured = and=20 confirmed to be exclusively generated by CD133⁺ glioma = cell