Jurkat and UC-MSCs

7/18/2019 Jurkat and UC-MSCs

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J South Med Univ, 2014, 34(4): 441-447 doi 10.3969/j.issn.1673-4254.2014.04.01

INTRODUCTION

Mesenchymal stem cells (MSCs) are non-hematopoietic progenitor cells first isolated from bonemarrow over 40 years ago ［1］. These cells possess aprofound immunosuppressive activity by modulating thefunctions of immune cells through a variety of mechanisms ［2-4］. Such unique immunological propertiesof MSCs, combined with their hematopoiesis-supportivefunction, have caused great enthusiasm about their potential for treatment of hematological malignancy andother disorders［5, 6］. Preliminary studies suggested thatco-infusion of MSCs could reduce the incidence of

graft-versus-host disease as well as promoting engra-ftment in patients undergoing hematopoietic stem cell(HSC) transplantation for hematologic malignancy［7, 8］.

In recent years, ex vivo expanded MSCs from other tissues, such as umbilical cord, have been activelyinvestigated. Human umbilical cord-derived MSCs(hUC-MSCs) have several advantages over bonemarrow-derived MSCs (BM-MSCs), such as a lower riskof viral contamination, painless collection procedures,better expandability, and possible source for autologous

cell therapy ［9］. However, cell-based therapy oftenrequires large unphysiological numbers of MSCs for achieving a clinical efficacy, and there is evidence thatco-transplantation of BM-MSCs and HSCs increased therisk of hematological malignancy relapse ［10］. In thiscontext, the potential influences of hUC-MSCs onhematologic malignant cells have to be evaluated inpreclinical models before it can be used in clinical trials.

T-cell acute lymphoblastic leukemia (T-ALL) isone of the aggressive hematological malignancies,constituting a large proportion of acute lymphoblasticleukemia (ALL) both in children and in adults［11］. It is

reported that Notch signaling is implicated in thepathogenesis of T-ALL［12, 13］. T-ALL patients commonlyhave mutations in Notch1, one of the mammalian Notchsignaling receptors, to cause the overactivation of Notchsignaling in T-ALL cells ［14］. In this study, weinvestigated the influence of co-culture with hUC-MSCson the proliferation as well as survival of Jurkat T-ALLcell line in vitro, and explored the possible mechanisminvolving Notch signaling to provide further insight intothe biological role of hUC-MSCs for their potentialclinical applications.

MATERIALSANDMETHODS

Cell line

Human T-ALL cell line Jurkat (CD4 + , clone E6-1)

Human umbilical cord-derived mesenchymal stem cells inhibit

proliferation but maintain survival of Jurkat leukemia cells in

vitro by activating Notch signaling

YUAN Yin1, CHEN Danliang2, CHEN Xuan1, SHAO Hongwei1, HUANG Shulin1*

1School of Biosciences and Biopharmaceutics, Guangdong Province Key Laboratory for Biotechnology Drug Candidates, Guangdong

Pharmaceutical University, Guangzhou 510006, China; 2 Department of Gynecology and Obstetrics, First Affiliated Hospital of Jinan

University, Guangzhou 510630, China

Original Article

Abstract: Objective To investigate the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on the

proliferation and survival of Jurkat leukemia cells in vitro and explore the possible mechanism. Methods Jurkat leukemia cellswere co-cultured with hUC-MSCs isolated from human umbilical cord tissues by plastic adherence at a ratio of 10∶1. The

proliferation and survival of the co-cultured Jurkat cells, separated by immunomagnetic bead cell sorting on day 4, were

evaluated by flow cytometry. Western blotting was performed to evaluate the activation of Notch signaling in the co-cultured

Jurkat cells. Results Jurkat leukemia cells co-cultured with hUC-MSCs for 4 days showed a lowered proliferation rate and cell

cycle arrest at G0/G1 phase with a reduction in the cell apoptotic rate. Notch signaling pathway was activated in the co-cultured

Jurkat cells as evidenced by an increased cellular expression of HES-1. Conclusion Co-culture with hUC-MSCs can inhibit the

proliferation of Jurkat leukemia cells in vitro and protect the cells from apoptosis by activating Notch signaling, indicating a

potential shielding effect of MSCs on leukemia cells.

Key words: mesenchymal stem cells; umbilical cord; leukemia; proliferation; Notch signaling

Received: 2013-09-23 Accepted: 2013-10-23

Supported by National "Key New Drug Creation" Special and

Major Project (2009ZX09103-708), National Natural Science

Foundation of China (31100664, 31300737, 81303292) and Medical

Scientific Research Foundation of Guangdong Province (B2013197).

*Corresponding author: HUANG Shulin, Professor, E-mail:

[email protected]

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purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China) were grown in suspension inRPMI 1640 medium (Gibco BRL, Grand Island, NY,USA) supplemented with 10% fetal bovine serum (FBS;Sijiqing, Hangzhou, China) and 1% penicillin/streptomycin (Gibco BRL). The cells were incubated at

37 ℃ in a humidified atmosphere of 5% CO2 andpassaged every 2 days to maintain a cell density in theculture not exceeding 1×106 cells/ml.

Isolation and expansion of hUC- MSCs

Umbilical cord tissues from full-term healthyneonates were collected immediately upon delivery withinformed consent from the donor, and stored in PBSsupplemented with 1% penicillin/streptomycin beforeprocessing. The hUC-MSCs were isolated by directplastic adherence method without enzymatic digestions.Briefly, the umbilical cord sample was cut into segments

2-3 cm long and the residual cord blood and bloodvessels (two arteries and one vein) were removed. Thetissue blocks were then minced into 1-2 mm3 fragmentsand plated separately in 6-cm polystyrene tissue culturedishes pre-coated with FBS. After the dishes were keptinverted for 20 min, growth medium [DMEM/F12medium (Gibco BRL) supplemented with 10% FBS and1% penicillin/streptomycin] was added carefully. Thecultures were maintained at 37 ℃ in a humidifiedatmosphere containing 5% CO2. On day 7, the non-adherent tissue fragments were removed by changing themedium. The culture medium was changed every 3 to 4days thereafter. Approximately 3 weeks later, the well-

developed colonies of fibroblast-like cells (80%-90%confluent) were harvested with 0.25% trypsin (GibcoBRL). The harvested cells were passed through a100-μm sterile mesh and then seeded in larger flasks for further expansion. The cells at passage 4 to 8 thatdisplayed a homogeneous MSC phenotype were used for experiments.

Co-culture of hUC- MSCs and leukemia cells

The hUC-MSCs were plated in 6-well plate at 2×105 per well and allowed to adhere at 37 ℃ for 24 h.Jurkat leukemia cells were then inoculated in

suspension or on the hUC-MSCs monolayer at a ratio of 10∶1, and on day 4, the co-cultured Jurkat cells wereseparated from hUC-MSCs by careful pipetting withice-cold PBS as previously described［15］. To eliminatepossible contamination with hUC-MSCs fraction, thecollected Jurkat cells were further purified using CD4 +

magnetic micro beads according to manufacturer 'sinstructions (Miltenyi Biotec, Bergisch Gladbach,Germany).

Cell proliferation assays

The proliferation of Jurkat leukemia cells was

assessed by two different methods. For cell cycleanalysis, Jurkat cells fixed in 70% ethanol overnight at4 ℃ were treated with 10 μg/ml RNase A (Sigma, St.Louis, MO, USA) and then stained with 50 μg/ml

propidium iodide (PI; Sigma) at room temperature for 30min. Following staining, the cellular DNA content wasimmediately analyzed on a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Alternatively, thecell proliferation was detected by carboxy fluoresceindiacetate succinimidyl ester (CFSE; eBioscience, San

Diego, CA, USA) labeling following the manufacturer 'sinstructions. The cell cycle distribution and cellgenerations were determined using ModFit software(Verity Software House, Topsham, ME, USA).

Cell apoptosis analysis

The cell apoptosis was analyzed using theMEBCYTO apoptosis kit (MBL, Nagoya, Japan). Jurkatcells were washed once with PBS and resuspended in 85μl binding buffer, followed by incubation with 10 μlAnnexin V-FITC and 5 μl PI at room temperature for 15min in the dark. After incubation, 400 μl of binding

buffer was added. The cell samples were then measuredby flow cytometry.

Detection of Notch signaling molecules

The expressions of Notch signaling molecules onJurkat cells and hUC-MSCs, including the Notch1receptor and one of its ligand, Jagged1, were confirmedby flow cytometry. The monoclonal antibodies usedincluded phycoerythrin (PE)-conjugated anti-Notch1(R&D systems, Minneapolis, MN) and carbo-xyfluorescein (CFS)-conjugated anti-Jagged1 (R&Dsystems). The intracellular level of HES-1, a classic

transcriptional target of Notch signaling, was examinedby Western blotting following standard procedures. Inbrief, the cells (106) were lysed in RIPA lysis buffer according to the manufacturer ’s instructions (Beyotime,Haimen, China). Equal amounts of protein from eachculture were loaded on polyacrylamide gels followed bytransfer to polyvinylidene difluoride (PVDF) membranes(Bio-Rad, Hercules, CA). The membranes were treatedwith rabbit anti-human HES-1 at 1∶1000 (Epitomics,Burlingame, CA, USA) or with anti-GAPDH (GoodhereBiotechnology, Hangzhou, China) and then with anti-rabbit horseradish peroxidase (HRP)-conjugated seco-ndary antibody (1∶5000, ZSGB-Bio, Beijing, China).

The protein bands were developed by chemilumi-nescence.

Statistical analysis

Statistical comparisons were carried out using theGraphPad Prism software. The data were presented as

Mean±SD. When applicable, Student's t-test was used toexamine the differences between groups. Differenceswere considered statistically significant for a P valueless than 0.05.

RESULTS

Characterization of hUC- MSCs

MSCs were successfully isolated from human

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http://www.j-smu.com J South Med Univ, 2014, 34(4): 441-447

umbilical cord tissues by direct plastic adherentmethod. Fibroblast-like cells around the umbilical cordtissue fragments were observed as early as 2 weeks of culture (Fig.1A). These cells formed whirlpool-like

arrays when a confluent monolayer was developed(Fig.1B), and showed a good homogeneity. The cellsexpressed MSC markers CD73, CD90, and CD105, butwere negative for CD34 and CD45 (data not shown).

Fig.1 Morphological features of hUC-MSCs and Jurkat leukemia cells under inverted microscope (Original magnification: ×

100). A : hUC-MSCs in primary culture. The black arrow indicates part of the adherent umbilical cord tissue fragment; B:

The confluent state of hUC-MSCs in subcultivation; C: Jurkat cells grown on hUC-MSC monolayer in the co-culture system.

BA C

hUC- MSCs inhibited Jurkat cell proliferation

We studied the influence of hUC-MSCs on theproliferative activity of Jurkat leukemia cells inco-culture. The spatial relationship of Jurkat cells andhUC-MSCs in the co-culture system was shown inFig.1C. Cell cycle analysis revealed a marked arrest incell cycle progression, characterized by a decrease of proliferating cells (S and G2 /M) and an accumulation of cells in G0 /G1 in Jurkat cells grown on hUC-MSCs as

compared with Jurkat cells cultured alone (Fig.2). Theinhibitory effect of hUC-MSCs on Jurkat cells wasfurther confirmed by cell division analysis using CFSElabeling. As shown in Fig.3, 27.57% of Jurkat cellscultured in suspension were already in the 7thgeneration, while most of the hUC-MSC-supportedJurkat cells (54.16% ) remained in the 6th generationand there were scarcely cells of the 7th generation,indicating a decreased proliferation of Jurkat cells incontact with hUC-MSCs.

Fig.2 Influence of hUC-MSCs on cell cycle distribution of Jurkat leukemia cells. A : Jurkat cells cultured with or without hUC-MSCs

were analyzed for cell cycle progression. Results shown are representative histograms of 3 independent experiments. Percentages

of cells in each phase are shown in the upper corner of each cell-cycle graph. B: Cell cycle fractions of 3 independent

measurements. In suspension: Jurkat cells cultured alone; On MSCs: Jurkat cells cultured on hUC-MSCs.

N u m b e r

0

3 0 0

6 0 0

9 0 0

1 2 0 0

N u m b e r

0

3 0 0

6 0 0

9 0 0

2 0 0

G0/G1: 39.7±3.7

S: 43.7±3.0

G2/M: 16.6±4.2

G0/G1: 56.6±3.6

S: 35.0±4.9

G2/M: 8.5±3.1

In suspension On MSCs

100%

80%

60%

40%

20%

0%

G2/MSG0/G1

PI fluorescence intensity PI fluorescence intensity0 50 100 150 200 0 50 100 150 200

In suspension On MSCsA B

hUC- MSCs suppressed Jurkat cell apoptosis

To investigate whether hUC-MSC-mediatedproliferation inhibition was associated with theinduction of apoptosis, we examined the influence of hUC-MSCs on the survival of Jurkat cells by Annexin-Vand PI staining. When maintained for 4 consecutive

days without changing or supplementing growthmedium, Jurkat cells exhibited spontaneous apoptoticfeatures (Fig.4A), while those grown on hUC-MSCsshowed a reduction in cell apoptosis by approximately50% (Fig.4). These data suggest that hUC-MSCsmediated the growth arrest of Jurkat cells withoutinducing their apoptosis and maintained their viability.

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Notch signaling is activated in Jurkat cells co-cultured with hUC- MSCs

To further explore the molecular mechanism for the

above observation, we examined Notch signaling in thecells, given its roles in regulating cell proliferation andapoptosis. As Notch signaling pathway is mainlytriggered by Notch receptor/ligand interactions through

In suspension On MSCsParentGeneration 2Generation 3Generation 4Generation 5Generation 6Generation 7

Generation 8Generation 9Generation 10

0 50 100 150 200 0 50 100 150 200

Channel (CFSE) Channel (CFSE)

54.16%

N u m b e r

0

8 0

1 6 0

2 4

0

3 2 0

N u m b e r

0

8 0

1 6 0

2 4 0

3 2 0

27.57%

Fig.3 Generation assay of Jurkat leukemia cells labeled with CFSE. Histogram deconvolution based on CFSE

fluorescence data were performed by ModFit software.

Fig.4 Influence of hUC-MSCs on spontaneous apoptosis of Jurkat leukemia cells evaluated by

annexin V staining and propidium iodide (PI) incorporation. A: Flow cytometry profiles of one

representative experiment; B: Percentage of apoptotic cells of 3 separate experiments. The

presence of hUC-MSCs protected Jurkat cells from apoptosis (P<0.05).

100 101 102 103


P I

Annexin V Annexin V

P=0.3

M e a n a p

o p

t o s

i s ( %

)

30

20

10

0


B

A

103

102

101

100

P I

103

102

101

100

100 101 102 103

5.6

3.1

11.5

2.3

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direct cell-cell contact, we first evaluated the presenceof specific Notch family members on hUC-MSCs andJurkat cells. Flow cytometry detected Notch1 receptor on Jurkat cells, and the hUC-MSCs expressed amoderate level of Jagged1 (Fig.5A), indicating thecapacity of hUC-MSCs to trigger Notch signaling in

Jurkat cells. We also assayed the expression of HES-1, aclassic transcriptional target of Notch signaling. HES-1was constitutively expressed in Jurkat cells, and thedirect contact with hUC-MSCs resulted in an increasedexpression of HES-1 in Jurkat cells as expected (Fig.5Band 5C).

Fig.5 Involvement of Notch signaling in the interaction between Jurkat leukemia cells and hUC-MSCs. A: Notch1 and

its ligand Jagged1 were expressed on Jurkat cells and hUC-MSCs, respectively. Open histograms: Isotype control;

Filled histograms: Cells labeled with specific monoclonal antibodies. B: Western blotting showing up-regulation of

HES-1 protein in Jurkat leukemia cells following contact with hUC-MSCs. C: Relative expression of HES-1 protein in

Jurkat cells from different cultures determined by Western blotting (Mean±SD, n=3). HES-1 protein levels were

quantified and normalized for GAPDH expression. *P<0.05 vs Jurkat cells cultured alone in suspension.

A

102 103 104 105

100

80

60

40

20

0

100

80

60

40

20

0

E v e n t s

102 103 104 105

Jagged1 (hUC-MSCs) Notch1 (Jurkat)

B C


HES-1

GAPDH

1.0

0.8

0.6

0.4

0.2

0.0

R e l a t i v e e x p r e s s i o n

HES-1/GAPDH


*

DISCUSSION

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells that can be isolated from avariety of tissues, most commonly from the bone marrow(BM). However, aspiration of BM involves invasiveprocedures, and the yield of bone marrow-derived MSCsdecreases significantly with the donor 's age ［16］.Therefore, the search for alternative sources of MSCs is

of significant value. MSCs from umbilical cord have inrecent years become a new focus in stem cell researchdue to their attractive features.

Similar to BM-MSCs, hUC-MSCs also exhibit a low

immunogenicity［17, 18］, suggesting their potential applic-ability in allogeneic HSC transplantations. However,considering the emerging evidence that bonemarrow-derived MSCs may increase the risk of cancer relapse［10, 19］, it is still necessary to evaluate the efficacyand safety of hUC-MSCs before it could be applied inclinical trials.

In the present study, we prepared MSCs fromhuman umbilical cord tissues and utilized a co-culture

system to evaluate their impact on Jurkat T-ALL cellline. Our data showed that hUC-MSCs had a dualfunction in vitro: they inhibited the proliferation of Jurkat cells and also prevented their death. A possible

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relationship is indicated between hUC-MSC-inducedproliferation inhibition and the survival of Jurkat cells.It was inferred that proliferation inhibition could confer cancer cells a better survival because proliferating cellsare more vulnerable to apoptotic stimuli ［20］. Thismechanism can preserve the self -renewal ability of cancer cells and thus sustain the malignant process.Therefore, the clinical use of MSCs for malignantconditions should be handled with extreme caution［21］.

To further explore the underlying mechanism, weexamined Notch signaling in co-cultured Jurkat cellsdue to its involvement in the pathogenesis of T-ALL［12, 13］

and its critical role in rescuing cells from apoptosis ［22, 23］.Notch pathway is evolutionarily conserved and plays akey role in cell fate determination in many tissues［13, 24］.The activation of Notch signaling depends on direct cell-cell contact. The binding of Notch receptor with itsligand on neighboring cells triggers proteolytic cleavage

and release of the intracellular domain of Notchreceptor, which enters the cell nucleus and regulates thedownstream target genes, such as Hes-1 and Deltex-1［13,

25］. Our results showed that Jurkat T-ALL cell lineconstitutively expressed Notch1 receptor as well as itstarget HES-1, and the level of Notch activation in Jurkatcells was further elevated following contact withhUC-MSCs, which may account for the anti-apoptoticeffect of hUC-MSCs in Jurkat leukemia cells. On theother hand, Jagged1, one of the Notch ligands, wasfound to be expressed by hUC-MSCs. Jagged1 is amembrane-spanning protein with a large extracellular domain for Notch receptor binding ［26］. Therefore, the

hUC-MSCs were able to initiate the stimulation of Notchsignaling in Jurkat leukemia cells through theinteraction between their Jagged1 ligand and the Notch1receptor on Jurkat cells. It has been reported thatJagged1-mediated Notch signaling is involved in thesuppressive effect of BM-MSCs on immune cells［27］, andMSC-derived osteoblasts regulate the HSC niche byusing the same Jagged1/Notch system ［28］. Besides,Jagged1 was also found necessary for the expression of various smooth muscle markers during differentiation of MSCs into smooth muscle cells［29］.

Overall, we observed anti-proliferation as well asanti-apoptotic effects of hUC-MSCs on Jurkat leukemiacells at the same time. Although the anti-proliferationeffect might have therapeutic implications, theanti-apoptotic effect constitutes a potential side effect of ex vivo expanded hUC-MSCs, which might maintainresidual leukemia cells and lead to disease recurrence.Therefore, the exploitation of MSCs in new therapeuticstrategies should be cautious for malignant conditions.Our results also point to the crucial role of Notchsignaling in the hUC-MSC-induced influence on Jurkatleukemia cells, indicating its role as a potential targetfor the reversion of MSC-mediated protection on T-ALLleukemia cells.

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脐带人脐带间充质干细胞通过激活间充质干细胞通过激活 otch

Notch信号通路促进白血病细胞株信号通路促进白血病细胞株

urkJurkatat的外存活的体外存活

袁茵 1，陈丹亮 2，陈璇 1，邵红伟 1，黄树林 1

1 广东药学院生命科学与生物制药学院//广东省生物技术候选药物研究重点实验室，广东广州 510006；2 暨南

大学附属第一医院妇产科，广东广州 510630

摘要：目的研究人脐带间充质干细胞对白血病细胞株Jurkat 体外增殖和存活能力的影响并探讨相关机制。方法组织块贴壁

法分离人脐带间充质干细胞，将其作为基质细胞与 Jurkat 细胞进行直接接触共培养。通过免疫磁珠法分离共培养体系中的

Jurkat细胞，流式细胞术检测Jurkat 细胞增殖能力、细胞周期和自发凋亡率的变化；Western blotting法检测共培养后 Jurkat 细胞

内 Notch信号通路的活化状况。结果共培养4 d 后，Jurkat 白血病细胞的增殖减缓、细胞周期被阻滞于 G0/G1期，而其自发凋亡

率则出现显著下降，并伴有 Notch 信号通路靶分子 HES-1 表达量的增加。结论人脐带间充质干细胞构成的微环境对 Jurkat白

血病细胞有保护作用， Notch信号通路可能参与了这一过程。

关键词：间充质干细胞；脐带；白血病；增殖； Notch信号通路

收稿日期：2013-09-23

基金项目：国家“重大新药创制”科技重大专项（2009ZX09103-708）；国家自然科学基金（31100664，31300737，81303292）；广东省医学科

研基金（B2013197）

作者简介：袁茵，博士研究生，讲师，E-mail: [email protected]

通信作者：黄树林，教授，博士生导师，E-mail: [email protected]

of JAGGED1 is essential for cellular transformation［J］. J Biol Chem,2003, 278(10): 8771-9.

［27］Liotta F, Angeli R, Cosmi L, et al. Toll-Like receptors 3 and 4 areexpressed by human bone marrow-derived mesenchymal stem cellsand can inhibit their T-Cell modulatory activity by impairing Notchsignaling［J］. Stem Cells, 2007, 26(1): 279-89.

［28］Calvi L, Adams G, Weibrecht K, et al. Osteoblastic cells regulate thehaematopoietic stem cell niche［J］. Nature, 2003, 425(6960): 841-6.

［29］Kurpinski K, Lam H, Chu J, et al. Transforming growth factor -betaand notch signaling mediate stem cell differentiation into smoothmuscle cells［J］. Stem Cells, 2010, 28(4): 734-42.

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Jurkat and UC-MSCs

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