Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells

Novi Silvia Hardiany; Purnamawati Huang; Syarifah Dewi; Reni Paramita; Septelia Inawati Wanandi

doi:10.12688/f1000research.13154.1

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Research Article

Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells

[version 1; peer review: 2 not approved]

Novi Silvia Hardiany¹, Purnamawati Huang², Syarifah Dewi¹, Reni Paramita¹, Septelia Inawati Wanandi ¹

Novi Silvia Hardiany¹, Purnamawati Huang², [...] Syarifah Dewi¹, Reni Paramita¹, Septelia Inawati Wanandi ¹

PUBLISHED 25 Jan 2018

Author details Author details

¹ Department of Biochemistry & Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
² Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Novi Silvia Hardiany
Roles: Formal Analysis, Funding Acquisition, Investigation, Methodology, Writing – Original Draft Preparation

Purnamawati Huang
Roles: Investigation, Methodology

Syarifah Dewi
Roles: Formal Analysis, Resources

Reni Paramita
Roles: Methodology, Validation

Septelia Inawati Wanandi
Roles: Conceptualization, Data Curation, Supervision, Writing – Original Draft Preparation

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: Glioblastoma multiforme (GBM) is the most aggressive form of malignant glioma and is also known as grade IV astrocytoma. This might be due to the presence of cancer stem cells with high pluripotency and ability of self-renewal. Recently, it has been reported that tumor stroma cells, including mesencyhmal stem cells (MSCs), secrete factors that affect cancer cell growth. Until now, the role of MSC secretomes in cancer stem cell pluripotency remains unclear. The aim of this study was to analyze the effect of MSC secretomes in conditioned medium (CM) on the expression of pluripotency markers of GBM cells.
Methods: Umbilical cord-derived MSCs (UCSCs) were grown on serum-free alphaMEM for 24 hours to prepare the UCSC-CM. Human GBM T98G cells were treated with UCSC-CM for 24 hours. Following this treatment, expression of pluripotency markers SOX2, OCT4 and NANOG genes was analyzed using quantitative RT-PCR.
Results: SOX2 and OCT mRNA expression was 4.7-fold (p=0.02) and 1.3-fold (p=0.03) higher in CM-treated cells compared to the control. However, there was no change in NANOG mRNA expression. This might be due to there being others factors regulating NANOG mRNA expression.
Conclusions: UCSC-CM could affect the expression of SOX2 and OCT4 in human glioblastoma multiforme T98G cells. Further research is needed to elucidate the mechanism by which pluripotency markers are expressed when induced by the UCSC secretome.

Keywords

conditioned medium, mesenchymal stem cells, glioblastoma multiforme, pluripotency expression

Corresponding author: Septelia Inawati Wanandi

Competing interests: No competing interests were disclosed.

Grant information: This study was funded by the Penelitian Unggulan Perguruan Tinggi (PUPT 2017) grant provided by the Direktorat Riset dan Pengabdian Masyarakat Universitas Indonesia (DRPM-UI).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2018 Hardiany NS et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

How to cite: Hardiany NS, Huang P, Dewi S et al. Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells [version 1; peer review: 2 not approved]. F1000Research 2018, 7:106 (https://doi.org/10.12688/f1000research.13154.1) First published: 25 Jan 2018, 7:106 (https://doi.org/10.12688/f1000research.13154.1) Latest published: 25 Jan 2018, 7:106 (https://doi.org/10.12688/f1000research.13154.1)

Introduction

Glioblastoma multiforme (GBM) is a primary brain tumor that arises from glial cells (glioma), and is the most aggressive form of malignant glioma. According to the WHO, GBM is also known as grade IV astrocytoma¹. The life expectancy of patients with GBM is very low, usually less than 1 year despite available options such as surgery and chemoradiation. Failure of therapy might be due to the presence of cancer stem cells with high pluripotency and ability of self-renewal². Cancer stem cells have an improved the ability to repair their own DNA³, and can be identified by the analysis of transcription factors found in embryonic stem cells such as OCT-4 (octamer-binding transcription factor 4), SOX2 [SRY (sex determining region Y)-box 2] and NANOG. Those transcription factors play an essential role in sustaining the pluripotency and self-renewal ability of embryonic stem cells^4,5.

Recently, it has been reported that tumor stroma cells, including mesencyhmal stem cells (MSCs), secrete factors that affect cancer cell growth. Previous studies have demonstrated that MSCs were recruited from bone marrow and that they home around the cancer cells to support tumor growth and metastasis⁶. Other studies have shown that MSCs can trigger cancer growth by inducing angiogenesis, suppressing the immune system, forming cancer-associated fibroblasts (CAF) that contributing to the tumor growth, epithelial mesenchymal transition (EMT) and metastasis⁷. The role of MSCs in cancer growth has been widely investigated. Nevertheless, the role of MSC secretomes in cancer stem cell pluripotency remains unclear. The aim of this study was to analyze the effect of MSC secretomes in conditioned medium (CM) on the expression of pluripotency markers of GBM cells.

Methods

Cell culture

The Human Glioblastoma Multiforme T98G cell line was grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) on T-25 cm² culture flasks (Corning). Medium was added with sodium bicarbonate, 10% fetal bovine serum (FBS, Biowest), 1% streptomycin - penicillin and 1% amphotericine at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The medium was changed 2 times in a week. T98G cells were sub-cultured after being 70–80% confluent⁸.

Preparation of conditioned medium of umbilical cord-derived MSCs (UCSC-CM)

Umbilical cord-derived MSCs (UCSCs) were kindly provided by Prof. Jeanne Adiwinata Pawitan (Cell Medical Technology Integrated Service Unit, Cipto Mangunkusumo Central Hospital, Jakarta)⁹. 125,000 UCSCs were cultured in Minimum Essential Medium alpha (αMEM, Gibco) supplemented with 10% FBS (Biowest)/Glutamax (Gibco), 1% streptomycin-penicillin and 1% amphotericin on T-25 cm² culture flasks (Corning) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. After the cells were 70–80% confluent, medium was removed and cells were washed 3 times using 1x Phosphate Buffer Saline (PBS). Then, cells were grown on serum free- αMEM for 24 hours to prepare the UCSC-CM. After 24 hours, UCSC-CM were collected, centrifuged to remove cell debris and passed through 0.22 µm filter. Concentration of UCSC-CM was 50%, diluting UCSC-CM in freshly high glucose DMEM.

UCSC-CM treatment of T98G cells

400,000 T98G cells were seeded in triplicate on a 12-well-plate in high glucose DMEM/10% FBS/1% streptomycin-penicillin/1% amphotericin and allowed to adhere overnight. The following day, medium of T98G cells was replaced by 50% (v/v) UCSC-CM and incubated for 24 hours.

Analysis of pluripotency marker expression

SOX2, OCT4 and NANOG genes expression was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). First, total RNA was extracted from CM-treated T98G cells using Tripure RNA Isolation kit (Roche). Total RNA was amplified using qPCR (PCRmax Eco48, United Kingdom) with KAPA SYBR FAST One step qRT-PCR (KAPA Biosystem) and primers for SOX2, OCT4, and NANOG (Table 1). Reaction protocol was as follows: cDNA synthesis for 10 minutes at 42°C, inactivation with iScript Reverse transcriptase for 5 minutes at 95°C, then 40 PCR cycles for 10 seconds at 95°C, followed by 30 seconds at 57°C for OCT4, 59°C for SOX2, 60°C for NANOG; then 30 seconds at 72°C. Following the PCR cycles, the protocol was subsequently continued with the melting curve analysis, i.e. 1 minute on 95°C; 1 minute on 55°C; 10 seconds on 55°C (80 cycles, increase 0.5°C every cycles).

Table 1. Primer sequences.

Human	Sense:	5’-GAGGAGTCCCAGGACATCAAA-3’
OCT4	Antisense:	5’-AGCTTCCTCCACCCACTTCT-3’
Human	Sense:	5’-GGAGAGTAAGAAACAGCATGGA-3’
SOX2	Antisense:	5’-GTGGATGGGATTGGTGTTCT-3’
Human	Sense:	5’-ACAGAAATACCTCAGCCTCCAGCA-3’
NANOG¹⁰	Antisense:	5’-CTCCAGGTTGAATTGTTCCAGGTC-3’

Statistical analysis

All data were presented as means ± SD from triplicate experiments. Statistical analysis was performed using Student’s t-test using PASW 18 software, with p < 0.05 as a cut-off for determining a significant difference.

Results

Cell morphology

Cell morphology was observed under an inverted microscope (100X magnification) after 24 hours of the CM treatment. There was no difference in morphology between control and CM-treated T98G cells, as shown in Figure 1. Both control T98G cells and CM-treated T98G cells appear with fibroblast morphology, adhering to the plate. There was no change in cell shape and the cell membrane was still intact.

Figure 1. T98G cell morphology.

About 4 × 10⁵cells were plated triplicate in a 12-well plate and grown in high glucose Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS/1% penicillin-streptomycin/1% amphotericin at 37°C, 5% CO₂ for 1 day. Afterwards, the medium of treated cells was replaced with 50% (v/v) conditioned medium of umbilical cord stem cells, while medium of control cells was replaced with 50% (v/v) Minimum Essential Medium alpha. After 24-hour incubation, cell morphology was observed under inverted microscope (100X magnification). (A). Control cells; (B). Conditioned Medium-treated cells.

Pluripotency marker expression

Pluripotency marker expression was analyzed by measuring SOX2, OCT4 and NANOG mRNA expression. Relative mRNA expression was calculated using the Livak method (2^-ΔΔCT) with 18S rRNA as the reference gene. SOX2 mRNA expression was significantly higher in CM-treated T98G cells compared to control (Figure 2). SOX2 mRNA expression was up-regulated 4.7-fold in CM-treated T98G cells. Moreover, OCT4 mRNA expression was also significantly increased in CM-treated T98G cells compared to the control (Figure 3). OCT4 mRNA expression increased 1.2 fold in CM-treated T98G cells. Nevertheless, there was no significant change in NANOG mRNA expression (Figure 4).

Figure 2. SOX2 mRNA expression.

100 ng of total RNA was amplified using quantitative reverse transcriptase polymerase chain reaction to detect SOX2 mRNA expression. The expression was relatively calculated using Livak formula with 18S rRNA gene as a reference gene. All values are means ± SE, n = 9. Significant differences at *(p<0.05). SOX2 expression was 4.7-fold (p=0.02) higher in the conditioned medium-treated T98G cells compared to the control.

Figure 3. OCT4 mRNA expression.

100 ng of total RNA was amplified using quantitative reverse transcriptase polymerase chain reaction to detect OCT4 mRNA expression. The expression was relatively calculated using Livak formula with 18S rRNA gene as a reference gene. All values are means ± SE, n = 9. Significant differences at *(p<0.05). OCT4 expression was 1.3-fold (p=0.03) higher in the conditioned medium-treated cells compared to the control.

Figure 4. NANOG mRNA expression.

100 ng of total RNA was amplified using quantitative reverse transcriptase polymerase chain reaction to detect NANOG mRNA expression. The expression was relatively calculated using Livak formula with 18S rRNA gene as a reference gene.All values are means ± SE, n = 9. Significant differences at *(p<0.05).There was no significant difference in NANOG expression between control and conditioned medium-treated T98G cells.

Dataset1. Cq value of 18S rRNA gene. 18S rRNA Cq was used to calculate SOX2,OCT4 and NANOG mRNA expression using Livak formula (K: control cells; CM: condition medium treated cells)

Well	Sample Name	Assay Name	Cq	Cq Mean	Cq Std. Dev.
A1	K1	18sRNA	19.054	19.123	0.080
A2	K1	18sRNA	19.210	19.123	0.080
A3	K1	18sRNA	19.104	19.123	0.080
A4	K2	18sRNA	17.071	17.104	0.038
A5	K2	18sRNA	17.146	17.104	0.038
A6	K2	18sRNA	17.096	17.104	0.038
A7	K3	18sRNA	17.202	17.175	0.247
A8	K3	18sRNA	16.915	17.175	0.247
B1	K3	18sRNA	17.406	17.175	0.247
B2	CM1	18sRNA	15.930	15.931	0.207
B3	CM1	18sRNA	15.724	15.931	0.207
B4	CM1	18sRNA	16.138	15.931	0.207
B5	CM2	18sRNA	19.439	19.406	0.260
B6	CM2	18sRNA	19.131	19.406	0.260
B7	CM2	18sRNA	19.648	19.406	0.260
B8	CM3	18sRNA	18.016	18.010	0.193
C1	CM3	18sRNA	18.201	18.010	0.193
C2	CM3	18sRNA	17.815	18.010	0.193
C3	NTC	18sRNA	30.218	29.966	0.357

Dataset 1.18S rRNA Cq values.

18S rRNA Cq was used to calculate SOX2, OCT4 and NANOG mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells).

Dataset2. Cq value of SOX2 gene. SOX2 Cq was used to calculate SOX2 mRNA expression using Livak formula as shown in figure 2 (K: control cells; CM: condition medium treated cells).

Well	Sample Name	Assay Name	Cq	Cq Mean	Cq Std. Dev.
A1	K1	SOX2	24.218	24.213	0.304
A2	K1	SOX2	24.514	24.213	0.304
A3	K1	SOX2	23.906	24.213	0.304
A4	K2	SOX2	25.765	25.767	0.673
A5	K2	SOX2	25.096	25.767	0.673
A6	K2	SOX2	26.441	25.767	0.673
A7	K3	SOX2	26.032	26.026	0.208
A8	K3	SOX2	26.231	26.026	0.208
B1	K3	SOX2	25.815	26.026	0.208
B2	CM1	SOX2	22.087	22.108	0.413
B3	CM1	SOX2	22.532	22.108	0.413
B4	CM1	SOX2	21.706	22.108	0.413
B5	CM2	SOX2	25.865	25.908	0.311
B6	CM2	SOX2	25.620	25.908	0.311
B7	CM2	SOX2	26.238	25.908	0.311
B8	CM3	SOX2	25.334	25.316	0.403
C1	CM3	SOX2	25.710	25.316	0.403
C2	CM3	SOX2	24.905	25.316	0.403
C3	NTC	SOX2	27.151	26.621	0.749

Dataset 2.SOX2 Cq values.

SOX2 Cq was used to calculate SOX2 mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells).

Dataset3. Cq value of OCT4 gene. OCT4 Cq was used to calculate OCT4 mRNA expression using Livak formula as shown in figure 3 (K: control cells; CM: condition medium treated cells).

Well	Sample Name	Assay Name	Cq	Cq Mean	Cq Std. Dev.
A1	K1	OCT4	26.886	27.216	0.287
A2	K1	OCT4	27.380	27.216	0.287
A3	K1	OCT4	27.384	27.216	0.287
A4	K2	OCT4	24.684	24.938	0.302
A5	K2	OCT4	25.848	24.938	0.302
A6	K2	OCT4	25.272	24.938	0.302
A7	K3	OCT4	25.745	25.602	0.140
A8	K3	OCT4	25.596	25.602	0.140
B1	K3	OCT4	25.466	25.602	0.140
B2	CM1	OCT4	24.331	24.428	0.111
B3	CM1	OCT4	24.549	24.428	0.111
B4	CM1	OCT4	24.403	24.428	0.111
B5	CM2	OCT4	27.510	27.216	0.582
B6	CM2	OCT4	26.546	27.216	0.582
B7	CM2	OCT4	27.593	27.216	0.582
B8	CM3	OCT4	26.171	25.895	0.287
C1	CM3	OCT4	25.914	25.895	0.287
C2	CM3	OCT4	25.599	25.895	0.287

Dataset 3.OCT4 Cq values.

OCT4 Cq was used to calculate OCT4 mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells).

Dataset4. Cq value of NANOG gene. NANOG Cq was used to calculate NANOG mRNA expression using Livak formula as shown in figure 4 (K: control cells; CM: condition medium treated cells).

Well	Sample Name	Assay Name	Cq	Cq Mean	Cq Std. Dev.
D4	K1	NANOG	32.057	32.490	1.056
D5	K1	NANOG	33.694	32.490	1.056
D6	K1	NANOG	31.720	32.490	1.056
D7	K2	NANOG	30.950	30.393	0.871
D8	K2	NANOG	29.389	30.393	0.871
E1	K2	NANOG	30.840	30.393	0.871
E2	K3	NANOG	31.850	30.982	1.043
E3	K3	NANOG	29.825	30.982	1.043
E4	K3	NANOG	31.272	30.982	1.043
E5	CM1	NANOG	29.211	29.724	0.452
E6	CM1	NANOG	30.064	29.724	0.452
E7	CM1	NANOG	29.896	29.724	0.452
E8	CM2	NANOG	33.018	32.678	0.373
F1	CM2	NANOG	32.279	32.678	0.373
F2	CM2	NANOG	32.738	32.678	0.373
F3	CM3	NANOG	34.004	31.234	2.645
F4	CM3	NANOG	28.736	31.234	2.645
F5	CM3	NANOG	30.962	31.234	2.645
F6		Blank		36.637	N/A
F7		Blank	36.637	36.637	N/A

Dataset 4.NANOG Cq values.

NANOG Cq was used to calculate NANOG mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells).

Dataset 5.Raw unedited images for Figures 1A and 1B.

Discussion

Similar to normal stem cells, stem cell-like properties such as pluripotency in glioma CSCs are maintained by a core set of transcription factors, including SOX2, OCT4, and NANOG. Up-regulation of this set of genes is associated with poor outcome in terms of tumor malignancy, recurrence and metastasis¹¹. Here, we demonstrated that the pluripotency markers SOX2 and OCT4 were up-regulated in UCSC-CM-treated cells. This indicates that UCSCs secrete certain factors that support the self-renewal capacity of GBM cells. Liu et al showed that bone marrow-derived MSCs produce a cytokine meshwork that stimulates CSCs¹².

There is accumulating evidence suggesting that the regulation of stem cell-like properties requires a two-way interaction between CSCs and their microenvironment, particularly the MSCs. For instance, secretion of interleukin-1 (IL-1) in colon cancer cells could stimulate MSCs to produce prostaglandin E2 (PGE2). Then, PGE2 collaborated with IL-1 to produce other cytokines and chemokines such as IL-6, CXCL1 & CXCL8 by MSCs, leading to enhancement of the cancer stem cell population¹³. Wu et al proved that cytokines (IL-6, CXCL-8) were detected in conditioned media from MSCs. Those cytokines stimulated the expression of pluripotency markers (SOX2, OCT4, cMyc), as well as NF-κB &AMPK/mTOR signaling pathways in colon cancer cells¹⁴. In another study by Luo et al., CCL5 secreted by recruited BM-MSCs were found to induce prostate CSCs via androgen receptor signaling¹⁵. Investigating the secreted components of our UCSC-CM could allow us to determine more targeted CSC therapy in GBM.

Unlike SOX2 and OCT4 expressions, NANOG mRNA expression in T98G cells was not affected by UCSC-CM in our study. This is might be due to the abundance of NANOG pseudogenes^16,17 and also due to the way NANOG mRNA undergoes unique N⁶-methyladenosine (m⁶A) posttranscriptional modification as part of its regulation¹⁸. Furthermore, microRNA-134 has been reported to suppress proliferation and invasion of T98G cells by reducing NANOG expression¹⁹. Further studies are required to elucidate the involvement of UCSC secretomes in inducing the differential expression of pluripotency markers.

Conclusions

The conditioned medium of umbilical cord-derived mesenchymal stem cells could affect the expression of SOX2 and OCT4 as pluripotency markers in human glioblastoma multiforme T98G cells.

Data availability

Dataset 1: 18S rRNA Cq values. 18S rRNA Cq was used to calculate SOX2, OCT4 and NANOG mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells). DOI, 10.5256/f1000research.13154.d191296²⁰

Dataset 2: SOX2 Cq values. SOX2 Cq was used to calculate SOX2 mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells). DOI, 10.5256/f1000research.13154.d191297²¹

Dataset 3: OCT4 Cq values. OCT4 Cq was used to calculate OCT4 mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells). DOI, 10.5256/f1000research.13154.d191298²²

Dataset 4: NANOG Cq values. NANOG Cq was used to calculate NANOG mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells). DOI, 10.5256/f1000research.13154.d191299²³

Dataset 5: Raw unedited images for Figures 1A and 1B. DOI, 10.5256/f1000research.13154.d191300²⁴

Competing interests

No competing interests were disclosed.

Grant information

This study was funded by the Penelitian Unggulan Perguruan Tinggi (PUPT 2017) grant provided by the Direktorat Riset dan Pengabdian Masyarakat Universitas Indonesia (DRPM-UI).

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgments

The authors would like to express our gratitude to Direktorat Riset dan Pengabdian Masyarakat Universitas Indonesia (DRPM-UI) for the Penelitian Unggulan Perguruan Tinggi (PUPT 2017) grant. Moreover, thank you to PT. Ecosains Hayati, Indonesia for the assistance in providing real time PCR equipment.

Faculty Opinions recommended

References

1. Louis DN, Ohgaki H, Wiestler OD, et al.: The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 2007; 114(2): 97–109. PubMed Abstract | Publisher Full Text | Free Full Text
2. Holmberg J, He X, Peredino I, et al.: Activation of neural and pluripotent stem cell signatures correlates with increased malignancy in human glioma. Plos One. 2011; 6(3): e18454. PubMed Abstract | Publisher Full Text | Free Full Text
3. Bao S, Wu Q, McLendon RE, et al.: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature Lett. 2006; 444(7120): 756–760. PubMed Abstract | Publisher Full Text
4. Fong H, Hohenstein KA, Donovan PJ: Regulation of self-renewal and pluripotency by Sox2 in human embryonic stem cells. Stem Cells. 2008; 26(8): 1931–1938. PubMed Abstract | Publisher Full Text
5. Loh YH, Wu Q, Chew JL, et al.: The Oct4 and nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nature Gen. 2006; 38(4): 431–440. PubMed Abstract | Publisher Full Text
6. Chaturvedi P, Gilkes DM, Wong CC, et al.: Hypoxia-inducible factor-dependent breast cancer-mesenchymal stem cell bidirectional signaling promotes metastasis. J Clin Invest. 2013; 123(1): 189–205. PubMed Abstract | Publisher Full Text | Free Full Text
7. Lee JK: Mesenchymal stem cell: a hard nut to crack in cancer development. Enliven Archive. 2014; 1. Reference Source
8. Hardiany NS, Sadikin M, Siregar NC, et al.: The suppression of manganese superoxide dismutase decreased the survival of human glioblastoma multiforme T98G cells. Med J Indones. 2017; 26(1): 19–25. Publisher Full Text
9. Pawitan JA, Kispa T, Mediana D, et al.: Simple production method of umbilical cord derived mesenchymal stem cell using xeno-free materials for translational research. J Chem Pharm Res. 2015; 7(8): 652–656. Reference Source
10. Tachi K, Shiraishi A, Bando H, et al.: FOXA1 expression affects the proliferation activity of luminal breast cancer stem cell populations. Cancer Sci. 2016; 107(3): 281–9. PubMed Abstract | Publisher Full Text | Free Full Text
11. Liu A, Yu X, Liu S: Pluripotency transcription factors and cancer stem cells: small genes make a big difference. Chin J Cancer. 2013; 32(9): 483–487. PubMed Abstract | Free Full Text
12. Liu S, Ginestier C, Ou SJ, et al.: Breast cancer stem cells are regulated by mesenchymal stem cells through cytokine networks. Cancer Res. 2011; 71(2): 614–624. PubMed Abstract | Publisher Full Text | Free Full Text
13. Li HJ, Reinhardt F, Herschman HR, et al.: Cancer-stimulated mesenchymal stem cells create a carcinoma stem cell niche via prostaglandin E₂ signaling. Cancer Discov. 2012; 2(9): 840–855. PubMed Abstract | Publisher Full Text | Free Full Text
14. Wu XB, Liu Y, Wang GH, et al.: Mesenchymal stem cells promote colorectal cancer progression through AMPK/mTOR-mediated NF-κB activation. Sci Rep. 2016; 6: 21420. PubMed Abstract | Publisher Full Text | Free Full Text
15. Luo J, Lee SO, Cui Y, et al.: Infiltrating bone marrow mesenchymal stem cells (BM-MSCs) increase prostate cancer cell invasion via altering the CCL5/HIF2α/androgen receptor signals. Oncotarget. 2015; 6(29): 27555–27565. PubMed Abstract | Publisher Full Text | Free Full Text
16. Jeter CR, Liu B, Liu X, et al.: NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation. Oncogene. 2011; 30(36): 3833–3845. PubMed Abstract | Publisher Full Text | Free Full Text
17. Kawamura N, Nimura K, Nagano H, et al.: CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells. Oncotarget. 2015; 6(26): 22361–22374. PubMed Abstract | Publisher Full Text | Free Full Text
18. Zhang C, Samanta D, Lu H, et al.: Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m⁶A-demethylation of NANOG mRNA. Proc Natl Acad Sci U S A. 2016; 113(14): E2047–E2056. PubMed Abstract | Publisher Full Text | Free Full Text
19. Niu CS, Yang Y, Cheng CD: MiR-134 regulates the proliferation and invasion of glioblastoma cells by reducing Nanog expression. Int J Oncol. 2013; 42(5): 1533–1540. PubMed Abstract | Publisher Full Text | Free Full Text
20. Hardiany NS, Huang P, Dewi S, et al.: Dataset 1 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source
21. Hardiany NS, Huang P, Dewi S, et al.: Dataset 2 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source
22. Hardiany NS, Huang P, Dewi S, et al.: Dataset 3 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source
23. Hardiany NS, Huang P, Dewi S, et al.: Dataset 4 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source
24. Hardiany NS, Huang P, Dewi S, et al.: Dataset 5 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source

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Author details Author details

¹ Department of Biochemistry & Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
² Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Novi Silvia Hardiany
Roles: Formal Analysis, Funding Acquisition, Investigation, Methodology, Writing – Original Draft Preparation

Purnamawati Huang
Roles: Investigation, Methodology

Syarifah Dewi
Roles: Formal Analysis, Resources

Reni Paramita
Roles: Methodology, Validation

Septelia Inawati Wanandi
Roles: Conceptualization, Data Curation, Supervision, Writing – Original Draft Preparation

Competing interests

No competing interests were disclosed.

Grant information

This study was funded by the Penelitian Unggulan Perguruan Tinggi (PUPT 2017) grant provided by the Direktorat Riset dan Pengabdian Masyarakat Universitas Indonesia (DRPM-UI).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (1)

version 1

Published: 25 Jan 2018, 7:106

https://doi.org/10.12688/f1000research.13154.1

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© 2018 Hardiany NS et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

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Hardiany NS, Huang P, Dewi S et al. Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells [version 1; peer review: 2 not approved]. F1000Research 2018, 7:106 (https://doi.org/10.12688/f1000research.13154.1)

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Version 1

VERSION 1

PUBLISHED 25 Jan 2018

Views

21

Reviewer Report 10 May 2018

Isabele C. Iser, Department of Basic Health Sciences, Laboratory of Cell Biology, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre, Brazil

Not Approved

https://doi.org/10.5256/f1000research.14268.r33378

In the study titled “Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells”, the authors investigated the effects of UCSC on the expression of pluripotency factors in GBM cells. ... Continue reading

In the study titled “Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells”, the authors investigated the effects of UCSC on the expression of pluripotency factors in GBM cells. The authors showed an increased expression of SOX4 and OCT4 in GBM cells treated with CM when compared to untreated cells.
The work presents numerous limitations, and does not have scientific relevance that justify its indexing. For example:

The authors evaluated the gene expression only at the RNA level, however, the ideal would be to evaluate also at the protein level in order to obtain more conclusive results. In addition, the authors evaluated a limited number of genes related to CSCs. I suggest evaluating a greater number of genes to confirm the results presented here.
The authors also should access other parameters in the cells besides gene expression, such as resistance to therapy, proliferation and viability.
It is also very important analyze which factors in the CM could be responsible for the effect presented in this work.
I strongly suggest that the authors use a positive control for qPCR reactions, such as GBM cells treated with CSC inducers.
In the discussion the authors say that “the up-regulation of this set of genes is associated with poor outcome in terms of tumor malignancy”, so in my opinion it would be interesting to perform an in vivo model to testing the effects of these treated cells in terms of tumor formation and malignancy.

In view of these considerations, I do not consider the work apt to be published in F1000 Research.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

CITE

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29

Reviewer Report 26 Mar 2018

Jie Sun, Department of Urology, Shanghai Children’s Medical Center, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

Not Approved

https://doi.org/10.5256/f1000research.14268.r32142

The present study mainly showed the effects of MSC conditioned medium in glioblastoma multiforme cells. Authors found that MSC-CM promote the expression of pluripotency markers in T98G cells, like SOX4 and OCT4, but not NANOG. The research has limited novelty ... Continue reading

The present study mainly showed the effects of MSC conditioned medium in glioblastoma multiforme cells. Authors found that MSC-CM promote the expression of pluripotency markers in T98G cells, like SOX4 and OCT4, but not NANOG. The research has limited novelty and integrity. Main concerns are listed below:

Serum free medium were used to acquire the MSC-CM for 24 hours in this study, authors should test the cell viability of MSC in this state for a better assess the quality of CM.
All the gene markers were tested in mRNA level in the manuscript, protein levels should be added.
Only a small number of cells were regarded as cancer stem cells, especially in commercial cell lines. MSC-CM increases the original stem cells to express pluripotency markers or it could induce non-stem cells to express these markers still unclear. What is more, whether CM would affects cell functions after the increase of these markers still unclear. All these date are needed to be completed or discussed in the manuscript.
As authors mentioned in the discussion, various researches has been tested to clarify the interactions between MSC and cancer cells. Based on the existed results in this paper, limited novelty also decrease the quality of the manuscript.
The background of the pictures in Figure 1 should be changed and the scale bar should be added.
The statistical analysis should not be used on its own, as the study is not good and qualified enough/

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

CITE

Report a concern

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Version 1

VERSION 1 PUBLISHED 25 Jan 2018

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Version 1 25 Jan 18	read	read

Jie Sun, Shanghai Jiao Tong University, Shanghai, China
Isabele C. Iser, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre, Brazil

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Reviewer Report

21 Views

10 May 2018 | for Version 1

Isabele C. Iser, Department of Basic Health Sciences, Laboratory of Cell Biology, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre, Brazil

21 Views Cite this report Responses(0)

Not Approved

In the study titled “Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells”, the authors investigated the effects of UCSC on the expression of pluripotency factors in GBM cells. The authors showed an increased expression of SOX4 and OCT4 in GBM cells treated with CM when compared to untreated cells.
The work presents numerous limitations, and does not have scientific relevance that justify its indexing. For example:

The authors evaluated the gene expression only at the RNA level, however, the ideal would be to evaluate also at the protein level in order to obtain more conclusive results. In addition, the authors evaluated a limited number of genes related to CSCs. I suggest evaluating a greater number of genes to confirm the results presented here.
The authors also should access other parameters in the cells besides gene expression, such as resistance to therapy, proliferation and viability.
It is also very important analyze which factors in the CM could be responsible for the effect presented in this work.
I strongly suggest that the authors use a positive control for qPCR reactions, such as GBM cells treated with CSC inducers.
In the discussion the authors say that “the up-regulation of this set of genes is associated with poor outcome in terms of tumor malignancy”, so in my opinion it would be interesting to perform an in vivo model to testing the effects of these treated cells in terms of tumor formation and malignancy.

In view of these considerations, I do not consider the work apt to be published in F1000 Research.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

29 Views

26 Mar 2018 | for Version 1

Jie Sun, Department of Urology, Shanghai Children’s Medical Center, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

29 Views Cite this report Responses(0)

Not Approved

The present study mainly showed the effects of MSC conditioned medium in glioblastoma multiforme cells. Authors found that MSC-CM promote the expression of pluripotency markers in T98G cells, like SOX4 and OCT4, but not NANOG. The research has limited novelty and integrity. Main concerns are listed below:

Serum free medium were used to acquire the MSC-CM for 24 hours in this study, authors should test the cell viability of MSC in this state for a better assess the quality of CM.
All the gene markers were tested in mRNA level in the manuscript, protein levels should be added.
Only a small number of cells were regarded as cancer stem cells, especially in commercial cell lines. MSC-CM increases the original stem cells to express pluripotency markers or it could induce non-stem cells to express these markers still unclear. What is more, whether CM would affects cell functions after the increase of these markers still unclear. All these date are needed to be completed or discussed in the manuscript.
As authors mentioned in the discussion, various researches has been tested to clarify the interactions between MSC and cancer cells. Based on the existed results in this paper, limited novelty also decrease the quality of the manuscript.
The background of the pictures in Figure 1 should be changed and the scale bar should be added.
The statistical analysis should not be used on its own, as the study is not good and qualified enough/

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Respond to this report

Responses (0)

[1] 1. Louis DN, Ohgaki H, Wiestler OD, et al.: The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 2007; 114(2): 97–109. PubMed Abstract | Publisher Full Text | Free Full Text

[2] 2. Holmberg J, He X, Peredino I, et al.: Activation of neural and pluripotent stem cell signatures correlates with increased malignancy in human glioma. Plos One. 2011; 6(3): e18454. PubMed Abstract | Publisher Full Text | Free Full Text

[3] 3. Bao S, Wu Q, McLendon RE, et al.: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature Lett. 2006; 444(7120): 756–760. PubMed Abstract | Publisher Full Text

[4] 4. Fong H, Hohenstein KA, Donovan PJ: Regulation of self-renewal and pluripotency by Sox2 in human embryonic stem cells. Stem Cells. 2008; 26(8): 1931–1938. PubMed Abstract | Publisher Full Text

[5] 5. Loh YH, Wu Q, Chew JL, et al.: The Oct4 and nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nature Gen. 2006; 38(4): 431–440. PubMed Abstract | Publisher Full Text

[6] 6. Chaturvedi P, Gilkes DM, Wong CC, et al.: Hypoxia-inducible factor-dependent breast cancer-mesenchymal stem cell bidirectional signaling promotes metastasis. J Clin Invest. 2013; 123(1): 189–205. PubMed Abstract | Publisher Full Text | Free Full Text

[7] 7. Lee JK: Mesenchymal stem cell: a hard nut to crack in cancer development. Enliven Archive. 2014; 1. Reference Source

[8] 8. Hardiany NS, Sadikin M, Siregar NC, et al.: The suppression of manganese superoxide dismutase decreased the survival of human glioblastoma multiforme T98G cells. Med J Indones. 2017; 26(1): 19–25. Publisher Full Text

[9] 9. Pawitan JA, Kispa T, Mediana D, et al.: Simple production method of umbilical cord derived mesenchymal stem cell using xeno-free materials for translational research. J Chem Pharm Res. 2015; 7(8): 652–656. Reference Source

[10] 10. Tachi K, Shiraishi A, Bando H, et al.: FOXA1 expression affects the proliferation activity of luminal breast cancer stem cell populations. Cancer Sci. 2016; 107(3): 281–9. PubMed Abstract | Publisher Full Text | Free Full Text

[11] 11. Liu A, Yu X, Liu S: Pluripotency transcription factors and cancer stem cells: small genes make a big difference. Chin J Cancer. 2013; 32(9): 483–487. PubMed Abstract | Free Full Text

[12] 12. Liu S, Ginestier C, Ou SJ, et al.: Breast cancer stem cells are regulated by mesenchymal stem cells through cytokine networks. Cancer Res. 2011; 71(2): 614–624. PubMed Abstract | Publisher Full Text | Free Full Text

[13] 13. Li HJ, Reinhardt F, Herschman HR, et al.: Cancer-stimulated mesenchymal stem cells create a carcinoma stem cell niche via prostaglandin E₂ signaling. Cancer Discov. 2012; 2(9): 840–855. PubMed Abstract | Publisher Full Text | Free Full Text

[14] 14. Wu XB, Liu Y, Wang GH, et al.: Mesenchymal stem cells promote colorectal cancer progression through AMPK/mTOR-mediated NF-κB activation. Sci Rep. 2016; 6: 21420. PubMed Abstract | Publisher Full Text | Free Full Text

[15] 15. Luo J, Lee SO, Cui Y, et al.: Infiltrating bone marrow mesenchymal stem cells (BM-MSCs) increase prostate cancer cell invasion via altering the CCL5/HIF2α/androgen receptor signals. Oncotarget. 2015; 6(29): 27555–27565. PubMed Abstract | Publisher Full Text | Free Full Text

[16] 16. Jeter CR, Liu B, Liu X, et al.: NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation. Oncogene. 2011; 30(36): 3833–3845. PubMed Abstract | Publisher Full Text | Free Full Text

[17] 17. Kawamura N, Nimura K, Nagano H, et al.: CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells. Oncotarget. 2015; 6(26): 22361–22374. PubMed Abstract | Publisher Full Text | Free Full Text

[18] 18. Zhang C, Samanta D, Lu H, et al.: Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m⁶A-demethylation of NANOG mRNA. Proc Natl Acad Sci U S A. 2016; 113(14): E2047–E2056. PubMed Abstract | Publisher Full Text | Free Full Text

[19] 19. Niu CS, Yang Y, Cheng CD: MiR-134 regulates the proliferation and invasion of glioblastoma cells by reducing Nanog expression. Int J Oncol. 2013; 42(5): 1533–1540. PubMed Abstract | Publisher Full Text | Free Full Text

[20] 20. Hardiany NS, Huang P, Dewi S, et al.: Dataset 1 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source

[21] 21. Hardiany NS, Huang P, Dewi S, et al.: Dataset 2 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source

[22] 22. Hardiany NS, Huang P, Dewi S, et al.: Dataset 3 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source

[23] 23. Hardiany NS, Huang P, Dewi S, et al.: Dataset 4 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source

[24] 24. Hardiany NS, Huang P, Dewi S, et al.: Dataset 5 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source

Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells

Abstract

Keywords

Introduction

Methods

Cell culture

Preparation of conditioned medium of umbilical cord-derived MSCs (UCSC-CM)

UCSC-CM treatment of T98G cells

Analysis of pluripotency marker expression

Table 1. Primer sequences.

Statistical analysis

Results

Cell morphology

Figure 1. T98G cell morphology.

Pluripotency marker expression

Figure 2. SOX2 mRNA expression.

Figure 3. OCT4 mRNA expression.

Figure 4. NANOG mRNA expression.

Discussion

Conclusions

Data availability

Competing interests

Grant information

Acknowledgments

References

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