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Cell Biology International (2012) 36, 793–801 (Printed in Great Britain)
Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) inhibit the proliferation of K562 (human erythromyeloblastoid leukaemic cell line)
Malini Fonseka*, Rajesh Ramasamy*, Boon Chong Tan† and Heng Fong Seow*1
*Immunology Laboratory, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia, and †Britannia Women and Children Specialist Centre, Kajang, Selangor, Malaysia


hUCB-MSC (human umbilical cord blood-derived mesenchymal stem cells) offer an attractive alternative to bone marrow-derived MSC for cell-based therapy by being less invasive a source of biological material. We have evaluated the effect of hUCB-MSC on the proliferation of K562 (an erythromyeloblastoid cell line) and the cytokine secretion pattern of hUCB-MSC. Co-culturing of hUCB-MSC and K562 resulted in inhibition of proliferation of K562 in a dose-dependent manner. However, the anti-proliferative effect was reduced in transwells, suggesting the importance of direct cell-to-cell contact. hUCB-MSC inhibited proliferation of K562, arresting them in the G0/G1 phase. NO (nitric oxide) was not involved in the hUCB-MSC-mediated tumour suppression. The presence of IL-6 (interleukin 6) and IL-8 were obvious in the hUCB-MSC conditioned media, but no significant increase was found in 29 other cytokines. Th1 cytokines, IFNγ (interferon γ), Th2 cytokine IL-4 and Th17 cytokine, IL-17 were not secreted by hUCB-MSC. There was an increase in the number of hUCB-MSC expressing the latent membrane-bound form of TGFβ1 co-cultured with K562. The anti-proliferative effect of hUCB-MSC was due to arrest of the growth of K562 in the G0/G1 phase. The mechanisms underlying increased IL-6 and IL-8 secretion and LAP (latency-associated peptide; TGFβ1) by hUCB-MSC remains unknown.


Key words: anti-proliferative, K562 leukaemic cells, mesenchymal stem cells, umbilical cord blood

Abbreviations: DMEM, Dulbecco's modified Eagle's medium, FBS, fetal bovine serum, hUCB-MSC, human umbilical cord blood-derived mesenchymal stem cells, ICAM-1, intercellular adhesion molecule 1, IFN, interferon, IL, interleukin, LAP, latency-associated peptide, MMP3, metalloproteinase 3, MSC, mesenchymal stem cells, NO, nitric oxide, TGFβ, transforming growth factor β, TIMP-1, tissue inhibitor of metalloproteinases-1, TNF, tumour necrosis factor, VEGF, vascular endothelial growth factor

1To whom correspondence should be addressed (email shf@medic.upm.edu.my).


1. Introduction

MSC (mesenchymal stem cells) are undifferentiated multipotent non-haematopoietic stem cells that are capable of self-renewal and differentiation into many cell types such as chrondrocytes, adipocytes, osteocytes, myocytes and neuron-like cells (Jiang et al., 2002). Sources of MSC include bone marrow (Friedenstein et al., 1970), umbilical cord blood (Erices et al., 2000; Lee et al., 2004), umbilical cord (Tong et al., 2011), peripheral blood (Zvaifler et al., 2000), adipose tissue (Gronthos et al., 2001) and placenta (Miao et al., 2006).

Due to the plasticity, migratory and relatively non-immunogenic properties, MSC are potentially useful for transplantation and treatment of degenerative diseases and cancer. Many of these studies have been conducted with bone marrow-derived MSC and less is known about hUCB-MSC (human umbilical cord blood-derived MSC). hUCB-MSC have a similar capacity for multi-lineage differentiation as bone marrow-derived MSC. Since collection of hUCB-MSC is less invasive than bone marrow, hUCB-MSC is an attractive source for clinical applications. However, more needs to be known of the activities of hUCB-MSC before use in patients.

Bone marrow-derived MSC and hUCB-MSC are immune suppressive to lymphocyte proliferation (Tong et al., 2008, 2011). There are no reports of tumour formation after transplantation with hUCB-MSC in animals and humans. However, bone marrow-derived MSC either favour or inhibit tumour growth. Bone marrow-derived MSC favour tumour growth either by protecting tumour cells from immune cell recognition (Djouad et al., 2003) or by enhancing their invasive abilities via extensive necrosis and angiogenesis (Zhu et al., 2006). However, others have found the opposite (Lazennec and Jorgensen, 2008; Sarmadi et al., 2010). Experimental models of Lewis lung carcinoma and B16 melanoma showed that co-injection of mouse MSC with tumour cells inhibited tumour growth, which was mediated by soluble factors (Maestroni et al., 1999). Khakoo et al. (2006) reported that MSC exerted anti-tumour effect on Kaposi's sarcoma cells in vivo through direct cell contact-dependent inhibition. Studeny et al. (2002) demonstrated that MSC could be modified to express IFNβ (interferon β) and inhibited tumour growth in vitro and in vivo.

Hence, there are discrepancies regarding the effect of bone marrow-derived MSC on tumour cells. These inconsistent results with their inadequate explanations are clear indicators that the effect of MSC on tumour cells and the mechanisms involved need resolution. Since the heterogeneity of MSC and the type of experimental tumour models used can influence the outcome of the interaction between MSC and tumour cells (Uccelli et al., 2007), the effect of hUCB-MSC on a leukaemic cell line was explored.

The objectives were to: (i) evaluate the effect of hUCB-MSC on the proliferation and survival of K562 (an erythromyeloblastoid leukaemic cell line) and (ii) determine the cytokines secreted by MSC when co-cultured with K562. An understanding of the molecular changes that occur when hUCB-MSC interact with tumour cells is required in the design of future strategies for cancer therapy with hUCB-MSC.

2. Materials and methods

2.1. Isolation and culture of MSC from human umbilical cord blood

hUCB-MSC were generated and characterized as described by Lee et al. (2004). hUCB-MSC are adherent cells that were cultured in complete MSC media consisting of DMEM (Dulbecco's modified Eagle's medium) with high glucose, l-glutamine, pyridoxine hydrochloride, with 2.0 g/l sodium bicarbonate and 25 mM Hepes (both from Sigma–Aldrich Co.), supplemented with 10% FBS (fetal bovine serum), 1% penicillin/streptomycin, 0.5% amphotericin B and 0.1% gentamycin (Calbiochem). Culture media and supplements were purchased from Gibco, unless otherwise specified. hUCB-MSC from passages 3–7 were used for the experiments, their morphology and immunophenotype being checked before use.

2.2. Cell culture of K562

K562 (ATCC CCL-243) is an undifferentiated erythroleukaemic cell line derived from a chronic myeloid leukaemia patient (Klein et al., 1976). Cells were grown in suspension in RPMI 1640 (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin.

2.3. 3H-thymidine assay

Five thousand K562 cells in suspension were removed from the treated and control experiments and re-plated into 96-well plates and pulsed with 3H-thymidine (PerkinElmer) at 0.5 μCi/well (0.037 MBq/well), during the final 18 h of incubation. The cells were collected with a harvester (Harvester 96 Mach III M). Intact DNA was then extracted on to filter mats, which were oven-dried for 10 min. Scintillation cocktail liquid was added to the filter mat and the sample bag was sealed. The radioactivity level of the incorporated thymidine was measured using a MicroBeta TriLux counter. Results were expressed in cpm (counts per minute).

2.4. Co-culture of hUCB-MSC and K562 leukaemic cells

hUCB-MSC were seeded on to 96 well plates at a ratio of 1:1, 1:5, 1:10 and 1:100 to a fixed number of K562 leukaemic cell (5000 cells/well). The final volume of the media was topped up to 200 μl. After 24 h incubation, MSC were attached to the bottom of the well. The culture supernatant containing K562 cells was harvested, centrifuged and the cell concentration adjusted to 2.5×104 cells/ml. Two hundred microlitres of K562 was aliquoted into each well containing hUCB-MSC. The plates were left to incubate for 48 h before the thymidine assay was carried out.

2.5. Co-culture of hUCB-MSC and K562 in a transwell system

In a transwell system, hUCB-MSC (5×104 cells/well) were seeded on the upper chamber of the transwell. The cells were incubated for 24 h, following which the remaining medium was removed and K562 (25×104 cells/well) were added either directly into the wells or into the lower chamber of the transwell. The cell cultures were incubated for 48 h before the thymidine assay was carried out.

2.6. Cell cycle assay

hUCB-MSC were co-cultured with K562 as described above. K562 suspension cells from the co-culture were re-plated for 1 or 2 h prior to fixation in 70% ice-cold ethanol. The cells were washed with PBS before incubation with 50 μl of 200 μg/ml of RNaseA at 37°C for 30 min. Next, 50 μl of 1 mg/ml PI (Sigma–Aldrich Co.) was added and incubated for a further 20 min. Approximately 20000 cells acquisitions were made using the CellQuest Pro software. The results were analysed using the mathematical models provided by FCS Express 3.0 software (BD Bioscience).

2.7. Griess assay

hUCB-MSC (5×104 cells/well) were cultured in the absence and presence of K562 tumour cells at a ratio 1:5 for 24, 48 and 72 h in 12-well plates, in complete DMEM (without Phenol Red). At every 24 h, 150 μl of culture supernatant was aspirated for the assay. The culture supernatant was equally distributed into 3 wells (50 μl/well) in 96 well plates. Fifty μl of freshly prepared Griess reagent [1% (w/v) sulfanilamide and 0.1% (w/v) NED in phosphoric acid, H3PO4] were added into each well and incubated in dark for 5 min before reading the absorbance at A530. A standard curve was generated with serial dilution of NaNO2 (sodium nitrate) (100, 50, 25, 12.5, 6.25, 3.13, 1.57 and 0 μM) for the estimation of NO2 concentration present in the samples.

2.8. Cytokine antibody array

A cytokine antibody array was analysed using the Human Antibody Array 3.0 kit (Panomics; Cat No. MA6160). The membrane array consisted of antibodies against 31 cytokines, epidermal growth factor, TNFR1 (tumour necrosis factor receptor 1), TNFRII (TNF receptor II), metalloproteinase MMP3 and adhesion molecules ICAM-1 (intercellular adhesion molecule 1) and VCAM-1. Conditioned media from hUCB-MSC (105 cells/well) culture, K562 (105 cells/well) culture and hUCB-MSC (105 cells/well) and K562 (105 cells/well) co-culture were collected after 48 h and stored at −80°C. The control was the culture media RPMI 1640 supplemented with 10% FBS. Membranes coated with cytokine antibodies were blocked with 5% BSA (Amresco) for 2 h. The membranes were washed 4 times in 1×Tris-buffered saline with Tween 20 prior to incubating with 2 ml of various conditioned media. The conditioned media were decanted and the membranes washed 4 times before incubation with 1.5 ml of secondary biotin-conjugated antibody for a further 2 h followed by washing steps. Two ml streptavidin-horseradish peroxidase (×1000 dilution) were added to the membrane and incubated for 1 h before being washed. Finally, 200 μl of chemiluminescent substrate (SuperSignal® West Femto Maximum Sensitivity Substrate; Thermo Fisher Scientific) was applied to the membrane and incubated for 5 min before visualization using the FluorChem™ 5500 imaging system (Alpha Innotech Corp.). The blot images were captured and their intensities measured densitometrically using the ImageJ software (National Institutes of Health). The average intensity value of duplicate dots of a cytokine was divided by that of internal positive control on the same or nearest row for normalization. The normalized value from each sample was divided by the value in the control, representing the fold change in comparison with that of the control which was set as 1. The dot intensities were quantified by ImageJ densitometric analysis and the values of cytokines normalized to those of internal positive control.

2.9. Immunostaining with anti-LAP (latency-associated peptide) conjugated to PE

hUCB-MSC and K562 cells were either cultured alone or co-cultured at 1:5 ratio for 48 h. K562 cells were harvested into 5 ml tubes and centrifuged at 2000 rev./min for 5 min. The adherent hUCB-MSC was trypsinized and both these cells were separately centrifuged, washed with PBS with 0.1% BSA and centrifuged. The pellets were resuspended in 100 μl of RPMI 1640 followed by addition of 2 μl of 1:50 diluted anti-human LAP (TGFβ1) antibody conjugated to PE (R&D System) before being incubated for 30 min in the dark at 4°C. Unstained and fluorochrome-matched non-specific isotype labelled cells were used as controls. Following incubation, cells were washed with 1 ml PBS containing 0.1% BSA and the pellet resuspended in 400 μl of PBS with 0.1% BSA for flow cytometric analysis.

3. Results

3.1. hUCB-MSC inhibit the proliferation of K562 in a dose-dependent manner

K562 cell proliferation rate was reduced by the presence of hUCB-MSC. Comparison of the proliferation of the K562 cells cultured with and without hUCB-MSC was 35.2% (P<0.05), 54.5% (P<0.05), 74.2% (P<0.05) and 90.9% at 1:1, 1:5, 1:10 and 1:100 ratios of hUCB-MSC: K562, respectively (Figure 1). Comparison of the proliferation rate of K562 co-cultured with hUCB-MSC at a ratio of 1:1 to ratios 1:10 (P<0.05) and 1:100 (P<0.05), showed that there was a significantly difference in the proliferation of K562, as indicated by an asterisk (*). Further comparison of 1:5 ratio of UCB-MSC:K562 to ratio 1:100 (P<0.05) showed that there was a significant difference as indicated by a double asterisk (**). The results suggest that hUCB-MSC are capable of inhibiting dose-dependently proliferation of the tumour cells (Figure 1).

3.2. Effect of loss of direct cell contact on inhibition of K562 proliferation by hUCB-MSC

K562 cells were cultured either in direct contact with hUCB-MSC or in a transwell system where they were physically separated by the transwell, or with culture supernatants harvested from 48 h cultures of hUCB-MSC. A lower proliferation rate was observed in all three systems compared with K562 alone. The highest degree of inhibition of proliferation of K562 cells (down to 45%) was in the direct co-culture system, followed by 53.3% reduction in the transwell system compared with control (Figure 2). Proliferation rate was 63% of K562 cells incubated with 48 h conditioned medium harvested from hUCB-MSC cultures. Thus proliferation of K562 is most effectively inhibited in direct contact with hUCB-MSC (Figure 2).

3.3. hUCB-MSC arrest the tumour cells in the G0/G1 phase of the cell cycle and prevent their entry into the S phase

Figure 3 shows the uptake of the PI dye, which correlates with the DNA content of the K562 cells in each phase of the cell cycle when cultured alone (Figure 3A) or with hUCB-MSC (Figures 3B and 3C). The histograms in Figure 3(C) were used to analyse and compare cell cycle progression. The profile of K562 in culture media only gave 32.7% in the G0/G1 phase, 51.0% in S phase and 16.3% in G2/M phase. In the presence of hUCB-MSC, there was an increased number of K562 cells (45.7%; P<0.05) in G0/G1. Fewer cells (40.3%; P<0.05) were detected in S phase and 14.0% in G2/M phase. hUCB-MSC arrested more K562 cells in G0/G1 phase, preventing many from progressing to S phase (Figure 3).

3.4. hUCB-MSC-mediated inhibition does not involve NO (nitric oxide)

A possible mechanism that might explain the anti-proliferative effect exhibited by MSC, is related to the production of NO, an important physiological messenger and effector molecule in many diverse systems, including immune system (Dawson and Dawson, 1995). Since NO quickly converts into NO2, the Griess assay is one of the simplest methods used to detect NO2 concentration in culture supernatant (Bredt and Snyder, 1994). Serial dilution of sodium nitrate was used to construct a standard curve of NO concentration in the Griess Assay. MSC cultured alone produced negligible amounts of NO at 24 h (0%), 48 h (9.1%) and 72 h (7.1%). In the supernatant derived from MSC and K562 co-cultures, insignificant NO was produced (0, 9.9 and 6.1 respectively) at the same time points (Figure 4B). This strongly suggests that NO production does not contribute to the inhibition of K562 tumour cells proliferation by hUCB-MSC.

3.5. Cytokine secretion profiling of hUCB-MSC, K562 and their co-culture

The possible role of soluble factors, such as cytokines, in contributing to the inhibitory effect of hUCB-MSC on the K562 cells was analysed by using the Cytokine Human Antibody Membrane Array. We compared the cytokine secretion profiles from 48 h conditioned media from hUCB-MSC culture, K562 culture and MSC co-cultured with K562. There was an obvious presence of IL-6 and IL-8 in the conditioned media of hUCB-MSC compared with the RPMI 1640+10% FBS (control) or K562. The secretion of IL (interleukin)-6 and IL-8 remained detectable when co-cultured with K562. Densitometric scanning analysis of the dots showed nearly a 20-fold increase of IL-6 from hUCB-MSC alone, which was reduced to only 10-fold when hUCB-MSC was co-cultured with K562 (Supplementary Figure S1 available online at http://www.cellbiolint.org/cbi/036/cbi0360793add.htm). Regarding IL-8, the corresponding increase was 5- and 6-fold (Supplementary Figure S1). Pro-inflammatory cytokines (TNF, IL-1 and IL-17), Th1 cytokines (IFNγ and IL-2) and Th2 cytokines (e.g. IL-4) were indetectable in all the conditioned media, like the control. There was no detectable increase in anti-inflammatory cytokines [IL-10 and TGFβ (transforming growth factor β)] secretion from the UCB-MSC culture and co-culture conditioned medium, like the control. Finally, there were no changes in chemokines [MCP-1, RANTES (regulated upon activation, normal T-cell expressed and secreted) and MIPs (macrophage inflammatory proteins)], adhesion molecules (ICAM and VCAM) and pro-angiogenic growth factor [e.g. VEGF (vascular endothelial growth factor); Figure 5]. A minor increase in Apol/Fas protein was found in conditioned media from K562.

3.6. MSC increased LAP/TGFβ1 expression when co-cultured together with K562 leukaemic cells

To further determine the identity of the factor that contributed to inhibition of K562 proliferation by UCB-MSC, the possible candidate, TGFβ, was investigated as a known mediator of cell growth that is involved in malignant progression (Stagg and Galipeau, 2007). TGFβ is synthesized as a precursor molecule with a C-terminal pro-peptide that binds to the N-terminal portion of the TGFβ (Young and Murphy-Ullrich, 2004). The C-terminal pro-peptide is known as LAP and the LAP portion is cleaved prior to secretion (Derynck et al., 1985; Khalil, 1999). Hence, expression of LAP (TGFβ1) protein was measured in MSC culture, K562 culture and their co-culture. The results shown in clearly indicated that 75.0% (P<0.05) of MSC from the co-culture system had the expression of LAP (TGFβ1), as compared with MSC cultured alone (Figures 6A and 6B), which had only 19.7% expression of LAP (TGFβ1). Although the percentage of MSC expressing LAP (TGFβ1), indicative of membrane-bound TGFβ1 in the co-culture system, was high, its intensity of expression remained low. Expression of LAP (TGFβ1) in K562 cells showed no obvious difference between K562 co-cultured with hUCB-MSC (54.3%) and hUCB-MSC alone (53.0%; Figure 6C).

4. Discussion

hUCB-MSC are capable of inhibiting the proliferation of leukaemic cells. When MSC were co-cultured with K562 leukaemic cells, proliferation was significantly inhibited (Figure 1). However, MSC-induced inhibition of the tumour cells was dose-dependent. suggesting hUCB-MSC are comparable as anti-tumour agents to human bone marrow-derived MSC (Ding et al., 2009). This is particularly significant in finding an alternative source of MSC without compromising anti-tumour activity.

Comparison of cell cycle progression of K562 cultured in the absence and presence of hUCB-MSC showed there was an accumulation predominantly in G0/G1 phase (Figure 3), slowing entry into S phase. This result agrees with other reports (Ramasamy et al., 2007; Liotta et al., 2008).

MSC are capable of suppressing immune reactions both in vitro and in vivo, in an MHC-independent manner. Soluble factors are heavily involved in this mechanism (Tyndall et al., 2007). Of these factors, HGF (hepatocyte growth factor), TGFβ1 (Di Nicola et al., 2002; Sotiropoulou et al., 2006), PGE2 (prostaglandin E2; Aggarwal and Pittenger, 2005), IDO (Meisel et al., 2004; Krampera et al., 2006) and NO (Sato et al., 2007) are possible candidates. Unlike MSC immunomodulation, the underlying mechanism involved in the interaction of MSC with tumour cells are just beginning to unravel, but at least some of the effects may be due to the same properties.

NO is an important biological signalling molecule involved in a variety of physiological and pathological processes (Hou et al., 1999). NO might also play a critical role in tumour inhibitory, but NO2 production measured by Griess assay in the both the culture systems (MSC and K562 cells cultured alone and co-cultured) proved negligible (Figure 4), suggesting that NO did not contribute to the inhibition of K562 proliferation by hUCB-MSC.

We hypothesized that the inhibition of proliferation by hUCB-MSC was governed by cytokines. Liu and Hwang (2005) had also used the cytokine antibody array to analyse cytokine expression by hUCB-MSC found that IL-6, IL-8, TIMP-1 (tissue inhibitor of metalloproteinases-1) and TIMP-2 were the most abundant proteins expressed, consistent with our data. The two prominent cytokines detected in co-culture most probably derived from hUCB-MSC as hUCB-MSC culture alone indicates a high secretion of these cytokines (20-fold and 5-fold increase for IL-6 and IL-8 respectively), but were absent in K562 culture (no data for TIMP-1 and TIMP-2 were obtained).

hUCB-MSC constitutively express pro-angiogenic factors, such as Ang1 (angiopoietin-1) and VEGF, as well as other growth factors and cytokines such as FGF (fibroblast growth factors), IL-6 and TNFα (Kogler et al., 2005). We did not detect TNF, which accords with another report (Salazar et al., 2009). Interestingly, we are the first to report that expression of Th1 cytokines, such as IFNγ, Th2 cytokines namely IL-4 and Th17 cytokines, namely, IL-17, were undetectable in conditioned media of hUCB-MSC, K562 and co-cultures.

Compared with the control, MSC produce generous amounts of both IL-6 and IL-8. IL-6 is a broad-acting cytokine for the development, differentiation, regeneration and degeneration of various stem cells (Klassen et al., 2003). In addition, IL-6 also plays a critical role in tissue remodelling within the connective tissue cells (Gadient and Otten, 1997). The reason for high expression of IL-6 in hUCB-MSC conditioned media remains to be investigated. IL-17 is known to induce production of pro-inflammatory cytokines such as TNF, IL-6 and IL-1β. Since IL-17 was undetectable in all conditioned media, increased production of IL-6 by hUCB-MSC is probably not due to induction by IL-17.

Our finding that hUCB-MSC produced high amount of IL-8 is consistent with a previous study, whereby, bone marrow-derived MSC co-cultured with K562 was found to express higher IL-8 mRNA as compared with MSC cultured alone (Wang et al., 2008). IL-8 is a member of the CXC chemokine family. It is a pro-inflammatory cytokine and functions as a chemoattractant (Modi et al., 1990). IL-8 has been referred as an important cytokine in the tumour progression and metastasis through its potential function in the modulation of angiogenesis, leucocyte infiltration and regulation of immune response (Miller et al., 1998).

TGFβ is a powerful pleiotropic immunosuppressive and anti-inflammatory cytokine (Derynck et al., 2001). It inherits both cytostatic and apoptotic functions to restrain cell growth and the loss of these effects leads to hyperproliferative disorders that are the hallmarks of tumours (Gold, 1999). Once tumour cells are released from the initial TGFβ growth constraints, tumour cells might overproduce TGFβ that can act as a pro-angiogenic factor and have potent immunosuppressive effects favourable for proliferation and metastasis (Gold, 1999). Hence, the growing interest in therapeutically targeting of TGFβ-mediated processes in tumour progression. However, we failed to detect TGFβ in the conditioned media using the cytokine membrane array method.

The possibility of the interaction between hUCB-MSC and K562 leukaemic cells being influenced by TGFβ was considered. The level of TGFβ on a cellular basis on the expression of LAP molecule via flow cytometry was measured. TGFβ is synthesized as a precursor molecule consisting of a C-terminal pro-peptide that is associated non-covalently with the N-terminal portion of the TGFβ homodimer (Young and Murphy-Ullrich, 2004). The C-terminal pro-peptide is referred to as LAP. The LAP portion is cleaved in a post-Golgi compartment prior to secretion (Derynck et al., 1985; Khalil, 1999).

A significantly elevated LAP expression was seen by co-culturing hUCB-MSC with K562 compared with MSC cultured alone (Figure 6). Detection of LAP indicates an increase in number of cells expressing the latent membrane form of TGFβ1 that has to be cleaved before release into the culture media. Acid activation of TGFβ1 to release the secreted form of TGFβ1 by hUCB-MSC was reported by Salazar et al. (2009).

5. Conclusion

We have shown that MSC exerted significant dose-dependent inhibition on K562 cells. MSC induced cell-cycle arrest by halting the progression of tumour cells in G0/G1 phase. The inhibition of proliferation was not due to the release of NO by hUCB-MSC. Hypothesizing that soluble factors might be highly involved in the anti-tumour effect mediated by MSC, we analysed the cytokine secretion profile. Significant production of IL-6 and IL-8 by hUCB-MSC was found, quantified by ELISA. The physiological role of IL-6 and IL-8 remains unclear. Increased IL-6 is not due to induction by Th17 cytokine because IL-17 was not detected in the conditioned media of all the cultures. The expression of LAP on hUCB-MSC suggests that the membrane-bound form of TGFβ could be involved in the inhibition of the tumour cell proliferation. The identity of molecules involved in the anti-proliferative effect of hUCB-MSC requires further investigation.

Author contribution

Malini Fonseka performed the experiments and planned the design of experiments. Rajesh Ramasamy assisted in the design of the study and technical aspects of the methodology. Wai Kien Yip assisted in the analysis of the antibody array data. Boon Chong Tan assisted in the collection of umbilical cord. Heng Fong Seow conducted the conceptualization of the project, design of study, grant application and writing of the draft of the manuscript.

Funding

This work was supported by the Research University Grants, Universiti Putra Malaysia [grant number 04/01/07/0113RU].

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Received 24 October 2011/18 January 2012; accepted 16 February 2012

Published as Cell Biology International Immediate Publication 16 February 2012, doi:10.1042/CBI20110595


© The Author(s) Journal compilation © 2012 International Federation for Cell Biology


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)