different effects of conditioned medium samples from

Доклади на Българската академия на науките Comptes rendus de l’Acad´ emie bulgare des Sciences Tome 63, No 1, 2010

BIOLOGIE Cytologie

DIFFERENT EFFECTS OF CONDITIONED MEDIUM SAMPLES FROM MESENCHYMAL STEM CELLS (CM-MSC) ISOLATED FROM DIFFERENT SOURCES Snejana Kestendjieva, Zlatka Popova, Gergana Cvetkova, Ivan Bochev, Ivan Kehayov, Stanimir Kyurkchiev, Milena Mourdjeva (Submitted by Corresponding Member O. Poljakova-Krusteva on July 31, 2009)

Abstract Stem cell-based therapies are promising new treatment approaches for a large number of diseases. Since paracrine mechanisms are primarily responsible for the organprotective actions of administered stem cells their conditioned medium has the potency to treat a variety of diseases in humans even without cell transplantation. Our previous work showed that bone marrow mesenchymal stem cells conditioned medium supports the growth of endothelial progenitor cells (EPCs) in vitro. Now we compare the effects of mesenchymal stem cells conditioned media from bone marrow (CM-BM-MSC) and adipose tissue (CM-AT-MSC) on EPCs survival. Our results suggest opposite effect of two conditioned media. EPCs cultured in CM-BM-MSC survived and were positive for mRNA of anti-apoptotic factors, while EPCs cultured in CM-AT-MSC were negative for the same factors. This strongly indicates that the secretion profiles of MSC from different origin have different biological effects. Exact factors and mechanisms of these effects have to be studied in future. Key words: endothelial progenitor cells, bone marrow mesenchymal stem cells, adipose tissue mesenchymal stem cells, mesenchymal stem cells conditioned media

Introduction. Mesenchymal stem cells (MSC) are cells that have the potential to differentiate into lineages of mesenchymal tissues including cartilage, bone, muscle and fat. They were initially isolated from bone marrow and characterized This work was partly supported by grants Genomika-4/1,2 and L-1517/2005 from the National Science Fund of the Ministry of Education and Science, Bulgaria. 7

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by the expression of various cell surface markers [1, 2 ]. MSC have more recently been obtained from adipose tissue, peripheral blood, cord blood, cartilage [3–6 ]. Transplantation of MSC has been used to treat a wide range of diseases, and the mechanism of action is postulated to be mediated by either differentiation into functional reparative cells that replace injured tissues or secretion of paracrine factors that promote tissue repair [7 ]. There are large number of MSC secretory products identified that have the potential to act as paracrine modulators of tissue repair and replacement in diseases of the cardiovascular, haematopoietic and skeletal tissues. Moreover, MSC-conditioned medium has the potency to treat a variety of diseases in humans without cell transplantation [8, 9 ]. Instead of using cells, repair of injured tissues will be mediated by enhancing endogenous tissue repair using biological factors secreted by MSCs. In our previous work we have shown that BM-MSC conditioned medium supports the growth of endothelial progenitor cells (EPCs) derived from umbilical cord blood mononuclear cells (UCBMNC) [10 ]. Here we compare the effects of conditioned media from bone marrow derived mesenchymal stem cells and adipose tissue derived mesenchymal stem cells on EPCs apoptosis. Materials and methods. Isolation of human umbilical cord blood mononuclear cells. Human umbilical cord blood (20–55 ml/sample) was obtained from 10 patients shortly after the end of full-term deliveries with the signed informed consent of the pregnant mothers. UCBMC were collected by centrifugation at 1200 rpm for 10 min, pellets were resuspended in DMEM (Cambrex Bio Science Verviers; Belgium) and overlaid onto Ficoll-PaqueTM Plus (Amersham Biosciences, Sweden), then the cells were centrifuged at 1750 rpm 4 ◦ C for 30 min. UCBMCs in the buffy coat were collected and washed twice with PBS. Pellets were resuspended in DMEM and cells were counted, diluted and plated. Endothelial progenitor cells culture. Cells were plated at density 1 × 108 cells per well in 6-well plates (Orange Scientific; Belgium) and maintained in DMEM (Cambrex Bio Science Verviers; Belgium) supplemented with 10% FCS (PAA; Austria) and 10% conditioned medium (CM) obtained from bone marrowderived (BM-MSC) or from adipose tissue derived (AT-MSC) mesenchymal stem cells in parallel. MSC were isolated and characterized in our laboratory [11 ]. The culture was maintained at 37 ◦ C and 5% CO2 in humidified atmosphere (Heraeus, Germany). The medium was changed on the 5th day after plating and subsequently on every 5th day of culture. Reverse transcription polymerase chain reaction. Total RNA from EPCs cultured for 20 days in CM from bone-marrow derived MSC (CM-BMMSC) or CM from adipose-derived MSC (CM-AT-MSC) was extracted using innuPREP Blood RNA Kit (aj ROBOSCREEN) according to manufacturer’s instructions. Total RNA was reverse transcribed using random hexamer primers and MultiScribe Reverse Transcriptase (GeneAmp RNA PCR Reagent Kit; Applied Biosystems). RT-PCR was done following a two-step protocol: composed 98

S. Kestendjieva, Z. Popova, G. Cvetkova et al.

hybridization at 25 ◦ C for 10 min and reverse transcription at 42 ◦ C for 12 min. PCR amplification of survivin, c-myc, hTERT, bcl-2 and β-actin was performed for 35 cycles as follows: denaturation at 95 ◦ C for 1 min, primer annealing at 60 ◦ C/61 ◦ C/65 ◦ C for 1 min and elongation at 72 ◦ C for 2 min. The reaction products were loaded on 2% agarose gels with 1 µg/ml EtBr, and run for 2 h at 15 V/cm. The gels were visualized at CAMAG, Reprostar 3 transilluminator. The following primers were used: Primer

Sequence

survivin Sense 5‘-ACAGCATCGAGCCAAGTCAT-3′ antisense 5‘-GAGCTGCAGGTTCCTTATC-3′

Amplicon bp 431 bp

Elong. T 60 ◦ C

Bcl-2

Sense 5′ -GTGGAGGAGCTCTTCAGGGA-3′ antisense 5′ -AGGCACCCAGGGTGATGCAA-3‘

304 bp

61 ◦ C

c-myc

Sense 5′ TACCCTCTCAACGACAGCAGCTCGCCCAACTCCT-3′ Antisense 5′ TCTTGACATTCTCCTCGGTGTCCGAGGACCT-3′

479 bp

60 ◦ C

264 bp

65 ◦ C

600 bp

60 ◦ C

hTERT Sense 5′ -GTGTGCTGCAGCTCCCATTTC-3′ antisense 5′ -GCTGCGTCTGGGCTGTCC-3′ β-actin

Sense 5′ TGACGGGGTCACCCACACTGTGCCCATCTA-3′ Antisense 5′ CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′

Results. As we have described previously [10 ], UCBMC cultured for one month in the presence of 10% conditioned media of BM-MSC showed specific changes in their growth characteristics. Colonies and cord-like structures were observed. Further, it was shown that the cells could bind lectin and were stained positive for vWF (von Willebrand factor) and KDR (Flk 1) but negative for endoglin (CD 105). This clearly shows that BM-MSC conditioned medium supports the growth of endothelial progenitor cells derived from UCBMNC. This work continued with comparing the effects of conditioned medium from MSC derived from bone marrow and from adipose tissue. It was unexpectedly that we observed the opposite effect of CM-BM-MSC versus CM-AT-MSC on expression of apoptosis related proteins. We used RT-PCR to investigate the expression of survivin, hTERT, c-myc and Bcl-2 in endothelial progenitor cells cultured in bone marrow derived mesenchymal stem cells conditioned media (EPCs/CM-BM-MSC) or in adipose tissue derived MSC (EPCs/CM-AT-MSC) or as control umbilical cord blood mononuclear cells (UCBMNC), cultured for one night after isolation. Expression of different forms of survivin depending on conditioned media used. There are five different survivin variants, wild-type (WT) survivin, survivin-2B, survivin-∆E × 3, survivin-2α, and survivin-3B [12 ]. Interestingly we observed the wild type survivin in EPCs/CM-BM-MSC (amplicon length of Compt. rend. Acad. bulg. Sci., 63, No 1, 2010

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431bp), which has anti-apoptotic properties while the survivin-2B observed in EPCs/CM-AT-MSC (500bp) has pro-apoptotic properties (Fig. 1). None of the survivin forms was observed in control UCBMNC cells. Survivin expression proves that the tested cells are not terminally differentiated endothelial progenitors since

Fig. 1. Survivin mRNA expression of EPCs cultured in conditioned media from MSC from bone marrow or from adipose tissue. Decidua cells were used as positive control for survivin expression. In EPCs cultured in the presence of CM-BM-MSC we found antiapoptotic wild type survivin, while in EPCs cultured in CM-AT-MSC there is 2B proapoptotic survivin. UCBMNC do not express survivin

Fig. 2. c-myc mRNA was expressed only when CM-BM-MSC was used for culturing of EPCs. EPCs/CM-AT-MSC do not express c-myc. UCBMNCs were strongly positive

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Fig. 3. EPCs/CM-BM-MSC expressed hTERT mRNA. hTERT mRNA was not found in EPCs/CM-AT-MSC, suggesting their apoptotic state. UCBMNCs were slightly positive which can be due of very low number of positive cells shortly after isolation

Fig. 4. Bcl-2 mRNA was the only one of the markers studied here found to be expressed in both EPCs/CM-BM-MSC and EPCs/CM-AT-MSC. Bone marrow MSC conditioned media cultured EPCs showed higher expression

survivin expression is typical for G2 /M phase. Mature endothelial cells are in G0 phase and are survivin negative. Expression of c-myc. c-myc was expressed in EPCs/CM-BM-MSC and was not detected in EPCs/CM-AT-MSC (Fig. 2). Lack of c-myc expression indirectly suggests that EPCs cultured in presence of adipose MSC conditioned media are apoptotic, since transcription factor c-myc has effects on genes related with cell growth and proliferation [13 ]. hTERT expression. hTERT, or “human Telomerase Reverse Transcriptase”, is a ribonucleoprotein that maintains telomere ends by addition of the telomere repeated sequence TTAGGG, which is activated in germline cells. UCBMNCs expressed high levels of hTERT as it was described in literature [14 ]. EPCs/ CM-BM-MSC were also positive for hTERT mRNA while EPCs/CM-AT-MSC were negative (Fig. 3). Compt. rend. Acad. bulg. Sci., 63, No 1, 2010

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Bcl-2. Both EPCs/CM-BM-MSC and EPCs/CM-AT-MSC expressed Bcl-2, the factor that assures cell’s survival by inhibiting apoptosis [15 ] (Fig. 4). We can speculate that Bcl-2 expression is stronger in cells cultured in the presence of bone marrow MSC conditioned media. We have to point out that the reported effects were observed in the cells isolated from cord blood and cultured in parallel both with conditioned media for 20 days and pro/anti apoptotic effects can be only due to the used conditioned media. We suppose that different factors in conditioned media are responsible for the observed induction of apoptosis or the survival of endothelial progenitor cells. We envisage elucidation of these key factors in our future investigations. Discussion. Some authors expect that significant similarity between hESCderived MSCs and adult tissue derived MSCs exist [16 ]. This means that conditioned medium of either MSC culture is likely to have similar biological activities. But in recent papers there are data about reproducible differences in the chemokine secretion profiles of various MSC preparations [17 ]. In vitro production of cytokines and growth factors was analysed and compared between MSC from bone marrow, from adipose tissue and normal human dermal fibroblasts (NHDFs). AT-MSC were shown to secrete IL-1Rα, IL-6, IL-8, G-CSF, GM-CSF, MCP-1, NGF and HGF in a volume higher than both BM-MSCs and NHDFs [18 ]. CM-MSC is known to have renoprotective action. Importantly, kidney cells in the vicinity of administered MSC were protected from apoptosis [19 ], which is further indication for the paracrine nature of the observed cytoprotection. These effects include secretion of growth factors, VEGF, HGF and IGF-I, i.e., renotropic factors that are known both to decrease apoptosis of endothelial and tubular cells and to stimulate proliferation of surviving cells. Furthermore, human adipose stromal cells, cell type that is very similar to MSC, secrete angiogenic and antiapoptotic factors that elicit cardiovascular protection [20 ]. While the predominant belief was that improvement in outcome following organ injury and stem cell administration was the result of replacing damaged cells by differentiated stem cells, some groups have shown that paracrine mechanisms are primarily responsible for the organprotective actions of administered stem cells [21, 22 ]. Gnecchi et al. [23 ] as well showed that protection from apoptosis by paracrine mechanisms contributes to the cardiac protection elicited by Akttransduced MSC. Together, these studies support the hypothesis that paracrine actions are of major importance in mediating the protective and regenerative effects of administered MSC after organ injury. Our results suggest opposite effect of conditioned media from BM-MSC and AT-MSC and this strongly indicates that the secretion profiles of MSC from different origin have different biological effects, on the contrary to some existing statements in the literature [16 ]. But our work is in agreement with findings that MSC secrete anti-apoptotic factors. Finding that bone marrow and adipose 102


tissue MSC have reversed properties on apoptosis of endothelial progenitor cells is intriguing and exact factors and mechanisms of these effects have to be studied.

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