Novel weapon to conquer human glioblastoma: G-quadruplexes and neuro-inducers

Cancer cell reprogramming based on aptamers with antiproliferative properties in combination with small molecules that are used for conversion iPSCs into neurons represents a new approach to reduce the probability of glioblastoma recurrence and tumor resistance to therapy. In this research we tested several combinations of factors on whole cell cultures, derived from tumor tissue after surgical resection, and on cell cultures divided in CD133 enriched and depleted populations, as CD133 marker is believed to be characteristic for glioblastoma stem cells. We showed that CD133 + and CD133 - cells have a different response to tested combinations of factors and CD133-positive cells are more stable and possess stemness properties. Thus, affecting these cells will lead to decrease of therapy resistance. Moreover, we found a combination of factors that is able to inhibit proliferation of both CD133 + and CD133 - cells. Our results reveal a promising strategy to improve treatment of patients with glioblastoma.


Introduction
Glioblastoma is one of the most severe forms of tumor genesis in a human body. Unfortunately, to date there are no effective ways of treatment for this malignant brain tumor. All medical treatments known and available today (chemotherapy, radiotherapy, surgical operations) provide only slight prolongation of a patient's lifespan. Perhaps, the approach to glioblastoma therapy should be reconsidered and changed. A commonlyknown fact that glioma has a heterogenic structure with an aberrant regulation of cell proliferation and differentiation should be primarily considered 1 . Such a feature of glioblastoma cell population makes it insensitive to various therapies, and the unaffected tumor recurs. Some cells of the tumor die under the influence of therapeutical factors, however, other cells with a different set of mutations appear to be resistant to these effectors, and their sustained proliferation promotes further tumor growth. Thus, the tumor is capable to actively increase in size and to withstand various types of therapies 2 . It is important to be specified, that the main goal of all approaches to glioma treatment is to make tumor cells die or to induce a toxic effect. Yet, this approach has not helped to cope with the problem. We suggest that other possible variants of glioma treatment should be considered. For example, an attempt to stimulate tumor cells' "maturation" may therefore promote the loss of their proliferative potential. Such an approach is beginning to attract scientists' attention in terms of different cancers therapy, for instance, a similar experience was described for rhabdomyosarcoma, rhabdoid tumors 3 and leukemia 4 . In the current work we attempt to generate a cocktail of a factor, which suppresses tumor proliferation, and neuro-inducers, which, we hope, can stimulate the maturation of tumor cells. While estimating the impact of non-toxic factors, it is extremely important to understand how exactly the proliferation state of tumor cells changes. It is generally accepted, that, according to one of the theories of glioma origin, the tumor derives and eternally extends from tumor stem cells. Several studies demonstrated that extracted and isolated tumor stem cells are capable of a new tumor formation and microenvironment modulation by themselves 5,6 . Hence, the understanding of the properties and characteristics of these cells is important for working out the effective means to influence and affect them. The researchers consistent with this approach concentrate on the biomarkers of cancer stem cells. It is worth to note, that despite the fact that all cancer stem cells are characterized by various biomarkers 7 , the proper identification of these cells remains an unsolved problem. Consequently, it becomes obvious that the investigations of this type of cells are of great importance for target cancer therapy development 8,9 . One of the markers arousing interest is CD133, which is found in glioblastoma (glioma Grade IV) cancer stem cells 10 . This protein has two names, CD133 and prominin-1 (Prom1): it is called CD133 when localized on the cell membrane surface, and Prom1 is used for its intracellular form. The role of CD133 protein in stem cells as well as its correlation with the properties of stem cells are unclear so far, which is striking, as the protein has been studied for quite a long time. It should be noticed, that CD133 is also a surface biomarker of normal brain stem cells 6 . Interestingly, the surface CD133 protein of neural stem cells is different from that of brain cancer stem cells in its structure. Kemper and coauthors demonstrated that the presence of non-glycosylated epitope of CD133, which is located in the second loop of the protein they named AC133, is characteristic of cancer stem cells only 11 . The same authors showed that the presence of the AC133 epitope on the cell surface correlates with clonogenicity 11 . It was also found that CD133 + stem cells, which possess an increased mutability and a predisposition to a higher mutation rate, are the main risk factor for tumor development not only in the brain, but also in many other organs 12 . Several researchers demonstrated that the decrease of CD133 + cells content in a cell culture led to a decrease in this culture's ability of selfrenewal and tumor potential 13 . This suggests the necessity of careful investigation of CD133 + glioblastoma cells. In addition, some studies evidence that, while CD133 is a biomarker of glioblastoma cancer stem cells, it functions as a protein associated with the maintenance of stem cells phenotype. For example, it was demonstrated that the tyrosine residue located in the C-terminus of CD133 interacts with the regulatory subunit of phosphoinositide 3-kinases (PI3K) p85 and activates PI3K-AKT signaling pathway in glioblastoma cells 14 . However, the data about CD133 significance are contradictory. For instance, several researchers disprove the correlation between CD133 expression and patients' survival 15 . In another study it was shown that CD133 expressed on the glioma cell surface was poorly immunoreactive for anti-AC133 antibodies, that are used to detect an epitope on CD133 16 . At the same time, these authors demonstrated that the concentration of CD133 in the membranes of glioma cells varies during the cell cycle. Based on this fact, they insist that the interpretation of AC133 epitope presence or absence in glioma cells should be done with caution 16 . Such contradictory results require careful investigation of CD133 + cells and the analysis of their significance for glioblastoma tumor genesis. In the current study we made and attempt to answer two questions: (i) whether the approach to the therapy of glioma can be changed via the application of proliferation-decreasing factors and neuro-inducers; and if it is possible, (ii) how significant the role of CD133 + cells is in providing cell population stability under a similar influence and their ability to preserve the "immature" state, which is based on their proliferation activity.

The search for glioblastoma cell culture characterized by CD133 high expression rate
Out of the 20 cell cultures obtained from surgical samples of tumor tissues, for the further investigation we selected a cell culture designated G01, which demonstrated the maximum expression level of CD133. G01 cell culture was derived from glioblastoma cancer tissue (Grade IV) from 37-old female patient with the tumor localized in the left frontal lobe. Figure 1a represents immunohistochemical staining of tumor transections for CD133, which demonstrates the enrichment of the tumor tissue in G01 CD133 + cells.
The cell culture G01 derived from glioblastoma tissue also had an elevated CD133 expression level. Figure 1b represents the comparative analysis of expression of genes known to correlate with the properties of neural stem cells. G01 cell culture possesses several characteristic features attractive for investigation, as this cell culture demonstrated high expression of DR4, GFAP, L1CAM in addition to the stable high CD133 expression.
Despite the fact that GFAP is known to be a biomarker of glial cells, it is also found in neural stem cells 17 . Therefore, the increased expression of both GFAP and CD133, as well as L1CAM, that is known to be characteristic for glioma cells 18 , should be considered as a feature of stem cells. An interesting fact to be discussed is the relatively low expression of Nestin, which is believed to be the main biomarker of neural stem cells.
For the analysis of the ability of G01 cell culture to form neuro-spheres, it was cultivated in a serum-free medium. G01 was shown to actively develop into neuro-spheres ( Figure  1c). It is possible that self-organizing of cells into a neuro-sphere is an attempt of a heterogenous population to make up a tumor with a structure similar to a natural tumor structure in vivo. a Immunohistochemical staining with anti-CD133 antibodies (left); bisbenzimide staining of cell nuclei of the patient's biopsy samples (middle); a and b merge (right); scale bar is 50 µm. b Real-time quantitative PCR. The expression of neural stem cells' genes in tested cell cultures. Data are represented as mean ± SEM. n=3 for each group. c Immunohistochemical staining of the neuro-spheres with anti-CD133 antibodies. CD133+ cells (arrow) are located in the outer layer of the neuro-spheres; scale bar is 20 µm.
This result demonstrates that CD133 + cells are located in the outer layer of the neurospheres, which is quite similar to the distribution of cancer stem cells in a developed brain tumor 19,20 The estimation of CD133 distribution in the cell membranes of G01 glioblastoma cells using СD133fr/peGFP-c1 recombinant DNA The estimation of the presence of cells with combined green fluorescence (GFP) and positive staining for anti-CD133 antibodies resulted in the observation of cells, which were intensively stained with both antibodies (Figure 2a). Interestingly, in these cells a combined fluorescence was observed both in cell cytoplasm and on the cell membrane. A colocalization of green (GFP) and red (anti-CD133 antibodies) fluorescence was proven by using a diagram, which demonstrated the distribution of the signals along the axis (Figure 2b). The graph (left) in the Figure 2b demonstrates the distribution of both proteins along the axis (right). As it can be observed from the Figure 2b, both CD133fr and GFP (red and green) are located together, i.e. glioblastoma cell culture transfection with the CD133fr/peGFP-c1 recombinant DNA succeeded. The same result was shown comparing signals from both proteins ( Figure 2c).
Because CD133 is a transmembrane isoform of Prom1, it was necessary to estimate the exact membrane colocalization of CD133fr and GFP (red and green). Figure 2d demonstrates that the CD133fr/peGFP-c1 recombinant DNA provides the visualization of colocalization of these proteins on the cell membrane. b Diagram of signal location along the axis (red) (right) through the areas of CD133fr localization (red) and CD133fr and GFP (red and green) (left). c Diagram of colocalization of CD133fr and GFP in fixed glioblastoma cells. To the left, signals from GFP and CD133fr are on the axes of both proteins (area 3). To the right, the area outlined in red is the co-location of CD133fr and GFP. d Micrographs of membrane localization of CD133fr/peGFP-c1 recombinant DNA, the arrows indicate the colocalization of GFP (green), CD133fr (red) and CD133/GFP (yellow) e Micrographs of the distribution of microbeads in glioblastoma cell culture transfected with реCD133fr/peGFP-c1. The arrows indicate magnetic beads.
We also examined the localization of microbeads that we used for CD133 + cells isolation and demonstrated that magnetic beads are indeed specific to CD133 localized in the cell membrane( Figure 2e) that is concordant with the data of P. Müller et al. 21 .
Therefore, we conclude that the anti-CD133 antibodies effectively bind to this protein located in the cell membranes. We also state that a significant amount of protein, which, nevertheless, does not tend to localize in the cell membrane, is situated in the cell cytoplasm.

G01 CD133-and G01 CD133+ cell cultures' analysis
Using magnetic beads with the immobilized anti-CD133 antibodies (Miltenyi Biotec, Germany) we obtained two cell fractions from G01 glioblastoma cell culture, that we conventionally named G01 CD133 + and G01 CD133 -. We underscore that the names are given by conventionality, as there is no unambiguous data so far whether this CD133 can be considered as a true biomarker of stem cells or cancer stem cells. If this is the case indeed, then the percentage of these cells will be constantly decreasing in a cell culture as a result of an asymmetric cell division, and such a cell culture should be considered as «enriched in G01 CD133 + cells». During our investigation we also found that it is impossible to completely deplete the cell culture of G01 CD133 + cells, as there were always some G01 CD133 + cells left in it, hence, the culture resulting from the depletion should be considered as «G01 CD133 + depleted». To simplify the terms, we introduce a standard designation of the cultures as G01 СD133 + and G01 СD133correspondingly. G01 СD133 + and G01 СD133cell cultures demonstrated different proliferation activity; at the beginning of the experiment G01 СD133cells proliferation rate was higher than that of G01 СD133 + both in 10 days and in 20 days following the separation (Figure 3a). Low proliferation activity of G01 СD133 + was observed as expected, as, firstly, the initial number of cells was quite small, and, secondly, if these cells possess the properties of stem cells indeed, the latter are known to lack high proliferation rate characteristic to immature progenitor cells.
As mentioned above, G01 CD133cell culture, on the contrary, possessed high proliferation activity. However, it is important to note that both cell cultures equaled in their proliferation rates in 30 days after separation, and by the 70 th day G01 CD133cell culture stopped proliferating and eventually died, while G01 CD133 + cells continued their growth and were passaged without a decrease in their proliferation activity. Therefore, we suggest that CD133cell culture has limited proliferation potential compared to CD133 + cell culture, which preserves stable proliferation rate 30 days after increasing the cell mass in culture. This experiment was repeated three times.
Using RT-qPCR analysis we investigated the changes in the expression of genes associated with the properties of stem cells in glioblastoma G01 CD133and G01 CD133 + cell cultures. Gene expression was estimated in 7, 21, 35 and 42 days after cell separation. The initial cell culture G01 (before separation to G01 CD133and G01 CD133 + ) was taken as a reference sample. DR4 and DR5 receptors of TRAIL-dependent apoptotic pathway were analyzed, however, the papers to date discussing whether this pathway is characteristic for stem cells are contradictory. At the same time, there are studies evidencing that DR5 also induces anti-apoptotic pathway and is characteristic for a number of tumor cells, possibly, stem cells, thus targeting this pathway may provide a new approach for cancer therapy 22 . Another gene in our panel was GFAP, which is a generally accepted biomarker of stem cells and a marker of mature glial cells at the same time. Interestingly, this gene is actively expressed in neural stem cells, both cancer and normal, while in daughter progenitor cells its expression disappears completely. Further expression of GFAP reappears in mature glial cells 17 . Hence, this marker should be estimated in a combination with others. Other genes in our panel, known to be associated with stem cells and their properties, included Nanog, Oct4, Sox2 17,23 . In addition, we included Notch2, as signal transduction via Notch-receptor family proteins is frequently reported in gliomas, and the increased signaling via Notch correlates with poorer prognosis. Several studies point to an important role of Notch2 in carcinogenesis and the development of the most malignant gliomas 24 . Recently, there appeared a great number of studies showing the significance of Notch2 as a marker of neural stem cells 25 . Further, we included L1CAM in the gene expression analysis, as it is also typical for neural stem cells and glioma stem cells 26 . Finally, we chose, as it may be expected, Nestin, which is one of the main biomarkers of neural stem cells 27 .
During the analysis of the expression of the panel of genes described above in CD133cell culture, it was shown that the expression of stem cell markers was either decreased (CD133, GFAP, Nanog, Oct4, Sox2, L1CAM, Notch2, Nestin) or did not change significantly (DR4 and DR5) ( Figure 3b).
We suggested that it would be interesting to estimate the expression of the same biomarkers in CD133 + cell culture and to compare it to CD133data. We noticed that the separation of the cell culture into CD133 + and CD133populations did not significantly affect the expression of DR4 and DR5. We also did not see a significant difference in the expression of GFAP, Nanog, Oct4, and Sox2. Notch2 demonstrated a slightly different expression profile in CD133 + cells: its expression started increasing by the 42 day of culture, which was not observed in CD133cells. CD133 + and CD133cell cultures differed significantly in the expression of L1CAM and Nestin ( Figure 3b). After separation of the cultures, L1CAM expression decreased severely, but by the 42 day of culture it was restored, moreover, it became significantly more intense in G01 CD133 + cell culture. The analysis of the expression of Nestin, one of the main markers of neural stem cells, also demonstrated intriguing results: it increased after separation of the cultures and was gradually rising as the culture proceeded Thus, in CD133 + cell culture we observe an obvious and expected increase in the expression of Nestin, the stem cell biomarker, and L1CAM, which also can be considered as a marker of glioblastoma stem cells. The difference between the initial G01 cell culture and G01 CD133 + is evidenced by Nestin expression, while the change of other markers of stem cells was insignificant. It is possible that there exist at least two populations of stem cells characterized by different patterns of the expression of the considered biomarkers.
Immunohistochemical analysis of the corresponding proteins in glioblastoma cell cultures G01 CD133and G01 CD133 + demonstrated that the cultures did not differ in the expression of Nestin and Oct4. At the same time, the content of Sox2 protein was higher in CD133 + cells (Figure 3c). We also observed a significant intensifying of the staining for Notch2 protein, as well as the increasing of Notch2 mRNA expression, in CD133 + cell culture compared to CD133cells.
Moreover, immunohistochemical analysis of Notch2 protein in G01 CD133 + and G01 CD133cell cultures showed the prevalence of this protein in G01 CD133 + cells starting from the 7 th day after separation, although there was no significant difference for Map2 content in the cultures from the 20 th day after separation (Figure 3d). During the analysis of CD133 in the cell cultures under investigation we noticed that the expression of CD133 was enough for detection, still low in G01 CD133cell culture. This fact suggested the need to determine Prom1 distribution inside the cell and its potential abundance in the cell membrane, as it is observed for CD133.

G01 CD133and G01 CD133 + exposure to an aptamer and a single neuro-inducing factor
With the analysis of molecular properties of the glioblastoma G01 CD133and G01 CD133 + cell cultures derived from G01 cell culture completed, from the mixture of previously tested DNA aptamers we selected two DNA aptamers demonstrated the most effective suppressing of glioblastoma cell cultures proliferation. It was shown that these aptamers decreased cell proliferation rates in a dose-dependent manner ( Figure S1a). One of them is designated biHD1 and reported by a group of scientists from MSU, and another one, currently being developed, is called bi-(AID-1-T). Both DNA aptamers were kindly given to us by the group of professor A. Kopylov. The aptamers arouse interest as they possess several advantages, namely, they are able to reduce proliferation of cancer cells, they are non-toxic and transiently acting, and they have a cytostatic effect.
10 days after the exposures to the aptamers in combination with a single neuro-inducer no significant effect on G01 CD133 + cell culture proliferation activity was observed. The proliferation rates of cells treated with biHD1, biHD1+SB, biHD1+BDNF, biHD1+PRM, bi-(AID-1-T), bi-(AID-1-T)+SB, bi-(AID-1-T)+BDNF, bi-(AID-1-T)+PRM were similar to that of the control (non-treated) cell culture. At the same time, glioblastoma G01 CD133cell culture demonstrated higher sensitivity in response to the exposures (Figure 4a). The aptamer bi-(AID-1-T) used alone and the combination of the bi-(AID-1-T) and BDNF were shown to significantly suppress cells proliferation. Interestingly, the cells did not die under the exposures, but stopped dividing, and the culture growth ceased. The different effects of the exposures on the two cell cultures were as expected, as CD133 + cell culture was believed to contain more stem cells, so that the response to the treatments, we thought, would be insignificant. However, we were inspired by the fact that the certain combinations of factors were able to substantially alter the state of tumor cells, at least, CD133cells.
The effect of the exposures changed dramatically after 20 days of culture. We observed significant changes in the proliferation rates of the G01 CD133 + cell culture treated with biHD1, biHD1+SB, biHD1+BDNF, biHD1+PRM, bi-(AID-1-T), bi-(AID-1-T)+SB, bi-(AID-1-T)+BDNF, bi-(AID-1-T)+PRM, and all of the options reduced the ability of the cells to divide compared to the non-treated cells. This means that CD133 + cells demonstrated sensitivity to the exposures only 20 days after the addition of the factors to the medium, i.e. the effect was deferred in time. As for the analysis of G01 CD133cell culture, several changes in the effects of the exposures were also detected 20 days after the initial treatment ( Figure 4b). It should be noted that by the 20 th day G01 CD133cell culture exposed to the combination of bi-(AID-1-T)+BDNF died completely and was not analyzed. At the same time, G01 CD133cell culture exposed to the aptamer bi-(AID-1-T) alone, which had demonstrated a significant decrease of the growth rates before, restored its ability to divide (apparently, when the aptamer's effect ceased), and thus, it was included in the test on the 20 th day along with the others. Since the significant recovery by the 20 th day for the G01 CD133cells treated with bi-(AID-1-T) alone was demonstrated, the importance of using the aptamer in a combination with a neuro-inducer became obvious. G01 CD133cell culture proliferation rates were significantly reduced in 20 days after the exposures to the biHD1, biHD1+SB, biHD1+BDNF, biHD1+PRM and bi-(AID-1-T)+SB, while bi-(AID-1-T)+PRM did not affect much the cell growth and division.
In summary, the experiment of the treatments of the cell cultures with different combinations of the DNA aptamers and neural differentiation inducing molecules demonstrated that the most interesting combination for further investigations was the bi-(AID-1-T)+BDNF.
The analysis of the expression of stem cells associated genes conducted in 20 days after the exposure to the factors showed that the surviving cells of the G01 CD133 + cell culture were characterized with the increased stem cells properties (increased expression of L1CAM, Nestin, Notch2 genes). In other words, those cells, which possessed the properties of stem cells, were able to survive. Therefore, we should search for the combinations of factors targeting the expression of stem cells biomarkers (Figure 4c). In addition to bi-(AID-1-T)+BDNF, a combination of bi-(AID-1-T)+PRM and biHD1+PRM might be used for this purpose.
During the analysis of G01 CD133cell culture we observed rather low expression rates of the genes associated with stem cells, with the increase in L1CAM and Notch2 expression after the exposure to biHD1+PRM being the only exception (Figure 4c). a MTT assay of G01 CD133 + cells and G01 CD133cells after the exposure to aptamers biHD1 and bi-(AID-1-T) and neural differentiation inducers SB431542, purmorphamine, BDNF in 10 days after the exposure. Data are represented as mean ± SD. n=5 for each group. b MTT assay of G01 CD133 + cells (left) and G01 CD133cells (right) after the exposure to aptamers biHD1 and bi-(AID-1-T) and neural differentiation inducers SB431542, purmorphamine, BDNF in 20 days after the exposure. Data are represented as mean ± SD. n=5 for each group. c Real-time quantitative PCR of stem cell genes in G01 CD133 + (left) and G01 CD133 -(right) cell cultures in 20 days after the exposure to the aptamers biHD1 and bi-(AID-1-T) and neural differentiation inducers SB431542, purmorphamine, BDNF. Data are represented as mean ± SD. n=3 for each group. SB -SB431542, PRM -purmorphamine In review, our results suggest that it is possible to make up a combination of factors able to reduce proliferation activity of glioblastoma tumor cells and even to make them switch from their immature state, which is in charge with the active cell division. To date, the most promising combinations, yet needed further investigations, are bi-(AID-1-T)+BDNF and bi-(AID-1-T)+PRM.

G01 CD133and G01 CD133 + cell cultures exposure to the bi-(AID-1-T) aptamer and a cascade of neural inducers commonly used for iPSCs
The results obtained inspired us to further test the combinations of anti-proliferative and neuro-inducing factors, which are capable of stable negative affecting glioblastoma cells ability to divide. However, we failed to get any worthy results in regard to CD133-positive cells. Obviously, the application of the BDNF neuro-inducer alone was enough to block cell division in the more mature CD133-negative cell culture, while for G01 CD133 + cells it appeared to be inappropriate. That is why next we tested a combination of the bi-(AID-1-T) DNA aptamer and the whole set of molecules used for neural differentiation of induced pluripotent stem cells (iPSCs). Therefore, we first applied bi-(AID-1-T) for primary reducing cell proliferation, and then SB431542, LDN-193189, Purmorphamine and finally BDNF (N cocktail) were added one by one with one-day interval between each of the treatments. This experiment included a wide panel of controls: a standard control of nontreated cells, and intermediate controls with the addition of either the aptamer alone or the aptamer in combination with one the factors from the cocktail. Table 1 represents the scheme of the treatments.
The effectiveness of the treatments was analyzed 10 days after the addition of the final factor (i.e. BDNF) via the MTT assay estimating cells proliferation activity. During the investigation of the effects of the N cocktail on the G01 CD133cell culture we observed a complete stopping of cells proliferation. The same effect was observed in bi-(AID-1-T)+BDNF treated cells, which is in concordance with the results of the previous experiment ( Figure 5a). An inhibiting effect on the cell growth and division was also demonstrated for the combinations of bi-(AID-1-T)+SB, and bi-(AID-1-T)+BDNF, but these treatments only slightly reduced cell proliferation.
When we analyzed the effects of the treatments on G01 CD133 + cell culture, it occurred that only N cocktail blocked cell proliferation (Figure 5a), while the rest of the combinations did not alter the culture's proliferation potential. Thus, we demonstrated that the N cocktail (bi-(AID-1-T)+SB431542, LDN-193189, Purmorphamine, BDNF) can significantly reduce the ability to proliferate of both G01 CD133and G01 CD133 + cell cultures.
Next, we repeated the experiment with the initial G01 cell culture, in which we compared the treatment of cells with the N cocktail and the combination of bi-(AID-1-T)+BDNF Figure 5b). The results of the treatments of the initial G01 cell culture demonstrated that bi-(AID-1-T) in combination with BDNF significantly reduced glioblastoma cell proliferation, however, did not completely suppress the tumor growth, while the N cocktail entirely blocked the ability of the cells to divide.
We also tested N cocktail on whole cell cultures including cell line U251 (Figure 5c) and showed that all cultures response to such treatment by decreasing their proliferation.

Discussion
Modern approaches to the treatment of malignant tumors have different success rates. At the same time, in this class of diseases there are tumors that are still practically not amenable to therapy. Glioma, and in particular, glioblastoma, its most severe form (Grade IV), is among these tumors. The types of therapy used today are limited to surgical removal of the tumor, exposure to radiotherapy, or chemotherapy, which is mainly based on toxic effects on cells. The cytotoxic effect of drugs is aimed at bringing tumor cells to death in one way or another. On the one hand, chemotherapy drugs, such as Temodal, kill proliferating tumor cells, but on the other hand, these medications also harm healthy proliferating cells of the body (cells of the intestinal lining, red bone marrow cells, epithelial cells etc). One of the disadvantages in the application of cytotoxic drugs is that they suppress the growth of actively dividing cells, which are thus more sensitive to such exposures, and which are, in fact, immature daughter cells of a tumor 28 . At the same time, cancer stem cells with relatively low proliferating potential, however, rather stable, appear to be insensitive to chemotherapy. We suggest that the approach to the regulation of tumor cells proliferating potential can be possibly changed to the applying of factors, which change cells differentiation status instead of using of death-inducing drugs. This approach seems promising, in particular, when taking into account the fact that every heterogeneous tumor population harbors cells which form the basis of glioma proliferating potential. In this regard, a tumor can be considered as an aggregate of "daughter" tumor cells possessing a high, still limited ability to proliferate, and cancer stem cells, which give rise to the whole population and maintain its growth. Several studies show that these are the cancer stem cells which have an increased resistance to any therapeutic effects. Therefore, chemotherapy and radiotherapy will primarily impact the tumor "daughter" cells, while cancer stem cells will survive and give rise to a new population 29 , restoring it the way a queen bee restores a swarm. For instance, Dawson and coauthors noticed that rhabdomyosarcoma and rhabdoid tumors harbor cells, which remain unaffected by chemotherapy, and which possess the properties of stem cells. The authors suggested a combined treatment of these cells, that made them sensitive to therapy 3 . Therefore, the idea of toxic influence on glioma cells should be reconsidered, and new approaches of tumor cells differentiation stimulation, thus blocking their proliferation potential, should be developed. Today such approaches are already being examined in regard with different cancers. For example, Xiong and co-authors reviewed tumor models as aggregates of cancer stem cell and cancer progenitor cells, as well as the possibilities of managing their differentiation potential 30 . Han and colleagues reported a similar approach to leukemia treatment, in which the aggression of cancer cells was reduces due to the use of combined therapy aimed at stimulating tumor cells differentiation 4 .
The aim of the current study was to search for the combination of factors able to stimulate neural differentiation of human glioblastoma cells. We were aware that there were cancer stem cells in the heterogeneous population of glioma cells, and that these cells were more immature then other cells of the tumor. It is generally accepted that these cells can be characterized as CD133-positive cells. For example, Mendiburu-Eliçabe et al. demonstrated that rapamycin significantly decreased the rate of cell growth, which correlated with the decrease in the expression of CD133 biomarker of stem cells in two cell cultures derived from GBM patients 31 . Moreover, Sunayama et al. showed that the combination of rapamycin and LY294002, an inhibitor of PI3K, suppressed the properties of stem cells while increasing the expression of βIII-tubulin neural marker, which suggests the differentiation effect of this double inhibition of PI3K/mTOR pathway 32 . However, this biomarker of stem cells and their properties is still ambiguous. Therefore, it was important to estimate to what extent CD133and CD133 + human glioblastoma cells proliferation could be restrained using the neuro-inducers. In this study we investigated G01 cell culture derived from human glioblastoma, which we separated into two cell cultures based on the CD133 content, namely, cell population enriched in CD133 and CD133 depleted cell population. The aim was to select a factor able to decrease or stop cell proliferation for a short period of time in order to immediately stimulate tumor cell differentiation. At the same time, it was important that the factor of choice would not be toxic for cells but only restrain their ability to divide. To reach this goal we took the aptamer bi-(AID-1-T), which was developed by professor A. Kopylov and his colleagues and showed a good anti-proliferation potential. We demonstrated that G01 CD133stopped proliferating 10 days after exposure to bi-(AID-1-T), and that without the following treatment with neuroinducers, the suppressing effect discontinued by the 20 th day and the cell culture growth recovered. As for G01 CD133 + cell culture, the aptamer did not alter the proliferation properties of the cells. At first, we did not consider the application of factors inducing neural differentiation of iPSC after proliferation suppression. However, preliminary experiments with different substances regarded as strong neuro-inducers -GDNF, IL6, RA ( Figure S1b; Figure S1c)did not produce a desired effect 33 . Then there appeared an idea to try molecules applied in iPSC neural differentiation 34 . First, we used SB431542, Purmorphamine and BDNF one at a time after ceasing cell proliferation with the help of the aptamer. It was demonstrated that the application of single BDNF neuro-inducer (after the exposure to bi-(AID-1-T)) was enough to block cell divisions in CD133population, but did not work in CD133 + cells. Obviously, CD133 + are immature compared to CD133 -, and this fact is in accordance with the assumption that CD133 is characteristic to stem cells or, at least, to some type of stem cells. BDNF is used for neural differentiation as a neurogenesis-encouraging factor, and it appeared to be applicable for CD133cells of glioblastoma. Therefore, next, we used a step-by-step method of G01 CD133 + and G01 CD133cell cultures exposure to neuro-inducers according to the protocol described by Wichterle 35 and modified by Lagarkova M. 34 .
It was clear that for an effective influence on G01 CD133 + a short-term blocking of cell proliferation was needed with subsequent exposure to neuro-inducers in order to manage the earliest stages of cell "maturation". Small molecule SB431542 taken for investigation inhibits Lefty/Activin/TGFβ signaling pathways via blocking the phosphorylation of the ALK4, ALK5, and ALK7 receptors 36 . SB431542 mechanism of action is based on the destabilization of the signaling network, which supports cells pluripotency and restricts differentiation.
Purmorfamin is another small molecule affecting midbrain cells ventralization 37 . LDN-193189, an inhibitor of BMP signaling pathway, was also chosen for investigation 38 . Finally, the application of BDNF, a neurotrophic factor known to stimulate neurons maturation, was a culmination of the experiment. It appeared that the use of this exact combination of factors was enough to block G01 CD133+ cell proliferation. It is important to note that 10 days following the exposure the cells stopped dividing and the culture further died. Therefore, we conclude that for G01 CD133 + cells a step-by-step treatment is required starting from the earliest differentiation stages. It is possible that socalled cancer stem cells possess a differentiation level comparable to that of embryonic stem cells or induced stem cells, and that is why a step-by-step influence is necessary.
In summary, we propose an alternative approach to glioma therapy, which will be able to change the tumor state of cells via stimulation of their maturation. An important point of the study is that we found at least one combination of factors able both to alter (stop) the proliferation of glioblastoma cells and to block the division of CD133 + cancer stem cells.

Primary cultures of glioblastoma cells
Cells were gained from human glioblastoma tissues after tumor resection. Cell culture G01 was chosen for this research as it showed high expression of CD133 cell surface marker. This culture was obtained by washing cells in Versen solution (Paneco), incubation with 0,25 % trypsin-EDTA (Ethylenediaminetetraacetic acid) solution (Gibco) at 37 o C for 40 min and disaggregation from tissue followed by centrifugation at 1000 rpm for 5 min. Cells were cultivated in DMEM/F12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) medium (Gibco) containing 1% L-glutamine (Paneco) and 10% FBS (Fetal Bovine Serum) (Thermo Scientific) and 1% HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Sigma-Aldrich) at 37°C with a humidified atmosphere of 5% CO2. Passaging was performed at 80% confluency. For passaging cells were washed in PBS (Phosphate Buffered Saline) (1x) (Gibco) and incubated with 0,25 % trypsin-EDTA solution. After trypsin inactivation by fresh medium cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was removed and fresh medium was added, cells were resuspended and plated into cultural flasks. Cell count was defined using trypan blue 1:1.

Cell separation
A separation was performed following a CD133 MicroBead Kit-Tumor tissue protocol. Before the separation cells were passed through 40 µm filters and centrifuged at 1000 rpm for 5 minutes. On the first step the cells were labeled with CD133 Microbeads (Miltenyi Biotec). Cells were centrifuged at 1000 rpm for 10 minutes then the supernatant was removed. The cell pellet was resuspended in 60 μl of buffer (PBS with 2 мМ EDTA and 0,5% BSA (Bovine Serum Albumin) (Amresco)). 20 μl of FcR Blocking Reagent was added to the suspension. After resuspending 20 μl of CD133 MicroBeads was added and resuspended. The mixture was placed at 4 o C for 30 minutes. 2 ml of the same buffer was added to the cell suspension, resuspended and centrifuged at 1000 rpm for 10 minutes. The supernatant was completely removed and 500 μl of fresh buffer was added and resuspended.
For magnetic separation MS Columns (Miltenyi Biotec) were used. After placing a column in the magnetic field of a MACS Separator (Miltenyi Biotec) and washing the column with 1 ml of buffer cell suspension was applied onto the column. The unlabeled cells that passed though the column were gained in the first collecting tube. The column was washed 3 more times in the second collecting tube to get rid of unlabeled cells. The fractions of unlabeled cells were combined in one collection tube. In a different collection tube magnetically labeled cells were eluted 3 times by pipetting 1 ml of buffer and firmly pushing the plunger into the column.
It should be mentioned that as the CD133 marker is believed to represent stem cells that divide asymmetrically there will always be a small amount of CD133cells in CD133 + cultures as a result of such division.

Cell cultivation with aptamers and factors of neurodifferentiation
We selected several factors, which are used to stimulate neural differentiation of iPSCs, as neuro-inducers. On the first stage of the experiment we used small molecules SB41542 and purmorphamine (PRM) (Miltenyi Biotec) and a neurotropic factor BDNF (Miltenyi Biotec). SB431542 (10 µM) (Miltenyi Biotec), known to destabilize signaling pathways, which maintains cells pluripotency. Another molecule used in our experiment is PRM that is commonly used as an analogue of recombinant Sonic hedgehog protein (SHH). The latter is known to play an important role in the nervous system development. SHH in the concentration of 200 ng/ml is capable to induce the development of lateral ganglionic eminence (LGE), and recent studies demonstrate that PRM concentrations of 0,65 µM have the same effect. Therefore, the two molecules (SB431542 and PRM), when applied to neural differentiation of iPSCs, influence different stages of neural "maturation" of progenitor cells. The third factor taken for investigation was the neurotropic factor BDNF (10 ng/ml), which is known as a neural inducer of the final stage of neural cells "maturation".
On the second stage of the experiment LDN-193189 (Miltenyi Biotec), a selective inhibitor of BMP (Bone Morphogenetic Proteins) signaling, that is used for neural induction together with SB431542, was added to the medium. It is shown that BMP4 expression is elevated during oncogenesis that leads to signal transduction by TGF-β/SMAD and canonical Wnt/β-catenin signal pathways resulting in proliferation and differentiation of glioma stem cells 39 . So, its blockage is essential for changing the fate of tumor stem cells.
Two aptamers named biHD1 and biGT3 were used in the experiments. They were added to the medium to a final concentration of 37,5 µM. Before adding to the cell cultures aptamers were preformed at 95 о С and then cooled at 4 о С overnight. On the first step G01-derived CD133and CD133 + cells were treated with DNA aptamers, in two days one of the three neuro-inducers was added to the medium. When combinations of aptamers and small molecules were tested on G01 CD133and G01 CD133 + cells neuronal inducers were consistently added to the growth medium in several combinations (see Table 1) by following scheme: in 2 days after adding of aptamer SB431542 or Purmorphamine or BDNF or combination of SB431542+LDN-193189 were added to the medium, on the 5 th day after partial changing of growth medium in all flasks Purmorphamine was added to the flasks with SB431542+LDN-193189, on the 7 th day BDNF was also added to the flasks with SB431542+LDN-193189+Purmorphamine. In the control flasks no additional factors were added. Proliferation activity of cells was tested using MTT assay 10 days after all of the exposures. Proliferation activity as well as gene expression (using RT-PCR method) were also analyzed 20 days after all of the exposures.
After testing all mentioned combinations on G01 CD133 + and G01 CD 133cultures the most effective ones were tested on G01 culture according to the same scheme.

Immunocytochemistry
Cells in concentration 1*10 4 cells / well were cultivated in a duplicate in 4-well plates for 24 hours in 500 µl of DMEM/F12 medium with 1% glutamine and 10% FBS. After washing twice in PBS (pH 7,3) cells were fixed in 500 µl of 4% paraformaldehyde solution for 30 min at 4 o C. Then cells were washed twice in PBS (pH 7,3).
After staining G01 CD133 + and G01 CD133cell cultures with anti-Nestin, anti-Sox2, anti-Oct4, anti-Notch1 and anti-Map2 antibodies we also evaluated the intensity of cell fluorescence in the microscope field of view. After evaluating the brightness of the pixels on the micrographs, graphs were plotted expressing the ratio of the fluorescence intensity during cell cultivation to control.
For RNA isolation TRIzolTM Reagent User Guide (Sigma-Aldrich) was used. First DNA strand synthesis was performed using MMLV RT kit (Evrogen). As a control initial G01 culture was used. The expression was estimated on 7, 21, 35 and 42 after the beginning of cell cultivation. The array was performed under the following conditions: preliminary warming for 5 min at 90°C, denaturation for 10 sec at 95°C, primer annealing and elongationfor 30 sec at 60°C. The number of cycles -40. The primers used in the assay are presented in Table S1. The house keeping genes used in the research are GAPDH, HPRT and GUSB.

MTT assay
Proliferative activity of G01 CD133 + and G01 CD133cell cultures was determined by MTT assay. It was also used to detect the changes in proliferative activity of the cells treated with specific aptamers. Glioblastoma cells were seeded in 96-well plates at cell density of 2000 cells per well in 200 µl of culture medium DMEM/F12. Cells were cultivated for 48 hours at 37 o C with a humidified atmosphere of 5% CO2. Before adding the oligonucleotides, they were performed by keeping at 95 o C for 5 minutes in a water bath. Cell medium was removed from the wells and the aptamers were added in prepared concentrations. For each concentration five repeats were done. Cells were incubated for 72 hours at 37 o C with a humidified atmosphere of 5% CO2. After removing the medium cells were washed with PBS and cultivated for 24 hours in 100 µl of culture medium. 20 µl of MTS reagent (Promega) was added to each well and the plate was incubated for 2 hours at 37 o C with a humidified atmosphere of 5% CO2. As a positive control the cells without aptamers were used. Cell medium was used as a blank. Optical density was measured on plate analyzer Tecan Infinite M200/Pro (Tecan) at λ=490 nm.

CD133 insert cloning
For isolation of total RNA from G01 cells RNAzol ® RT (Sigma-Aldrich) was used. First RNA strand synthesis was performed using MMLV RT kit. In order to get the CD133 DNA fragment of interest -CD133frthree consistent PCRs were run followed by 1% agarose gel electrophoresis and DNA extraction using QIAquick® Gel Extraction Kit (Qiagen). As an insert a fragment of CD133 protein from 485 to 865 amino acids was used. Primers were designed using NCBI (National Center for Biotechnological Information) Blast, melting temperature (Tm) and annealing temperature were estimated using NEB Tm Calculator (New England Biolabs melting temperature calculator). The primers used in the assay are presented in Table S2. Annealing sites of the used primers on the target gene Prom1 are shown in Figure S2a.
For PCR1-3 Q5 TM High-Fidelity DNA Polymerase (Thermo Scientific) was used. As a marker for 1% agarose gel electrophoresis in TAE (Trisacetate-EDTA) buffer GeneRuler 100 bp Plus DNA Ladder (Thermo Scientific) was taken. DNA concentration and purity were measured using NanoDrop 2000.
The first PCR (PCR1) was run using cd133 f2-cd133 r1 and cd133 f3-Prom1 r0 primer pairs. Amplification of the nucleotide sequence correspond to the second extracellular loop of CD133 (PCR2) was run using cd133 f2-Prom1 r0 primer pair. For ligation with peGFP-c1 vector PCR3 was run using XhoI f-BamH1 r primer pair. The PCR amplification in all the reactions was carried out in the following conditions: preliminary warming at 98°C for 30 sec, denaturation for 10 sec at 98°C, primer annealing for 15 sec at 58°C in PCR1-2 and at 51-58°C in PCR3, elongation at 72°C for 25 sec in PCR1-2 and for 40 sec in PCR2-3, followed by final elongation at 72°C for 2 min in PCR1-2 and for 5 min in PCR2-3. The number of cycles: in PCR1 and PCR3 -30, in PCR2 -27.
Restriction of insert (CD133fr) and peGFP-c1 vector was done by XhoI and BamH1 sites of restriction. Ligation of CD133fr and peGFP-c1 vector ( Figure S2b) was performed using 50 µg of each with T4 DNA High Concentration ligase (New England BioLabs), mixture was incubated overnight at 16 о С.
Transformation of E. coli TOP10 cells (Invitrogen) with CD133fr/peGFP-c1 was done using heat shock. Screening of overnight grown colonies after transformation with CD133fr/peGFP-c1 was performed using cd133 f3-cd133 r0 primer pairs with Taqpolymerase (5 units/µl) ( Thermo Scientific). The PCR was carried out under the following conditions: preliminary warming for 30 sec at 98°C, denaturation for 10 sec at 98°C, primer annealing at 58°C for 15 sec, elongation at 72°C for 15 sec and final elongation at 72°C for 2 min. The number of cycles -27. For positive control probe with CD133fr/pGEM-T easy recombinant DNA was used (intermediate results).
After PCR 1% agarose gel electrophoresis in TAE buffer with GeneRuler 100 bp Plus DNA Ladder was run. For following experiments probe that corresponded to 552 bp was used. This probe was incubated on a shaker overnight at 37 о С in LB (Lysogeny Broth) medium.
Bacterial DNA was isolated using GeneJET Plasmid Miniprep (Thermo Scientific). Sanger sequencing was done to verify the nucleotide sequence of DNA that corresponded to the second extracellular loop of CD133.
Glioblastoma cell transfection was performed using TurboFect Transfection Reagent (Thermo Scientific). For confocal microscopy human glioblastoma cells were seeded in 4-well chamber slide and after transfection they were incubated at 37 о С with a humidified atmosphere of 5% CO2 for 48 hours.

Quantification and statistical analysis
Imaging of fixed samples was performed on an inverted confocal microscope (Olympus IX81) with a computer-controlled motorized stage and an Olympus DP72 digital camera. For confocal microscopy of live and fixed cells laser scanning microscope Zeiss LSM 900 was used. Images analysis was performed using ZEN software and Microsoft Excel. For evaluation of the intensity of cell fluorescence after staining with anti-Nestin, anti-Sox2, anti-Oct4, anti-Notch1, anti-Map2 antibodies in certain periods of time cells were selected (Table S3) and their intensity of fluorescence was established in percent of control. Images analysis was performed using ZEN software and Microsoft Excel.
MTT assay was performed using Tecan Infinite M200/Pro plate analyzer at λ=490 nm. For each sample 5 replicates were accomplished. Analysis was done with i-control 1.10 software.
To measure the levels of target genes' expression CFX96 Real-Time PCR System was used. Each sample was done in triplicate. The analysis was performed using CFX96 Real-Time PCR Detection System. The concentration of samples during DNA recombinant vectors obtaining was measured with Nanodrop 2000 microvolume spectrophotometer for DNA samples. The number of biological replicates (n) is indicated in the corresponding Figure legends. Data are plotted as mean ± standard error of the mean (SEM) or Standard Deviation (SD).