Direct Reprogramming of Glioblastoma Cells into Neurons Using

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Direct Reprogramming of Glioblastoma Cells into Neurons Using Small Molecules Christopher Lee, Meghan Robinson, and Stephanie Michelle Willerth ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00365 • Publication Date (Web): 09 Aug 2018 Downloaded from http://pubs.acs.org on August 10, 2018

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ACS Chemical Neuroscience

Direct Reprogramming of Glioblastoma Cells into Neurons Using Small Molecules Christopher Lee1, Meghan Robinson3, Stephanie M. Willerth2,3,4,5* 1. Department of Biology, University of Victoria, Victoria, B.C. 2. Department of Mechanical Engineering, University of Victoria, Victoria, B.C. 3. Division of Medical Sciences, University of Victoria, Victoria, B.C. 4. Centre for Biomedical Research, University of Victoria, Victoria, B.C. 5. International Collaboration on Repair Discoveries, University of British Columbia, Vancouver, B.C.

Keywords: tissue engineering, cellular reprogramming, differentiation, neuroscience, regenerative medicine

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Abstract Glioblastoma multiforme, a type of deadly brain cancer, originates most commonly from astrocytes found in the brain. Current multimodal treatments for glioblastoma minimally increase life expectancy, but significant advancements in prognosis have not been made in the past few decades. Here we investigate cellular reprogramming for inhibiting the aggressive proliferation of glioblastoma cells. Cellular reprogramming converts one differentiated cell type into another type based on the principles of regenerative medicine. In this study, we used cellular reprogramming to investigate whether small molecule mediated reprogramming could convert glioblastoma cells into neurons. We investigated a novel method for reprogramming U87MG human glioblastoma cells into terminally differentiated neurons using a small molecule cocktail consisting of forskolin, ISX9, CHIR99021 I-BET 151, and DAPT. Treating U87MG glioblastoma cells with this cocktail successfully reprogrammed the malignant cells into early neurons over 13 days. The reprogrammed cells displayed morphological and immunofluorescent characteristics associated with neuronal phenotypes. Genetic analysis revealed that the chemical cocktail upregulates the Ngn2, Ascl1, Brn2 and MAP2 genes, resulting in neuronal reprogramming. Furthermore, these cells displayed decreased viability and lacked the ability to form high numbers of tumor-like spheroids. Overall, this study validates the use of a novel small molecule cocktail for reprogramming glioblastoma into non-proliferating neurons.


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Introduction Glioblastoma multiforme (GBM), a deadly primary central nervous system (CNS) tumor, originates most often from the glial cells of the brain and exhibits invasive proliferation of brain tissue.1 GBM occurs at an incidence rate of 6.95 per 100,000 people aged 40 years or older and has a 5-year survival rate of 5.5%, respectively, making it the most common malignant CNS tumor and deadliest primary brain cancer.2 The current standard of care for GBM consists of a multi-model treatment approach comprised of aggressive surgical resection of the tumor followed by radio- and/or chemotherapy.3 The multi-model treatment method minimally increases life expectancy while inducing unpleasant side effects. Additionally, the disease displays an almost universal recurrence after a median survival time of 32-36 weeks,4,5 highlighting the need for more advanced treatments. Much of the current molecular research focuses on inhibiting GBM cell proliferation.6 Reprogramming GBM cells into terminally differentiated cells could serve as a potential strategy for preventing their invasive proliferation. Current therapeutic agents, including retinoic acid, bone morphogenetic proteins, and histone deacetylase inhibitors7,8 currently target cancer cell differentiation into a glial cell fate for inhibiting proliferation. However, reprogramming GBM cells into their precursor cell type raises concerns, including possible tumorigenic reactivation of the differentiated glial cells.9 Direct reprogramming GBM into a terminal neuronal fate could potentially serve as a more effective treatment than the aforementioned strategies because reprogrammed neurons do not proliferate.10,11 Direct reprogramming converts one distinct cell type into another. Exogenous expression of transcription factors implicated in neuronal development, such as Ascl1, Brn2, Myt1l, and Ngn2, can directly reprogram somatic cells, glial cells and pluripotent stem cells (PSC) to different types of neurons.12,13,14,15,16,17 Furthermore, forced expression of the 3 ACS Paragon Plus Environment


transcription factors Ascl1, Brn2, Myt1l, and Ngn2 directly reprograms malignant glioma cells into mature MAP2 positive neurons with a high conversion rate, and that pre-existing tumors in an in vivo model could be successfully reduced via stereotactical injections of these pro-neural transcription factor containing lentiviruses.11,18 However, viral vectors do not allow for control over protein expression levels and they can potentially induce oncogenesis when integrating into the host genome.19 Small molecule-based reprogramming protocols have recently emerged as novel method of direct reprogramming. In these protocols, cocktails of small molecules activate the pathways associated with neural transcription factors. Such cocktails can directly reprogram somatic and glial cell lines to neurons.20,21,22,23,24,25 Rat glioma cells can be directly reprogrammed to mature neuronal-like cells using the small molecules forskolin and CHIR99021, supporting the hypothesis that small molecule cocktails can directly convert glioma cells to neurons in the absence of exogenous transcription factors.26 In this study, we investigated a novel cocktail of small molecules for directly reprogramming metastatic human GBM cells into neurons. The combination of forskolin, ISX9, CHIR99021 I-BET 151, and DAPT rapidly differentiated GBM cells into neuronal-like cells with a high rate of efficiency without passing through an intermediate pluripotent state. These reprogrammed cells showed high expression of neuronal markers, high gene expression of proneural transcription factors, and were unable to proliferate in extended culture periods. Furthermore, the cocktail can inhibit spheroid formation, confirming that the cocktail functions to supress GBM invasiveness. Our results provide supporting evidence that reprogramming GBM cells into terminally differentiated neurons could serve as a promising therapeutic approach for treating brain tumors.


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Results and Discussion Experimental Plan We developed a cocktail of small molecules for reprogramming GBM cells to neurons with high efficiency based the literature. We chose to use the small molecule mix consisting of forskolin, ISX9, CHIR99021, I-BET 151 and DAPT (FICBD) (Figure S1). Forskolin, a cAMP agonist, can act as a chemical substitute for Oct4, one of the Yamanaka factors that maintains pluripotency in stem cells.27,28 ISX9 induces neuronal differentiation through the myocyteenhancer factor 2 (Mef2), a vital pathway for neural differentiation and maturation.29,30 CHIR99021 serves as a potent inhibitor of glycogen synthesis kinase 3 (GSK3) and induces neural development from PSCs.31 I-BET 151 inhibits the BET family of proteins and promotes neural stem cells to differentiate into a neuronal fate.32 Each of these molecules individually increases the reprogramming efficiency of virally expressed Ascl1, and together as a cocktail can reprogram fibroblasts and astrocytes to a neuronal fate in the absence of ectopic transcription factor expression.22,25 DAPT was added to the cocktail due to its property as a γ-secretase inhibitor, which induces neural reprogramming in GBM stem cells virally expressing Ascl1.33 While each individual chemical has supporting evidence for neuronal reprogramming of GBM or related cell types, it is unlikely that the FICBD cocktail is the ideal reprogramming cocktail. The addition of other small molecules, such as valproic acid, could increase efficiency. However, many of the omitted neuronal reprogramming small molecule require complex cocktail formula changes over the course of reprogramming cell types related to GBM,23 and an aim of this work was to identify a cocktail formula that required no alteration during the reprogramming process.



Overall, FICBD cocktail serves as a starting point for GBM reprogramming investigation. Here we investigated its ability to reprogram U87MG human GBM cells into neurons. The FICBD Cocktails Reprograms U87MG Cells into a Neuronal Morphology Without Passing Through an Intermediate Pluripotent State U87MG cells were grown in defined media in the presence or absence of the FICBD cocktail with the concentrations: 10 µM Forskolin, 10 µM ISX9, 3 µM CHIR99021, 2 µM I-BET 151, and 5 µM DAPT over 13 days. U87MG cells lacking FICBD supplementation appeared unchanged morphologically during the early stages of treatment (Figure 1A-1C). U87MG cells treated with the FICBD cocktail showed rapid change in morphology in as little as 2 days following FICBD supplementation, with cells displaying elongated bipolar neurite-like extensions (Figure 1D). More cells exhibited bipolar extensions by Day 5, while few gained multipolar extensions (Figure 1E). Significant neuronal-like morphology was observed at days 7 and 9, as the cell bodies appeared more compact, neurite extensions appeared further elongated, more cells displayed multipolar branching, and cells displayed higher order branching (Figure 1F, 2A). Furthermore, few cells at day 7 resembled either the control group grown in defined media for 7 days, or the morphology typically associated with U87MG cells. Quantification of morphological confirmed physical signs of reprograming, as FICBD treated cells exhibited increased neurite length by day 2, and increased cell body compactness, increased cell body roundness, and decreased cell body size for day 9 FICBD-treated cells (Table S1). Culture confluency decreased in FICBD-treated cells over the course of treatment while control cell confluency increased, suggesting that the FICBD-cocktail inhibited cell proliferation. FICBDtreated cells exhibited highly complex neuronal morphology, with cells displaying 2nd and 3rd degree branching by day 13 (Figure 2B,2C). The development of compact cell bodies and


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extended neurite-like structures appeared as a rapid and continuous change from initial day 0 supplementation, suggesting that these cells were directly reprogrammed into a neuronal fate and that they did not pass through an intermediate multipotent neural progenitor cell (NPC) state.34,35 Overall, cells treated with the small molecule cocktail displayed significant morphological evidence of reprogramming to a neuronal fate without passing through an intermediate pluripotent state. This was accompanied by a decrease in confluence indicating possibly inhibited proliferation. Reprogrammed U87 Glioblastoma Cells Express Early Neuronal Markers We next confirmed neuronal fate reprogramming by investigating the expression of proteins that are markers for neural cell fate commitment. Immunocytochemistry was performed on day 9 control and FICBD treated cells for the early neuronal markers β-tubulin III (TUJ1) and doublecortin (DCX), the mature neuronal marker microtubule-associated protein 2 (MAP2), and the astrocyte marker glial fibrillary acidic protein (GFAP). It was observed that FICBD-treated cells showed a significant increase in TUJ1 and DCX when compared to control cells (Figure 3A,3B). The number of cells highly expressing TUJ1 and DCX along with a neuronal morphology was quantified. It was observed that 71.4 ± 4.6% of FICBD-treated cells expressed TUJ1 and a neuronal morphology, while 39 ± 4.4% expressed DCX and a neuronal morphology. This was significantly higher than the 3.8 ± 0.4% TUJ1 expression and 2.7 ± 0.3% DCX expression measured in day 9 untreated U87MG cells (Figure 3C). This high level of reprogramming efficiency is comparable to previous studies where U87MG cells were reprogrammed into neurons using viral expression of the transcription factors Ngn2 and Sox11,11 and higher than the previously reported 20-40% observed when glioma cells were forced to express Ascl1, Brn2, and Ngn2.18 Due to the high recurrence rate of GBM, a high conversion rate



is essential for terminal differentiation-based therapeutic strategies.7,8,10,18,36 Small molecule treatment strategies have an advantage over viral expression as it does not require transfection of the cells. Indeed, previously reported TUJ1+ conversion efficiencies of 95% at 14 days post infection11 is based upon the number of cells successfully virally infected. Viral infection of specific cells in vivo in the mammalian brain remains difficult to perform with consistency.37 Additional drawbacks include a lack of control over protein expression levels and the potential for further oncogenesis stemming from viral integration into the host genome.19 Conversely, the small molecules present in the FICBD cocktail are 234-465 Da and do not require successful transfection to exert their reprogramming effect, providing a simpler path to therapeutic application when compared to viral vectors. Interestingly, cells did not show any change in GFAP expression levels. No significant change in GFAP expression between the day 9 controls and chemically treated cells indicates no concomitant astrocyte differentiation caused by the small molecule treatment (Figure 3D). This effect is desirable, as reprogramming GBM cells to a glial fate would leave chemically reprogrammed cells able to proliferate and put them at risk of reversion to cancerous cells.9 While no change in GFAP expression is an encouraging result, it was expected that reprogramming towards a neuronal fate would be coupled with a decrease in GFAP expression, as has been seen during previous astrocyte to neuron reprogramming.38 Day 9 small molecule treated cells also lacked significant expression of MAP2, indicating that cells at this stage had not yet reached a state of maturity despite complex branching shown morphologically (Figure 3E). In terms of reprogramming GBM cells to impede tumorgenicity, it is not required that cells reach functional maturation to impede proliferation and invasiveness.11,26 However, it remains a possibility that GBM cells reprogrammed to early neurons can revert to a metastatic state.


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Development of functional maturation would likely be more indicative of irreversible epigenetic changes within the cell and indicate permanent commitment,9 and could likely be achieved through longer cell culture conditions. Flow cytometry performed on day 12 cells revealed that FICBD-treated cells were 72.2 ± 2.3% TUJ1 positive, compared to the control value of 29.6 ± 0.3%. (Figure 4B). Furthermore, it was found that TUJ1 expression in small molecule treated cells showed an elongated peak of expression, indicating that cells were at different stages of neuronal fate reprogramming (Figure 4A). Necessity of Chemicals in the FICBD Chemical Cocktail Following confirmation of reprogramming to an early neuronal fate by the FICBD cocktail, we sought to optimize the cocktail by withdrawing discrete chemicals to determine if any molecules were redundant, improved, or impaired the reprogramming process. In addition, we also wished to investigate the effects of removing individual chemicals on cell morphology to analyze the effects of each chemical within the cocktail. Cells were grown in the presence of the FICBD cocktail minus individual chemicals, e.g. FICB, -D, over a 7-day period and imaged using ICC for the marker TUJ1. Morphology of TUJ1 expressing cells varied slightly after removal of each chemical, however, removal of DAPT, I-BET 151, CHIR99021, and Forskolin still allowed the remaining cocktail to reprogram U87MG cells to a generally neuronal morphology, although effectiveness and confluence were varied (Figure 5). However, the removal of ISX9 inhibited the formation of the compact cell bodies and extended neurites seen in the other withdrawal groups and in FICBD-treated cells. FCBD-treated cells instead appeared to have mostly enlarged cell bodies regardless of the level of TUJ1 expression. ISX9 alters gene expression by acting through calcium-calmodulin kinase (CamK) to trigger a calcium activated signalling pathway to phosphorylate histone deacetylase 5, thereby de-repressing myocyte



enhancer factor-2 (MEF2), this has been shown to cause robust neuronal differentiation in adult neuronal stem cells and malignant astrocytes.39,40 Furthermore, triggering of CamK activity has been reported to reverse epigenetic silencing of tumor suppressor genes by causing the release of the methyl-binding protein MeCP2 from silenced promoter, causing inhibited cell proliferation and tumorigenesis. However, the effects of ISX9 on neuronal differentiation of GBM cells or GBM stem cells (GSCs) had not yet been confirmed. Our results suggest that ISX9 is vital for neuronal conversion via the FICBD cocktail, and that U87MG cells fail to reprogram in its absence. It is possible that calcium dependent signalling pathway activation is required in some cases of neuronal differentiation of GBM cells, and might act through induced activation of the MEF2 pathway or the triggered release of MeCP2 from genes. It is of interest to note that previous chemical-based neuronal reprogramming protocols have shown neuronal reprogramming of fibroblasts, astrocytes, and rat glioma cells in the absence of ISX9, indicating that MEF2 activation via ISX9 is not required in all cases of reprogramming, although it has yet to be investigated whether the reprogramming protocols would benefit upon the addition of ISX9 or another similar MEF2 activating molecule. While minor changes were observed upon removal of DAPT, I-BET 151, CHIR99021, and Forskolin, the withdrawal of these chemical did not show significant improvement in neuronal reprogramming, nor clear enough redundancy to warrant removal for this current study. However, if chemical cocktail reprogramming, specifically FICBD, is to be further investigated for its viability as clinical treatment, it is vital that an optimized high efficacy, minimal cocktail be identified for reprogramming GBM cells. Removal of ISX9 inhibits neuronal reprogramming, making it likely that removal of more than one of DAPT, I-BET 151, CHIR99021, and Forskolin would incur similar effects. Another important consideration is the ability to deliver these


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molecules across the blood-brain barrier and DAPT shows limited ability to cross the bloodbrain barrier, making long-term treatment inadvisable. 41 Identification of a minimal high efficacy GBM reprogramming cocktail should be followed by concentration screens allowing for further optimization, and assays on various healthy non-cancerous neural tissue to test for reprogramming specificity. Furthermore, additional drug screens for reprogramming chemicals not present in this study could identify small molecules that could increase GBM reprogramming efficiency or confirm efficiency of the identified minimal cocktail. Chemical Reprogramming of U87MG Cells Increases Expression of Pioneering Transcription Factors Implicated in Neuronal Development Real-time quantitative-PCR (qPCR) was performed for small molecule reprogrammed U87MG cells and control cells that did not receive small molecule supplementation at days 0, 2, 9, and 12 for the genes Ascl1, MAP2, Ngn2, GFAP, and Brn2 to analyze changes in gene expression that the FICBD cocktail causes GBM cells to undergo, driving cells to a neuronal fate. Relative expression changes between the small molecule treated group and the control group was measured for the different time points and then compared to day 0 to test for statistical significance (Figure 6A-E). Day 2 FICBD treated cells showed a significant decrease in GFAP expression, and further days did not show a significant difference from the expression change at day 0, supporting previous ICC evidence that the FICBD cocktail does not revert GBM cells to a glial fate. Chemically treated cells at day 2 also showed a significantly increased expression in the neuron-enriched gene MAP2, which supports the idea that the FICBD cocktail may be able to generate mature neurons under specific conditions. However, further qPCR analysis did not reveal a significant change in MAP2 expression up to day 12, disputing this claim. Interestingly, both MAP2 and GFAP displayed dynamic expression patterns by day 2 followed by an increase



to levels similar to controls in the following days, and either gene was only affected at most below 5-fold. The FICBD cocktail’s effect on structural genes may be dynamic during the early phases of reprogramming and requires analysis beyond day 12 to identify overall trends. Indeed, dynamic gene expression for GFAP has been found in the small molecule reprogramming of astrocytes to neurons, supporting this hypothesis.25 Day 12 chemically treated cells showed a significant upregulation for the transcription factors Ascl1 and Ngn2. These pioneering neuronal reprogramming genes have previously been found to be both essential and sufficient for forced neuronal reprogramming of multiple cell types,12,13,14,15,42,43 indicating that FICBD treatment causes the upregulation of these genes to drive reprogramming to a neuronal fate. Furthermore, Brn2, a transcription factor associated with neuronal differentiation and reprogramming of induced neuronal cells was upregulated on day 12 chemically treated cells, showing that the FICBD cocktail upregulates supporting neuronal reprogramming genes as well as pioneering reprogramming transcription factors. Generation of Chemically Induced Neuronal Cells Do Not Pass Though an Intermediate Pluripotent Stage ICC for the markers SOX2 and NESTIN was performed on day 5 for FICBD-treated U87MG cells, control U87MG cells, and positive control neural progenitor cells (NPC) to confirm the lack of an NPC intermediate state during reprogramming. No changes in expression between control cells, FICBD-treated cells, or day 0 cells were observed (Figure 7A, B). Furthermore, neither condition showed expression that resembled that of non-malignant human NPCs (Figure 7B). Overall, these results indicate that the reprogramming of U87MG cells by use of the FICBD cocktail is direct and does not pass through an intermediate pluripotent state. It is ideal that direct reprogramming of GBM cells avoids passing through a multipotent state, for it


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is possible that passing through a multipotent state could contribute to the population of GSC and increase tumorigenesis.44,45 FICBD Cocktail Suppresses Growth of U87MG Cells and Impedes Tumor-Like Spheroid Formation We next determined whether the FICBD cocktail-based reprogramming of GSCs into neurons would impede the formation of GSC tumor-like spheroids. GSC spheroids isolated from U87MG cells were formed in the presence of the FICBD cocktail and displayed a significant reduction in both the number of neurospheres formed (Figure 8A, C) and the total area that spheroids were comprising (Figure 8A, D). However, FICBD treated neurospheres showed an increased average neurosphere area (Figure 8A, E), displaying both a lack of smaller neurospheres, and neurospheres larger than those found in control cells. A possible explanation for this would be the observed result of individual reprogrammed cells and reprogrammed cells along the perimeter of GSC spheroids displaying the ability to adhere to ultra-low adherence cell culture plates (Figure 8A,B). This capability was not observed in any capacity in control GSC spheroids or individual cells, and had not previously been reported in separate GSC formation assays.46 These perimeter FICBD reprogrammed cells displayed morphological evidence of neuronal-fate adoption, with long neurite-like structures and compact cell bodies similar to that seen in FICBD reprogrammed cells grown in 2d cell culture (Figure 2A, 8B). It was not expected that FICBD induced cells entering a terminal neuronal fate would display increased adherence, as it is normally required that both neuroblasts cells to undergo neuronal differentiation and differentiated neuronal cells be seeded on substrate-coated adherent cell culture plates for cultivation.47 The reduced neurosphere number and total neurosphere area formed indicates that the FICBD cocktail is able to inhibit the stem cell spheroid generating



ability of U87MG cells. However, the FICBD cocktail causing an increased average neurosphere area suggests that GSC spheroids are not inhibited in their ability to increase in size once formed and are able to reach larger sizes likely either through increased initial integration of more GBM cells into the GSC spheroid or higher proliferation in rates in the outermost region of formed spheroids.48 Furthermore, the observation of cell adhesion to ultra-low adherence cell culture plates is possibly detrimental to the clinical significance of neuronal reprogramming of GBM cells, as the link between cell adhesion and the survival of tumor cell types is well established,49 although it is currently unclear whether FICBD-treated cells were adhering due to increased expression of adhesive surface proteins or by induced secretion of extracellular matrix. It is possible that if only a subset of GBM cells reprogramed to induced neuronal cells, the increased adhesion of those cells could allow for non-reprogrammed GBM cells to form larger GSC tumor spheroids in vivo, contributing to the cancers tumorgenicity. Overall, the FICBD cocktail impedes GSC spheroid formation.

Conclusions The aggressive proliferation of glioblastoma cells can potentially be stopped by reprogramming these cells into a terminally differentiated state. Advances in small molecule neuronal reprogramming have opened the door to direct reprogramming as a possible treatment for glioblastoma. This study investigated the ability of a small molecule cocktail of forskolin, ISX9, CHIR99021 I-BET 151, and DAPT (FICBD) to reprogram U87MG cells to neuronal cells. The FICBD cocktail directly reprogrammed glioblastoma cells to early neurons rapidly with a high rate of efficiency and without passing through an intermediate pluripotent state. This small molecule mediated reprogramming was dependent on the chemical activation of pioneering 14 ACS Paragon Plus Environment

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neuronal transcription factors. Furthermore, we demonstrated that ISX9-induced calcium channel activation was fundamental for neuronal-fate adoption of human glioblastoma cells, confirming the significance of calcium channel activation for the treatment of glioblastoma. Importantly, we also demonstrated that the FICBD cocktail inhibits the formation of glioblastoma stem cell spheroids, and that reprogrammed cells displayed increased adhesion. Overall, this study applied chemical-mediated cellular reprogramming as a novel method of glioblastoma reprogramming into early neurons.

Methods Materials Laminin from Egelbreth-Holm-Swarm murine sarcoma basement membrane, normal goat serum (NGS), Poly-L-ornithine (PLO) 0.01% solution, N-2 100X supplement, B-27 50X supplement, cAMP, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Forskolin, ISX9, CHIR99021, I-BET 151 and DAPT were purchased from MedChemExpress (South Brunswick, NJ, USA). Goat anti-rabbit IgG (H+L) highly crossadsorbed secondary antibody Alexa Fluor 408, Goat anti-mouse IgG (H+L) highly crossadsorbed secondary antibody Alexa Fluor 488, Goat anti-rabbit IgG (H+L) highly crossadsorbed secondary antibody Alexa Fluor 568, 4,6-diamindino- 2-phenylindole, dihydrochloride (DAPI) nucleic acid stain, Fast SYBR Green Master Mix, High-Capacity cDNA Reverse Transcription Kit, UltraPure DNase/RNase-free distilled water, and phosphatebuffered saline (PBS) were purchased from ThermoFisher Scientific (Waltham, MA, USA). U87MG glioblastoma cells were obtained from the American Type Culture Collection 15 ACS Paragon Plus Environment


(Manassas, VA, USA). RNeasy Mini Kit was purchased from QIAGEN (Venlo, Netherlands). Anti-doublecortin (DCX) rabbit antibody, anti-sex determining region Y-box 2 (SOX2) rabbit antibody, anti-glial fibrillatory acidic protein (GFAP) rabbit antibody, anti-microtubuleassociated protein 2 (MAP2) rabbit antibody were purchased from Millipore (St. Louis, MO, USA). Astrocyte Medium was purchased from ScienCell (San Diego, CA, USA). Brainphys Neuronal Medium, STEMdiff™ Neural Progenitor Medium, Gentle Cell Dissociation Reagent, and anti-beta-tubulin III mouse monoclonal [clone AA10] IgG2a antibody were purchased from STEMCELL Technologies (Vancouver, BC, Canada). Flow cytometry fixation buffer, flow cytometry permeabilization/Wash Buffer I, and Neuron-specific beta tubulin-III PerCPconjugated Antibody were purchased from R&D systems (Minneapolis, MN, USA). Custom forward and reverse primer oligos for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), TUJ1, and Ascl1 were purchased from Eurofins Genomics (Luxembourg City, Luxembourg). Brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), insulin-like growth factor (IGF), and platelet-derived growth factor AA homodimer (PDGF-AA) were purchased from PeproTech (Rocky Hill, NJ, USA). GBM Cell-Line Culture and FICBD Induction U87MG glioblastoma cells were obtained from the American Type Culture Collection (U.S.A). Cells were maintained in Astrocyte Media (ScienCell) with media being was changed every 3-4 days. Upon reaching >90% confluence, cells were passaged onto an experimental plate at a rate of approximately 3.3 x 104 cells/cm2. Cells were grown in astrocyte media with media changes every 3-4 days until cells reached 90% confluence, at which point media was changed to a neuronal induction medium, which consists of Brainphys, 1% N-2, and 1% B-27 and the following small molecules: 10 µM Forskolin, 10 µM ISX9, 3 µM CHIR99021, 2 µM I-BET 151,


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and 5 µM DAPT. Neuronal induction medium was half changed every 2 days or fully changed every 3-4 days. For cells to undergo immunocytochemistry on day 9, on day 7 media was changed 50% to neuronal maturation media, which consists of Brainphys containing 1% N-2, 1% B27, 0.01% BDNF, 0.01% GDNF, 0.01% IGF, 1 µM cAMP, PDGF-AA. For all other cells, media was half changed on day 7 to Brainphys, 1% N-2, and 1% B-27 and the small molecule 10 µM Forskolin. Media was then half changed every 2 days or fully changed every 3-4 days. Cells were imaged using phase microscopy at indicated time points using a Leica DMI3000B (Leica Biosystems, Wetzlar, Germany) microscope and QImaging RETIGA 2000R camera (QImaging, Surrey, BC, Canada) at 100X magnification. Images were captured using QCapture Software 2.9.12. For measurements of cellular morphology, adherent cells with identifiable cell shape were manually outlined and scored for area, perimeter, and circularity using the ImageJ program. Neurite length were outlined and scored using the semi-automatic measurement plug-in NeuriteGrowth for ImageJ.50 Immunocytochemistry of Cultures and Fluorescent Imaging At days 5, 7 and 9 for FICBD-treated and control U87MG cells, and the day following initial plating for NPC cells, ICC samples were prepared as previously described51,52 using 1:500 dilutions of Primary mouse antibody anti-TUJ1, primary rabbit antibodies for either MAP2, DCX, or GFAP, Alexa Fluor® 408 goat anti-rabbit, Alexa Fluor® 488 goat anti-mouse and Alexa Fluor® 568 goat anti-rabbit antibodies. Immunofluorescence was visualized using a Leica DMI300 B microscope with an X-Cite® Series 120Q fluorescent light source (Excelitas Technologies, Waltham, MA, USA) and a QImaging RETIGA 2000R camera at 100X magnification. Images were captured using a QCapture Software 2.9.12. TUJ1+ cells and DCX+ with a neuronal morphology, indicated by a round or pyramidal soma with a thin process at least



three times longer than the cell body, were counted at days 7 and 911. The conversion efficiency was estimated by dividing the total number of neuron-like cells by the total number of cells indicated by counting DAPI stained cells. RNA Extraction, cDNA Synthesis, and Real-Time qPCR At days 0, 2, 9, and 12, cells were removed from the culture and spun down at 300 xg in a conical tube for 5 minutes to pellet the cells. RNA was extracted from the cells using the RNAeasy® Mini Kit according to protocol. RNA content was measured using a NanoVue Plus (GE Healthcare, Chicago, IL, USA) and was subsequently diluted to 0.1 µg/µL in UltraPure water. RNA was then reverse transcribed by the High-Capacity cDNA Reverse Transcription Kit using an Eppendorf Mastercycler® pro (Eppendorf, Hamburg, Germany). cDNA levels were measured by a NanoVue Plus and cDNA was subsequently diluted to 10ng/mL. mRNA levels were quantified for the primers listed in table S1 using the Fast SYBR® Green Master Mix protocol and an Applied Biosystems StepOnePlus Real-Time PCR System for 3 biological samples for each test conditions with 2 technical replicates each (Foster City, CA, USA). DeltaCt was used to measure fold expression change. For replicates where amplification was not seen, Ct value was assumed to be LOD (+1) Flow Cytometry After 12 days of culture, cells were then lifted from the plate, transferred to a conical tube, and spun down at 300 xg for 5 minutes to compact cells at the bottom of the tube. Flow cytometry sample preparation was performed as previously described.53 Flow cytometric analysis was then performed using a Guava® easyCyte flow cytometer (Millipore, St. Louis, MO, USA). Data was collected and analysed using GuavaSoft v2.6 software. Neurosphere Formation Assay


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The ability of cells of monolayer cultures to initiate neurosphere formation was assessed by adapting the protocol of Daniele et al.46 Upon growth of U87MG cells to >90% confluency, cells were removed from the culture and spun down at 300 xg in a conical tube for 5 minutes to pellet the cells. Cells were then washed and resuspended in Neural Progenitor Medium (StemCell). Cells were then seeded into 6 well suspension culture plates at 1 x 106 cells/well and incubated with DMSO (0.5%, control), or with the small molecule mix: 10 µM Forskolin, 10 µM ISX9, 3 µM CHIR99021, 2 µM I-BET 151, and 5 µM DAPT for 9 days without disturbing the plates and without replenishing the medium. Neurosphere number and diameter was counted and scored using the ImageJ program. Statistical Analysis Results are reported as mean values ± standard error of the mean unless otherwise stated. Statistical significance was determined using two-tailed students t-test and one-way ANOVA with Turkey's post hoc analysis with p < 0.05 indicating minimal significance.

Supporting Information Chemical structures of drugs used in the FICBD cocktail; morphological measurements of control and FICBD reprogrammed cells; list of RT-qPCR primers used in this study

Author Information *Corresponding author Dr. Stephanie Willerth Email: [email protected] Phone: (250) 721-7303 Address: P.O. Box 1700 Stn Csc Victoria, B.C. V8W 2Y2 Canada



Author Contributions C.L. performed a majority of biological experiments; M.R. assisted in biological experiments; C.L. analyzed the data and wrote the manuscript; C.L., M.R., and S.M.W. edited the manuscript. C.L. and S.M.W. designed the study. M.R. and S.M.W. provided valuable mentorship; S.M.W. served as project supervisor. All authors approved the final manuscript. Funding Sources This work was supported by an NSERC Discovery Grant (S.M.W.), an NSERC Collaborative Research Development grant (S.M.W.) and the Canada Research Chairs program (S.M.W.) Conflict of Interest The authors declare no conflict of interest.


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Day 5

Day 7

Control

Day 2

FICBD

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Figure 1. Effects of the FICBD chemical cocktail on U87MG glioblastoma cell morphology grown in defined media over 7 days. Control cells displayed a consistent increase in confluence between day 2 (A), day 5 (B), and day 7 (C) with minimal signs of neurite-like processes. (D) FICBD treated cells displayed a rapid adoption of bipolar neurite-like processes at day 2. (E) Day 5 FICBD-treated cells displayed multipolar neurite outgrowths. (F) Day 7 FICBD-treated cells showed decreased confluence in addition to multiple multipolar cells, cells also began to show signs of complex branching. Scale bars represent 100 µm.


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Day 9

Day 13

Figure 2. Adoption of complex morphologies by U87MG cells treated with the FICBD cocktail. (A) Complex branching can be seen by multiple cells at day 9. (B and C) Complex morphologies adoption of distinct complex branching neuronal/astrocytic morphology by day 13 FICBD-treated U87MG glioblastoma cells. Scale bars represent 100 µm.



A

DCX/TUJ1/DAPI

Control Day 9

C

% of Cells Marker+

B

D

80

DCX

*

TUJ1

70

DCX/TUJ1/DAPI

FICBD Day 9

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60 50

*

40 30 20 10 0 Control

GFAP

E

FICBD

MAP2

Figure 3. Immunocytochemistry for day 9 FICBD-treated cells showed an increase in early neuronal marker proteins. (A) Control cells grown for 9 days in defined media absent of the FICBD small molecules showed minimal increase in the expression of the early neuronal genes DCX and TUJ1. (B) Cells grown in defined media supplemented with the FICBD small molecule cocktail showed an increase in the expression of DCX and TUJ1. (C) The number of FICBD-treated cells expressing TUJ1 and DCX were quantified and compared to control cells for the same marker. (mean ± SEM, N=10 random fields from triplicate samples, *P

Direct Reprogramming of Glioblastoma Cells into Neurons Using

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