Adherent Monolayer Culture Research Articles

An easy and cost-effective method for the isolation and culturing of neural stem/progenitor cells from the subventricular (SVZ) and dentate gyrus (DG) of adult mouse brain

BackgroundIsolation of adult Neural Stem/Progenitor Cells (NSPCs) from their neurogenic niches, is a prerequisite for studies involving culturing of NSPCs as neurospheres or attached monolayers in vitro. The currently available protocols involve the use of multiple animals and expensive reagents to establish the NSPCs culture. New methodThis unit describes a method to isolate and culture NSPCs from the two neurogenic niches in the mouse brain, the Subventricular Zone (SVZ) and Dentate gyrus (DG)/subgranular zone (SGZ), in an easy and cost-effective manner. ResultsNSPCs from SVZ and DG regions of adult mouse brains were isolated and cultured up to passage 15 without losing their stem/progenitor characteristics. These NSPCs could be differentiated into neurons, astrocytes, and oligodendrocytes, revealing its trilineage potential. Comparison with existing methodsThis protocol eliminates the need for multiple animals as well as the use of many expensive reagents mentioned in previous protocols, adding to the cost-effectiveness of experiments. In addition, we have effectively reduced the number of steps involved in isolation and propagation, thereby minimizing the chances of contamination. ConclusionOur simplified protocol for the isolation and culturing of adult NSPCs from the SVZ and DG demonstrates a cost-effective and efficient alternative to existing methods, reducing the need for sacrificing many animals and the usage of expensive reagents. This method permits the long-term maintenance of NSPCs' stem/progenitor characteristics and their effective differentiation into the major types of cells in the brain, making it a valuable resource for researchers in the field. Basic protocolIsolation and Culturing of Neural Stem/Progenitor cells from the Sub ventricular Zone and the Dentate Gyrus of the adult mouse brain. Support protocol 1Cryopreservation, and revival of frozen NSPCs. Support protocol 2Preparation of adherent monolayer cultures of neural stem/progenitor cells for the differentiation into multiple lineages Support protocol 3Differentiation of NSPCs to neuronal and glial lineages Support protocol 4Characterization of differentiated cells by immunocytochemistry

Comparison of Resection Assisting Devices in the Process of Collecting Brain Tumor Tissue for Basic Research: Microdebrider Versus Ultrasonic Aspirator

Brain tumors display significant inter and intratumoral heterogeneity, impacting disease progression and outcomes. Preserving surgically resected tissue is vital for ensuring accurate research results to enhance understanding of tumor pathophysiology. This study evaluates tissue integrity and viability of tissue resected using 2 surgical devices for tumor resection: a mechanical microdebrider (MD) and an ultrasonic aspirator (UA). Tumor samples were obtained from patients undergoing surgical resection of primary and secondary intracranial tumors. Cell viability was assessed, and histopathological analysis of Hematoxylin and Eosin -stained tissues was performed. Adherent monolayer and neurospheres cell cultures were established from paired samples. RNA isolation and quantitative polymerase chain reaction of housekeeping genes were conducted to compare genetic integrity. The cellular viability was comparable between samples obtained using both the MD and the UA, with a mean viability of 75.2% ± 15.6 and 70.7% ± 16.8, respectively (P = 0.318). Histopathological evaluation indicated no discernible differences in cellular integrity between the devices. Cell culture success rates and growth characteristics were similar for both devices. RNA concentration and integrity were well-maintained in both MD and UA samples, with no significant differences (P= 0.855). Quantitative polymerase chain reaction analysis of housekeeping genes showed consistent results across matched tissues from both devices and different tumor pathologies. Surgical handheld devices provide valuable, high-quality tissue samples for research. Surgeon preference, tumor pathology, and anatomical location dictate device choice. Both MD and UA devices are reliable for obtaining quality tissue specimens, facilitating translational neuro-oncology research.

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

Abstract 6373: Spheroid culture of ER+ breast cancer patient-derived tumor cells enriches cancer stem-likecells with EpCAM-/CD49f+and high ALDH activity and poor prognostic gene signatures

Abstract Background: Cancer stem-like cells (CSCs) represent a critical subset of the tumor population, which contributes to tumor recurrence, metastasis and therapy resistance. Spheroid culture is unique method for CSC enrichment. Patient-derived breast cancer cells (PDBCCs) has provided an invaluable tool in preclinical and translational research. The objective of this study is to characterize the immunophenotype and transcriptomes of PDBCCs grown in the adherent monolayer and spheroid condition. Methods: PDBCCs were harvested from residual tumor of fresh surgical specimens of patients with ER-positive subtype (8 cases). The adherent monolayer(M) and spheroid(S) culture of PDBCCs were applied on collagen I-coated plate and an ultra-low attachment polymer-X coated plate. Flow cytometry and immunocytochemistry was performed by using fluorescent-dye conjugated antibodies for EpCAM, CD49f, CD24, CD44, and cytokeratin. ALDEFLUOR® assay to detect ALDH activity was used. Total RNA-sequencing was applied to investigate differentially expressed genes (DEGs), followed by GO and KEGG pathway enrichment analysis. Real-time RT-PCR was performed to evaluate BCSC-related RNA levels. The Kaplan-Meier Plotter online database was used to evaluate the prognostic value of DEGs (2-fold change, p<0.05). Results: Under adherent monolayer condition, more than 95% of PDBCCs passaged for up to 5 passages on collagen I-coated plate sustained EpCAM+/cytokeratin+/fibroblast marker_ phenotype. On polymer-X coated plates, PDBCCs formed compact multicellular spheroids with a diameter of less than 300 µm. As compared with monolayer, CSCs with EpCAM-/CD49f+ phenotype (M vs S; 0.16±0.06 vs 2.34±0.65, p=0.015) and high ALDH activity (M vs S; 0.73±0.43 vs 7.05±1.48, p=0.038) were significantly increased by spheroid culture. In whole-transcriptome analysis (3 cases) between spheroid and monolayer, a total of 561 DEGs were identified, including 290 upregulated and 271 downregulated DEGs (2- fold change, p<0.05) in spheroid. The upregulated and downregulated DEGs in spheroids were enriched in 8 and 19 KEGG pathways, respectively. In recurrence-free survival analysis based on the Kaplan-Meier Plotter database of the genes corresponding to Oncotype Dx, Mammaprint, and Prosigna and top 30 ranked DEGs identified in spheroids, 15 upregulated and 14 downregulated DEGs were associated with poor prognosis of breast cancer patients. Conclusion: Spheroid culture of PDBCCs with ER+ subtype enriches CSCs and poor prognostic gene signatures. Future studies to explore the specific molecular mechanisms underlying these observations may provide new molecular targets for the development of therapies for targeting CSCs in breast cancer patient. Citation Format: Hoe Suk Kim, Seungyeon Ryu, So-Hyun Yoon, Sangeun Lee, Junhyuk Song, Yoonjung Choi, Moonjou Baek, Han-Byoel Lee, Sangyong Jon, Daeyoup Lee, Wonshik Han. Spheroid culture of ER+ breast cancer patient-derived tumor cells enriches cancer stem-likecells with EpCAM-/CD49f+and high ALDH activity and poor prognostic gene signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6373.

Mesenchymal Stromal Cells (MSCs) Isolated from Various Tissues of the Human Arthritic Knee Joint Possess Similar Multipotent Differentiation Potential

(1) Background: The mesenchymal stromal cells (MSCs) of different tissue origins are applied in cell-based chondrogenic regeneration. However, there is a lack of comparability determining the most suitable cell source for the tissue engineering (TE) of cartilage. The purpose of this study was to compare the in vitro chondrogenic potential of MSC-like cells from different tissue sources (bone marrow, meniscus, anterior cruciate ligament, synovial membrane, and the infrapatellar fat pad removed during total knee arthroplasty (TKA)) and define which cell source is best suited for cartilage regeneration. (2) Methods: MSC-like cells were isolated from five donors and expanded using adherent monolayer cultures. Differentiation was induced by culture media containing specific growth factors. Transforming growth factor (TGF)-ß1 was used as the growth factor for chondrogenic differentiation. Osteogenesis and adipogenesis were induced in monolayer cultures for 27 days, while pellet cell cultures were used for chondrogenesis for 21 days. Control cultures were maintained under the same conditions. After, the differentiation period samples were analyzed, using histological and immunohistochemical staining, as well as molecularbiological analysis by RT-PCR, to assess the expression of specific marker genes. (3) Results: Plastic-adherent growth and in vitro trilineage differentiation capacity of all isolated cells were proven. Flow cytometry revealed the clear co-expression of surface markers CD44, CD73, CD90, and CD105 on all isolated cells. Adipogenesis was validated through the formation of lipid droplets, while osteogenesis was proven by the formation of calcium deposits within differentiated cell cultures. The formation of proteoglycans was observed during chondrogenesis in pellet cultures, with immunohistochemical staining revealing an increased relative gene expression of collagen type II. RT-PCR proved an elevated expression of specific marker genes after successful differentiation, with no significant differences regarding different cell source of native tissue. (4) Conclusions: Irrespective of the cell source of native tissue, all MSC-like cells showed multipotent differentiation potential in vitro. The multipotent differentiation capacity did not differ significantly, and chondrogenic differentiation was proven in all pellet cultures. Therefore, cell suitability for cell-based cartilage therapies and tissue engineering is given for various tissue origins that are routinely removed during total knee arthroplasty (TKA). This study might provide essential information for the clinical tool of cell harvesting, leading to more flexibility in cell availability.

Establishment of patient‑derived lung tumorspheres and their response to internal irradiation by Auger electrons.

The high recurrence rate of lung cancer is a major clinical challenge associated with therapy‑resistant cancer stem cells (CSCs), which are rare subpopulations. Future successful treatment is required to also eradicate these subpopulations. Furthermore, the majority of anti‑cancer treatments are being tested in adherent monolayer cultures with the limitations this entails in the translation of results into clinical practice. The present study aimed to establish and characterize patient‑derived long‑term primary lung cancer tumorspheres enriched in CSCs and evaluate the effects of Auger electrons on them. These electrons are emitted from radionuclides that decay by electron capture or internal conversion and have demonstrated promising therapeutic potential. Their low energy (<1keV) is sufficiently potent to induce DNA double‑strand breaks and eventually cell death while minimizing irradiation of non‑targeted surrounding cells. Labeling a thymidine analog (deoxyuridine) with the Auger electron‑emitting radionuclide [125I], which is exclusively incorporated into the DNA of proliferating cells during the S‑phase, ensures a close distance to the DNA. Primary cell cultures grown as tumorspheres were established and characterized. The tumorspheres were morphologically distinct and differed concerning their proliferation rate and fraction of CSCs. Surface markers associated with CSCs were upregulated and 5‑[125I]iodo‑2'‑deoxyuridine was incorporated in the tumorspheres. The Auger electrons induced DNA double‑strand breaks, G2/M arrest and apoptosis in the tumorspheres; however, the tumorspheres derived from different patients exhibited heterogeneities in their sensitivity to Auger electron irradiation.

In vitro teratogenicity testing using a 3D, embryo-like gastruloid system

Pharmaceuticals intended for use in patients of childbearing potential need to be tested for teratogenicity before marketing. Several pharmaceutical companies use animal-free in vitro models which allow a more rapid selection of lead compounds and contribute to 3Rs principles (‘replace, reduce and refine’) by streamlining the selection of promising compounds submitted to further regulatory studies in animals. Currently available in vitro models typically rely on adherent monolayer cultures or disorganized 3D structures, both of which lack the spatiotemporal and morphological context of the developing embryo. A newly developed 3D ‘gastruloid’ model has the potential to achieve a more reliable prediction of teratogenicity by providing a robust recapitulation of gastrulation-like events alongside morphological coordination at relatively high-throughput. In this first proof-of-concept study, we used both mouse and human gastruloids to examine a panel of seven reference compounds, with associated in vivo data and known teratogenic risk, to quantitatively assess in vitro teratogenicity. We observed several gross morphological effects, including significantly reduced elongation or decreased size of the gastruloids, upon exposure to several of the reference compounds. We also observed aberrant gene expression using fluorescent reporters, including SOX2, BRA, and SOX17, suggestive of multi-lineage differentiation defects and disrupted axial patterning. Finally, we saw that gastruloids recapitulated some of the known in vivo species-specific susceptibilities between their mouse and human counterparts. We therefore suggest that gastruloids represent a powerful tool for teratogenicity assessment by enabling relevant physiological recapitulation of early embryonic development, demonstrating their use as a novel in vitro teratogenic model system.

Comparative transcriptome analysis of transcultured human skin-derived precursors (tSKPs) from adherent monolayer culture system and tSKPs-derived fibroblasts (tFBs) by RNA-Seq.

Transcultured human skin derived precursors (tSKPs) from adherent monolayer culture system have similar characteristics as traditional skin derived precursors (SKPs), making tSKPs a suitable candidate for regenerative medicine. tSKPs can differentiate into fibroblasts. However, little is known about the molecular mechanism of the transition from tSKPs to fibroblasts. Here, we compared the transcriptional profiles of human tSKPs and tSKPs-derived fibroblasts (tFBs) by RNA-Sequence aiming to determine the candidate genes and pathways involving in the differentiation process. A total of 1042 differentially expressed genes (DEGs) were identified between tSKPs and tFBs, with 490 genes up-regulated and 552 genes down-regulated. Our study showed that these DEGs were significantly enriched in tumor necrosis factor signaling pathway, focal adhesion, extracellular matrix-receptor interaction and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway. A further transcription factors (TFs) analysis of DEGs revealed the significantly down-expressed TFs (p21, Foxo1and Foxc1) in tFBs were mostly the downstream nodes of PI3K-Akt signaling pathway, which suggested PI3K-Akt signaling pathway might play an important role in tSKPs differentiation. The results of our study are useful for investigating the molecular mechanisms in tSKPs differentiation into tFBs, making it possible to take advantage of their potential application in regenerative medicine.

Primary Spinal Cord Neurons Better Attach to Non-Coated Surfaces Than to Poly(D-lysine)-Coated Surfaces

Background: Cell culture is an important technique in cellular and molecular biology. There are two basic systems for culturing cells, adherent (monolayer) culture and suspension culture. In adherent cell culture, cellular adhesion molecules play a vital role in many physiological processes. Many cells, especially primary-obtained cells, hardly attach to plates, so coating agents are used for cell attachment and growth. There is no striking report on whether all cells need coated plates. Objectives: We investigated whether a coating plate is essential for primary spinal cord-obtained neuron culture. Methods: Cells were separated and seeded in tissue culture plates coated with either Poly(L-lysine) or Poly(D-lysine), as well as without coating containing DMEM-F12 media with 10% FBS. Results: Interestingly, we found that neuron cells more potently attached to coating free plates. Conclusions: These results provide strong evidence helping the researchers to optimize primary spinal cord cultures.

BMP Inhibition in the Presence of LIF Differentiates Murine Embryonic Stem Cells to Early Neural Stem Cells

Early mouse neural stem cells (NSCs) first appear in embryonic day E5.5 and express pluripotency markers Oct4, Sox2, Nanog and early neural marker Sox1. Early NSCs are a good model for understanding the role of various pathways that control initial neural commitment. However, a protocol for differentiation of mouse embryonic stem cells (ESCs) into early NSCs by adherent monolayer culture has not yet been established. Hence, in this study, we identified the combination of growth factors and small molecules that differentiated mouse ESCs into early NSCs and supported their proliferation. Leukaemia inhibitory factor (LIF) was the first factor to be tested and it was found that ESCs can differentiate into early neurogenic lineage in the presence of LIF. However, we found that the induction is weaker in the presence of LIF as compared to cells differentiated in its absence. GSK-3 inhibitor, along with BMP and TGF-β pathway inhibitor (dual SMAD inhibition), are commonly used to sequentially direct ESCs towards NSCs. However, when we used this combination, mouse ESCs failed to differentiate into early NSCs. We observed that by adding Wnt inhibitor to the combination of GSK-3 inhibitor, BMP inhibitor, TGF-β inhibitor and LIF, it was possible to differentiate ESCs into early NSCs. qRT-PCR analysis of early NSCs illustrated that they expressed key pluripotency genes Oct4 and Nanog, albeit at levels lower than non-differentiated ESCs, along with early neural markers Sox1 and Pax6.

Bone marrow-derived neural crest precursors improve nerve defect repair partially through secreted trophic factors

BackgroundEmerging evidence suggests that neural crest-derived cells (NCCs) present important functions in peripheral nerve regeneration to correct the insufficiency of autogenous Schwann cells. Postmigratory NCCs have been successfully isolated from adult rat bone marrow in our previous work. In this study, we aim to provide neural crest-derived Schwann cell precursors (SCPs) for repair of nerve defects in adult rats, and partially reveal the mechanisms involved in neuroregeneration of cell therapy.MethodsA clonal cell line of neural crest precursors of rat bone marrow origin (rBM-NCPs) with SCP identity was expanded in adherent monolayer culture to ensure the stable cell viability of NCPs and potentiate the repair of nerve defects after rBM-NCPs implantation based on tissue engineering nerve grafts (TENG). Here the behavioral, morphological, and electrophysiological detection was performed to evaluate the therapy efficacy. We further investigated the treatment with NCP-conditioned medium (NCP-CM) to sensory neurons after exposure to oxygen-glucose-deprivation (OGD) and partially compared the expression of trophic factor genes in rBM-NCPs with that in mesenchymal stem cells of bone marrow origin (rBM-MSCs).ResultsIt was showed that the constructed TENG with rBM-NCPs loaded into silk fibroin fiber scaffolds/chitosan conduits repaired 10-mm long sciatic nerve defects more efficiently than conduits alone. The axonal regrowth, remyelination promoted the reinnervation of the denervated hind limb muscle and skin and thereby alleviated muscle atrophy and facilitated the rehabilitation of motor and sensory function. Moreover, it was demonstrated that treatment with NCP-CM could restore the cultured primary sensory neurons after OGD through trophic factors including epidermal growth factor (EGF), platelet-derived growth factor alpha (PDGFα), ciliary neurotrophic factor (CNTF), and vascular endothelial growth factor alpha (VEGFα).ConclusionsIn summary, our findings indicated that monolayer-cultured rBM-NCPs cell-based therapy might effectively repair peripheral nerve defects partially through secreted trophic factors, which represented the secretome of rBM-NCPs differing from that of rBM-MSCs.

Isolation, Characterization, and Safety Evaluation of Human Skin-Derived Precursors from an Adherent Monolayer Culture System.

Background Skin-derived precursors (SKPs) are promising candidates for regenerative medicine. Several studies have transcultured human SKPs (termed tSKPs) from fibroblasts (FBs) expanded in monolayer culture. Herein, we optimized the procedure by treating flasks with poly-2-hydroxyethyl methacrylate (poly-HEMA). Methods tSKPs generated from our adherent monolayer culture system were investigated for protein expression and differentiation capacity. The aggregated cells and the proliferative cells within tSKP spheres were detected by mix-culturing FBs expressing two different fluorescent proteins and BrdU- or EdU-positive cells, respectively. To distinguish tSKPs from FBs, we compared their phenotypes and transcriptomes. The tumorigenicity of tSKPs and FBs was also assessed in our study. Results tSKPs expressed Versican, Fibronectin, Vimentin, Sox2, and Nestin. Under appropriate stimuli, tSKPs could differentiate to mesenchymal or neural lineages. While these spheres were heterogeneous populations consisting of both proliferative and aggregated cells, the rate of proliferative cells correlated with a seeding density. tSKPs, isolated from FBs, were distinctive from FBs in cell cycle, marker expression, neural differentiation potential, and transcript profiles despite the two sharing partial similarity in certain properties. As for tumorigenesis, both tSKPs and FBs could be considered as nontumorigenic ex vivo and in vivo. Conclusion tSKPs were heterogeneous populations presenting similar characteristics as traditional SKPs, while being different from FBs. The potential mixture of FBs in spheres did not affect the biosafety of tSKPs, as both of which had normal karyotype and nontumorigenicity. Taken together, we suggested tSKPs had potential applications in regenerative medicine.

Selective enrichment of CD133+/SOX2+ glioblastoma stem cells via adherent culture.

Most of the brain tumors are malignant with an extremely poor survival rate. Recent progress in identifying cancer stem cells (CSCs) within the brain tumors is starting to revolutionize our understanding in the imitation and progression of tumors as well as relapse and the development of therapeutic strategies. Suspension spheroid body culture paradigm is a routine method in enriching CSCs. While, it was reported recently that CSCs within the brain tumor may also be enriched through adherent monolayer culture with optimized properties. In the present study, 18 surgically resected brain tumors were used for analyzing the feasibility of adherent enrichment of CSCs. The results indicated that 50% of glioblastomas were able to generate adherent CSCs, which were uniformly positive for Sox2, CD133, GFAP and Nestin. However, adherent culture paradigm failed to yield CSCs in secondary brain tumors, including neurocytomas, ependymomas, germ cell tumors or low-grade astrocytomas, which is most likely due to a lack of CD133+/Sox2+ cells within the original biopsies. Therefore, it was concluded that the adherent culture paradigm may serve as a reliable method in enriching brain CSCs, but this method is more suitable for enriching CD133+/Sox2+ CSCs in glioblastomas.

Abstract LB-171: Potential chemopreventative treatments in Fanconi anemia related HNSCC

Abstract Fanconi anemia (FA) is a DNA repair mechanism genetic disorder which leads to bone marrow failure and cancer. Risk of solid tumor malignancy in FA patients occurs decades earlier and at a rate several hundred-fold higher than the general population, necessitating regular cancer surveillance. Development of an adjunct therapy or preventative survival-enhancing therapy with a minimal side effect profile will benefit this population in which chemotherapy and radiation are particularly damaging. To this end, we established a cell line from a post bone marrow transplant FA patient with a T2N2bM0 oral SCC grown as an adherent monolayer culture. We tested pioglitazone, metformin, and N1-hexyl-N5-benzyl-biguanide (HBB), emerging chemopreventative therapies for head and neck squamous carcinoma, for growth inhibition in head and neck cancer cell lines, including our FA SCC cell line. Additional cell lines include UMSCC 11A (laryngeal squamous cell carcinoma), CA 9-22 (oral squamous cell carcinoma), Beas2B (SV40 immortalized normal bronchial epithelium), and MSK Leuk1 (oral leukoplakia cell line). All cell lines are HPV negative. MTT assays determined cell viability after 1, 2, and 3 days of treatment. Pioglitazone at all concentrations (5, 10, 20, 40 μM) decreases cell proliferation compared to solvent controls (P&amp;lt;0.0001). Metformin (1 μM - 1 mM) unexpectedly increased cell proliferation or was otherwise equal in growth with the controls, and at higher doses (10 mM - 100 mM) metformin decreased cell proliferation or resulted in cytotoxicity (P&amp;lt;0.0001). Pioglitazone (10 μM) and metformin (above 1 mM) resulted in larger, cell line dependent, decreases in cell proliferation than either agent alone. In the FA cell line, pioglitazone and HBB resulted in increased efficacy. HBB treatment resulted in decreases in proliferation and percent live cells at 20 μM equivalent to the effect of 10 to 100 mM metformin. Combining HBB with 10 μM pioglitazone decreased cell proliferation and percent live cells over pioglitazone alone. HBB also caused S phase blockade in a pilot FA cell cycle experiment. Pioglitazone combined with HBB appears to be a promising chemopreventative adjunct treatment for HNSCC in FA patients. Further studies are underway to evaluate the relative contribution of HBB to decreased cell proliferation versus apoptotic mechanisms. Recent studies of HBB in breast cancer indicate cytochrome P450 arachidonic acid epoxygenase activity as an HBB target and suggest further exploration of this target in head and neck squamous cell carcinoma. Citation Format: Kimberly A. Miller, Beverly R. Wuertz, Anna M. Haynes, David A. Potter, Frank G. Ondrey. Potential chemopreventative treatments in Fanconi anemia related HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-171.

Transcriptional control of O6 -methylguanine DNA methyltransferase expression and temozolomide resistance in glioblastoma.

O6 -methylguanine DNA methyltransferase (MGMT) promoter methylation is a predictive biomarker for benefit from alkylating chemotherapy, specifically temozolomide (TMZ), in glioblastoma, the most common malignant intrinsic brain tumor. Glioma-initiating cells (GIC) with stem-like properties have been associated with resistance to therapy and progression. We assessed the levels of MGMT mRNA and MGMT protein by real-time PCR and immunoblot and evaluated the impact of MGMT on TMZ sensitivity in clonogenicity assays in GIC sphere cultures (S) or differentiated adherent monolayer cultures (M). Nuclear factor kappa B (NF-κB) signaling was assessed by reporter assay and immunoblot. Compared to M cells, S cells expressed higher levels of MGMT. Differentiation of GIC induced by S-to-M transition resulted in a gradual loss of MGMT expression and increased TMZ sensitivity. This transcriptional regulation of MGMT was restricted to cell lines without MGMT promoter methylation and was not coupled to any specific neurobasal (NB) stem cell medium supplement or loss of cell adhesion. Expression levels of p50/p65 subunits of NF-κB, a transcriptional regulator of MGMT, were increased in S cells. Inhibition of NF-κB by the small molecule inhibitor, BAY 11-7082, or siRNA-mediated gene silencing, reduced MGMT levels. In summary, alkylator resistance of S cells is mainly promoted by over-expression of MGMT which results from increased activity of the NF-κB pathway in this cell culture model of glioma stem-like cells. Read the Editorial Highlight for this article on page 688.

Establishing Primary Human Glioblastoma Adherent Cultures from Operative Specimens.

This chapter describes a method for isolation, maintenance, and propagation of primary glioblastoma (GBM) cells in adherent monolayer cultures from patient tumor specimens. This method enables the establishment of GBM cultures with stem or progenitor-like cell characteristics, including self-renewal capacity, differentiation along restricted neural lineages, and tumor-initiating potential when orthotopically injected into immunocompromised mice. This experimentally tractable model system is therefore suitable for a wide variety of analyses in vitro as well as in vivo. Key examples of biological analyses that can be performed using these cells are also described.

A morphology-based assay platform for neuroepithelial-like cells differentiated from human pluripotent stem cells.

Cell morphology is recognized as an important hallmark of neural cells. During the differentiation of human pluripotent stem cells (hPSCs) into neural cells, cell morphology changes dynamically. Therefore, characterization of the morphology of cells during this period is important to improve our understanding of the differentiation and development of neural cells. General methods for the directed induction of hPSCs include the steps of multi-cellular aggregation or high-density cell culture, particularly at the early phase of neural differentiation, and therefore, the morphology of each differentiating cell is difficult to recognize. Here, we have developed a new method for the directed differentiation of neuroepithelial-like cells (NELCs) from hPSCs at a low cell density in an adherent monolayer culture, as well as an image-processing algorithm to evaluate the cell morphology of differentiating NELCs, in order to follow cell morphology during the differentiation of hPSCs into NELCs. Using these methods, the morphological transition of differentiating cells was observed in real time using phase contrast imaging and then quantified. Because cell morphology is also considered an inherent biological marker of neural cells cultured in vitro, this method is potentially useful to study the mechanisms underlying neural cell differentiation.

Establishing Primary Human Glioblastoma Tumorsphere Cultures from Operative Specimens.

In vitro propagation of patient-derived glioblastoma (GBM) cells can be achieved either by adherent monolayer culture, already described in Chapter 3 , or by tumorsphere culture in suspension. Here, we provide a detailed protocol for establishing patient-derived tumorsphere cultures. Such cultures are enriched for GBM stem cells (GSCs) and can be used to generate orthotopic tumor xenografts in the brain of immunocompromised mice. We also point out nuances in the protocol that can increase the yield of successful cultures from operative specimens.

Nicotinamide alone accelerates the conversion of mouse embryonic stem cells into mature neuronal populations.

IntroductionVitamin B3 has been shown to play an important role during embryogenesis. Specifically, there is growing evidence that nicotinamide, the biologically active form of vitamin B3, plays a critical role as a morphogen in the differentiation of stem cells to mature cell phenotypes, including those of the central nervous system (CNS). Detailed knowledge of the action of small molecules during neuronal differentiation is not only critical for uncovering mechanisms underlying lineage-specification, but also to establish more effective differentiation protocols to obtain clinically relevant cells for regenerative therapies for neurodegenerative conditions such as Huntington’s disease (HD). Thus, this study aimed to investigate the potential of nicotinamide to promote the conversion of stem cells to mature CNS neurons.MethodsNicotinamide was applied to differentiating mouse embryonic stem cells (mESC; Sox1GFP knock-in 46C cell line) during their conversion towards a neural fate. Cells were assessed for changes in their proliferation, differentiation and maturation; using immunocytochemistry and morphometric analysis methods.ResultsResults presented indicate that 10 mM nicotinamide, when added at the initial stages of differentiation, promoted accelerated progression of ESCs to a neural lineage in adherent monolayer cultures. By 14 days in vitro (DIV), early exposure to nicotinamide was shown to increase the numbers of differentiated βIII-tubulin-positive neurons. Nicotinamide decreased the proportion of pluripotent stem cells, concomitantly increasing numbers of neural progenitors at 4 DIV. These progenitors then underwent rapid conversion to neurons, observed by a reduction in Sox 1 expression and decreased numbers of neural progenitors in the cultures at 14 DIV. Furthermore, GABAergic neurons generated in the presence of nicotinamide showed increased maturity and complexity of neurites at 14 DIV. Therefore, addition of nicotinamide alone caused an accelerated passage of pluripotent cells through lineage specification and further to non-dividing mature neurons.ConclusionsOur results show that, within an optimal dose range, nicotinamide is able to singly and selectively direct the conversion of embryonic stem cells to mature neurons, and therefore may be a critical factor for normal brain development, thus supporting previous evidence of the fundamental role of vitamins and their metabolites during early CNS development. In addition, nicotinamide may offer a simple effective supplement to enhance the conversion of stem cells to clinically relevant neurons.

MiR‑146b‑5p promotes the neural conversion of pluripotent stem cells by targeting Smad4.

Pluripotent stem cells (PSCs) are regarded as potential sources that provide specific neural cells for cell therapy in some nervous system diseases. However, the mechanisms underlying the neural differentiation of PSCs remain largely unknown. MicroRNAs (miRNAs or miRs) are a class of small non-protein-coding RNAs that act as critical regulatory molecules in many cellular processes. In this study, we found that miR-146b-5p expression was markedly increased following the neural induction of mouse embryonic stem cells (ESCs) or induced PSCs (iPSCs). In this study, to further identify the role of miR-146b-5p, we generated stable miR-146b-5p- overexpressing ESC and iPSC cell lines, and induced the differentiation of these cells by the adherent monolayer culture method. In the miR-146b-5p-overexpressing ESC- or iPSC- derived cultures, RT-qPCR analysis revealed that the mRNA expression levels of neuroectoderm markers, such as Sox1, Nestin and Pax6, were markedly increased, and flow cytometric analysis verified that the number of Nestin-positive cells was higher in the miR-146b-5p-overexpressing compared with the control cells. Mechanistically, the miR-146b-5p-overexpressing ESCs or iPSCs exhibited a significant reduction in Oct4 expression, which may be an explanation for these cells having a tendency to differentiate towards the neural lineage. Moreover, we confirmed that miR-146b-5p directly targeted Smad4 and negatively regulated the transforming growth factor (TGF)-β signaling pathway, which contributed to the neural commitment of PSCs. Collectively, our findings uncover the essential role of miR-146b-5p in the neural conversion of PSCs.