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Retention of Somatic Memory Associated with Cell Identity, Age and Metabolism in Induced Pluripotent Stem (iPS) Cells Reprogramming

Khoo TS, Jamal R, Abdul Ghani NA, Alauddin H, Hussin NH, Abdul Murad NA

Stem Cell Rev Rep, 2020 04;16(2):251-261.
PMID: 32016780 DOI: 10.1007/s12015-020-09956-x

The discovery of induced pluripotent stem (iPS) cells in 2006 marked a major breakthrough in regenerative medicine, enabling reversal of terminally differentiated somatic cells into pluripotent stem cells. The embryonic stem (ES) cells-like pluripotency and unlimited self-renewal capability of iPS cells have granted them enormous potential in many applications, particularly regenerative therapy. Unlike ES cells, however, iPS cells exhibit somatic memories which were carried over from the tissue of origin thus limited its translation in clinical applications. This review provides an updated overview of the retention of various somatic memories associated with the cellular identity, age and metabolism of tissue of origin in iPS cells. The influence of cell types, stage of maturation, age and various other factors on the retention of somatic memory has been discussed. Recent evidence of somatic memory in the form of epigenetic, transcriptomic, metabolic signatures and its functional manifestations in both in vitro and in vivo settings also have been reviewed. The increasing number of studies which had adopted isogenic cell lines for comparisons in recent years had facilitated the identification of genuine somatic memories. These memories functionally affect iPS cells and its derivatives and are potentially tumorigenic thus, raising concerns on their safety in clinical application. Various approaches for memory erasure had since being reported and their efficacies were highlighted in this review.

Matched MeSH terms: Induced Pluripotent Stem Cells/metabolism*
Augmentation of angiogenic and endothelial associated gene expression by EDM50 in human chorion-derived stem cells

Manzor NF, Chua KH, Tan GC, Tan AE, Abdul Rahman H

Med J Malaysia, 2008 Jul;63 Suppl A:11-2.
PMID: 19024960

The objective of this study was to investigate the angiogenic potential of human chorion-derived stem cells (CDSC) cultured in medium containing bFGF and VEGF (EDM50). Total RNA was extracted from cells cultured in FD+10% FBS and EDM50. Quantitative RT-PCR was carried out to score the differential mRNA expression of genes involve in angiogenesis and endothelial differentiation. Our finding demonstrated that all angiogenic and endothelial associated genes were expressed higher in EDM50. Expression level of ANG-1, eNOS and VEGFR2 were significantly higher in EDM50 compared to FD+10% FBS. Our results suggested that human CDSC cultured in EDM50 can be used for angiogenesis purpose in regenerative medicine.

Matched MeSH terms: Stem Cells/metabolism*
Revolutionizing colorectal cancer treatment: unleashing the potential of miRNAs in targeting cancer stem cells

Osei GY, Adu-Amankwaah J, Koomson S, Beletaa S, Ahmad MK, Asiamah EA, et al.

Future Oncol, 2023 Nov;19(35):2369-2382.
PMID: 37970643 DOI: 10.2217/fon-2023-0426

Colorectal cancer (CRC) is a significant contributor to cancer mortality worldwide, and the presence of cancer stem cells (CSC) represents a major challenge for achieving effective treatment. miRNAs have emerged as critical regulators of gene expression, and recent studies have highlighted their role in regulating stemness and therapeutic resistance in CRC stem cells. This review highlights the mechanisms of CSC development, therapy resistance and the potential of miRNAs as therapeutic targets for CRC. It emphasizes the promise of miRNAs as a novel approach to CRC treatment and calls for further research to explore effective miRNA-based therapies and strategies for delivering miRNAs to CSCs in vivo.

Matched MeSH terms: Neoplastic Stem Cells/metabolism
Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells

Alhaji SY, Nordin N, Ngai SC, Al Abbar A, Mei L, Abdullah S

Gene, 2020 Oct 20;758:144958.
PMID: 32683073 DOI: 10.1016/j.gene.2020.144958

Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.

Matched MeSH terms: Embryonic Stem Cells/metabolism*; Induced Pluripotent Stem Cells/metabolism*
Fulltext Dental derived stem cell conditioned media for hair growth stimulation

Gunawardena TNA, Masoudian Z, Rahman MT, Ramasamy TS, Ramanathan A, Abu Kasim NH

PLoS One, 2019;14(5):e0216003.
PMID: 31042749 DOI: 10.1371/journal.pone.0216003

Alopecia is a clinical condition caused by excessive hair loss which may result in baldness, the causes of which still remain elusive. Conditioned media (CM) from stem cells shows promise in regenerative medicine. Our aim was to evaluate the potential CM of dental pulp stem cells obtained from human deciduous teeth (SHED-CM) to stimulate hair growth under in vitro and in vivo conditions. SHED and hair follicle stem cells (HFSCs) (n = 3) were cultured in media combinations; i) STK2, ii) DMEM-KO+10% FBS, iii) STK2+2% FBS and profiled for the presence of positive hair growth-regulatory paracrine factors; SDF-1, HGF, VEGF-A, PDGF-BB and negative hair growth-regulatory paracrine factors; IL-1α, IL-1β, TGF-β, bFGF, TNF-α, and BDNF. The potential of CM from both cell sources to stimulate hair growth was evaluated based on the paracrine profile and measured dynamics of hair growth under in vitro conditions. The administration of CM media to telogen-staged synchronized 7-week old C3H/HeN female mice was carried out to study the potential of the CM to stimulate hair growth in vivo. SHED and HFSCs cultured in STK2 based media showed a shorter population doubling time, higher viability and better maintenance of MSC characteristics in comparison to cells cultured in DMEM-KO media. STK2 based CM contained only two negative hair growth-regulatory factors; TNF-α, IL-1 while DMEM-KO CM contained all negative hair growth-regulatory factors. The in vitro study confirmed that treatment with STK2 based media CM from passage 3 SHED and HFSCs resulted in a significantly higher number of anagen-staged hair follicles (p<0.05) and a significantly lower number of telogen-staged hair follicles (p<0.05). Administration of SHED-CM to C3H/HeN mice resulted in a significantly faster stimulation of hair growth in comparison to HFSC-CM (p<0.05), while the duration taken for complete hair coverage was similar for both CM sources. Thus, SHED-CM carries the potential to stimulate hair growth which can be used as a treatment tool for alopecia.

Matched MeSH terms: Adult Stem Cells/metabolism
Neuroimmunomodulatory properties of DPSCs in an in vitro model of Parkinson's disease

Gnanasegaran N, Govindasamy V, Mani V, Abu Kasim NH

IUBMB Life, 2017 09;69(9):689-699.
PMID: 28685937 DOI: 10.1002/iub.1655

In neurodegenerative diseases, such as Alzheimer's and Parkinson's, microglial cell activation is thought to contribute to their degeneration by producing neurotoxic compounds. While dental pulp stem cells (DPSCs) have been regarded as the next possible cell source for cell replacement therapy (CRT), their actual role when exposed in such harsh environment remains elusive. In this study, the immunomodulatory behavior of DPSCs from human subjects was investigated in a coculture system consisting of neuron and microglia which were treated with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine, which mimics the inflammatory conditions and contribute to degeneration of dopaminergic (DA-ergic) neurons. Assessments were performed on their proliferation, extent of DNA damage, productions of reactive oxygen species (ROS) and nitric oxide (NO), as well as secretion of inflammatory mediators. Notably, DPSCs were shown to attenuate their proliferation, production of ROS, and NO significantly (P

Matched MeSH terms: Stem Cells/metabolism
Effects of cell cycle phases on the induction of dental pulp stem cells toward dopaminergic-like cells

Gnanasegaran N, Govindasamy V, Kathirvaloo P, Musa S, Abu Kasim NH

J Tissue Eng Regen Med, 2018 02;12(2):e881-e893.
PMID: 28079995 DOI: 10.1002/term.2401

Parkinson's disease (PD) is characterized by tremors and cognitive issues, and is due to the death of dopaminergic (DA-ergic) neurons in brain circuits that are responsible for producing neurotransmitter dopamine (DA). Currently, cell replacement therapies are underway to improve upon existing therapeutic approaches such as drug treatments and electrical stimulation. Among the widely available sources, dental pulp stem cells (DPSCs) from deciduous teeth have gained popularity because of their neural crest origin and inherent propensity toward neuronal lineage. Despite the various pre-clinical studies conducted, an important factor yet to be elucidated is the influence of growth phases in a typical trans-differentiation process. This study selected DPSCs at three distinct time points with variable growth phase proportions (G0/G1, S and G2/M) for in vitro trans-differentiation into DA-ergic-like cells. Using commercially available PCR arrays, we identified distinct gene profiles pertaining to cell cycles in these phases. The differentiation outcomes were assessed in terms of morphology and gene and protein expression, as well as with functional assays. It was noted that DPSCs with the highest G0/G1 phase were comparatively the best, representing at least a 2-fold up regulation (p

Matched MeSH terms: Stem Cells/metabolism
CD133: beyond a cancer stem cell biomarker

Barzegar Behrooz A, Syahir A, Ahmad S

J Drug Target, 2019 03;27(3):257-269.
PMID: 29911902 DOI: 10.1080/1061186X.2018.1479756

CD133 (prominin-1), a pentaspan membrane glycoprotein, is one of the most well-characterized biomarkers used for the isolation of cancer stem cells (CSCs). The presence of CSCs is one of the main causes of tumour reversal and resilience. Accumulating evidence has shown that CD133 might be responsible for CSCs tumourigenesis, metastasis and chemoresistance. It is now understood that CD133 interacts with the Wnt/β-catenin and PI3K-Akt signalling pathways. Moreover, CD133 can upregulate the expression of the FLICE-like inhibitory protein (FLIP) in CD133-positive cells, inhibiting apoptosis. In addition, CD133 can increase angiogenesis by activating the Wnt signalling pathway and increasing the expression of vascular endothelial growth factor-A (VEGF-A) and interleukin-8. Therefore, CD133 could be considered to be an 'Achilles' heel' for CSCs, because by inhibiting this protein, the signalling pathways that are involved in cell proliferation will also be inhibited. By understanding the molecular biology of CD133, we can not only isolate stem cells but can also utilise it as a therapeutic strategy. In this review, we summarise new insights into the fundamental cell biology of CD133 and discuss the involvement of CD133 in metastasis, metabolism, tumourigenesis, drug-resistance, apoptosis and autophagy.

Matched MeSH terms: Neoplastic Stem Cells/metabolism*
Cancer-associated fibroblasts of colorectal cancer and their markers: updates, challenges and translational outlook

Musa M, Ali A

Future Oncol, 2020 Oct;16(29):2329-2344.
PMID: 32687721 DOI: 10.2217/fon-2020-0384

Accumulation of cancer-associated fibroblasts (CAFs) in the tumor microenvironment is associated with poor prognosis and recurrence of colorectal cancer (CRC). Despite their prominent roles in colorectal carcinogenesis, there is a lack of robust and specific markers to classify the heterogeneous and highly complex CAF populations. This has resulted in confusing and misleading definitions of CAFs in cancer niche. Advancements in molecular biology approaches have open doors to reliable CAF marker detection methods in various solid tumors. These discoveries would contribute to more efficient screening, monitoring and targeted therapy of CRC thus potentially will reduce cancer morbidity and mortality rates. This review highlights current scenarios, dilemma, translational potentials of CAF biomarker and future therapeutic applications involving CAF marker identification in CRC.

Matched MeSH terms: Neoplastic Stem Cells/metabolism
Fulltext Evaluation of Indonesian mangrove Xylocarpus granatum leaves ethyl acetate extract as potential anticancer drug

Darmadi J, Batubara RR, Himawan S, Azizah NN, Audah HK, Arsianti A, et al.

Sci Rep, 2021 Mar 16;11(1):6080.
PMID: 33727582 DOI: 10.1038/s41598-021-85383-3

Local Xylocarpus granatum leaves were extracted by ethyl acetate solvent and characterized by TLC fingerprinting and 2D 1H NMR spectroscopy to contain phenolic compounds as well as several organic and amino acids as metabolic byproducts, such as succinic acid and acetic acid. Traces of flavonoids and other non-categorized phenolic compounds exhibited intermediate antioxidant activity (antioxidant IC50 84.93 ppm) as well as anticancer activity against HeLa, T47D, and HT-29 cell lines; which the latter being most effective against HT-29 with Fraction 5 contained the strongest activity (anticancer IC50 23.12 ppm). Extracts also behaved as a natural growth factor and nonlethal towards brine shrimps as well as human adipose-derived stem cell hADSC due to antioxidative properties. A stability test was performed to examine how storage conditions factored in bioactivity and phytochemical structure. Extracts were compared with several studies about X. granatum leaves extracts to evaluate how ethnogeography and ecosystem factored on biologically active compounds. Further research on anticancer or antioxidant mechanism on cancer cells is needed to determine whether the extract is suitable as a candidate for an anticancer drug.

Matched MeSH terms: Stem Cells/metabolism
Amniotic Membrane Enhance the Effect of Vascular Endothelial Growth Factor on the Angiogenic Marker Expression of Stem Cells from Human Exfoliated Deciduous Teeth

Yusof MFH, Hashim SNM, Zahari W, Chandra H, Noordin KBAA, Kannan TP, et al.

Appl Biochem Biotechnol, 2020 May;191(1):177-190.
PMID: 32096060 DOI: 10.1007/s12010-020-03266-1

Previously, it was reported that human amniotic membrane (AM) induced stem cells from human deciduous exfoliated teeth (SHED) endothelial-like-cell differentiation. This interesting effect of AM matrix on SHED demands further elucidation. Objective of this in vitro work was to study the effect of 24-h VEGF induced on SHED endothelial differentiation when seeded on acellular stromal side (SS) of AM matrix. Stemness of SHED was identified by flow cytometry. Cell attachment and morphological changes towards the matrix was observed by scanning electron microscopy. Protein expression of endothelial marker was examined by Western blot. The expression of stem cells and endothelial-specific gene markers of VEGF-induced SHED cultured on human AM was inspected via reverse transcriptase-polymerase chain reaction. Results showed SHED at both passages retain stemness property. Ang-1 protein was expressed in SHED. Cells treated with VEGF and cultured on AM transformed attached well to AM. VEGF-induced SHED expressed both stem cell and endothelial-specific markers throughout the treatments and timeline. Interestingly, prolonged VEGF treatment increased the expression of Cox-2 and VE-Cadherin genes in all treated groups when compared to SHED. It was concluded that the VEGF-induced SHED showed better expression of endothelial-specific markers when cultured on SS of AM, with prolonged VEGF treatment.

Matched MeSH terms: Stem Cells/metabolism*
Fulltext Stearoyl CoA Desaturase Is Essential for Regulation of Endoplasmic Reticulum Homeostasis and Tumor Growth in Glioblastoma Cancer Stem Cells

Pinkham K, Park DJ, Hashemiaghdam A, Kirov AB, Adam I, Rosiak K, et al.

Stem Cell Reports, 2019 04 09;12(4):712-727.
PMID: 30930246 DOI: 10.1016/j.stemcr.2019.02.012

Inherent plasticity and various survival cues allow glioblastoma stem-like cells (GSCs) to survive and proliferate under intrinsic and extrinsic stress conditions. Here, we report that GSCs depend on the adaptive activation of ER stress and subsequent activation of lipogenesis and particularly stearoyl CoA desaturase (SCD1), which promotes ER homeostasis, cytoprotection, and tumor initiation. Pharmacological targeting of SCD1 is particularly toxic due to the accumulation of saturated fatty acids, which exacerbates ER stress, triggers apoptosis, impairs RAD51-mediated DNA repair, and achieves a remarkable therapeutic outcome with 25%-100% cure rate in xenograft mouse models. Mechanistically, divergent cell fates under varying levels of ER stress are primarily controlled by the ER sensor IRE1, which either promotes SCD1 transcriptional activation or converts to apoptotic signaling when SCD1 activity is impaired. Taken together, the dependence of GSCs on fatty acid desaturation presents an exploitable vulnerability to target glioblastoma.

Matched MeSH terms: Neoplastic Stem Cells/metabolism*
Co-culture of mesenchymal-like stromal cells derived from human foreskin permits long term propagation and differentiation of human embryonic stem cells

Mamidi MK, Pal R, Mori NA, Arumugam G, Thrichelvam ST, Noor PJ, et al.

J Cell Biochem, 2011 May;112(5):1353-63.
PMID: 21337383 DOI: 10.1002/jcb.23052

Among the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal-derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF-MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin-C treatment. Growth pattern, gene profiling (Oct-4, Nanog, Sox-2, Rex-1), cytoskeletal protein expression (vimentin, nestin) and tri-lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF-MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA-4, and negative for CD34, CD45, HLA-DR cell-surface markers and were found to exhibit BM-MSC-like characteristics. hESC lines co-cultured with HF-MSC feeders showed expression of expected pluripotent transcription factors Oct-4, Nanog, Sox-2, GDF-3, Rex-1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA-4, TRA-1-81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical-grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications.

Matched MeSH terms: Pluripotent Stem Cells/metabolism; Embryonic Stem Cells/metabolism
Fulltext Understanding Lung Carcinogenesis from a Morphostatic Perspective: Prevention and Therapeutic Potential of Phytochemicals for Targeting Cancer Stem Cells

Heng WS, Kruyt FAE, Cheah SC

Int J Mol Sci, 2021 May 27;22(11).
PMID: 34071790 DOI: 10.3390/ijms22115697

Lung cancer is still one of the deadliest cancers, with over two million incidences annually. Prevention is regarded as the most efficient way to reduce both the incidence and death figures. Nevertheless, treatment should still be improved, particularly in addressing therapeutic resistance due to cancer stem cells-the assumed drivers of tumor initiation and progression. Phytochemicals in plant-based diets are thought to contribute substantially to lung cancer prevention and may be efficacious for targeting lung cancer stem cells. In this review, we collect recent literature on lung homeostasis, carcinogenesis, and phytochemicals studied in lung cancers. We provide a comprehensive overview of how normal lung tissue operates and relate it with lung carcinogenesis to redefine better targets for lung cancer stem cells. Nine well-studied phytochemical compounds, namely curcumin, resveratrol, quercetin, epigallocatechin-3-gallate, luteolin, sulforaphane, berberine, genistein, and capsaicin, are discussed in terms of their chemopreventive and anticancer mechanisms in lung cancer and potential use in the clinic. How the use of phytochemicals can be improved by structural manipulations, targeted delivery, concentration adjustments, and combinatorial treatments is also highlighted. We propose that lung carcinomas should be treated differently based on their respective cellular origins. Targeting quiescence-inducing, inflammation-dampening, or reactive oxygen species-balancing pathways appears particularly interesting.

Matched MeSH terms: Neoplastic Stem Cells/metabolism
An insight into the associations between microRNA expression and mitochondrial functions in cancer cell and cancer stem cell

Tan WL, Subha ST, Mohtarrudin N, Cheah YK

Mol Biol Rep, 2023 Jun;50(6):5395-5405.
PMID: 37074612 DOI: 10.1007/s11033-023-08421-5

The self-renew ability of cancer stem cells (CSCs) continues to challenge our determination for accomplishing cancer therapy breakthrough. Ineffectiveness of current cancer therapies to eradicate CSCs has contributed to chemoresistance and tumor recurrence. Yet, the discoveries of highly effective therapies have not been thoroughly developed. Further insights into cancer metabolomics and gene-regulated mechanisms of mitochondria in CSCs can expedite the development of novel anticancer drugs. In cancer cells, the metabolism is reprogrammed from oxidative phosphorylation (OXPHOS) to glycolysis. This alteration allows the cancer cell to receive continuous energy supplies and avoid apoptosis. The pyruvate obtained from glycolysis produces acetyl-coenzyme A (Acetyl-CoA) via oxidative decarboxylation and enters the tricarboxylic acid cycle for adenosine triphosphate generation. Mitochondrial calcium ion (Ca2+) uptake is responsible for mitochondrial physiology regulation, and reduced uptake of Ca2+ inhibits apoptosis and enhances cell survival in cancer. There have been many discoveries of mitochondria-associated microRNAs (miRNAs) stimulating the metabolic alterations in mitochondria via gene regulation which promote cancer cell survival. These miRNAs are also found in CSCs where they regulate genes and activate different mechanisms to destroy the mitochondria and enhance CSCs survival. By targeting the miRNAs that induced mitochondrial destruction, the mitochondrial functions can be restored; thus, it triggers CSCs apoptosis and completely eliminates the CSCs. In general, this review article aims to address the associations between miRNAs with mitochondrial activities in cancer cells and cancer stem cells that support cancer cell survival and self-renewal.

Matched MeSH terms: Neoplastic Stem Cells/metabolism
Therapeutic potentials of neural stem cells in Alzheimer's disease

Wong RSY, Cheong SK

Malays J Pathol, 2020 Aug;42(2):157-170.
PMID: 32860368

The commonest cause of dementia among the elderly population is Alzheimer's disease (AD). It is a health concern globally as the number of people affected by dementia worldwide is rapidly increasing. Several genes have been linked to AD and the pathogenesis of the disease has been extensively and vigorously examined. Thus far, only a few drugs have been approved by the Food and Drug Administration (FDA) for the pharmacological treatment of AD and a growing body of research has turned to alternative options such as stem cell therapy. This review will give an overview of the pathological and clinical aspects of AD. Although researchers have explored the suitability and feasibility of using various types of stems cells to treat AD, this review will focus mainly on neural stem cells (NSCs)/ neural progenitor cells (NPCs). The behaviour and properties of NSCs will be described, accompanied by a comprehensive discussion of the therapeutic strategies involving the use of NSCs/NPCs in the treatment of the disease.

Matched MeSH terms: Stem Cells/metabolism
Circular RNA ZNF800 (hsa_circ_0082096) regulates cancer stem cell properties and tumor growth in colorectal cancer

Rengganaten V, Huang CJ, Wang ML, Chien Y, Tsai PH, Lan YT, et al.

BMC Cancer, 2023 Nov 10;23(1):1088.
PMID: 37950151 DOI: 10.1186/s12885-023-11571-1

BACKGROUND: Cancer stem cells form a rare cell population in tumors that contributes to metastasis, recurrence and chemoresistance in cancer patients. Circular RNAs (circRNAs) are post-transcriptional regulators of gene expression that sponge targeted microRNA (miRNAs) to affect a multitude of downstream cellular processes. We previously showed in an expression profiling study that circZNF800 (hsa_circ_0082096) was up-regulated in cancer stem cell-enriched spheroids derived from colorectal cancer (CRC) cell lines.
METHODS: Spheroids were generated in suspension spheroidal culture. The ZNF800 mRNA, pluripotency stem cell markers and circZNF800 levels were determined by quantitative RT-PCR. CircZNF800-miRNA interactions were shown in RNA pulldown assays and the miRNA levels determined by stem-loop qRT-PCR. The effects of circZNF800 on cell proliferation were tested by EdU staining followed by flowcytometry. Expression of stem cell markers CD44/CD133, Lgr5 and SOX9 was demonstrated in immunofluorescence microscopy. To manipulate the cellular levels of circZNF800, circZNF800 over-expression was achieved via transfection of in vitro synthesized and circularized circZNF800, and knockdown attained using a CRISPR-Cas13d-circZNF800 vector system. Xenografted nude mice were used to demonstrate effects of circZNF800 over-expression and knockdown on tumor growth in vivo.
RESULTS: CircZNF800 was shown to be over-expressed in late-stage tumor tissues of CRC patients. Data showed that circZNF800 impeded expression of miR-140-3p, miR-382-5p and miR-579-3p while promoted the mRNA levels of ALK/ACVR1C, FZD3 and WNT5A targeted by the miRNAs, as supported by alignments of seed sequences between the circZNF800-miRNA, and miRNA-mRNA paired interactions. Analysis in CRC cells and biopsied tissues showed that circZNF800 positively regulated the expression of intestinal stem cell, pluripotency and cancer stem cell markers, and promoted CRC cell proliferation, spheroid and colony formation in vitro, all of which are cancer stem cell properties. In xenografted mice, circZNF800 over-expression promoted tumor growth, while circZNF800 knockdown via administration of CRISPR Cas13d-circZNF800 viral particles at the CRC tumor sites impeded tumor growth.
CONCLUSIONS: CircZNF800 is an oncogenic factor that regulate cancer stem cell properties to lead colorectal tumorigenesis, and may be used as a predictive marker for tumor progression and the CRISPR Cas13d-circZNF800 knockdown strategy for therapeutic intervention of colorectal cancer.

Matched MeSH terms: Neoplastic Stem Cells/metabolism
Fulltext Mitochondrial DNA alterations may influence the cisplatin responsiveness of oral squamous cell carcinoma

Aminuddin A, Ng PY, Leong CO, Chua EW

Sci Rep, 2020 May 12;10(1):7885.
PMID: 32398775 DOI: 10.1038/s41598-020-64664-3

Cisplatin is the first-line chemotherapeutic agent for the treatment of oral squamous cell carcinoma (OSCC). However, the intrinsic or acquired resistance against cisplatin remains a major obstacle to treatment efficacy in OSCC. Recently, mitochondrial DNA (mtDNA) alterations have been reported in a variety of cancers. However, the role of mtDNA alterations in OSCC has not been comprehensively studied. In this study, we evaluated the correlation between mtDNA alterations (mtDNA content, point mutations, large-scale deletions, and methylation status) and cisplatin sensitivity using two OSCC cell lines, namely SAS and H103, and stem cell-like tumour spheres derived from SAS. By microarray analysis, we found that the tumour spheres profited from aberrant lipid and glucose metabolism and became resistant to cisplatin. By qPCR analysis, we found that the cells with less mtDNA were less responsive to cisplatin (H103 and the tumour spheres). Based on the findings, we theorised that the metabolic changes in the tumour spheres probably resulted in mtDNA depletion, as the cells suppressed mitochondrial respiration and switched to an alternative mode of energy production, i.e. glycolysis. Then, to ascertain the origin of the variation in mtDNA content, we used MinION, a nanopore sequencer, to sequence the mitochondrial genomes of H103, SAS, and the tumour spheres. We found that the lower cisplatin sensitivity of H103 could have been caused by a constellation of genetic and epigenetic changes in its mitochondrial genome. Future work may look into how changes in mtDNA translate into an impact on cell function and therefore cisplatin response.

Matched MeSH terms: Neoplastic Stem Cells/metabolism
The Promises and Pitfalls of CRISPR-Mediated Base Editing in Stem Cells

Wong PK, Mohamad Zamberi NN, Syafruddin SE, Cheah FC, Azmi N, Law JX, et al.

CRISPR J, 2023 Jun;6(3):196-215.
PMID: 37219623 DOI: 10.1089/crispr.2023.0013

Stem cells such as induced pluripotent stem cells, embryonic stem cells, and hematopoietic stem and progenitor cells are growing in importance in disease modeling and regenerative medicine. The applications of CRISPR-based gene editing to create a mélange of disease and nondisease stem cell lines have further enhanced the utility of this innately versatile group of cells in the studies of human genetic disorders. Precise base edits can be achieved using a variety of CRISPR-centric approaches, particularly homology-directed repair and the recently developed base editors and prime editors. Despite its much-touted potential, editing single DNA bases is technically challenging. In this review, we discuss the strategies for achieving exact base edits in the creation of various stem cell-based models for use in elucidating disease mechanisms and assessing drug efficacy, and the unique characteristics of stem cells that warrant special considerations.

Matched MeSH terms: Embryonic Stem Cells/metabolism
Alteration of gene expression levels during osteogenic induction of human adipose derived stem cells in long-term culture

Safwani WK, Makpol S, Sathapan S, Chua KH

Cell Tissue Bank, 2013 Jun;14(2):289-301.
PMID: 22476937 DOI: 10.1007/s10561-012-9309-1

Adipose tissue is a source of multipotent stem cells and it has the ability to differentiate into several types of cell lineages such as neuron cells, osteogenic and adipogenic cells. Most studies on human adipose-derived stem cells (ASCs) have been carried out at the early passages. For clinical usage, ASCs need to be expanded in vitro for a period of time to get sufficient cells for transplantation into patients. However, the impact of long-term culture on ASCs molecular characteristics has not been established yet. Several studies have also shown that osteogenic and adipogenic cells have the ability to switch pathways during in vitro culture as they share the same progenitor cells. This data is important to ensure their functionality and efficacy before being used clinically in the treatment of bone diseases. Therefore, we aim to investigate the effect of long-term culture on the adipogenic, stemness and osteogenic genes expression during osteogenic induction of ASCs. In this study, the molecular characteristics of ASCs during osteogenic induction in long-term culture was analysed by observing their morphological changes during induction, analysis of cell mineralization using Alizarin Red staining and gene expression changes using quantitative RT-PCR. Morphologically, cell mineralization at P20 was less compared to P5, P10 and P15. Adipogenesis was not observed as negative lipid droplets formation was recorded during induction. The quantitative PCR data showed that adipogenic genes expression e.g. LPL and AP2 decreased but PPAR-γ was increased after osteogenic induction in long-term culture. Most stemness genes decreased at P5 and P10 but showed no significant changes at P15 and P20. While most osteogenic genes increased after osteogenic induction at all passages. When compared among passages after induction, Runx showed a significant increased at P20 while BSP, OSP and ALP decreased at later passage (P15 and P20). During long-term culture, ASCs were only able to differentiate into immature osteogenic cells.

Matched MeSH terms: Pluripotent Stem Cells/metabolism

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