Functional outcome of the anterior vaginal wall in a pelvic surgery injury rat model after treatment with stem cell-derived progenitors of smooth muscle cells.

Background: Stem-cell-derived therapy is a promising option for tissue regeneration. Human iPSC-derived progenitors of smooth muscle cells (pSMCs) have limited proliferation and differentiation, which may minimize the risk of in vivo tumor formation while restoring smooth muscle cell deficiencies. Up to 30 % of women who suffer from recurrence of vaginal prolapse after prolapse surgery are faced with reoperation. Therefore, there is an unmet need for therapies that can restore vaginal tissue function. We hypothesize that human pSMCs can restore vaginal function in a vaginal-injury rat model. Methods: Female immune-compromised RNU rats were divided into 5 groups: intact controls (n=12), VSHAM (surgery + saline injection, n=33), and cell-injection group (surgery + cell injection using three patient pSMCs lines, n=14/cell line). The surgery, similar to what is done in vaginal prolapse surgery, involved ovariectomy, urethrolysis, and vagina injury. The vagina, urethra, bladder dome and trigone were harvested 10 weeks after surgery (5 weeks after injection). Organ bath myography was performed to evaluate the contractile function of vagina, and smooth muscle thickness was examined by tissue immunohistochemistry. Collagen I, collagen III, and elastin mRNA and protein expressions in tissues were assessed. Results: When compared to the VSHAM group, cell-injection groups showed significantly increased vaginal smooth muscle contractions induced by carbachol (groups A and C) and by KCl (group C), and significantly higher collagen I protein expression in the vagina (groups A and B). Elastin mRNA and protein expressions in the vagina did not correlate with injection group. In the urethra, mRNA expressions of collagen I, collagen III, and elastin were all significantly higher in the cell-injection groups compared to the VSHAM group. Collagen I protein expression of the urethra was also higher in the cell-injection group compared to the VSHAM group. Elastin protein expression in the urethra did not correlate with injection group. Conclusions: Human iPSC-derived pSMCs improved contractile function of the post-surgery vagina. Additionally, pSMC injection modulated collagen I, collagen III and elastin mRNA and protein expressions in the vagina and urethra. These findings suggest that pSMCs may be a possible therapy for vaginal prolapse recurrence after surgical intervention.

Generation of an induced pluripotent stem cell line from a patient with arrhythmogenic right ventricular cardiomyopathy harboring a TMEM43 splice-site variant.

Arrhythmogenic cardiomyopathy (ACM) is a cardiomyopathy that is predominantly inherited and characterized by cardiac arrhythmias and structural abnormalities. TMEM43 (transmembrane protein 43) is one of the well-known genetic culprits behind ACM. In this study, we successfully generated an induced pluripotent stem cell (iPSC) line, YCMi010-A, derived from a male patient diagnosed with ACM. Although these iPSCs harbored a heterozygous intronic splice variant, TMEM43 c.443-2A > G, they still displayed normal cellular morphology and were confirmed to express pluripotency markers. YCMi010-A iPSC line is a promising model for investigating the pathomechanisms associated with ACM and exploring potential therapeutic strategies.

Oxidative DNA damage contributes to usnic acid-induced toxicity in human induced pluripotent stem cell-derived hepatocytes.

Dietary supplements containing usnic acid have been increasingly marketed for weight loss over the past decades, even though incidences of severe hepatotoxicity and acute liver failure due to their overuse have been reported. To date, the toxic mechanism of usnic acid-induced liver injury at the molecular level still remains to be fully elucidated. Here, we conducted a transcriptomic study on usnic acid using a novel in vitro hepatotoxicity model employing human induced pluripotent stem cell (iPSC)-derived hepatocytes. Treatment with 20 µM usnic acid for 24 h caused 4272 differentially expressed genes (DEGs) in the cells. Ingenuity Pathway Analysis (IPA) based on the DEGs and gene set enrichment analysis (GSEA) using the whole transcriptome expression data concordantly revealed several signaling pathways and biological processes that, when taken together, suggest that usnic acid caused oxidative stress and DNA damage in the cells, which further led to cell cycle arrest and eventually resulted in cell death through apoptosis. These transcriptomic findings were subsequently corroborated by a variety of cellular assays, including reactive oxygen species (ROS) generation and glutathione (GSH) depletion, DNA damage (pH2AX detection and 8-hydroxy-2'-deoxyguanosine [8-OH-dg] assay), cell cycle analysis, and caspase 3/7 activity. Collectively, the results of the current study accord with previous in vivo and in vitro findings, provide further evidence that oxidative stress-caused DNA damage contributes to usnic acid-induced hepatotoxicity, shed new light on molecular mechanisms of usnic acid-induced hepatotoxicity, and demonstrate the usefulness of iPSC-derived hepatocytes as an in vitro model for hepatotoxicity testing and prediction.

Generation of induced pluripotent stem cell lines from patients with LQT1 caused by heterozygous mutations in the KCNQ1 gene.

Long QT Syndrome (LQTS) is a genetic heart disorder that can induce cardiac arrhythmias. The most prevalent subtype, LQT1, stems from rare variants in the KCNQ1 gene. Utilizing induced pluripotent stem cells (iPSCs) enables detailed cellular studies and personalized medicine approaches for this life-threatening condition. We generated two LQT1 iPSC lines with single nucleotide nonsense mutations, c.1031 C > T and c.1121 T > A in KCNQ1. Both lines exhibited typical iPSC morphology, expressed high levels of pluripotent markers, maintained normal karyotype, and possessed the capability to differentiate into three germ layers. These cell lines serve as important tools for investigating the biological mechanisms underlying LQT1 due to mutations in the KCNQ1 gene.

Mutations in the postsynaptic density signaling hub TNIK disrupt PSD signaling in human models of neurodevelopmental disorders.

A large number of synaptic proteins have been recurrently associated with complex brain disorders. One of these proteins, the Traf and Nck interacting kinase (TNIK), is a postsynaptic density (PSD) signaling hub, with many variants reported in neurodevelopmental disorder (NDD) and psychiatric disease. While rodent models of TNIK dysfunction have abnormal spontaneous synaptic activity and cognitive impairment, the role of mutations found in patients with TNIK protein deficiency and TNIK protein kinase activity during early stages of neuronal and synapse development has not been characterized. Here, using hiPSC-derived excitatory neurons, we show that TNIK mutations dysregulate neuronal activity in human immature synapses. Moreover, the lack of TNIK protein kinase activity impairs MAPK signaling and protein phosphorylation in structural components of the PSD. We show that the TNIK interactome is enriched in NDD risk factors and TNIK lack of function disrupts signaling networks and protein interactors associated with NDD that only partially overlap to mature mouse synapses, suggesting a differential role of TNIK in immature synapsis in NDD.

Advances in the Generation of Constructed Cardiac Tissue Derived from Induced Pluripotent Stem Cells for Disease Modeling and Therapeutic Discovery.

The human heart lacks significant regenerative capacity; thus, the solution to heart failure (HF) remains organ donation, requiring surgery and immunosuppression. The demand for constructed cardiac tissues (CCTs) to model and treat disease continues to grow. Recent advances in induced pluripotent stem cell (iPSC) manipulation, CRISPR gene editing, and 3D tissue culture have enabled a boom in iPSC-derived CCTs (iPSC-CCTs) with diverse cell types and architecture. Compared with 2D-cultured cells, iPSC-CCTs better recapitulate heart biology, demonstrating the potential to advance organ modeling, drug discovery, and regenerative medicine, though iPSC-CCTs could benefit from better methods to faithfully mimic heart physiology and electrophysiology. Here, we summarize advances in iPSC-CCTs and future developments in the vascularization, immunization, and maturation of iPSC-CCTs for study and therapy.

MEA-NAP compares microscale functional connectivity, topology, and network dynamics in organoid or monolayer neuronal cultures.

Microelectrode array (MEA) recordings are commonly used to compare firing and burst rates in neuronal cultures. MEA recordings can also reveal microscale functional connectivity, topology, and network dynamics-patterns seen in brain networks across spatial scales. Network topology is frequently characterized in neuroimaging with graph theoretical metrics. However, few computational tools exist for analyzing microscale functional brain networks from MEA recordings. Here, we present a MATLAB MEA network analysis pipeline (MEA-NAP) for raw voltage time-series acquired from single- or multi-well MEAs. Applications to 3D human cerebral organoids or 2D human-derived or murine cultures reveal differences in network development, including topology, node cartography, and dimensionality. MEA-NAP incorporates multi-unit template-based spike detection, probabilistic thresholding for determining significant functional connections, and normalization techniques for comparing networks. MEA-NAP can identify network-level effects of pharmacologic perturbation and/or disease-causing mutations and, thus, can provide a translational platform for revealing mechanistic insights and screening new therapeutic approaches.

Urine-derived cells from the aged donor for the 2D/3D modeling of neural cells via iPSCs.

Human neural cell models derived from induced pluripotent stem cells (iPSCs) have been widely accepted to model various neurodegenerative diseases such as Alzheimer's disease (AD) in vitro. Although the most common sources of iPSCs are fibroblasts and peripheral blood mononuclear cells, the collection of these cells is invasive. To reduce the donor's burden, we propose the use of urine-derived cells (UDCs), which can be obtained non-invasively from a urine sample. However, the collection of UDCs from elderly donors suffering from age-related diseases such as AD has not been reported, and it is unknown whether these UDCs from the donor aged over 80 years old can be converted into iPSCs and differentiated into neural cells. In this study, we reported a case of using the UDCs from the urine sample of an 89-year-old AD patient, and the UDCs were successfully reprogrammed into iPSCs and differentiated into neural cells in four different ways: (i) the dual SMAD inhibition with small-molecules via the neural progenitor precursor stage, (ii) the rapid induction method using transient expression of Ngn2 and microRNAs without going through the neural progenitor stage, (iii) the cortical brain organoids for 3D culture, and (iv) the human astrocytes. The accumulation of phosphorylated Tau proteins, which is a pathological hallmark of AD, was examined in the neuronal models generated from the UDCs of the aged donor. The application of this cell source will broaden the target population for disease modeling using iPS technology.

Induced pluripotent stem cells-based disease modeling, drug screening, clinical trials, and reverse translational research for amyotrophic lateral sclerosis.

It has been more than 10 years since the hopes for disease modeling and drug discovery using induced pluripotent stem cell (iPSC) technology boomed. Recently, clinical trials have been conducted with drugs identified using this technology, and some promising results have been reported. For amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, several groups have identified candidate drugs, ezogabine (retigabine), bosutinib, and ropinirole, using iPSCs-based drug discovery, and clinical trials using these drugs have been conducted, yielding interesting results. In our previous study, an iPSCs-based drug repurposing approach was utilized to show the potential of ropinirole hydrochloride (ROPI) in reducing ALS-specific pathological phenotypes. Recently, a phase 1/2a trial was conducted to investigate the effects of ropinirole on ALS further. This double-blind, randomized, placebo-controlled study confirmed the safety and tolerability of and provided evidence of its ability to delay disease progression and prolong the time to respiratory failure in ALS patients. Furthermore, in the reverse translational research, in vitro characterization of patient-derived iPSCs-motor neurons (MNs) mimicked the therapeutic effects of ROPI in vivo, suggesting the potential application of this technology to the precision medicine of ALS. Interestingly, RNA-seq data showed that ROPI treatment suppressed the sterol regulatory element-binding protein 2-dependent cholesterol biosynthesis pathway. Therefore, this pathway may be involved in the therapeutic effect of ROPI on ALS. The possibility that this pathway may be involved in the therapeutic effect of ALS was demonstrated. Finally, new future strategies for ALS using iPSCs technology will be discussed in this paper.

Nicotine-mediated effects in neuronal and mouse models of synucleinopathy.

Introduction: Alpha-synuclein (α-Syn) aggregation, transmission, and contribution to neurotoxicity represent central mechanisms underlying Parkinson's disease. The plant alkaloid "nicotine" was reported to attenuate α-Syn aggregation in different models, but its precise mode of action remains unclear. Methods: In this study, we investigated the effect of 2-week chronic nicotine treatment on α-Syn aggregation, neuroinflammation, neurodegeneration, and motor deficits in D-line α-Syn transgenic mice. We also established a novel humanized neuronal model of α-Syn aggregation and toxicity based on treatment of dopaminergic neurons derived from human induced pluripotent stem cells (iPSC) with α-Syn preformed fibrils (PFF) and applied this model to investigate the effects of nicotine and other compounds and their modes of action. Results and discussion: Overall, our results showed that nicotine attenuated α-Syn-provoked neuropathology in both models. Moreover, when investigating the role of nicotinic acetylcholine receptor (nAChR) signaling in nicotine's neuroprotective effects in iPSC-derived dopaminergic neurons, we observed that while α4-specific antagonists reduced the nicotine-induced calcium response, α4 agonists (e.g., AZD1446 and anatabine) mediated similar neuroprotective responses against α-Syn PFF-provoked neurodegeneration. Our results show that nicotine attenuates α-Syn-provoked neuropathology in vivo and in a humanized neuronal model of synucleinopathy and that activation of α4ß2 nicotinic receptors might mediate these neuroprotective effects.

Functional evaluation of rare OASL variants by analysis of SLE patient-derived iPSCs.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by genetic heterogeneity and an interferon (IFN) signature. The overall landscapes of the heritability of SLE remains unclear. OBJECTIVES: To identify and elucidate the biological functions of rare variants underlying SLE, we conducted analyses of patient-derived induced pluripotent stem cells (iPSCs) in combination with genetic analysis. METHODS: Two familial SLE patient- and two healthy donor (HD)-derived iPSCs were established. Type 1 IFN-secreting dendritic cells (DCs) were differentiated from iPSCs. Genetic analyses of SLE-iPSCs, and 117 SLE patients and 107 HDs in the ImmuNexUT database were performed independently. Genome editing of the variants on iPSCs was performed with the CRISPR/Cas9 system. RESULTS: Type 1 IFN secretion was significantly increased in DCs differentiated from SLE-iPSCs compared to HD-iPSCs. Genetic analyses revealed a rare variant in the 2'-5'-Oligoadenylate Synthetase Like (OASL) shared between SLE-iPSCs and another independent SLE patient, and significant accumulation of OASL variants among SLE patients (HD 0.93%, SLE 6.84%, OR 8.387) in the database. Genome editing of mutated OASL 202Q to wild-type 202 R or wild-type OASL 202 R to mutated 202Q resulted in reduced or enhanced Type 1 IFN secretion of DCs. Three other OASL variants (R60W, T261S and A447V) accumulated in SLE patients had also capacities to enhance Type 1 IFN secretion in response to dsRNA. CONCLUSIONS: We established a patient-derived iPSC-based strategy to investigate the linkage of genotype and phenotype in autoimmune diseases. Detailed case-based investigations using patient-derived iPSCs provide information to unveil the heritability of the pathogenesis of autoimmune diseases.

A pilot investigation of differential hydroxymethylation levels in patient-derived neural stem cells implicates altered cortical development in bipolar disorder.

Introduction: Bipolar disorder (BD) is a chronic mental illness characterized by recurrent episodes of mania and depression and associated with social and cognitive disturbances. Environmental factors, such as maternal smoking and childhood trauma, are believed to modulate risk genotypes and contribute to the pathogenesis of BD, suggesting a key role in epigenetic regulation during neurodevelopment. 5-hydroxymethylcytosine (5hmC) is an epigenetic variant of particular interest, as it is highly expressed in the brain and is implicated in neurodevelopment, and psychiatric and neurological disorders. Methods: Induced pluripotent stem cells (iPSCs) were generated from the white blood cells of two adolescent patients with bipolar disorder and their same-sex age-matched unaffected siblings (n = 4). Further, iPSCs were differentiated into neuronal stem cells (NSCs) and characterized for purity using immuno-fluorescence. We used reduced representation hydroxymethylation profiling (RRHP) to perform genome-wide 5hmC profiling of iPSCs and NSCs, to model 5hmC changes during neuronal differentiation and assess their impact on BD risk. Functional annotation and enrichment testing of genes harboring differentiated 5hmC loci were performed with the online tool DAVID. Results: Approximately 2 million sites were mapped and quantified, with the majority (68.8%) located in genic regions, with elevated 5hmC levels per site observed for 3' UTRs, exons, and 2-kb shorelines of CpG islands. Paired t-tests of normalized 5hmC counts between iPSC and NSC cell lines revealed global hypo-hydroxymethylation in NSCs and enrichment of differentially hydroxymethylated sites within genes associated with plasma membrane (FDR = 9.1 × 10-12) and axon guidance (FDR = 2.1 × 10-6), among other neuronal processes. The most significant difference was observed for a transcription factor binding site for the KCNK9 gene (p = 8.8 × 10-6), encoding a potassium channel protein involved in neuronal activity and migration. Protein-protein-interaction (PPI) networking showed significant connectivity (p = 3.2 × 10-10) between proteins encoded by genes harboring highly differentiated 5hmC sites, with genes involved in axon guidance and ion transmembrane transport forming distinct sub-clusters. Comparison of NSCs of BD cases and unaffected siblings revealed additional patterns of differentiation in hydroxymethylation levels, including sites in genes with functions related to synapse formation and regulation, such as CUX2 (p = 2.4 × 10-5) and DOK-7 (p = 3.6 × 10-3), as well as an enrichment of genes involved in the extracellular matrix (FDR = 1.0 × 10-8). Discussion: Together, these preliminary results lend evidence toward a potential role for 5hmC in both early neuronal differentiation and BD risk, with validation and more comprehensive characterization to be achieved through follow-up study.

Progress and emerging techniques for biomaterial-based derivation of mesenchymal stem cells (MSCs) from pluripotent stem cells (PSCs).

The use of mesenchymal stem cells (MSCs) for clinical purposes has skyrocketed in the past decade. Their multilineage differentiation potentials and immunomodulatory properties have facilitated the discovery of therapies for various illnesses. MSCs can be isolated from infant and adult tissue sources, which means they are easily available. However, this raises concerns because of the heterogeneity among the various MSC sources, which limits their effective use. Variabilities arise from donor- and tissue-specific differences, such as age, sex, and tissue source. Moreover, adult-sourced MSCs have limited proliferation potentials, which hinders their long-term therapeutic efficacy. These limitations of adult MSCs have prompted researchers to develop a new method for generating MSCs. Pluripotent stem cells (PSCs), such as embryonic stem cells and induced PSCs (iPSCs), can differentiate into various types of cells. Herein, a thorough review of the characteristics, functions, and clinical importance of MSCs is presented. The existing sources of MSCs, including adult- and infant-based sources, are compared. The most recent techniques for deriving MSCs from iPSCs, with a focus on biomaterial-assisted methods in both two- and three-dimensional culture systems, are listed and elaborated. Finally, several opportunities to develop improved methods for efficiently producing MSCs with the aim of advancing their various clinical applications are described.

BMP4-SMAD1/5/9-RUNX2 pathway activation inhibits neurogenesis and oligodendrogenesis in Alzheimer's patients' iPSCs in senescence-related conditions.

In addition to increasing ß-amyloid plaque deposition and tau tangle formation, inhibition of neurogenesis has recently been observed in Alzheimer's disease (AD). This study generated a cellular model that recapitulated neurogenesis defects observed in patients with AD, using induced pluripotent stem cell lines derived from sporadic and familial AD (AD iPSCs). AD iPSCs exhibited impaired neuron and oligodendrocyte generation when expression of several senescence markers was induced. Compound screening using these cellular models identified three drugs able to restore neurogenesis, and extensive morphological quantification revealed cell-line- and drug-type-dependent neuronal generation. We also found involvement of elevated Sma- and Mad-related protein 1/5/9 (SMAD1/5/9) phosphorylation and greater Runt-related transcription factor 2 (RUNX2) expression in neurogenesis defects in AD. Moreover, BMP4 was elevated in AD iPSC medium during neural differentiation and cerebrospinal fluid of patients with AD, suggesting a BMP4-SMAD1/5/9-RUNX2 signaling pathway contribution to neurogenesis defects in AD under senescence-related conditions.

Characterization of cellular phenotypes in neurons derived from induced pluripotent stem cells of male patients with Fabry disease.

Fabry disease (FD) is an X-linked inherited lysosomal metabolism disorder in which globotriaosylceramide (Gb3) accumulates in various organs resulting from a deficiency in alpha-galactosidase A. The clinical features of FD include progressive impairments of the renal, cardiac, and peripheral nervous systems. In addition, patients with FD often develop neuropsychiatric symptoms, such as depression and dementia, which are believed to be induced by the cellular injury of cerebrovascular and partially neuronal cells due to Gb3 accumulation. Although the analysis of autopsy brain tissue from patients with FD showed no accumulation of Gb3, abnormal deposits of Gb3 were found in the neurons of several brain areas, including the hippocampus. Therefore, in this study, we generated induced pluripotent stem cells (iPSCs) from patients with FD and differentiated them into neuronal cells to investigate pathological and biological changes in the neurons of FD. Neural stem cells (NSCs) and neurons were successfully differentiated from the iPSCs we generated; however, cellular damage and morphological changes were not found in these cells. Immunostaining revealed no Gb3 accumulation in NSCs and neurons. Transmission electron microscopy did not reveal any zebra body-like structures or inclusion bodies, which are characteristic of FD. These results indicated that neuronal cells derived from FD-iPSCs exhibited normal morphology and no Gb3 accumulation. It is likely that more in vivo environment-like cultures are needed for iPSC-derived neurons to reproduce disease-specific features.

Research progress of methodological techniques of generation of platelets from induced pluripotent stem cells in vitro / 中国输血杂志

Platelets play a role in hemostasis in vivo, and platelet transfusion is the main means to treat bleeding diseases caused by thrombocytopenia or platelet dysfunction. However, platelets are in short supply due to the increasing demand for platelet products in clinical, the limited number of blood donors and the disadvantages of platelet products such as short shelf life and bacteria contamination. Currently, induced pluripotent stem cells are considered an ideal source for producing platelets in vitro. They have the potential for self-renewal and differentiation into any cell type, and can be obtained and manipulated easily. Given the recent advances in megakaryocytic series, bioreactors, feeder-free cell production and large-scale propagation research, platelet preparations derived from induced pluripotent stem cells have gradually shown great potential for clinical applications. Considering the minimal risk of alloimmunization and tumorigenesis with these blood products, they are promising to become the standard source of future blood transfusions. This paper reviews the research progress of the methodological techniques of in vitro generation of platelets from induced pluripotent stem cells.

Endocardium in Hypoplastic Left Heart Syndrome: Implications from In Vitro Study.

Endocardium lines the inner layer of the heart ventricle and serves as the source of valve endothelial cells and interstitial cells. Previously, endocardium-associated abnormalities in hypoplastic left heart syndrome (HLHS) have been reported, including endocardial fibroelastosis (EFE) and mitral and aortic valve malformation. However, few mechanistic studies have investigated the molecular pathological changes in endocardial cells. Recently, the emergence of a powerful in vitro system-induced pluripotent stem cells (iPSCs)-was applied to study various genetic diseases, including HLHS. This review summarized current in vitro studies in understanding the endocardial pathology in HLHS, emphasizing new findings of the cellular phenotypes and underlying molecular mechanisms. Lastly, a future perspective is provided regarding the better recapitulation of endocardial phenotypes in a dish.

Capability of Human Dendritic Cells Pulsed with Autologous Induced Pluripotent Stem Cell Lysate to Induce Cytotoxic T Lymphocytes against HLA-A33-Matched Cancer Cells.

Irradiated murine induced-pluripotent stem cells (iPSCs) elicit the antitumor response in vivo. However, it is unclear whether human iPSCs would elicit antitumor effects. In the present study, we investigated the capability of human iPSC lysate (iPSL)-pulsed dendritic cells (DCs) (iPSL/DCs) to induce cancer-responsive cytotoxic T lymphocytes (CTLs) in vitro. iPSCs and DCs were induced from peripheral blood mononuclear cells isolated from a human leukocyte antigen (HLA)-A33 homozygous donor. The iPSL was pulsed with immature DCs, which were then stimulated to allow full maturation. The activated DCs were co-cultured with autologous CTLs and their responses to SW48 colorectal carcinoma cells (HLA-A32/A33), T47D breast cancer cells (HLA-A33/A33), and T98G glioblastoma cells (HLA-A02/A02) were tested with enzyme-linked immunospot (ELISPOT) assays. Comprehensive gene expression analysis revealed that the established iPSCs shared numerous tumor-associated antigens with the SW48 and T47D cells. Immunofluorescent analysis demonstrated that the fluorescent-labeled iPSL was captured by the immature DCs within 2 h. iPSL/DCs induced sufficient CTL numbers in 3 weeks for ELISPOT assays, which revealed that the induced CTLs responded to SW48 and T47D cells. Human iPSL/DCs induced cancer-responsive CTLs on HLA-A33-matched cancer cells in vitro and could be a promising universal cancer vaccine for treating and preventing cancer.