Adult cardiomyocytes-derived EVs for the treatment of cardiac fibrosis.

Cardiac fibrosis is a common pathological feature of cardiovascular diseases that arises from the hyperactivation of fibroblasts and excessive extracellular matrix (ECM) deposition, leading to impaired cardiac function and potentially heart failure or arrhythmia. Extracellular vesicles (EVs) released by cardiomyocytes (CMs) regulate various physiological functions essential for myocardial homeostasis, which are disrupted in cardiac disease. Therefore, healthy CM-derived EVs represent a promising cell-free therapy for the treatment of cardiac fibrosis. To this end, we optimized the culture conditions of human adult CMs to obtain a large yield of EVs without compromising cellular integrity by using a defined combination of small molecules. EVs were isolated by ultracentrifugation, and their characteristics were analysed. Finally, their effect on fibrosis was tested. Treatment of TGFß-activated human cardiac fibroblasts with EVs derived from CMs using our culture system resulted in a decrease in fibroblast activation markers and ECM accumulation. The rescued phenotype was associated with specific EV cargo, including multiple myocyte-specific and antifibrotic microRNAs, although their effect individually was not as effective as the EV treatment. Notably, pathway analysis showed that EV treatment reverted the transcription of activated fibroblasts and decreased several signalling pathways, including MAPK, mTOR, JAK/STAT, TGFß, and PI3K/Akt, all of which are involved in fibrosis development. Intracardiac injection of CM-derived EVs in an animal model of cardiac fibrosis reduced fibrotic area and increased angiogenesis, which correlated with improved cardiac function. These findings suggest that EVs derived from human adult CMs may offer a targeted and effective treatment for cardiac fibrosis, owing to their antifibrotic properties and the specificity of cargo.

Extracellular vesicles as novel uro-oncology biomarkers: insights toward clinical applications.

PURPOSE OF REVIEW: We discussed the challenges associated with the clinical application of extracellular vesicles and summarized their potential impact on oncological clinical practice in urology. RECENT FINDINGS: Despite extensive research on extracellular vesicles, their clinical applications remain limited; this is likely to be because of small study cohorts, a lack of large-scale analyses, and the impact of variable extraction and storage methods on analysis outcomes. However, promising results have emerged from clinical trials targeting urinary extracellular vesicles in prostate cancer using ExoDx Prostate Test. The ExoDx Prostate Test has demonstrated its efficacy in diagnosing prostate cancer in previous studies and is the only FDA-approved kit for this purpose. Moreover, recent trials have investigated the use of the ExoDx Prostate Test to determine the optimal timing for biopsies in prostate cancer patients undergoing active surveillance. SUMMARY: We summarized recent studies on the potential of extracellular vesicles in the management of urological cancers. Particularly, the diagnosis of prostate cancer using the ExoDx Prostate Test has yielded positive results in several clinical trials. Additionally, while there are other studies suggesting its efficacy, most of these are based on retrospective analyses. These findings warrant further large-scale studies to optimize extracellular vesicle-based diagnostic and monitoring strategies. Although further research is required, extracellular vesicles would be attractive for early detection and surveillance.

Mitigation of acute lung injury by human bronchial epithelial cell-derived extracellular vesicles via ANXA1-mediated FPR signaling.

Acute lung injury (ALI) is characterized by respiratory failure resulting from the disruption of the epithelial and endothelial barriers as well as immune system. In this study, we evaluated the therapeutic potential of airway epithelial cell-derived extracellular vesicles (EVs) in maintaining lung homeostasis. We isolated human bronchial epithelial cell-derived EVs (HBEC-EVs), which endogenously express various immune-related surface markers and investigated their immunomodulatory potential in ALI. In ALI cellular models, HBEC-EVs demonstrated immunosuppressive effects by reducing the secretion of proinflammatory cytokines in both THP-1 macrophages and HBECs. Mechanistically, these effects were partially ascribed to nine of the top 10 miRNAs enriched in HBEC-EVs, governing toll-like receptor-NF-κB signaling pathways. Proteomic analysis revealed the presence of proteins in HBEC-EVs involved in WNT and NF-κB signaling pathways, pivotal in inflammation regulation. ANXA1, a constituent of HBEC-EVs, interacts with formyl peptide receptor (FPR)2, eliciting anti-inflammatory responses by suppressing NF-κB signaling in inflamed epithelium, including type II alveolar epithelial cells. In a mouse model of ALI, intratracheal administration of HBEC-EVs reduced lung injury, inflammatory cell infiltration, and cytokine levels. Collectively, these findings suggest the therapeutic potential of HBEC-EVs, through their miRNAs and ANXA1 cargo, in mitigating lung injury and inflammation in ALI patients.

Extracellular vesicles and prostate cancer management: a narrative review.

Background and Objective: Prostate cancer (PCa) is the second most common male cancer in the United States. Although new drugs have recently been approved, clinical challenges remain, notably the precise detection and prognostic implications of drug-resistant PCa. Extracellular vesicles (EVs), nanoscale lipid membrane vesicles, are actively secreted into the extracellular milieu by a variety of cell types. Over the past decade, interest in EVs has grown, and emerging evidence suggests that EVs play pivotal roles in cancer biology. In this review, we would like to summarize recent reports on EVs in PCa and discuss the potential clinical applications. Methods: We performed a non-systematic literature review using the PubMed database to identify articles specifically related to EVs and PCa management. Key Content and Findings: EVs contain pathogenic components, such as proteins, DNA fragments, mRNA, non-coding RNA, and lipids, all of which can trigger intercellular signaling within tumor microenvironments. Thereby, EVs exert significant effects on several stages of cancer progression, influencing the immune system, angiogenesis, and the establishment of pre-metastatic niches. Furthermore, as EVs are encapsulated, their contents are stable in bodily fluids, and thus EVs have recently attracted attention as a novel kind of liquid biopsy. Conclusions: We have summarized recent research on how EVs may aid PCa management. To date, we have discovered only the tip of the iceberg. We anticipate that further research will yield innovative therapeutic modalities, thereby aiding all PCa patients.

Potential utility of pretreatment serum miRNAs for optimal treatment selection in advanced high-grade serous ovarian cancer.

OBJECTIVE: The primary treatment of patients with advanced ovarian cancer is selected from whether primary debulking surgery or neoadjuvant chemotherapy. We investigated whether pretreatment serum microRNA profiles are useful for selecting patients with advanced high-grade serous ovarian cancer who obtain better outcomes from undergoing primary debulking surgery or neoadjuvant chemotherapy. METHODS: Consecutive patients with clinical stage IIIB-IVB and serum microRNA data were selected. Patients who underwent primary debulking surgery or neoadjuvant chemotherapy were subjected to 1:1 propensity score matching before comparing their progression-free survival using Cox modelling. Progression-free probabilities for the selected microRNA profiles were calculated, and the estimated progression-free survival with the recommended primary treatment was determined and compared with the actual progression-free survival of the patients. RESULTS: Of the 108 patients with stage IIIB-IVB disease, the data of 24 who underwent primary debulking surgery or neoadjuvant chemotherapy were compared. Eleven and three microRNAs were independent predictors of progression-free survival in patients who underwent primary debulking surgery and neoadjuvant chemotherapy, respectively. Two microRNAs correlated significantly with complete resection of the tumours in primary debulking surgery. No differences were found between the actual and estimated progression-free survival in the primary debulking surgery and neoadjuvant chemotherapy groups (P > 0.05). The recommended and actual primary treatments were identical in 27 (56.3%) of the 48 patients. The median improved survival times between recommended and actual treatment were 11.7 and 32.6 months for patients with actual primary debulking surgery and neoadjuvant chemotherapy, respectively. CONCLUSIONS: Pretreatment microRNA profiles could be used to select subgroups of patients who benefited more from primary debulking surgery or neoadjuvant chemotherapy and might contribute to selecting the optimal primary treatment modality in advanced high-grade serous ovarian cancer patients.

Liquid biopsy using non-coding RNAs and extracellular vesicles for breast cancer management.

This article examines liquid biopsy using non-coding RNAs and extracellular vesicles in detail. Liquid biopsy is emerging as a prominent non-invasive diagnostic tool in the treatment of breast cancer. We will elucidate the roles of these molecules in early detection, monitoring treatment effectiveness, and prognostic assessment of breast cancer. Additionally, the clinical significance of these molecules will be discussed. We aim to delve into the distinct characteristics of these molecules and their possible roles in breast cancer management, with an anticipation of their contribution to future diagnostic and therapeutic advancements.

Plasma extracellular vesicle microRNAs reflecting the therapeutic effect of the CBP/ß-catenin inhibitor PRI-724 in patients with liver cirrhosis.

There is an unmet need for antifibrotic therapies to prevent the progression of liver cirrhosis. Previously, we conducted an exploratory trial to assess the safety and antifibrotic efficacy of PRI-724, a selective CBP/ß-catenin inhibitor, in patients with liver cirrhosis. PRI-724 was well tolerated and exerted a potential antifibrotic effect. Here, we investigated whether the profiles of circulating microRNAs packaged in extracellular vesicles (EV-miRNAs) are associated with responses to liver fibrosis treatments. Eighteen patients who received PRI-724 for 12 weeks in a phase 1/2a study were classified as responders (n = 10) or non-responders (n = 8) based on changes in liver stiffness. Plasma samples were obtained before and after PRI-724 administration and the levels of EV-miRNAs were analyzed. Three miRNAs (miR-6510-5p, miR-6772-5p, and miR-4261) were identified as predictors of response or non-response to PRI-724, and the levels of three other miRNAs (miR-939-3p, miR-887-3p, and miR-7112-5p) correlated with the efficacy of treatment. Expression of miR-887-3p was detected in hepatocytes and was decreased significantly in liver tissue following PRI-724 treatment. In addition, transfection of a miR-887-3p mimic activated hepatic stellate cells. Thus, decreases in the miR-887-3p level in blood may reflect recovery from liver fibroses in patients with liver cirrhosis treated with PRI-724, although further validation studies are warranted to confirm this.

Prospects for liquid biopsy using microRNA and extracellular vesicles in breast cancer.

Among the analytes circulating in body fluids, microRNAs, a type of non-coding RNA and known to exist 2655 in primates, have attracted attention as a novel biomarker for cancer screening. MicroRNAs are signaling molecules with important gene expression regulatory functions that can simultaneously control many gene functions and multiple different pathways in living organisms. These microRNAs are transported in extracellular vesicles (EVs), which are lipid bilayers with 50-150 nm in diameter, and are used as communication tools between cells. Furthermore, the EVs that carry these microRNAs circulate in the bloodstream and have other important implications for understanding the pathogenesis and diagnosis of breast cancer. The greatest benefit from cancer screening is the reduction in breast cancer mortality rate through early detection. Other benefits include reduced incidence of breast cancer, improved quality of life, prognosis prediction, contribution to personalized medicine, and relative healthcare cost containment. This paper outlines the latest developments in liquid biopsy for breast cancer, especially focusing on microRNA and EV diagnostics.

Impacts of tissue context on extracellular vesicles-mediated cancer-host cell communications.

Tumor tissue is densely packed with cancer cells, non-cancerous cells, and ECM, forming functional structures. Cancer cells transfer extracellular vesicles (EVs) to modify surrounding normal cells into cancer-promoting cells, establishing a tumor-favorable environment together with other signaling molecules and structural components. Such tissue environments largely affect cancer cell properties, and so as EV-mediated cellular communications within tumor tissue. However, current research on EVs focuses on functional analysis of vesicles isolated from the liquid phase, including cell culture supernatants and blood draws, 2D-cultured cell assays, or systemic analyses on animal models for biodistribution. Therefore, we have a limited understanding of local EV transfer within tumor tissues. In this review, we discuss the need to study EVs in a physiological tissue context by summarizing the current findings on the impacts of tumor tissue environment on cancer EV properties and transfer and the techniques required for the analysis. Tumor tissue environment is likely to alter EV properties, pose physical barriers, interactions, and interstitial flows for the dynamics, and introduce varieties in the cell types taken up. Utilizing physiological experimental settings and spatial analyses, we need to tackle the remaining questions on physiological EV-mediated cancer-host cell interactions. Understanding cancer EV-mediated cellular communications in physiological tumor tissues will lead to developing interaction-targeting therapies and provide insight into EV-mediated non-cancerous cells and interspecies interactions.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Profiling miRNAs in tear extracellular vesicles: a pilot study with implications for diagnosis of ocular diseases.

PURPOSE: To estimate the roles of extracellular vesicles (EVs) in tears and to determine whether their profiles are associated with the type of ocular disease. STUDY DESIGN: Cross-sectional study. METHODS: Tear EVs were extracted from 14 healthy participants and from 21 patients with retinal diseases (age-related macular degeneration [AMD] or diabetic macular edema [DME]). The surface marker expression of tear EVs was examined, and microRNAs (miRNAs) were extracted and profiled by use of real-time PCR array. The stability of the expression of the miRNAs was determined, and their functions were assessed by network analyses. Classification accuracy was evaluated by use of a random forest classifier and k-fold cross-validation. RESULTS: The miRNAs that were highly expressed in tear EVs were miR-323-3p, miR-548a-3p, and miR-516a-5p. The most stably expressed miRNAs independent of diseases were miR-520h and miR-146b-3p. The primary networks of the highly stably expressed endogenous miRNAs were annotated as regulation of organismal injury and abnormalities. The highly expressed miRNAs for severe retinal disease were miR-151-5p for AMD and miR-422a for DME, suggesting potential roles of tear EVs in liquid biopsy. Nine miRNAs (miR-25, miR-30d, miR-125b, miR-132, miR-150, miR-184, miR-342-3p, miR-378, and miR-518b) were identified as distinguishing individuals with AMD from healthy individuals with a classification accuracy of 91.9%. CONCLUSIONS: The finding that tear EVs contain characteristic miRNA species indicates that they may help in maintaining homeostasis and serve as a potential tool for disease diagnosis.

Characterization of circulating miRNAs in the treatment of primary liver tumors.

BACKGROUND AND AIM: Circulating micro RNAs (miRNAs) indicate clinical pathologies such as inflammation and carcinogenesis. In this study, we aimed to investigate whether miRNA expression level patterns in could be used to diagnose hepatocellular carcinoma (HCC) and biliary tract cancer (BTC), and the relationship miRNA expression patterns and cancer etiology. METHODS: Patients with HCC and BTC with indications for surgery were selected for the study. Total RNA was extracted from the extracellular vesicle (EV)-rich fraction of the serum and analyzed using Toray miRNA microarray. Samples were divided into two cohorts in order of collection, the first 85 HCC were analyzed using a microarray based on miRBase ver.2.0 (hereafter v20 cohort), and the second 177 HCC and 43 BTC were analyzed using a microarray based on miRBase ver.21 (hereafter v21 cohort). RESULTS: Using miRNA expression patterns, we found that HCC and BTC could be identified with an area under curve (AUC) 0.754 (v21 cohort). Patients with anti-hepatitis C virus (HCV) treatment (SVR-HCC) and without antiviral treatment (HCV-HCC) could be distinguished by an AUC 0.811 (v20 cohort) and AUC 0.798 (v21 cohort), respectively. CONCLUSIONS: In this study, we could diagnose primary hepatic malignant tumor using miRNA expression patterns. Moreover, the difference of miRNA expression in SVR-HCC and HCV-HCC can be important information for enclosing cases that are prone to carcinogenesis after being cured with antiviral agents, but also for uncovering the mechanism for some carcinogenic potential remains even after persistent virus infection has disappeared.

Extracellular vesicles in the breast cancer brain metastasis: physiological functions and clinical applications.

Breast cancer, which exhibits an increasing incidence and high mortality rate among cancers, is predominantly attributed to metastatic malignancies. Brain metastasis, in particular, significantly contributes to the elevated mortality in breast cancer patients. Extracellular vesicles (EVs) are small lipid bilayer vesicles secreted by various cells that contain biomolecules such as nucleic acids and proteins. They deliver these bioactive molecules to recipient cells, thereby regulating signal transduction and protein expression levels. The relationship between breast cancer metastasis and EVs has been extensively investigated. In this review, we focus on the molecular mechanisms by which EVs promote brain metastasis in breast cancer. Additionally, we discuss the potential of EV-associated molecules as therapeutic targets and their relevance as early diagnostic markers for breast cancer brain metastasis.

Analysis of distribution, collection, and confirmation of capacity dependency of small extracellular vesicles toward a therapy for liver cirrhosis.

BACKGROUND: The progression of liver fibrosis leads to portal hypertension and liver dysfunction. However, no antifibrotic agents have been approved for cirrhosis to date, making them an unmet medical need. Small extracellular vesicles (sEVs) of mesenchymal stem cells (MSCs) are among these candidate agents. In this study, we investigated the effects of sEVs of MSCs, analyzed their distribution in the liver post-administration, whether their effect was dose-dependent, and whether it was possible to collect a large number of sEVs. METHODS: sEVs expressing tdTomato were generated, and their uptake into constituent liver cells was observed in vitro, as well as their sites of uptake and cells in the liver using a mouse model of liver cirrhosis. The efficiency of sEV collection using tangential flow filtration (TFF) and changes in the therapeutic effects of sEVs in a volume-dependent manner were examined. RESULTS: The sEVs of MSCs accumulated mostly in macrophages in damaged areas of the liver. In addition, the therapeutic effect of sEVs was not necessarily dose-dependent, and it reached a plateau when the dosage exceeded a certain level. Furthermore, although ultracentrifugation was commonly used to collect sEVs for research purposes, we verified that TFF could be used for efficient sEV collection and that their effectiveness is not reduced. CONCLUSION: In this study, we identified some unknown aspects regarding the dynamics, collection, and capacity dependence of sEVs. Our results provide important fundamentals for the development of therapies using sEVs and hold potential implications for the therapeutic applications of sEV-based therapies for liver cirrhosis.

Extracellular vesicle-based liquid biopsies in cancer: Future biomarkers for oral cancer.

Oral cancer is the sixth most common cancer worldwide, with approximately 530,000 new cases and 300,000 deaths each year. The process of carcinogenesis is complex, and survival rates have not changed significantly in recent decades. Early detection of cancer, prognosis prediction, treatment selection, and monitoring of progression are important to improve survival. With the recent significant advances in analytical technology, liquid biopsy has made it possible to achieve these goals. In this review, we report new results from clinical and cancer research applications of liquid biopsy, focusing on extracellular vesicles (EVs) among the major targets of liquid biopsy, namely, circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and EVs. In addition, the potential application of EVs derived from gram-negative bacteria (outer membrane vesicles; OMVs) among oral bacteria, which have recently attracted much attention, to liquid biopsy for oral cancer will also be addressed.

Identifying high-grade serous ovarian carcinoma-specific extracellular vesicles by polyketone-coated nanowires.

Cancer cell-derived extracellular vesicles (EVs) have unique protein profiles, making them promising targets as disease biomarkers. High-grade serous ovarian carcinoma (HGSOC) is the deadly subtype of epithelial ovarian cancer, and we aimed to identify HGSOC-specific membrane proteins. Small EVs (sEVs) and medium/large EVs (m/lEVs) from cell lines or patient serum and ascites were analyzed by LC-MS/MS, revealing that both EV subtypes had unique proteomic characteristics. Multivalidation steps identified FRα, Claudin-3, and TACSTD2 as HGSOC-specific sEV proteins, but m/lEV-associated candidates were not identified. In addition, for using a simple-to-use microfluidic device for EV isolation, polyketone-coated nanowires (pNWs) were developed, which efficiently purify sEVs from biofluids. Multiplexed array assays of sEVs isolated by pNW showed specific detectability in cancer patients and predicted clinical status. In summary, the HGSOC-specific marker detection by pNW are a promising platform as clinical biomarkers, and these insights provide detailed proteomic aspects of diverse EVs in HGSOC patients.

Osteoblast-derived extracellular vesicles exert osteoblastic and tumor-suppressive functions via SERPINA3 and LCN2 in prostate cancer.

Clinically, the osteolytic phenotype is rare in prostate cancer (PCa), and the prognosis is generally worse than that of the osteoblastic phenotype. Osteoblastic prostate cancer (BPCa) is a major type of bone metastasis. Several factors responsible for osteogenesis have been identified, but the molecular mechanism of osteoblastic bone metastasis in PCa is not fully understood. Here, we show the osteogenic and tumor-suppressive roles of SERPINA3 and LCN2 in BPCa. In a co-culture of osteoblasts (OBs) and BPCa cells, SERPINA3 and LCN2 were remarkably upregulated in BPCa via OB-derived extracellular vesicles, while they were not in the co-culture of OBs and osteolytic prostate cancer (LPCa) cells. In both the co-culture system and mouse xenograft experiments with intracaudal injection, enhanced expression of SERPINA3 and LCN2 in PCa led to osteogenesis. Additionally, the addition of SERPINA3 and LCN2 to BPCa cells significantly suppressed the proliferative potential. Retrospective analysis also confirmed that high expression levels of SERPINA3 and LCN2 were significantly correlated with a better prognosis. Our results may partially explain how osteoblastic bone metastasis develops and why the prognosis for BPCa is relatively better than that for LPCa.

Small extracellular vesicles derived from human adipose-derived mesenchymal stromal cells cultured in a new chemically-defined contaminate-free media exhibit enhanced biological and therapeutic effects on human chondrocytes in vitro and in a mouse osteoarthritis model.

Human small extracellular vesicles (sEVs) derived from adipose-derived mesenchymal stromal cells (ASC) have been reported to suppress the progression of osteoarthritis (OA) in animal studies and subsequently, translation of this potential to assess their clinical efficacy is anticipated. However, fabrication protocols for sEVs to eliminate potential contamination by culture medium-derived components need to be established prior to their clinical use. The purpose of the present studies was to elucidate the influence of medium-derived contaminants on the biological effects of sEVs, and to establish isolation methods for sEVs using a new clinical grade chemically-defined media (CDM). The quantity and purity of ASC-derived sEVs cultured in four different CDMs (CDM1, 2, 3 and 4) were evaluated. The concentrates of the four media incubated without cells were used as the background (BG) control for each set of sEVs. The biological effect of sEVs fabricated in the four different CDMs on normal human articular chondrocytes (hACs) were evaluated in vitro using a variety of methodological assessments. Finally, the sEVs with the highest purity were tested for their ability to suppress the progression of knee OA mouse model. Analysis of the BG controls revealed that CDM1-3 contained detectable particles, while there was no visible contamination of culture media-derived components detected with CDM4. Accordingly, the sEVs fabricated with CDM4 (CDM4-sEVs) exhibited the highest purity and yield. Notably, the CDM4-sEVs were the most efficient in promoting the cellular proliferation, migration, chondrogenic differentiation, and anti-apoptotic activity of hACs. Furthermore, CDM4-sEVs significantly suppressed the osteochondral degeneration in vivo model. Small EVs derived from ASCs cultured in a CDM without detectable contaminants demonstrated enhanced biological effects on hACs and the progression of OA. Thus, sEVs isolated with CDM4 most optimally meet the requirements of efficacy and safety for assessment in their future clinical applications.

Evidence for in vitro extensive proliferation of adult hepatocytes and biliary epithelial cells.

Over the last several years, a method has emerged that endows adult hepatocytes with in vitro proliferative capacity, producing chemically induced liver progenitors (CLiPs). However, there is a growing controversy regarding the origin of these cells. Here, we provide lineage tracing-based evidence that adult hepatocytes acquire proliferative capacity in vitro using rat and mouse models. Unexpectedly, we also found that the CLiP method allows biliary epithelial cells to acquire extensive proliferative capacity. Interestingly, after long-term culture, hepatocyte-derived cells (hepCLiPs) and biliary epithelial cell-derived cells (bilCLiPs) become similar in their gene expression patterns, and they both exhibit differentiation capacity to form hepatocyte-like cells. Finally, we provide evidence that hepCLiPs can repopulate injured mouse livers, reinforcing our earlier argument that CLiPs can be a cell source for liver regenerative medicine. This study advances our understanding of the origin of CLiPs and motivates the application of this technique in liver regenerative medicine.