METHODS: Cardiac organoids derived from the self-assembled human induced pluripotent stem cells were constructed by regulating the Wnt signaling pathway. Flow cytometry was used to detect the proportion of cardiomyocytes in the cardiac organoids, and RT-qPCR was employed to detect the mRNA expressions. Immunofluorescence staining was used to detect the protein expressions of TNNT2, CD31, and vimentin. The beating amplitude of the cardiac organoids was determined with calcium transient. In vitro myocardial injury models and ischemia-reperfusion models were established, and the cell injuries were examined using Masson staining. TUNEL staining and calcium transient detection were used to evaluate the adverse effects of doxorubicin and trastuzumab in the cardiac organoids. RESULTS: The cardiac organoids began to beat on the 8th day of culture and consisted of 32.4% cardiomyocytes with high expressions of the myocardial markers TNNT2, NKX2.5, RYR2 and KCNJ2. No significant differences in morphological size, beating frequency, proportion of cardiomyocytes, or myocardial contractility were observed in the cardiac organoids differentiated from different batches. These cardiac organoids could be maintained in in vitro culture conditions for at least 50 days. Captopril treatment could obviously alleviate liquid nitrogen-induced myocardial injury in the cardiac organoids. Hypoxia/reoxygenation induced ischemia-reperfusion injury and promoted myocardial fibrosis and apoptosis in the cardiac organoids. Treatment with doxorubicin for 24 h resulted in significantly increased cell death and reduced beating frequency and cell viability in the cardiac organoids in a dose-dependent manner. Trastuzumab significantly impaired the contractile and calcium handling abilities of the cardiac organoids. CONCLUSIONS Cardiac organoids derived from human induced pluripotent stem cells have been successfully constructed and can be used for cardiac disease modeling and drug evaluation.
Humans ; Induced Pluripotent Stem Cells/cytology* ; Organoids/cytology* ; Myocytes, Cardiac/cytology* ; Cell Differentiation ; Heart Diseases

Frostbite is the most common cold injury and is caused by both immediate cold-induced cell death and the gradual development of localized inflammation and tissue ischemia. Delayed healing of frostbite often leads to scar formation, which not only causes psychological distress but also tends to result in the development of secondary malignant tumors. Therefore, a rapid healing method for frostbite wounds is urgently needed. Herein, we used a mouse skin model of frostbite injury to evaluate the recovery process after frostbite. Moreover, single-cell transcriptomics was used to determine the patterns of changes in monocytes, macrophages, epidermal cells, and fibroblasts during frostbite. Most importantly, human-induced pluripotent stem cell (hiPSC)-derived skin organoids combined with gelatin-hydrogel were constructed for the treatment of frostbite. The results showed that skin organoid treatment significantly accelerated wound healing by reducing early inflammation after frostbite and increasing the proportions of epidermal stem cells. Moreover, in the later stage of wound healing, skin organoids reduced the overall proportions of fibroblasts, significantly reduced fibroblast-to-myofibroblast transition by regulating the integrin α5β1-FAK pathway, and remodeled the extracellular matrix (ECM) through degradation and reassembly mechanisms, facilitating the restoration of physiological ECM and reducing the abundance of ECM associated with abnormal scar formation. These results highlight the potential application of organoids for promoting the reversal of frostbite-related injury and the recovery of skin functions. This study provides a new therapeutic alternative for patients suffering from disfigurement and skin dysfunction caused by frostbite.
Animals ; Organoids/metabolism* ; Mice ; Humans ; Wound Healing ; Frostbite/metabolism* ; Skin/pathology* ; Induced Pluripotent Stem Cells/cytology* ; Cicatrix/pathology* ; Fibroblasts/metabolism* ; Disease Models, Animal ; Mice, Inbred C57BL ; Extracellular Matrix/metabolism* ; Male

In multiple areas such as science, technology, and economic activities, it is necessary to unify the management of repetitive tasks or concepts by standardization to obtain the best order and high efficiency. Organoids, as living tissue models, have rapidly developed in the past decade. Organoids can be used repetitively for in vitro culture, cryopreservation, and recovery for further utilization. Because organoids can recapitulate the parental tissues' morphological phenotypes, cell functions, biological behaviors, and genomic profiles, they are known as renewable "living biobanks". Organoids cover two mainstream fields: Adult stem cell-derived organoids (also known as patient-derived organoids) and induced pluripotent stem cell-derived and/or embryonic stem cell-derived organoids. Given the increasing importance of organoids in the development of new drugs, standardized operation, and management in all steps of organoid construction is an important guarantee to ensure the high quality of products. In this review, we systematically introduce the standardization of organoid construction operation procedures, the standardization of laboratory construction, and available standardization documents related to organoid culture that have been published so far. We also proposed the challenges and prospects in this field.
Organoids ; Humans ; Biological Specimen Banks/standards* ; Induced Pluripotent Stem Cells/cytology* ; Cryopreservation/methods*

OBJECTIVE: To establish an efficient protocol for directed differentiation of human induced pluripotent stem cells (hiPSCs) into functional midbrain dopaminergic progenitor cells (DAPs) in vitro. METHODS: hiPSCs were induced to differentiate into DAPs in two developmental stages. In the first stage (the first 13 days), hiPSCs were induced into intermediate cells morphologically similar to primitive neuroepithelial cells (NECs) in neural induction medium containing a combination of small molecule compounds. In the second stage, the intermediate cells were further induced in neural differentiation medium until day 28 to obtain DAPs. After CM-DiI staining, the induced DAPs were stereotactically transplanted into the right medial forebrain bundle (MFB) of rat models of Parkinson's disease (PD). Eight weeks after transplantation, the motor behaviors of PD rats was evaluated. Immunofluorescence assay of brain sections of the rats was performed at 2 weeks after transplantation to observe the survival, migration and differentiation of the transplanted cells in the host brain microenvironment. RESULTS: hiPSCs passaged stably on Matrigel showed a normal diploid karyotype, expressed the pluripotency markers OCT4, SOX2, and Nanog, and were positive for alkaline phosphatase. The primitive neuroepithelial cells obtained on day 13 formed dense cell colonies in the form of neural rosettes and expressed the neuroepithelial markers (SOX2, Nestin, and PAX6, 91.3%-92.8%). The DAPs on day 28 highly expressed the specific markers (TH, FOXA2, LMX1A and NURR1, 93.3-96.7%). In rat models of PD, the hiPSCs-DAPs survived and differentiated into TH⁺, FOXA2⁺ and Tuj1⁺ neurons at 2 weeks after transplantation. Eight weeks after transplantation, the motor function of PD rats was significantly improved as shown by water maze test (P < 0.0001) and apomorphine-induced rotation test (P < 0.0001) compared with rats receiving vehicle injection. CONCLUSION HiPSCs can be effectively induced to differentiate into DAPs capable of differentiating into functional neurons both in vivo and in vitro. In rat models of PD, the transplanted hiPSCs-DAPs can survive for more than 8 weeks in the MFB and differentiate into multiple functional neurocytes to ameliorate neurological deficits of the rats, suggesting the potential value of hiPSCs-DAPs transplantation for treatment of neurological diseases.
Humans ; Rats ; Animals ; Induced Pluripotent Stem Cells ; Cell Differentiation/physiology* ; Neurons ; Parkinson Disease ; Mesencephalon ; Cells, Cultured

Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.
Humans ; Induced Pluripotent Stem Cells ; Sirolimus/metabolism* ; Caspase 9/metabolism* ; RNA, Guide, CRISPR-Cas Systems ; Pluripotent Stem Cells/metabolism* ; Cell Differentiation ; Puromycin/metabolism*

Accurate assessment of the risks associated with traditional Chinese medicine(TCM), such as the potential to induce serious cardiovascular adverse reactions including cardiac arrhythmias, is crucial. This article introduced the pharmacological evaluation strategies for cardiac safety and the progress in cardiac organ research, with a focus on discussing the application prospects of human induced pluripotent stem cells(hiPSCs) and organoids in assessing the risks of TCM-induced cardiac arrhythmias. Compared with traditional animal models, hiPSCs and organoid models provide better reference and predictive capabilities, allowing for more accurate simulation of human cardiac responses. Researchers have successfully generated various cardiac tissue models that mimic the structure and function of the heart to evaluate the effects of TCM on the heart. The hiPSCs model, by reprogramming adult cells into pluripotent stem cells and differentiating them into cardiac cells, enables the generation of personalized cardiac tissue, which better reflects individual differences and drug responses. This provides guidance for the assessment of TCM cardiac toxicity risks. By combining organoid model with cardiac safety pharmacology strategies such as electrocardiogram monitoring and ion channel function assessment, the impact of TCM on the heart can be comprehensively evaluated. In addition, the application of the Comprehensive in Vitro Proarrhythmia Assay(CiPA) approach improves the accuracy of evaluation. Applying the CiPA approach to TCM research reveals potential risks and provides a scientific basis for the clinical application and industrial development of TCM. In conclusion, organoid model and cardiac safety pharmacology evaluation strategies provide important tools for assessing the cardiac toxicity risks of TCM. The combination of hiPSCs model, comprehensive assessment methods, and the CiPA strategy enables an accurate assessment of the risks of TCM-induced cardiac arrhythmias, thus providing a scientific basis for the safe use and international recognition of TCM in clinical practice. This contributes to ensuring the safety and efficacy of TCM and promoting its clinical application and global acceptance.
Animals ; Humans ; Medicine, Chinese Traditional/adverse effects* ; Cardiotoxicity ; Induced Pluripotent Stem Cells ; Arrhythmias, Cardiac/chemically induced* ; Myocytes, Cardiac ; Organoids ; Drugs, Chinese Herbal/adverse effects*

Understanding the fundamental processes of human brain development and diseases is of great importance for our health. However, existing research models such as non-human primate and mouse models remain limited due to their developmental discrepancies compared with humans. Over the past years, an emerging model, the "brain organoid" integrated from human pluripotent stem cells, has been developed to mimic developmental processes of the human brain and disease-associated phenotypes to some extent, making it possible to better understand the complex structures and functions of the human brain. In this review, we summarize recent advances in brain organoid technologies and their applications in brain development and diseases, including neurodevelopmental, neurodegenerative, psychiatric diseases, and brain tumors. Finally, we also discuss current limitations and the potential of brain organoids.
Animals ; Mice ; Humans ; Induced Pluripotent Stem Cells ; Brain/pathology* ; Disease Models, Animal ; Neurodegenerative Diseases/pathology* ; Organoids/pathology*

Objective: To investigate the cytotoxic effects of induced pluripotent stem (iPS) cells of anti-mesothelin (MSLN)-chimeric antigen receptor natural killer (CAR-NK) cells (anti-MSLN-iCAR-NK cells) on ovarian epithelial cancer cells. Methods: Twenty cases of ovarian cancer patients who underwent surgical treatment at Henan Provincial People's Hospital from September 2020 to September 2021 were collected, and 20 cases of normal ovarian tissues resected during the same period due to other benign diseases were also collected. (1) Immunohistochemistry and immunofluorescence were used to verify the expression of MSLN protein in ovarian cancer tissues. (2) Fresh ovarian cancer tissues were extracted and cultured to obtain primary ovarian cancer cells. Recombinant lentiviral vectors targeting anti-MSLN-CAR-CD₂₄₄ were constructed and co-cultured with iPS cells to obtain anti-MSLN-iCAR cells. These cells were differentiated into anti-MSLN-iCAR-NK cells using cytokine-induced differentiation method. The cell experiments were divided into three groups: anti-MSLN-iCAR-NK cell group, natural killer (NK) cell group, and control group. (3) Flow cytometry and live cell staining experiment were used to detect the apoptosis of ovarian cancer cells in the three groups. (4) Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granzyme B (GZMB), perforin 1 (PRF1), interleukin (IL)-6, and IL-10 in the three groups of ovarian cancer cells. Results: (1) Immunohistochemistry analysis showed that a positive expression rate of MSLN protein in ovarian cancer tissues of 65% (13/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ²=4.912, P=0.027). Immunofluorescence analysis revealed that the positive expression rate of MSLN protein in ovarian cancer tissues was 70% (14/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ²=6.400, P=0.011). (2) Flow cytometry analysis showed that the apoptotic rate of ovarian cancer cells in the anti-MSLN-iCAR-NK cell group was (29.27±0.85)%, while in the NK cell group and control group were (8.44±0.34)% and (6.83±0.26)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.01). Live cell staining experiment showed that the ratio of dead cells to live cells in the anti-MSLN-iCAR-NK cell group was (36.3±8.3)%, while in the NK cell group and control group were (5.4±1.4)% and (2.0±1.3)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.001). (3) ELISA analysis revealed that the expression levels of IFN-γ, TNF-α, GZMB, PRF1, IL-6, and IL-10 in ovarian cancer cells of the anti-MSLN-iCAR-NK cell group were significantly higher than those in the NK cell group and the control group (all P<0.05). Conclusion: The anti-MSLN-iCAR-NK cells exhibit a strong killing ability against ovarian cancer cells, indicating their potential as a novel immunotherapy approach for ovarian cancer.
Humans ; Female ; Carcinoma, Ovarian Epithelial/metabolism* ; Ovarian Neoplasms/metabolism* ; Interleukin-10/pharmacology* ; Induced Pluripotent Stem Cells/metabolism* ; Iron-Dextran Complex/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism* ; Cell Line, Tumor ; Killer Cells, Natural ; Interleukin-6

Adoptive therapeutic immune cells, such as chimeric antigen receptor (CAR)-T cells and natural killer cells, have established a new generation of precision medicine based on which dramatic breakthroughs have been achieved in intractable lymphoma treatments. Currently, well-explored approaches focus on autologous cells due to their low immunogenicity, but they are highly restricted by the high costs, time consumption of processing, and the insufficiency of primary cells in some patients. Induced pluripotent stem cells (iPSCs) are cell sources that can theoretically produce indefinite well-differentiated immune cells. Based on the above facts, it may be reasonable to combine the iPSC technology and the CAR design to produce a series of highly controllable and economical "live" drugs. Manufacturing hypoimmunogenic iPSCs by inactivation or over-expression at the genetic level and then arming the derived cells with CAR have emerged as a form of "off-the-shelf" strategy to eliminate tumor cells efficiently and safely in a broader range of patients. This review describes the reasonability, feasibility, superiority, and drawbacks of such approaches, summarizes the current practices and relevant research progress, and provides insights into the possible new paths for personalized cell-based therapies.
Humans ; Receptors, Chimeric Antigen/genetics* ; Induced Pluripotent Stem Cells ; Killer Cells, Natural ; Cell- and Tissue-Based Therapy ; T-Lymphocytes ; Immunotherapy, Adoptive ; Neoplasms/genetics*

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.
Swine ; Animals ; Induced Pluripotent Stem Cells/metabolism* ; Receptors, Cell Surface/genetics* ; Antigens, CD/metabolism* ; Porcine respiratory and reproductive syndrome virus/genetics*