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AIM: To investigate the potential of human induced pluripotent stem cells(hiPSCs)differentiating into corneal epithelial cells in the simulated limbal stem cells(LSCs)microenvironment.METHODS: The hiPSC cell lines were established in vitro, and hiPSCs were co-cultured with corneal stromal cells in transwell system, which simulated the LSC microenvironment. Bone morphogenetic protein 4(BMP4)and a specific transforming growth factor β inhibitor(SB431542)were added to improve the differentiation efficacy. The expression of corneal epithelial cell-specific markers CK3 and CK12, corneal epithelial cell precursor CK15, and the limbal stem cell markers ABCG5 were determined by immunofluorescence staining and flow cytometry.RESULTS: The hiPSCs were actively proliferated in vitro, and immunofluorescence staining showed positive stem cell-specific markers OCT4, SOX2, TRA-1-60 and NANOG. Furthermore, hiPSCs co-cultured with corneal stromal cells exhibited LSCs markers ABCG5 and corneal epithelial cell precursor markers CK15 were positive; however, corneal epithelial cell markers CK3 and CK12 were negative. With the addition of BMP4 and SB431542, hiPSCs showed positive expression of CK3, and the CK3 expression increased over the time.CONCLUSION: With the addition of SB431542 and BMP4, hiPSCs cultured in simulated LSCs microenvironment could differentiate into corneal epithelial cells.
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Cardiovascular diseases are fatal threats to human health and also important fields in drug discovery. Organoid is a miniature with the structure and function similar to the organ, which is formed by the self-updating and specific differentiation of stem cells during the in vitro culture. Considering its characteristics of human origin, physical features, self-assembling and genetic stability, heart organoid has attracted much attention in the study of cardiogenesis, cardiovascular diseases modeling and related drug research. Hence, this article will review the development of heart organoids and its construction strategies, highlighting its application and prospects in drug discovery.
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Testicular aging is mainly characterized by a gradual decline in the capability of testosterone synthesis and spermatogenesis, which not only affects male fertility, but also correlates with aging-related chronic diseases intimately. Therefore, delaying testicular aging plays a significant role in improving the health and quality of life of middle-aged and elderly men. Stem cells are a cell group with potent self-renewal capability and multi-directional differentiation potential. In recent years, the research of stem cells in basic and clinical application has been carried out in-depth, which has accelerated the development of cell therapy. Currently, stem cell transplantation has been employed to treat multiple diseases, which has captivated widespread attention in the field of aging and regenerative medicine. Stem cell transplantation has demonstrated promising prospects in the treatment of testicular aging. In this article, research profile and progress of stem cell transplantation in the treatment of testicular aging were reviewed, and bottleneck issues encountered in clinical translation and strategies for optimizing clinical efficacy were discussed, aiming to provide novel ideas for the research and development and clinical translation of stem cell therapy for testicular aging.
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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.
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Humans , Rats , Animals , Induced Pluripotent Stem Cells , Cell Differentiation/physiology , Neurons , Parkinson Disease , Mesencephalon , Cells, CulturedABSTRACT
Efforts have been made to establish various human pluripotent stem cell lines. However, such methods have not yet been duplicated in non-human primate cells. Here, we introduce a multiplexed single-cell sequencing technique to profile the molecular features of monkey pluripotent stem cells in published culture conditions. The results demonstrate suboptimized maintenance of pluripotency and show that the selected signaling pathways for resetting human stem cells can also be interpreted for establishing monkey cell lines. Overall, this work legitimates the translation of novel human cell line culture conditions to monkey cells and provides guidance for exploring chemical cocktails for monkey stem cell line derivation.
Subject(s)
Animals , Haplorhini , Single-Cell Gene Expression Analysis , Pluripotent Stem Cells/metabolism , Cell Line , Signal Transduction , Cell Differentiation , TranscriptomeABSTRACT
@#Objective To provide experimental data and theoretical support for further studying the maturity of cardiac patches in other in vitro experiments and the safety in other in vivo animal experiments, through standard chemically defined and small molecule-based induction protocol (CDM3) for promoting the differentiation of human induced pluripotent stem cells (hiPSCs) into myocardium, and preliminarily preparing cardiac patches. Methods After resuscitation, culture and identification of hiPSCs, they were inoculated on the matrigel-coated polycaprolactone (PCL). After 24 hours, the cell growth was observed by DAPI fluorescence under a fluorescence microscope, and the stemness of hiPSCs was identified by OCT4 fluorescence. After fixation, electron microscope scanning was performed to observe the cell morphology on the surface of the patch. On the 1st, 3rd, 5th, and 7th days of culture, the cell viability was determined by CCK-8 method, and the growth curve was drawn to observe the cell growth and proliferation. After co-cultured with matrigel-coated PCL for 24 hours, hiPSCs were divided into a control group and a CDM3 group, and continued to culture for 6 days. On the 8th day, the cell growth was observed by DAPI fluorescence under a fluorescence microscope, and hiPSCs stemness was identified by OCT4 fluorescence, and cTnT and α-actin for cardiomyocyte marker identification. Results Immunofluorescence of hiPSCs co-cultured with matrigel-coated PCL for 24 hours showed that OCT4 emitted green fluorescence, and hiPSCs remained stemness on matrigel-coated PCL scaffolds. DAPI emitted blue fluorescence: cells grew clonally with uniform cell morphology. Scanning electron microscope showed that hiPSCs adhered and grew on matrigel-coated PCL, the cell outline was clearly visible, and the morphology was normal. The cell viability assay by CCK-8 method showed that hiPSCs proliferated and grew on PCL scaffolds coated with matrigel. After 6 days of culture in the control group and the CDM3 group, immunofluorescence showed that the hiPSCs in the control group highly expressed the stem cell stemness marker OCT4, but did not express the cardiac markers cTnT and α-actin. The CDM3 group obviously expressed the cardiac markers cTnT and α-actin, but did not express the stem cell stemness marker OCT4. Conclusion hiPSCs can proliferate and grow on matrigel-coated PCL. Under the influence of CDM3, hiPSCs can be differentiated into cardiomyocyte-like cells, and the preliminary preparation of cardiac patch can provide a better treatment method for further clinical treatment of cardiac infarction.
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Objective:To establish and identify a high-throughput culture platform for induced pluripotent stem cells to differentiated kidney organoids.Methods:Human urine-derived induced pluripotent stem cells were selected and plated at a suitable cell density, and differentiated using small molecule compounds such as CHIR99021/fibroblast growth factor 9/heparin during day 1-6. On day 7, cells with appropriate density were digested and resuspended, than added to a 96-well 3D culture plate for 24 hours. After the cells formed spheroids, fibroblast growth factor 9 and heparin were added to induce differentiation till day 24. The immunofluorescence and transmission electron microscopy were used to compare the differences of kidney organoids obtained by the reported differentiation protocol (transwell protocol) method and high-throughput culture platform.Results:Kidney organoids were successfully differentiated by two protocols. Immunofluorescence results showed that LTL, GATA-3, and synaptopodin, which were major kidney cell markers, were all expressed, and mature renal organoids were formed. The results of transmission electron microscopy showed that the kidney organoids successfully developed foot processes, the unique cellular feature of the glomerular podocytes, which were evenly distributed and neatly interspersed with each other. At the same intermediate mesodermal cell count of 1.0×10 7, approximately 7 renal organoids were obtained by the transwell protocol, while approximately 1 000 renal organoids were obtained by the high-throughput culture platform. Conclusion:A high-throughput culture platform for kidney organoids is successfully established, and a large amount of mature kidney organoids with complete structure and function can be obtained. The differentiation efficiency of kidney organoids is greatly improved.
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Objective:To establish the hepatic organoid of hepatitis B virus (HBV) infection on the basis of induced pluripotent stem cells (iPSC) and an inverted colloidal crystal polyethylene glycol scaffold (ICC), and to evaluate the antiviral effect of nucleoside drugs.Methods:iPSC was differentiated into hepatocyte-like cells (HLC), and inoculated into ICC to construct a hepatic organoid. The relative mRNA expressions of Nanog homeobox (NANOG), sex determining region Y-box (SOX) 2, SOX17, forkhead box protein A2 (FOXA2), alpha fetoprotein (AFP) and albumin (ALB) were detected by real time quantitative polymerase chain reaction (RT-qPCR). Confocal laser microscopy was used to photograph the three-dimension (3D) structure of organs. The expression of sodium taurocholate cotransporting polypeptide (NTCP) in HLC was analyzed by Western blot and immunofluorescence. HepG2.2.15 cells were used to extract HBV virus particles to infect hepatic organoid. The relative expression of HBV pregenome RNA (pgRNA) in cells was detected by RT-qPCR. The expressions of hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) in cytoplasm were observed under confocal laser microscopy. A total of 0.5 μmol/L entecavir and 0.5 μmol/L lamivudine were used to treat the infected cells respectively. The relative expression of HBV pgRNA in infected and uninfected cells was detected by RT-qPCR. Independent sample t test and one-way analysis of variance were used for statistical analysis. Results:Within 21 days of iPSC differentiation, the mRNA expressions of NANOG and SOX2 in stem cells markers decreased ( F=158.90 and 8.31, respectivley; P<0.001 and P=0.002, respectively), while the mRNA expressions of SOX17 and FOXA2 in the endoderm increased first and then decreased ( F=37.23 and 82.57, respectively, both P<0.001). In the later stage of differentiation, the mRNA expressions of AFP and ALB in liver cells increased ( F=4.65 and 34.64, respectively, P=0.012 and P<0.001, respectively), and all differences were statistically significant. NTCP was highly expressed in differentiated cells detected by Western blot and fluorescence microscopy, the protein expression level was 0.803±0.099. Confocal laser microscopy confirmed that the differentiated cells expressed ALB and presented spherical structure in ICC. The expression of HBV pgRNA and the immunostaining of HBsAg and HBcAg confirmed that HBV successfully infected hepatic organoid. Three days after the application of entecavir and lamivudine, the HBV pgRNA level decreased significantly both in entecavir group (0.665±0.220) and lamivudine group (0.503±0.117) compared to the uninfected cells (3.347±0.454), and the differences were both statistically significant ( t=10.53 and 12.72, respectively, both P<0.001). Conclusions:HLC display hepatic specific genes ALB and NTCP. Hepatic organoids constructed with iPSC and ICC have human liver function and can be infected by HBV. Entecavir and lamivudine could effectively inhibit the replication of HBV in hepatic organoids.
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Objective:To investigate the effects of induced pluripotent stem cell-derived exosome (iPSC-Exo) on releasing inflammatory factors from microglia induced by lipopolysaccharide (LPS).Methods:iPSC derived from the tubular epithelial cells of sepsis encephalopathy patients were resuscitated and cultured. The iPSC-Exo was isolated by low-temperature ultracentrifugation and analyzed by transmission electron microscopy, Western blot and high sensitivity flow cytometry (HSFCM). Based on the concentration of iPSC-Exo, human microglia line HMO6 cells activated by LPS (100 ng/mL) were divided into four groups randomly: LPS+ phosphate buffer solution (PBS) group, LPS+iPSC-Exo 10 5 group, LPS+iPSC-Exo 10 6 group and LPS+iPSC-Exo 10 7 group. The control group was added equal PBS but not LPS. After culture for 24 h, the concentrations of malondialdehyde in cells were detected. Quantitative RT-PCR was used to measure the mRNA expression levels of macrophage inflammatory protein 2 (MIP2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in the cells and enzyme-linked immunosorbent assay (ELISA) was used to assess the concentration of these cytokines in the supernatant. Under the same concentration of iPSC-Exo, one-way ANOVA and SNK- q test were used for comparison between groups. Results:The extracts showed spherical membrane structure by transmission electron microscopy. HSFCM showed the mean diameter of the extracts was (74.66±15.60) nm and the concentration around 2.98×10 10/mL. Western blot analysis showed high expression of exosome markers CD63, Alix and TSG101, but not GM130. Intracellular MDA concentration and mRNA expression levels and protein concentration of MIP2, TNF-α, IL-1β and IL-6 in the LPS+PBS group were significantly higher than those in the control group (all P<0.01). With the increase of iPSC-Exo concentration, the intracellular MDA concentration decreased gradually ( P<0.01), the mRNA expression levels of inflammatory factors showed a gradual downward trend (all P<0.05). Each inflammatory cytokine in the supernatant declined in a manner that was almost consistent with mRNA. Concentrations of MDA remained constant in the control group. Conclusions:iPSC-Exo derived from the tubular epithelial cells of sepsis encephalopathy patients alleviate oxidative stress and inflammation effect of microglia induced by LPS, and the modulatory effect is dose-dependent.
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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.
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Animals , Swine , Induced Pluripotent Stem Cells/metabolism , Receptors, Cell Surface/genetics , Antigens, CD/metabolism , Porcine respiratory and reproductive syndrome virus/geneticsABSTRACT
OBJECTIVE To study the role of phosphatidylinositol-3-kinase （PI3K） on sunitinib-induced myocardial systolic dysfunction. METHODS Using human-induced pluripotent stem cell-derived cardiomyocytes （hiPSC-CMS） as objects， the contractile force of cardiomyocytes was measured by CardioExcyte 96 system， and IC50 of sunitinib was calculated after hiPSC- CMS were treated with sunitinib at different concentrations [0 （control）， 0.5， 1， 3， 5， 10 μmol/L] for 24 hours. The effects of sunitinib （3.14 μmol/L） on the contractile frequency of cardiomyocytes， calcium transient amplitude and calcium transient recovery time course， mRNA expression of myocardial injury markers atrial natriuretic peptide （ANP）， brain natriuretic peptide （BNP） and β-myosin heavy chain （β-MHC） were detected. PI3K activator 3，4，5-triphosphate phos-phatidylinositol （PIP3， 1 μmol/L） and sunitinib were used to intervene in hiPSC-CMs jointly， so as to investigate the role of PI3K in the myocardial systolic dysfunction induced by sunitinib. RESULTS Sunitinib inhibited the contractile force of hiPSC-CMs in a concentration-dependent manner. IC50 of sunitinib was 3.14 μmol/L. After intervention with 3.14 μmol/L sunitinib， the contractile frequency of hiPSC-CMs and calcium transient amplitude were decreased significantly （P＜0.05 or P＜0.01）； the duration of calcium transient recovery was prolonged significantly （P＜0.05）， and mRNA expressions of ANP， BNP and β-MHC were significantly increased （P＜0.01）. After PI3K was activated with PIP3， the contractile force of hiPSC-CMs was increased significantly （P＜0.01）. CONCLUSIONS Activating PI3K activity is a potential molecular mechanism to improve myocardial toxicity induced by sunitinib.
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Objective To understand the characteristics and developmental differences between cerebral organoids in vitro and normal cerebral cortices in vivo. Methods 1. Grouping: cerebral cortices in vivo group and cultured cerebral organoids in vitro group. 2. Sample collection: cortical tissues were collected from Kunming mouse embryos at embryonic day 7.5(E7.5), E9.5, E11.5, E14.5, and postnatal day 3 (P3) or P7. Three specimens were taken from each group. Meanwhile, cerebral organoids were cultured with mouse induced pluripotent stem cells (iPSCs), and samples at different culture time point were collected, and more than 3 samples were collected at each time point. 3. Detection method: the distribution of different types of cells in each group of specimens was analyzed by immunofluorescent staining. Results While relative similarities between in vivo cerebral cortical development and the cerebral organoids in vitro were observed, including the histogenesis, and the morphological differentiation of nerve cells and glial cells, the lamellar architecture of cerebral cortex in mouse brain was not observed in cerebral organoids. Conclusion The development of cerebral organoids in vitro has some similarity with body's cortical development. Therefore, cerebral organoids can be used to a substitution of cortex and diseases' models, but improvement of the existing technologies is necessary.
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Cardiomyocytes are highly differentiated terminal cells with poor self-renewal ability. Therefore, after myocardial infarction necrotic cardiomyocytes cannot be effectively replenished, and the infarcted area is quickly replaced by fibrous tissue, which seriously affects cardiac function. The reduction of the number of myocardial cells and the destruction of the structural integrity of the heart have caused cardiovascular diseases such as myocardial infarction and heart failure, which continue to endanger human life and health. At present, the treatment of coronary heart disease has made great progress. The commonly used treatment options for myocardial repair after myocardial infarction mainly include stem cell transplantation, exosome mediation and microenvironment construction, but all of them are difficult to solve to varying degrees. Cardiac fibroblasts occupy the majority of cardiac cells, and the distribution characteristics of fibroblasts and their role in the process of myocardial infarction make them important effector cells after myocardial infarction. Therefore, this article reviews the source, distribution, post-infarction status of myocardial fibroblasts and the effect of fibroblasts on cardiomyocytes, in order to provide new treatment ideas and solutions for fibroblasts in the repair and regeneration of myocardial cells after myocardial infarction.
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While Mek1/2 and Gsk3β inhibition ("2i") supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naïve state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of DNMT3s that prevent DNA hypomethylation. Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female "all-ESC" mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.
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Male , Animals , Female , Mice , Mouse Embryonic Stem Cells , Embryonic Stem Cells , Genomic Instability , Lipids , DNA/metabolism , Cell DifferentiationABSTRACT
Epilepsy is a common, chronic neurological disorder that has been associated with impaired neurodevelopment and immunity. The chemokine receptor CXCR5 is involved in seizures via an unknown mechanism. Here, we first determined the expression pattern and distribution of the CXCR5 gene in the mouse brain during different stages of development and the brain tissue of patients with epilepsy. Subsequently, we found that the knockdown of CXCR5 increased the susceptibility of mice to pentylenetetrazol- and kainic acid-induced seizures, whereas CXCR5 overexpression had the opposite effect. CXCR5 knockdown in mouse embryos via viral vector electrotransfer negatively influenced the motility and multipolar-to-bipolar transition of migratory neurons. Using a human-derived induced an in vitro multipotential stem cell neurodevelopmental model, we determined that CXCR5 regulates neuronal migration and polarization by stabilizing the actin cytoskeleton during various stages of neurodevelopment. Electrophysiological experiments demonstrated that the knockdown of CXCR5 induced neuronal hyperexcitability, resulting in an increased number of seizures. Finally, our results suggested that CXCR5 deficiency triggers seizure-related electrical activity through a previously unknown mechanism, namely, the disruption of neuronal polarity.
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Animals , Humans , Mice , Actin Cytoskeleton/metabolism , Actins/metabolism , Epilepsy/metabolism , Neurons/metabolism , Receptors, CXCR5/metabolism , Seizures/metabolismABSTRACT
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.
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Animals , Mice , Humans , Induced Pluripotent Stem Cells , Brain/pathology , Disease Models, Animal , Neurodegenerative Diseases/pathology , Organoids/pathologyABSTRACT
ObjectiveDirected differentiation of human induced pluripotent stem cells （hiPSCs） into spinal cord γ-aminobutyric acid （GABA）-ergic progenitor cells were implanted into an decellularized optical nerve （DON） bioscaffold to construct a hiPSC-derived inhibitory neural network tissue with synaptic activities. This study aimed to provide a novel stem cell-based tissue engineering product for the study and the repair of central nervous system injury. MethodsThe combination of stepwise directional induction and tissue engineering technology was applied in this study. After hiPSCs were directionally induced into human neural progenitor cells （hNPCs） in vitro， they were seeded into a DON for three-dimensional culture， allowing further differentiation into inhibitory GABAergic neurons under the specific neuronal induction environment. Transmission electron microscopy and whole cell patch clamp technique were used to detect whether the hiPSCs differentiated neurons could form synapse-like structures and whether these neurons had spontaneous inhibitory postsynaptic currents， respectively， in order to validate that the hiPSC-derived neurons would form neural networks with synaptic transmission potentials from a structural and functional perspective. ResultsThe inhibitory neurons of GABAergic phenotype were successfully induced from hiPSCs in vitro， and maintained good viability after 28 days of culture. With the transmission electron microscopy， it was observed that many cell junctions were formed between hiPSC-derived neural cells in the three-dimensional materials， some of which presented a synapse- like structure， manifested as the slight thickness of cell membrane and a small number of vesicles within one side of the cell junctions， the typical structure of a presynatic component， and focal thickness of the membrane of the other side of the cell junctions， a typical structure of a postsynaptic component. According to whole-cell patch-clamp recording， the hiPSC-derived neurons had the capability to generate action potentials and spontaneous inhibitory postsynaptic currents were recorded in this biotissue. ConclusionsThe results of this study indicated that hiPSCs can be induced to differentiate into GABAergic progenitor cells in vitro and can successfully construct iPSC-derived inhibitory neural network tissue with synaptic transmission after implanted into a DON for three-dimensional culture. This study would provide a novel neural network tissue for future research and treatment of central nervous system injury by stem cell tissue engineering technology.
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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.
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Humans , Induced Pluripotent Stem Cells , Sirolimus/metabolism , Caspase 9/metabolism , RNA, Guide, CRISPR-Cas Systems , Pluripotent Stem Cells/metabolism , Cell Differentiation , Puromycin/metabolismABSTRACT
@#Somatic cell reprogramming has developed rapidly in the field of modern biology. Induced pluripotent stem cells(iPSCs)obtained through somatic cell reprogramming are not only capable of self-renewal,but also have multidirectional differentiation potential,which plays an important role in disease modeling and regenerative medicine. This paper reviewed the gene reprogramming technology,the disease models of iPSCs and the application prospects of iPSCs in childhood genetic diseases,so as to provide a reference for the application of iPSCs in the research of mechanism and treatment of various genetic diseases.
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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.