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Induced pluripotent stem cells (iPSCs) are a type of cells similar to embryonic stem cells but produced by reprogramed somatic cells. Through in vitro differentiation of iPSCs, we can interrogate the evolution history as well as the various characteristics of macrophages. iPSCs derived macrophages are not only a good model for drug screening, but also an important approach for immunotherapy. This review summarizes the advances, challenges, and future directions in the field of iPSCs-derived macrophages.
Subject(s)
Cell Differentiation , Embryonic Stem Cells , Induced Pluripotent Stem Cells , MacrophagesABSTRACT
Background Pluripotent stem cell-derived retinal pigment epithelial (RPE) cells holds great promise for the treatment of age-related macular degeneration (AMD) and retinitis pigmentosa (RP),but the poor induction efficiency and the according high cost of RPE differentiation hindere its clinical applications.Curcumin is proved to have a promoting effect on the induced differentiation of embryonic stem cells (ESCs).However,the mechanism of curcumin on differentiation of human ESCs into RPE-like cells remains unclear.Objective This study aimed to explore the underlying molecular mechanism of curcumin on directed differentiation of human ESCs into RPE-like cells.Methods Human ESCs strains were cultured in the Matrigel-coated 6-well plate with mTeSRTM 1 medium until over-confluence,and basic fibroblast growth factor was withdrawn there after to induce automatic differentiation.Curcumin at the final concentration 1 μmol/L was added in the first day of differentiation for 24 hours,and the cells without curcumin in the medium served as the control group.Total RNA and protein were extracted at 3 weeks and 5 weeks after induction.RT-PCR,Western blot and immunofluorescence were performed to examine the expressions of the biomarks of stem cells and RPE cells as well as Wnt/β-catenin signaling pathway components.The endocytosis of polystyrene microsphere by induced RPE (iRPE) cells was investigated to verify their function of phagocytosis which features RPE cells.Results Pigmented cells were found from 3 weeks through 5 weeks after induction in the curcumin group,but only less pigmented cells were seen in the fifth week after induction in the control group.In the third and fifth week after induction,the relative expression levels of NANOG mRNA in the iRPE cells were significantly lower than those in the control group (t =13.086,P =0.022;t =34.186,P =0.004),and the relative expression levels of Pax6,RX,CRALBP and RPE65 mRNA were higher in the curcumin group than those of the control group (all at P＜0.01).Western blot assay showed that the expressing bands for CRALBP,RPE65 and MITF enhanced in iRPE cells with a similar appearance in human RPE cells.However,these expressions were all absent in human ESCs.Immunofluorescence staining showed the positive expressions of Pax6,MITF and ZO-1 in cytoplasm of iRPE cells in the curcumin group with a purified efficacy 100％.The fluorescence dye-doped polystyrene microspheres in cytoplasm were obvious in the iRPE cells like positive controls,but the polystyrene microsphere was absent in the negative controls.From 3 weeks through 5 weeks after induced,the relative expression levels of Lef1,MYC and TCF7 mRNA (the dwnstream target genes of Wnt signaling pathway),FZD3 mRNA (Wnt receptor),Wnt2B mRNA (Wnt ligand) and Wnt7B mRNA were significantly reduced in the curcumin group compared with the control group (all at P＜0.01).Conclusions Curcumin promotes the differentiation of human ESCs into RPE-like cells by stimulating the activation of Wnt signaling pathway,and therefore accelerate the differentiation and mature of iRPE cells.
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BACKGROUND:There have been a large number of reports on establishing induced pluripotent stem celllines, but studies concerning large-scale in vitro induced differentiation of induced pluripotent stem cels into hematopoietic progenitor cels stil have a lack of in-depth discussion. OBJECTIVE:To develop methods to induce differentiation of induced pluripotent stem cels into hematopoietic progenitor cels in vitro. METHODS: Using the method of infection with lentivirus particles containing four transcriptionfactor genes, which are Oct4, Sox2, Nanog and Lin28, human skin fibroblasts are transduced into induced pluripotent stem cels. In the induced differentiation system, Y-27632, a kind of tyrosine kinase inhibitor-ROCK (p160-Rho-associated coiled-coil kinase), was added, which obviously suppressed apoptosis of cels. Based on conditioned medium with OP9 cels, a differentiation system of inducing induced pluripotent stem cels differentiating into hematopoietic progenitor cels was established. RESULTS AND CONCLUSION:(1) Apoptosis of induced pluripotent stem cels at the first three passages was very obvious, and the cels were difficult in a large-scale expansion. After Y-27632 was added, the apoptosis of embryonic stem cels was obviously inhibited. (2) During embryoid body differentiation, induced pluripotent stem cels cultured in OP9 conditional growth medium differentiated into hematopoietic progenitor celsin vitro that were positive for CD34.
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An embryonic stem cell (ESC) is a good tool to generate neurons in vitro and can be used to mimic neural development in vivo. It has been widely used in research to examine the role of cell signalling during neuronal development, test the effects of drugs on neurons, and generate a large population of functional neurons. So far, a number of protocols have been established to promote the differentiation of ESCs, such as direct and indirect differentiation. One of the widely used protocols to generate neurons is through the spontaneous formation of multicellular aggregates known as embryonic bodies (EBs). However, for some, it is not clear why EB protocol could be the protocol of choice. EB also is known to mimic an early embryo; hence, knowing the similarities between EB and an early embryo is essential, particularly the information on the players that promote the formation of EBs or the aggregation of ESCs. This review paper focuses on these issues and discusses further the generation of neural cells from EBs using a well-known protocol, the 4−/4+ protocol.
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Debido al auge de la medicina regenerativa, las Células Madre (SC) representan una fuente de reemplazo celular para cualquier tejido, decidiendo emprender este trabajo de investigación con el objetivo de diferenciar células madre embrionarias de ratón (mESC) a células pancreáticas tempranas, realizando su caracterización génica y morfológica. Primeramente se cultivaron y arrestaron en su ciclo celular fibroblastos embrionarios de ratón (MEF) con mitomicina, posteriormente se expandieron las mESC y se sometieron a un protocolo de diferenciación de 21 días hacía células pancreáticas tempranas, evaluándose durante la diferenciación su morfología y expresión relativa de los genes sox-17, pdx-1, ins-1 e ins-2, determinando además la producción de las proteínas insulina y glucagón mediante inmunocitoquímica y citometría de flujo. Se obtuvieron cuerpos embrionarios (EBs) a partir de mESC, con características morfológicas diferentes de acuerdo a su diferenciación, los cuales expresaron genes de la línea germinal endodérmica (sox-17 y pdx-1) a los días 0, 11 y 17 de diferenciación, gen inductor del desarrollo embrionario pancreático (pdx-1) al día 11 de diferenciación y, genes de expresión pancreática (ins-1 e ins-2) a los días 17 y 21 de diferenciación. Finalmente se detectó la producción de proteínas insulina y glucagón en los EBs al día 21 de diferenciación. Se logró diferenciar mESC. El análisis morfológico evidenció cúmulos celulares tridimensionales correspondientes a EBs. Con el análisis de los patrones de expresión génica, se distinguieron inicialmente células con características genéticas de endodermo y posteriormente a partir del día 17 células pancreáticas tempranas, las cuales al día 21 de diferenciación expresaron las proteínas insulina y glucagón...
Due to the boom in regenerative medicine, Stem Cells (SC) represent a source of cell replacement to any tissue, we decided to undertake this research with the objective of differentiating mouse embryonic stem cells (mESC) to early pancreatic cells, developing their genetic and morphological characterization. Initially Mouse embryonic fibroblasts (MEF) were grown and arrested in their cell cycle with mitomycin, subsequently mouse embryonic SC (mESC) were expanded and subjected in to a pancreatic cell differentiation protocol of 21 days. During differentiation, morphology and the relative expression of sox-17, pdx-1, Ins-1 and Ins-2 genes were assessed, also the production of insulin and glucagon proteins was determinated by fluorescence microscopy and flow cytometry. Embryoid bodies (EBs) were obtained from mESC, with different morphological characteristics according to their differentiation, which expressed endodermal germ line genes (sox-17 y pdx-1) at days 0, 11 and 17 of differentiation, an inductor gene of embryonic pancreas development (pdx-1) was detected at day 11 of differentiation. Pancreas genes (ins-1 e ins-2) were expressed at day 17 and 21 of differentiation. Finally the production of insulin and glucagon proteins was detected on the EBS at day 21 of differentiation. In conclusion, the mESC differentiation was achieved. The morphological analysis evidenced three-dimensional cell clusters corresponding to EBs. Analysis of the gene expression patterns in the differentiation process, cells initially showed genetic characteristics of endoderm and thereafter from day 17 of differentiation characteristics of early pancreatic cells which by day 21 of differentiation expressed insulin and glucagon proteins...
Subject(s)
Animals , Mice , Cell Differentiation , Embryonic Stem Cells/physiology , Insulin-Secreting Cells/physiology , Flow Cytometry , Immunohistochemistry , Insulin/biosynthesis , Pancreas/cytology , Real-Time Polymerase Chain ReactionABSTRACT
Objective To investigate the role of different culture densities of embryoid bodies (EBs) in cardiac dif-ferentiation of mouse embryonic stem cells (ESCs). Methods The mouse ESCs were cultured in hanging drops for 3 days, followed by another 2 days for suspension culture to form EBs. Suspended EBs of different densities (60 or 120 EBs/60 mm tissue culture dish) were transferred onto tissue culture plates. The cardiac specific troponin T (TnT) was detected by immu-nocytochemistry. The percentage of beating EBs was calculated at different time points. The mRNA expression of cardiac spe-cific transcriptional factors Nkx2.5, GATA4 and cardiac specific proteinsβ-MHC and ANF were detected by RT-PCR. The protein expressions of TnT and p38 were detected by Western-blot assay. Results Spontaneously beating EBs were posi-tively stained with TnT. There were significantly higher percentage of beating EBs, higher gene expression levels of Nkx 2.5, GATA4,β-MHC and ANF and higher protein expression of TnT in high density culture group than those of low density cul-ture group (P＜0.05). Furthermore, there was significantly higher activity of p38 pathway in high density culture group than that of low density culture group. And the percentages of beating EBs and TnT protein expression were decreased by p 38 pathway inhibitor SB203580. Conclusion The culture density of EBs is important in regulating the cardiac differentiation of ESCs. The high cell-density density culture of EBs enhances the cardiac differentiation of ESCs, which might be mediated by p38 signaling pathway.
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Objective To explore the correlation between the amount of residual undifferentiated embryonic stem cells (ESCs) in embryoid bodies and its tumorigenicity.Methods Mouse R1 ESCs were cultured in suspension to form embryoid bodies (EBs).Ten days later,EBs were digested into single cells and then re-plated in standard ESCs culture condition.The residual undifferentiated embryonic stem cells surface maker SSEA-1 was examined by flow cytometry in EBs.The morphology of residual undifferentiated cells in EBs were observed,meanwhile the surface marker SSEA-1 was examined by immunofluorescent staining.EBs were digested into single cells and grouped into 104,105,106,2×106,and then injected into limb muscle of nude mice.The correlation of the amount of cells and its tumorigenicity was observed.Results Residual undifferentiated ESCs were observed after EBs differentiated for 10 days,which displayed clonal morphology and expressed undifferentiated ceil markers of ESCs,such as SSEA-1.The expression rate of undifferentiated cells surface marker SSEA-1 was (13.5±0.75)％ in EBs differentiated for 10 days.Only two millions single cells harvested from EBs were able to form teratoma after being injected into muscle of nude mice for 6 weeks.Mature endoderm,mesoderm and ectoderm tissues could be found in teratoma.No teratoma formed in other groups.Conclusion A certain amount residual undifferentiated ESCs still exist after differentiation of ESCs into EBs.About 2.7× 105 undifferentiated cells are able to form teratoma by iniecting into muscle of nude mice.
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BACKGROUND AND OBJECTIVES: SIRT1, a histone diacetylase, modify transactivation function of various transcription factor including p53 and NF-kappaB. p53 and NF-kappaB is involved in in vitro differentiation of mouse embryonic stem cells (mESC) into mouse embryoid body (mEB). These suggest that SIRT1 might affect in vitro differentiation of mESC into mEB by regulation of p53 and NF-kappaB. METHODS AND RESULTS: In this study we analyzed the effect of SIRT1 in in vitro differentiation of mESC into mEB using wild and SIRT1 knockout mESC. To examine SIRT1-specific gene in mESC, this study conducted microarray-based differential gene expression analysis between wild and SIRT1 knockout mESC. Comparing their gene expression patterns, this study determined a list of genes regulated by SIRT1. cDNA microarray data-set analysis revealed that genes associated with transcription and signal transduction are significantly modified in SIRT1 knockout mESC. cDNA microarray data-set analysis between mESC and EB in wild and SIRT1 showed that SIRT1 inhibits p53 signaling pathway but not affect NF-kappaB signaling pathway. CONCLUSIONS: This study suggests that SIRT1 modify mESC differentiation by regulation of p53 transcriptional activity.
Subject(s)
Animals , Mice , Embryoid Bodies , Embryonic Stem Cells , Gene Expression , Histones , NF-kappa B , Oligonucleotide Array Sequence Analysis , Signal Transduction , Transcription Factors , Transcriptional ActivationABSTRACT
PURPOSE: Human embryonic stem cells (hESCs) can proliferate for a prolonged period and differentiate into cardiomyocytes in vitro. Recent studies used bone morphogenetic protein 2 (BMP2) to generate cardiomyocytes from hESCs, however, all those studies used early embryoid bodies (EBs) and did not retrieve cardiomyocytes with a high yield. In this study, we treated long-term cultured EBs with BMP2 in order to promote differentiation into cardiomyocytes from hESCs. MATERIALS AND METHODS: hESC lines, including SNUhES3 and SNUhES4, were used in this study. Undifferentiated hESC colonies were detached to form EBs and cultured for up to 30 days. These long-term cultured EBs were differentiated into cardiomyocytes in serum-containing media. In our protocol, BMP2 was applied for 5 days after attachment of EBs. Cardiac specific markers, beating of differentiated cells and electron microscopic (EM) ultrastructures were evaluated and analyzed. RESULTS: Compared to 10-day or 20-day EBs, 30-day EBs showed a higher expression level of cardiac specific markers, Nkx2.5 and a-myosin heavy chain (alphaMHC). Treatment of BMP2 increased expression of cardiac troponin (cTn) I and a-actinin when evaluated at 20 days after attachment of 30-day EBs. Beating of differentiated cells was observed from 7 to 20 days after attachment. Moreover, EM findings demonstrated fine structures such as Z bands in these differentiated cardiomyocytes. These long-term cultured EBs yielded cardiomyocytes with an efficiency of as high as 73.6% when assessed by FACS. CONCLUSION: We demonstrated that the use of long-term cultured EBs may enhance differentiation into cardiomyocytes from hESCs when treated with BMP2.