Journal articles on the topic 'Hesci'
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1
Yeo, C. X., J. Rathjen, and D. K. Gardner. "112. FETAL CALF SERUM AFFECTS hESC METABOLISM AND GENE EXPRESSION LEADING TO DIFFERENTIATION IN CULTURE." Reproduction, Fertility and Development 22, no. 9 (2010): 30. http://dx.doi.org/10.1071/srb10abs112.
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Fetal calf serum (FCS) has conventionally been used to support the growth and maintenance of human embryonic stem cells (hESCs). FCS however, is an undefined complex mixture containing factors which potentially alter the functionality of hESCs. Inclusion of FCS during embryo culture negatively impacts embryo metabolism and viability but comparative studies on hESCs have been hindered by the lack of serum and feeder independent culture systems. Using a recently available defined culture system, the effects of FCS on hESC metabolism and pluripotentcy were investigated. Mel2 hESCs were grown at 37°C in 5% CO2 for 3 days on matrigel (hESC-qualified) coated tissue culture wells in mTeSR1 medium. hESCs were then cultured in mTeSR1 (control) or in mTeSR1 supplemented with 20% FCS from different manufacturers or knockout serum replacement (KOSR), for 96 hours. Media was renewed daily. At the end of the culture period, spent media was collected and cells were trypsinised and counted and/or collected for gene expression analysis. FCS decreased cell survival and altered hESC morphology from densely packed colonies with distinct borders into non-uniform heterogeneous populations comprising of hESC-like cells and fibroblastic-like cells with high cytoplasmic to nuclear ratios. Media analysis revealed altered cell metabolism with increased glucose consumption rates per cell (P < 0.01) with FCS supplementation, compared to cells cultured in mTeSR1 alone. Gene expression analysis revealed that FCS, regardless of its manufacturer, decreased the expression of some pluripotent markers and increased differentiation markers. A decrease in pluripotent gene expression was also observed in hESCs cultured with KOSR compared to mTeSR1 alone. Maintenance of homogeneity in hESC populations is crucial for the advancement of hESC clinical therapies. This study demonstrates that FCS promotes heterogeneity and impacts the metabolic function and gene expression of hESCs thereby supporting the need for serum-free culture systems as standard practice in hESC culture.
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Wang, Lisheng, Li Li, Pablo Menendez, Chantal Cerdan, and Mickie Bhatia. "Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development." Blood 105, no. 12 (June 15, 2005): 4598–603. http://dx.doi.org/10.1182/blood-2004-10-4065.
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Abstract To date, hematopoietic development of human embryonic stem cells (hESCs) has been limited to cell lines cultured in the presence of either mouse embryonic fibroblasts (MEFs) or MEF-conditioned media (MEF-CM). Anonymous xenogenic factors from MEFs or MEF-CM complicate studies of hESC self-renewal and also raise concerns for the potential clinical applications of generating primitive hematopoietic cells from hESC lines maintained under these ambiguous conditions. Here, we demonstrate that hESCs can be cultured over 30 passages in defined conditions in the absence of MEFs or MEF-CM using only serum replacement (SR) media and high concentrations of basic fibroblast growth factor (SR-bFGF). Similar to hESCs cultured in MEF-CM, hESCs cultured in SR-bFGF sustained characteristics of undifferentiated hESCs, proliferative potential, normal karyotype, in vitro and in vivo 3 germ-layer specification and gave rise to hemogenic-endothelial precursors required for subsequent primitive hematopoietic development. Our report demonstrates that anonymous factors produced by feeder cells are not necessary for hESC maintenance and subsequent hematopoietic specification, thereby providing a defined system for studies of hESC self-renewal and hESC-derived hematopoiesis. (Blood. 2005;105:4598-4603)
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Martin, Colin H., Petter S. Woll, Zhenya Ni, Juan Carlos Zúñiga-Pflücker, and Dan S. Kaufman. "Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood–derived hematopoietic progenitor cells." Blood 112, no. 7 (October 1, 2008): 2730–37. http://dx.doi.org/10.1182/blood-2008-01-133801.
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Abstract Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34+ cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34+ cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34+ cells also did not produce B cells in vitro. In contrast, CD34+ cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor–induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs.
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Li, Zongjin, Bryan Smith, Mei Huang, Xiaoyan Xie, Sanjiv Sam Gambhir, and Joseph Wu. "Endothelial Differentiation of Human Embryonic Stem Cell and Functional Blood Vessels Formation in Vivo." Blood 112, no. 11 (November 16, 2008): 5455. http://dx.doi.org/10.1182/blood.v112.11.5455.5455.
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Abstract Background: Human embryonic stem (hES) cells are distinguished by their capacity for self-renewal and pluripotency. Here we characterize the differentiation of hES cell-derived endothelial cells (hESC-ECs), use molecular imaging techniques to examine their survival and function in vivo. Methods and Results: Here we introduced two-step procedures to increase endothelial differentiation efficiency of hESCs by subcultured embryoid bodies (EBs) in collagen. Single cell suspensions from EBs sprouting in collagen were obtained by treatment with collagenase and Liberase Blendzyme IV and CD31/CD144 positive cells were isolated by FACScan. After isolation, these hESC-ECs express endothelial cell markers similar to HUVEC, form vascular-like channels, and incorporate DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) in vitro. Real time PCR array described increasing endothelial transcription. Using whole genome microarrays, we investigated the hESCs derived endothelial cells (hESC-ECs) transcriptome that occur among sequenced hESCs differentiation processes and human umbilical vein endothelial cells (HUVECs). We found that hESC-ECs expressed endothelial gene at pattern similar to HUVECs. By intravital microscope, we demonstrated that hESC-ECs can form function vessels with blood flow. Conclusion: Taken together, two-steps procedures increased the endothelial differentiation efficiency hESCs, and hESC-ECs can form functional vessel in vivo.
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Khan, Firdos Alam, Dana Almohazey, Munthar Alomari, and Sarah Ameen Almofty. "Isolation, Culture, and Functional Characterization of Human Embryonic Stem Cells: Current Trends and Challenges." Stem Cells International 2018 (August 26, 2018): 1–8. http://dx.doi.org/10.1155/2018/1429351.
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Human embryonic stem cells (hESCs) hold great potential for the treatment of various degenerative diseases. Pluripotent hESCs have a great ability to undergo unlimited self-renewal in culture and to differentiate into all cell types in the body. The journey of hESC research is not that smooth, as it has faced several challenges which are limited to not only tumor formation and immunorejection but also social, ethical, and political aspects. The isolation of hESCs from the human embryo is considered highly objectionable as it requires the destruction of the human embryo. The issue was debated and discussed in both public and government platforms, which led to banning of hESC research in many countries around the world. The banning has negatively affected the progress of hESC research as many federal governments around the world stopped research funding. Afterward, some countries lifted the ban and allowed the funding in hESC research, but the damage has already been done on the progress of research. Under these unfavorable conditions, still some progress was made to isolate, culture, and characterize hESCs using different strategies. In this review, we have summarized various strategies used to successfully isolate, culture, and characterize hESCs. Finally, hESCs hold a great promise for clinical applications with proper strategies to minimize the teratoma formation and immunorejection and better cell transplantation strategies.
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Kim, Yoon Young, Seung-Yup Ku, Zev Rosenwaks, Hung Ching Liu, Sun Kyung Oh, Shin Yong Moon, and Young Min Choi. "Red Ginseng Extract Facilitates the Early Differentiation of Human Embryonic Stem Cells into Mesendoderm Lineage." Evidence-Based Complementary and Alternative Medicine 2011 (2011): 1–8. http://dx.doi.org/10.1155/2011/167376.
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Human embryonic stem cells (hESCs) have capacities to self-renew and differentiate into all cell typesin vitro. Red ginseng (RG) is known to have a wide range of pharmacological effectsin vivo; however, the reports on its effects on hESCs are few. In this paper, we tried to demonstrate the effects of RG on the proliferation and differentiation of hESCs. Undifferentiated hESCs, embryoid bodies (EBs), and hESC-derived cardiac progenitors (CPs) were treated with RG extract at 0.125, 0.25, and 0.5 mg/mL. After treatment of undifferentiated hESCs from day 2 to day 6 of culture, BrdU labeling showed that RG treatment increased the proliferation of hESCs, and the expression of Oct4 and Nanog was increased in RG-treated group. To find out the effects of RG on early differentiation stage cells, EBs were treated with RG extract for 10 days and attached for further differentiation. Immunostaining for three germ layer markers showed that RG treatment increased the expressions of Brachyury and HNF3βon EBs. Also, RG treatment increased the expression of Brachyury in early-stage and of Nkx2.5 in late-stage hESC-derived CPs. These results demonstrate facilitating effects of RG extract on the proliferation and early differentiation of hESC.
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Kumar, Deepak, Saniya Gupta, Ying Yang, and Nicholas R. Forsyth. "αVβ5 and CD44 Are Oxygen-Regulated Human Embryonic Stem Cell Attachment Factors." BioMed Research International 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/729281.
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Human embryonic stem cells (hESCs) have great potential for clinical therapeutic use. However, relatively little is known of the mechanisms which dictate their specificity of adhesion to substrates through adhesion proteins including integrins. Previous observations demonstrated enhanced clonogenicity in reduced oxygen culture systems. Here, we demonstrated via antibody blocking experiments thatαVβ5 andα6 significantly promoted hESC attachment in 2% O2only, whereas blockage of CD44 inhibited cell attachment in 21% O2alone. Immunofluorescence confirmed expression ofαVβ5 and CD44 in both 2% O2and 21% O2cultured hESCs while flow cytometry revealed significantly higherαVβ5 expression in 2% O2versus 21% O2cultured hESCs and higher CD44 expression in 21% O2versus 2% O2cultured hESCs. Adhered hESCs following blockage ofαVβ5 in 2% O2displayed a reduction in nuclear colocalisation of Oct-4 and Nanog with little effect observed in 21% O2. Blockage of CD44 had the converse effect with dramatic reductions in nuclear colocalisation of Oct-4 and Nanog in 21% O2cultured hESC which retained adherence, but not in 2% O2cultured cells. Identification of oxygen-dependent substrate attachment mechanisms in hESCs has the potential to play a role in the development of novel substrates to improve hESC attachment and culture.
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Svensson, Bengt, Srinivasa R. Nagubothu, Christoffer Nord, Jessica Cedervall, Isabell Hultman, Lars Ährlund-Richter, Anna Tolf, and Stellan Hertegård. "Stem Cell Therapy in Injured Vocal Folds: A Three-Month Xenograft Analysis of Human Embryonic Stem Cells." BioMed Research International 2015 (2015): 1–7. http://dx.doi.org/10.1155/2015/754876.
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We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (p≤0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (p≤0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives.
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Son, Mi-Young, Janghwan Kim, Hyo-Won Han, Sun-Mi Woo, Yee Sook Cho, Yong-Kook Kang, and Yong-Mahn Han. "Expression profiles of protein tyrosine kinase genes in human embryonic stem cells." REPRODUCTION 136, no. 4 (October 2008): 423–32. http://dx.doi.org/10.1530/rep-08-0080.
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Complex signaling pathways operate in human embryonic stem cells (hESCs) and are coordinated to maintain self-renewal and stem cell characteristics in them. Protein tyrosine kinases (PTKs) participate in diverse signaling pathways in various types of cells. Because of their functions as key molecules in various cellular processes, PTKs are anticipated to have important roles also in hESCs. In this study, we investigated the roles of PTKs in undifferentiated and differentiated hESCs. To establish comprehensive PTK expression profiles in hESCs, we performed reverse transcriptase PCR using degenerate primers according to the conserved catalytic PTK motifs in both undifferentiated and differentiated hESCs. Here, we identified 42 different kinases in two hESC lines, including 5 non-receptor tyrosine kinases (RTKs), 24 RTKs, and 13 dual and other kinases, and compared the protein kinase expression profiles of hESCs and retinoic acid-treated hESCs. Significantly, up- and downregulated kinases in undifferentiated hESCs were confirmed by real-time PCR and western blotting. MAP3K3, ERBB2, FGFR4, and EPHB2 were predominantly upregulated, while CSF1R, TYRO3, SRC, and GSK3A were consistently downregulated in two hESC lines. Western blot analysis showed that the transcriptional levels of these kinases were consistent with the translational levels. The obstruction of upregulated kinases’ activities using specific inhibitors disturbed the undifferentiated status and induced the differentiation of hESCs. Our results support the dynamic expression of PTKs during hESC maintenance and suggest that specific PTKs that are consistently up- and downregulated play important roles in the maintenance of stemness and the direction of differentiation of hESCs.
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Denham, Mark, Brock J. Conley, Fredrik Olsson, Lerna Gulluyan, Timothy J. Cole, and Richard Mollard. "A murine respiratory-inducing niche displays variable efficiency across human and mouse embryonic stem cell species." American Journal of Physiology-Lung Cellular and Molecular Physiology 292, no. 5 (May 2007): L1241—L1247. http://dx.doi.org/10.1152/ajplung.00440.2006.
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Human embryonic stemlike cells (hESCs) are pluripotent cells derived from blastocysts. Differentiating hESCs into respiratory lineages may benefit respiratory therapeutic programs. We previously demonstrated that 24% of all mouse embryonic stem cell (mESC) derivatives cocultured with embryonic day 11.5 (E11.5) mouse lung rudiments display immunoreactivity to the pneumonocyte II specific marker surfactant-associated protein C (Sftpc). Here we further investigate the effects of this inductive niche in terms of its competence to induce hESC derivative SFTPC immunoreactivity and the expression of other markers of terminal lung secretory units. When hESCs were cocultured as single cells, clumps of ∼10 cells or embryoid bodies (EBs), hESC derivatives formed pan-keratin-positive epithelial tubules at high frequency (>30% of all hESC derivatives). However, human-specific SFTPC immunoreactivity associated with tubule formation only at low frequency (<0.1% of all hESC derivatives). Human-specific SFTPD and secretoglobin family 1A member 1 ( SCGB1A1, also known as CC10) transcripts were detected by PCR after prolonged culture. Expression of other terminal lung secretory unit markers ( TITF1, SFTPA, and SFTPB) was not detected at any time point analyzed. On the other hand, hESC derivatives cultured as plated EBs in media previously demonstrated to induce Sftpc expression in isolated mouse fetal tracheal epithelium expressed all terminal lung secretory unit markers examined. mESCs and hESCs thus display fundamental differences in their response to the E11.5 mouse lung inductive niche, and these data provide an important step in the delineation of signaling mechanisms capable of efficiently inducing hESC differentiation into terminal secretory units of the lung.
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Islam, Intekhab, Gopu Sriram, Mingming Li, Yu Zou, Lulu Li, Harish K. Handral, Vinicus Rosa, and Tong Cao. "In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors." Stem Cells International 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/1659275.
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Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.
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Xie, Xiaoling, Xiaoling Zhou, Tingdang Liu, Zhiqian Zhong, Qi Zhou, Waqas Iqbal, Qingdong Xie, et al. "Direct Differentiation of Human Embryonic Stem Cells to 3D Functional Hepatocyte-like Cells in Alginate Microencapsulation Sphere." Cells 11, no. 19 (October 5, 2022): 3134. http://dx.doi.org/10.3390/cells11193134.
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Background: The lack of a stable source of hepatocytes is one of major limitations in hepatocyte transplantation and clinical applications of a bioartificial liver. Human embryonic stem cells (hESCs) with a high degree of self-renewal and totipotency are a potentially limitless source of a variety of cell lineages, including hepatocytes. Many techniques have been developed for effective differentiation of hESCs into functional hepatocyte-like cells. However, the application of hESC-derived hepatocyte-like cells (hESC-Heps) in the clinic has been constrained by the low yield of fully differentiated cells, small-scale culture, difficulties in harvesting, and immunologic graft rejection. To resolve these shortcomings, we developed a novel 3D differentiation system involving alginate-microencapsulated spheres to improve current hepatic differentiation, providing ready-to-use hESC-Heps. Methods: In this study, we used alginate microencapsulation technology to differentiate human embryonic stem cells into hepatocyte-like cells (hESC-Heps). Hepatic markers of hESC-Heps were examined by qPCR and Western blotting, and hepatic functions of hESC-Heps were evaluated by indocyanine-green uptake and release, and ammonia removal. Results: The maturity and hepatic functions of the hESC-Heps derived from this 3D system were better than those derived from 2D culture. Hepatocyte-enriched genes, such as HNF4α, AFP, and ALB, were expressed at higher levels in 3D hESC-Heps than in 2D hESC-Heps. 3D hESC-Heps could metabolize indocyanine green and had better capacity to scavenge ammonia. In addition, the 3D sodium alginate hydrogel microspheres could block viral entry into the microspheres, and thus protect hESC-Heps in 3D microspheres from viral infection. Conclusion: We developed a novel 3D differentiation system for differentiating hESCs into hepatocyte-like cells by using alginate microcapsules.
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Schultz, Sherri S., Sabrina C. Desbordes, Zhuo Du, Settapong Kosiyatrakul, Inna Lipchina, Lorenz Studer, and Carl L. Schildkraut. "Single-Molecule Analysis Reveals Changes in the DNA Replication Program for the POU5F1 Locus upon Human Embryonic Stem Cell Differentiation." Molecular and Cellular Biology 30, no. 18 (July 20, 2010): 4521–34. http://dx.doi.org/10.1128/mcb.00380-10.
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ABSTRACT Human embryonic stem cells (hESCs), due to their pluripotent nature, represent a particularly relevant model system to study the relationship between the replication program and differentiation state. Here, we define the basic properties of the replication program in hESCs and compare them to the programs of hESC-derived multipotent cells (neural rosette cells) and primary differentiated cells (microvascular endothelial cells [MECs]). We characterized three genomic loci: two pluripotency regulatory genes, POU5F1 (OCT4) and NANOG, and the IGH locus, a locus that is transcriptionally active specifically in B-lineage cells. We applied a high-resolution approach to capture images of individual replicated DNA molecules. We demonstrate that for the loci studied, several basic properties of replication, including the average speed of replication forks and the average density of initiation sites, were conserved among the cells analyzed. We also demonstrate, for the first time, the presence of initiation zones in hESCs. However, significant differences were evident in other aspects of replication for the DNA segment containing the POU5F1 gene. Specifically, the locations of centers of initiation zones and the direction of replication fork progression through the POU5F1 gene were conserved in two independent hESC lines but were different in hESC-derived multipotent cells and MECs. Thus, our data identify features of the replication program characteristic of hESCs and define specific changes in replication during hESC differentiation.
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Bigdeli, Narmin, Giuseppe Maria de Peppo, Camilla Karlsson, Maria Lennerås, Raimund Strehl, Johan Hyllner, and Anders Lindahl. "Upregulation of Adhesion Molecules Sustains Matrix-Free Growth of Human Embryonic Stem Cells." Open Stem Cell Journal 5, no. 1 (November 23, 2018): 14–30. http://dx.doi.org/10.2174/1876893801805010014.
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Background: Despite recent advances in culture techniques for undifferentiated human Embryonic Stem Cells (hESCs), further improvements are required to facilitate research and translation of these cells in clinical settings. We have previously derived hESC lines that can be cultured in their undifferentiated state on regular plastic culture dishes, without the need for feeder cells or other coating supports, denoted Matrix-Free Growth hESCs (MFG-hESCs). Objective: In this study, we further characterize and compare MFG-hESCs to hESCs in order to understand the molecular differences responsible for the unique ability of MFG-hESCs. Results: Microarray analysis demonstrated that MFG-hESCs highly resemble feeder-cultured hESCs in global gene expression profile. Two identified groups of genes with differential expression were those encoding for ribosomal proteins and attachment proteins, such as the RGD (Arg-Gly-Asp)-associated proteins. Real-time PCR and flow cytometry corroborated the microarray results. Culture of MFG-hESCs in the presence of RGD peptides resulted in decreased attachment ability compared to cells cultured in the presence of RGES (Arg-Gly-Asp-Ser) peptides. Conclusion: This study demonstrates that MFG-hESC lines overexpress cell attachment proteins but retain the typical characteristics of undifferentiated feeder-cultured hESCs. The ability to culture high-quality pluripotent stem cells in feeder- and matrix-free conditions creates a new opportunities for their large-scale manufacturing for experimental research and translational applications.
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Kiprilov, Enko N., Aashir Awan, Romain Desprat, Michelle Velho, Christian A. Clement, Anne Grete Byskov, Claus Y. Andersen, et al. "Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery." Journal of Cell Biology 180, no. 5 (March 10, 2008): 897–904. http://dx.doi.org/10.1083/jcb.200706028.
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Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC differentiation, demonstrating the existence of primary cilia and the localization of signaling components in undifferentiated hESCs establishes a mechanistic basis for the regulation of hESC differentiation. Using electron microscopy (EM), immunofluorescence, and confocal microscopies, we show that primary cilia are present in three undifferentiated hESC lines. EM reveals the characteristic 9 + 0 axoneme. The number and length of cilia increase after serum starvation. Important components of the hedgehog (Hh) pathway, including smoothened, patched 1 (Ptc1), and Gli1 and 2, are present in the cilia. Stimulation of the pathway results in the concerted movement of Ptc1 out of, and smoothened into, the primary cilium as well as up-regulation of GLI1 and PTC1. These findings show that hESCs contain primary cilia associated with working Hh machinery.
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Bueno, Clara, Verónica Ayllón, Rosa Montes, Oscar Navarro-Montero, Verónica Ramos-Mejia, Pedro J. Real, Damià Romero-Moya, Marcos J. Araúzo-Bravo, and Pablo Menendez. "FLT3 activation cooperates with MLL-AF4 fusion protein to abrogate the hematopoietic specification of human ESCs." Blood 121, no. 19 (May 9, 2013): 3867–78. http://dx.doi.org/10.1182/blood-2012-11-470146.
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Key Points FLT3 activation cooperates with the MLL-AF4 fusion gene to fully abolish blood formation from hESCs. FLT3 activation does not cooperate with the MLL-AF4 fusion oncogene to transform hESCs or hESC-derived hematopoietic progeny.
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Bagherpoor, Alireza Jian, Martin Kučírek, Radek Fedr, Soodabeh Abbasi Sani, and Michal Štros. "Nonhistone Proteins HMGB1 and HMGB2 Differentially Modulate the Response of Human Embryonic Stem Cells and the Progenitor Cells to the Anticancer Drug Etoposide." Biomolecules 10, no. 10 (October 15, 2020): 1450. http://dx.doi.org/10.3390/biom10101450.
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HMGB1 and HMGB2 proteins are abundantly expressed in human embryonic stem cells(hESCs) and hESC-derived progenitor cells (neuroectodermal cells, hNECs), though their functionalroles in pluripotency and the mechanisms underlying their dierentiation in response to the anticancerdrug etoposide remain to be elucidated. Here, we show that HMGB1 and/or HMGB2 knockdown(KD) by shRNA in hESCs did not aect the cell stemness/pluripotency regardless of etoposidetreatments, while in hESC-derived neuroectodermal cells, treatment resulted in dierential eects oncell survival and the generation of rosette structures. The objective of this work was to determinewhether HMGB1/2 proteins could modulate the sensitivity of hESCs and hESC-derived progenitorcells (hNECs) to etoposide. We observed that HMGB1 KD knockdown (KD) and, to a lesser extent,HMGB2 KD enhanced the sensitivity of hESCs to etoposide. Enhanced accumulation of 53BP1 ontelomeres was detected by confocal microscopy in both untreated and etoposide-treated HMGB1KD hESCs and hNECs, indicating that the loss of HMGB1 could destabilize telomeres. On the otherhand, decreased accumulation of 53BP1 on telomeres in etoposide-treated HMGB2 KD hESCs(but not in HMGB2 KD hNECs) suggested that the loss of HMGB2 promoted the stability of telomeres.Etoposide treatment of hESCs resulted in a significant enhancement of telomerase activity, withthe highest increase observed in the HMGB2 KD cells. Interestingly, no changes in telomerase activitywere found in etoposide-treated control hNECs, but HMGB2 KD (unlike HMGB1 KD) markedlydecreased telomerase activity in these cells. Changes in telomerase activity in the etoposide-treatedHMGB2 KD hESCs or hNECs coincided with the appearance of DNA damage markers and couldalready be observed before the onset of apoptosis. Collectively, we have demonstrated that HMGB1or HMGB2 dierentially modulate the impact of etoposide treatment on human embryonic stem cellsand their progenitor cells, suggesting possible strategies for the enhancement of the ecacy of thisanticancer drug.
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Kaufman, Dan S., Petter S. Woll, Colin H. Martin, and Jeffrey S. Miller. "Human Embryonic Stem Cells Differentiate into Functional Natural Killer Cells with the Capacity To Mediate Anti-Tumor Activity." Blood 106, no. 11 (November 16, 2005): 763. http://dx.doi.org/10.1182/blood.v106.11.763.763.
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Abstract Hematopoiesis from human embryonic stem cells (hESCs) follows developmental kinetics similar to what is observed during normal human ontogeny. Myeloid, erythroid and megakaryocytic progenitors can be routinely generated from hESCs. However, little is known about the ability of hESCs to differentiate into the lymphoid lineage. Natural killer (NK) cells are important mediators of donor anti-host alloreactivity seen after allogeneic transplant for myeloid leukemias. Our studies use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. CD34+ and CD34+CD45+ hESC-derived hematopoietic progenitor cells were co-cultured with inactivated AFT024 stromal cells in medium supplemented with IL-7, IL-15, SCF and FL. Generation of NK cells was established by phenotypic and functional analysis. CD34+ umbilical cord blood (UCB) cells were utilized as a positive control. After 14 days of culture of CD34+ hESC-derived cells, more than 90% of the cells express CD45, a pan-hematopoietic cell marker, but few CD56+ cells are observed. At 21 days of culture a distinct CD56+CD45+ cell population develops (14.9%), which increases to 37.5% of cells after 28 days of culture. Similar results are observed for CD34+CD45+ hESC-derived cells, characterizing that both CD34+ and CD34+CD45+ cell populations contain hematopoietic progenitors with NK cell developmental potential. Limiting dilution analysis of hESC-derived progenitor cells demonstrates CD34+ hESC-derived cells have a low NK cell progenitor frequency. However, sorting for CD34+CD45+ hESC-derived cells significantly increased the NK cell cloning frequency (1.92% ± 1.20%) to a level comparable to the frequency observed for CD34+ UCB cells cultured in the same manner (3.57% ± 1.68%). The hESC-derived NK cells also express receptors known to regulate NK cell cytolytic activity, including killer-Ig-like receptors (KIRs), C-type lectin-like receptors (CD94 and NKG2A) and natural cytotoxicity receptors (NKp30, NKp44, and NKp46). Furthermore, hESC-derived NK cells also express CD16, an Fc-receptor typically expressed on more mature NK cells. The expression of KIRs is significantly higher for the hESC-derived NK cells compared to the UCB-derived NK cells. This may lead to future strategies to generate selective alloreactive NK cell populations for therapy. To investigate the functional properties of the hESC-derived NK cells, cytolytic activity was tested against K562 erythroleukemia cells and Raji B-lymphoblastoid cells. hESC-derived NK cells effectively killed K562 cells, with activity similar to that seen with UCB-derived NK cells. As expected, Raji cells were resistant to direct cytotoxicity by both hESC and UCB-derived NK cells. However, treatment of Raji cells with anti-CD20 antibody results in effective antibody-dependent cell-mediated cytoxicity by the hESC-derived NK cells. The hESC-derived NK cells also demonstrate ability to upregulate production of cytokines such as IFN-γ upon stimulation. Furthermore, we also find that hESC-derived progenitors also have T cell and/or B cell potential based on cells that express Ikaros, Rag1, and IL7Rα. These results demonstrate that the CD34+ and CD34+CD45+ hESC-derived cell populations contain lymphoid progenitor cells that can develop into both innate and adaptive immune cells. The ability to generate functional NK cells that can target and lyse human tumor cells via two distinct mechanisms suggests potentially novel anti-cancer therapy applications of hESCs.
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Linehan, Jonathan L., Xinghui Tian, Julie K. Morris, and Dan S. Kaufman. "Improved Analysis of Hematopoietic Engraftment by Non-Invasive In Vivo Bioluminescent Imaging of Transplanted Human Embryonic Stem Cell-Derived Hematopoietic Cells." Blood 106, no. 11 (November 16, 2005): 1270. http://dx.doi.org/10.1182/blood.v106.11.1270.1270.
Full textAbstract:
Abstract Animal transplantation models are essential to characterize the long-term in vivo engraftment capacity of putative hematopoietic stem cells derived from human embryonic stem cells (hESCs). We have previously demonstrated that hESCs can be routinely utilized to derive multiple hematopoietic cell lineages. Here, we use in vivo bioluminescence imaging (BLI) of stable luciferase (luc)-expressing hESCs to noninvasively monitor the dynamics of transplantation, engraftment, and growth of hESC-derived hematopoietic cells within individual animals over an extended time course. Luc expression under control of an EF1α promoter was introduced into the H1 hESC line using a self-inactivating lentiviral vector. Undifferentiated hESC colonies that stably expressed luciferase were established and selected, and the pluripotent capability of luc+ hESCs was first explored by teratoma formation. Undifferentiated luc+ human ES cells were intramuscularly injected into NOD/SCID mice. The dynamics of survival and growth of the hESCs was monitored by BLI using the IVIS Imaging System (Xenogen) at regular time points post-transplantation. There was a decrease of luminescent signal during the first 1–2 weeks. This was followed by a dramatic increase in luminescent signals after about 5 weeks, which correlated with teratoma size. Immunohistochemical analysis confirmed stable luc-expression in multiple differentiated cell types within the teratomas. We next used BLI to examine luc+ H1 hESCs that were induced to undergo hematopoietic differentiation by co-culture with S17 cells, to give rise to H1/S17 cells. Flow cytometric studies confirmed hematopoietic cells (CD34+, CD45+, CD31+, and c-kit+ cells) were derived from these differentiated luc+ hESCs, with 5–10% of H1/S17 cells being CD34+. Hematopoietic progenitors that gave rise to colonies of mature luc+ blood cells in a standard CFU assay were also observed from the H1/S17 cells. Luc-expression of differentiated hESCs was maintained at similar levels to those of the undifferentiated ES cells. To define the in vivo potential of luc+ hESC-derived hematopoietic cells, hESCs were allowed to differentiate on S17 cells for two weeks. SCID-repopulating cell studies were done by intravenous (iv) injection into sublethally irradiated NOD/SCID mice. After iv injection of 2–3 x106 unsorted luc+ H1/S17 cells, BLI showed the brightest signal in the lung at day 0 (within 2 hours), followed by a rapid decline in signal on the next day (day1). On day 8, most luc+ cells were detected in the abdomen and liver. Subsequently, after 6–12 weeks, multiple engraftment loci were identified in hematopoietic tissues. Flow cytometric analysis of bone marrow from these mice confirmed the presence of hESC-derived human CD45+ cells. Engraftment was also demonstrated after direct intra-bone marrow injection with as few as 60,000 CD34+ cells sorted from luc+ H1/S17 cells. Again, stable engraftment can be monitored by BLI for 8+ weeks. These results demonstrate that BLI has several important advantages as an effective non-invasive approach to track and quantitatively monitor in vivo engraftment of hematopoietic or other cell lineages derived from hESCs.
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Wang, Lisheng, Pablo Menendez, Farbod Shojaei, Li Li, Frederick Mazurier, John E. Dick, Chantal Cerdan, Krysta Levac, and Mickie Bhatia. "Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression." Journal of Experimental Medicine 201, no. 10 (May 9, 2005): 1603–14. http://dx.doi.org/10.1084/jem.20041888.
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Despite the need for alternative sources of human hematopoietic stem cells (HSCs), the functional capacity of hematopoietic cells generated from human embryonic stem cells (hESCs) has yet to be evaluated and compared with adult sources. Here, we report that somatic and hESC-derived hematopoietic cells have similar phenotype and in vitro clonogenic progenitor activity. However, in contrast with somatic cells, hESC-derived hematopoietic cells failed to reconstitute intravenously transplanted recipient mice because of cellular aggregation causing fatal emboli formation. Direct femoral injection allowed recipient survival and resulted in multilineage hematopoietic repopulation, providing direct evidence of HSC function. However, hESC-derived HSCs had limited proliferative and migratory capacity compared with somatic HSCs that correlated with a distinct gene expression pattern of hESC-derived hematopoietic cells that included homeobox (HOX) A and B gene clusters. Ectopic expression of HOXB4 had no effect on repopulating capacity of hESC-derived cells. We suggest that limitations in the ability of hESC-derived HSCs to activate a molecular program similar to somatic HSCs may contribute to their atypical in vivo behavior. Our study demonstrates that HSCs can be derived from hESCs and provides an in vivo system and molecular foundation to evaluate strategies for the generation of clinically transplantable HSC from hESC lines.
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Souralova, Tereza, Daniela Rehakova, Michal Jeseta, Lenka Tesarova, Jindrich Beranek, Pavel Ventruba, Ales Hampl, and Irena Koutna. "The Manufacture of Xeno- and Feeder-Free Clinical-Grade Human Embryonic Stem Cell Lines: First Step for Cell Therapy." International Journal of Molecular Sciences 23, no. 20 (October 18, 2022): 12500. http://dx.doi.org/10.3390/ijms232012500.
Full textAbstract:
Human embryonic stem cells (hESCs) are increasingly used in clinical trials as they can change the outcome of treatment for many human diseases. They are used as a starting material for further differentiation into specific cell types and to achieve the desirable result of the cell therapy; thus, the quality of hESCs has to be taken into account. Therefore, current good manufacturing practice (cGMP) has to be implemented in the transport of embryos, derivation of inner cell mass to xeno-free, feeder-free and defined hESC culture, and cell freezing. The in-depth characterization of hESC lines focused on safety, pluripotency, differentiation potential and genetic background has to complement this process. In this paper, we show the derivation of three clinical-grade hESC lines, MUCG01, MUCG02, and MUCG03, following these criteria. We developed and validated the system for the manufacture of xeno-free and feeder-free clinical-grade hESC lines that present high-quality starting material suitable for cell therapy according to cGMP.
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Woll, Petter S., Bartosz Grzywacz, Xinghui Tian, Rebecca K. Marcus, David A. Knorr, Michael R. Verneris, and Dan S. Kaufman. "Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity." Blood 113, no. 24 (June 11, 2009): 6094–101. http://dx.doi.org/10.1182/blood-2008-06-165225.
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Abstract Natural killer (NK) cells serve as important effectors for antitumor immunity, and CD56+CD45+ NK cells can be routinely derived from human embryonic stem cells (hESCs). However, little is know about the ability of hESC-derived NK cells to mediate an effective in vivo antitumor response. Using bioluminescent imaging, we now demonstrate that H9 line hESC-derived NK cells mediate effective clearance of human tumor cells in vivo. In addition to increased in vitro killing of diverse tumor targets, the in vivo tumor clearance by H9 hESC-derived NK cells was more effective compared with NK cells derived from umbilical cord blood (UCB). Phenotypic analysis demonstrates the hESC-derived NK cells are uniformly CD94+CD117low/−, an NK-cell population characterized by potent cytolytic activity and thus more competent to mediate tumor clearance. These studies demonstrate that hESCs provide an important model to study human lymphocyte development and may serve as a novel source for antitumor immunotherapy.
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Arlier, Sefa, Umit A. Kayisli, Nihan Semerci, Asli Ozmen, Kellie Larsen, Frederick Schatz, Charles J. Lockwood, and Ozlem Guzeloglu-Kayisli. "Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation." International Journal of Molecular Sciences 24, no. 13 (June 23, 2023): 10532. http://dx.doi.org/10.3390/ijms241310532.
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Progestin-only long-acting reversible-contraceptive (pLARC)-exposed endometria displays decidualized human endometrial stromal cells (HESCs) and hyperdilated thin-walled fragile microvessels. The combination of fragile microvessels and enhanced tissue factor levels in decidualized HESCs generates excess thrombin, which contributes to abnormal uterine bleeding (AUB) by inducing inflammation, aberrant angiogenesis, and proteolysis. The- zinc finger and BTB domain containing 16 (ZBTB16) has been reported as an essential regulator of decidualization. Microarray studies have demonstrated that ZBTB16 levels are induced by medroxyprogesterone acetate (MPA) and etonogestrel (ETO) in cultured HESCs. We hypothesized that pLARC-induced ZBTB16 expression contributes to HESC decidualization, whereas prolonged enhancement of ZBTB16 levels triggers an inflammatory milieu by inducing pro-inflammatory gene expression and tissue-factor-mediated thrombin generation in decidualized HESCs. Thus, ZBTB16 immunostaining was performed in paired endometria from pre- and post-depo-MPA (DMPA)-administrated women and oophorectomized guinea pigs exposed to the vehicle, estradiol (E2), MPA, or E2 + MPA. The effect of progestins including MPA, ETO, and levonorgestrel (LNG) and estradiol + MPA + cyclic-AMP (E2 + MPA + cAMP) on ZBTB16 levels were measured in HESC cultures by qPCR and immunoblotting. The regulation of ZBTB16 levels by MPA was evaluated in glucocorticoid-receptor-silenced HESC cultures. ZBTB16 was overexpressed in cultured HESCs for 72 h followed by a ± 1 IU/mL thrombin treatment for 6 h. DMPA administration in women and MPA treatment in guinea pigs enhanced ZBTB16 immunostaining in endometrial stromal and glandular epithelial cells. The in vitro findings indicated that: (1) ZBTB16 levels were significantly elevated by all progestin treatments; (2) MPA exerted the greatest effect on ZBTB16 levels; (3) MPA-induced ZBTB16 expression was inhibited in glucocorticoid-receptor-silenced HESCs. Moreover, ZBTB16 overexpression in HESCs significantly enhanced prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP1), and tissue factor (F3) levels. Thrombin-induced interleukin 8 (IL-8) and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels in control-vector-transfected HESCs were further increased by ZBTB16 overexpression. In conclusion, these results supported that ZBTB16 is enhanced during decidualization, and long-term induction of ZBTB16 expression by pLARCs contributes to thrombin generation through enhancing tissue factor expression and inflammation by enhancing IL-8 and PTGS2 levels in decidualized HESCs.
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Trivedi, Parul, and Peiman Hematti. "Derivation of SSEA4-/CD73+ Mesenchymal Stem Cells from Human Embryonic Stem Cells." Blood 108, no. 11 (November 16, 2006): 2579. http://dx.doi.org/10.1182/blood.v108.11.2579.2579.
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Abstract Human embryonic stem cells (hESCs) could potentially provide a renewable source of different types of cells for cell therapy applications. Recently, mesenchymal stem cells (MSCs) have been derived from hESCs either through co-culturing with murine OP9 bone marrow stromal cell line or directly from hESCs without co-culturing with OP9 cells. Although the latter methodology is clinically advantageous over co-culturing with an animal cell layer those mesenchymal cells were reported to be positive for SSEA4. SSEA4 is a marker of undifferentiated hESCs and thus the presence of this marker on hESC-derived cells could potentially be problematic for clinical applications. We have recently achieved a novel and reproducible methodology for deriving a pure population of SSEA4-/CD73+ MSCs from federally approved hESC lines H1 and H9. To initiate the differentiation of hESCs to MSCs, we cultured undifferentiated hESCs on matrigel plates in murine embryonic fibroblast conditioned media with media changes every 3 days. Under these culture conditions a portion of embryonic stem cells differentiated into fibroblast looking cells. Through a multi-step process which involved the use of a culture methodology similar to what is being used to culture bone marrow (BM)-derived MSCs, and passaging cultured cells at defined time points we were able to derive a pure population of cells that were uniformly positive for MSC marker CD73 in about a 4-weeks period. These cells had fibroblast/mesenchymal looking morphology, and expressed cell surface marker antigens similar to what has been reported for adult human BM-derived MSCs: they are positive for CD29, CD44, CD54, CD71, CD90, glycophorin A, CD105, and were negative for hematopoietic markers such as CD34 and CD45. Similar to adult BM-derived MSCs these cells express HLA class-I antigens but not class-II antigens. Using established differentiation protocols we could differentiate the hESC-derived CD73+ MSCs into adipocytes, osteocytes, and chondrocytes as verified by immunohistochemistry and RT-PCR assays. So far we have grown these CD73+ MSCs up to passages 15–18. These cells retained their differentiation potential, and were karotypically normal when tested at passage 12. Most importantly, we did not observe any MSCs that were double positive for CD73 and SSEA4 antigen at any time point during our experiments. MSCs from a variety of fetal and adult sources are in various stages of clinical trials with some encouraging preliminary results. Our hESC-derived MSCs that are very similar to adult BM-derived MSCs regarding their growth and morphologic properties, immunophenotypic characteristics, differentiation potential, and importantly are devoid of hESC marker SSEA4 could potentially provide a novel source of MSCs for clinical applications.
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Wang, Linlin, Thomas C. Schulz, Eric S. Sherrer, Derek S. Dauphin, Soojung Shin, Angelique M. Nelson, Carol B. Ware, et al. "Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling." Blood 110, no. 12 (December 1, 2007): 4111–19. http://dx.doi.org/10.1182/blood-2007-03-082586.
Full textAbstract:
Abstract Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1β (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.
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Yokoyama, Yasuhisa, Takahiro Suzuki, Mamiko Sakata-Yanagimoto, Keiki Kumano, Katsumi Higashi, Tsuyoshi Takato, Mineo Kurokawa, Seishi Ogawa, and Shigeru Chiba. "Derivation of functional mature neutrophils from human embryonic stem cells." Blood 113, no. 26 (June 25, 2009): 6584–92. http://dx.doi.org/10.1182/blood-2008-06-160838.
Full textAbstract:
Abstract Human embryonic stem cells (hESCs) proliferate infinitely and are pluripotent. Only a few reports, however, describe specific and efficient methods to induce hESCs to differentiate into mature blood cells. It is important to determine whether and how these cells, once generated, behave similarly with their in vivo–produced counterparts. We developed a method to induce hESCs to differentiate into mature neutrophils. Embryoid bodies were formed with bone morphogenic protein-4, stem cell factor (SCF), Flt-3 ligand (FL), interleukin-6 (IL-6)/IL-6 receptor fusion protein (FP6), and thrombopoietin (TPO). Cells derived from the embryoid bodies were cultured on a layer of irradiated OP9 cells with a combination of SCF, FL, FP6, IL-3, and TPO, which was later changed to granulocyte–colony-stimulating factor. Morphologically mature neutrophils were obtained in approximately 2 weeks with a purity and efficiency sufficient for functional analyses. The population of predominantly mature neutrophils (hESC-Neu's) showed superoxide production, phagocytosis, bactericidal activity, and chemotaxis similar to peripheral blood neutrophils from healthy subjects, although there were differences in the surface antigen expression patterns, such as decreased CD16 expression and aberrant CD64 and CD14 expression in hESC-Neu's. Thus, this is the first description of a detailed functional analysis of mature hESC-derived neutrophils.
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Li, Dong, Jiaxi Zhou, Lu Wang, Myung Eun Shin, Pei Su, Xiaohua Lei, Haibin Kuang, et al. "Integrated biochemical and mechanical signals regulate multifaceted human embryonic stem cell functions." Journal of Cell Biology 191, no. 3 (October 25, 2010): 631–44. http://dx.doi.org/10.1083/jcb.201006094.
Full textAbstract:
Human embryonic stem cells (ESCs [hESCs]) proliferate as colonies wherein individual cells are strongly adhered to one another. This architecture is linked to hESC self-renewal, pluripotency, and survival and depends on epithelial cadherin (E-cadherin), NMMIIA (nonmuscle myosin IIA), and p120-catenin. E-cadherin and p120-catenin work within a positive feedback loop that promotes localized accumulation of E-cadherin at intercellular junctions. NMMIIA stabilizes p120-catenin protein and controls E-cadherin–mediated intercellular adhesion. Perturbations of this signaling network disrupt colony formation, destabilize the transcriptional regulatory circuitry for pluripotency, and impair long-term survival of hESCs. Furthermore, depletion of E-cadherin markedly reduces the efficiency of reprogramming of human somatic cells to an ESC-like state. The feedback regulation and mechanical–biochemical integration provide mechanistic insights for the regulation of intercellular adhesion and cellular architecture in hESCs during long-term self-renewal. Our findings also contribute to the understanding of microenvironmental regulation of hESC identity and somatic reprogramming.
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Kimbrel, Erin A., and Shi-Jiang Lu. "Potential Clinical Applications for Human Pluripotent Stem Cell-Derived Blood Components." Stem Cells International 2011 (2011): 1–11. http://dx.doi.org/10.4061/2011/273076.
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The ability of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to divide indefinitely without losing pluripotency and to theoretically differentiate into any cell type in the body makes them highly attractive cell sources for large scale regenerative medicine purposes. The current use of adult stem cell-derived products in hematologic intervention sets an important precedent and provides a guide for developing hESC/iPSC based therapies for the blood system. In this review, we highlight biological functions of mature cells of the blood, clinical conditions requiring the transfusion or stimulation of these cells, and the potential for hESC/iPSC-derivatives to serve as functional replacements. Many researchers have already been able to differentiate hESCs and/or iPSCs into specific mature blood cell types. For example, hESC-derived red blood cells and platelets are functional in tasks such as oxygen delivery and blood clotting, respectively and may be able to serve as substitutes for their donor-derived counterparts in emergencies. hESC-derived dendritic cells are functional in antigen-presentation and may be used as off-the-shelf vaccine therapies to stimulate antigen-specific immune responses against cancer cells. However,in vitrodifferentiation systems used to generate these cells will need further optimization before hESC/iPSC-derived blood components can be used clinically.
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Wang, LiYun, RuiNa Zhang, RongHua Ma, GongXue Jia, ShengYan Jian, XiangHui Zeng, ZhengFang Xiong, et al. "Establishment of a feeder and serum-free culture system for human embryonic stem cells." Zygote 28, no. 3 (January 22, 2020): 175–82. http://dx.doi.org/10.1017/s0967199419000625.
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SummaryStem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cells in vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and α-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and α-fetoprotein were expressed in H9 cells, suggesting that the cells maintained in vitro differentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs.
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Ishii, Takamichi, Ken Fukumitsu, Kentaro Yasuchika, Keiko Adachi, Eihachiro Kawase, Hirofumi Suemori, Norio Nakatsuji, Iwao Ikai, and Shinji Uemoto. "Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 295, no. 2 (August 2008): G313—G321. http://dx.doi.org/10.1152/ajpgi.00072.2008.
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Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human α-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of growth factors including hepatocyte growth factor (HGF), bone morphogenetic protein 4, fibroblast growth factor 4, all- trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and 20 ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.
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Zhu, Hailin, Jinliang Yang, Yuquan Wei, and Harry Huimin Chen. "Development of a Xeno-Free Substrate for Human Embryonic Stem Cell Growth." Stem Cells International 2015 (2015): 1–8. http://dx.doi.org/10.1155/2015/621057.
Full textAbstract:
Traditionally, human embryonic stem cells (hESCs) are cultured on inactivated live feeder cells. For clinical application using hESCs, there is a requirement to minimize the risk of contamination with animal components. Extracellular matrix (ECM) derived from feeder cells is the most natural way to provide xeno-free substrates for hESC growth. In this study, we optimized the step-by-step procedure for ECM processing to develop a xeno-free ECM that supports the growth of undifferentiated hESCs. In addition, this newly developed xeno-free substrate can be stored at 4°C and is ready to use upon request, which serves as an easier way to amplify hESCs for clinical applications.
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Woll, Petter S., Rebecca Marcus, and Dan S. Kaufman. "NK Cells Derived from Human Embryonic Stem Cells Demonstrate More Effective In Vivo Clearance of Xenografted Human Tumor Cells Compared to NK Cells Derived from Cord Blood." Blood 110, no. 11 (November 16, 2007): 2745. http://dx.doi.org/10.1182/blood.v110.11.2745.2745.
Full textAbstract:
Abstract The derivation of both myeloid and lymphoid cells from human embryonic stem cells (hESCs) clearly establish hESCs as an important model system to study human hematopoietic ontogeny. However, the potential for clinical applications of hESC-derived hematopoietic cells still remain poorly characterized. Here we have analyzed the efficacy of hESC-derived natural killer (NK) cells in a model of anti-tumor immunotherapy. hESC-derived NK cells were compared to NK cells derived from human umbilical cord blood (UCB) for ability to clear both established human tumors and metastatic disease in an in vivo model. Using a two-step differentiation process, we have demonstrated effective derivation of NK cells from hESCs. The hESC-derived NK cells express activating and inhibitory receptors similar to NK cells derived from UCB. These receptors include C-type lectin-like receptors, natural cytotoxicity receptors, CD16 and diverse killer-cell Ig-like receptors. More importantly, the hESC-derived NK cells also demonstrate cytokine production and potent direct cytolytic activity against multiple types of tumors, including leukemia, lymphoma, glioma, testicular cancer and breast cancer cells lines. This in vitro cytolytic activity is similar to what found for UCB-derived NK cells cultured in identical conditions. To advance these studies to a more relevant pre-clinical model, we have now investigated the in vivo activity of hESC-derived NK cells in a xenogeneic mouse model. Here, K562 erythroleukemia cells stably expressing firefly luciferase (luc) were injected subcutaneously into sub-lethally irradiated NOD/SCID mice. The luc+ K562 cells allows serial bioluminescent imaging to follow growth of the tumor cells non-invasively over a prolonged time course, as well as sensitive detection of micro-metastasis. Three days after tumor-inoculation, mice received one of three treatment courses: NK cells derived from hESCs, NK cells derived from UCB, or no cells. Each group received ip injections of IL-15 every 2–3 days for the first 7 days after treatment, then IL-2 every 2–3 days for an additional 2 weeks. In this model, mice that received cytokine treatment but did not receive NK cells (n=11) consistently developed large tumors within three weeks. Remarkably, all mice treated with hESC-derived NK cells demonstrated complete clearance of the primary tumor two weeks after tumor inoculation (n=8). In contrast, mice treated with UCB-derived NK cells had significantly less anti-tumor activity in vivo, with only 50% tumor-free animals treated with UCB-derived NK cells (n=8). Some mice treated with hESC-derived NK cells were monitored up to 8 weeks with no evidence of tumor development. Furthermore, liver, lungs, spleen and kidneys were harvested at the time of sacrifice and analyzed for presence of micro-metastasis by detection of luc. In animals receiving cytokines alone, 50% of the organs analyzed displayed metastatic presence of luc+ cells. However, there was a significant reduction of metastases in UCB-NK-treated (9%) and hESC-NK-treated (4%) animals. These results suggest that hESC-derived NK cells are capable of clearing human tumor cells in vivo more effectively than UCB-derived NK cells. Current studies are underway to investigate in vivo activity of hESC-derived NK cells in other tumor models, and to evaluate specific mechanisms that might regulate improved in vivo activity of hESC- compared to UCB-derived NK cells, focusing on in vivo cell migration, cell survival and proliferation.
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Yu, Qing C., Claire E. Hirst, Magdaline Costa, Elizabeth S. Ng, Jacqueline V. Schiesser, Karin Gertow, Edouard G. Stanley, and Andrew G. Elefanty. "APELIN promotes hematopoiesis from human embryonic stem cells." Blood 119, no. 26 (June 28, 2012): 6243–54. http://dx.doi.org/10.1182/blood-2011-12-396093.
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Abstract Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein–coupled APELIN receptor (APLNR). APLNR-positive cells, identified by binding of the fluoresceinated peptide ligand, APELIN (APLN), or an anti-APLNR mAb, were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs), increased the expression of hematoendothelial genes in differentiating hESCs, and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore, APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells.
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Woll, Petter S., Colin H. Martin, and Dan S. Kaufman. "Characterization of Hematopoietic Progenitor Cells Derived from Human Embryonic Stem Cells That Differentiate into Natural Killer Cells Capable of In Vivo Anti-Tumor Activity." Blood 108, no. 11 (November 16, 2006): 645. http://dx.doi.org/10.1182/blood.v108.11.645.645.
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Abstract Derivation of multiple blood cell lineages from human embryonic stem cells (hESCs) clearly establishes these cells as an important model system to better characterize human hematopoietic ontogeny. Previously we have demonstrated development of myeloid, erythroid and lymphoid cells, as well as endothelial cells, from hESCs. However, the progenitor cells that give rise to these more mature cells remain poorly characterized. Here we use combined phenotypic and genetic analysis to discriminate between distinct progenitor cell populations. We also demonstrate that natural killer (NK) cells derived from these hESC-derived progenitors are capable of killing diverse tumors both in vitro and in vivo. By detailed flow cytometric analysis of differentiated hESCs over a defined time course, we identify development of two waves of CD34+ cells. The first wave consists of CD34brightCD31+Flk1+ cells, whereas the second wave consists of CD34dimCD45+ cells. Novel combined morphologic and phenotypic analysis by image scanning flow cytometry demonstrates these phenotypically different cell populations also are morphologically distinct. CD34brightCD31+ cells derived from hESCs appear to have a heterogeneous cell morphology, with an irregular cell shape, higher mean cell size and complex cytoplasmic organization. In contrast, CD34dimCD45+ cells are a more homogeneous cell population with a uniform spherical morphology and smaller cell size. Purified CD34brightCD31+Flk1+ cells express transcription factors associated with both the hematopoietic and endothelial lineages and can differentiate into both these lineages in vitro. In contrast, CD34dimCD45+ cells display a transcription profile suggestive of hematopoietic commitment and are significantly enriched for hematopoietic progenitors. Taken together, these results suggest that the CD34brightCD31+Flk1+ and CD34dimCD45+ cell populations are distinct cell lineages that represent early hemato-endothelial and hematopoietic precursors, respectively. Next, we better characterized the lymphoid developmental potential of the CD34dimCD45+ cells by the ability of these cells to differentiate into NK cells. Using a two-step differentiation process we find that hESC-derived NK cells express the wide repertoire of activating and inhibitory receptors similar to NK cells derived from other sources. We now demonstrate that these hESC-derived NK cells acquire cytolytic activity against breast cancer and glioma cell lines, as well as leukemia and lymphoma cells, in vitro. Finally, we have begun to test the in vivo efficacy of hESC-derived NK cells against established tumors. Here, luciferase (luc)-expressing K562 cells are inoculated into NOD/SCID mice, and several days later 2x106 NK cells derived from hESCs or from umbilical cord blood (UCB) cells are injected iv. The luc+ K562 cells allows serial bioluminescent imaging to follow growth of the tumor cells non-invasively over a prolonged time course. These studies demonstrate a beneficial effect of hESC-derived NK cells on tumor growth and metastasis, comparable to the effect of UCB-derived NK cells. Taken together, these studies characterize the earliest hematopoietic progenitors as they develop from hESCs during in vitro differentiation, providing a starting point to evaluate the effect of endogenous and exogenous factors on differentiation of distinct human hematopoietic lineages. This will translate into even more efficient derivation of NK cells from hESCs with a potential use for cancer immunotherapy.
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Liu, Ying, Man Ryul Lee, Khalid Timani, Johnny J. He, and Hal E. Broxmeyer. "Tip110 Maintains Expression of Pluripotent Factors in and Pluripotency of Human Embryonic Stem Cells." Blood 118, no. 21 (November 18, 2011): 2353. http://dx.doi.org/10.1182/blood.v118.21.2353.2353.
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Abstract Abstract 2353 Embryonic stem cells (ESCs) are pluripotent, self-renew and can be differentiated into cells of all three germ layers, and nanog, Oct4 and Sox2, form a core of the self-renewal transcription network. Nanog expression is restricted to pluripotent cells and is down regulated upon differentiation; little is known about its regulation. Expression of the OCT4 gene maintains cell pluripotency via a stringent dose-dependent regulation with OCT4 levels above or below required dosages producing cellular differentiation; thus maintenance of a critical amount of OCT4 is necessary to prevent ESC differentiation. Sox2, a high-mobility group domain containing transcription factor, binds to the consensus motif CATTGTT. We recently reported in Blood that Tip110 is an essential gene expressed in earliest cells of adult bone marrow hematopoietic development. Increased TIP110 expression enhanced hematopoietic progenitor cell (HPC) numbers, survival, and cell cycling; decreased Tip110 expression manifested the opposite effect, demonstrating a role for TIP110 in regulation of hematopoiesis. Herein, we investigated TIP110 expression and actions in human (h)ESCs. Quantitative RT-PCR showed that TIP110, as well as Nanog, Oct4 and Sox2 were expressed in a hESC line. hESCs were removed from feeder layers and b-FGF for 5 days, to allow ESC differentiation. TIP110 expression levels were dramatically reduced (by 77%); this was associated with large decreases in expression of NANOG (82%), OCT4 (80%), and SOX2 (85%). We then assessed whether TIP110 might regulate hESC pluripotency. We exogenously over-expressed TIP110 in hESC cells. Feeder layers and b-FGF were withdrawn upon introducing the TIP110 vector and cells cultured for 5 days to test whether sustained TIP110 expression rendered ESCs less sensitive to differentiation. Compared with controls, TIP110 over-expressing cells stained positive for OCT4, NANOG and were negative for Tuji, SMA and AFP, demonstrating that over-expression of TIP110 rendered ESCs less responsive to differentiation. Next, we reduced TIP110 expression by transfection of the hESCs with TIP110 siRNA. Cells were cultured in mTeSR medium on Matrigel-coated dishes for an additional 5 days in order to maintain cells under undifferentiation conditions. TIP 110 siRNA vector expressing cells were negative for OCT4, NANOG, and positive for Tuji, SMA and AFP expression compared with control cells, demonstrating that enforced reduction of TIP110 expression in hESCs causes hESC differentiation. This demonstrated the importance of TIP110 in maintenance of ESC pluripotency. We speculated that TIP110 maintenance of hESC pluripotency might be through regulation of NANOG, OCT4 and SOX2. We silenced TIP110 expression in hESCs by transfection with a TIP110 siRNA vector, previously shown to reduce TIP110 expression by 70%. Cells were cultured in complete 20% KSR hESC medium for an additional 5 days. Expression of these three transcription factors was dramatically decreased, demonstrating that TIP110 is required for maintaining NANOG, OCT4 and SOX2 levels in this hESC line. Reduction of TIP110 expression caused hESC differentiation directly or indirectly through down-regulation of NANOG, OCT4 and SOX2 expression. Thus, TIP110 is preferentially expressed in the undifferentiated state in hESCs and plays a key role in regulating OCT4, SOX2, and NANOG, factors required to maintain pluripotency. Together, our present and previous studies suggest TIP110 expression as a useful marker to distinguish early from more differentiated cells. Modulating TIP110 expression in a controlled fashion may be relevant for cellular engineering and regenerative medicine. Disclosures: No relevant conflicts of interest to declare.
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Ma, Feng, Yasuhiro Ebihara, Katsutsugu Umeda, Hiromi Sakai, Sachiyo Hanada, Hong Zhang, Eishun Tsuchida, Tatsutoshi Nakahata, Hiromitsu Nakauchi, and Kohichiro Tsuji. "Clonal Analysis of Progressive Maturation of Erythroid Cells from Human Embryonic Stem Cell-Derived Definitive Hematopoiesis." Blood 110, no. 11 (November 16, 2007): 2236. http://dx.doi.org/10.1182/blood.v110.11.2236.2236.
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Abstract A critical issue for utilization of human embryonic stem cells (hESCs) in regenerative medicine is whether they can derive terminally mature progenies with normal function. We recently developed an efficient co-culture system of hESCs with murine fetal liver-derived stromal cells (mFLSCs), which enabled us to produce a large quantity of hESC-derived erythroid progenitor cells. By combining the hESC/mFLSC co-culture and a clonal culture, we analyzed the development of hESC-derived erythroid cells at a clone level. From day 10 of the co-culture, hESCs generated both erythroid colony- and erythroid burst-forming cells (E-CFCs and E-BFCs, respectively), and their numbers rapidly increased and reached a peak at day 14. By a clone-culture, we analyzed the erythroid cells in individually colonies and bursts by immunostaining. All erythroid cells in the colonies and bursts expressed alpha and gama globins, while beta and epsilon globins were expressed in a portion of these erythroid cells, but all the colonies and bursts contained a substantial number of beta and epsilon globin-expressing cells. We then examined the globin expression in individual bursts at day 12, 14, 16 and 18 of the co-culture. All erythroid cells in the bursts contained alpha and gama globins while beta globin-expressing erythroid cells gradually increased up to 100% until day 18, along with a decrease of epsilon globin-expressing erythroid cells in individual bursts. We then examined the globin expression of erythroid cells through day 12 to 18 in clonal culture of E-BFC at day 14 of hESC/mFLSC co-culture. Resultantly, beta globin-positive cells gradually increased up to 100% at day 18 of clonal culture, while epsilon globin-positive cells decreased. We equally divided erythroid cells in single bursts at day 12 of clonal culture; half was processed to the analysis of globin expression, and the other half was re-cultured for additional 6 days, and then their globin expression was examined. We again observed the time-dependent increase of beta globin and decrease of epsilon globin in hESC-derived erythroid cells at a clone level. On the other hand, hESC-derived erythroid cells could undergo encleation and their differentiation, when defined by co-expression of Glycophorin A, could be confirmed by up-regulation of CD71 in association with down-regulation of CD81 by flow cytometry, showing a similar pattern to human cord blood-derived erythroid cells. Furthermore, these hESC-derived erythroid cells could function as oxygen carrier, and had a sufficient glucose-6-phosphate dehydrogenase activity. The present study provides an experimental model for investigating early development of human erythropoiesis at a clone level. It enables us to conduct subtle and detailed experiment on hemoglobin switching, and the pathogenesis and discovery of drugs for hereditary diseases in human erythrocyte development. More importantly, the time-dependent maturation of hESC-derived erythroid cells indicates that hESCs can be a novel source for therapeutic transfusion.
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Dvorakova, Dana, Stanislava Koskova, Martina Vodinska, Ales Hampl, Jiri Mayer, and Petr Dvorak. "Fibroblast Growth Factor Receptors in Human Embryonic Stem Cells." Blood 104, no. 11 (November 16, 2004): 4168. http://dx.doi.org/10.1182/blood.v104.11.4168.4168.
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Abstract Signals via fibroblast growth factor receptors (FGFRs) are involved in mesoderm induction events and may be also critical for early hematopoietic specification and proliferation of the hemangioblast. In vitro differentiated embryonic stem cells represent excellent system for the study of early hematopoietic commitment, particularly for understanding signals regulating the onset of hematopoietic differentiation. We have used human embryonic stem cells (hESCs) to study the expression of FGFR1, 2, 3, and 4 in undifferentiated cells and their differentiated progeny. Culturing hESCs i/ in high densities (protocol 1), ii/ without feeder layer of mouse embryonal fibroblasts and basic fibroblast growth factor (protocol 2), and iii/ in three-dimensional aggregates called embryoid bodies (protocol 3), was used to induce the differentiation. To achieve more directed and homogenous differentiation feeder-free hESCs were first subjected to the aggregation step (formation of embryoid bodies) that resembles the gastrulation process. This was followed by differentiation in monolayer in the presence of basic fibroblast growth factor (protocol 4). Such two-step differentiation protocol (5 + 10 days) was shown to activate ectodermal and mesodermal genes and form ectodermal and mesodermal cells (Schuldiner et al., PNAS97:11307, 2000). The gene expression levels for all FGFRs were determined by quantitative real-time RT-PCR. Real-time RT-PCR results were normalized by comparison to the expression of ABL gene. We revealed that undifferentiated hESCs that were cultured with feeder cells and in low density express all four FGFRs in the following pattern: FGFR1 is highly expressed and dominant; FGFR3 is also strongly expressed; FGFR4 shows lower expression; and FGFR2 is only weakly expressed. This expression pattern was changed when hESCs grew and started to differentiate in high densities (protocol 1) or have initiated differentiation either by feeder cells and basic fibroblast growth factor withdrawal or by aggregation step (protocol 2 and 3). Two-fold upregulation of FGFR1 and FGFR4, and downregulation of FGFR3 characterize such changed expression pattern. Notably, the expression levels for all four FGFRs were increased when hESCc were subjected to the two-step differentiation protocol (protocol 4). Compared to the undifferentiated hESCs, FGFR1 and 4 exhibited 7-fold increase, and FGFR2 and 3 were found to be upregulated more than twice. In summary our results show that the expression of FGFRs tightly follows changing culture conditions that may direct hESCs to differentiate. Furthermore, strong upregulation of FGFR1 and 4 in prospective hESC-derived mesodermal cells suggests their involvement in the earliest stages of hematopoiesis. This research was supported in part by the Grant Agency of the Czech Republic (301/03/1122), Ministry of Health (MZ 00065269705), Ministry of Education, Youth, and Sports (MSM 432100001, LN 00A065), and Academy of Sciences of the Czech Republic (AV 0Z5039906).
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Freude, Kristine K., Mahmud Penjwini, Joy L. Davis, Frank M. LaFerla, and Mathew Blurton-Jones. "Soluble Amyloid Precursor Protein Induces Rapid Neural Differentiation of Human Embryonic Stem Cells." Journal of Biological Chemistry 286, no. 27 (May 23, 2011): 24264–74. http://dx.doi.org/10.1074/jbc.m111.227421.
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Human embryonic stem cells (hESCs) offer tremendous potential for not only treating neurological disorders but also for their ability to serve as vital reagents to model and investigate human disease. To further our understanding of a key protein involved in Alzheimer disease pathogenesis, we stably overexpressed amyloid precursor protein (APP) in hESCs. Remarkably, we found that APP overexpression in hESCs caused a rapid and robust differentiation of pluripotent stem cells toward a neural fate. Despite maintenance in standard hESC media, up to 80% of cells expressed the neural stem cell marker nestin, and 65% exhibited the more mature neural marker β-3 tubulin within just 5 days of passaging. To elucidate the mechanism underlying the effects of APP on neural differentiation, we examined the proteolysis of APP and performed both gain of function and loss of function experiments. Taken together, our results demonstrate that the N-terminal secreted soluble forms of APP (in particular sAPPβ) robustly drive neural differentiation of hESCs. Our findings not only reveal a novel and intriguing role for APP in neural lineage commitment but also identify a straightforward and rapid approach to generate large numbers of neurons from human embryonic stem cells. These novel APP-hESC lines represent a valuable tool to investigate the potential role of APP in development and neurodegeneration and allow for insights into physiological functions of this protein.
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Janson, Christine, Kristine Nyhan, and John P. Murnane. "Replication Stress and Telomere Dysfunction Are Present in Cultured Human Embryonic Stem Cells." Cytogenetic and Genome Research 146, no. 4 (2015): 251–60. http://dx.doi.org/10.1159/000441245.
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Replication stress causes DNA damage at fragile sites in the genome. DNA damage at telomeres can initiate breakage-fusion-bridge cycles and chromosome instability, which can result in replicative senescence or tumor formation. Little is known about the extent of replication stress or telomere dysfunction in human embryonic stem cells (hESCs). hESCs are grown in culture with the expectation of being used therapeutically in humans, making it important to minimize the levels of replication stress and telomere dysfunction. Here, the hESC line UCSF4 was cultured in a defined medium with growth factor Activin A, exogenous nucleosides, or DNA polymerase inhibitor aphidicolin. We used quantitative fluorescence in situ hybridization to analyze individual telomeres for dysfunction and observed that it can be increased by aphidicolin or Activin A. In contrast, adding exogenous nucleosides relieved dysfunction, suggesting that telomere dysfunction results from replication stress. Whether these findings can be applied to other hESC lines remains to be determined. However, because the loss of telomeres can lead to chromosome instability and cancer, we conclude that hESCs grown in culture for future therapeutic purposes should be routinely checked for replication stress and telomere dysfunction.
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Meng, Guoliang, Shiying Liu, Xiangyun Li, Roman Krawetz, and Derrick E. Rancourt. "Derivation of human embryonic stem cell lines after blastocyst microsurgery." Biochemistry and Cell Biology 88, no. 3 (June 2010): 479–90. http://dx.doi.org/10.1139/o09-188.
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Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types, human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently, although several hundred hESC lines are available in the word, only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here, we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage, and used to isolate ICM via microsurgery. Unlike previous microsurgery methods, which use specialized glass or steel needles, our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated, cut into several cell clumps, and transferred onto fresh feeders. After more than 30 passages, the two hESC lines established using this method exhibited normal morphology, karyotype, and growth rate. Moreover, they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions, including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders.
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Cheng, Chih-Lun, Shang-Chih Yang, Chien-Ying Lai, Cheng-Kai Wang, Ching-Fang Chang, Chun-Yu Lin, Wei-Ju Chen, et al. "CXCL14 Maintains hESC Self-Renewal through Binding to IGF-1R and Activation of the IGF-1R Pathway." Cells 9, no. 7 (July 16, 2020): 1706. http://dx.doi.org/10.3390/cells9071706.
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Human embryonic stem cells (hESCs) have important roles in regenerative medicine, but only a few studies have investigated the cytokines secreted by hESCs. We screened and identified chemokine (C-X-C motif) ligand 14 (CXCL14), which plays crucial roles in hESC renewal. CXCL14, a C-X-C motif chemokine, is also named as breast and kidney-expressed chemokine (BRAK), B cell and monocyte-activated chemokine (BMAC), and macrophage inflammatory protein-2γ (MIP-2γ). Knockdown of CXCL14 disrupted the hESC self-renewal, changed cell cycle distribution, and further increased the expression levels of mesoderm and endoderm differentiated markers. Interestingly, we demonstrated that CXCL14 is the ligand for the insulin-like growth factor 1 receptor (IGF-1R), and it can activate IGF-1R signal transduction to support hESC renewal. Currently published literature indicates that all receptors in the CXCL family are G protein-coupled receptors (GPCRs). This report is the first to demonstrate that a CXCL protein can bind to and activate a receptor tyrosine kinase (RTK), and also the first to show that IGF-1R has another ligand in addition to IGFs. These findings broaden our understanding of stem cell biology and signal transduction.
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Zhao, Hong-Xi, Feng Jiang, Ya-Jing Zhu, Li Wang, Ke Li, Yang Li, Xiao-Hong Wang, Ling-Song Li, and Yuan-Qing Yao. "Enhanced Immunological Tolerance by HLA-G1 from Neural Progenitor Cells (NPCs) Derived from Human Embryonic Stem Cells (hESCs)." Cellular Physiology and Biochemistry 44, no. 4 (2017): 1435–44. http://dx.doi.org/10.1159/000485539.
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Background: Despite the great potential of utilizing human embryonic stem cells (hESCs)-derived cells as cell source for transplantation, these cells were often rejected during engraftment by the immune system due to adaptive immune response. Methods: We first evaluated HLA-G expression level in both hESCs and differentiated progenitor cells. After that, we generated modified hESC lines that over-express HLA-G1 using lentiviral infection with the construct contains both HLA-G1 and GFP tag. The lentivirus was first produced by co-transfecting HLA-G1 expressing lentiviral vector together with packaging vectors into packaging cell line 293T. Then the produced virus was used for the infection of selected hESC lines. We characterized the generated cell lines phenotype, including pluripotency and self-renewal abilities, as well as immune tolerance ability by mixed lymphocyte reaction (MLR) and cytotoxicity assays. Results: Although the hESCs do not express high levels of HLA-G1, over-expression of HLA-G1 in hESCs still retains their stem cell characteristics as determined by retaining the expression levels of OCT4 and SOX2, two critical transcriptional factors for stem cell function. Furthermore, the HLA-G1 overexpressing hESCs retain the self-renewal and pluripotency characteristics of stem cells, which can differentiate into different types of cells, including pigment cells, smooth muscle cells, epithelia-like cells, and NPCs. After differentiation, the differentiated cells including NPCs retain the high levels of HLA-G1 protein. In comparison with conventional NPCs, these HLA-G1 positive NPCs have enhanced immune tolerance ability. Conclusions: Ectopic expression of HLA-G1, a non-classical major histocompatibility complex class I (MHC I) antigen that was originally discovered involving in engraftment tolerance during pregnancy, can enhance the immunological tolerance in differentiated neural progenitor cells (NPCs). Our study shows that stably overexpressing HLA-G1 in hESCs might be a feasible strategy for enhancing the engraftment of NPCs during transplantation.
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Kim, Annie, Kun-Gu Lee, Yeongbeen Kwon, Kang-In Lee, Heung-Mo Yang, Omer Habib, Jihun Kim, et al. "Off-the-Shelf, Immune-Compatible Human Embryonic Stem Cells Generated Via CRISPR-Mediated Genome Editing." Stem Cell Reviews and Reports 17, no. 3 (January 9, 2021): 1053–67. http://dx.doi.org/10.1007/s12015-020-10113-7.
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AbstractHuman embryonic stem cells (hESCs) hold promise in regenerative medicine but allogeneic immune rejections caused by highly polymorphic human leukocyte antigens (HLAs) remain a barrier to their clinical applications. Here, we used a CRISPR/Cas9-mediated HLA-editing strategy to generate a variety of HLA homozygous-like hESC lines from pre-established hESC lines. We edited four pre-established HLA-heterozygous hESC lines and created a mini library of 14 HLA-edited hESC lines in which single HLA-A and HLA-B alleles and both HLA-DR alleles are disrupted. The HLA-edited hESC derivatives elicited both low T cell- and low NK cell-mediated immune responses. Our library would cover about 40% of the Asian-Pacific population. We estimate that HLA-editing of only 19 pre-established hESC lines would give rise to 46 different hESC lines to cover 90% of the Asian-Pacific population. This study offers an opportunity to generate an off-the-shelf HLA-compatible hESC bank, available for immune-compatible cell transplantation, without embryo destruction.
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Mamidi, Murali Krishna, Rajarshi Pal, Ramesh Bhonde, Zubaidah Zakaria, and Satish Totey. "Application of Multiplex PCR for Characterization of Human Embryonic Stem Cells (hESCs) and Its Differentiated Progenies." Journal of Biomolecular Screening 15, no. 6 (June 8, 2010): 630–43. http://dx.doi.org/10.1177/1087057110370211.
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Techniques to evaluate gene expression profiling, including real-time quantitative PCR, TaqMan® low-density arrays, and sufficiently sensitive cDNA microarrays, are efficient methods for monitoring human embryonic stem cell (hESC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a paucity of rapid, cost-effective, robust, yet sensitive methods for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all germ layers, including ecto-, meso-, and endoderm. To quantify the modulation of gene expression in hESCs during their propagation, expansion, and differentiation via embryoid body (EB) formation, the authors developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising set 1, and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2 to 4, respectively. The authors found that these 4 sets were not only effective in determining the relative differentiation in hESCs, but were easily reproducible. In this study, they used the HUES-7 cell line to standardize the technique, which was subsequently validated with HUES-9, NTERA-2, and mouse embryonic fibroblast cells. This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.
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Trounson, Alan. "The Production and Directed Differentiation of Human Embryonic Stem Cells." Endocrine Reviews 27, no. 2 (April 1, 2006): 208–19. http://dx.doi.org/10.1210/er.2005-0016.
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Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The methods for deriving hESCs are well established and repeatable and are relatively successful with a ratio of 1:10 to 1:2 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of early progenitors in this lineage. There are reports of enriched cultures of keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurons, hepatic progenitors, and cells that have some markers of gut tissue and pancreatic islet-like cells. The prospects for use of hESC derivatives in regenerative medicine are significant, and there is much optimism for their potential contributions to human regenerative medicine.
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Kaufman, Dan S. "Human Pluripotent Stem Cell-Derived Blood Cells for Therapies." Blood 132, Supplement 1 (November 29, 2018): SCI—14—SCI—14. http://dx.doi.org/10.1182/blood-2018-99-109424.
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Abstract It has now been twenty years since human embryonic stem cells (hESCs) were first isolated and described in 1998. In the next decade, induced pluripotent stem cells (iPSCs) were produced first from mouse somatic cells and then from human cells. Since these landmark advances, hESCs and iPSCs have been utilized to advance our understanding of basic human developmental biology and cellular plasticity. These lessons are crucial to fulfill the goal to use human pluripotent stem cells to derive new cellular therapies to better treat and repair organs and tissues damaged by disease, trauma or aging. Clinical trials are underway to utilize differentiated cells derived from hESCs or iPSCs for treatment of retinal disease, spinal cord injury, diabetes, cardiac failure, and other disorders. Production of therapeutic blood cells such as transplantable hematopoietic stem cells (HSCs) from hESCs and iPSCs remains a key goal. However, despite intensive research efforts by our group and many others, there remain challenging to achieve long-term multi-lineage engraftment in vivo with HSCs derived from unmodified hESCs/iPSCs. More successful approaches have used genetic modification or teratoma formation, though these strategies cannot be directly translated to clinical cell products. Reasons for this continued challenge and novel solutions such as use of a Runx1 genetic reporter system will be discussed. In contrast to production of transplantable HSCs, the ability use hESCs/iPSCs to produce functional lymphocytes with anti-tumor and anti-viral activity has been quite successful. Our group has defined methods to efficiently differentiate and expand clinical-scale quantities of natural killer (NK) cells. These hESC/iPSC-derived NK cells have phenotypic and genetic profiles similar to NK cells isolated from peripheral blood. Additionally, hESC/iPSC-derived NK cells are able to kill diverse tumor cells in vitro and in vivo. The hESCs/iPSCs also serve as a versatile platform to engineer genetic enhancements to produce NK cells with improved anti-tumor activity. For example, we have produced hESC/iPSC-derived NK cells that express novel chimeric antigen receptors (CARs) that are able to better target tumors that are more refractory to NK cell-mediated killing. This optimized NK-CAR construct utilizes the NKG2D transmembrane domain, 2B4 co-stimulatory domain, and the CD3ζ signaling domain to activate key NK cell-specific intracellular signaling pathways and increase NK cell survival and expansion in vivo. In one direct comparison between CAR-expressing-iPSC-derived NK cells and "conventional" CAR-expressing T cells, demonstrates the CAR-NK cells have similar ability to kill ovarian tumors in vivo, but with less toxicity, suggesting a safer approach. We have engineered other modifications into iPSC-NK cells to enhance NK cell targeting, proliferation, expansion and survival -- all key qualities to improve in vivo anti-tumor activity. These studies demonstrate that hESC/iPSC-provide an ideal platform to produce standardized, targeted, "off-the-shelf" cellular immunotherapies to treat refractory hematological malignancies and solid tumors. Finally, iPSC-derived NK cells are now being produced at clinical scale under current good manufacturing practices (cGMP) conditions with clinical trials scheduled to start by the end of 2018. Disclosures Kaufman: Fate Therapeutics: Consultancy, Research Funding.
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Vij, Shubha, Lai ZhenYang, Wong Pui Mun, Chak Li-Ling, Wang Yue, Chan Woon-Khiong, and Ariff Bongso. "The Human Embryonic Stem Cells Transcriptome: How Much Do We Know?" Open Biotechnology Journal 2, no. 1 (February 29, 2008): 56–62. http://dx.doi.org/10.2174/1874070700802010056.
Full textAbstract:
The establishment of human embryonic stem cell (hESC) lines in 1998 served to set the pace for understanding the molecular biology behind the two hallmark features of stem cells: self renewal and pluripotency. The excitement was generated in the hope that understanding the molecular biology of hESCs would provide a good model for studying early human development, disease and drug discovery and also hold the promise for providing a cure for degenerative human diseases. In spite of the large number of studies, the molecular basis of pluripotency has remained a matter of intrigue ever since the embryonic stem cells (ESCs) were first identified. A considerable percentage of these studies have been transcriptome-based. Interestingly, significant differences are seen not only between mouse and human ESC transcriptomes but also amongst the hESC studies. Nevertheless, a key set of pluripotency genes seem to be common, reinforcing the utility of transcriptome-based approaches in identifying the molecular basis of pluripotency in hESCs.
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Silk, Kathryn M., Alison J. Leishman, Kevin P. Nishimoto, Anita Reddy, and Paul J. Fairchild. "Rapamycin Conditioning of Dendritic Cells Differentiated from Human ES Cells Promotes a Tolerogenic Phenotype." Journal of Biomedicine and Biotechnology 2012 (2012): 1–11. http://dx.doi.org/10.1155/2012/172420.
Full textAbstract:
While human embryonic stem cells (hESCs) may one day facilitate the treatment of degenerative diseases requiring cell replacement therapy, the success of regenerative medicine is predicated on overcoming the rejection of replacement tissues. Given the role played by dendritic cells (DCs) in the establishment of immunological tolerance, we have proposed that DC, rendered tolerogenic during their differentiation from hESC, might predispose recipients to accept replacement tissues. As a first step towards this goal, we demonstrate that DC differentiated from H1 hESCs (H1-DCs) are particularly responsive to the immunosuppressive agent rapamycin compared to monocyte-derived DC (moDC). While rapamycin had only modest impact on the phenotype and function of moDC, H1-DC failed to upregulate CD40 upon maturation and displayed reduced immunostimulatory capacity. Furthermore, coculture of naïve allogeneic T cells with rapamycin-treated H1-DC promoted an increased appearance of CD25hiFoxp3+regulatory T cells, compared to moDC. Our findings suggest that conditioning of hESC-derived DC with rapamycin favours a tolerogenic phenotype.
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Xu, Zhuojin, Aaron M. Robitaille, Jason D. Berndt, Kathryn C. Davidson, Karin A. Fischer, Julie Mathieu, Jennifer C. Potter, Hannele Ruohola-Baker, and Randall T. Moon. "Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells." Proceedings of the National Academy of Sciences 113, no. 42 (October 3, 2016): E6382—E6390. http://dx.doi.org/10.1073/pnas.1613849113.
Full textAbstract:
In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/β-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/β-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of β-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/β-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/β-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.
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Prajumwongs, Piya, Oratai Weeranantanapan, Thiranut Jaroonwitchawan, and Parinya Noisa. "Human Embryonic Stem Cells: A Model for the Study of Neural Development and Neurological Diseases." Stem Cells International 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/2958210.
Full textAbstract:
Although the mechanism of neurogenesis has been well documented in other organisms, there might be fundamental differences between human and those species referring to species-specific context. Based on principles learned from other systems, it is found that the signaling pathways required for neural induction and specification of human embryonic stem cells (hESCs) recapitulated those in the early embryo developmentin vivoat certain degree. This underscores the usefulness of hESCs in understanding early human neural development and reinforces the need to integrate the principles of developmental biology and hESC biology for an efficient neural differentiation.