To see the other types of publications on this topic, follow the link: HPSC.
Journal articles on the topic 'HPSC'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'HPSC.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Hayashi, Yohei, and Miho Kusuda Furue. "Biological Effects of Culture Substrates on Human Pluripotent Stem Cells." Stem Cells International 2016 (2016): 1–11. http://dx.doi.org/10.1155/2016/5380560.
Full textAbstract:
In recent years, as human pluripotent stem cells (hPSCs) have been commonly cultured in feeder-free conditions, a number of cell culture substrates have been applied or developed. However, the functional roles of these substrates in maintaining hPSC self-renewal remain unclear. Here in this review, we summarize the types of these substrates and their effect on maintaining hPSC self-renewal. Endogenous extracellular matrix (ECM) protein expression has been shown to be crucial in maintaining hPSC self-renewal. These ECM molecules interact with integrin cell-surface receptors and transmit their cellular signaling. We discuss the possible effect of integrin-mediated signaling pathways on maintaining hPSC self-renewal. Activation of integrin-linked kinase (ILK), which transmits ECM-integrin signaling to AKT (also known as protein kinase B), has been shown to be critical in maintaining hPSC self-renewal. Also, since naïve pluripotency has been widely recognized as an alternative pluripotent state of hPSCs, we discuss the possible effects of culture substrates and integrin signaling on naïve hPSCs based on the studies of mouse embryonic stem cells. Understanding the role of culture substrates in hPSC self-renewal and differentiation enables us to control hPSC behavior precisely and to establish scalable or microfabricated culture technologies for regenerative medicine and drug development.
2
Vojnits, Kinga, Mio Nakanishi, Deanna Porras, et al. "Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening." Molecules 27, no. 8 (2022): 2434. http://dx.doi.org/10.3390/molecules27082434.
Full textAbstract:
Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC–OCT4–EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC–AAVS1–EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC–AAVS1–EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.
3
Hai, Nan, Dong Shin, Huanjing Bi, Kaiming Ye, and Sha Jin. "Mechanistic Analysis of Physicochemical Cues in Promoting Human Pluripotent Stem Cell Self-Renewal and Metabolism." International Journal of Molecular Sciences 19, no. 11 (2018): 3459. http://dx.doi.org/10.3390/ijms19113459.
Full textAbstract:
We have previously reported that a porous membrane of polyethylene terephthalate (PET) enables significant augmentation of human pluripotent stem cell (hPSC) proliferation and differentiation. The interaction between hPSCs and the PET surface induces β-catenin-mediated wingless/integrated (Wnt) signaling, leading to upregulation of the expression of adhesion molecules in hPSCs. In this study, we sought to unveil mechanisms underlying the role of the PET membrane in hPSC self-renewal and metabolism. We discovered that physicochemical cues of the PET membrane considerably alter hPSC metabolism by increasing the cell yield and suppressing the generation of toxic byproduct, indicating an effective cell self-renewal and a less apoptotic culture environment in the membrane culture system. Furthermore, we discovered that a caspase-8 medicated apoptotic pathway plays a profound role in obstructing hPSCs grown on a traditional tissue culture plate (TCP). Treating hPSCs seeded on a TCP surface with a caspase-8 inhibitor significantly suppressed cellular apoptotic pathway and improved cell proliferation and metabolism. Our experimental results provided valuable insights into signal pathways influencing hPSC self-renewal during routine maintenance and expansion, which would shed light on large-scale preparation of hPSCs for clinical applications.
4
Lim, Seakcheng, Rachel A. Shparberg, Jens R. Coorssen, and Michael D. O’Connor. "Application of the RBBP9 Serine Hydrolase Inhibitor, ML114, Decouples Human Pluripotent Stem Cell Proliferation and Differentiation." International Journal of Molecular Sciences 21, no. 23 (2020): 8983. http://dx.doi.org/10.3390/ijms21238983.
Full textAbstract:
Retinoblastoma binding protein 9 (RBBP9) is required for maintaining the expression of both pluripotency and cell cycle genes in human pluripotent stem cells (hPSCs). An siRNA-based study from our group showed it does so by influencing cell cycle progression through the RB/E2F pathway. In non-pluripotent cells, RBBP9 is also known to have serine hydrolase (SH) activity, acting on currently undefined target proteins. The role of RBBP9 SH activity in hPSCs, and during normal development, is currently unknown. To begin assessing whether RBBP9 SH activity might contribute to hPSC maintenance, hPSCs were treated with ML114—a selective chemical inhibitor of RBBP9 SH activity. Stem cells treated with ML114 showed significantly reduced population growth rate, colony size and progression through the cell cycle, with no observable change in cell morphology or decrease in pluripotency antigen expression—suggesting no initiation of hPSC differentiation. Consistent with this, hPSCs treated with ML114 retained the capacity for tri-lineage differentiation, as seen through teratoma formation. Subsequent microarray and Western blot analyses of ML114-treated hPSCs suggest the nuclear transcription factor Y subunit A (NFYA) may be a candidate effector of RBBP9 SH activity in hPSCs. These data support a role for RBBP9 in regulating hPSC proliferation independent of differentiation, whereby inhibition of RBBP9 SH activity de-couples decreased hPSC proliferation from initiation of differentiation.
5
Buchholz, David E., Thomas S. Carroll, Arif Kocabas, et al. "Novel genetic features of human and mouse Purkinje cell differentiation defined by comparative transcriptomics." Proceedings of the National Academy of Sciences 117, no. 26 (2020): 15085–95. http://dx.doi.org/10.1073/pnas.2000102117.
Full textAbstract:
Comparative transcriptomics between differentiating human pluripotent stem cells (hPSCs) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate human pluripotent stem cell-derived Purkinje cells (hPSC-PCs) that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we usedTg(Pcp2-L10a-Egfp)TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenicPcp2-L10a-EgfpTRAP hPSC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides a direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders.
6
Armstrong, Alison J., Hal Hansen, and Roger Gauthier. "Development of a Model for Evaluating High Performance Sport Centers in Canada." Journal of Sport Management 5, no. 2 (1991): 153–76. http://dx.doi.org/10.1123/jsm.5.2.153.
Full textAbstract:
A theory based model was developed for the evaluation of high performance sport centers (HPSCs) in Canada. The model was developed according to de Groot’s (1969) four-phase interpretative-theoretical methodology. The phases of exploration, analysis, classification, and explanation guided the collection of current program evaluation literature and information on the nature of the HPSC program and its past evaluation practices. Appropriate evaluation models from the literature were assessed with respect to the HPSC program’s nature, and a single theoretical-integrative model was developed with corresponding guidelines for HPSC evaluation. The model is described with reference to (a) the role of evaluation at each stage of the HPSC life cycle, (b) the evaluators and decision makers, (c) utilization of the evaluation information, and (d) a general format for guiding the responsible national sport organizations through the important process of evaluation.
7
Yamada, Daisuke, Tomoka Takao, Masahiro Nakamura, Toki Kitano, Eiji Nakata, and Takeshi Takarada. "Identification of Surface Antigens That Define Human Pluripotent Stem Cell-Derived PRRX1+Limb-Bud-like Mesenchymal Cells." International Journal of Molecular Sciences 23, no. 5 (2022): 2661. http://dx.doi.org/10.3390/ijms23052661.
Full textAbstract:
Stem cell-based therapies and experimental methods rely on efficient induction of human pluripotent stem cells (hPSCs). During limb development, the lateral plate mesoderm (LPM) produces limb-bud mesenchymal (LBM) cells that differentiate into osteochondroprogenitor cells and form cartilage tissues in the appendicular skeleton. Previously, we generated PRRX1-tdTomato reporter hPSCs to establish the protocol for inducing the hPSC-derived PRRX1+ LBM-like cells. However, surface antigens that assess the induction efficiency of hPSC-derived PRRX1+ LBM-like cells from LPM have not been identified. Here, we used PRRX1-tdTomato reporter hPSCs and found that high pluripotent cell density suppressed the expression of PRRX1 mRNA and tdTomato after LBM-like induction. RNA sequencing and flow cytometry suggested that PRRX1-tdTomato+ LBM-like cells are defined as CD44high CD140Bhigh CD49f−. Importantly, other hPSC lines, including four human induced pluripotent stem cell lines (414C2, 1383D2, HPS1042, HPS1043) and two human embryonic stem cell lines (SEES4, SEES7), showed the same results. Thus, an appropriate cell density of hPSCs before differentiation is a prerequisite for inducing the CD44high CD140Bhigh CD49f− PRRX1+ LBM-like cells.
8
Ghosheh, Nidal, Barbara Küppers-Munther, Annika Asplund, et al. "Comparative transcriptomics of hepatic differentiation of human pluripotent stem cells and adult human liver tissue." Physiological Genomics 49, no. 8 (2017): 430–46. http://dx.doi.org/10.1152/physiolgenomics.00007.2017.
Full textAbstract:
Hepatocytes derived from human pluripotent stem cells (hPSC-HEP) have the potential to replace presently used hepatocyte sources applied in liver disease treatment and models of drug discovery and development. Established hepatocyte differentiation protocols are effective and generate hepatocytes, which recapitulate some key features of their in vivo counterparts. However, generating mature hPSC-HEP remains a challenge. In this study, we applied transcriptomics to investigate the progress of in vitro hepatic differentiation of hPSCs at the developmental stages, definitive endoderm, hepatoblasts, early hPSC-HEP, and mature hPSC-HEP, to identify functional targets that enhance efficient hepatocyte differentiation. Using functional annotation, pathway and protein interaction network analyses, we observed the grouping of differentially expressed genes in specific clusters representing typical developmental stages of hepatic differentiation. In addition, we identified hub proteins and modules that were involved in the cell cycle process at early differentiation stages. We also identified hub proteins that differed in expression levels between hPSC-HEP and the liver tissue controls. Moreover, we identified a module of genes that were expressed at higher levels in the liver tissue samples than in the hPSC-HEP. Considering that hub proteins and modules generally are essential and have important roles in the protein-protein interactions, further investigation of these genes and their regulators may contribute to a better understanding of the differentiation process. This may suggest novel target pathways and molecules for improvement of hPSC-HEP functionality, having the potential to finally bring this technology to a wider use.
9
Walasek, Marta A., Crystal Chau, Christian Barborini, et al. "A Reproducible and Simple Method to Generate Red Blood Cells from Human Pluripotent Stem Cells." Blood 134, Supplement_1 (2019): 1189. http://dx.doi.org/10.1182/blood-2019-128830.
Full textAbstract:
Erythroid cells generated from human pluripotent stem cells (hPSCs) can potentially offer an unlimited and safe supply of red blood cells (RBCs) for transfusion. Human PSC-derived erythroid cells at various stages of differentiation can also be used to model blood diseases, test new drug candidates, and develop cellular and genetic therapies. Although several protocols for deriving RBCs from hPSCs have been described, these are typically complex, involving multiple culture steps that may include co-culture with feeder cells, and exhibit large variability in erythroid cell yields between hPSC lines and replicate experiments. We have developed a straightforward, serum-free and feeder-free culture method to generate erythroid cells from hPSCs with high yields and high purity. The method has been validated on multiple human embryonic stem (ES) cell lines (H1, H7, H9) and induced pluripotent stem (iPS) cell lines (WLS-1C, STiPS-F016, STiPS-B004). The protocol involves two steps: hematopoietic specification of hPSCs, followed by differentiation of hPSC-derived hematopoietic stem and progenitor cells (HSPCs) into erythroid cells. Lineage specification and differentiation is driven by only three supplements that combine cytokines and other factors to support optimal differentiation efficiency and cell yield across cell lines. In the first step, small hPSC aggregates routinely maintained in feeder-free maintenance medium, are plated onto matrigel-coated microwells, and specification to mesoderm and subsequent hematoendothelial differentiation is induced by addition of successive expansion supplements. This phase promotes extensive hematopoietic progenitor cell generation, with a single hPSC producing on average 142 HSPCs (range: 50 - 360, n = 3 experiments) by day 10 across all six ES and iPS cell lines tested. The average frequency of cells expressing CD43, an embryonic pan-hematopoietic marker, is 92% (range: 85 - 95%), and the frequency of CD34+ cells ranges between 24-55%. In the second, erythroid differentiation step, hPSC-derived HSPCs expand on average 300-fold (range: 80 - 1000) within 10 - 14 days, and the average frequency of GlyA+ cells is 75% (range: 70 - 85%). Cumulatively, this results in the generation of on average 30,000 GlyA+ cells (range: 10,000 - 80,000) per initial hPSC after 20 - 24 days. Further maturation in 7-day cultures containing EPO and human serum resulted in a > 90% pure population of GlyA+ erythroid cells. Notably, no cell loss was observed during the maturation phase, resulting in an average yield of 50,000 GlyA+ cells (range: 10,000 - 220,000) per single initial hPSC on day 31. Differentiated cells were characterized by orthochromatic normoblast morphology and decreased CD71 expression, consistent with erythroid maturation. Erythroid cells generated in this differentiation culture system expressed a mix of 'primitive' and 'definitive' hemoglobin types, but with adult and fetal hemoglobin being expressed at higher levels than embryonic hemoglobin. The observed enucleation rates of hPSC-derived erythroid cells are consistent with current reports and are subject to further optimization. In summary, we have developed a two-step, serum- and feeder-free erythroid differentiation method to generate large numbers of erythroid cells from multiple hPSC lines. This culture system provides a simple, standardized and reproducible platform to generate RBCs from hPSCs with high yields and efficiency for basic and translational research. Disclosures Walasek: STEMCELL Technologies, Inc: Employment. Chau:STEMCELL Technologies, Inc: Employment. Barborini:STEMCELL Technologies, Inc: Employment. Richardson:STEMCELL Technologies, Inc: Employment. Szilvassy:STEMCELL Technologies, Inc: Employment. Louis:STEMCELL Technologies, Inc: Employment. Thomas:STEMCELL Technologies, Inc: Employment. Eaves:STEMCELL Technologies, Inc: Employment. Wognum:STEMCELL Technologies, Inc: Employment.
10
Dick, Emily, Divya Rajamohan, Jonathon Ronksley, and Chris Denning. "Evaluating the utility of cardiomyocytes from human pluripotent stem cells for drug screening." Biochemical Society Transactions 38, no. 4 (2010): 1037–45. http://dx.doi.org/10.1042/bst0381037.
Full textAbstract:
Functional cardiomyocytes can now be derived routinely from hPSCs (human pluripotent stem cells), which collectively include embryonic and induced pluripotent stem cells. This technology presents new opportunities to develop pharmacologically relevant in vitro screens to detect cardiotoxicity, with a view to improving patient safety while reducing the economic burden to industry arising from high drug attrition rates. In the present article, we consider the need for human cardiomyocytes in drug-screening campaigns and review the strategies used to differentiate hPSCs towards the cardiac lineage. During early stages of differentiation, hPSC-cardiomyocytes display gene expression profiles, ultra-structures, ion channel functionality and pharmacological responses reminiscent of an embryonic phenotype, but maturation during extended time in culture has been demonstrated convincingly. Notably, hPSC-cardiomyocytes have been shown to respond in a highly predictable manner to over 40 compounds that have a known pharmacological effect on the human heart. This suggests that further development and validation of the hPSC-cardiomyocyte model as a tool for assessing cardiotoxicity is warranted.
11
Wang, Zongjie, Mark Gagliardi, Reza M. Mohamadi, et al. "Ultrasensitive and rapid quantification of rare tumorigenic stem cells in hPSC-derived cardiomyocyte populations." Science Advances 6, no. 12 (2020): eaay7629. http://dx.doi.org/10.1126/sciadv.aay7629.
Full textAbstract:
The ability to detect rare human pluripotent stem cells (hPSCs) in differentiated populations is critical for safeguarding the clinical translation of cell therapy, as these undifferentiated cells have the capacity to form teratomas in vivo. The detection of hPSCs must be performed using an approach compatible with traceable manufacturing of therapeutic cell products. Here, we report a novel microfluidic approach, stem cell quantitative cytometry (SCQC), for the quantification of rare hPSCs in hPSC-derived cardiomyocyte (CM) populations. This approach enables the ultrasensitive capture, profiling, and enumeration of trace levels of hPSCs labeled with magnetic nanoparticles in a low-cost, manufacturable microfluidic chip. We deploy SCQC to assess the tumorigenic risk of hPSC-derived CM populations in vivo. In addition, we isolate rare hPSCs from the differentiated populations using SCQC and characterize their pluripotency.
12
Ghosheh, Nidal, Björn Olsson, Josefina Edsbagge, et al. "Highly Synchronized Expression of Lineage-Specific Genes duringIn VitroHepatic Differentiation of Human Pluripotent Stem Cell Lines." Stem Cells International 2016 (2016): 1–22. http://dx.doi.org/10.1155/2016/8648356.
Full textAbstract:
Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, andCYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models.
13
Maysubara, Hiroyuki, Akira Niwa, Tatsutoshi Nakahata, and Megumu K. Saito. "NK Cells from Human Pluripotent Stem Cells for Immunotherapy." Blood 132, Supplement 1 (2018): 4955. http://dx.doi.org/10.1182/blood-2018-99-115499.
Full textAbstract:
Abstract Natural Killer (NK) cells are a one of innate lymphocytes and show cytotoxicity against tumour cells without prior antigen specific stimulation. . NK cells can demonstrate stronger cytotoxicity than T cells in the absence of MHC Class I, and survive short lifespan from several weeks to one month. It suggested that NK cells show low risk of cytokine long-term secretion inside patient's body. Previous studies have developed peripheral blood mononuclear cells (PBMC) derived NK cells expansions or NK cells differentiation from cord blood (CB) cells for immunotherapy. Expansion trial using K562 tumor cell line, or with IL-15, or an anti-tumor antibody dasatinib is not sufficient to obtain NK cells with high cytotoxicity.More recently, NK cells induction from human pluripotent stem cells (hPSCs), taking the advantage of their unlimited growth potential, has been reported. Although previous studies regarding hPSC-derived NK cells seems impressive and successful, most systems used a bovine and human serum, which might result in the unstable yield and efficiency in the production of Hematopoietic progenitor cells (HPCs) and NK cells for immunotherapy. To resolve those problems, we tried to induce functional NK cells from hPSCs in xeno and serum free condition. This study used three hPSC cell lines; human ES cell (cell line: KhES1) and iPS cells (cell line: 409B2 and CB-A11) to check reproducibility. To differentiate hPSCs into hematopoietic cells, changed cytokine combinations and chemically defined medium in step-wise manner. We first induced HPC from hPSCs over 90% purity by 12 days culture. At this point, we selected two media to induce NK cells. We compared serum-containing medium that previous report used (Medium A) and chemically-defined medium (Medium B) by evaluating the differentiation efficiency and function of NK cells. NK cell marker CD56 (NCAM) was gradually expressed after additional 16 days culture (28 days of differentiation). Until hPSC-derived NK cells were maturated, we traced the expression of NK specific markers and transcriptional factors. On day48, the frequency of CD56 positive cells showed no significant differences between medium A (79.15 ± 5.30%) and medium B (80.90 ± 1.27%). In both conditions, NK cells expressed specific receptors such as CD161, NKG2D, killer immunoglobulin-like receptors (KIRs), NKG2a (CD94/CD159a heterodimeric inhibitory receptor), NKp44 and NKp46. hPSC-derived NK cells showed the compatible size and morphology to NK cells isolated from peripheral blood NK (PB-NK) cells: their nucleus was kidney-like shape and cytoplasm contained azurophilic granules. For functional assay, leukemia cell line K562 was incubated with 51 chromium (51Cr) for 1 hour at 37 degrees. After that, K562 was co-cultured with purified CD56 positive hPSC-derived NK cells for 4 hours at 37 degrees. The cytotoxic activity of NK cells was confirmed by 51Cr release from K562. PBMC-NK cells showed 49.65 ± 3.46% of killing activity against K562 target cells, while the killing potential of PSC-derived NK cell showed killing potential against K562 cells (Medium A: 25.4 ± 5.52%, Medium B: 23.25 ± 9.26%) which was slightly lower than that of PB-NK cells. Next trial, we are going to transplant hPSC-derived NK cells into immune deficiency mice. In detail, this mice was infected luciferase expressed K562. Using IVIS imaging system to detect intensity of luciferase, we characterized hPSC-derived NK cells potential in vivo. Here we have developed a novel and robust method to facilitate efficient NK cells differentiation in serum and xeno-free condition in all clones. They showed similar phenotypes compare to PBMC derived NK cells in terms of morphology, surface markers, translational factors and cytotoxicity against leukemia cell line K562 in vitro. This technology expected to be applicable not only to immunotherapy but also to model studies of the NK cells associating diseases. Disclosures No relevant conflicts of interest to declare.
14
Park, Seo-Yeon, and Yong Kyun Kim. "Strategies for vascularization in kidney organoids." Organoid 1 (August 15, 2021): e14. http://dx.doi.org/10.51335/organoid.2021.1.e14.
Full textAbstract:
The establishment of protocols for differentiating kidney organoids from human pluripotent stem cells (hPSCs) has potential for the application of kidney organoids in regenerative medicine. However, the primary obstacle to the regenerative application of hPSC-derived kidney organoids is precise vascularization due to the lack of vasculature in hPSC-derived kidney organoids. In this article, we review the recent methodologies for developing vasculature of kidney organoids to overcome this limitation of kidney organoids, together with a discussion of their clinical applications.
15
Le, Minh Nguyen Tuyet, and Kouichi Hasegawa. "Expansion Culture of Human Pluripotent Stem Cells and Production of Cardiomyocytes." Bioengineering 6, no. 2 (2019): 48. http://dx.doi.org/10.3390/bioengineering6020048.
Full textAbstract:
Transplantation of human pluripotent stem cell (hPSCs)-derived cardiomyocytes for the treatment of heart failure is a promising therapy. In order to implement this therapy requiring numerous cardiomyocytes, substantial production of hPSCs followed by cardiac differentiation seems practical. Conventional methods of culturing hPSCs involve using a 2D culture monolayer that hinders the expansion of hPSCs, thereby limiting their productivity. Advanced culture of hPSCs in 3D aggregates in the suspension overcomes the limitations of 2D culture and attracts immense attention. Although the hPSC production needs to be suitable for subsequent cardiac differentiation, many studies have independently focused on either expansion of hPSCs or cardiac differentiation protocols. In this review, we summarize the recent approaches to expand hPSCs in combination with cardiomyocyte differentiation. A comparison of various suspension culture methods and future prospects for dynamic culture of hPSCs are discussed in this study. Understanding hPSC characteristics in different models of dynamic culture helps to produce numerous cells that are useful for further clinical applications.
16
Yuzuriha, Akinori, Sou Nakamura, Toshie Kusunoki, et al. "Environmental Cell-Matrix Adhesion Modulates Pluripotent Stem Cell Fate Toward Definitive Hemogenic Endothelium and Hematopoietic Progenitor Cells." Blood 132, Supplement 1 (2018): 3845. http://dx.doi.org/10.1182/blood-2018-99-114505.
Full textAbstract:
Abstract [Background] Human pluripotent stem cell (hPSC) technology enables regenerative transplantation therapy and establishment of in vitro disease models. hPSCs can be maintained to proliferate limitlessly and also differentiated to specific lineage cells, but hematopoietic cell differentiation efficiency varies among each hPSC lines. It has been reported the intrinsic expression level of transcription factors in hPSCs determine the commitment capacity toward hematopoietic cells (i.e. c-MYC [J Exp Med. 2010; 207: 2817-2830], IGF2 [Cell Stem Cell. 2016; 19: 341-354]). However, whether extracellular scaffold factors that contribute to maintain hPSCs influence hematopoietic differentiation potential remain elusive. [Aim] In this study, we hypothesized that the extracellular environment such as cell-matrix adhesion signal regulates the commitment capacity toward hematopoietic cells in hPSCs. Moreover, we sought to improve the conventional hematopoietic differentiation PSC-sac method, which is simple but inefficient. [Results] The recombinant fragment of a laminin isoform, LM511-E8, [Neurosci Res. 2018; 96: 863-874, Nature. 2016; 531: 376-380, Nat commun. 2012; 3: 1236] is used in culture to maintain pluripotency of cGMP grade hPSCs instead of Matrigel in research grade hPSCs. However, cGMP grade hPSCs maintained on LM511-E8 showed decreased differentiation towards CD34+CD43+ Hematopoietic progenitor cells (HPCs) than hPSCs maintained on Matrigel or mouse embryonic fibroblasts as feeder cells. Meanwhile, hPSCs maintained on human recombinant laminin fragments LM121-E8 or LM421-E8 yielded significantly more HPCs than hPSCs maintained on LM511-E8. Microarray analysis revealed that hPSCs maintained on those "hematopoietic laminins (HLs)" showed upregulated activity of canonical Wnt/β-catenin signaling pathway. Activation of canonical Wnt signaling pathway by GSK3β inhibitor significantly improved the yield of HPC from hPSCs on LM511-E8. We then found using flow cytometry that hPSCs on HLs had higher cell surface expression of integrin β1 subunit (ITGB1), which is a part of integrin a6b1 complex, the laminin receptor, and also found the higher expression of ILK, pGSK3β (Ser9) and β-catenin by Western blot analysis. These results accordingly suggest that the ITGB1-ILK-pGSK3β-β-catenin axis modulate the commitment of hPSCs towards hematopoietic lineage. We also found lower concentrations of LM511-E8 increased ITGB1 surface expression and HPC differentiation on day14, without influencing the activity of pluripotency of hPSCs. These results indicate the modulation of hematopoietic capacity by extrinsic laminin at pluripotent state is determined by the affinity of laminin-integrin, not by the specificity of the laminin subunits, and this extrinsic modulation might be regulated through the expression level of ITGB1. Next, we revised hPSC-sac method, a conventional hematopoietic differentiation method which we originally proposed [Cell Stem Cell. 2017; 20(3) : 329-344.e7. Stem Cells. 2016; 34: 1541-1552. Blood. 2011; 111(11): 5298-5306.] By adding basic fibroblast growth factor (bFGF), TGFβ inhibitor SB431542 and heparin during differentiation period, the number of HPC increased dramatically on differentiation day14. The number of definitive hemogenic endothelium (CD34+ CD31+ CD73- CD144+ CD117+ KDR+ CD41- CD43- CD45- [Cell Reports, 2012; 2: 553-567][Blood, 2013; 121(5): 770-780]) on differentiation day 9 was also increased. Simultaneously, transcription factors specific for hematopoietic lineage, ERG, SPI1, Runx1c, GFI1b, TAL1, and TEK were upregulated in hPSCs derived cells on day 9. [Conclusion] This study revealed a mechanism in which the signals from cell-matrix adhesion by cell surface integrins and extracellular laminins modulate hematopoietic differentiation potential in hPSCs. Moreover, the revised hPSC-Sac differentiation method enables a simple, efficient and economical differentiation of hematopoietic cells. These findings thus should contribute to development of the fundamental research on hematopoietic differentiation from hPSCs towards further application. Disclosures Sekiguchi: Matrixome, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Nippi, Inc: Research Funding. Eto:Megakaryon co. Ltd.: Research Funding.
17
Schwach, Verena, Carla Cofiño-Fabres, Simone A. ten Den та Robert Passier. "Improved Atrial Differentiation of Human Pluripotent Stem Cells by Activation of Retinoic Acid Receptor Alpha (RARα)". Journal of Personalized Medicine 12, № 4 (2022): 628. http://dx.doi.org/10.3390/jpm12040628.
Full textAbstract:
Human pluripotent stem cell (hPSC)-derived cardiomyocytes have proven valuable for modeling disease and as a drug screening platform. Here, we depict an optimized protocol for the directed differentiation of hPSCs toward cardiomyocytes with an atrial identity by modulating the retinoic acid signaling cascade in spin embryoid bodies. The crucial steps of the protocol, including hPSC maintenance, embryoid body (EB) differentiation, the induction of cardiac mesoderm, direction toward the atrial phenotype, as well as molecular and functional characterization of the cardiomyocytes, are described. Atrial cardiomyocytes (AMs) can be generated within 14 days. Most importantly, we show that induction of the specific retinoic acid receptor alpha (RARα) increased the efficiency of atrial differentiation to 72% compared with 45% after modulating the retinoic acid (RA) pathway with all-trans RA (atRA). In contrast, the induction of RARβ signaling only had a minor impact on the efficiency of atrial differentiation (from about 45% to 50%). Similarly, the total yield of AM per EB of 5000 hPSCs was increased from 10,350 (2.07 per hPSC) to 16,120 (3.22 per hPSC) while selectively modulating RARα signaling. For further purification of the AMs, we describe a metabolic selection procedure that enhanced the AM percentage to more than 90% without compromising the AM yield (15,542 per EB, equal to 3.11 per hPSC) or functionality of the AMs as evaluated by RNAseq, immunostaining, and optical action potential measurement. Cardiomyocytes with distinct atrial and ventricular properties can be applied for selective pharmacology, such as the development of novel atrial-specific anti-arrhythmic agents, and disease modeling, including atrial fibrillation, which is the most common heart rhythm disorder. Moreover, fully characterized and defined cardiac subtype populations are of the utmost importance for potential cell-based therapeutic approaches.
18
Nii, Takenobu, Hiroshi Kohara, Tomotoshi Marumoto, Tetsushi Sakuma, Takashi Yamamoto, and Kenzaburo Tani. "Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency." Blood 126, no. 23 (2015): 2037. http://dx.doi.org/10.1182/blood.v126.23.2037.2037.
Full textAbstract:
Abstract Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the potential to self-renew indefinitely and differentiate into various cell types. hPSCs can differentiate into various stem or progenitor cell populations used for regenerative medicine and drug development. Newly developed genome editing technology has advanced the use of hPSCs for such purposes. However, to fully utilize hPSCs to achieve this goal, more efficient gene transfer methods under defined conditions are required. Development of efficient genome editing methods, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9), for use in hPSCs holds great promise in the fields of basic and clinical research. Among these methods, TALENs are more efficient and safer for use in hPSCs to achieve specific gene editing, as ZFNs had a low gene editing efficiency and CRISPR/Cas9 was accompanied by more severe off-target effects than TALENs. Electroporation is a widely used transfection method for hPSC genome editing; however, this method results in reduced cell viability and gene editing efficiency. In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a culture method of hPSCs at a single-cell-state to reduce cell density, and investigated gene transfection efficiency followed by gene editing efficiency. hPSCs cultured in a single-cell-state were transfected using non-liposomal transfection reagents with plasmid DNA driven by the human elongation factor 1-alpha 1 (EF1α) promoter or mRNA encoding enhanced green fluorescent protein (eGFP). The proportion of eGFP+ cells considerably increased in single-cell-state cultures (DNA: 95.80 ± 2.51%, mRNA: 99.70 ± 0.10%). Moreover, most of the cells were viable (control: 93.10 ± 0.40%, DNA: 83.40 ± 2.03%, mRNA: 86.71 ± 0.19%). The mean fluorescence intensity (MFI) was approximately three-fold higher than that in cells transfected by electroporation (electroporation (EPN): 6631 ± 992; transfection (TFN): 17933 ± 1595). eGFP expression was detected by fluorescence microscopy until day seven post-transfection. Our results also demonstrate an inverse correlation between cell density and transfection efficiency. To test whether transfection using this method affected the "stemness" of hPSCs, we examined SSEA4 and NANOG expression in eGFP-transfected cells by flow cytometry analysis. The percentage of both SSEA4+ and NANOG+ cells was greater than 90%. Moreover, transplantation of eGFP-transfected cells into immunodeficient mice led to the formation of teratomas. These results strongly suggested that single-cell-state hPSC culture improved transfection efficiency without inducing differentiation or loss of pluripotency. Moreover, we used our efficient transfection method to edit the hPSC genome using TALENs. We constructed a Platinum TALEN driven by the EF1α promoter targeting the adenomatous polyposis coli (APC) gene and analyzed the efficiency of gene editing using the Cel-1 assay. Our efficient transfection method induced mutations more efficiently than electroporation (Transfection: 11.1 ± 1.38%, Electroporation: 3.2 ± 0.89). These results showed that TALENs increased gene editing efficiency in single-cell-state hPSC cultures. Overall, our efficient hPSC transfection method using single-cell-state culture provides an excellent experimental system to investigate the full potential of hPSCs. We expect that this method may contribute to the fields of hPSC-based regenerative medicine and drug discovery. Disclosures No relevant conflicts of interest to declare.
19
Nemade, Harshal, Aviseka Acharya, Umesh Chaudhari, et al. "Cyclooxygenases Inhibitors Efficiently Induce Cardiomyogenesis in Human Pluripotent Stem Cells." Cells 9, no. 3 (2020): 554. http://dx.doi.org/10.3390/cells9030554.
Full textAbstract:
Application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited by the challenges in their efficient differentiation. Recently, the Wingless (Wnt) signaling pathway has emerged as the key regulator of cardiomyogenesis. In this study, we evaluated the effects of cyclooxygenase inhibitors on cardiac differentiation of hPSCs. Cardiac differentiation was performed by adherent monolayer based method using 4 hPSC lines (HES3, H9, IMR90, and ES4SKIN). The efficiency of cardiac differentiation was evaluated by flow cytometry and RT-qPCR. Generated hPSC-CMs were characterised using immunocytochemistry, electrophysiology, electron microscopy, and calcium transient measurements. Our data show that the COX inhibitors Sulindac and Diclofenac in combination with CHIR99021 (GSK-3 inhibitor) efficiently induce cardiac differentiation of hPSCs. In addition, inhibition of COX using siRNAs targeted towards COX-1 and/or COX-2 showed that inhibition of COX-2 alone or COX-1 and COX-2 in combination induce cardiomyogenesis in hPSCs within 12 days. Using IMR90-Wnt reporter line, we showed that inhibition of COX-2 led to downregulation of Wnt signalling activity in hPSCs. In conclusion, this study demonstrates that COX inhibition efficiently induced cardiogenesis via modulation of COX and Wnt pathway and the generated cardiomyocytes express cardiac-specific structural markers as well as exhibit typical calcium transients and action potentials. These cardiomyocytes also responded to cardiotoxicants and can be relevant as an in vitro cardiotoxicity screening model.
20
Hayes, Kevin, Yun-Kyo Kim, and Martin F. Pera. "A Case for Revisiting Nodal Signaling in Human Pluripotent Stem Cells." Stem Cells 39, no. 9 (2021): 1137–44. http://dx.doi.org/10.1002/stem.3383.
Full textAbstract:
Abstract Nodal is a transforming growth factor-β (TGF-β) superfamily member that plays a number of critical roles in mammalian embryonic development. Nodal is essential for the support of the peri-implantation epiblast in the mouse embryo and subsequently acts to specify mesendodermal fate at the time of gastrulation and, later, left-right asymmetry. Maintenance of human pluripotent stem cells (hPSCs) in vitro is dependent on Nodal signaling. Because it has proven difficult to prepare a biologically active form of recombinant Nodal protein, Activin or TGFB1 are widely used as surrogates for NODAL in hPSC culture. Nonetheless, the expression of the components of an endogenous Nodal signaling pathway in hPSC provides a potential autocrine pathway for the regulation of self-renewal in this system. Here we review recent studies that have clarified the role of Nodal signaling in pluripotent stem cell populations, highlighted spatial restrictions on Nodal signaling, and shown that Nodal functions in vivo as a heterodimer with GDF3, another TGF-β superfamily member expressed by hPSC. We discuss the role of this pathway in the maintenance of the epiblast and hPSC in light of these new advances.
21
Li, Sen, Wendy Keung, Heping Cheng, and Ronald A. Li. "Structural and Mechanistic Bases of Nuclear Calcium Signaling in Human Pluripotent Stem Cell-Derived Ventricular Cardiomyocytes." Stem Cells International 2019 (April 1, 2019): 1–17. http://dx.doi.org/10.1155/2019/8765752.
Full textAbstract:
The loss of nonregenerative, terminally differentiated cardiomyocytes (CMs) due to aging or diseases is generally considered irreversible. Human pluripotent stem cells (hPSCs) can self-renew while maintaining their pluripotency to differentiate into all cell types, including ventricular (V) cardiomyocytes (CMs), to provide a potential unlimited ex vivo source of CMs for heart disease modeling, drug/cardiotoxicity screening, and cell-based therapies. In the human heart, cytosolic Ca2+ signals are well characterized but the contribution of nuclear Ca2+ is essentially unexplored. The present study investigated nuclear Ca2+ signaling in hPSC-VCMs. Calcium transient or sparks in hPSC-VCMs were measured by line scanning using a spinning disc confocal microscope. We observed that nuclear Ca2+, which stems from unitary sparks due to the diffusion of cytosolic Ca2+ that are mediated by RyRs on the nuclear reticulum, is functional. Parvalbumin- (PV-) mediated Ca2+ buffering successfully manipulated Ca2+ transient and stimuli-induced apoptosis in hPSC-VCMs. We also investigated the effect of Ca2+ on gene transcription in hPSC-VCMs, and the involvement of nuclear factor of activated T-cell (NFAT) pathway was identified. The overexpression of Ca2+-sensitive, nuclear localized Ca2+/calmodulin-dependent protein kinase II δB (CaMKIIδB) induced cardiac hypertrophy through nuclear Ca2+/CaMKIIδB/HDAC4/MEF2 pathway. These findings provide insights into nuclear Ca2+ signal in hPSC-VCMs, which may lead to novel strategies for maturation as well as improved systems for disease modeling, drug discovery, and cell-based therapies.
22
Chang, Xuyao, Mingxia Gu, and Jason Tchieu. "Harnessing the Power of Stem Cell Models to Study Shared Genetic Variants in Congenital Heart Diseases and Neurodevelopmental Disorders." Cells 11, no. 3 (2022): 460. http://dx.doi.org/10.3390/cells11030460.
Full textAbstract:
Advances in human pluripotent stem cell (hPSC) technology allow one to deconstruct the human body into specific disease-relevant cell types or create functional units representing various organs. hPSC-based models present a unique opportunity for the study of co-occurring disorders where “cause and effect” can be addressed. Poor neurodevelopmental outcomes have been reported in children with congenital heart diseases (CHD). Intuitively, abnormal cardiac function or surgical intervention may stunt the developing brain, leading to neurodevelopmental disorders (NDD). However, recent work has uncovered several genetic variants within genes associated with the development of both the heart and brain that could also explain this co-occurrence. Given the scalability of hPSCs, straightforward genetic modification, and established differentiation strategies, it is now possible to investigate both CHD and NDD as independent events. We will first overview the potential for shared genetics in both heart and brain development. We will then summarize methods to differentiate both cardiac & neural cells and organoids from hPSCs that represent the developmental process of the heart and forebrain. Finally, we will highlight strategies to rapidly screen several genetic variants together to uncover potential phenotypes and how therapeutic advances could be achieved by hPSC-based models.
23
Wei, Yanxing, Tianyu Wang, Lishi Ma, et al. "Efficient derivation of human trophoblast stem cells from primed pluripotent stem cells." Science Advances 7, no. 33 (2021): eabf4416. http://dx.doi.org/10.1126/sciadv.abf4416.
Full textAbstract:
Human trophoblast stem cells (hTSCs) provide a valuable model to study placental development and function. While primary hTSCs have been derived from embryos/early placenta, and transdifferentiated hTSCs from naïve human pluripotent stem cells (hPSCs), the generation of hTSCs from primed PSCs is problematic. We report the successful generation of TSCs from primed hPSCs and show that BMP4 substantially enhances this process. TSCs derived from primed hPSCs are similar to blastocyst-derived hTSCs in terms of morphology, proliferation, differentiation potential, and gene expression. We define the chromatin accessibility dynamics and histone modifications (H3K4me3/H3K27me3) that specify hPSC-derived TSCs. Consistent with low density of H3K27me3 in primed hPSC-derived hTSCs, we show that knockout of H3K27 methyltransferases (EZH1/2) increases the efficiency of hTSC derivation from primed hPSCs. Efficient derivation of hTSCs from primed hPSCs provides a simple and powerful model to understand human trophoblast development, including the pathogenesis of trophoblast-related disorders, by generating disease-specific hTSCs.
24
McIntyre, Brendan A. S., Veronica Ramos-Mejia, Shravanti Rampalli, et al. "Gli3-mediated hedgehog inhibition in human pluripotent stem cells initiates and augments developmental programming of adult hematopoiesis." Blood 121, no. 9 (2013): 1543–52. http://dx.doi.org/10.1182/blood-2012-09-457747.
Full textAbstract:
Key Points Transient inhibition of hedgehog signaling augments hematopoiesis in hPSC-derived EBs. Hedgehog inhibition initiates an advancement in the developmental state of hematopoietic cells derived from hPSCs.
25
Porras, Deanna P., Jennifer C. Reid, Borko Tanasijevic, Diana Golubeva, Allison L. Boyd, and Mickie Bhatia. "Challenges in Cell Fate Acquisition to Scid-Repopulating Activity from Hemogenic Endothelium of hiPSCs Derived from AML Patients Using Forced Transcription Factor Expression." Cells 11, no. 12 (2022): 1915. http://dx.doi.org/10.3390/cells11121915.
Full textAbstract:
The generation of human hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) represents a major goal in regenerative medicine and is believed would follow principles of early development. HSCs arise from a type of endothelial cell called a “hemogenic endothelium” (HE), and human HSCs are experimentally detected by transplantation into SCID or other immune-deficient mouse recipients, termed SCID-Repopulating Cells (SRC). Recently, SRCs were detected by forced expression of seven transcription factors (TF) (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1) in hPSC-derived HE, suggesting these factors are deficient in hPSC differentiation to HEs required to generate HSCs. Here we derived PECAM-1-, Flk-1-, and VE-cadherin-positive endothelial cells that also lack CD45 expression (PFVCD45−) which are solely responsible for hematopoietic output from iPSC lines reprogrammed from AML patients. Using HEs derived from AML patient iPSCs devoid of somatic leukemic aberrations, we sought to generate putative SRCs by the forced expression of 7TFs to model autologous HSC transplantation. The expression of 7TFs in hPSC-derived HE cells from an enhanced hematopoietic progenitor capacity was present in vitro, but failed to acquire SRC activity in vivo. Our findings emphasize the benefits of forced TF expression, along with the continued challenges in developing HSCs for autologous-based therapies from hPSC sources.
26
Chen, Yu-Fan, Yi-Shuan J. Li, Chih-Hung Chou, et al. "Control of matrix stiffness promotes endodermal lineage specification by regulating SMAD2/3 via lncRNA LINC00458." Science Advances 6, no. 6 (2020): eaay0264. http://dx.doi.org/10.1126/sciadv.aay0264.
Full textAbstract:
During endoderm formation, cell identity and tissue morphogenesis are tightly controlled by cell-intrinsic and cell-extrinsic factors such as biochemical and physical inputs. While the effects of biochemical factors are well studied, the physical cues that regulate cell division and differentiation are poorly understood. RNA sequencing analysis demonstrated increases of endoderm-specific gene expression in hPSCs cultured on soft substrate (Young’s modulus, 3 ± 0.45 kPa) in comparison with hard substrate (Young’s modulus, 165 ± 6.39 kPa). Further analyses revealed that multiple long noncoding RNAs (lncRNAs) were up-regulated on soft substrate; among them, LINC00458 was identified as a stiffness-dependent lncRNA specifically required for hPSC differentiation toward an early endodermal lineage. Gain- and loss-of-function experiments confirmed that LINC00458 is functionally required for hPSC endodermal lineage specification induced by soft substrates. Our study provides evidence that mechanical cues regulate the expression of LINC00458 and induce differentiation of hPSC into hepatic lineage progenitors.
27
Hyakumura, Tomoko, Stuart McDougall, Sue Finch, Karina Needham, Mirella Dottori, and Bryony A. Nayagam. "Organotypic Cocultures of Human Pluripotent Stem Cell Derived-Neurons with Mammalian Inner Ear Hair Cells and Cochlear Nucleus Slices." Stem Cells International 2019 (November 20, 2019): 1–14. http://dx.doi.org/10.1155/2019/8419493.
Full textAbstract:
Stem cells have been touted as a source of potential replacement neurons for inner ear degeneration for almost two decades now; yet to date, there are few studies describing the use of human pluripotent stem cells (hPSCs) for this purpose. If stem cell therapies are to be used clinically, it is critical to validate the usefulness of hPSC lines in vitro and in vivo. Here, we present the first quantitative evidence that differentiated hPSC-derived neurons that innervate both the inner ear hair cells and cochlear nucleus neurons in coculture, with significantly more new synaptic contacts formed on target cell types. Nascent contacts between stem cells and hair cells were immunopositive for both synapsin I and VGLUT1, closely resembling expression of these puncta in endogenous postnatal auditory neurons and control cocultures. When hPSCs were cocultured with cochlear nucleus brainstem slice, significantly greater numbers of VGLUT1 puncta were observed in comparison to slice alone. New VGLUT1 puncta in cocultures with cochlear nucleus slice were not significantly different in size, only in quantity. This experimentation describes new coculture models for assessing auditory regeneration using well-characterised hPSC-derived neurons and highlights useful methods to quantify the extent of innervation on different cell types in the inner ear and brainstem.
28
Ran, Dan, Wei-Jong Shia, Miao-Chia Lo, et al. "RUNX1a enhances hematopoietic lineage commitment from human embryonic stem cells and inducible pluripotent stem cells." Blood 121, no. 15 (2013): 2882–90. http://dx.doi.org/10.1182/blood-2012-08-451641.
Full textAbstract:
Abstract Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo–expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with β-globin production. Moreover, HPCs generated from RUNX1a EBs possess ≥9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.
29
Zhang, Fengzhi, Hui Qiu, Xiaohui Dong, et al. "Transferrin improved the generation of cardiomyocyte from human pluripotent stem cells for myocardial infarction repair." Journal of Molecular Histology 52, no. 1 (2020): 87–99. http://dx.doi.org/10.1007/s10735-020-09926-0.
Full textAbstract:
AbstractHuman pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) hold great promise for the repair of the injured heart, but optimal cell production in a fully chemically defined and cost-effective system is essential for the efficacy and safety of cell transplantation therapies. In this study, we provided a simple and efficient strategy for cardiac differentiation from hPSCs and performed functional evaluation in a rat model of myocardial infarction. Using a chemically defined medium including four components, recombinant human albumin, ascorbic acid, human transferrin, and RPMI 1640, we developed a manageable and cost-effective protocol for robust generation of CMs from hPSCs. Interestingly, the addition of transferrin helped hPSCs to transit from TeSR-E8 medium to the simple cardiac differentiation medium and successfully initiated mesoderm differentiation without significant cell death. The CM generation efficiency was up to 85% based on cTnT expression. We performed transcriptome profiling from differentiation day 0 to 35, and characterized interesting dynamic change of cardiac genes. CMs derived from transferrin-supplemented simple medium have similar transcriptome and the maturation level compared to those generated in B27 minus insulin medium as well as their in vivo counterparts. Importantly, after transplantation, hPSC-derived CMs survived in the infarcted rat heart, significantly improved the physiological function and reduced fibrosis. Our study offers an easy-to-use and cost-effective method for cardiac differentiation and facilitates the translational application of hPSC-derived CMs for heart repair.
30
Souidi, Monia, Yvonne Sleiman, Ivana Acimovic, et al. "Oxygen Is an Ambivalent Factor for the Differentiation of Human Pluripotent Stem Cells in Cardiac 2D Monolayer and 3D Cardiac Spheroids." International Journal of Molecular Sciences 22, no. 2 (2021): 662. http://dx.doi.org/10.3390/ijms22020662.
Full textAbstract:
Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.
31
Souidi, Monia, Yvonne Sleiman, Ivana Acimovic, et al. "Oxygen Is an Ambivalent Factor for the Differentiation of Human Pluripotent Stem Cells in Cardiac 2D Monolayer and 3D Cardiac Spheroids." International Journal of Molecular Sciences 22, no. 2 (2021): 662. http://dx.doi.org/10.3390/ijms22020662.
Full textAbstract:
Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.
32
Roberts, R. Michael, Kyle M. Loh, Mitsuyoshi Amita, et al. "Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?" REPRODUCTION 147, no. 5 (2014): D1—D12. http://dx.doi.org/10.1530/rep-14-0080.
Full textAbstract:
It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.
33
Go, Young-Hyun, Jumee Kim, Ho-Chang Jeong, et al. "Luteolin Induces Selective Cell Death of Human Pluripotent Stem Cells." Biomedicines 8, no. 11 (2020): 453. http://dx.doi.org/10.3390/biomedicines8110453.
Full textAbstract:
Despite recent advances in clinical stem cell therapy applications based on human pluripotent stem cells (hPSCs), potential teratoma formation due to the presence of residual undifferentiated hPSCs remains a serious risk factor that challenges widespread clinical application. To overcome this risk, a variety of approaches have been developed to eliminate the remaining undifferentiated hPSCs via selective cell death induction. Our study seeks to identify natural flavonoids that are more potent than quercetin (QC), to selectively induce hPSC death. Upon screening in-house flavonoids, luteolin (LUT) is found to be more potent than QC to eliminate hPSCs in a p53-dependent manner, but not hPSC-derived smooth muscle cells or perivascular progenitor cells. Particularly, treating human embryonic stem cell (hESC)-derived cardiomyocytes with LUT efficiently eliminates the residual hESCs and only results in marginal effects on cardiomyocyte (CM) functions, as determined by calcium influx. Considering the technical limitations of isolating CMs due to a lack of exclusive surface markers at the end of differentiation, LUT treatment is a promising approach to minimize teratoma formation risk.
34
Hazeltine, Laurie B., Chelsey S. Simmons, Max R. Salick, et al. "Effects of Substrate Mechanics on Contractility of Cardiomyocytes Generated from Human Pluripotent Stem Cells." International Journal of Cell Biology 2012 (2012): 1–13. http://dx.doi.org/10.1155/2012/508294.
Full textAbstract:
Human pluripotent stem cell (hPSC-) derived cardiomyocytes have potential applications in drug discovery, toxicity testing, developmental studies, and regenerative medicine. Before these cells can be reliably utilized, characterization of their functionality is required to establish their similarity to native cardiomyocytes. We tracked fluorescent beads embedded in 4.4–99.7 kPa polyacrylamide hydrogels beneath contracting neonatal rat cardiomyocytes and cardiomyocytes generated from hPSCs via growth-factor-induced directed differentiation to measure contractile output in response to changes in substrate mechanics. Contraction stress was determined using traction force microscopy, and morphology was characterized by immunocytochemistry for α-actinin and subsequent image analysis. We found that contraction stress of all types of cardiomyocytes increased with substrate stiffness. This effect was not linked to beating rate or morphology. We demonstrated that hPSC-derived cardiomyocyte contractility responded appropriately to isoprenaline and remained stable in culture over a period of 2 months. This study demonstrates that hPSC-derived cardiomyocytes have appropriate functional responses to substrate stiffness and to a pharmaceutical agent, which motivates their use in further applications such as drug evaluation and cardiac therapies.
35
Lipsitz, Yonatan Y., Curtis Woodford, Ting Yin, Jacob H. Hanna, and Peter W. Zandstra. "Modulating cell state to enhance suspension expansion of human pluripotent stem cells." Proceedings of the National Academy of Sciences 115, no. 25 (2018): 6369–74. http://dx.doi.org/10.1073/pnas.1714099115.
Full textAbstract:
The development of cell-based therapies to replace missing or damaged tissues within the body or generate cells with a unique biological activity requires a reliable and accessible source of cells. Human pluripotent stem cells (hPSC) have emerged as a strong candidate cell source capable of extended propagation in vitro and differentiation to clinically relevant cell types. However, the application of hPSC in cell-based therapies requires overcoming yield limitations in large-scale hPSC manufacturing. We explored methods to convert hPSC to alternative states of pluripotency with advantageous bioprocessing properties, identifying a suspension-based small-molecule and cytokine combination that supports increased single-cell survival efficiency, faster growth rates, higher densities, and greater expansion than control hPSC cultures. ERK inhibition was found to be essential for conversion to this altered state, but once converted, ERK inhibition led to a loss of pluripotent phenotype in suspension. The resulting suspension medium formulation enabled hPSC suspension yields 5.7 ± 0.2-fold greater than conventional hPSC in 6 d, for at least five passages. Treated cells remained pluripotent, karyotypically normal, and capable of differentiating into all germ layers. Treated cells could also be integrated into directed differentiated strategies as demonstrated by the generation of pancreatic progenitors (NKX6.1+/PDX1+ cells). Enhanced suspension-yield hPSC displayed higher oxidative metabolism and altered expression of adhesion-related genes. The enhanced bioprocess properties of this alternative pluripotent state provide a strategy to overcome cell manufacturing limitations of hPSC.
36
Oh, Jisun, Oh-Bin Kwon, Sang-Wook Park, et al. "Advancing Cardiovascular Drug Screening Using Human Pluripotent Stem Cell-Derived Cardiomyocytes." International Journal of Molecular Sciences 25, no. 14 (2024): 7971. http://dx.doi.org/10.3390/ijms25147971.
Full textAbstract:
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a promising tool for studying cardiac physiology and drug responses. However, their use is largely limited by an immature phenotype and lack of high-throughput analytical methodology. In this study, we developed a high-throughput testing platform utilizing hPSC-CMs to assess the cardiotoxicity and effectiveness of drugs. Following an optimized differentiation and maturation protocol, hPSC-CMs exhibited mature CM morphology, phenotype, and functionality, making them suitable for drug testing applications. We monitored intracellular calcium dynamics using calcium imaging techniques to measure spontaneous calcium oscillations in hPSC-CMs in the presence or absence of test compounds. For the cardiotoxicity test, hPSC-CMs were treated with various compounds, and calcium flux was measured to evaluate their effects on calcium dynamics. We found that cardiotoxic drugs withdrawn due to adverse drug reactions, including encainide, mibefradil, and cetirizine, exhibited toxicity in hPSC-CMs but not in HEK293-hERG cells. Additionally, in the effectiveness test, hPSC-CMs were exposed to ATX-II, a sodium current inducer for mimicking long QT syndrome type 3, followed by exposure to test compounds. The observed changes in calcium dynamics following drug exposure demonstrated the utility of hPSC-CMs as a versatile model system for assessing both cardiotoxicity and drug efficacy. Overall, our findings highlight the potential of hPSC-CMs in advancing drug discovery and development, which offer a physiologically relevant platform for the preclinical screening of novel therapeutics.
37
Barra, Jessie, Rob Robino, Roberto Castro-Gutierrez, Leonardo Ferreira, and Holger Russ. "Combinatorial genome engineering strategy for CAR-Treg antigen-specific immune protection of stem cell-derived cells." Journal of Immunology 212, no. 1_Supplement (2024): 1538_4544. http://dx.doi.org/10.4049/jimmunol.212.supp.1538.4544.
Full textAbstract:
Abstract Improved methods for generating human pluripotent stem cell (hPSC)-derived cells and tissues have rapidly expanded the field of regenerative medicine. Yet, in most cases, hPSC-derived cells need to be protected, ideally in a localized manner, from an antagonistic host immune response. Chimeric antigen receptor (CAR) technology can confer new antigen specificities to effector T cells and, more recently, regulatory T cells (Tregs), potent immunosuppressive cells. However, one challenge in CAR design is identifying a target molecule uniquely expressed by the cells of interest to prevent unwanted off-target effects. Here, we developed a combinatorial genetic engineering approach to confer CAR-Treg-mediated localized immune protection specifically to transplanted stem cell-derived cells using a humanized type 1 diabetes preclinical mouse model. We engineered hPSCs to express a truncated epidermal growth factor receptor (EGFRt) and differentiated functional stem cell derived beta-like cells (sBCs) from them. In parallel, we generated EGFR CAR-Tregs that were activated by EGFRt-expressing sBCs without disrupting Treg identity. Strikingly, activated CAR-Tregs suppressed innate and adaptive effector immune responses in vitro and prevented effector CAR-T-cell-mediated sBC graft destruction in vivo. These data provide evidence that combinatorial genome engineering of hPSCs and Tregs can be harnessed to protect hPSC-derived tissues from immune attack via immune tolerance induction.
38
Qian, Shiguang, Hong-Shiue Chou, Ronald Charles, John Fung, and Lina Lu. "Induction of cell transplant tolerance: a roadmap from bench to bedside (P2220)." Journal of Immunology 190, no. 1_Supplement (2013): 69.48. http://dx.doi.org/10.4049/jimmunol.190.supp.69.48.
Full textAbstract:
Abstract Organ transplants have worked for decades, but outcomes of cell transplants remain disappointing. Enlightened by mouse liver allografts being spontaneously accepted, but hepatocyte transplants acutely rejected (suggesting lack of non-parenchymal cell protection), we identified strong regulatory activity of hepatic stellate cells (HpSC). Islet allografts (300) mixed with HpSC (3x105) achieved long survival without immunosuppression, associated with apoptosis of CD8 T cells, enhancement of Tregs, which depends on IFNγ/B7-H1 pathway. However, co-transplanted HpSC have to be syngeneic to recipients - a hurdle for clinical application. We revealed that HpSC did not directly induce Tregs, but induce MDSC. Addition of HpSC in DC culture (1:40) generated MDSC (instead of DC) with high iNOS and argenase 1 and potent suppressive activity. The induction of MDSC was found to be mediated by soluble factors, particularly retinoic acid and iC3b. Islet allografts mixed with (or followed by iv injection of) generated MDSC (3x106) were protected as effectively as HpSC, associated with elimination of effector T and enhancement of Tregs, which also required intact IFNγ/B7-H1 signaling. Human HpSC were tested showing markedly T cell suppression in MLR, which was reversed by B7-H1 blocking Ab. Human HpSC also induced MDSC in vitro. We will reexamined these in vivo in a humanized NOD/SCID mouse model before pursue to clinical trial.
39
Weis, Victoria G., Anna C. Deal, Gehad Mekkey, et al. "Human placental-derived stem cell therapy ameliorates experimental necrotizing enterocolitis." American Journal of Physiology-Gastrointestinal and Liver Physiology 320, no. 4 (2021): G658—G674. http://dx.doi.org/10.1152/ajpgi.00369.2020.
Full textAbstract:
These studies demonstrate a human placental-derived stem cell (hPSC) therapeutic strategy for necrotizing enterocolitis (NEC). In an experimental model of NEC, hPSC administration improved macroscopic intestinal health, ameliorated epithelial morphology, and supported the intestinal stem cell niche. Our data suggest that hPSC are a potential therapeutic approach to attenuate established intestinal NEC damage. Further, we show hPSC are a novel research tool that can be utilized to elucidate critical neonatal repair mechanisms to overcome NEC.
40
Yang, Hui, Weiyi Zhong, Mohammad Rafi Hamidi, Gaojun Zhou, and Chen Liu. "Functional improvement and maturation of human cardiomyocytes derived from human pluripotent stem cells by barbaloin preconditioning." Acta Biochimica et Biophysica Sinica 51, no. 10 (2019): 1041–48. http://dx.doi.org/10.1093/abbs/gmz090.
Full textAbstract:
Abstract The development of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is a significant advancement in our ability to obtain cardiomyocytes in vitro for regenerative therapies and drug discovery. However, hPSC-CMs obtained via existing protocols usually exhibit a markedly immature phenotype, compared with adult cardiomyocytes, thereby limiting their application. Here we report that barbaloin preconditioning dramatically improves the morphology, structure-related cardiac gene expression, calcium handling, and electrophysiological properties of hPSC-CMs, which means that barbaloin may have the potential to induce the maturation of hPSC-CMs, providing a novel strategy to generate more adult-like cardiomyocytes and promoting the application of hPSC-CMs in regenerative medicine, drug development, and disease modeling.
41
Rungsiwiwut, Ruttachuk, Praewphan Ingrungruanglert, Pranee Numchaisrika, Pramuan Virutamasen, Tatsanee Phermthai, and Kamthorn Pruksananonda. "Human Umbilical Cord Blood-Derived Serum for Culturing the Supportive Feeder Cells of Human Pluripotent Stem Cell Lines." Stem Cells International 2016 (2016): 1–12. http://dx.doi.org/10.1155/2016/4626048.
Full textAbstract:
Although human pluripotent stem cells (hPSCs) can proliferate robustly on the feeder-free culture system, genetic instability of hPSCs has been reported in such environment. Alternatively, feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS) prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. Recently, human cord blood-derived serum (hUCS) showed a positive effect on culture of mesenchymal stem cells. It is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs) prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGFβ1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSCs culture system.
42
Goldsteen, Pien A., Christina Yoseif, Amalia M. Dolga, and Reinoud Gosens. "Human pluripotent stem cells for the modelling and treatment of respiratory diseases." European Respiratory Review 30, no. 161 (2021): 210042. http://dx.doi.org/10.1183/16000617.0042-2021.
Full textAbstract:
Respiratory diseases are among the leading causes of morbidity and mortality worldwide, representing a major unmet medical need. New chemical entities rarely make it into the clinic to treat respiratory diseases, which is partially due to a lack of adequate predictive disease models and the limited availability of human lung tissues to model respiratory disease. Human pluripotent stem cells (hPSCs) may help fill this gap by serving as a scalable human in vitro model. In addition, human in vitro models of rare genetic mutations can be generated using hPSCs. hPSC-derived epithelial cells and organoids have already shown great potential for the understanding of disease mechanisms, for finding new potential targets by using high-throughput screening platforms, and for personalised treatments. These potentials can also be applied to other hPSC-derived lung cell types in the future. In this review, we will discuss how hPSCs have brought, and may continue to bring, major changes to the field of respiratory diseases by understanding the molecular mechanisms of the pathology and by finding efficient therapeutics.
43
Long, Hong-Yan, Zu-Ping Qian, Qin Lan, et al. "Human pluripotent stem cell-derived kidney organoids: Current progress and challenges." World Journal of Stem Cells 16, no. 2 (2024): 114–25. http://dx.doi.org/10.4252/wjsc.v16.i2.114.
Full textAbstract:
Human pluripotent stem cell (hPSC)-derived kidney organoids share similarities with the fetal kidney. However, the current hPSC-derived kidney organoids have some limitations, including the inability to perform nephrogenesis and lack of a corticomedullary definition, uniform vascular system, and coordinated exit pathway for urinary filtrate. Therefore, further studies are required to produce hPSC-derived kidney organoids that accurately mimic human kidneys to facilitate research on kidney development, regeneration, disease modeling, and drug screening. In this review, we discussed recent advances in the generation of hPSC-derived kidney organoids, how these organoids contribute to the understanding of human kidney development and research in disease modeling. Additionally, the limitations, future research focus, and applications of hPSC-derived kidney organoids were highlighted.
44
Cotovio, João P., and Tiago G. Fernandes. "Production of Human Pluripotent Stem Cell-Derived Hepatic Cell Lineages and Liver Organoids: Current Status and Potential Applications." Bioengineering 7, no. 2 (2020): 36. http://dx.doi.org/10.3390/bioengineering7020036.
Full textAbstract:
Liver disease is one of the leading causes of death worldwide, leading to the death of approximately 2 million people per year. Current therapies include orthotopic liver transplantation, however, donor organ shortage remains a great challenge. In addition, the development of novel therapeutics has been limited due to the lack of in vitro models that mimic in vivo liver physiology. Accordingly, hepatic cell lineages derived from human pluripotent stem cells (hPSCs) represent a promising cell source for liver cell therapy, disease modelling, and drug discovery. Moreover, the development of new culture systems bringing together the multiple liver-specific hepatic cell types triggered the development of hPSC-derived liver organoids. Therefore, these human liver-based platforms hold great potential for clinical applications. In this review, the production of the different hepatic cell lineages from hPSCs, including hepatocytes, as well as the emerging strategies to generate hPSC-derived liver organoids will be assessed, while current biomedical applications will be highlighted.
45
Ashok, Preeti, and Emmanuel S. Tzanakakis. "Proteomic Analysis of Exosomes during Cardiogenic Differentiation of Human Pluripotent Stem Cells." Cells 10, no. 10 (2021): 2622. http://dx.doi.org/10.3390/cells10102622.
Full textAbstract:
Efforts to direct the specification of human pluripotent stem cells (hPSCs) to therapeutically important somatic cell types have focused on identifying proper combinations of soluble cues. Yet, whether exosomes, which mediate intercellular communication, play a role in the differentiation remains unexplored. We took a first step toward addressing this question by subjecting hPSCs to stage-wise specification toward cardiomyocytes (CMs) in scalable stirred-suspension cultures and collecting exosomes. Samples underwent liquid chromatography (LC)/mass spectrometry (MS) and subsequent proteomic analysis revealed over 300 unique proteins from four differentiation stages including proteins such as PPP2CA, AFM, MYH9, MYH10, TRA2B, CTNNA1, EHD1, ACTC1, LDHB, and GPC4, which are linked to cardiogenic commitment. There was a significant correlation of the protein composition of exosomes with the hPSC line and stage of commitment. Differentiating hPSCs treated with exosomes from hPSC-derived CMs displayed improved efficiency of CM formation compared to cells without exogenously added vesicles. Collectively, these results demonstrate that exosomes from hPSCs induced along the CM lineage contain proteins linked to the specification process with modulating effects and open avenues for enhancing the biomanufacturing of stem cell products for cardiac diseases.
46
Pelkonen, Anssi, Cristiana Pistono, Pamela Klecki, et al. "Functional Characterization of Human Pluripotent Stem Cell-Derived Models of the Brain with Microelectrode Arrays." Cells 11, no. 1 (2021): 106. http://dx.doi.org/10.3390/cells11010106.
Full textAbstract:
Human pluripotent stem cell (hPSC)-derived neuron cultures have emerged as models of electrical activity in the human brain. Microelectrode arrays (MEAs) measure changes in the extracellular electric potential of cell cultures or tissues and enable the recording of neuronal network activity. MEAs have been applied to both human subjects and hPSC-derived brain models. Here, we review the literature on the functional characterization of hPSC-derived two- and three-dimensional brain models with MEAs and examine their network function in physiological and pathological contexts. We also summarize MEA results from the human brain and compare them to the literature on MEA recordings of hPSC-derived brain models. MEA recordings have shown network activity in two-dimensional hPSC-derived brain models that is comparable to the human brain and revealed pathology-associated changes in disease models. Three-dimensional hPSC-derived models such as brain organoids possess a more relevant microenvironment, tissue architecture and potential for modeling the network activity with more complexity than two-dimensional models. hPSC-derived brain models recapitulate many aspects of network function in the human brain and provide valid disease models, but certain advancements in differentiation methods, bioengineering and available MEA technology are needed for these approaches to reach their full potential.
47
Bai, Hao, Yin-Liang Xie, Yong-Xing Gao, Tao Cheng, and Zack Z. Wang. "The Balance of Positive and Negative Effects of TGF-Beta Signaling Regulates the Hemangioblast Development in Human Pluripotent Stem Cells." Blood 120, no. 21 (2012): 1220. http://dx.doi.org/10.1182/blood.v120.21.1220.1220.
Full textAbstract:
Abstract Abstract 1220 Derived from mesoderm precursors, hemangioblasts are bipotential common progenitors of hematopoietic cells and endothelial cells. The regulatory events controlling human hemangioblast development are largely unknown. Our previous study demonstrated that CD34 progenitors from human embryonic stem cells (hESCs) contain a population of cells that give rise to hematopoietic cells and endothelial cells. In this study, we established a serum-free and feeder-free system to investigate the signals that direct differentiation of human pluripotent stem cells (hPSCs), including hESCs and induced-pluripotent stem cells (hiPSCs). The hemangioblast development in hPSCs was established in three major steps: i) the formation of uniformed embryoid bodies (EBs) in hanging-drops, ii) the development of multipotential mesoderm precursors in cultivation of EBs, and iii) the generation of CD34+CD31+VE-cadherin+ progenitors, which have hemangioblast potential to give rise to both hematopoietic and endothelial cells (via hemogenic endothelium). The hESC-derived hematopoietic cells expressed CD41, CD61, CD117, CD71, and CD235a. The endothelial cells were CD31+ and VE-cadherin+ cells. We found that inhibition of Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) by Y27632 was critical for hanging-drop EB formation. Transforming growth factor beta (TGF-beta) is a key regulator of hematopoiesis with potent inhibitory effects on progenitor and stem cell proliferation. The expression of TGF-beta was increased during hPSC differentiation. Inhibition of TGF-beta signaling by the TGF-beta inhibitor, SB431542, significantly decreased the efficiency of CD34+ progenitors, suggesting a crucial role of TGF-beta signaling in hPSC differentiation. To further investigate when TGF-beta signaling affects hPSC differentiation to CD34+ progenitors, we added TGF-beta inhibitor at different stages of hPSC differentiation. We found that TGF-beta signaling is required for the early mesoderm induction. Inhibition of TGFb signaling before hemangioblast commitment during PSC differentiation significantly decreases the frequency of hemangioblasts (CD34+/CD31+/VE-cadherin+ cells) and the expression of mesoderm markers, such as Brachyury, Tie1, EOMES, and MIXL1. On the other hand, inhibition of TGF-beta signaling after mesoderm induction increases the number of CD34+/CD31+/VE-cadherin+ cells. EdU cell proliferation assays indicated that the proliferation of CD34+ progenitors were not affected by SB431542, suggesting that TGF-beta signaling suppresses hemangioblast generation. Our study demonstrated that TGF-beta signaling has the double-edged effect on hPSC differentiation to hemangioblasts. Disclosures: No relevant conflicts of interest to declare.
48
Ribeiro, Alexandre J. S., Yen-Sin Ang, Ji-Dong Fu, et al. "Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness." Proceedings of the National Academy of Sciences 112, no. 41 (2015): 12705–10. http://dx.doi.org/10.1073/pnas.1508073112.
Full textAbstract:
Single cardiomyocytes contain myofibrils that harbor the sarcomere-based contractile machinery of the myocardium. Cardiomyocytes differentiated from human pluripotent stem cells (hPSC-CMs) have potential as an in vitro model of heart activity. However, their fetal-like misalignment of myofibrils limits their usefulness for modeling contractile activity. We analyzed the effects of cell shape and substrate stiffness on the shortening and movement of labeled sarcomeres and the translation of sarcomere activity to mechanical output (contractility) in live engineered hPSC-CMs. Single hPSC-CMs were cultured on polyacrylamide substrates of physiological stiffness (10 kPa), and Matrigel micropatterns were used to generate physiological shapes (2,000-µm2 rectangles with length:width aspect ratios of 5:1–7:1) and a mature alignment of myofibrils. Translation of sarcomere shortening to mechanical output was highest in 7:1 hPSC-CMs. Increased substrate stiffness and applied overstretch induced myofibril defects in 7:1 hPSC-CMs and decreased mechanical output. Inhibitors of nonmuscle myosin activity repressed the assembly of myofibrils, showing that subcellular tension drives the improved contractile activity in these engineered hPSC-CMs. Other factors associated with improved contractility were axially directed calcium flow, systematic mitochondrial distribution, more mature electrophysiology, and evidence of transverse-tubule formation. These findings support the potential of these engineered hPSC-CMs as powerful models for studying myocardial contractility at the cellular level.
49
Al-attar, Rasha, Joseph Jargstorf, Rocco Romagnuolo, et al. "Casein Kinase 1 Phosphomimetic Mutations Negatively Impact Connexin-43 Gap Junctions in Human Pluripotent Stem Cell-Derived Cardiomyocytes." Biomolecules 14, no. 1 (2024): 61. http://dx.doi.org/10.3390/biom14010061.
Full textAbstract:
The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has shown promise in preclinical models of myocardial infarction, but graft myocardium exhibits incomplete host–graft electromechanical integration and a propensity for pro-arrhythmic behavior. Perhaps contributing to this situation, hPSC-CM grafts show low expression of connexin 43 (Cx43), the major gap junction (GJ) protein, in ventricular myocardia. We hypothesized that Cx43 expression and function could be rescued by engineering Cx43 in hPSC-CMs with a series of phosphatase-resistant mutations at three casein kinase 1 phosphorylation sites (Cx43-S3E) that have been previously reported to stabilize Cx43 GJs and reduce arrhythmias in transgenic mice. However, contrary to our predictions, transgenic Cx43-S3E hPSC-CMs exhibited reduced Cx43 expression relative to wild-type cells, both at baseline and following ischemic challenge. Cx43-S3E hPSC-CMs showed correspondingly slower conduction velocities, increased automaticity, and differential expression of other connexin isoforms and various genes involved in cardiac excitation–contraction coupling. Cx43-S3E hPSC-CMs also had phosphorylation marks associated with Cx43 GJ internalization, a finding that may account for their impaired GJ localization. Taken collectively, our data indicate that the Cx43-S3E mutation behaves differently in hPSC-CMs than in adult mouse ventricular myocytes and that multiple biological factors likely need to be addressed synchronously to ensure proper Cx43 expression, localization, and function.
50
Esfahani, Sajedeh Nasr, Yi Zheng, Auriana Arabpour, et al. "Derivation of human primordial germ cell-like cells in an embryonic-like culture." Nature Communications 15, no. 1 (2024). http://dx.doi.org/10.1038/s41467-023-43871-2.
Full textAbstract:
AbstractPrimordial germ cells (PGCs) are the embryonic precursors of sperm and eggs. They transmit genetic and epigenetic information across generations. Given the prominent role of germline defects in diseases such as infertility, detailed understanding of human PGC (hPGC) development has important implications in reproductive medicine and studying human evolution. Yet, hPGC specification remains an elusive process. Here, we report the induction of hPGC-like cells (hPGCLCs) in a bioengineered human pluripotent stem cell (hPSC) culture that mimics peri-implantation human development. In this culture, amniotic ectoderm-like cells (AMLCs), derived from hPSCs, induce hPGCLC specification from hPSCs through paracrine signaling downstream of ISL1. Our data further show functional roles of NODAL, WNT, and BMP signaling in hPGCLC induction. hPGCLCs are successfully derived from eight non-obstructive azoospermia (NOA) participant-derived hPSC lines using this biomimetic platform, demonstrating its promise for screening applications.