Generation of Functional Lymphoid (Natural Killer) Cells From Human ESC-Derived Hemangioblasts.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1502-1502 ◽  
Author(s):  
Erin A. Kimbrel ◽  
Shi-Jiang Lu ◽  
Robert Lanza
Keyword(s):  
Stem Cell ◽  
Regenerative Medicine ◽  
Nk Cells ◽  
Natural Killer ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Cell Detection ◽  
Natural Cytotoxicity ◽  
Intermediary Cell ◽  
Hesc Lines

Abstract Abstract 1502 Poster Board I-525 Studies with human and mouse embryonic stem cells (ESCs) have shown that a common precursor to both vascular (endothelial and smooth muscle cells) and hematopoietic cell lineages called the hemangioblast can be produced from ESC-derived embryoid bodies in culture. We have developed a simple strategy to efficiently and reproducibly generate hemangioblasts from multiple hESC lines under serum- and stromal-free conditions, which will be important for their productive use in regenerative medicine. Previous work has shown that hESC-derived hemangioblasts can effectively differentiate into erythroid and myeloid lineages, but their ability to produce lymphoid lineage cells, including those with immunotherapeutic potential, is relatively unknown. Natural killer (NK) cells, which are part of the innate immune system, provide rapid, non-specific responses against viral infection and are involved in tumor cell detection and elimination. Interplay between various activating and inhibitory signals control the three main functions of NK cells, which are cytokine release, natural cytotoxicity, and antibody-dependent cellular cytotoxicity. Using hemangioblasts generated from both H7 and HuES-3 hESC lines, we have been able to produce mature CD56low/−CD16+ NK cells and found that their production does not require the use of stromal feeder layers. The differentiation procedure involves an initial 4 day culture to generate embryoid bodies, followed by a 12-14 day culture in methylcellulose supplemented with a set of cytokines and growth factors for the production and expansion of a hemangioblastic population. An additional 14-17 days in liquid culture plus human serum and a cocktail of cytokines allows for the differentiation of NK cells as assessed by flow cytometry. A non-radioactive cytotoxicity assay similar to the 51Cr release assay shows that these hemangioblast-derived NK cells harbor natural cytotoxicity function as they are able to effectively induce apoptosis in target K562 erythroblastic leukemia cells after a standard 4 hr co-culture. Using hemangioblasts as an intermediary cell source may enhance the capability and/or efficiency of hESCs to differentiate in vitro and importantly, allow for the development of feeder-free systems for the production of cells with immunotherapeutic potential. Disclosures: Kimbrel: Stem Cell & Regenerative Medicine International: Employment. Lu: Stem Cell & Regenerative Medicine International: Employment. Lanza: Stem Cell & Regenerative Medicine International/Advanced Cell Technology: Employment.

scholarly journals The Production and Directed Differentiation of Human Embryonic Stem Cells

2006 ◽  
Vol 27 (2) ◽  
pp. 208-219 ◽  
Author(s):  
Alan Trounson
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Regenerative Medicine ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Directed Differentiation ◽  
Hesc Lines

Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The methods for deriving hESCs are well established and repeatable and are relatively successful with a ratio of 1:10 to 1:2 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of early progenitors in this lineage. There are reports of enriched cultures of keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurons, hepatic progenitors, and cells that have some markers of gut tissue and pancreatic islet-like cells. The prospects for use of hESC derivatives in regenerative medicine are significant, and there is much optimism for their potential contributions to human regenerative medicine.

scholarly journals Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing

2021 ◽  
Author(s):  
Karin Lauschke ◽  
Andreas Frederik Treschow ◽  
Mikkel Aabech Rasmussen ◽  
Nichlas Davidsen ◽  
Bjørn Holst ◽  
...  
Keyword(s):  
Stem Cell ◽  
Reporter Gene ◽  
Luminescence Intensity ◽  
Developmental Toxicity ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Luciferase Reporter ◽  
Reporter Cell ◽  
Gene Assay ◽  
Induced Pluripotent

AbstractTo test large numbers of chemicals for developmental toxicity, rapid in vitro tests with standardized readouts for automated data acquisition are needed. However, the most widely used assay, the embryonic stem cell test, relies on the counting of beating embryoid bodies by visual inspection, which is laborious and time consuming. We previously developed the PluriBeat assay based on differentiation of human induced pluripotent stem cells (hiPSC) that we demonstrated to be predictive for known teratogens at relevant concentrations using the readout of beating cardiomyocytes. Here, we report the development of a novel assay, which we term the PluriLum assay, where we have introduced a luciferase reporter gene into the locus ofNKX2.5of our hiPSC line. This enabled us to measure luminescence intensities instead of counting beating cardiomyocytes, which is less labor intensive. We established twoNKX2.5reporter cell lines and validated their pluripotency and genetic stability. Moreover, we confirmed that the genetically engineeredNKX2.5reporter cell line differentiated into cardiomyocytes with the same efficiency as the original wild-type line. We then exposed the cells to valproic acid (25–300 μM) and thalidomide (0.1–36 µM) and compared the PluriBeat readout of the cardiomyocytes with the luminescence intensity of the PluriLum assay. The results showed that thalidomide decreased luminescence intensity significantly with a higher potency and efficacy compared to the beating readout. With this, we have developed a novel hiPSC-based assay with a standardized readout that may have the potential for higher throughput screening for developmental toxicity.

scholarly journals Cytokine-Mediated Natural Killer Cells Effects Impair Hematopoietic Stem Cell Function

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2641-2641
Author(s):  
Lorena Lobo Figueiredo-Pontes ◽  
Robert S. Welner ◽  
Miroslava Kardosova ◽  
Hong Zhang ◽  
Meritxell Alberich-Jorda ◽  
...  
Keyword(s):  
Stem Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Cytokine Production ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Cytokine Signaling ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Nk Cytotoxicity

Abstract Natural killer (NK) cells participate in innate and adaptive immune responses, and upon activation rapidly produce cytokines, chemokines, and growth factors, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10, and others, which can affect the function of other hematopoietic cells. Considering the recent evidences that hematopoietic stem cells (HSCs) respond to cytokine signaling, we hypothesized that NK cell-mediated cytokine production could mediate HSC function. By the use of co-cultures of purified Ly5.1 murine NK cells and congenic Ly5.2 HSCs, we concluded that NK activity affects HSC frequency in vitro as well as hematopoietic reconstitution in vivo. Sorted NK cells (CD3- NK1.1+) and HSCs (Lin-, Sca1+, ckithi, CD48-, CD150+) were co-cultured in the presence or absence of IL2 over an OP9 stromal cells layer for 14 to 28 days. After 14 days, the addition of NK cells to HSC cultures resulted in an approximate 2-fold reduction of lineage negative cells (Lin-) recovered cells, as compared to control HSC cultures, as determined by flow cytometry analysis. Lin- counts were even lower in HSC+NK long-term cultures when compared to HSC only cultures. Ly5.1 HSCs and/or Ly5.2 NK cells were injected into sublethally irradiated Ly5.1/2 chimeric mice in a ratio of 105 NK to 103 HSCs per mouse. The addition of IL2-stimulated NK to injected HSCs reduced engraftment from 15.7% to 1.82% when the 16 weeks bone marrow (BM) chimerism was analyzed. In agreement, donor CD45.1 cells contribution to the LSK and HSC subpopulations was reduced in the HSC+NK transplanted mice. To test whether NK depletion from BM grafts would affect HSC function, we performed limiting dilution transplantation assays where whole BM from Ly5.2 mice was submitted to immunonagnetic NK1.1 or IgG depletion and injected into lethally irradiated Ly5.1 animals. Donor chimerism after 8 and 16 weeks of transplant showed that depleting NK cells improves the engraftment ability of HSC in a cell dose-dependent manner. When 25 x104 BM cells were injected, chimerism increased from 40 to more than 90% in NK depleted group. Of note, HSC frequency was 1 in 1595 in the control and 1 in 95 in the NK depleted group. In order to understand the mechanisms by which NK cells could regulate HSCs, we took advantage of a CCAAT/enhancer-binding protein gamma (C/ebpg) knockout (KO) conditional mouse model generated in our laboratory, considering that C/ebpg had been previously shown to regulate NK cytotoxicity. Using similar culture conditions, HSCs and NK cells isolated from control (CT) or Cebpg KO mice were injected into congenic sublethally irradiated recipients. Results showed that Cebpg-deficient NK cells do not harm HSC engraftment as CT NK cells do. For instance, after 8 weeks, the addition of CT non-stimulated and IL-2-stimulated NK cells to normal transplanted HSCs reduced the engraftment from 40% to 20% and 10%, respectively. In contrast, chimerism was not different when HSCs only or HSCs + stimulated KO NK cells were transplanted. Gene expression and cytokine profiles of deficient and normal NK cells revealed the potential players of this HSC-NK regulation. Of these, interferon gamma (IFNg), was lower produced by the C/ebpg deficient NK cells. Therefore, besides controlling NK cytotoxicity, we showed here that C/ebpg also plays a role in the regulation of HSCs by NK-mediated cytokine production. Next, we investigated whether depletion of NK cells from human BM samples would improve transplantation efficiency. NK cells were removed using CD56 antibody and transplanted into sublethally irradiated NSG mice. Sixteen weeks after transplantation, animals were sacrificed and the percentage of human CD45 cells in blood, BM, and spleen demonstrated that NK depletion from human BM favors engraftment. Altogether, these findings provide new insights to the knowledge of HSC regulation by NK cells, which are present in BM transplantation (BMT) grafts. Although the alloreactive effect of NK cells against non-identical tumor cells from BMT recipients is well known, its cytokine-mediated effects over identical progenitor cells from the graft were not previously explored. We show that NK-secreted cytokines harm stem cell function, thus suggesting that depletion of NK cells from BM donor cells preparations can improve stem cell engraftment, particularly in the setting of alternative transplants with limiting cell numbers or non-myeloablative conditioning regimens. Disclosures No relevant conflicts of interest to declare.

Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

2001 ◽  
Vol 114 (4) ◽  
pp. 671-676 ◽  
Author(s):  
E. Gustafsson ◽  
C. Brakebusch ◽  
K. Hietanen ◽  
R. Fassler
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Cre Recombinase ◽  
Embryoid Bodies ◽  
Lymphoid Cells ◽  
Adult Brain ◽  
Specific Gene

Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth, LacZ activity was detected in most endothelial cells within the embryo and of extra-embryonic tissues such as yolk sac and chorioallantoic placenta. Ectopic expression of Cre was observed in approximately 12–20% of the adult erythroid, myeloid and lymphoid cells and in subregions of the adult brain. These results show that the tie-1-Cre transgenic strain can efficiently direct deletion of floxed genes in endothelial cells in vivo.

scholarly journals Identification of genes involved in VEGF-mediated vascular morphogenesis using embryonic stem cell-derived cystic embryoid bodies

2004 ◽  
Vol 84 (9) ◽  
pp. 1209-1218 ◽  
Author(s):  
Yin-Shan Ng ◽  
Markus Ramsauer ◽  
Robyn M B Loureiro ◽  
Patricia A D'Amore
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Vascular Morphogenesis

Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 1935-1947 ◽  
Author(s):  
Sherif S. Farag ◽  
Todd A. Fehniger ◽  
Loredana Ruggeri ◽  
Andrea Velardi ◽  
Michael A. Caligiuri
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Killer Cell ◽  
Natural Cytotoxicity ◽  
Receptor Ligand ◽  
Cell Receptors ◽  
Nk Cell Receptors ◽  
Ligand Interactions ◽  
Nk Receptor

AbstractNatural killer (NK) cells have held great promise for the immunotherapy of cancer for more than 3 decades. However, to date only modest clinical success has been achieved manipulating the NK cell compartment in patients with malignant disease. Progress in the field of NK cell receptors has revolutionized our concept of how NK cells selectively recognize and lyse tumor and virally infected cells while sparing normal cells. Major families of cell surface receptors that inhibit and activate NK cells to lyse target cells have been characterized, including killer cell immunoglobulinlike receptors (KIRs), C-type lectins, and natural cytotoxicity receptors (NCRs). Further, identification of NK receptor ligands and their expression on normal and transformed cells completes the information needed to begin development of rational clinical approaches to manipulating receptor/ligand interactions for clinical benefit. Indeed, clinical data suggest that mismatch of NK receptors and ligands during allogeneic bone marrow transplantation may be used to prevent leukemia relapse. Here, we review how NK cell receptors control natural cytotoxicity and novel approaches to manipulating NK receptor-ligand interactions for the potential benefit of patients with cancer.

scholarly journals Isolation, Characterization and Differentiation Potential of Chicken Spermatogonial Stem Cell Derived Embryoid Bodies

2016 ◽  
Vol 16 (1) ◽  
pp. 115-128 ◽  
Author(s):  
Thanh Luan Nguyen ◽  
Jae Gyu Yoo ◽  
Neelesh Sharma ◽  
Sung Woo Kim ◽  
Yong Jun Kang ◽  
...  
Keyword(s):  
Developmental Biology ◽  
Regenerative Medicine ◽  
Connexin 43 ◽  
Es Cells ◽  
Periodic Acid ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Significant Finding ◽  
Differentiation Potential

Abstract Human, murine and monkey spermatogonial stem cells (SSCs) have the capability to undergo self-renewal and differentiation into different body cell types in vitro, which are expected to serve as a powerful tool and resource for the developmental biology and regenerative medicine. We have successfully isolated and characterized the chicken SSCs from 3-day-old chicken testicular cells. The pluripotency was using Periodic Acid-Schiff (PAS ) staining or alkaline phosphatase staining, and antibodies to stage-specific embryonic antigens. In suspension culture conditions SSCs formed embryoid bodies (EBs) like embryonic stem (ES) cells. Subsequently EB differentiated into osteoblasts, adipocytes and most importantly into cardiomyocytes under induced differentiation conditions. The differentiation potential of EBs into cardiomyocyte-like cells was confirmed by using antibodies against sarcomeric α-actinin, cardiac troponin T and connexin 43. Cardiomyocytes-like cells were also confirmed by RT-PCR analysis for several cardiac cell genes like GATA-4, Nkx2-5, α-MHC, and ANF. We have successfully established an in vitro differentiation system for chicken SSCs into different body cells such as osteoblasts, adipocytes and cardiomyocytes. The most significant finding of this study is the differentiation potential of chicken SSCs into cardiomyocytes. Our findings may have implication in developmental biology and regenerative medicine by using chicken as the most potential animal model.

RESEARCH FINDINGS

2011 ◽  
Vol 15 (12) ◽  
pp. 43-44
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Kawasaki Disease ◽  
Regenerative Medicine ◽  
Embryonic Stem Cell ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Medicine Research ◽  
Research Findings ◽  
Cell Study

Singapore Scientists Lead Human Embryonic Stem Cell Study to Advance Regenerative Medicine Research. Singapore Scientists Discover Genetic Link in Kawasaki Disease. Stem Cells Engineered to Kill Cancer.

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