scholarly journals Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

2008 ◽  
Vol 180 (5) ◽  
pp. 897-904 ◽  
Author(s):  
Enko N. Kiprilov ◽  
Aashir Awan ◽  
Romain Desprat ◽  
Michelle Velho ◽  
Christian A. Clement ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Embryonic Stem ◽  
Serum Starvation ◽  
Mechanistic Basis ◽  
Patched 1 ◽  
Hesc Lines

Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC differentiation, demonstrating the existence of primary cilia and the localization of signaling components in undifferentiated hESCs establishes a mechanistic basis for the regulation of hESC differentiation. Using electron microscopy (EM), immunofluorescence, and confocal microscopies, we show that primary cilia are present in three undifferentiated hESC lines. EM reveals the characteristic 9 + 0 axoneme. The number and length of cilia increase after serum starvation. Important components of the hedgehog (Hh) pathway, including smoothened, patched 1 (Ptc1), and Gli1 and 2, are present in the cilia. Stimulation of the pathway results in the concerted movement of Ptc1 out of, and smoothened into, the primary cilium as well as up-regulation of GLI1 and PTC1. These findings show that hESCs contain primary cilia associated with working Hh machinery.

scholarly journals Expression profiles of protein tyrosine kinase genes in human embryonic stem cells

Reproduction ◽  
2008 ◽  
Vol 136 (4) ◽  
pp. 423-432 ◽  
Author(s):  
Mi-Young Son ◽  
Janghwan Kim ◽  
Hyo-Won Han ◽  
Sun-Mi Woo ◽  
Yee Sook Cho ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Signaling Pathways ◽  
Human Embryonic Stem Cells ◽  
Tyrosine Kinases ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Degenerate Primers ◽  
Protein Tyrosine ◽  
Hesc Lines

Complex signaling pathways operate in human embryonic stem cells (hESCs) and are coordinated to maintain self-renewal and stem cell characteristics in them. Protein tyrosine kinases (PTKs) participate in diverse signaling pathways in various types of cells. Because of their functions as key molecules in various cellular processes, PTKs are anticipated to have important roles also in hESCs. In this study, we investigated the roles of PTKs in undifferentiated and differentiated hESCs. To establish comprehensive PTK expression profiles in hESCs, we performed reverse transcriptase PCR using degenerate primers according to the conserved catalytic PTK motifs in both undifferentiated and differentiated hESCs. Here, we identified 42 different kinases in two hESC lines, including 5 non-receptor tyrosine kinases (RTKs), 24 RTKs, and 13 dual and other kinases, and compared the protein kinase expression profiles of hESCs and retinoic acid-treated hESCs. Significantly, up- and downregulated kinases in undifferentiated hESCs were confirmed by real-time PCR and western blotting. MAP3K3, ERBB2, FGFR4, and EPHB2 were predominantly upregulated, while CSF1R, TYRO3, SRC, and GSK3A were consistently downregulated in two hESC lines. Western blot analysis showed that the transcriptional levels of these kinases were consistent with the translational levels. The obstruction of upregulated kinases’ activities using specific inhibitors disturbed the undifferentiated status and induced the differentiation of hESCs. Our results support the dynamic expression of PTKs during hESC maintenance and suggest that specific PTKs that are consistently up- and downregulated play important roles in the maintenance of stemness and the direction of differentiation of hESCs.

Step-Wise Differentiation of CD34+ Cell Derived from Nuclear Transfer-Human Embryonic Stem Cells into Myeloid and Lymphoid Precursors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3614-3614
Author(s):  
Gabsang Lee ◽  
Jae-hung Sheih ◽  
Woosuk Hwang ◽  
Malcolm A.S. Moore
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Cell Marker ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Hesc Lines

Abstract Human embryonic stem cells derived after nuclear transfer (NT-hESCs) may be used to study the earliest events in human hematopoietic stem cell development. They may also be potential candidates for cell therapy in degenerative and genetic diseases because of immunological compatibility, important for eventual transplantation. We developed an efficient, step-wise system for differentiating hESC to CD34+ hematopoietic stem cells from embryoid bodies (EB) in liquid cultures and, subsequently, further differentiating these into myeloid and lymphoid precursors using different combinations of cytokines and stromal cells. The H1 and NT-hESC-1 lines were maintained on mouse embryonic fibroblasts and NT-hESC-2 was cultured on autologous human fibroblasts with basic fibroblast growth factor in serum-replacement containing medium. In all three lines initiation of hematopoiesis begins during the first week of EB differentiation in the presence of different combinations of hematopoietic growth factors and in the absence of stromal support. Gene expression analysis, using RT-PCR methods, revealed that the first wave of embryonic hematopoiesis at 4–8 days of EB development was associated with expression of SCL/TAL1, flt3, KDR and GATA2. In FACS analysis, CD34+, KDR+, CD31+ cells were firstly detected in 6–10 days and CD34+, KDR+, CD45+, CD14+ (monocyte/macrophage marker), CD33+ (early myeloid cell marker), CD56+ (natural killer cell marker), Glycoporin A+ (erythroid cell marker), CD86 + (dendritic cell marker) cells were observed up to 15–20 days of EB development. CD34+ cell derived from 10–12 day EB’s from all three hESC lines, formed multiple lineage colonies in methylcellulose culture. These CD34+ cells can be maintained on OP9- stroma transduced with an adenovector expressing thrombopoietin (Tpo). We developed selective culture conditions for differentiation of CD34+ cells into different lineages. Isolated CD34+ cells from 10 to 12 Day EB’s differentiated in CD86+ dendritic cells in the presence of SCF, Tpo, Flt3 ligand, G-CSF, and TNF-alpha. Mature multinucleated CD41a+ megakaryocytes developed in cultures with SCF, Flt3L, and Tpo. Glycophorin A+ erythroid differentiation through to enucleated red cell stage was obtained with SCF, Epo, and IGF-1. B cell precursors (CD19+) developed in MS-5 stromal co-culture with SCF and G-SCF while CD44+CD25+ (early T progenitor marker) cells were detected in co-culture with OP9 transduced with the Notch ligand delta like-1 and IL-7. We have developed GFP and luciferase-expressing H1 and NT-hES-2 lines by lentiviral-vector-mediated gene transfer. These lines will allow us to monitor ES-derived cell localization and quantitation in immunodeficient mice using whole animal luciferase imaging systems. We are examining the potential for GFP-luciferase-transduced NT-hES-derived hematopoietic cells to engraft in NOD/SCID beta2−/ − mice. Our data demonstrate that NT-hESC lines as well as H1 lines are capable of differentiation to various lymphoid and hematopoietic lineages.

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.

Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells

2008 ◽  
Vol 295 (2) ◽  
pp. G313-G321 ◽  
Author(s):  
Takamichi Ishii ◽  
Ken Fukumitsu ◽  
Kentaro Yasuchika ◽  
Keiko Adachi ◽  
Eihachiro Kawase ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factors ◽  
Embryonic Stem Cells ◽  
Growth Factor ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Activin A ◽  
Immunocytochemical Staining ◽  
Hepatic Differentiation ◽  
Hesc Lines

Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human α-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of growth factors including hepatocyte growth factor (HGF), bone morphogenetic protein 4, fibroblast growth factor 4, all- trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and 20 ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.

scholarly journals Role of Sonic hedgehog signaling and the expression of its components in human embryonic stem cells

2010 ◽  
Vol 4 (1) ◽  
pp. 38-49 ◽  
Author(s):  
Selena Meiyun Wu ◽  
Andre B.H. Choo ◽  
Miranda G.S. Yap ◽  
Ken Kwok-Keung Chan
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Sonic Hedgehog ◽  
Human Embryonic Stem Cells ◽  
Hedgehog Signaling ◽  
Embryonic Stem ◽  
Sonic Hedgehog Signaling

scholarly journals Identification and characterization of small-molecule ligands that maintain pluripotency of human embryonic stem cells

2010 ◽  
Vol 38 (4) ◽  
pp. 1058-1061 ◽  
Author(s):  
Peter Burton ◽  
David R. Adams ◽  
Achamma Abraham ◽  
Robert W. Allcock ◽  
Zhong Jiang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Large Scale ◽  
Pharmaceutical Research ◽  
Embryonic Stem ◽  
Good Manufacturing Practice ◽  
Exogenous Cytokine ◽  
Small Molecule Ligands ◽  
Hesc Lines

hESCs (human embryonic stem cells) offer great potential for pharmaceutical research and development and, potentially, for therapeutic use. However, improvements in cell culture are urgently required to allow the scalable production of large numbers of cells that maintain pluripotency. Supplementing feeder-free conditions with either EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine] or readily synthesized analogues of this compound maintains hESC pluripotency in the absence of exogenous cytokines. When the hESC lines SA121 or SA461 were maintained in feeder-free conditions with EHNA they displayed no reduction in stem-cell-associated markers such as Nanog, Oct4 (octamer-binding protein 4) and SSEA4 (stage-specific embryonic antigen 4) when compared with cells maintained in full feeder-free conditions that included exogenously added bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but EHNA did not limit efficient spontaneous or directed differentiation following its removal. We conclude that EHNA or related compounds offers a viable alternative to exogenous cytokine addition in maintaining hESC cultures in a pluripotent state and might be a particularly useful replacement for bFGF for large-scale or GMP (good manufacturing practice)-compliant processes.

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