generation-integration-free-human-induced-pluripotent-stem-cells

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

University of Melbourne

Gap junctions are intercellular channels that allow both chemical and electrical signaling between two adjacent cells. Gap junction intercellular communication has been implicated in the regulation of various cellular processes, including cell migration, cell proliferation, cell differentiation, and cell apoptosis. This study aimed to determine the presence and functionality of gap junctions in human embryonic stem cells (hESCs). Using reverse transcription--polymerase chain reaction and immunocytochemistry, we demonstrate that human ES cells express two gap junction proteins, connexin 43 and connexin 45. Western blot analysis revealed the presence of three phosphorylated forms (nonphosphorylated [NP], P1, and P2) of connexin 43, NP being prominent. Moreover, scrape loading/dye transfer assay indicates that human ES cells are coupled through functional gap junctions that are inhibited by protein kinase C activation and extracellular signal-regulated kinase inhibition.

Presence of functional gap junctions in human embryonic stem cells.

Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum--sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF)--in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S1P and PDGF in a serum-free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri-potentiality.

Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells.

We investigated the gap junctional properties of human embryonic stem cells (hESC) cultivated in a serum-free system using sphingosine-1-phosphate and platelet-derived growth factor (S1P/PDGF). We compared this condition to hESC grown on Matrigel in mouse embryonic fibroblast conditioned medium (MEF-CM) or unconditioned medium (UM). We show that in all culture systems, hESC express connexins 43 and 45. hESC maintained in S1P/PDGF conditions and hESC grown in presence of MEF-CM are coupled through gap junctions while hESC maintained on Matrigel in UM do not exhibit gap junctional intercellular communication. In this latter condition, coupling was retrieved by addition of noggin, suggesting that BMP-like activity in UM inhibits gap junctional communication. Last, our data indicate that the closure of gap junctions by the decoupling agent alpha-glycyrrhetinic acid increases cell apoptosis and inhibits hESC colony growth. Altogether, these results suggest that gap junctions play an important role in hESC maintenance.

Gap junctions modulate apoptosis and colony growth of human embryonic stem cells maintained in a serum-free system.

Human embryonic stem (hES) cells hold great promise for use in regenerative medicine. However, technologies first need to be established to maintain hES cells efficiently in vitro. Understanding the signaling networks involved in hES cell maintenance will prove to be essential to the development of such culture systems. Previously, we described a serum-free medium capable of supporting prolonged hES cell maintenance using sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF). Here, we describe an anti-apoptotic effect of S1P and PDGF in hES cells and demonstrate a direct effect of S1P in preventing hES cell apoptosis. Western blot analysis shows that S1P stimulates the phosphorylation of the mitogen-activated protein (MAP) kinases Erk1/2 but not of Akt, whereas PDGF stimulates both Erk1/2 and Akt phosphorylation. Moreover, our study suggests that the Erk1/2 and PI3K/Akt signaling pathways act independently of each other. Furthermore, neither S1P nor PDGF modify intracellular calcium concentration ([Ca(2+)]( i )) and Smad2 phosphorylation. Using pharmacological inhibitors of Erk1/2 and PI3K, our results demonstrate a critical role of the Erk1/2 and PI3K/Akt signaling pathways in mediating the anti-apoptotic effect of S1P and PDGF on hES cells. However, inhibition of the mammalian target of rapamycin (mTOR), a common downstream effector of Erk1/2 and PI3K/Akt, has no effect on hES cell apoptosis.

Anti-apoptotic effect of sphingosine-1-phosphate and platelet-derived growth factor in human embryonic stem cells.

Stem cells provide an invaluable tool to develop cell replacement therapies for a range of serious disorders caused by cell damage or degeneration. Much research in the field is focused on the identification of signals that either maintain stem cell pluripotency or direct their differentiation. Understanding how stem cells communicate within their microenvironment is essential to achieve their therapeutic potentials. Gap junctional intercellular communication (GJIC) has been described in embryonic stem cells (ES cells) and various somatic stem cells. GJIC has been implicated in regulating different biological events in many stem cells, including cell proliferation, differentiation and apoptosis. This review summarizes the current understanding of gap junctions in both embryonic and somatic stem cells, as well as their potential role in growth control and cellular differentiation.

Role of gap junctions in embryonic and somatic stem cells.

Gap junctional intercellular communication (GJIC) has been described in different cell types including stem cells and has been involved in different biological events. GJIC is required for mouse embryonic stem cell maintenance and proliferation, and various studies suggest that functional GJIC is a common characteristic of human embryonic stem cells (hESC) maintained in different culture conditions. This chapter introduces methods to study gap junctions in hESC, from expression of gap junction proteins to functional study of GJIC in hESC proliferation, apoptosis, colony growth, and pluripotency.

Study of gap junctions in human embryonic stem cells.

Endothelin (ET) family comprises three isoforms, ET-1, ET-2 and ET-3 that bind to two receptors ET-A and ET-B. Upon hESC differentiation, ET-1 and ET-B are respectively up- and down-regulated, suggesting a potential role of ETs in hESC biology. Here we show expression of ET receptors in hESC and demonstrate that ET-1 and ET-2 inhibit gap junctional intercellular communication (GJIC), while ET-3 does not. Pre-incubation of the cell cultures with the two specific antagonists of ET-A and ET-B, BQ123 and BQ788 respectively, demonstrate that inhibition of GJIC by ETs is mediated by ET-A. Long-term treatment of hESC with ET-1 indicates no visible effect on hESC maintenance of pluripotency markers, as assessed by expression of the hESC markers Oct-4, GCTM-2 and TG-30. Altogether these data show that hESC are target cells of ETs.

Acute effect of endothelins on intercellular communication of human embryonic stem cells.

Embryonic stem cells are pluripotent and capable of indefinite self-renewal in vitro. Human embryonic stem cells (hESC) have generally been cultivated on feeder layers of primary mouse embryonic fibroblasts (MEF) in media supplemented with fetal calf serum (FCS). However, serum contains a wide variety of biologically active compounds that might adversely affect hESC growth and differentiation. Thus, cultivation of stem cells in FCS complicates experimental approaches to define the intracellular mechanisms required for hESC maintenance. This chapter describes the serum-free maintenance of hESC in culture by addition of sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF). This complete protocol provides a chemically defined serum-free system that is advantageous for studying signaling pathways involved in hESC pluripotency.

Maintenance of human embryonic stem cells by sphingosine-1-phosphate and platelet-derived growth factor.

Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1-Eif1al10) and 9 pseudogenes (Eif1al-ps1-Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state.

Repression of global protein synthesis by Eif1a-like genes that are expressed specifically in the two-cell embryos and the transient Zscan4-positive state of embryonic stem cells.

Gap junctional intercellular communication (GJIC) has been described in different cell types including stem cells and has been involved in different biological events. GJIC is required for mouse embryonic stem cell maintenance and proliferation and various studies suggest that functional GJIC is a common characteristic of human embryonic stem cells (hESC) maintained in different culture conditions. This chapter introduces methods to study gap junctions in hESC, from expression of gap junction proteins to functional study of GJIC in hESC proliferation, apoptosis, colony growth, and pluripotency.

Study of Gap Junctions in Human Embryonic Stem Cells.

Glaucoma, the worldwide leading cause of irreversible blindness, is characterized by progressive degeneration of the optic nerve and loss of retinal ganglion cells. Research into glaucoma pathogenesis has been hampered by difficulties in isolating and culturing retinal ganglion cells in vitro. However, recent improvements in laboratory techniques have enabled the generation of a variety of mature cell types from pluripotent stem cells, including retinal ganglion cells. Indeed, stem cell-based approaches have the potential to revolutionize the field by providing an unlimited source of cells for replacement therapies and by enabling development of in vitro disease models for drug screening and research. Consequently, research aimed at directing pluripotent stem cells to differentiate into retinal ganglion cells has expanded dramatically during the past decade, resulting in significant advances in technique and efficiency. In this paper, we review the methodology for retinal ganglion cell differentiation from pluripotent stem cells of both mouse and human origin and summarize how these techniques have opened up new avenues for modelling glaucoma. Generation of stem cell-derived retinal ganglion cells will have significant translational values, providing an in vitro platform to study the mechanisms responsible for pathogenesis and for drug screening to improve treatment options, as well as for the development of cell therapies for optic neuropathies such as glaucoma.

Raymond C.B. Wong

Centre for Eye Research Australia & Department of Ophthalmology

University of Melbourne

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes