Regulation of human embryonic stem cell fate by different forms of the Polycomb transcriptional silencing machinery.

Funding Type: 
SEED Grant
Grant Number: 
RS1-00200
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
We propose to investigate the mechanisms that maintain embryonic stem cells in the undifferentiated, pluripotent state and how those mechanisms change to allow cells to initiate the first steps of embryonic development. Work in mouse embryonic stem cells suggests that genes that will be turned on during early embryonic development are primed for expression but kept silenced by a complex of regulatory proteins termed the Polycomb group machinery (PcG). We will investigate whether different forms of the PcG machinery components are expressed in human embryonic stem cells compared to differentiating early embryonic cells and whether different forms of the PcG complex bind to and silence different target genes. Our proposed work will uncover fundamental mechanisms that allow human embryonic stem cells to grow and proliferate in the undifferentiated state while maintaining their pluripotent character. These properties are critical for the ability to expand embryonic stem cell populations in order to be able to use the cells for screens for potential therapeutic treatments (if derived from disease carrying individuals). Maintenance of the pluripotent state is also critical for expanding stem cell populations for use in possible cell based transplantation therapies. In addition, our work may reveal mechanisms that help guide early embryonic cells toward particular differentiation pathways, such as neuronal or endoderm or muscle precursors, during the early stages of development. Knowledge of these mechanisms will help in the design of strategies for inducing human embryonic stem cells to initiate differentiation into specific cell types, a key step for cell based regenerative medicine.
Statement of Benefit to California: 
The proposed research will lay important groundwork for the ability to grow and guide the differentiation of human embryonic stem cells. These capabilities underlie all ability to use embryonic stem cells for screens for therapies and for embryonic stem cell based regenerative medicine. The proposed experiments may uncover fundamental mechanisms that allow human embryonic stem cells to grow and proliferate in the undifferentiated state while maintaining their pluripotent character. Maintenance of the pluripotent state is critical for expanding stem cell populations for use in possible cell based transplantation therapies. These properties are also critical for the ability to expand embryonic stem cell populations in order to be able to use the cells for screens for potential therapeutic treatments. In addition, the proposed research may reveal mechanisms that help guide early embryonic cells toward particular differentiation pathways, such as neuronal or endoderm (precursor to pancreas and liver) or muscle precursors, during the early stages of development. Knowledge of these mechanisms will help in the design of strategies for inducing human embryonic stem cells to initiate differentiation into specific cell types, a key step for cell based regenerative medicine.
Progress Report: 
  • Complex sugar chains decorate the cell surface. The cell makes and organizes these complicated molecules on their surface, for reasons best known to the cells themselves. We want to understand their function of sugar chains (glycans).
  • We know they help cells communicate about their past and future journeys within the developing body, exactly where they go and what they become during development. Human genetic disorders where just one step in their assembly is missing, and mental retardation, seizures, blindness and poor motor skills often result.
  • Embryonic stem cells have a particular set of glycans on their surface and it changes as the cells develop into different cell types. We have two major goals in this project. First, to identify the human embryonic stem (hES) cell glyco-signatures and then determine what changes as they differentiate into neural precursor cells (NPC). Second, to ask whether these glycan changes are important for correct timing and normal differentiation of the cells. What happens if we change or keep the original sugar coating? Does it change the cell’s fate?
  • We can produce substantial amounts of hES cells that uniformly transition into NPC. Because these are early days of understanding stem cells, it was important to analyze different hES cell lines that differentiate into NPC under two different conditions. Glycan analysis can be done indirectly using sugar-binding proteins called lectins, or directly by mass spectrometry of glycans released from proteins or lipids. Another way to infer an alteration in glycan composition is by changes in the expression of genes encoding enzymes that make glycan chains. This is called transcriptional profiling. If more glycan is needed, more enzyme may be needed to produce that glycan; less glycan involves reduced expression of those genes. Sometimes more than one enzyme (gene product) carries out the very synthetic same step, but one of the enzymes may prefer a certain kind of glycan over another.
  • We used both approaches (lectin binding and transcriptional profiling) in parallel. Lectins bind to various types of N- and O-linked glycans and we used an instrument that detects small changes in the binding of cell fragments to a battery of 40 lectins. Carefully worked out and standardized conditions showed that only a few lectins consistently increased binding during transitions from hES cells into NPC. These lectins recognized some changes, but were in the 2-3 fold range at most. Comparison of hES cells grown on mouse feeder-layers to those grown without feeders, showed mostly similar (not identical) patterns. The changes were subtle, so instead of continuing glycan analysis by mass spectrometry, we chose to analyze transcriptional profiles. Our goal was to identify genes whose expression changed quite substantially.
  • Initially, we used results obtained by Dr. Terskikh to profile the entire genome when hES cells developed into NPC. Here, subtle changes in glycosylation gene expression (2-3 fold increases/decreases) were near baseline making it hard to know if they were accurate. An exception was the chondroitin sulfate core protein, decorin, which increased >30-fold during differentiation. Since many glycosylation-related genes are expressed at low levels, we went to colleagues at the University of Georgia for analysis of >700 genes using Real Time PCR. Their 5 order of magnitude dynamic range and reproducibility of triplicate samples (+/-15-20%) is impressive.
  • These results showed no (<2-fold) changes in sugar precursor metabolism, N-linked glycan precursor synthesis, and only a decrease in tetra-branched chains N-glycans. Considerable increases (5->100 fold) in terminal modifications common to both N- and O-linked glycans (Sda, polysialic acids, Lewisa) in the different hES cells prepared by either method. Fringe genes encoding glycans that modify Notch signaling (LFNG, especially) increased. GPI anchor biosynthesis gene that adds a final mannose (PIGZ) is decreased as is palmitoylation of the anchors. Fine differences in HS-6 sulfation changed and one especially prominent gene that appears only in neural tissue (NDST4) showed a substantial increase.
  • These transcriptional changes are more wide-ranging and different than what we could see using lectin profiles or direct analysis of N- and O-glycans. The two approaches do not necessarily give congruent results. These transcriptional changes allow us to focus on genes more likely to be affected by the knockdown strategies we planned for aim two. In that aim, we want to prevent the up-regulation of those specific genes to see if it affects differentiation. If preventing transcriptional increase (by siRNA) does have developmental consequences then we will be in a better position to analyze which specific glycans are important. Our top knockdown candidates are: Decorin, Sd antigen (B4GALNT2), polysialic acid (STaSia1, 2, 3), NDST4 and Lewis antigen (FT3). We hope to cover these in the no-cost extension.
  • Complex sugar chains decorate the cell surface. The cell makes and organizes these complicated molecules on their surface, for reasons best known to the cells themselves. We want to understand their function of sugar chains (glycans).
  • We know they help cells communicate about their past and future journeys within the developing body, exactly where they go and what they become during development. Human genetic disorders where just one step in their assembly is missing, and mental retardation, seizures, blindness and poor motor skills often result.
  • Embryonic stem cells have a particular set of glycans on their surface and it changes as the cells develop into different cell types. We have two major goals in this project. First, to identify the human embryonic stem (hES) cell glyco-signatures and then determine what changes as they differentiate into neural precursor cells (NPC). Second, to ask whether these glycan changes are important for correct timing and normal differentiation of the cells. What happens if we change or keep the original sugar coating? Does it change the cell’s fate?
  • We can produce substantial amounts of hES cells that uniformly transition into NPC. Because these are early days of understanding stem cells, it was important to analyze different hES cell lines that differentiate into NPC under two different conditions. Glycan analysis can be done indirectly using sugar-binding proteins called lectins, or directly by mass spectrometry of glycans released from proteins or lipids. Another way to infer an alteration in glycan composition is by changes in the expression of genes encoding enzymes that make glycan chains. This is called transcriptional profiling. If more glycan is needed, more enzyme may be needed to produce that glycan; less glycan involves reduced expression of those genes. Sometimes more than one enzyme (gene product) carries out the very synthetic same step, but one of the enzymes may prefer a certain kind of glycan over another.
  • We used both approaches (lectin binding and transcriptional profiling) in parallel. Lectins bind to various types of N- and O-linked glycans and we used an instrument that detects small changes in the binding of cell fragments to a battery of 40 lectins. Carefully worked out and standardized conditions showed that only a few lectins consistently increased binding during transitions from hES cells into NPC. These lectins recognized some changes, but were in the 2-3 fold range at most. Comparison of hES cells grown on mouse feeder-layers to those grown without feeders, showed mostly similar (not identical) patterns. The changes were subtle, so instead of continuing glycan analysis by mass spectrometry, we chose to analyze transcriptional profiles. Our goal was to identify genes whose expression changed quite substantially.
  • Initially, we used results obtained by Dr. Terskikh to profile the entire genome when hES cells developed into NPC. Here, subtle changes in glycosylation gene expression (2-3 fold increases/decreases) were near baseline making it hard to know if they were accurate. An exception was the chondroitin sulfate core protein, decorin, which increased >30-fold during differentiation. Since many glycosylation-related genes are expressed at low levels, we went to colleagues at the University of Georgia for analysis of >700 genes using Real Time PCR. Their 5 order of magnitude dynamic range and reproducibility of triplicate samples (+/-15-20%) is impressive.
  • These results showed no (<2-fold) changes in sugar precursor metabolism, N-linked glycan precursor synthesis, and only a decrease in tetra-branched chains N-glycans. Considerable increases (5→100 fold) in terminal modifications common to both N- and O-linked glycans (Sda, polysialic acids, Lewisa) in the different hES cells prepared by either method. Fringe genes encoding glycans that modify Notch signaling (LFNG, especially) increased. GPI anchor biosynthesis gene that adds a final mannose (PIGZ) is decreased as is palmitoylation of the anchors. Fine differences in HS-6 sulfation changed and one especially prominent gene that appears only in neural tissue (NDST4) showed a substantial increase.
  • These transcriptional changes are more wide-ranging and different than what we could see using lectin profiles or direct analysis of N- and O-glycans. The two approaches do not necessarily give congruent results. These transcriptional changes allow us to focus on genes more likely to be affected by the knockdown strategies we planned for aim two. In that aim, we want to prevent the up-regulation of those specific genes to see if it affects differentiation. If preventing transcriptional increase (by siRNA) does have developmental consequences then we will be in a better position to analyze which specific glycans are important. Our top knockdown candidates are: Decorin, Sd antigen (B4GALNT2), polysialic acid (STaSia1, 2, 3), NDST4 and Lewis antigen (FT3).

© 2013 California Institute for Regenerative Medicine