REST/NRSF Knockdown Alters Survival, Lineage Differentiation and Signaling in Human Embryonic Stem Cells.

REST (RE1 silencing transcription factor), also known as NRSF (neuron-restrictive silencer factor), is a well-known transcriptional repressor of neural genes in non-neural tissues and stem cells. Dysregulation of REST activity is thought to play a role in diverse diseases including epilepsy, cancer, Down's syndrome and Huntington's disease. The role of REST/NRSF in control of human embryonic stem cell (hESC) fate has never been examined. To evaluate the role of REST in hESCs we developed an inducible REST knockdown system and examined both growth and differentiation over short and long term culture. Interestingly, we have found that altering REST levels in multiple hESC lines does not result in loss of self-renewal but instead leads to increased survival. During differentiation, REST knockdown resulted in increased MAPK/ERK and WNT signaling and increased expression of mesendoderm differentiation markers. Therefore we have uncovered a new role for REST in regulation of growth and early differentiation decisions in human embryonic stem cells

REST/NRSF regulates genetic stability and cell fate in human embryonic stem cells

REST (RE1 silencing transcription factor), also known as NRSF (neuron-restrictive silencer factor), is well-known as a transcriptional repressor of neural genes in non-neural tissues. Dysregulation of REST activity is thought to play a role in diverse diseases including cardiac hypertrophy, Down Syndrome, Huntington's disease and cancer. Previous studies examining the pluripotency transcriptional network in mouse as well as human embryonic stem cells (hESCs) have revealed that REST is regulated by the pluripotency factors OCT4, NANOG and SOX2. The goal of the present study was to evaluate the role of REST in hESCs. An inducible REST knockdown system was used to examine growth and differentiation over short and long-term culture. Interestingly, altering REST levels in multiple hESC lines did not result in loss of self-renewal, but instead led to aneuploidy. During differentiation, reduced REST levels led to altered MAPK/ERK and WNT signaling, as well as upregulation of endoderm and mesoderm markers. Critical hurdles for the translation of the clinical potential of hESCs into practice are their tumorigenic capacity, and the inefficiency in tailoring lineage differentiation. Elucidating the role of REST in regulating cell fate and genetic stability of hESCs could enable development of robust methods to stably culture and tailor lineage differentiation of these cells for use in regenerative medicine applications

REST/NRSF regulates genetic stability and cell fate in human embryonic stem cells

REST (RE1 silencing transcription factor), also known as NRSF (neuron-restrictive silencer factor), is well-known as a transcriptional repressor of neural genes in non-neural tissues. Dysregulation of REST activity is thought to play a role in diverse diseases including cardiac hypertrophy, Down Syndrome, Huntington's disease and cancer. Previous studies examining the pluripotency transcriptional network in mouse as well as human embryonic stem cells (hESCs) have revealed that REST is regulated by the pluripotency factors OCT4, NANOG and SOX2. The goal of the present study was to evaluate the role of REST in hESCs. An inducible REST knockdown system was used to examine growth and differentiation over short and long-term culture. Interestingly, altering REST levels in multiple hESC lines did not result in loss of self-renewal, but instead led to aneuploidy. During differentiation, reduced REST levels led to altered MAPK/ERK and WNT signaling, as well as upregulation of endoderm and mesoderm markers. Critical hurdles for the translation of the clinical potential of hESCs into practice are their tumorigenic capacity, and the inefficiency in tailoring lineage differentiation. Elucidating the role of REST in regulating cell fate and genetic stability of hESCs could enable development of robust methods to stably culture and tailor lineage differentiation of these cells for use in regenerative medicine applications

REST/NRSF Knockdown Alters Survival, Lineage Differentiation and Signaling in Human Embryonic Stem Cells.

REST (RE1 silencing transcription factor), also known as NRSF (neuron-restrictive silencer factor), is a well-known transcriptional repressor of neural genes in non-neural tissues and stem cells. Dysregulation of REST activity is thought to play a role in diverse diseases including epilepsy, cancer, Down's syndrome and Huntington's disease. The role of REST/NRSF in control of human embryonic stem cell (hESC) fate has never been examined. To evaluate the role of REST in hESCs we developed an inducible REST knockdown system and examined both growth and differentiation over short and long term culture. Interestingly, we have found that altering REST levels in multiple hESC lines does not result in loss of self-renewal but instead leads to increased survival. During differentiation, REST knockdown resulted in increased MAPK/ERK and WNT signaling and increased expression of mesendoderm differentiation markers. Therefore we have uncovered a new role for REST in regulation of growth and early differentiation decisions in human embryonic stem cells

REST KD results in altered signaling including an increase in MEK and WNT activity in embryoid bodies (EBs).

<p><b>A</b>. Western blot showing that REST KD H9 hESCs have increased pMEK1/2 (S217/221) expression compared to control NT H9 hESCs. MEK1/2 and β-ACTIN were used as loading controls. <b>B</b>. QPCR showing that REST KD H9 hESCs have increased expression of CFOS, a down-stream target of pMEK1/2. CFOS expression was examined in cells collected under four conditions: 24 hours after last feeding with media containing knockout serum (hESC); 24 hours after last feeding with media devoid of knockout serum (Starve); 30 minutes after switching from ‘Starve’ condition to knockout serum media; 4 hours after switching from ‘Starve’ condition to knockout serum media. REST KD hESCs had increased CFOS expression under all four conditions. <b>C</b>. Western Blot Analysis of Day 5 EBs for changes in MEK signaling revealed that pMEK 1/2 was increased in REST KD EBs. MEK ½ and GAPDH were used as loading controls. <b>D</b>. In order to evaluate WNT signaling, we evaluated expression of the WNT target genes AXIN2, Beta-CATENIN (B-CAT), FRIZZLED2 (FZD2) AND TROY in NT and REST KD hESCs as well as EBs, in H9 and H1 lines, via qPCR. Significant changes in expression of WNT target genes in REST KD hESCs are shown with a single asterisk (*). For both H1 and H9 REST KD hESCs, FZD2 was statistically significantly decreased (p<0.05). Significant changes in expression of WNT target genes in REST KD Day 5 EBs are shown with two asterisks (**). Across both lines of REST KD Day 5 EBs, we found a statistically significant increase in expression of WNT target genes AXIN2, B-CAT and TROY (p<0.022). Error bars represent SEM of three independent experiments and asterisks denote a p value < .05 using Student’s t-test analysis.</p

REST KD hESCs have increased survival.

<p><b>A</b>. REST KD and NT hESCs (H1 and H9) were evaluated for BrdU incorporation by FACS analysis for TRA-1-81 and BrdU double positive cells. <b>B</b>. Percentage of TRA-1-81and BrdU positive cells were not statistically significantly different in REST KD compared to control NT cells. Error bars represent standard error of three independent experiments (performed at p52, p54 and p60 for H9 cells, and at p47, p49, and p50 for H1 cells). <b>C</b>. REST KD and NT hESCs (H1 and H9) were evaluated for the percentage of Annexin V and DAPI positive cells by FACS analysis. <b>D</b>. FACS analysis for Annexin V and DAPI staining demonstrated that REST KD hESCs have statistically significant improvements in survival as demonstrated by reduced levels of apoptotic cells (p<0.032 in H9 and p<0.002 in H1) compared to control NT hESCs. Error bars represent standard error of three independent experiments (performed at p55, p57 and p59 for H9 cells, and at p55, p56, and p57 for H1 cells) and asterisks denote a p value < .05 using Student’s t-test analysis.</p

REST KD cells have increased mesendoderm lineage differentiation bias <i>in vitro</i>.

<p><b>A-B</b>. Evaluation of <i>in vitro</i> differentiation potential in Day 10 embryoid bodies (EBs). QPCR analysis revealed an increase in expression of endoderm and/or mesoderm markers in both H9 (<b>A</b>.) and H1 (<b>B</b>.) REST KD day 10 EBs. Shown are representative graphs of lineage marker analysis for each of the three germ layers. Error bars represent standard error of the mean (SEM) from three technical replicates. ND = Not detected. <b>C-E</b>. Evaluation of protein changes using FACS analysis across two independent REST KD lines (H9, H1) revealed an increase in expression of the mesendoderm marker BRACHYURY compared to control NT lines but no change in the ectoderm marker PAX6 or endoderm marker SOX17. Error bars represent SEM of three independent experiments and asterisks denote a p value < .05 using Student’s t-test analysis.</p