Signal Transduction and Stem Cell Pluripotency: A CQuential Solutions Review

By Ric Ian Cone, PhD All rights reserved, Copyright 2011

Abstract: Pathway-specific studies pertaining to pluripotency and self-renewal are reviewed, followed by sections on microRNA studies and chemical induction of reprogramming. The review concludes with global analyses comparing the phosphoproteome and kinome during pluripotent and differentiated states using mass spectrometry (MS) endpoints. Bibliography and definitions follow review.

Outline Introduction ( pg. 1) Pathway Specific Studies ( pg. 3)

TGF/Activin/Nodal and BMP Pathways (pg. 4)

WNT/Catenin Pathway (pg. 6) PI3K/AKt & MAPK/ERK Signaling (pg. 8) Growth Factors Insulin Receptor/IGF1-R Cell Adhesion (pg. 9)

Notch (pg. 14) Sonic Hedgehog (pg. 14)

MicroRNA Studies (pg. 15) Chemical induction of reprogramming by small molecules (pg. 16) Phosphoprotein analysis of the pluripotent versus differentiated states (pg. 17) Summation (pg. 26) Bibliography ( pg 29) Definitions ( pg. 38)

Introduction

Progress in research towards the clinical utilization of pluripotent stem cells led to the first FDA-

approved human trial of stem cell therapy in a patient with spinal cord injury in January , 2009, and

enrollment of the first patient in October, 2010. (1) (2) Since then, there have been additional FDA approvals

such as for treatment of cerebral palsy and macular degeneration. (3)

Two major sources of human pluripotent cells originate from either: (1) isolating the inner cell mass

from an early human blastocyst and culturing those cells to generate hESC; or (2) induction of pluripotency in

human somatic cells by artificial expression of transcriptional factors such as OCT 4, SOX2, c-Myc and Klf4, or

OCT4, SOX2, NANOG and Lin28 to form and maintain iPSC. (4) (5) Published accounts of approved clinical

use of stems cells describe use of hESC rather than iPSC sources. (1-3)

hESC have the advantage of conferring normal karyotype and totipotency and passing the most

stringent developmental assay, tetraploid embryo complementation. (6) Nevertheless, preparation of iPSCs

from a patient’s own somatic cells could minimize or eliminate immunologic reactivity, and potentially

provide robust and stable cell populations for clinical use. And iPSCs from patients with genetic or acquired

diseases can provide powerful tools for disease modeling. (4) The limitations for hESC are variability, source

equivalency and reprogramming efficiency. (4) (7) The conventional iPSC technique has limitations such as the

introduction of permanent genome modification of target cells by exogenous oncogenes ( ie. any of the four

reprogramming genes); or introduction of other integrated sequences from expression vectors. This risks the

expression of genetic and epigenetic abnormalities during reprogramming. (7) (8) iPSC methods that avoid this

problem, such as transient transfection or other non-integrating gene delivery or transduction using

reprogramming proteins, result in processes that are too slow and efficiencies that are too low .

Since human pluripotent cell growth and maintenance is a starting point for the designed production

of specific populations of differentiated cells for clinical use, a more detailed understanding of the key

signaling pathways involved in both pluripotency and initial differentiation will be important regardless of

whether hESC or iPSC are contemplated as source for clinical use. As noted by Chng (9), current knowledge on

direct differentiation of hESCs could be adapted for human iPSCs since similar signaling pathways were

shown by Vallier (10) to control early fate decisions of hiPSCs and hESCs. However, induction of pluripotency

characteristics upon reprogramming versus maintenance of self-renewal in ESCs may not necessarily result

in acquisition of identical targeted steady-states. As noted by Stadtfeld, (4) pluripotent cell lines exist in two

distinct states characterized by different growth factor requirements and developmental properties.

Acquisition of pluripotency may not necessarily be complete upon expression of the necessary transcription

factors as there are notable differences in telomere length, and transcriptional and DNA methylation patterns

between early and late stage iPSCs. (11) (12) (13)

Reviewing the status of signaling pathways in pluripotent cells during self-renewal versus lineage-

specific differentiation may seem daunting in scope, and may require oversimplification to some extent.

However, the exercise may reveal new approaches to consider, and offer perspective on how far research has

progressed and where it is headed. We’ll begin with a brief review of those signaling pathways known to

participate in pluripotency and self-renewal, provide additional background from genomic and chemical

induction research, and then focus on recent results from global analyses based upon phosphoproteomic

analysis. We’ll look at research focused on different subsets of these signaling pathways, and their activity,

and we’ll compare what has been learned from those studies, with very recent data obtained using mass

spectrometry (MS) endpoints for the system –wide comparison and analysis of the phosphoproteome and

kinome during pluripotent and differentiated states. This review will not focus on neuronal development,

hematopoesis, development of the immune system, angiogensis, apoptosis, autophagy, chromatin regulation

or cell cycle control, per se. However some research relevant to these areas may be included in the course of

discussion on signaling and stemness.

Pathway-Specific Studies

Broadly speaking, cellular signaling pathways that influence differentiation and pluripotency/ self-

renewal include: (1) TGF/Activin; (2) Wnt/-Catenin; (3) growth factors such as insulin, IGF, bFGF, EGFR

family, and PDGF; (4) cell adhesion (14); and (5) Notch. (4) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) Some of these signaling

pathways involve regulation and transport of compounds between the cell nucleus and cytosol, resulting in

alterations in gene expression that influence pluripotency transcription factors such as Oct4, Sox2, and Nanog.

Others may act through promiscuous influence on other signaling pathways in an autoregulatory fashion.

Although human tissue and cell models are preferred for most clinically-relevant research using stem

cells, much of what we do know about signaling and pluripotency was initially based upon research in mice.

mESCs and hESCs respond very differently to several key signaling pathways in self renewal and

differentiation. (24) mESCs self-renew under LIF and BMP, whereas hESCs appear dependant upon FGF and

TGF/Activin/Nodal pathway activity for self renewal. However, as noted by Stadtfeld, (4) mESCs established

from the ICM of preimplantation blastocysts in the presence of LIF and BMP exist in a more primitive or

‘naïve’ pluripotent state and fulfill all criteria of pluripotency, whereas EpiSCs from mice derived from post-

implantation embryos in the presence of bFGF and Activin represent a more advanced or primed pluripotent

state that exhibits some pluripotency critieria such as teratoma formation, but fails to contribute efficiently to

tissues in mice. hESCs and iPSCs more closely resemble mEPiSCs than mESCs, suggesting most research

using hESC reflects a primed rather than a naïve pluripotent state. ESCs exposed to bFGF readily give rise to

EpiSCs, (24) whereas EpiSCs cultured in LIF and BMP reprogram into ESC-like cells at low frequency, (25)

possibly by forced expression of Klf4 and activation of the LIF/Stat3 pathway. LIF, acting through the receptor

gp130, stimulates two separate signaling axis: Stat3/Klf4/Sox2 and PI3K/Tbx3/NANOG, which converge in the

regulation of OCT4 expression. (14) (26) LIF signaling is not required in vivo for self renewal because the

canonical Wnt pathways results in direct regulation of Tbx3, NANOG, and OCT4. (14)

TGF/Activin/Nodal and BMP Pathways

The TGFreceptor signaling pathway is involved in diverse cell fate decisions during development

through control of proliferation, differentiation, apoptosis, and cell adhesion. (9) The TGF receptor superfamily

ligands include TGF, BMPs, Activin, Nodal, and other growth and differentiation factors. These extracellular

ligands bind to TGFActivin, or BMP type II receptors, which are serine/threonine receptor kinases, and then

phosphorylate TGF type I receptors, also known as ALK receptors. Following type I receptor activation,

signaling is propagated by intracellular mediators, the receptor-activated SMADs (R-SMADs) . Nodal,

Activin, TGF, and BMP act through different cascading phosphorylated receptor systems upstream of R-

SMADS, which converge to either a BMP branch, acting on R-SMADs 1, 5,8 , or an Activin/Nodal/TGF

branch acting on R-SMADS 2 & 3. Upon activation by phosphorylation, R-SMADS from either branch,

associate with SMAD 4 and the complexes translocate to the nucleus regulating targeted gene expression in

concert with other transcription factors. SMADS interact with a large number of proteins and can also act as

autoregulators of either the BMP or TGF/Activin/Nodal branches at the level of complex

formation/dissociation and translocation. SMAD 6 inhibits the BMP branch, and SMAD 7 inhibits both

branches.

There is conflicting data regarding theTGF signaling pathway and pluripotency in hESC versus iPSC.

One the one hand, TGF/Activin/Nodal participate in hESC self-renewal. (15) (27) (28) (18) On the other hand,

addition of recombinant TGF to fibroblast cultures can block iPSC formation, and chemical inhibition of

TGF signaling can increase iPSC reprogramming and eliminate the requirement for c-Myc and to a lesser

extent SOX2 expression in mice. (29) (30) Increased iPSC formation following inhibition of TGF signaling may

be associated with mesenchymal-to-epithelial transition (MET) morphological changes (31) (32) or fascilitation of

NANOG activation. (33) Activation of Activin/Nodal signaling maintains stem cell pluripotency through

SMAD2 and SMAD3 signal transduction in hESC, hiPSCs, and mEpiSCs by blocking spontaneous

neuroectoderm differentiation, (27) whereas BMP acts upstream of SMAD 1/5/8, which upon translocation to

the nucleus, blocks NANOG on the one hand and induces differentiation on the other hand. (9) (34) Inhibition of

Nodal (35) or blockade of Activin/Nodal signaling with the pharmaceutical SB4315452 (9) (18) will induce

differentiation of hESCs. On the other hand, high doses of Activin in combination with serum or BMP can

also induce differentiation of hESCs into mesoderm and endoderm. (36) (37) (38) There is evidence that BMPs

and growth factors interact with NF-kB transcription factors to modulate the expression of target genes

involved in cell growth, survival, adhesion and cell death . For example, BMP2 mediates the growth

promoting effect of NK-kB on longitudinal bone growth,(39) may interact in growth plate chondrocytes.

Further understanding of TGF/Activin/Nodal and BMP signaling, the autoregulatory network associated

with translocation and recycling of both SMAD4 complexes to the nucleus, and identification of the target

genes controlled by SMAD2/3 versus SMAD 1/5/8 transcriptional complexes should provide additional insight

regarding potential dual functions of these signaling cascades in pluripotency and self-renewal versus

differentiation. An inhibitor of TGFβ signal transduction, Noggin, binds to TGF-β family ligands and prevents

them from binding to their corresponding receptors. Noggin also plays a key role in neural induction by

inhibiting BMP4, along with other TGF-β signaling inhibitors such as chordin and follistatin. (40) (41)

Wnt/β-Catenin Pathway

Wnt/β-Catenin activation through the canonical pathway results in cytoplasmic accumulation of

Catenin and subsequent translocation to the nucleus, where it participates in sustaining the self-renewal

state of hESC through maintenance of OCT4, NANOG and other transcription factors (42) (43) (19) (44) (45) (14) or

increases reprogramming efficiency to iPSCs from fibroblasts in the absence of c-Myc. (46) Using β-Catenin-/-

and Lrh-1 -/- knock-out ESC, Lrh-1 was identified as a β-catenin target gene, and it’s regulation required to

maintain proper levels of OCT4, NANOG and Tbx3. (14) Nuclear -catenin replaces the transcriptional

inhibitor Groucho to activate Wnt target genes by binding to the LEF / (TCF) transcription factor complex at

Wnt responsive elements (WRE), (47) (43) regulating genes associated with cell cycle control and epithelial

differentiation. (48) Nuclear -catenin up-regulates Nanog expression through interaction with OCT 3 / 4 in

embryonic stem cells (49) and forms diverse complexes with a large number of transcription factors. (42) In the

absence of Wnt extracellular activation of the LRP/Frizzled receptor complex, -Catenin is phosphorylated in

the cytoplasm by a complex of APC protein, axin, and GSK3, and is rapidly degraded by the ubiquitin-

proteosome system. (42)

Regulation of the APC/Axin/GSK3 complex activity and it’s availability in the cytosol influences -

catenin accumulation and nuclear translocation, and may therefore influence self-renewal . Dishevelled (DSh)

is a cytosolic phosphoprotein involved in transduction of the Wnt signal upstream of the APC/Axin/GSK3b

complex. (42) Par-1 and CKII directly phosphorylates DSh, (50) which may recruit DSH to Frizzled. Membrane

recruitment of Axin to LRP is mediated by LRP phosphorylation by kinases CK1gamma and GSK-3. (51) (52)

GSK3 & GSK3 are serine/threonine protein kinases implicated in control of glycogen and protein synthesis

by insulin, and influence both the canonical and non-canonical Wnt pathways , PI3K signaling, and

modulation of a number of transcription factors. Understanding and isolating effects of GSK-3 on -Catenin

accumulation versus degradation may be difficult. Sata (44) found that GSK-3 inhibition by a small molecule

inhibitor was sufficient to maintain pluripotency in mouse and human ESCs. Li (53) found that chemical

inhibition of GSK3 allowed for reprogramming of human fibroblasts without exogenous SOX-2. GSK3

inhibition promotes stemness in ESC, possibly through chromatin remodeling. (43) However, high levels of

Wnt activity in GSK-3 double knockout cells led to differentiation of ESCs. (54)

As with TGF family signaling, dual effects of Wnt signaling on pluripotency versus differentiation

are evident, as Wnt signaling is also important in stem cell differentiation. (55) Tcf3, a transcription factor

regulated by Wnt signaling represses Nanog. (56) Over expression of OCT4 leads to increased β-Catenin

activity. (57) Some influences of Wnt signaling on differentiation may be mediated through non-canonical Wnt

branches, such as the PCP pathway that includes jun kinase, or the Wnt/calcium pathway involved in

regulation of intracellular calcium. (43) Wnt signaling induces gastrulation and formation of the primitive

streak in embryoid bodies by an unknown mechanism, mimicking the in vivo process in human embryos.

Activation of the Wnt pathway in mouse embryonic stem cells induces differentiation into multipotent

mesoderm and endoderm cells. (58) Expression of high levels of T-brachyury and Flk-1, markers associated

with mesoderm development, and high levels of Foxa2, Lhx1, and AFP, are associated with endoderm

development. The progenitor cells created via Wnt activation seemed to have particularly high potential to

differentiate into bone and cartilage, suggesting that β-Catenin plays an important role in skeletal

development. Wnt signaling can also induce hemato-endothelial cell development from hESCs. (59) In contrast

to Wnt3, which is associated with mesoderm and endoderm differentiation, Wnt1 appears to be a major factor

in self-renewal of neural stem cells. This suggests a likely path for Wnt’s role in promoting neural stem cell

proliferation. (55)

PI3K/Akt and MAPK/ERK Signaling

PI3Ks are lipid kinases that are activated by many different growth factor receptor kinases such as

FGF, EGF and PDGF, and promote a signaling cascade that includes Akt1. Akt1 is a serine/threonine kinase

that modifies numerous substrates including MDM2 and IKK, both of which have roles in self-renewal, the

former upstream of p53 modification of BCl-2 and the latter involved in NK-kB signaling . (19) A current model

for growth factor influences on self renewal and differentiation suggest MAPK/ERK and PI3k/Akt signaling

pathways act in concert with the canonical Wnt/Catenin pathway in an autoregulatory fashion through

inhibitory influence of Akt1 on MEK in the MAPK/ERK pathway and GSK3beta in Wnt/-catenin pathway. (19)

(4) Combined inhibition of GSK-3 and MAPK signaling maintains self-renewal of mESCs, (54) (60) and activation

of Akt1 is sufficient to induce pluripotency of ESCs without LIF and feeder cells. (61) (62) Dual inhibition of MEK

and GSK3 promoted transformation of pre-iPSCs into the ‘I’ pluripotent state, also referred to as a metastable

ESC-like state, (63) which can also be induced by forced expression of Klf4 and c-Myc. (4) (64) PI3k/Akt activation,

inhibition of the ERK pathway, and GSK3 inhibition all improve reprogramming efficiency based on either

nuclear transfer or cell fusion mediated reprogramming. (19) (44) Akt1 fascilitates cell fusion mediated

reprogramming through inhibition of GSK-3, (44) but was also shown to negatively influence reprogramming

via nuclear transfer. (65) Differentiation of pluripotent ESCs to lineage commitment can be triggered by ERK

signaling, which is downstream of MEK. (66) The inhibitor pD98059 reduces ERK activity and promotes ESC

production. (67) Inhibition of MAPK/ERK signaling disrupts differentiation in mouse blastocycsts. (68) (69)

PI3K/Akt signaling has known epigenetic effects during reprogramming possibly through phosphorylation of

the histone methylase enhancer of Zeste homolog 2. (70)

The insulin receptor ( InsR) and related insulin-like growth factor 1 receptor (IGF-1R) are among the

most potent PI3K receptor tyrosine kinases. Adams (71) provides a concise description of the molecular

mechanisms associated with InsR and IGF-1R PI3K signaling linked to growth control and transformation.

Intended as an introduction to PI3K signaling and breast cancer, Adams (71) notes that p53, a well-known

tumor suppressor protein, and the PI3K pathway intersect at multiple levels , and that genes coding for most

of the proteins in the PI3K pathway are either oncogenes or tumor suppressor genes, depending on whether

they function to activate signaling through the PI3Kpathway or act to inhibit it. Given the level of cross-talk

between PI3K, MAPK, TGF, and Wnt signaling pathways, a better understanding of specific differences

between pluripotent and differentiated cells in InsR and IGF-1R signaling activity versus general downstream

influences of PI3K/Akt on MEK and GSK3 may prove useful.

Cell Adhesion

Maintenance of cellular association and colony integrity is a widely accepted indicator of ESC state. (72)

hESC and iPSC proliferate in culture as tight and compact colonies having strong intercellular association, and

often propogate better when passaged as small colonies of cells rather than when fully dissociated as single

cells. Stewart (6) recently showed that hESC cultures contained subpopulations with distinct morphologies.

Cells with clonigenic potential that grew well as single cells and were able to form autologous feeder layers

differentiated poorly as embryoid bodies, but formed teratomas in high frequency. Another sub-population

did not clone well as single cells, self-renewed at higher populations and did not form fibroblast feed cell

layers. This latter sub-population differentiated well into embryoid bodies but formed teratomas in lower

frequency. Clearly profound changes in cell and colony morphology occur associated with either

differentiation or changes in the cell culture extracellular environment that impact development and survival.

Cellular interactions with the extracellular matrix and with neighboring cells profoundly influence a variety of

intracellular signaling events, some regulated by cell adhesion receptors. (48)

The four main families of transmembrane cell adhesion molecules ( CAM) that are classified as plasma

membrane receptors, cadherins, integrins, IgCAMs and selectins, influence cell morphology through specific

interactions with their ligands, occurring outside the cell , such as extracellular matrices ( ECM) or with

molecular components of adjacent cells. (48) Upon receptor activation, information about the extracellular

environment is then transduced inside the cell through specific interactions between the cytoplasmic

domains of these receptors and receptor-specific cytoskeletal proteins such as - and - Catenins, -actinin,

talin, vinculin, ankyrin, spectrin, ezrin, or other proteins. These interactions form signaling cascades to actin

and other cytoskeletal elements within the cell that influence cell morphology and motility. ECM includes

fibronectin, vitronectin, laminin, or other components such as those associated with the basal lamina, and

adjacent cells interact through association of cadherins, which form tight junctions between cells expressing

the appropriate cell-specific cadherin subtypes.

Epithelial-cadherin ( E-cadherin) plays an important role in tissue morphogenesis, development,

tumorigenesis and signal transduction. (48) (73) -catenin and p-120 catenin bind the cytoplasmic domain of E-

cadherin, and are critical regulators of E-cadherin functions. As noted earlier, -catenin is also associated with

LRP/Frizzled receptor complex in the membrane , with the APC/axin/GSK3 complex in the cytoplasm and

with OCT, NANOG, LEF/TCF. Association with other transcription factors upon translocation to the nucleus,

provides a basis for cadherin influence on Wnt signaling, activation of pluripotency, and cell cycle control.

-Catenin is an actin-binding protein that regulates interaction of the E-cadherin-Catenin complex with the

actin cytoskeleton . (48) p120 catenin is implicated in signaling to Rho GTPases, (74) and inhibition of the Rho-

associated kinase (ROCK) –non muscular myosinIIA (NMMIIA) cascade, (72) improving survival of dissociated

hESC (75) (76) and stabilizing OCT4-SOX2-NANOG transcriptional regulation. NMMIIs are actin binding

proteins that regulate contractile functions in cells and are necessary for E-cadherin mediated intercellular

contact formation, adhesion, and mechanical tension. Overexpression of p120 catenin can lead to decreased

RhoA activity, increased RAC and CDC42 activity, and dramatic morphological changes. (74) Depletion of p120

catenin by gene silencing (shRNAs) reduced hESC colony formation. E-cadherins have also been linked to

PI3K and PKB, (77) signaling by the VEGF-2 receptor, (78) (79) cross-talk with GPCRs, (80) and cross-talk with

integrins via an integrin linked kinase(ILK). (81) ILK is an ankryn-repeat containing serine/threonine kinase

that phosphorylates GSK3, increasing Catenin translocation to the nucleas and contributing to cell cycle

progression. (81)

Integrins can regulate MAPK/ERK signaling at three locations: (1) at the level of membrane receptor

tyrosine kinases; (2) coupling of upstream and downstream elements at the level of RAS to Raf coupling

upstream of MEK/ERK; and (3) associated with ERK translocation to the nucleus . (48) At the membrane level,

integrins influence a number of systems through a variety of mechanisms. Tyrosine phosphorylation of

growth factor receptors for EGF, PDGF or FGF can be triggered by ligand occupancy plus activation of

integrin-cytoskeletal complexes, often through activation and autophosphorylation of FAK. (48) Direct

association of integrin beta cytoplasmic tails with some receptor tyrosine kinases may also occur; such as for

insulin and VEGF, or direct activation of MAPK/ERK through caveolin-1, Fyn ( a src-kinase), and adaptor

protein Shc. (48) In addition to MAPK/ERK, integrins activate pathways involving JNK, Rho, RAC or CDC42,

the latter three known to influence actin-myosin contractility, formation of stress fibers, and motility.

Inhibition of Rho associated ROCK expression and resultant stabilization of E-cadherin may be important for

improved human ESC and iPSC survival following single cell dissociation in the absence of ECM

supplements. (48) GPCR agonists and muscarinic agents can trigger autophosphorylation of FAK, a process

requiring Rho-GTPase. (82) Integrin mediated cell anchorage can modulate signaling between GPCRs and

MAPK/ERK, most likely through RAS/Raf coupling, as well as signaling between NFkB and MAPK/ERK

initiated by cytokines. (48) Integrin cross-talk with MAPK/ERK and PI3K/ERK signaling is extensive outside

the nucleus.

Anchorage regulation of ERK activation involves cortical actin filaments rather than focal contacts and

stress fibers. (83) Inactive ERK associates with MEK and remains in the cytoplasm. Upon activation, ERK is

phosphorylated, dissociates from MEK, and enters the nucleus, where ERK phosphorylates the ETS family of

transcription factors such as ELK1. (84) Dephosphorylation of ERK results in export from the nucleus and

reassociation with MEK. Forced activation of MEK or Raf does not induce ERK translocation to the nucleus,

and ERK activation is not sufficient to drive cells into the cell cycle. (48) The mechanism by which actin-

filaments might influence ERK trafficking or cell cycle/checkpoint control remain uncertain. (48)

Xu (24) recently described results from studies aimed at identifying small molecular regulators of

pluripotent stem cell survival and self-renewal. The results suggest an effective approach to sorting integrin

and cadherin influence on cell cycle/checkpoint control. They also implicate an essential role for E-cadherin

signaling in ESC survival. Differences in the requirements for self-renewal in mouse ESC ( ie LIF & BMP)

verus hESC ( ie. FGF and TGFb/Activin/Nodal pathway activity) were also addressed. Xu (24) showed that the

primary cause of increased hESC cell death following enzymatic dissociation comes from irreparable

disruption of E-cadherin signaling, which then leads to a fatal perturbation of integrin signaling. The small

molecule 2,4-disubstituted thiazole (Tzv) inhibits Rho-associated kinase (ROCK) expression, which then

inhibits endocytosis of E-cadherin on the cell surface, thereby stabilizing newly formed E-cadherin. This was

essential for hESC survival in ECM-free conditions following single-cell dissociation, possibly because

dissociation disrupts E-cadherin . Another small molecule, 2,4, disubstituted pyrimidine ( Ptn) only enhanced

survival of hESC dissociated cells on matrigel-coated plates, and not under ECM-free conditions ( ie. matrigel

provides an undefined ECM, and is obtained from a mouse tumour). Both Tzn and Ptn increased cell-ECM

adhesion mediated integrin activity, substituting for growth factor expression via cross-talk effects of integrins

on PI3K/Akt and MAPK/ERK signaling. Such cross-talk may also promote cell survival when hESCs are

grown on matrigel. In contract to hESCs, mESCs are more tolerant to single cell dissociation , possibly

resulting from a non-anchorage dependant mechanism that inhibits ROCK as postulated by Xu. (24) hESCs

were converted to survive under mouse-like conditions , in which media was supplemented with LIF and

inhibitors of p38 and MEK, the latter effectively replacing BMP through suppression of SHP-2 and MAPK/

ERK signaling. (85) (86) Converted hESCs relied more on E-cadherin to maintain self-renewal , and significantly

less on integrin signaling. The converted hESCs resemble mESCs and have higher levels of E-cadherin and

lower levels of phosphorylated FAK ( ie. integrin signaling marker). Given that conventional mESCs require

different cytokines and may represent an earlier pluripotent state (ie. preimplantation epiblast) than hESCs (

ie. postimplantation epiblast) , these converted hESC may prove useful for understanding a molecular basis for

distinguishing these stages. Such studies may also help clarify a discrepancy that conventional hSCs can

differentiate into trophoblasts, which first diverge prior to epiblast formation.

Notch Pathway

The Notch pathway mediates short-range interaction with neighboring cells in which contact between a

cell expressing a membrane associated ligand and a cell expressing the Notch transmembrane receptor

induces a regulatory signal that may induce cell fate in one or both cells. Once the Notch extracellular domain

interacts with a ligand, a metalloprotease cleaves the Notch protein such that the extracellular portion

continues to interact with the ligand and is then endocytosed within the ligand-expressing cell. At the Notch

receptor expressing cell, an enzyme called γ-secretase then cleaves the cytoplasmic portion of the remaining

Notch receptor protein ( ie NIC) , which then moves to the nucleus where it can either regulate gene

expression by forming a complex with and activating the transcription factor CSL, or become phosphorylated

by CDK8, ubiquitinated and degraded . NIC- CSL target genes are numerous and include HES family, c-Myc,

p21, and Cyclin D3. Non-canonical signaling may also result from NIC complexing with IKK, p50, or p85

PI3-kinase , resulting in cross-talk with NF-kB or Akt. Pannuti (87) recently reviewed the Notch pathway

with regard to cancer stem cells.

Sonic Hedgehog Pathway

Sonic Hedgehog Signaling (SHH) pathway has a pivotal function in cell development and

tumorigenesis in a wide variety of tissues, both processes sustained by stem cells. (88) Patched 1, the receptor of

the SHH ligands inhibits the transducer Smoothened (Smo), and Patched 1 mutation leads to constitutive

activity. Downstream effectors of Smo activity are Gli transcription factors, which act on a set of target genes

promoting cell proliferation and reducing cell differentiation. These target genes include Gli1 itself, thus

autoreinforcing the signaling strength. The identity of SHH/Gli-target genes involved in the control of

stemness in neural stem cells and cancer stem cells is poorly understood. Wu (89) examined the gene and

protein expression of key components of the SHH signaling pathway in hESC and differentiated embryoid

bodies. Po (88) showed that both NANOG and Gli1 are highly expressed in postnatal cerebellar neural stem

cells, and in SHH-dependent mouse and human medulloblastoma stem cells. NANOG is required as a critical

mediator of SHH-driven self-renewal of neural stem cells , as SHH acts through transcriptional activation of

both mouse and human Nanog. P53 suppresses both the SHH pathway and Nanog expression. In the model

suggested by Po, (88) p53 suppresses GL1 expression, however, GL1 can autoregulate by suppressing p53

through activation of MDM2. Despite the presence of functioning pathway components, SHH played a

minimal role in maintaining pluripotency and regulating proliferation of undifferentiated hESC. However,

during differentiation with retinoic acid, a GLI-responsive luciferase assay and expression of the target genes

PTCH1 and GLI1 revealed that the SHH signaling pathway was highly activated. Po (88) notes that SHH

signaling may be important during hESC differentiation toward the neuroectodermal lineage.

MicroRNA

MicroRNAs are required for the proper function of both proliferation and differentiation pathways in

ESCs, which are characterized by a defined microRNA (miRNA) signature among the 500 plus miRNAs that

have been identified. (90) Knockdown experiments in mouse and human block processing of pri-miRNAs to

miRNAs resulting in delayed G1-S and G2-M transition. (91) ESCs express a very limited repertoire of

miRNAs whose levels decrease as the stem cells differentiate.. (92) This includes the miR-371 family in hESCs

and the mi-290 family and mi-302 cluster in mESCs. On the other hand, the let-7 family of miRNAs are widely

expressed in most differentiated cell types, and present in low levels in ESCs. (93)

OCT 4, SOX2, NANOG, c-Myc, and Tcf3 directly bind the promoters of, and upregulate the expression

of mouse miRNA clusters that share similar seed sequence and regulate embryonic stem cell cyle ( ESCC),

including mi-290 and mi-302 families. (94) ESCC miRNAs directly repress key regulators of the cell cycle to

ensure a fast G1-S transition. For example mi-290 repress de nova DNA methyl-transferases expression by

silencing the transcriptional repressor Rbl2. (92) (95) The role of miRNAs in cell cycle progression seems to be

conserved between mouse and human. (90) MiR-92a regulates G1-S transition in hESC by repressing the

Cdkn1c checkpoint gene. (96) MiR-372 ( ie miR-371 family) regulates the G1-S checkpoint inhibitor Cdkn1a,

and mi-195 regulates the G2-M checkpoint inhibitory kinase WEE1, one of three kinases that regulate the G2-

cyclin B-Cdk complex in mammalian cells. (91)

Additional studies support the model that let-7 miRNAs oppose the function of ESCC miRNAs by

repressing expression of many of the same downstream target genes that are indirectly activated by ESCC

miRNAs. (90)

Chemical Induction

In recent years, researchers have identified chemical compounds that can enhance reprogramming

efficiency and kinetics and/or functionally replace exogenous reprogramming factors. (97) These studies may

eventually lead to or improve clinical use of iPSCs. Meanwhile, some of these studies provide supportive

information on the role of signaling pathways in pluripotency. The GSK3 inhibitors Kenpaullone (98) and

CHIR99021 (53) enhance reprogramming, the former replacing Klf4. This is consistent with MAPK/ERK and

PI3k/Akt signaling pathways acting in concert with the canonical Wnt/-Catenin pathway in an autoregulatory

fashion through the inhibitory influence of Akt1. PS48, a small molecule activator of 3 phosphoinositide

dependant protein kinase 1 (PDK1) also facilitated reprogramming through PI3k/Akt signaling. (99) Enhanced

reprogramming following treatment with ROCK inhibitors Thiazovivin (29) (24) or Y27632, (100) or treatment with

the E-cadherin inducers, Apigenin or luteolin, (101) confirmed the importance of E-cadherin, and influence of

cell-cell interaction upon contractile elements for enhanced reprogramming, including survival of human

pluripotent cells following dissociation.

Phosphoproteome Analysis

The phosphorylation status of specific kinases or other proteins within signaling pathways offers

useful information about a given pathway’s activation status. At least one third of all cellular proteins are

estimated to be phosphorylated. Their levels of phosphorylation vary widely and specific sites may be

phosphorylated from less than 1% to greater than 90% . (102) In signal transduction, protein phosphorylation

may be associated with the active or inactive status of a given protein. Intracellular signal transduction,

mediated by receptor-protein tyrosine kinases, is initiated by time-ordered tyrosine phosphorylation of

intracellular receptor chains, which immediately leads to the recruitment of adaptor proteins and other

signaling molecules, including serine/threonine kinases and phoshophatases. (103) Phosphorylation plays an

equally important role in the attenuation and termination of the signal in the later stages of signal progression.

(104) (105) The complexity with respect to the number of proteins involved , the number and variety of

phosphorylation sites on these proteins, and the diverse timing of metabolic events makes identifying post-

translational modification associated with self-renewal versus lineage-specific differentiation a formidable

task.

Over the past two years, significant progress has been made using MS-based system-wide quantitative

proteome and phosphoproteome analyses, generating new data and perspectives upon which future pathway-

specific and disease-specific studies of kinome regulation are likely to be based. Excellent reviews of current

phosphoproteomic analysis and quantification strategies are provided by Macek, (102) by Thingholm (106) and by

Babtschoff. (107) In particular, quantitative phosphoproteomic analysis is being used to identify up and down

regulation of phosphorylation at specific sites upon stimulation. This should provide insight into the specific

regulation of different phosphorylation pathways, and help identify the different target proteins affected by

phosphorylation upon stimulation. (106)

Brill (16) compared hESC under conditions of self-renewal versus differentiated state four days after

treatment with retinoic acid. Van Hoof (108) compared hESC during the first 4 hours following differentiation

with BMP-4. Rigbolt (109) compared hESCs in the presence of media conditioned for self-renewal, with hESCs

following removal of conditioned media ( NCM). Mouse embryo fibroblasts (MEF) were used to condition

medium, and after removal of MEF, differentiation occurred due to the absence of factors contributed to the

medium by the feeder cells. In a separate protocol, Rigbolt treated hESCs with phorbol 12-myristate 13-

acetate (PMA) , a diacylglycerol analog that activates PKCs and induces differentiation. Rigbolt (109) used two

separate time-course studies, one at T=0, 30 min and 6 hours, and the other at T=0, 1 hour and 24 hours for

each protocol, and applied protocols to two separate hESC cell lines.

Brill (16) used strong cation exchange (SCX) and immobilized metal affinity chromatography ( IMAC)

for enrichment of phosphopeptides from unlabelled tryptic digests and LC-MS/MS based phosphopeptide

analysis. Both Van Hoof and Rigbolt used stable isotope labeling by amino acids in cell culture (SILAC) prior

to experimental treatments. At the selected times following treatment, metabolically labeled cells were

trypsinized, and phosphopeptides enriched, isolated and analyzed by LC-MS/MS following strong cation

exchange ( SCX) /TiO2 chromatography.

Van Hoof and Rigbolt used similar computer programs to analyze and present their results, including

: (1) MaxQuant, designed for proteome-wide protein quantification of MS data; (2) Motif –X to identify

protein phosphorylation motifs; (3) NetworKIN to identify the kinases likely to be responsible for

phosphorylating these motifs; and (4) Gene Ontology ( GO) to functionally classify phosphorylated proteins.

The results from these three studies will be discussed collectively. The reader is referred to the

original articles and supplemental data for all three studies for additional specifics and details regarding each

author’s use of bioinformatics and computer generated data analysis.

The Van Hoof (108) study revealed decreased activity of cyclin-dependant kinase (CDK2 ) associated

with differentiation to trophoblasts following induction with BMP4. The Brill (16) study identified a role for

platelet-derived growth factor ( PDGF) in hESC self-renewal. The Rigbolt (109) study showed good correlation

with the Van Hoof study regarding major fingings. Both Van Hoof and Rigbolt reported three pS sites on

SOX2 and no changes in phosphorylation status at these sites upon differentiation induced by either BMP4 or

PMA. VanHoof further identified these sites as important for SUMOylation of SOX2. Although decreased

SOX2 abundance was noted by VanHoof following BMP4 treatment, and by Rigbolt after PMA treatment,

following withdrawal of conditioned medium (NCM), Rigbolt noted an increased abundance of SOX2, and

decreased phosphorylation of SOX2 in the SUMOylation motif. Rigbolt suggested increased abundance may

be associated with either reduced degradation or an increase in neural stem cells ( ie. SOX2 is a reliable neural

stem cell marker). Both Van Hoof and Rigbolt noted changes in the expression of either one or both genes

encoding the self-renewal transcription factors OCT4 and NANOG, changes in the phosphorylation status of

DNA methyltransferases (DNMTs) which silence these genes, and increased interaction of DNMTs with a

transcriptional elongation complex, polymerase-associated factor 1 ( PAF-1) within 24 hours of induced

differentiation. All three authors identified significant changes in the phosphorylation of tyr15 on CDCK2

and CDC2. Phosphorylation at this site is inhibitory, and is associated with decreased cell cycle progression.

VanHoof and Rigbolt found increased phosphorylation at this site associated with differentiation (BMP4,

PMA, or NCM treatments), however Brill found decreased phosphorylation associated with retinoic acid

treatment. Rigbolt describes a model where increased expression of WEE1, noted in his results, leads to

increased inhibitory phosphorylation of CDC2 and CDK2, which then inhibits cell cycle progression. Rigbolt

suggests the decreased CDC2 and CDK2 phosphorylation noted by Brill may be due to influences of retinoic

acid treatment unrelated to cell cycle progression.

One of the benefits of system-wide phosphoproteomic analysis associated with pluripotent versus

differentiated states is the potential to identify relevant enzymes, proteins or substrates that are

phosphorylated or dephosphorylated, and have not previously been associated with either pluripotency or

lineage-specific differentiation. Generally phosphoproteome analysis results begin with the numbers of

proteins, phosphoproteins, and phosphorylation sites identified. Brill identified 2546 phosphorylation sites on

1602 phosphoproteins, of which 389 proteins contained more phosphorylation sites associated with

pluripotency, and 540 proteins contained more phosphorylation sites associated with the differentiated state

(ie 4 days after induction). Van Hoof identified 5222 proteins, with 1399 proteins phosphorylated on 3067

amino acid residues (2,431 serines, 582 threonines and 54 tyrosines) . These proteins were then classified by

molecular function, biological process and subcellular localization. The two most abundant classifications

were 18% nucleic-acid binding proteins and 6.6% transcription factors, suggesting that chromatin remodeling

and transcription are highly active in hESCs.

Van Hoof noted that the most dramatic phosphorylation changes took place during the first hour

following BMP treatment, coincident with SMAD 1/5/8 phosphorylation in hESC, and consistent with

previously noted BMP influence on SMAD 1/5/8 . By examining temporal patterns of phosphorylation

following BMP treatment across four times points ( 0, 30, 60, 240 minutes) and classifying them into 7

different temporal categories , Van Hoof established criteria for ‘regulated’ phosphorylation sites, which

were, for the most part, sites undergoing dramatic temporal increase in phosphorylation. For example, four

temporal patterns were described for tumor suppressor p53-binding protein 1, suggesting different kinetics.

There were two coincident patterns for transcription factor AP-1 showing increased phosphorylation during

the first hour followed by a drop-off, and one pattern for GSK3showing rapid increased phosphorylation

within 30 minutes that was then sustained. Van Hoof tabulated 70 pathways represented by proteins with

dynamic phosphorylation, and suggested these as activated signaling cascades. He noted target sites on

SMAD 5, SMAD 8, PDPK1, AKT1, MDM2, CHK, c-Jun, Wnt, GSK3 and casein kinase 1. Van Hoof also

charted 60 marker categories related to signaling pathways illustrating for each category the number of

regulated phosporylated proteins, non-regulated phosphorylated proteins and non-phosphorylated proteins.

Notably, the greatest number of regulated phosphorylated proteins were from the Wnt pathway (ie. 20) and

p53 (ie. 16) . Other notable categories of signaling having between 4- 10 regulated phosphorylated proteins

were for secretase (Alzheimers) and presenillin (Alzheimers), angiogenesis, apoptosis, cadherin, cytoskeletal

regulation by Rho GTPase, FAS, Hedgehog, Huntington Disease, Insulin/IGF/ PKB signaling, integrin,

interleukin, oxidative stress response, Parkinson Disease, PDGF, PI3K , RAS, TGFubiquitin, and Notch.

Phosphosites identified with ESC-associated genes defined by the International Stem Cell initiative (110)

were mapped by Van Hoof to Lin28, DNMT3B, Pou5F1 ( ie OCT4) , SOX2, GAL, UTF, FOXD3, GDF3,

NODAL, PDXL, and NANOG. Of these, some phosphosites on LIN28, DNMT3B, GAL, and UTF were

regulated during differentiation. One hundred phosphorylation sites regulated during differentiation were

identified as downstream targets for OCT4, SOX2, and NANOG, based upon mapping to chromatin

immunoprecipitation data sets. (111) Van Hoof suggests many components of the hESC transcriptional circuitry

are regulated by phosphorylation.

NetworKIN, developed by Linding, (112) combines probabilistic modeled network context with linear

motifs recognized by the catalytic domain of kinases, and was used to predict kinases for every

phosphorylation site identified. The result was an hESC kinome, described by Van Hoof, comprising 107

kinases that could be rank-ordered based upon the number of phosphopeptide substrates identified. The

more prominent kinases included: CDK1/2 (25.7%); MAPK14 (7.7%) ; CK2A1 (5.7%) ; MAPK8 (5.7%); TGFR2

(4.4%) ; GSK3 (3.8%); NEK2 (3.5%) and DMPK (2.7%). Using the temporal profiles of phosphorylation at 30,

60, 240 minutes after BMP treatment, Van Hoof linked specific phosphosites identified on regulated kinases to

their predicted upstream kinases to create a temporal kinase cascade model subsequent to differentiation. The

number of phosphosites expanded with increased time after BMP treatment . Although many of the identified

links could be found at all three times points, there were increasing numbers of unique links associated with

each time point following BMP treatment.

Protein phosphatase (PP) activity was also tracked in the Van Hoof study. Forty-three PPs or 30% of all

phosphatases reported in SwissProt were identified, and four presented differentially regulated phosphosites:

PPMH1; DUSP19; PTPN13 and PTPN14.

Rigbolt profiled 6,521 proteins and 23,522 phosphorylation sites, most single (66.6%) or double (23.4%)

phosphorylated on 4335 proteins. Rigbolt found that about 50% displayed dynamic changes in

phosphorylation status during 24 hours differentiation. 15,004 phosphorylation sites could be assigned as

class 1 sites based on localization probability of at least 0.75 (113). Of these sites, 79.7% were phosphorylated

serine ( pS) , 17.4% were phosphorylated threonine (pT) and 2.9% were phosphorylated tyrosine ( pY) sites.

Rigbolt also noted that changes in phosphorylation state associated with differentiation status were more

extensive than changes in protein abundance. Of the class 1 phosphorylation sites, 45% of 10,066 sites

dynamically changed after PMA treatment, and 30% of 11,104 sites dynamically changed after NCM

treatment.

Rigbolt assessed results from QPCR analyses of the different cell lines (ie. HUES9 and Odense-3) and

differentiation protocols (ie. PMA versus NCM) to examine whether or not there were notable differences in

lineage-specific differentiation. Apart from differences in expression of transcripts for ectodermal marker

PAX6 and mesodermal marker BRACHYURY between the two cell lines, Rigbolt found that differentiation

occurred randomly in both cell lines and that both PMA and NCM triggered hESCs towards two distinct, but

lineage-independent differentiation programs .

Using GO analysis, Rigbolt’s data on proteins, phosphorylation sites and class 1 phosphorylation sites

were classified as either associated with functional activities attributed to molecular function (ie protein kinase

activity, transcription regulatory activity, receptor activity) , or associated with cellular components ( ie.

cytoskeleton, nucleus, extracellular matrix). Altogether 714 class 1 phosphorylation sites were identified on

transcription factors, including 282 sites on 106 distinct proteins not previously reported as phosphorylated.

Detailed examination of 216 transcription factors identified families comprising most of the well known

factors implicated in pluripotency, self-renewal, and differentiation, including OCT4, SOX2, SOX13, SOX15,

UTF1, FOXO1, FOXO3,FOXO4, SALL1, SALL2, and SALL4. The direction of change in protein abundance, or

phosphorylation status for these factors was then evaluated among transcription factors that had homologous

DNA binding domains, and the results were organized according to homology, and ordered by phylogenetic

distance of their DNA binding domains, with five transcription factor groups noted ; HLH, HMG box , basic

leucine zipper ( bZIP), Forkhead, and Homeobox . The abundance of only 26 and 20 of these factors changed

within 24 hours of PMA and NCM treatment, respectively. Decreased abundance of POUSF1 ( ie. OCT4) , in

the Homebox group was observed for both PMA and NMA induced differentiation. Decreased abundance of

SOX2 occurred after PMA treatment, but increased after NCM treatment. In addition to it’s functions

maintaining hESC pluripotency, SOX2 is also a neural stem cell marker. PMA trestment and withdrawal of

factors from MEF probably induce distinct differentiation programs in hESCs. Within a group comprising the

bZIP domain, increased abundance and number of phosphorylation sites was noted for c-Jun, JunB and FRA2

( ie. FOSL2) transcription factors which belong to the activator protein 1 (AP-1) heterodimer family, consistent

with the transient increased AP-1 phosphorylation noted by Van Hoof.

Transcription factors where the number of class 1 phosphorylation sites decreased by two or more

following one or the other treatment included: PMA ZNF44, ZSCAN10, SMAD2, SOX11, TGIF2, GPBP1,

ETV3, SALL1, ZNF217, TRIM25, KIAA0415, FOX04, ZNF219, RUNX1T1, HNRNPAB, ZNF446, UTF1, REX04,

TCF9, ARID3A, SOX2, SOX13, TCF7L2, CTCF, MAX1, MYC, HMGA1-Y, HMGA1-I, UBTF, and MBD1.

Transcription factors where the number of class 1 phosphorylation sites increased by two or more were noted

for CUX2, TCF20, ZFP36L2, c-Jun, SOX 15, and TFAM.

Using Motif-X, Rigbolt identified 33 significantly enriched pS motifs and 11 pT motifs. The

NetworKin algorithm was then used to identify the kinases likely to phosphorylate these motifs. Of the

motifs that became increasingly phosphorylated after PMA and NCM treatment, a significant portion of

basic-rich motifs were predicted to be substrates of CDC like Kinase 2 (CLK2) , p21-activated kinase 7 (PAK7),

PIM2, or PKA. Acid-rich motifs were predicted to be phosphorylated by members of the casein kinase family

and myotonic dystrophy protein kinase (DMPK). Substrates having decreased phosphorylation following

induction of differentiation were motifs phosphorylated by proline-directed kinases such as MAPKs and

CDKs .

Rigbolt identified more than 200 protein kinases with 654 class 1 phosphorylation sites, including 195

previously unreported sites on 86 kinases. He grouped these results by kinase family and similarity of their

kinase domains to illustrate increases and decreases in kinase abundance following PMA and NCM treatments

as well changes in phosphorylation status based on increases or decreases in the number of class 1

phosphorylation sites detected on a given kinase following treatment. Increased kinase abundance upon

differentiation was noted for at least one of the two treatments for YES, EPHB4, EPHA2, LIMK2, PKR, WEE1,

NRK, LOK, MAP2K1, NEK7, CAMK2D, AMPKA1, CDK6, and CLK1. CDK6 was the only kinase

demonstrating increased abundance after both PMA or NCM treatments. Since NANOG controls the gene

encoding CDK6, the 2-3 fold increase during either differentiation paradigm was unexpected. Decreased

kinase abundance was noted for at least one of the two treatments for IGF1R, FLT1, MLK3, BRAF, BRD2,

PAK1, MAP3K4, MAP2K4, NDR2, DMPK, AURA,VACAMKL, CHK1, and PCTAIRE1.

Rigbolt defined regulated phosphorylated sites from linear phosphorylation motif analysis of the hESC

phosphoproteome. Significantly overexpressed linear phosphorylation motifs were identified using Motif X,

matched to kinases with NetworKin, and then clustered according to the average phosphorylation dynamics

of the sites matching each motif.

Observed increased phosphorylation of Thr14 and Tyr 15 on both CDK2 and CDC2 (ie CDK1), and

increased abundance of WEE1 following induction of differentiation is consistent with Rigbolt’s suggestion ,

noted earlier, that these changes relate to repression of cell cycle progression. According to Chen, (114) WEE1 is

repressed by Klf4 during self-renewal, and reduction of Klf4 during differentiation (115) would be expected to

lead to increased abundance in WEE1, followed by increased phosphorylation of CDK2 and CDC2 at Tyr15,

which inhibits cell cycle progression, (116) inhibits CDK2 and CDC2 kinase activity, (117) and should also result

in observed decrease in phosphorylation sites on CDK targets. The latter is consistent with Rigbolt’s

observation of extensive reduction of phosphorylation sites following PMA and NCM treatment identified by

Motif X analysis, and predicted by NetworKin to be targeted by the CDK, MAPK, GSK and CDK-like (CMGC)

family of kinases.

Kinases where the number of class 1 phosphorylation sites increased by two or more following at least

one treatment included PEK, ATR, SMG1, FRAP, SGK269, sGK223, MAP3K7, CRIK (a DMPK) , CRIK-3HS (a

DMPL) , DCLK1, DAPK1, CDC2, CDK2, GSK3A, GSK3B,and SRPK2. Kinases where the number of class 1

phosphorylation sites decreased by two or more following at least one treatment included KDR, RAF1, GCN2,

WEE1, RIOK1, DNAPK, BCR, PAK2, PKN1, RSK3, MRCKB, GPRK6, ULK1, TLK2, and CHED .

Some of the largest increases or decreases in phosphorylation sites were noted, for several kinases

following PMA and NCM treatments, however for these kinases some phosphorylation sites increased while

others decreased. For example, after NCM treatment there was an increase on CRK7 (a CDK kinase), of 20

class 1 phosphorylation sites and a decrease of 8 different class 1 phosphorylation sites. After PMA treatment

there was a decrease on CRK7 of 4 class 1 phosphorylation sites. After NCM treatment there was a decrease

on WNK1 of 13 phosphorylation sites. But following PMA treatment there was an increase of 7 sites and a

decrease of 4 different sites. WNK1 contains a putative AKT1 phosphorylation site, and is a substrate for

AKT1. PI3K/Akt pathway activation is required for insulin to stimulate WNK1 phosphorylation. (118) Removal

of MEK factors in the media (ie. NCM treatment) may result in withdrawal of insulin, thereby decreasing

WNK1 phosphorylation status, whereas the PMA induced increase would likely represent a separate process

related to that treatment. The other kinases having dramatic changes in phosphorylation are worth further

consideration based upon their known roles in signaling cascades.

Although other protein modifications such as acetylation and methylation, as well as epigenetic

changes associated with DNA methylation, including the role of DNA methylation in expression of

pluripotency markers OCT4 and NANOG have not been a subject of this review, it is worth noting that Rigbolt

identified extensive changes in the phosphorylation of sites in the N-terminal regions of DNMTs involved both

in preserving methylation patterns in nascent DNA during replication , and in de novo DNA methylation, and

identified increased interaction of DNMTs with a transcriptional elongation complex, polymerase-associated

factor 1 (PAF-1) within 24 hours of induced differentiation.

Summation

In this review, the role of signaling in pluripotency, and in induction of differentiation from the

pluripotent state has been examined using a wide variety of orthogonal approaches and methodologies. How

well does data from these different approaches fit together to provide the information required to harness

what we know about signaling and stemness for clinical use? What future research directions should be

taken?

How well did the results of the phosphoprotein studies correlate with the pathway-specific studies,

microRNA profiling, knock-down and small molecule studies? Some of what we generally understand

about the role of signaling pathways and pluripotency was corroborated. Following induction of

differentiation, increased phosphorylation of GSK phosphoproteins was observed by both Van Hoof and

Rigbolt. Van Hoof noted a transient increase in GSK3 phosphorylation at Y216 within 30 minutes of BMP4

treatment, and Rigbolt indicated increased phosphorylation of GSK3 , GSK3, and SRPK2, a closely related

kinase. Does increased GSK3 phosphorylation result in increased phosphorylation of -Catenin by the

APC/AXIN/GSK3 complex, and subsequent degradation, thus turning off self-renewal through reductionof

nuclear -Catenin? Van Hoof notes that several key peptides of -Catenin were identified, however the N-

terminal phosphopeptide marking activity was not detected, and so the activation of -catenin could not be

determined. However, Van Hoof notes that several co-activators and repressors of -catenin were

differentially phosphorylated at different positions suggesting a role for the WNT pathway. Dual inhibition

of ERK and GSK3 facilitates reprogramming and combined inhibition by MAPK, and GSK3 maintains self

renewal. Perhaps increased GSK3 phosphorylation is acting on these systems through Akt autoregulation of

PI3K and MAPK signaling. Following PMA treatment, decreased phosphorylation of SMAD2 occurs. Is this

sufficient to decrease Activin/Nodal/TGF activation of R-SMADS 2 & 3, and turn off self renewal? Brill’s

phosphoproteome analysis support the role for growth factor activation of the pluripotent state. Rigbolt and

Van Hoof’s phosphoproteome results fit well with a role for CDK and WEE1 in repressing cell cycling, and a

role for DNMT phosphorylation in regulating the effect of DNA methylation on OCT4 and NANOG

expression. And regulation of phosphorylation in the SUMOylation motif within SOX2 was also noted as

generally consistent, except for the increases in phosphorylation noted after withdrawal of conditioned media.

Could phosphoproteome analysis reach a level of detail and consistency with regard to proteome

activity where every change subsequent to perturbation of signaling was measureable and understood within

the context of it’s clinical influence upon the patient? The difficulty of attempting to correlate measureable

results of phosphoproteomeanalysis with respect to pluripotency or differentiation status, and assign those

results to specific signaling components is that one cannot be certain whether induction of differentiation is

acting directly to turn off self-renewal at the nuclear level, or is acting on processes at the cytoplasmic or

membrane level that engage or disrupt feedback mechanisms to maintain self-renewal.

Please see Part 2 for Bibliography and Definitions

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