review article alkaline phosphatase in stem...

Review ArticleAlkaline Phosphatase in Stem Cells

Katelina Štefkovaacute1 Jilina Prochaacutezkovaacute23 and Jiliacute Pacherniacutek14

1 Institute of Experimental Biology Faculty of Science Masaryk University Kotlarska 2 611 37 Brno Czech Republic2Institute of Biophysics Academy of Sciences of the Czech Republic vvi Kralovopolska 135 612 65 Brno Czech Republic3Department of Histology and Embryology Faculty of Medicine Masaryk University Kamenice 5 625 00 Brno Czech Republic4International Clinical Research Center Center of Biomolecular and Cellular Engineering St Annersquos University Hospital BrnoPekarska 53 656 91 Brno Czech Republic

Correspondence should be addressed to Jirı Pachernık jipascimunicz

Received 10 November 2014 Accepted 21 January 2015

Academic Editor Kenneth R Boheler

Copyright copy 2015 Katerina Stefkova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Alkaline phosphatase is an enzyme commonly expressed in almost all living organisms In humans and other mammalsdeterminations of the expression and activity of alkaline phosphatase have frequently been used for cell determination indevelopmental studies andor within clinical trials Alkaline phosphatase also seems to be one of the key markers in theidentification of pluripotent embryonic stem as well as related cells However alkaline phosphatases exist in some isoenzymesand isoforms which have tissue specific expressions and functions Here the role of alkaline phosphatase as a stem cell marker isdiscussed in detail First we briefly summarize contemporary knowledge of mammalian alkaline phosphatases in general Secondwe focus on the known facts of its role in and potential significance for the identification of stem cells

1 Alkaline Phosphatase

Alkaline phosphatase (AP EC 3131 orthophosphoric-monoesterase alkaline optimum) is a membrane boundenzyme which occurs in almost all living organisms Mam-malian APs have low sequence identity with E coli butresidues involved in the active site of the enzyme and theligands coordinating the two zinc atoms and the magnesiumion are conserved thus the catalytic mechanism is consid-ered to be similar in prokaryotic and eukaryotic APs [1 2]Individual mammalian alkaline phosphatases differ in theirheat stabilities and uncompetitive inhibition properties Thisis the result of their nonhomologous disulphide bonds theirstructural significance and nonconserved residues [3 4]In humans and probably also in other mammals fourisoenzymes have developed during evolution and are codedby up to four genes These four isoenzymes can be foundin various tissues where they have different physiologicalfunctions We know three isoenzymes of AP which arespecific for certain tissues in humans these are tissue-specific alkaline phosphatases (TSAP) They are intestinalalkaline phosphatase (IAP Kasahara isoenzyme) placental

alkaline phosphatase (PLAP Regan isoenzyme) and germcell alkaline phosphatase (GCAP Nagao isoenzyme) whichare expressed by embryonic cells but also carcinoma cellsThe fourth isoenzyme is ubiquitous and we call it tissuenonspecific alkaline phosphatase (TNAP) TNAP occurs inthree isoforms which are bone liver and kidney TNAP [5]

In mice there are also four APs three of them aretissue specific and the fourth is tissue nonspecific Intestinalintestinal-like (IAP-like) and embryonicAP (EAP) are TSAPThe IAP-like AP was identified in mice which have a knock-out gene for IAP and still measurable residual activity in thegutMurine EAP is similar to humanGCAP [6]Many impor-tant facts about the structure physical and chemical proper-ties gene localisation regulation function and tissue expres-sion ofAPs can be obtained fromMillan (2006) andMcCombet al (2011) [7 8] In the field of stem cell biology TNAPare the main focus of interest but EAP or GCAP which areexpressed in pluripotent inner call mass and primordial cellsrespectively cannot be ignored (see below) In general TNAPand EAPGCAP are frequently associated with germ layersprogenitors and naive nondifferentiating cells

Hindawi Publishing CorporationStem Cells InternationalVolume 2015 Article ID 628368 11 pageshttpdxdoiorg1011552015628368

2 Stem Cells International

In contrast TSAPs are expressed with the increasingdifferentiation and maturation of particular cell lineages andtissues because of their connection to the functions of suchcells for example enterocytes (IAP) [9ndash11]

2 Expression of AP during Development

Similar to other lineages and developmental-specific genesrecognized as determinants of stem cells the expressionpattern of AP during ontogenesis in some parts of tissuesalso corresponds with particular stem cell precursors andtheir niches Thus here we briefly summarize the expressionand presumable function of particular AP associated withcell differentiation potential (such as pluripotency eg) andstemness during development in general

The expression of alkaline phosphatase can already bedetected in 2-cell stage preimplantation embryos in miceand it is equally expressed in each embryonic cell to thestage of early blastocyst At first it is expressed by both thetrophectoderm and inner cell mass (ICM) In the stage oflate blastocyst it seems that AP is strictly expressed in ICMLepire and Ziomek [12] described the isoenzyme of APin the preimplantation stage of embryo-embryonic alkalinephosphatase (EAP) Until this discovery only two isoenzymesofAPhad been defined inmice EAP andTNAP TNAP is alsoexpressed in the preimplantation embryo but 10 times lessthan EAP Approximately 7 days post coitum (dpc) the expres-sion of Akp5 (EAP) decreases rapidly and Akp2 (TNAP)becomes themajor gene of AP which is expressed between 7ndash14 dpc in primordial germ (PG) cells [6] The PG cells exhibithigh activity of TNAP during migration to the developinggonadThe activity of TNAP decreases 14-15 dpc in these cells[13] In humans it is known that the expression of alkalinephosphatases is detectable prior to 4 weeks of gestation [1415]

In contrast to mice PG cells (which express TNAP)GCAP activity is observed in human migrating PG cells[15ndash17] In the adult it is primarily synthesized in thetestes cervix and thymus Trace amounts are synthesizedin placenta and lung tissues [18] It is not known whetherAP is expressed in the preimplantation stage of humanembryos Also little is known about the expression of otherisoenzymes in human embryonic development because ofethical limitations On the 8th day post coitum (in mice)TNAP is also expressed in the neuroectoderm [19] Later(95 dpc) TNAP activity is observed in the area of the brainand spinal cord Between 105 and 145 dpc TNAP activity isobserved in the mesencephalon and the rhombencephalonalong the entire spinal cord and cranial nerves and at the endof this stage TNAP positive fibres are in the pons Fourteen-and-a-half days post coitum TNAP expression is decreased inthe neuroepithelium In the adult TNAP positive clusters areobserved in the subependymal layer where the neural pro-genitors are localized It is suggested that TNAP is involvedin the development of the nervous system partly as anectonucleotidase in neurogenic zones [20 21] or specificallyby interacting with collagen during neuronal migration [2223] However TNAP activity is detectable in the choroidplexus up to adult age [23]

However amongst all vertebrates (including humans)TNAP activity is dominant in the developing skeletonbecause TNAP is involved in the mineralization of tissuesThe activity within the developing skeleton is associated withthe expression of TNAP in chondrocytes and osteoblasts Inmice this arises between 13 and 14 dpc [19]

The expression of another AP TSAP is also mostlyassociated with cell differentiation and such AP activity isgenerally recognized as amarker of cell differentiation [9 24ndash26]

3 Role and Function of AP

If we ask why some stem cells express AP we must know howAPs are utilized by particular cells In general AP catalyzesthe hydrolysis of phosphate esters AP exhibits three types ofactivity [27ndash31]

(1) hydrolytic activity RndashP + HndashOHrarrRndashOH + HndashP

(2) phosphotransferase activity RndashP + R1015840ndashOHrarrRndashOH+ R1015840P

(3) pyrophosphatase activity RndashPndashPndashR1015840+ HndashOHrarrRndashP+ R1015840ndashP

Hydrolytic activity is considered a general reaction which iscatalysed by AP

The role of individual APs is also apparent from the phe-notype of organisms with nonfunctional AP EAP depletionis not associated with any detectable abnormalities Also thedepletion of TNAP has no effect on the differentiation ormigration of PGC so we may suggest a similar state also forGCAP in human PGC however experimental data are unob-tainable due to ethical limitations PLAPwhich is only knownin primates enables the transport of maternal IgG throughthe placenta and by an unknown mechanism improves thegrowth and development of embryos and cells in generalHigh levels of GCAP and PLAP are also markers of tumordiseases typically such neoplasia as germ cell tumours [3233] squamous cell carcinoma of the lung [34] and carcinomaof the gastrointestinal tract and uterus [35 36]

The key roles of TNAP are in the mineralization ofhard tissue (it provides free phosphate for the creation ofhydroxyapatite crystals and hydrolyses pyrophosphate aninhibitor of bonematrix formation) and in themetabolism ofvitamin B6 and thus in the metabolism of neurotransmitter120574-aminobutyric acid (GABA) Therefore the depletion orrecessive mutation of TNAP leads to defects in both themineralization of hard tissue and the development of thenervous system

IAP plays a role in the transport of fatty acids and triglyc-erides from the intestinal tract to the circulation [37ndash40]IAP also regulates duodenal surface pH [41] and detoxifiesbacterial endotoxins in the colon by means of dephosphory-lation [42 43]

Stem Cells International 3

4 Regulation of the Expression andActivity of AP

AP activity clearly correlates with its expression and itsfine regulation is mediated by the actual microenvironmentrather than by some individual signaling pathways ThusAP expression and activity are regulated mainly through thedevelopmental status of cells or tissues Therefore as men-tioned above AP expression is a generally suitable markerof differentiation processes both in vivo and in vitro forparticular cell types Although some data on the role of p38kinase (mitogen-activate protein kinase (MAPK) p38) in theregulation of TNAP expression exist the precise mechanismremains unknown [44ndash47] Similarly we observed both adecreased level of AP and decreasedAP activity in p38minusminusEScells [48 49] in comparison with their wt counterparts whilethe expressions of pluripotent markers such as Oct-4 Nanogand Zfp42 remained unchanged (our unpublished data)

5 Alkaline Phosphatase and Stem Cells

51 Pluripotent Stem Cells A high level of AP and highAP activity are traditional markers of pluripotent embryonicstem (ES) cells both mouse and human This is based on thefact that ICM is highly positive for AP activity in contrast totrophoblast cells at the blastocyst stage As ICM is committedto lineage differentiation AP expression is downregulatedand it appears in discrete specialized cell populations suchas PG cells and later also in other tissues for example inosteoblasts (see above) High AP activity is associated withthe majority of pluripotent stem cells Embryonal cancer(EC also called teratocarcinoma stem cells) embryonic germ(EG) the already mentioned embryonic stem (ES) andinduced pluripotent stem (iPS) cells express high activityof AP Interestingly the absence of AP activity has beenreported in pluripotent epiblast stem (EpiS) cells which arederived from epiblasts of later developmental stages of theembryo than those from which ES cells are derived Thepluripotency of EpiS cells is partially restricted in comparisonwith other pluripotent stem cells which correspond to theirmore differentiated phenotype compared to ES cells [50 51]

Mouse ES cells are derived from pluripotent cells of ICMof early blastocyst 35ndash4 dpc At this time theAkp5 gene (cod-ing EAP) is dominantly expressed in the embryo Howevermouse ES cells express the Akp2 gene (coding TNAP) whichis used for determination of their undifferentiated state [52]Mouse PG EG and EC cells derived from teratocarcinomaalso show high activity of TNAP [13 53 54] The dominantexpression of TNAP but not EAP in mouse ES cells supportsthe hypothesis concerning a shared predecessor of ES andPGEG cells [55] On the other hand the shift in expressionof AP isoenzymes may also correspond to the fact that theexpression of AP is important for cells but that as cellenvironments are modified during developmental processesparticular AP may work more effectively in new conditionsThis we may suggest is based on the sensitivity of particularAP to various amino acids and small peptides which weredetermined as inhibitors of AP activity these are presented

in Table 1 [5 56ndash62] However further experiments will berequired to verify such a hypothesis

In human embryos GCAP activity is found in PG cells incontrast to human ES cells where TNAP is detected [63 64]However it is uncertain whether GCAP is also expressedwhich might be expected Human EC cells express bothisoenzymes TNAP and GCAP [65] In human EG cells theGCAP isoenzyme is expected because GCAP is expressed inmigrating human primordial germ cells and gonocytes [1863] However precise studies of this phenomenon in humansare lacking

In addition some experimental results point to greatersimilarity betweenmouse ESPGEGECandhumanPGEGEC cells but not human ES cells which raises anotherquestion about the significance and role of AP in these cells[66]

However this inconsistency in the phenotype of thesecells was partially resolved by Brons et al [51] and Tesaret al [50] Both these groups described so-called epiblaststem (EpiS) cells which correspond to pluripotent cells ofepiblast Mouse EpiS cells are more differentiated thanmouseES cells Mouse and human ES cells should be equivalent intheir properties However human ES cells correspond moreclosely to mouse EpiS cells Interestingly EpiS cells do notexhibit detectable AP activity [50] Thus we can suggest thatthe precise determination of AP isoform expression patternsin human ES in comparison to the abovementioned cellpopulations may further improve our picture of its identityregulation of self-renewal and phenotype

The specific importance of AP for pluripotent cells as wellfor pluripotent stem cells remains questionable Andang et alsuggest the importance of GABA synthesis in ES cells for theregulation of their proliferation and self-renewal [67] and thatAP participates in GABA synthesis (see above) We may alsosuggest the increased need for substrate dephosphorylationassociated with the quickly proliferating metabolism of EScells In ICM and other types of pluripotent cells we can alsohypothesise a similar role for AP as in ES cells

Interestingly as mentioned above and expected in EpiScells all known mouse and human pluripotent stem cellsexpress TNAP preferentially The expression of TNAP is alsoquickly upregulated during the process of reprogrammingsomatic cells into iPS cells In the original protocol theiPS phenotype is induced through the exogenous expressionof four genes Oct-4 (Pouf5) Sox-2 and Klf4 which areresponsible for the maintenance of pluripotency and c-myc responsible for the induction andor increasing rate ofproliferation [68] TNAP expression increases directly afterthe transfection of somatic cells by these four genes Thiscorresponds with the conception of the direct regulation ofTNAP expression by Oct-4 and Sox-2 [69] see Table 2ThusTNAP expression appears long before real cell reprogramingand the reexpression of endogenous genes for Oct-4 or Sox-2[70] Further our in silico analysis identified several bindingsites for both Oct-4 and Sox-2 and further factors associatedwith pluripotency such as Nanog Tcf3 Sa4b and FoxD3 inpromoters of TNAP (Table 2) For all the above-mentionedfacts AP activity is a widely accepted marker of pluripotentstem cells and it has been shown that the maintenance


Table1Alkalinep

hosphatasese

xpressed

inhu

mansa

ndmicew

iththeirb

asicattributes

Isoenzym

eAP

Gene

Molecular

weight

Tissue

localization

Temperature

ofinactiv

ation

Specificinh

ibito

rs

PLAP

ALPP

90ndash120

kDa

Sync

ytiotrop

hoblast

Stableat70∘C

neuram

inidase[

5]L-phenylalanine

[58]

L-leucine[60]L-leucyl-glycyl-g

lycine

[56]L-phenylalanyl-g

lycyl-g

lycine

[56]

GCA

PAL

PPL2

PGC

testescervixthym

usEA

P(m

ouse)

Akp5

(Alppl2)

Preimplan

tatio

nstageo

fembryo

IAP

IAP-lik

e(m

ouse)

ALPI

(hum

an)A

kp3

(mou

se)

Akp6

(Alpi)

70ndash9

0kDa

Smallintestin

e(du

odenum

)Stableat56∘C

L-Ph

enylalanine[

58]L-tryptoph

an[59]

Bone

isoform

TNAP

ALPL

(hum

an)

Alpl(m

ouse)

120ndash

150k

Da

Bone

(preosteob

lasticc

ells

basolateralsite

ofosteob

lasts

)chon

drocytesodo

ntob

lasts

PGC(m

ouse)brain

(sub

ependymallayer)

embryotestes(mou

se)

Non

stablea

t55∘C

L-Hom

oarginine[61]neuram

inidase[

5]

L-levamiso

le[57]imidazole[62]

Liverisoform

TNAP

Liver(bille

epith

elium

)At

55∘Cmores

table

than

bone

isoform

Kidn

eyiso

form

TNAP

Kidne

y(epithelialcells

ofproxim

altubu

les)

Non

stablea

t45∘C

PLAP

placentalA

PGCA

Pgerm

cellAP

EAP

embryonicA

PIAP

intestinalA

PTN

AP

tissuen

onspecificA

P


Table 2 Transcription factor binding site (TFBS) analysis of promoters of tissue nonspecific alkaline phosphatase isoforms

Species Gene symbol(name)

Matrix familymatrixV$HOXF V$STEM V$SORY V$FKHD V$RXRF

V$NANOG01 V$OCT3 402 (Oct-4) V$OSNT01 V$SOX201 V$HFH201 (FoxD3) V$RAR RXR01

HumanALPP (PLAP) 1 mdash mdash mdash mdash 2ALPPL2(GCAP) mdash 1 1 1 mdash 1

ALPL (TNAP) 1 3 4 mdash mdash 3

MouseAlppl2 (EAP) 1 mdash mdash mdash mdash mdashAlpl(Akp2TNAP) 1 6lowast 2 1 1 7

NANOG Oct3-4 SOX-2 FoxD3 and RARRXR binding motifs were predicted by the Genomatix software tool MatInspector (1) (digits denote numbersof matches) and further analysed using the rVista algorithm (based on Transfac Professional Library v102) (2) for evolutionary conserved motifs betweenmouse and human (grey fields)The asterisk ldquolowastrdquo symbol indicates previously observed Oct-4 binding in Alpl (Akp2) regulatory sequences using the Chip-PETmethodology (3 4) Matrix V$OSNT01 represents composed binding sites for Oct-4 Sox-2 Nanog Tcf3 (Tcf7l1) and Sall4b in pluripotent cells

of AP activity in AP-positive colony formation positivelycorrelates with the clonogenic and self-renewal potential ofundifferentiated human ES cells in cultures [64] In additionAP activity is downregulated reciprocally with differentiationprocesses involving pluripotent stem cells [71] On the otherhand depletion of the TNAP gene Akp2 has no effect on theformation of either ICM or PG cells or their expansions inmouse [52] Surprisingly at the same time both ICM andPG cells may be considered at least precursors of pluripotentstem cells as are ES or EG cells Unfortunately little is knownabout the inability to isolate ES cells fromTNAPminusminus embryosMore importantly a consequence of TNAP depletion is anegative effect on bone development and vitamin B turnover(see above)

Interestingly although EAP is the dominant form of APin pluripotent ICM we recognized only one Nanog bindingsite within the mouse EAP gene and no binding sites wereobserved for the other pluripotent genesmentioned above Inhuman GCAP one binding site for the mentioned pluripo-tent genes apart from FoxD3 was recognized (Table 2)The mechanism concerning the shift from EAPGCAPexpression in pluripotent ICM to TNAP expression in theother pluripotent cells and PG cells is still not understood

However it seems clear that TNAP expression is notclosely associated with pluripotent stem cells or with pluripo-tency alone because ICM cells express another AP domi-nantly and they have no pure self-renewal potential (and suchICM cells cannot be recognized as stem cells) [19] Further-more PG cells are other cells which strongly express TNAPand other so-called pluripotent markers (Oct-4 Nanog) butthey are not pluripotent

Paradoxically the regulation of TNAP expression inpluripotent stem cells is also influenced by the effectof retinoic acid (RA) RA has a prodifferentiation andpleiotropic effect on many cells both in vivo and in vitro[72 73] The RA effect is fast and strong Cells undergo thedifferentiation process after the addition of RA to a cultureindependently of other conditions The final phenotypedepends on the RA concentration type of culture (adherentsuspension) and presence of particular signaling molecules

[72 74ndash77] It is interesting that TNAP activity increases withthis differentiation although both stemness and pluripotentmarkers decrease This has been proven in both EC andES cells ([78 79] and Figure 2) The upregulation of TNAPexpression is clearly mediated by RA receptors bindingmotifs in the TNAP gene (Table 2) The role and significanceof this RA-induced TNAP expression in EC and ES celldifferentiation are unknown However this observation goesagainst the hypothesis concerning the role of TNAP in fastproliferating cells RA induces a decrease in the proliferationand accumulation of cells in the G1 phase of the cell cycle inboth ES and EC cells [80ndash82]

Further the in vitro study of the differentiation of ES cellsto PG cells yields a remarkable observation RA is required forthe induction of PG cell formation during the EBs-mediateddifferentiation of ES cells [83] In this system 5-day-oldEBs in which TNAP Oct-4 and Nanog expression havebeen downregulated are treated by RA After this treatmentPG cells can be determined within EBs However PG cellsalso express high levels of Oct-4 Nanog and TNAP [84]Increased TNAP may simply be explained by the presenceof RA receptor (RARRXR) binding sites in the TNAP geneas mentioned above However the expression of Oct-4 isdirectly inhibited by RA [85] The expression of Nanog thesecond most important pluripotent gene can be positivelyregulated by RA as has been shown in some recent studies[77 86] In this case Nanog expression is probably inducedthrough the induction of insulin-like growth factor (IGF)expression by RA or other retinoids [86] IGF is a stronginductor of the PI3-K pathway whose activity leads to theincreasing expression of Nanog in ES cells [87 88] ThusRA mediates the auto- and paracrine stimulation of PI3-K pathways by IGF Importantly Nanog itself regulates theexpression of Oct-4 [89] Thus we suppose that in thementioned model of the RA-induced formation of PG cellsOct-4 expression is upregulated and maintained by NanogThe feedback in the regulation network Oct-4Nanog andthe positive role of IGF in the maintenance of ES cells arewell known [87 89 90] However the factors that enable theabove supposed RA-induced creation of Oct-4 positive cells


AP activity

00ES bmMSC aMSC NSC

05

10

15

20

Rela

tive a

ctiv

ity o

f AP

lowast

lowast

(a)

TNAP

000

005

010

015

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast lowastlowast

lowast

ES bmMSC aMSC NSC

(b)

Oct-4

00000

00005

00010010

015

020

Tran

scrip

t lev

el re

lativ

e to

GA

PDH lowastlowast

ES bmMSC aMSC NSC

(c)

Nanog

000

002

004

006

008

010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES bmMSC aMSC NSC

(d)

OstC

0000

0002

0004

0006

0008

0010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowast

lowast

ES bmMSC aMSC NSC

(e)

OstP

0

2

4

6

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowast

lowast

ES bmMSC aMSC NSC

(f)

Figure 1 Relative alkaline phosphatase activity (a) and mRNA expression of TNAP ((b) qRT-PCR) in various stemprogenitor cellsBone marrow-derived MSC (bmMSC) has both high AP activity and TNAP expression followed by ES cells Relative expression (qRT-PCR) of common pluripotent (Oct-4 (c) and Nanog (d)) and osteogenic (Osteocalcin OstC (e) and Osteopontin OstP (f)) genes Ahigh level of Oct-4 and Nanog documented the real pluripotent status of ES cells A higher expression of OstC and OstP mRNA inbmMSC documented their osteogenic propertieslineages specification (ES mouse embryonic stem cells NSC neural stemprogenitorcells bmMSC bone marrow mesenchymal stem cells aMSC adipose tissue mesenchymal stem cells) Details of the presented assay may befound in our previous work [9 77] Primers and conditions for OstC and OstP were as follows OstC 51015840-CTTGGGTTCTGACTGGGTGT-3101584051015840-GCCCTCTGCAGGTCATAGAG-31015840 (212 bp 60∘C) OstP 51015840-TCACCATTCGGATGAGTCTG-31015840 51015840-ACTTGTGGCTCTGATGTTCC-31015840(436 bp 60∘C) Error bars indicate plusmnSD (119899 ge 3 lowast119875 lt 005 lowastlowast119875 lt 001 ANOVA post hoc Bonferronirsquos Multiple Comparison Test)


AP activity

0

1

2

3Re

lativ

e act

ivity

of A

P

lowastlowast

ES dES raES

(a)

TNAP

0000

0002

0004

0006

0008

0010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH lowastlowast

lowast

ES dES raES

(b)

Oct-4

000

005

010

015

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES dES raES

(c)

Nanog

000

002

004

006

008

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES dES raES

(d)

Figure 2 Retinoic acid increasedAP activity (a) andmRNAexpression of TNAP ((b) qRT-PCR) inmouse ES cells On the other hand retinoicacid downregulated the expression of pluripotent markers Oct-4 (c) and Nanog (d) in the same manner as the spontaneous differentiationof ES cells through leukemia inhibitory factor withdrawal [75] (ES mouse embryonic stem cells dES spontaneously differentiating ES cellsfor two days raES retinoic acid-treated (02 120583M) ES cells for two days) Details of the presented assay are as in Figure 1 Error bars indicateplusmnSD (119899 = 4 lowast119875 lt 005 lowastlowast119875 lt 001 ANOVA post hoc Bonferronirsquos Multiple Comparison Test)

(PG cells) in contrast to the fast differentiation of RA-treatedpluripotent stem cells are still unknown We can hypothesizea small degree of balance between the expression of RA-targeted genes and modulation of the Oct-4Nanog networkThe reciprocal ordering of Oct-4 and Nanog expressionduring the establishment of their network may also play animportant role here Recently the preferential role of Nanogin PG cell formation rather than in pluripotency itself has alsobeen demonstrated [91]

52 Other Stem Cells The high expressionactivity of AP inES cells leads to the presumption that it is a universal markerof SC Recent research has not confirmed the accuracy ofsuch an assumption Except for spermatogonia stem cellsa high level of AP has not been definitively demonstratedas a general SC marker in other recognized tissue-specificstem (embryonal or adult) cells or stem cell rich populationsNevertheless Langer et al [20] determined some TNAPpositive cells in embryonal and adult central nervous systemsThese neural TNAP positive cells were observed within cell

populations of the subventricular zonewhich is rich in neuralstem cells and various neural progenitors Some of theseprogenitors were TNAP positive Here TNAPwas not associ-ated with particular progenitors but with subpopulations ofseveral identified progenitors It seems that TNAP expressionin the subventricular zone (SVZ) is not associated with aspecific cell phenotype but with cell behaviour for exampleproliferation or migration [20] This hypothesis is supportedby the mitogenic effect of adenosine generated by TNAPthrough the hydrolyzation of nucleoside triphosphates anddiphosphates [20 92] Further examples of AP activity insomatic stem cells can be found in reports of various setsof mesenchymal stem cells (MSC) [93ndash95] Sobiesiak et al[96] identified TNAP as a stemness marker ofMSC signed asMACS1 Recently however Kim et al [97] presented proost-eogenic properties of MSC with higher TNAP activity MSCderived from adipose tissue exhibit significantly lower levelsof TNAP transcript and activity (Figure 1) This is in contrastto the situation in pluripotent stem cells in which it may besuggested that improved stemness correlates with high TNAP


activity excluding RA effects (see above) The notion thatTNAP in mesenchymal cells is a mark of progenitors ratherthan MSC themselves is also supported by further studiesdocumenting high TNAP in differentiated osteoblasts andodontoblasts [97 98] (Figure 1) In addition side population(SP) cells derived from human dental pulp tissue which arerich in stem cells exhibit lower levels of TNAP mRNA thanthemajor population [98] However further detailed study inthis field and on tissue-derived SP cells would be useful Datacomparing the expressions and activities of TNAP in variousstem cell-enriched populations are presented in Figure 1

6 Conclusion

TNAP expression is a suitable marker of pluripotent stemcells but with some limitations A high level of AP correlatesvery well with pluripotency and undifferentiated pluripotentstem cell phenotypes A low level of AP activity denotes therestriction of pluripotency which was also observed in EpiScells [50 51] and with differentiation Some other limitationsare mentioned above In all other cases that is in adult SCand so forth a high level of AP is associated with the processof differentiation rather than with stemness Thus particularcell types are able to regulate the expression of TNAP andprobably also other APs through various combinations oftranscriptional regulatory networks Accordingly AP such asTNAP for example may be expressed under the control ofOct-4 in pluripotent cells and also in Oct-4 negative cellssuch as mesenchymal cells and their progeny Unfortunatelythe importance of AP activity for pluripotent cells andorstem cells (principally for pluripotent stem cells) is stillgenerally unclear Similarly we do not understand the role ofthe shift in expression of EAPGCAP to TNAP between ICMand ES cells Does it play a role in the pluripotency of stemcells or does it only represent a marker of the common ances-torprecursor of ES and PG cells Curiously enough no PGor ICM cells are considered genuine pluripotent stem cellsPG cells are not pluripotent and both ICM and PG cells havelimited self-renewal potential [55] To answer the question ofwhether APTNAP level and activity are common markersof pluripotent stem cells further work is required

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ministryof Education Youth and Sport of the Czech Repub-lic (MSM0021622430 HistoPARK CZ1072300200185)and the European Regional Development Fund (KI-MUCZ1072300200180)

References

[1] E E Kim and HWWyckoff ldquoReaction mechanism of alkalinephosphatase based on crystal structures Two-metal ion catal-ysisrdquo Journal of Molecular Biology vol 218 no 2 pp 449ndash4641991

[2] M H Le Du T Stigbrand M J Taussig A Menez and E AStura ldquoCrystal structure of alkaline phosphatase from humanplacenta at 18 A resolution implication for a substrate speci-ficityrdquo The Journal of Biological Chemistry vol 276 no 12 pp9158ndash9165 2001

[3] A Kozlenkov T Manes M F Hoylaerts and J L MillanldquoFunction assignment to conserved residues in mammalianalkaline phosphatasesrdquoThe Journal of Biological Chemistry vol277 no 25 pp 22992ndash22999 2002

[4] M-H Le Du and J LMillan ldquoStructural evidence of functionaldivergence in human alkaline phosphatasesrdquo The Journal ofBiological Chemistry vol 277 no 51 pp 49808ndash49814 2002

[5] DWMoss ldquoAlkaline phosphatase isoenzymesrdquo Clinical Chem-istry vol 28 no 10 pp 2007ndash2016 1982

[6] A C Hahnel D A Rappolee J L Millan et al ldquoTwo alkalinephosphatase genes are expressed during early development inthe mouse embryordquo Development vol 110 no 2 pp 555ndash5641990

[7] J L Millan Mammalian Alkaline Phosphatases Wiley-VCHVerlag GmbH amp Co KGaA Weinheim Germany 2006

[8] R M McComb G N Bowers and S Posen Alkaline Phos-phatase Springer New York NY USA 2011

[9] M Kovarıkova J Pachernık J Hofmanova Z Zadak and AKozubık ldquoTNF-alpha modulates the differentiation induced bybutyrate in the HT-29 human colon adenocarcinoma cell linerdquoEuropean Journal of Cancer vol 36 no 14 pp 1844ndash1852 2000

[10] D Krejcova J Prochazkova L Kubala and J Pachernık ldquoMod-ulation of cell proliferation and differentiation of human lungcarcinoma cells by the interferon-alphardquoGeneral Physiology andBiophysics vol 28 no 3 pp 294ndash301 2009

[11] C McCormick R I Freshney and V Speirs ldquoActivity ofinterferon 120572 interleukin 6 and insulin in the regulation ofdifferentiation in A549 alveolar carcinoma cellsrdquo British Journalof Cancer vol 71 no 2 pp 232ndash239 1995

[12] M L Lepire and C A Ziomek ldquoPreimplantation mouseembryos express a heat-stable alkaline phosphataserdquo Biology ofReproduction vol 41 no 3 pp 464ndash473 1989

[13] MGinsburgMH L Snow andAMcLaren ldquoPrimordial germcells in the mouse embryo during gastrulationrdquo Developmentvol 110 no 2 pp 521ndash528 1990

[14] J H Pinkerton D G McKay E C Adams and A T HertigldquoDevelopment of the human ovarymdasha study using histochem-ical technicsrdquo Obstetrics and Gynecology vol 18 pp 152ndash1811961

[15] H Stoop F Honecker M Cools R de Krijger C Bokemeyerand L H J Looijenga ldquoDifferentiation and development ofhuman female germ cells during prenatal gonadogenesis animmunohistochemical studyrdquoHumanReproduction vol 20 no6 pp 1466ndash1476 2005

[16] F E Franke K Pauls R Rey A Marks M Bergmann andK Steger ldquoDifferentiation markers of Sertoli cells and germcells in fetal and early postnatal human testisrdquo Anatomy andEmbryology vol 209 no 2 pp 169ndash177 2004

[17] J Hustin Y Gillerot P Franchimont and J Collette ldquoPlacentalalkaline phosphatase in developing normal and abnormalgonads and in germ-cell tumoursrdquo Virchows Archiv A vol 417no 1 pp 67ndash72 1990

[18] J Hustin J Collette and P Franchimont ldquoImmunohistochem-ical demonstration of placental alkaline phosphatase in variousstates of testicular development and in germ cell tumoursrdquoInternational Journal of Andrology vol 10 no 1 pp 29ndash35 1987


[19] G R MacGregor B P Zambrowicz and P Soriano ldquoTissuenon-specific alkaline phosphatase is expressed in both embry-onic and extraembryonic lineages duringmouse embryogenesisbut is not required for migration of primordial germ cellsrdquoDevelopment vol 121 no 5 pp 1487ndash1496 1995

[20] D Langer Y Ikehara H Takebayashi R Hawkes and H Zim-mermann ldquoThe ectonucleotidases alkaline phosphatase andnucleoside triphosphate diphosphohydrolase 2 are associatedwith subsets of progenitor cell populations in themouse embry-onic postnatal and adult neurogenic zonesrdquo Neuroscience vol150 no 4 pp 863ndash879 2007

[21] V Kermer M Ritter B Albuquerque C Leib M Stanke andH Zimmermann ldquoKnockdown of tissue nonspecific alkalinephosphatase impairs neural stem cell proliferation and differen-tiationrdquo Neuroscience Letters vol 485 no 3 pp 208ndash211 2010

[22] M Bossi M F Hoylaerts and J L Millan ldquoModifications in aflexible surface loop modulate the isozyme-specific propertiesof mammalian alkaline phosphatasesrdquo Journal of BiologicalChemistry vol 268 no 34 pp 25409ndash25416 1993

[23] SNarisawaHHasegawa KWatanabe and J LMillan ldquoStage-specific expression of alkaline phosphatase during neural devel-opment in the mouserdquoDevelopmental Dynamics vol 201 no 3pp 227ndash235 1994

[24] M Hyzdrsquoalova J Hofmanova J Pachernık A Vaculova and AKozubık ldquoThe interaction of butyrate with TNF-alpha duringdifferentiation and apoptosis of colon epithelial cells Role ofNF-kappaB activationrdquo Cytokine vol 44 no 1 pp 33ndash43 2008

[25] J A Barnard andGWarwick ldquoButyrate rapidly induces growthinhibition and differentiation in HT-29 cellsrdquo Cell Growth ampDifferentiation vol 4 no 6 pp 495ndash501 1993

[26] R A Hodin S Meng S Archer and R Tang ldquoCellulargrowth state differentially regulates enterocyte gene expressionin butyrate-treated HT-29 cellsrdquo Cell Growth amp Differentiationvol 7 no 5 pp 647ndash653 1996

[27] W E Hull S E Halford H Gutfreund and B D Sykes ldquo31Pnuclear magnetic resonance study of alkaline phosphatase therole of inorganic phosphate in limiting the enzyme turnover rateat alkaline pHrdquo Biochemistry vol 15 no 7 pp 1547ndash1561 1976

[28] L Zhang M Balcerzak J Radisson et al ldquoPhosphodiesteraseactivity of alkaline phosphatase inATP-initiatedCa+2 and phos-phate deposition in isolated chicken matrix vesiclesrdquo The Jour-nal of Biological Chemistry vol 280 no 44 pp 37289ndash372962005

[29] R P Cox P Gilbert Jr and M J Griffin ldquoAlkaline inorganicpyrophosphatase activity of mammalian-cell alkaline phos-phataserdquo Biochemical Journal vol 105 no 1 pp 155ndash161 1967

[30] J G Georgatsos ldquoSpecificity and phosphotransferase activity ofpurified placental alkaline phosphataserdquo Archives of Biochem-istry and Biophysics vol 121 no 3 pp 619ndash624 1967

[31] R A Stinson J L McPhee and H B Collier ldquoPhosphotrans-ferase activity of human alkaline phosphatases and the role ofenzyme Zn2+rdquo Biochimica et Biophysica ActamdashProtein Structureand Molecular vol 913 no 3 pp 272ndash278 1987

[32] M C Hofmann and J L Millan ldquoDevelopmental expression ofalkaline phosphatase genes reexpression in germ cell tumoursand in vitro immortalized germ cellsrdquo European Urology vol23 no 1 pp 38ndash45 1993

[33] T M Ulbright ldquoGerm cell tumors of the gonads a selectivereview emphasizing problems in differential diagnosis newlyappreciated and controversial issuesrdquo Modern Pathology vol18 no 2 pp S61ndashS79 2005

[34] W H Fishman N R Inglis S Green et al ldquoImmunology andbiochemistry of Regan isoenzyme of alkaline phosphatase inhuman cancerrdquo Nature vol 219 no 5155 pp 697ndash699 1968

[35] M Li J Gao R Feng et al ldquoGeneration ofmonoclonal antibodyMS17-57 targeting secreted alkaline phosphatase ectopicallyexpressed on the surface of gastrointestinal cancer cellsrdquo PLoSONE vol 8 no 10 Article ID e77398 2013

[36] J D Goldsmith B Pawel J R Goldblum et al ldquoDetectionand diagnostic utilization of placental alkaline phosphatase inmuscular tissue and tumors with myogenic differentiationrdquoTheAmerican Journal of Surgical Pathology vol 26 no 12 pp 1627ndash1633 2002

[37] D H Alpers A Mahmood M Engle F Yamagishi and KDeSchryver-Kecskemeti ldquoThe secretion of intestinal alkalinephosphatase (IAP) from the enterocyterdquo Journal of Gastroen-terology vol 29 supplement 7 pp 63ndash67 1994

[38] A Mahmood F Yamagishi R Eliakim K DeSchryver-Kecskemeti T L Gramlich and D H Alpers ldquoA possiblerole for rat intestinal surfactant-like particles in transepithelialtriacylglycerol transportrdquo The Journal of Clinical Investigationvol 93 no 1 pp 70ndash80 1994

[39] J-S Shao M Engle Q Xie et al ldquoEffect of tissue non-specificalkaline phosphatase in maintenance of structure of murinecolon and stomachrdquoMicroscopy Research and Technique vol 51no 2 pp 121ndash128 2000

[40] S Narisawa L Huang A Iwasaki H Hasegawa D H Alpersand J LMillan ldquoAccelerated fat absorption in intestinal alkalinephosphatase knockout micerdquo Molecular and Cellular Biologyvol 23 no 21 pp 7525ndash7530 2003

[41] MMizumoriMHam PHGuth E Engel J DKaunitz andYAkiba ldquoIntestinal alkaline phosphatase regulates protective sur-face microclimate pH in rat duodenumrdquo The Journal of Physi-ology vol 587 no 14 pp 3651ndash3663 2009

[42] J M Bates J Akerlund E Mittge and K Guillemin ldquoIntestinalalkaline phosphatase detoxifies lipopolysaccharide and pre-vents inflammation in zebrafish in response to the gut micro-biotardquo Cell Host and Microbe vol 2 no 6 pp 371ndash382 2007

[43] R F Goldberg W G Austen Jr X Zhang et al ldquoIntestinalalkaline phosphatase is a gutmucosal defense factormaintainedby enteral nutritionrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 9 pp 3551ndash3556 2008

[44] A Suzuki J Guicheux G Palmer et al ldquoEvidence for a role ofp38 MAP kinase in expression of alkaline phosphatase duringosteoblastic cell differentiationrdquo Bone vol 30 no 1 pp 91ndash982002

[45] A Suzuki G Palmer J-P Bonjour and J Caverzasio ldquoReg-ulation of alkaline phosphatase activity by p38 MAP kinasein response to activation of Gi protein-coupled receptors byepinephrine in osteoblast-like cellsrdquo Endocrinology vol 140 no7 pp 3177ndash3182 1999

[46] A Rey D Manen R Rizzoli S L Ferrari and J CaverzasioldquoEvidences for a role of p38 MAP kinase in the stimulationof alkaline phosphatase and matrix mineralization induced byparathyroid hormone in osteoblastic cellsrdquo Bone vol 41 no 1pp 59ndash67 2007

[47] J Caverzasio and D Manen ldquoEssential role of Wnt3a-mediatedactivation of mitogen-activated protein kinase p38 for thestimulation of alkaline phosphatase activity and matrix miner-alization in C3H10T12 mesenchymal cellsrdquo Endocrinology vol148 no 11 pp 5323ndash5330 2007


[48] M Allen L Svensson M Roach J Hambor J McNeish and CA Gabel ldquoDeficiency of the stress kinase p38120572 results in embry-onic lethality characterization of the kinase dependence ofstress responses of enzyme-deficient embryonic stem cellsrdquoTheJournal of Experimental Medicine vol 191 no 5 pp 859ndash8702000

[49] J M Kim J M White A S Shaw and B P SleckmanldquoMAPK p38120572 is dispensable for lymphocyte development andproliferationrdquo Journal of Immunology vol 174 no 3 pp 1239ndash1244 2005

[50] P J Tesar J G Chenoweth F A Brook et al ldquoNew celllines from mouse epiblast share defining features with humanembryonic stem cellsrdquo Nature vol 448 no 7150 pp 196ndash1992007

[51] I GM Brons L E SmithersMW B Trotter et al ldquoDerivationof pluripotent epiblast stem cells from mammalian embryosrdquoNature vol 448 no 7150 pp 191ndash195 2007

[52] SNarisawaN Frohlander and J LMillan ldquoInactivation of twomouse alkaline phosphatase genes and establishment of amodelof infantile hypophosphatasiardquo Developmental Dynamics vol208 no 3 pp 432ndash446 1997

[53] M Buehr ldquoThe primordial germ cells of mammals somecurrent perspectivesrdquo Experimental Cell Research vol 232 no2 pp 194ndash207 1997

[54] N Shimada K Yamada T Tanaka et al ldquoAlterations of geneexpression in endoderm differentiation of F9 teratocarcinomacellsrdquo Molecular Reproduction and Development vol 60 no 2pp 165ndash171 2001

[55] T P Zwaka and J AThomson ldquoA germ cell origin of embryonicstem cellrdquo Development vol 132 no 2 pp 227ndash233 2005

[56] R A Mulivor L I Plotkin and H Harris ldquoDifferentialinhibition of the products of the human alkaline phosphataselocirdquo Annals of Human Genetics vol 42 no 1 pp 1ndash13 1978

[57] H van Belle ldquoAlkaline phosphatase I Kinetics and inhibitionby levamisole of purified isoenzymes from humansrdquo ClinicalChemistry vol 22 no 7 pp 972ndash976 1976

[58] T Komoda S Hokari M Sonoda Y Sakagishi and T TamuraldquoL-phenylalanine inhibition of human alkaline phosphataseswith p-nitrophenyl phosphate as substraterdquo Clinical Chemistryvol 28 no 12 pp 2426ndash2428 1982

[59] C W Lin H G Sie and W H Fishman ldquoL-tryptophan Anon-allosteric organ-specific uncompetitive inhibitor of humanplacental alkaline phosphataserdquo Biochemical Journal vol 124no 3 pp 509ndash516 1971

[60] G J Doellgast andWH Fishman ldquoL leucine a specific inhibitorof a rare human placental alkaline phosphatase phenotyperdquoNature vol 259 no 5538 pp 49ndash51 1976

[61] WH Fishman andH-G Sie ldquoL-Homoarginine an inhibitor ofserum ldquobone and liverrdquo alkaline phosphataserdquo Clinica ChimicaActa vol 29 no 2 pp 339ndash341 1970

[62] C Brunel and G Cathala ldquoImidazole aan inhibitor of l-phenylalanine-insensitive alkaline phosphatases of tissues otherthan intestine and placentardquo Biochimica et Biophysica ActamdashEnzymology vol 268 no 2 pp 415ndash421 1972

[63] E J Nouwen P G Hendrix S Dauwe M W Eerdekens andM E de Broe ldquoTumor markers in the human ovary and itsneoplasms A comparative immunohistochemical studyrdquo TheAmerican Journal of Pathology vol 126 no 2 pp 230ndash242 1987

[64] M D OrsquoConnor M D Kardel I Iosfina et al ldquoAlkalinephosphatase-positive colony formation is a sensitive specificand quantitative indicator of undifferentiated human embry-onic stem cellsrdquo Stem Cells vol 26 no 5 pp 1109ndash1116 2008

[65] M F Pera B Reubinoff and A Trounson ldquoHuman embryonicstem cellsrdquo Journal of Cell Science vol 113 no 1 pp 5ndash10 2000

[66] I Ginis Y Luo TMiura et al ldquoDifferences between human andmouse embryonic stem cellsrdquo Developmental Biology vol 269no 2 pp 360ndash380 2004

[67] M Andang J Hjerling-Leffler A Moliner et al ldquoHistoneH2AX-dependent GABA(A) receptor regulation of stem cellproliferationrdquo Nature vol 451 no 7177 pp 460ndash464 2008

[68] S Yamanaka and K Takahashi ldquoInduction of pluripotent stemcells from mouse fibroblast culturesrdquo Tanpakushitsu KakusanKoso Protein Nucleic Acid Enzyme vol 51 no 15 pp 2346ndash2351 2006

[69] Y-H Loh Q Wu J-L Chew et al ldquoThe Oct4 and Nanog tran-scription network regulates pluripotency in mouse embryonicstem cellsrdquo Nature Genetics vol 38 no 4 pp 431ndash440 2006

[70] F Gonzalez S Boue and J C I Belmonte ldquoMethods formaking induced pluripotent stem cells reprogramming a lacarterdquo Nature Reviews Genetics vol 12 no 4 pp 231ndash242 2011

[71] R L Williams D J Hilton S Pease et al ldquoMyeloid leukaemiainhibitory factor maintains the developmental potential ofembryonic stem cellsrdquo Nature vol 336 no 6200 pp 684ndash6871988

[72] J Rohwedel K Guan and A M Wobus ldquoInduction of cellulardifferentiation by retinoic acid in vitrordquo Cells Tissues Organsvol 165 no 3-4 pp 190ndash202 1999

[73] M Mark N B Ghyselinck and P Chambon ldquoFunction ofretinoic acid receptors during embryonic developmentrdquoNuclear Receptor Signaling vol 7 article e002 2009

[74] C L Mummery A Feyen E Freund and S Shen ldquoCharac-teristics of embryonic stem cell differentiation a comparisonwith two embryonal carcinoma cell linesrdquo Cell Differentiationand Development vol 30 no 3 pp 195ndash206 1990

[75] J Pachernık M Esner V Bryja P Dvorak and A HamplldquoNeural differentiation ofmouse embryonic stem cells grown inmonolayerrdquo Reproduction Nutrition Development vol 42 no 4pp 317ndash326 2002

[76] J Pachernık V Bryja M Esner L Kubala P Dvorak and AHampl ldquoNeural differentiation of pluripotentmouse embryonalcarcinoma cells by retinoic acid inhibitory effect of serumrdquoPhysiological Research vol 54 no 1 pp 115ndash122 2005

[77] H Kotasova I Vesela J Kucera et al ldquoPhosphoinositide 3-kinase inhibition enables retinoic acid-induced neurogenesis inmonolayer culture of embryonic stem cellsrdquo Journal of CellularBiochemistry vol 113 no 2 pp 563ndash570 2012

[78] M Giannirsquo M Studer G Carpani M Terao and E Garat-tini ldquoRetinoic acid induces liverbonekidney-type alkalinephosphatase gene expression in F9 teratocarcinoma cellsrdquo TheBiochemical Journal vol 274 no 3 pp 673ndash678 1991

[79] R J Scheibe I Moeller-Runge and W H Mueller ldquoRetinoicacid induces the expression of alkaline phosphatase in P19teratocarcinoma cellsrdquoThe Journal of Biological Chemistry vol266 no 31 pp 21300ndash21305 1991

[80] H Preclıkova V Bryja J Pachernık P Krejcı P Dvorak and AHampl ldquoEarly cycling-independent changes to p27 cyclin D2and cyclin D3 in differentiating mouse embryonal carcinomacellsrdquoCell Growth andDifferentiation vol 13 no 9 pp 421ndash4302002

[81] V Bryja J Pachernık J Vondracek et al ldquoLineage specificcomposition of cyclin D-CDK4CDK6-p27 complexes revealsdistinct functions of CDK4 CDK6 and individual D-typecyclins in differentiating cells of embryonic originrdquo Cell Prolif-eration vol 41 no 6 pp 875ndash893 2008


[82] V Bryja J Pachernık K Soucek V Horvath P Dvorak andA Hampl ldquoIncreased apoptosis in differentiating p27-deficientmouse embryonic stem cellsrdquo Cellular and Molecular LifeSciences vol 61 no 11 pp 1384ndash1400 2004

[83] K Nayernia J Nolte H W Michelmann et al ldquoIn vitro-differentiated embryonic stem cells give rise to male gametesthat can generate offspringmicerdquoDevelopmental Cell vol 11 no1 pp 125ndash132 2006

[84] A M Elliott M P de Miguel V I Rebel and P J DonovanldquoIdentifying genes differentially expressed between PGCs andES cells reveals a role for CREB-binding protein in germ cellsurvivalrdquo Developmental Biology vol 311 no 2 pp 347ndash3582007

[85] E Pikarsky H Sharir E Ben-Shushan and Y BergmanldquoRetinoic acid represses Oct-34 gene expression through sev-eral retinoic acid-responsive elements located in the promoter-enhancer regionrdquoMolecular and Cellular Biology vol 14 no 2pp 1026ndash1038 1994

[86] J S Kim B S Kim J Kim C-S Park and I Y Chung ldquoThephosphoinositide-3-kinaseAkt pathway mediates the transientincrease in Nanog expression during differentiation of F9 cellsrdquoArchives of Pharmacal Research vol 33 no 7 pp 1117ndash1125 2010

[87] D Hallmann K Trumper H Trusheim et al ldquoAltered signalingand cell cycle regulation in embryonal stem cells with adisruption of the gene for phosphoinositide 3-kinase regulatorysubunit p85120572rdquoThe Journal of Biological Chemistry vol 278 no7 pp 5099ndash5108 2003

[88] N R D Paling H Wheadon H K Bone and M J WelhamldquoRegulation of embryonic stem cell self-renewal by phospho-inositide 3-kinase-dependent signalingrdquoThe Journal of Biologi-cal Chemistry vol 279 no 46 pp 48063ndash48070 2004

[89] D J Rodda J-L Chew L-H Lim et al ldquoTranscriptionalregulation of Nanog by OCT4 and SOX2rdquo Journal of BiologicalChemistry vol 280 no 26 pp 24731ndash24737 2005

[90] G Pan J Li Y ZhouH Zheng andD Pei ldquoA negative feedbackloop of transcription factors that controls stem cell pluripotencyand self-renewalrdquoThe FASEB Journal vol 20 no 10 pp 1730ndash1732 2006

[91] S Yamaguchi K Kurimoto Y Yabuta et al ldquoConditionalknockdown of Nanog induces apoptotic cell death in mousemigrating primordial germ cellsrdquo Development vol 136 no 23pp 4011ndash4020 2009

[92] P Heine N Braun A Heilbronn and H ZimmermannldquoFunctional characterization of rat ecto-ATPase and ecto-ATPdiphosphohydrolase after heterologous expression in CHOcellsrdquo European Journal of Biochemistry vol 262 no 1 pp 102ndash107 1999

[93] R K Jaiswal N Jaiswal S P Bruder G Mbalaviele D R Mar-shak andM F Pittenger ldquoAdult humanmesenchymal stem celldifferentiation to the osteogenic or adipogenic lineage is regu-lated by mitogen-activated protein kinaserdquo The Journal of Bio-logical Chemistry vol 275 no 13 pp 9645ndash9652 2000

[94] H-J Buhring S Treml F Cerabona P De Zwart L Kanz andM Sobiesiak ldquoPhenotypic characterization of distinct humanbone marrow-derived MSC subsetsrdquo Annals of the New YorkAcademy of Sciences vol 1176 pp 124ndash134 2009

[95] C E Gargett and H Masuda ldquoAdult stem cells in theendometriumrdquo Molecular Human Reproduction vol 16 no 11pp 818ndash834 2010

[96] M Sobiesiak K Sivasubramaniyan C Hermann et al ldquoThemesenchymal stem cell antigen MSCA-1 is identical to tissue

non-specific alkaline phosphataserdquo Stem Cells and Develop-ment vol 19 no 5 pp 669ndash677 2010

[97] Y H Kim D S Yoon H O Kim and J W Lee ldquoCharacter-ization of different subpopulations from bone marrow-derivedmesenchymal stromal cells by alkaline phosphatase expressionrdquoStemCells andDevelopment vol 21 no 16 pp 2958ndash2968 2012

[98] M Kenmotsu K Matsuzaka E Kokubu T Azuma and TInoue ldquoAnalysis of side population cells derived from dentalpulp tissuerdquo International Endodontic Journal vol 43 no 12 pp1132ndash1142 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


In contrast TSAPs are expressed with the increasingdifferentiation and maturation of particular cell lineages andtissues because of their connection to the functions of suchcells for example enterocytes (IAP) [9ndash11]

2 Expression of AP during Development

Similar to other lineages and developmental-specific genesrecognized as determinants of stem cells the expressionpattern of AP during ontogenesis in some parts of tissuesalso corresponds with particular stem cell precursors andtheir niches Thus here we briefly summarize the expressionand presumable function of particular AP associated withcell differentiation potential (such as pluripotency eg) andstemness during development in general

The expression of alkaline phosphatase can already bedetected in 2-cell stage preimplantation embryos in miceand it is equally expressed in each embryonic cell to thestage of early blastocyst At first it is expressed by both thetrophectoderm and inner cell mass (ICM) In the stage oflate blastocyst it seems that AP is strictly expressed in ICMLepire and Ziomek [12] described the isoenzyme of APin the preimplantation stage of embryo-embryonic alkalinephosphatase (EAP) Until this discovery only two isoenzymesofAPhad been defined inmice EAP andTNAP TNAP is alsoexpressed in the preimplantation embryo but 10 times lessthan EAP Approximately 7 days post coitum (dpc) the expres-sion of Akp5 (EAP) decreases rapidly and Akp2 (TNAP)becomes themajor gene of AP which is expressed between 7ndash14 dpc in primordial germ (PG) cells [6] The PG cells exhibithigh activity of TNAP during migration to the developinggonadThe activity of TNAP decreases 14-15 dpc in these cells[13] In humans it is known that the expression of alkalinephosphatases is detectable prior to 4 weeks of gestation [1415]

In contrast to mice PG cells (which express TNAP)GCAP activity is observed in human migrating PG cells[15ndash17] In the adult it is primarily synthesized in thetestes cervix and thymus Trace amounts are synthesizedin placenta and lung tissues [18] It is not known whetherAP is expressed in the preimplantation stage of humanembryos Also little is known about the expression of otherisoenzymes in human embryonic development because ofethical limitations On the 8th day post coitum (in mice)TNAP is also expressed in the neuroectoderm [19] Later(95 dpc) TNAP activity is observed in the area of the brainand spinal cord Between 105 and 145 dpc TNAP activity isobserved in the mesencephalon and the rhombencephalonalong the entire spinal cord and cranial nerves and at the endof this stage TNAP positive fibres are in the pons Fourteen-and-a-half days post coitum TNAP expression is decreased inthe neuroepithelium In the adult TNAP positive clusters areobserved in the subependymal layer where the neural pro-genitors are localized It is suggested that TNAP is involvedin the development of the nervous system partly as anectonucleotidase in neurogenic zones [20 21] or specificallyby interacting with collagen during neuronal migration [2223] However TNAP activity is detectable in the choroidplexus up to adult age [23]

However amongst all vertebrates (including humans)TNAP activity is dominant in the developing skeletonbecause TNAP is involved in the mineralization of tissuesThe activity within the developing skeleton is associated withthe expression of TNAP in chondrocytes and osteoblasts Inmice this arises between 13 and 14 dpc [19]

The expression of another AP TSAP is also mostlyassociated with cell differentiation and such AP activity isgenerally recognized as amarker of cell differentiation [9 24ndash26]

3 Role and Function of AP

If we ask why some stem cells express AP we must know howAPs are utilized by particular cells In general AP catalyzesthe hydrolysis of phosphate esters AP exhibits three types ofactivity [27ndash31]

(1) hydrolytic activity RndashP + HndashOHrarrRndashOH + HndashP

(2) phosphotransferase activity RndashP + R1015840ndashOHrarrRndashOH+ R1015840P

(3) pyrophosphatase activity RndashPndashPndashR1015840+ HndashOHrarrRndashP+ R1015840ndashP

Hydrolytic activity is considered a general reaction which iscatalysed by AP

The role of individual APs is also apparent from the phe-notype of organisms with nonfunctional AP EAP depletionis not associated with any detectable abnormalities Also thedepletion of TNAP has no effect on the differentiation ormigration of PGC so we may suggest a similar state also forGCAP in human PGC however experimental data are unob-tainable due to ethical limitations PLAPwhich is only knownin primates enables the transport of maternal IgG throughthe placenta and by an unknown mechanism improves thegrowth and development of embryos and cells in generalHigh levels of GCAP and PLAP are also markers of tumordiseases typically such neoplasia as germ cell tumours [3233] squamous cell carcinoma of the lung [34] and carcinomaof the gastrointestinal tract and uterus [35 36]

The key roles of TNAP are in the mineralization ofhard tissue (it provides free phosphate for the creation ofhydroxyapatite crystals and hydrolyses pyrophosphate aninhibitor of bonematrix formation) and in themetabolism ofvitamin B6 and thus in the metabolism of neurotransmitter120574-aminobutyric acid (GABA) Therefore the depletion orrecessive mutation of TNAP leads to defects in both themineralization of hard tissue and the development of thenervous system

IAP plays a role in the transport of fatty acids and triglyc-erides from the intestinal tract to the circulation [37ndash40]IAP also regulates duodenal surface pH [41] and detoxifiesbacterial endotoxins in the colon by means of dephosphory-lation [42 43]














Table1Alkalinep

hosphatasese

xpressed

inhu

mansa

ndmicew

iththeirb

asicattributes

Isoenzym

eAP

Gene

Molecular

weight

Tissue

localization

Temperature

ofinactiv

ation

Specificinh

ibito

rs

PLAP

ALPP

90ndash120

kDa

Sync

ytiotrop

hoblast

Stableat70∘C

neuram

inidase[

5]L-phenylalanine

[58]


lycine


lycyl-g

lycine

[56]

GCA

PAL

PPL2

PGC

testescervixthym

usEA

P(m

ouse)

Akp5

(Alppl2)

Preimplan

tatio

nstageo

fembryo

IAP

IAP-lik

e(m

ouse)

ALPI

(hum

an)A

kp3

(mou

se)

Akp6

(Alpi)

70ndash9

0kDa

Smallintestin

e(du

odenum

)Stableat56∘C

L-Ph

enylalanine[

58]L-tryptoph

an[59]

Bone

isoform

TNAP

ALPL

(hum

an)

Alpl(m

ouse)

120ndash

150k

Da

Bone

(preosteob

lasticc

ells

basolateralsite

ofosteob

lasts

)chon

drocytesodo

ntob

lasts

PGC(m

ouse)brain

(sub

ependymallayer)

embryotestes(mou

se)

Non

stablea

t55∘C

L-Hom

oarginine[61]neuram

inidase[

5]

L-levamiso

le[57]imidazole[62]

Liverisoform

TNAP

Liver(bille

epith

elium

)At

55∘Cmores

table

than

bone

isoform

Kidn

eyiso

form

TNAP

Kidne

y(epithelialcells

ofproxim

altubu

les)

Non

stablea

t45∘C

PLAP

placentalA

PGCA

Pgerm

cellAP

EAP

embryonicA

PIAP

intestinalA

PTN

AP

tissuen

onspecificA

P

















AP activity

00ES bmMSC aMSC NSC

05

10

15

20

Rela

tive a

ctiv

ity o

f AP

lowast

lowast

(a)

TNAP

000

005

010

015

Tran

scrip

t lev

el re

lativ

e to

GA

PDH


lowast

ES bmMSC aMSC NSC

(b)

Oct-4

00000

00005

00010010

015

020

Tran

scrip

t lev

el re

lativ

e to

GA

PDH lowastlowast

ES bmMSC aMSC NSC

(c)

Nanog

000

002

004

006

008

010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES bmMSC aMSC NSC

(d)

OstC

0000

0002

0004

0006

0008

0010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowast

lowast

ES bmMSC aMSC NSC

(e)

OstP

0

2

4

6

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowast

lowast

ES bmMSC aMSC NSC

(f)



AP activity

0

1

2

3Re

lativ

e act

ivity

of A

P

lowastlowast

ES dES raES

(a)

TNAP

0000

0002

0004

0006

0008

0010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH lowastlowast

lowast

ES dES raES

(b)

Oct-4

000

005

010

015

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES dES raES

(c)

Nanog

000

002

004

006

008

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES dES raES

(d)







6 Conclusion




Acknowledgments


References














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Table1Alkalinep

hosphatasese

xpressed

inhu

mansa

ndmicew

iththeirb

asicattributes

Isoenzym

eAP

Gene

Molecular

weight

Tissue

localization

Temperature

ofinactiv

ation

Specificinh

ibito

rs

PLAP

ALPP

90ndash120

kDa

Sync

ytiotrop

hoblast

Stableat70∘C

neuram

inidase[

5]L-phenylalanine

[58]


lycine


lycyl-g

lycine

[56]

GCA

PAL

PPL2

PGC

testescervixthym

usEA

P(m

ouse)

Akp5

(Alppl2)

Preimplan

tatio

nstageo

fembryo

IAP

IAP-lik

e(m

ouse)

ALPI

(hum

an)A

kp3

(mou

se)

Akp6

(Alpi)

70ndash9

0kDa

Smallintestin

e(du

odenum

)Stableat56∘C

L-Ph

enylalanine[

58]L-tryptoph

an[59]

Bone

isoform

TNAP

ALPL

(hum

an)

Alpl(m

ouse)

120ndash

150k

Da

Bone

(preosteob

lasticc

ells

basolateralsite

ofosteob

lasts

)chon

drocytesodo

ntob

lasts

PGC(m

ouse)brain

(sub

ependymallayer)

embryotestes(mou

se)

Non

stablea

t55∘C

L-Hom

oarginine[61]neuram

inidase[

5]

L-levamiso

le[57]imidazole[62]

Liverisoform

TNAP

Liver(bille

epith

elium

)At

55∘Cmores

table

than

bone

isoform

Kidn

eyiso

form

TNAP

Kidne

y(epithelialcells

ofproxim

altubu

les)

Non

stablea

t45∘C

PLAP

placentalA

PGCA

Pgerm

cellAP

EAP

embryonicA

PIAP

intestinalA

PTN

AP

tissuen

onspecificA

P

















AP activity

00ES bmMSC aMSC NSC

05

10

15

20

Rela

tive a

ctiv

ity o

f AP

lowast

lowast

(a)

TNAP

000

005

010

015

Tran

scrip

t lev

el re

lativ

e to

GA

PDH


lowast

ES bmMSC aMSC NSC

(b)

Oct-4

00000

00005

00010010

015

020

Tran

scrip

t lev

el re

lativ

e to

GA

PDH lowastlowast

ES bmMSC aMSC NSC

(c)

Nanog

000

002

004

006

008

010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES bmMSC aMSC NSC

(d)

OstC

0000

0002

0004

0006

0008

0010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowast

lowast

ES bmMSC aMSC NSC

(e)

OstP

0

2

4

6

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowast

lowast

ES bmMSC aMSC NSC

(f)



AP activity

0

1

2

3Re

lativ

e act

ivity

of A

P

lowastlowast

ES dES raES

(a)

TNAP

0000

0002

0004

0006

0008

0010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH lowastlowast

lowast

ES dES raES

(b)

Oct-4

000

005

010

015

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES dES raES

(c)

Nanog

000

002

004

006

008

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES dES raES

(d)







6 Conclusion




Acknowledgments


References














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















AP activity

00ES bmMSC aMSC NSC

05

10

15

20

Rela

tive a

ctiv

ity o

f AP

lowast

lowast

(a)

TNAP

000

005

010

015

Tran

scrip

t lev

el re

lativ

e to

GA

PDH


lowast

ES bmMSC aMSC NSC

(b)

Oct-4

00000

00005

00010010

015

020

Tran

scrip

t lev

el re

lativ

e to

GA

PDH lowastlowast

ES bmMSC aMSC NSC

(c)

Nanog

000

002

004

006

008

010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES bmMSC aMSC NSC

(d)

OstC

0000

0002

0004

0006

0008

0010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowast

lowast

ES bmMSC aMSC NSC

(e)

OstP

0

2

4

6

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowast

lowast

ES bmMSC aMSC NSC

(f)



AP activity

0

1

2

3Re

lativ

e act

ivity

of A

P

lowastlowast

ES dES raES

(a)

TNAP

0000

0002

0004

0006

0008

0010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH lowastlowast

lowast

ES dES raES

(b)

Oct-4

000

005

010

015

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES dES raES

(c)

Nanog

000

002

004

006

008

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES dES raES

(d)







6 Conclusion




Acknowledgments


References














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


AP activity

0

1

2

3Re

lativ

e act

ivity

of A

P

lowastlowast

ES dES raES

(a)

TNAP

0000

0002

0004

0006

0008

0010

Tran

scrip

t lev

el re

lativ

e to

GA

PDH lowastlowast

lowast

ES dES raES

(b)

Oct-4

000

005

010

015

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES dES raES

(c)

Nanog

000

002

004

006

008

Tran

scrip

t lev

el re

lativ

e to

GA

PDH

lowastlowast

ES dES raES

(d)







6 Conclusion




Acknowledgments


References














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



6 Conclusion




Acknowledgments


References














































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article alkaline phosphatase in stem...

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