Abdulrazzak et al. - 2010 - Biological characteristics of stem cells from foetal, cord blood and...

doi: 10.1098/rsif.2010.0347.focus published online 25 August 2010J. R. Soc. Interface

Hassan Abdulrazzak, Dafni Moschidou, Gemma Jones and Pascale V. Guillot and extraembryonic tissuesBiological characteristics of stem cells from foetal, cord blood

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doi:10.1098/rsif.2010.0347.focusPublished online

REVIEW

*Author for c

One contribcommercializa

Received 30 JAccepted 5 A

Biological characteristics of stem cellsfrom foetal, cord blood and

extraembryonic tissuesHassan Abdulrazzak, Dafni Moschidou, Gemma Jones

and Pascale V. Guillot*

Institute of Reproductive and Developmental Biology, Imperial College London,London W12 0NN, UK

Foetal stem cells (FSCs) can be isolated during gestation from many different tissues such asblood, liver and bone marrow as well as from a variety of extraembryonic tissues such asamniotic fluid and placenta. Strong evidence suggests that these cells differ on many biolo-gical aspects such as growth kinetics, morphology, immunophenotype, differentiationpotential and engraftment capacity in vivo. Despite these differences, FSCs appear to bemore primitive and have greater multi-potentiality than their adult counterparts. Forexample, foetal blood haemopoietic stem cells proliferate more rapidly than those found incord blood or adult bone marrow. These features have led to FSCs being investigated forpre- and post-natal cell therapy and regenerative medicine applications. The cells havebeen used in pre-clinical studies to treat a wide range of diseases such as skeletal dysplasia,diaphragmatic hernia and respiratory failure, white matter damage, renal pathologies aswell as cancers. Their intermediate state between adult and embryonic stem cells alsomakes them an ideal candidate for reprogramming to the pluripotent status.

Keywords: stem cell; foetal; regenerative medicine; induced pluripotent stem

1. INTRODUCTION

Stem cells are undifferentiated cells with the capacity toself-renew, differentiate and repopulate a host in vivo(Weissman et al. 2001). Their plasticity or potency ishierarchical ranging from totipotent (differentiatinginto all cell types including placenta), pluripotent (dif-ferentiating into cells of the three germ layers,ectoderm, mesoderm and endoderm, but not tropho-blastic cells), multipotent (differentiating into cells ofmore than one type but not necessarily into all thecells of a given germ layer) to unipotent (differentiatinginto one type of cell only, e.g. muscle or neuron).

Pluripotent stem cells can be derived from the innercell mass of the pre-implantation embryo (i.e. embryo-nic stem (ES) cells) or isolated from the foetalprimordial germ cell pool (PGC) above the allantois(i.e. embryonic germ (EG) cells and embryonic carci-noma (EC) cells; Thomson et al. 1998; Andrews et al.2005). The destruction of the blastocyst or earlyfoetus necessary for their derivation/isolation raisesethical concerns (Lo & Parham 2009), although recent

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ution to a Theme Supplement ‘Translation andtion of regenerative medicines’.

une 2010ugust 2010 1

work has shown that ES cells can be derived fromsingle blastomeres isolated using procedures similar tothose routinely used for pre-implantation genetic diag-nosis (Klimanskaya et al. 2007). However, safetyconcerns still remain because of the tumorigenicity ofES cells. Alternatively, adult stem cells can be foundin almost all tissues examined including brain, dentalpulp, muscle, bone marrow, skin and pancreas andhave been extensively characterized for their thera-peutic potential. The adult stem cell could be multi-potent (e.g. haematopoietic stem cells (HSCs) givingrise to all blood cells and adherent stromal/mesenchy-mal stem cells (MSCs) that give rise to bone, fat,cartilage and muscle) or unipotent (e.g. progenitorcells). Adult MSCs have the problem of being difficultto extract in sufficient numbers for therapy and/or pre-senting restricted plasticity and limited proliferativecapacity compared with ES cells.

In recent years, foetal stem cells (FSCs) and stemcells isolated from cord blood or extraembryonic tissueshave emerged as a potential ‘half way house’ betweenES cells and adult stem cells. FSCs can be foundin foetal tissues such as blood, liver, bone marrow,pancreas, spleen and kidney, and stem cells are alsofound in cord blood and extraembryonic tissues suchas amniotic fluid, placenta and amnion (Marcus &

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2 Review. Characteristics of stem cells H. Abdulrazzak et al.


Woodbury 2008). Their primitive properties, expansionpotential and lack of tumorogenicity make them anattractive option for regenerative medicine in celltherapy and tissue engineering settings. While extraem-bryonic tissues could be used with few ethicalreservations, the isolation of FSCs from abortuses issubject to significant public unease. We review herethe phenotypic characteristics of stem cells fromfoetal, cord blood and extraembryonic tissues, theirapplication in cell therapy and their potential for repro-gramming towards pluripotentiality.

2. PHENOTYPIC CHARACTERISTICS OFFOETAL STEM CELLS

Stem cells are collected from abortal foetal tissue,surplus of pre-natal diagnostic tissues or tissues atdelivery, subject to informed consent, institutionalethics approval and compliance with national guidelinescovering foetal tissue research. Until recently theywere thought to be multi-potent (O’Donoghue & Fisk2004), but this picture is now changing as evidence ismounting regarding the existence of pluripotent sub-populations in some foetal and extraembryonic tissues(Cananzi et al. 2009). Some of the properties of thecells are summarized in table 1.

2.1. Foetal tissues

2.1.1. Foetal MSCs. MSCs are multi-potent stem cellsthat can differentiate towards mesoderm-derivedlineages (i.e. osteogenic, adipogenic, chondrogenic andmyogenic; Pittenger et al. 1999). Their potential torepair damaged tissue and their immunomodulatoryproperties make them very attractive for a wide rangeof regenerative medicine applications such as celltherapy and tissue engineering for hollow and solidorgans (Horwitz et al. 2002; Abdallah & Kassem2008; Le Blanc et al. 2008). They were first identifiedin adult bone marrow where they represent 0.001–0.01% of total nucleated cells (Owen & Friedenstein1988). Adherent stromal cells with similar characteristicswere subsequently isolated from other tissues such as adi-pose, dental pulp, muscle, liver and brain (Porada et al.2006; Huang et al. 2009). They are also found in foetaland extraembryonic tissues, where they seem to possessgreater proliferation capacity and differentiation poten-tial than their adult counterparts (Campagnoli et al.2001; Guillot et al. 2007b; Pappa & Anagnou 2009).

2.1.1.1. Common characteristics. MSCs isolated fromfoetal tissues such as blood, liver, bone marrow, lungand pancreas all share common characteristics. Forexample, they are spindle-shaped cells with the capacityto differentiate into the standard mesenchymallineages, i.e. bone, fat and cartilage. They do notexpress haematopoietic or endothelial markers (e.g.they are CD452/342/142 and von Willebrand factornegative; Gucciardo et al. 2009), but express stroma-associated markers CD29 (b1-integrin), CD73 (SH3and SH4), CD105 (SH2), CD44 (HCAM1), the earlybone marrow progenitor cell marker CD90 (thy-1) andthe extracellular matrix proteins vimentin, laminin and


fibronectin (Guillot et al. 2006, 2007a). Contrary toadult bone marrow MSCs, first-trimester foetal blood,liver and bone marrow MSCs express baseline levels ofthe pluripotency stem cell markers Oct-4, Nanog, Rex-1,SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81 (Guillot et al.2007b; Zhang et al. 2009). Regardless of their tissue oforigin, first-trimester foetal MSCs self-renew faster in cul-ture than adult MSCs (30–35 h versus 80–100 h) andsenesce later while retaining a stable phenotype (Guillotet al. 2007b). Foetal MSCs are therefore more readilyexpandable to therapeutic scales for either pre- or post-natal ex vivo gene or cell therapy and tissue engineering.Having greater multi-potentiality and differentiatingmore readily than adult MSCs into cells of mesodermalorigin such as bone and muscle, they can also differentiateinto cells from other lineages such as oligodendrocytesand haematopoietic cells (MacKenzie et al. 2001; Chanet al. 2006; Kennea et al. 2009). In terms of engraftment,they have a competitive advantage compared with adultcells (Rebel et al. 1996; Harrison et al. 1997; Taylor et al.2002). Other advantageous characteristics relevant to celltherapy include having active telomerase (Guillot et al.2007b), expressing low levels of HLA I and lacking intra-cellular HLA II, and taking longer to express this uponinterferon g stimulation than adult MSCs (Gotherstromet al. 2004). Foetal MSCs also express a shared a2, a4and a5b1 integrin profile with first-trimester HSCs, impli-cating them in homing and engraftment, and, consistentwith this, they have significantly greater binding totheir respective extracellular matrix ligands than adultMSCs (de la Fuente et al. 2003). Finally, human foetalMSCs are readily transducible with transduction efficien-cies of greater than 95 per cent using lentiviral vectorswith stable gene expression at both short- and long-timepoints without affecting self-renewal or multi-potentiality(Chan et al. 2005).

2.1.1.2. Specific characteristics. Although the abovecharacteristics are shared among the various types offoetal stem, they also have specific traits that are depen-dent on tissue of origin and gestational age. For example,foetal MSCs are present in foetal blood from the earliestgestation tested, seven weeks, where they account forapproximately 0.4 per cent of foetal nucleated cells.Their numbers decline to very low levels after 13weeks’ gestation (Campagnoli et al. 2001). MSC derivedfrom foetal bone marrow also decline with age with oneMSC found among 10 000 in mid-trimester as comparedwith one MSC per 250 000 cells in adult marrow.

Significant variations have been found among foetaltissue cells in terms of cell surface markers. Forexample, foetal lung has a higher proportion ofCD34þ/CD452 cells (44%) than bone marrow (4.8%),spleen (12.6%) and liver (7.5%) (in’t Anker et al.2003a,b). Also foetal metanephric MSCs do not expressCD45, CD34 or other haematopoietic markers; instead,they express high levels of mesenchymal markers suchas vimentin, laminin and type I collagen.

There is also variation among the cells in terms ofdifferentiation capacity. MSCs derived from foetalliver during the first and second trimester have areduced osteogenic differentiation potential in


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etal

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Review. Characteristics of stem cells H. Abdulrazzak et al. 3






comparison with first-trimester foetal blood and withsecond-trimester spleen, lung and bone marrow MSCs,possibly because of a reduction in osteogenic progenitornumbers (in‘t Anker et al. 2003a,b). Foetal pancreaticMSCs, shown to be capable of differentiating into chon-drogenic, osteogenic or adipogenic lineages (Hu et al.2003), can also engraft, differentiate and secretehuman insulin in a sheep model (Ersek et al. 2010).Moreover, foetal pancreatic ductal stem cells candifferentiate into insulin-producing cells in vitro (Yaoet al. 2004).

Foetal metanephric MSCs can be induced in vitro toadopt an osteogenic or myogenic phenotype and, whentransplanted in a foetal lamb model, they show long-term persistence and site-specific differentiation(Almeida-Porada et al. 2002).

Altogether these data indicate a high level ofheterogeneity within the foetal stem cell pool.

2.1.2. Foetal HSCs. HSCs are multi-potent stem cellsthat maintain functional haematopoiesis by generationof all haematopoietic lineages throughout foetal andadult life (Weissman & Shizuru 2008). They are charac-terized by the expression of CD34 and CD45 antigens,and the absence of markers such as CD38 and humanleucocyte antigen (HLA)/DRE (Huss 2000; Tayloret al. 2002). During ontogeny the site of haematopoiesisis modified several times (Cumano et al. 2001). An areaalong the dorsal embryonic aorta termed the aorta–gonad–mesonephros (AGM) is a rich source of HSCs,which then migrate to the embryonic liver and laterseed other haematopoietic tissues such as the bonemarrow (McGrath & Palis 2008). HSCs are usuallyassayed in animal models based on their capacity torepopulate the entire haematopoietic system in con-ditioned recipients after transplantation. Similar toMSCs, it is possible to transplant human cells into xeno-geneic recipients such as foetal sheep that act as modelsof human haematopoiesis (O’Donoghue & Fisk 2004).

First-trimester foetal blood contains more CD34þ

cells than term gestation blood, yet CD34þ cellsaccount for only 5 per cent of CD45þ cells in theblood at that stage (Campagnoli et al. 2001). Thenumber of circulating HSCs increases from the first tri-mester to peak in the second trimester in utero,probably because of cells migrating from the foetalliver to establish haematopoiesis in the foetal bonemarrow (Clapp et al. 1995). Some HSCs remain in theumbilical cord at delivery, where they can be collectedfor allogeneic or occasionally autologous cell transplan-tation (see §2.2.1.).

In the second trimester, CD34þ cells constitute 4 percent of cells in blood, 16.5 per cent in bone marrow,6 per cent in liver, 5 per cent in spleen and 1.1 percent in the thymus (Lim et al. 2005). This frequencyof CD34þ cells in the blood gradually diminishesduring the third trimester, probably reflecting establish-ment of the marrow as the primary site ofhaematopoiesis and the declining role of the foetalliver in that regard (Jones et al. 1994; Wagers et al.2002). The number of cells negative for CD38 withinthe CD34þ population is also higher in early foetal


blood, suggesting that these cells are more primitiveand have greater potential than HSCs circulating laterin ontogeny (O’Donoghue & Fisk 2004). Even relativelydifferentiated erythroblasts are more primitive in firstcompared with later trimester foetal blood. Foetalblood HSCs proliferate more rapidly than those incord blood or adult bone marrow, and produce allhaematopoietic lineages (Campagnoli et al. 2001).

Foetal liver is also a source of HSCs, and, despitetheir relatively small number, it is possible to generatesufficient numbers of cells for transplantation in fourweeks (Rollini et al. 2004), although, to our knowledge,it has not been proved that foetal liver HSCs can suc-cessfully transplant a human recipient. The phenotypeof foetal liver HSCs changes with gestation age. Forexample, their differentiation potential decreases mark-edly with gestational age, and cells that expressed bothCD34 and c-kit (CD117) were identified in the first-trimester foetal liver whereas the cells expressing morecommitted hepatic markers only appeared during thesecond trimester (Nava et al. 2005).

2.2. Extraembryonic tissue

2.2.1. Umbilical cord blood. Umbilical cord blood is nowan established source of transplantable HSCs that havea greater proliferative capacity, lower immunologicalreactivity and lower risk of graft-versus-host disease(GVHD) than those derived from adult bone marrow(Broxmeyer et al. 1989; Yu et al. 2001; Ballen 2005;Schoemans et al. 2006; Brunstein et al. 2007; Hwanget al. 2007; Broxmeyer 2010). These cells are capableof repopulating bone following intra-bone injection ofsevere combined immunodeficiency mice (Mazurieret al. 2003; Wang et al. 2003) and are used clinicallyas an alternative to adult bone marrow HSCs in somecases (Delaney et al. 2010). The cells are CD34þ andCD382 and their frequency is greater than that ofbone marrow or peripheral blood following cytokinemobilization (Pappa & Anagnou 2009). It has alsobeen demonstrated that cord blood contains MSCs(Weiss & Troyer 2006; Secco et al. 2008) that can sup-port the in vivo expansion of HSCs and function as anaccessory cell population for engraftment (Javazonet al. 2004; Wang, J. et al. 2004). The frequency ofMSCs in umbilical cord blood is low however, withMSCs successfully isolated from only a third of allsamples collected (Bieback et al. 2004).

Cord blood stem cells expressing baseline levels of EScell markers such as Oct-4, Nanog, SSEA-3 and SSEA-4have also been described (Zhao et al. 2006). Using atwo-stage isolation approach whereby the erythrocytesin cord blood are lysed and the remaining cells aresorted using flow cytometry, it has been possible to iso-late and enrich for a subpopulation of cells that areCXCR4þ, CD133þ, CD34þ, Lin2 and CD452.

These cells have been described as very smallembryonic-like (VSEL) stem cells because they areonly 3–5 mm in diameter and express Oct-4, Nanogand SSEA-4 (Kucia et al. 2007; Zuba-Surma et al.2010). Compared with MSCs, VSEL cells are smaller,have a large nucleus to cytoplasm ratio and open-typechromatin. As a similar population has also been




found in adult bone marrow, where they also express thesame primitive markers, it has been hypothesized thatVSEL cells are related to a population of early PGCsthat are retained during development (Kucia et al.2006).

2.2.2. Wharton’s jelly. Wharton’s jelly is the connectivetissue surrounding the umbilical vessels and includesthe perivascular, intervascular and subamnion regions(Troyer & Weiss 2008). MSCs have been isolated fromall three regions and it remains unclear whether theyrepresent distinct cell populations (Karahuseyinogluet al. 2007). The cells have been given different namesby different groups (Troyer & Weiss 2008). MSCs iso-lated from Wharton’s jelly share similar propertieswith other cord blood MSCs as well as adult bonemarrow MSCs on the expression of markers, differen-tiation potential and cytokine production (McElreaveyet al. 1991; Wang, H.-S. et al. 2004; Anzalone et al.2010). Wharton’s jelly MSCs can also be induced todifferentiate into cells similar to neural, expressingneuron-specific enolase and other neural markers(Mitchell et al. 2003; Anzalone et al. 2010).

2.2.3. Amniotic fluid. In recent years, amniotic fluid hasemerged as a major source of putative pluripotent stemcells that avoid many of the problems associated withES cells such as their non-suitability for autologoususe, their capacity for tumour formation and the ethicalconcerns they raise.

In humans, the amniotic fluid starts to appear at thebeginning of week 2 of gestation as a small film ofliquid between the cells of the epiblast. The fluid expandsseparating the epiblast (i.e. the future embryo) from theamnioblasts (i.e. the future amnion), thus forming theamniotic cavity (Miki & Strom 2006). The origin ofamniotic fluid cells is still very much debatable(Kunisaki et al. 2007; Cananzi et al. 2009). However,what is known is that the majority of cells present areterminally differentiated and have limited proliferativecapacity (Gosden & Brock 1978; Siegel et al. 2007). How-ever, a number of studies have demonstrated the presenceof a subset of cells with a proliferative and differentiationpotential (Torricelli et al. 1993; Streubel et al. 1996).

A variety of different types of stem cells have been iso-lated and characterized from amniotic fluid. Theseinclude cells found in mid-gestation expressing the haema-topoietic marker CD34 (Da Sacco et al. 2010) as well ascells with mesenchymal features, able to proliferate invitro more rapidly than comparable foetal and adultcells (Kaviani et al. 2001; in‘t Anker et al. 2003a,b;Nadri & Soleimani 2007; Roubelakis et al. 2007; Sessaregoet al. 2008). Amniotic fluid MSCs are negative forhaematopoietic markers such as CD45, CD34 and CD14(Prusa & Hengstschlager 2002; in‘t Anker et al. 2003a,b;Prusa et al. 2003; Tsai et al. 2004). Despite their high pro-liferation rate, these cells display a normal karyotypewhen expanded in vitro and do not form tumours invivo (Sessarego et al. 2008). They exhibit a broad differen-tiation potential towards mesenchymal lineages (in‘tAnker et al. 2003a,b; Kolambkar et al. 2007; Nadri &Soleimani 2007; Tsai et al. 2007).


De Coppi et al. isolated c-kit-positive (CD117) cellsthat represent about 1 per cent of cells present insecond-trimester amniotic fluid. These cells werenamed amniotic fluid stem (AFS) cells. They can becultured without feeders, double in 36 h, are nottumorigenic, have long telomeres and retain a normalkaryotype for over 250 population doublings (DeCoppi et al. 2007a). Cultured human AFS cells are posi-tive for ES cell (e.g. Oct-4, Nanog and SSEA-4) andmesenchymal cell markers such as CD90, CD105(SH2), CD73 (SH3/4) and several adhesion molecules(e.g. CD29 and CD44; Tsai et al. 2006; Chambers et al.2007; De Coppi et al. 2007a). Furthermore, it was poss-ible to generate clonal human lines from these cells,verified by retroviral marking, which were capable of dif-ferentiating into lineages representative of all three EGlayers. Almost all clonal AFS cell lines express Oct-4and Nanog, markers of a pluripotent undifferentiatedstate (De Coppi et al. 2007a). c-kitþ Lin2 cells fromhuman and mouse amniotic fluid display a multi-lineagehaematopoietic potential in vitro and in vivo, despitehaving low or negative CD34 expression (Ditadi et al.2009). c-kit-positive human AFS cell lines can alsoform embryoid bodies (EB) with an incidence of18–82%. EB formation was accompanied by a decreasein Oct-4 and Nodal and the induction of differentiationmarkers such as Pax 6, Nestin (ectodermal), GATA 4and HBE1 (mesodermal) (Valli et al. 2010). The poten-tial for a wide range of regenerative medicine and tissueengineering applications as well as their relatively fewethical concerns makes them an attractive sourcefor cell therapy, particularly considering that a bank of100 000 amniotic fluid specimens could potentiallysupply 99 per cent of the US population with a perfectmatch for transplantation (Atala 2009).

2.2.4. Placenta. The placenta is a fetomaternal organinvolved in maintaining foetal tolerance and allowsnutrient uptake and gas exchange with the mother,but also contains a high number of progenitor cells orstem cells (Parolini et al. 2010). It has two sides: onefoetal (amnion and chorion) and one maternal (decid-uas). The amnion membrane contains two cell types:the amniotic epithelial cells (AECs) derived from theepiblast; and amniotic mesenchymal cells derived fromthe hypoblast. The chorion layer consists of cytotropho-blasts and syncytiotrophoblasts that are derived fromthe outer layer of the blastocyst (trophectoderm)(Ilancheran et al. 2009; Insausti et al. 2010).

The availability, phenotypic plasticity and immuno-modulatory properties of placenta-derived progenitor/stem cells are useful characteristics for cell therapyand tissue engineering. Cells can be isolated duringongoing pregnancy using minimally invasive techniquessuch as chorionic villus sampling (CVS) and placentaltissues are readily available at delivery for allogeneicor autologous use. Cells that have been isolated fromplacenta include the human AECs, human amnionmesenchymal stromal stem cells (AMSCs), humanchorionic mesenchymal/stromal stem cells (CMSCs),human chorionic trophoblastic cells and HSCs (Paroliniet al. 2008).




Mesenchymal placenta cells are plastic adherent,share a similar immunophenotype and have lineagedifferentiation potential. They express stromal markerssuch as CD166, CD105, CD73, CD90 and others, whilethey are negative for the haematopoietic markers CD14,CD34 and CD45 (Igura et al. 2004; Sudo et al. 2007).Additionally, they express pluripotency markers such asSSEA3, SSEA4, Oct-4, Nanog, Tra-1-60 and Tra-1-81(Yen et al. 2005; Battula et al. 2007). They lack or havevery low expression of MHC class I antigens and do notexpress MHC class II antigens or the T-cell co-stimulatorymolecule B7, giving the cells immunomodulatoryproperties (Bailo et al. 2004; Miao et al. 2006).

2.2.4.1. Amniotic epithelial cells. AECs are commonlyisolated from the amniotic membrane using digestiveenzymes. The cells are plastic adherent and grow underMSC conditions, i.e. Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10–20% FBS,with the addition of growth factors such as epidermalgrowth factor (EGF) (Miki et al. 2010). c-Kit and CD90(Thy-1) expression is either negative or at a low level(Miki et al. 2005; Miki & Strom 2006). However, evidencesuggests that they express pluripotency markers and havethe ability to form xenogeneic chimera with mouse EScells in vitro (Tamagawa et al. 2004). The cells have sub-sequently been differentiated in vitro into cell types fromall three germ layers (Miki et al. 2005; Miki & Strom2006; Ilancheran et al. 2007; Parolini et al. 2008).However, in contrast with ES cells, Miki et al. (2005)demonstrated that human AE cells did not form grossor histological tumours up to seven months post-transplantation in SCID/Beige mice.

2.2.4.2. Amnion-derived mesenchymal stromal cells.Both amnion and chorion MSCs have been extensivelycharacterized and can be isolated throughout gestationfrom first trimester to delivery. Both cells sharecommon characteristics, such as plastic adherence,and, despite limited proliferation capacity, show invitro differentiation down the osteogenic, adipogenic,chrondrogenic and neurogenic lineages, although somereport and show in vivo multi-organ engraftmentcapacity. In addition, the volume of term placentamakes it an attractive source of stem cells, as on averagehuman term placenta weighs more than 590 g(Bolisetty et al. 2002). Human placenta contains var-ious types of cells of different developmental origin.Cells of the chorion and decidua are derived from tro-phectoderm, while amnion is derived from the epiblastof the developing embryo (Crane & Cheung 1988).

MSCs have been successfully isolated from first-,second- and third-trimester placental compartments,including the amnion, chorion, decidua parietalis anddecidua basalis (in‘t Anker et al. 2004; Portmann-Lanz et al. 2006; Soncini et al. 2007; Poloni et al.2008), and represent less than 1 per cent of cells presentin the human placenta (Zhang et al. 2004; Alviano et al.2007). First-trimester placenta stem cells generally growfaster than those found in the third trimester(Portmann-Lanz et al. 2006), and the majority of


term placenta-derived stem cells are found at thequiescent G0/G1 phase of the cell cycle.

2.2.4.3. Chorion-derived stem cells. CMSCs have beenisolated during pregnancy (CVS) or from the term pla-centa at delivery. The immunophenotype of termplacenta cells is similar to that of adult bone marrowMSCs, although renin and flt-1 have been shown tobe expressed uniquely in placenta MSCs (Fukuchiet al. 2004). Similarly to human amnion isolated fromterm placenta, term chorion MSCs successfully engraftin neonatal swine and rats and failed to induce a xeno-geneic response (Bailo et al. 2004), indicating that thesecells may have an immunoprivileged status consistentwith their low level of HLA I and absence of HLA IIexpression (Kubo et al. 2001). Of relevance for pre-natal autologous and allogeneic therapy, chorionMSCs can be successfully isolated from chorionic villiduring first-trimester gestation. The chorionic villi donot express typical MSC cell surface antigens andhave the capacity to differentiate into lineages of thethree germ layers, with a subset of cells expressing thepluripotency markers Oct-4, ALP, Nanog and Sox2(Spitalieri et al. 2009). In addition to differentiatinginto adipogenic, chondrogenic and osteogenic cells invitro (Portmann-Lanz et al. 2006; Ilancheran et al.2007), they can also differentiate into cells with somecharacteristics of hepatocytes in vitro and have the abil-ity to store glycogen (Chien et al. 2006; Huang 2007;Tamagawa et al. 2007). Differentiation into other celltypes such as endothelial cells (Alviano et al. 2007),cardiomyocytes (Zhao et al. 2005), neurons, oligoden-drocytes and glial cells (Miao et al. 2006; Yen et al.2008; Portmann-Lanz et al. 2010) has also beenreported, but these findings have mostly relied on upre-gulation of tissue-specific markers without providingrobust functionality tests.

3. THERAPUTIC APPLICATIONS OFFOETAL STEM CELLS

Regenerative medicine aims to restore diseased ordamaged tissue using the body’s own cells in order toovercome the shortage of donated organs and the riskassociated with their rejection (Atala 2009). The useof stem cells from foetal, cord blood and extraembryonictissues for regenerative medicine applications hasincreased over the past few years. However, the surpris-ing picture that has emerged is that cell replacement isprobably not the major mechanism by which celltherapy confers functional benefit. Rather, stem cellsact beneficially by exerting trophic effects on host tis-sues. Moreover, because they can be expanded to verylarge numbers compared with adult stem cells, theyare ideal candidates for seeding scaffolds and matricesused for tissue engineering applications such as therepair of hollow and solid organs. In this section, wewill consider some of the therapeutic applicationsFSCs have been put to in pre-clinical studies for repair-ing diseased or damaged tissue as well as in cancertreatment (summarized examples are listed in table 2).


Tab

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7)







3.1. Bone

Osteogenesis imperfecta (OI) is characterized by osteo-penia and bone fragility due to abnormal collagenproduction caused by mutations in the a chains of col-lagen type I. First-trimester allogeneic HLA-mismatched male foetal liver MSCs were transplantedin utero into a female foetus diagnosed with severeOI. Bone biopsy showed regularly arranged and config-ured bone trabeculae and no adverse immune reactionwas observed (Le Blanc et al. 2005). Using a mousemodel of OI, our group has shown that in utero trans-plantation of foetal blood MSCs ameliorated thedisease phenotype, producing a clinically relevanttwo-thirds reduction in fracture incidence along withan improvement in bone structure and mechanicalproperties (Guillot et al. 2008a). Foetal bonemarrow MSCs loaded on a highly porous scaffold wereable to reach confluence within the scaffold morequickly than umbilical cord and adult MSCs. Further-more, they demonstrated higher in vitro and in vivoosteogenic differentiation capacity, highlighting theirsuitability for bone tissue engineering applications(Zhang et al. 2009).

3.2. Muscle

Duchenne muscular dystrophy (DMD) is an X-linkedmyopathy affecting one in 3500 boys. The main geneticdefect leads to absence of dystrophin, resulting inmuscle damage and wasting. The affected boysbecome wheelchair-bound by 12 years of age and suc-cumb to respiratory failure or cardiopmyopathy bythe third or forth decade of life (Manzur & Muntoni2009). The main animal model for studying DMD isthe mdx mouse that has a premature stop codon result-ing in a termination in exon 23 of the dystrophin gene(Grounds et al. 2008). Galectin-1-treated foetal bloodand bone marrow MSCs, transplanted into postnatalsevere combined immunodeficiency/dystrophic mice(scid/mdx) as well immunodeficient mice whosemuscle was induced to regenerate by cryodamage,formed fourfold more human muscle fibres than non-stimulated MSCs (Chan et al. 2006). Intrauterinetransplantation of foetal MSCs into immunocompetentE14-16 mdx mice resulted in widespread long-termengraftment in multiple organs with a predilection formuscle compared with non-muscle tissues. However,the engraftment level observed (0.5–1%) falls signifi-cantly below the levels required for functionalimprovement in DMD. The low engraftment might belinked to an absence of muscle pathology at the timeof transplantation (Chan et al. 2007).

AFS cells have been used in an ovine model ofdiaphragmatic hernia: repair of the muscle deficitusing grafts engineered with autologous mesenchymalamniocytes leads to better structural and functionalresults than equivalent foetal myoblast-based and acel-lular grafts (Fuchs et al. 2004; Kunisaki et al. 2006). Ina rat model of bladder cryo-injury, AFS cells also showthe ability to differentiate into smooth muscle and toprevent the compensatory hypertrophy of survivingsmooth muscle cells (De Coppi et al. 2007b).


Human AFS cells, transplanted into an immune-competent and ciclosporin immune-suppressed ratmodel of myocardial infarction, were rejected probablybecause of the expression of B7 co-stimulatorymolecules as well as macrophage marker CD68.Furthermore, chondro-osteogenic cell masses wereobserved in the infarcted hearts of some of the trans-planted animals (Chiavegato et al. 2007). However, inanother study it was found that these masses occurredindependently of AFS cell injection and that the cellsdid not increase the presence of these masses (Deloet al. 2010).

Human AMSCs are able not only to express cardiac-specific genes under specific culture conditions, but alsoto integrate into normal and infarcted rat cardiac tissuewhere they differentiate into cardiomyocyte-like cells(Zhao et al. 2005). The cells result in considerableimprovements to ventricular function, capillary densityand scar tissue (Ventura et al. 2007) and similar find-ings have been reported using native rat AMSCs(Fujimoto et al. 2009). Stem cells derived from pre-natal chorionic villi were also successfully engineeredinto a living autologous heart valve, which could havepostnatal applications for repairing congenital cardiacmalformations (Schmidt et al. 2006).

3.3. Kidney

In a mouse model of collagen deficiency characterizedby abnormal collagen deposition in renal glomeruli(Phillips et al. 2002; Brodeur et al. 2007), we recentlyshowed that intrauterine transplantation of foetalblood MSCs led to a reduction of abnormal homotri-meric collagen type I deposition in the glomeruli of4–12 week old col1a2-deficient mice. Furthermore, weshowed that the damaged kidneys preferentiallyrecruited donor cells in the glomeruli. The study sup-ports the feasibility of pre-natal treatment for renaldiseases such as Alport syndrome and the polycystickidney diseases (Guillot et al. 2008b).

Acute tubular necrosis could also be potentially trea-ted with foetal cells. Human AFS cells injected into animmunodeficient mouse model of the disease decreasedthe number of damaged tubules and reduced apoptosis.The cells also promoted the proliferation of tubular epi-thelial cells and appeared to have a beneficialimmunomodulatory effect (Perin et al. 2010).

3.4. Lung

Human AFS cells injected into the tail vein of nudemice following hyperoxia injury show the capacity tohome to the lung and engraft. They also expressedspecific alveolar and bronchiolar epithelial markers(e.g. TFF1, SPC and CC10; Carraro et al. 2008).

AECs and AMSCs from either human or mouse havebeen used in a mouse model of bleomycin-induced lunginjury and shown to cause a reduction in severity oflung fibrosis. This occurs regardless of cell source (allo-geneic or xenogeneic) or administration route (systemic,intravenous or intraperitoneal; local, intratracheal;Cargnoni et al. 2009).




3.5. Liver

Second-trimester foetal liver cells, enriched for CD326(hepatocyte progenitor marker) and labelled with Tc-d,l-hexamethyl-propylene-amine oxime (Tc-HMPAO)were infused into the hepatic artery of 25 patientswith end stage liver cirrhosis. The cells restored thelost liver function in the patients without provokingan immune reaction, probably because of the absenceof HLA class II expression of the infused cells (Khanet al. 2010).

Human AECs can secrete albumin in culture and,when b-galactosidase-tagged AECs were transplantedinto immunodeficient mice, the cells integrated intothe liver parenchyma and could be detected until day7 post transplant (duration of study; Sakuragawaet al. 2000). In another study, the engrafted cellsresulted in the detection of human a-1 antitrypsin inthe serum of recipient animals, confirming that AECsare capable of performing this important hepaticfunction in vivo (Miki & Strom 2006).

3.6. Brain

Human Wharton’s jelly MSCs ameliorate apomorphine-induced behavioural deficits in a hemiparkinsonian ratmodel (Weiss et al. 2006). There was a significantdecrease in apomorphine-induced rotations at fourweeks continuing up to 12 weeks post transplantationin Parkinson’s disease (PD) rats that received humanWharton’s jelly MSCs transplants compared with thePD rats that received a sham transplant. The behav-ioural findings correlated with the numbers of tyrosinehydroxylase-positive cell bodies observed in the mid-brain following sacrifice at 12 weeks, indicating a‘rescue from a distance’ phenomenon. One explanationfor this effect may be that Wharton’s jelly MSCssynthesize glial cell-derived neurotrophic factor(GDNF), a potent survival factor for dopaminergicneurons, as well as other trophic factors such asvascular endothelial growth factor (VEGF) and ciliaryneurotrophic factor. In another report, Wharton’sjelly MSCs were first induced towards dopaminergicneurons using neuron-conditioned media, sonic hedge-hog and fibroblast growth factor 8, and thentransplanted into hemiparkinsonian rats (Fu et al.2006). Despite the fact that the rats were not immunesuppressed, Wharton’s jelly MSCs were identified fivemonths later and prevented the progressive degener-ation/behavioural deterioration seen in control ratswith unilateral lesions. Similarly, rat Wharton’s jellycells transplanted into the brains of rats with global cer-ebral ischaemia caused by cardiac arrest andresuscitation significantly reduced neuronal loss, appar-ently owing to a rescue phenomenon (Jomura et al.2007). Lund et al. administered Wharton’s jelly MSCsinto the eyes of a rodent model of retinal disease.Here, Wharton’s jelly MSCs were compared with bonemarrow-derived mesenchymal stem cells (BMSCs) andplacental stem cells. They reported that the Wharton’sjelly MSCs exhibited the best histological evidence ofphotoreceptor rescue with increased production oftrophic factors such as brain-derived neurotrophic


factor and fibroblast growth factor 2 (Lund et al.2007). Interestingly, Wharton’s jelly MSCs trans-planted into the vitreous demonstrated a rescue effect,indicating that they could enhance the survival ofphotoreceptor cells without being in close proximityto them. This effect was presumably the result ofdiffusible growth factors.

Very similar findings have also been reported forAECs that confer neuroprotection and functional recov-ery in animal models of Parkinson’s disease (Bankiewiczet al. 1994; Kakishita et al. 2000, 2003; Kong et al.2008) and ischaemia (Liu et al. 2008). The observedtherapeutic effects are probably mediated by secretionof diffusible factors, including neurotransmitters(Elwan & Sakuragawa 1997; Sakuragawa et al. 1997,2001) and neurotrophic and growth factors (Koizumiet al. 2000; Uchida et al. 2000).

AFS cells can harbour trophic and protective effectsin the central and the peripheral nervous systems. Panet al. (2006, 2007, 2009) showed that AFS cells facilitateperipheral nerve regeneration after injury, particularlyif accompanied with suppressors of inflammatory cyto-kines such as fermented soya bean extracts. Aftertransplantation into the striatum, AFS cells are capableof surviving and integrating in the rat adult brain andcan migrate towards areas of ischaemic damage(Cipriani et al. 2007). Moreover, the intra-ventricularadministration of AFS cells in mice with focal cerebralischaemia–reperfusion injuries significantly reversesneurological deficits in the treated animals (Rehniet al. 2007).

3.7. Cancer therapy

HSCs from bone marrow or umbilical cord blood havelong been used to re-establish the haematopoieticsystem following radiation and/or chemotherapy(Osawa et al. 1996; Bhatia et al. 1997). More recently,research has shown that MSCs are attracted to tumoursbecause the latter often act as unresolved wounds pro-ducing a continuous source of chemoattractantinflammatory mediators. This property is beingexploited by using exogenously delivered MSCs asvehicles for delivering anti-cancer molecules to tumours(Loebinger & Janes 2010). Wharton’s jelly cells, likeneural stem cells and MSCs (Aboody et al. 2000;Studeny et al. 2004), appear to migrate to areas oftumour growth. Human breast carcinoma cells wereintravenously injected into SCID mice, followed byintravenous transplantation of fluorescently labelledWharton’s jelly MSCs. One week after transplantWharton’s jelly MSCs were found near or within lungtumours and not in other tissues. They were engineeredto express human interferon beta and were administeredintravenously into SCID mice bearing tumours. Thistreatment significantly reduced the tumour burden(Rachakatla et al. 2007). Similarly, human umbilicalcord blood-derived MSCs (UCB–MSCs) have beenused to deliver a secretable trimeric form of tumournecrosis factor-related apoptosis-inducing ligand(stTRAIL), via adenoviral transduction mediated bycell-permeable peptides, to human glioma cells innude mice. The genetically modified cells showed




greater efficacy in terms of inhibiting tumour growthand prolonging the survival of glioma-bearing micecompared with direct injection of adenovirus encodingthe stTRAIL gene into the tumour mass (Kim et al.2008).

4. USE OF FSCs FOR REPROGRAMMING

The major advantage of human ES cells over other stemcell types is their capacity to differentiate into lineagesfrom the three germ layers. However, there are twomajor objections to their use. The moral objection isthat their derivation requires the destruction ofembryos. The practical objection is that they have alimited clinical application as they can only be used inan allogeneic fashion. This is also true for some foetaltissues such as liver and bone marrow. However, extra-embryonic tissues can be used autologously.

Thus, it was seen as advantageous to develop amethod of creating ES-like cells from somatic cells.Early attempts at achieving this were nuclear trans-plantation and fusion of somatic cells and ES cells(Jaenisch 2009). However, a considerable step forwardwas the generation of the induced pluripotent stem(iPS) cells. The production of iPS cells with quasi-identical genetic and functional properties offers thepossibility to bypass both moral conflicts and differentgenetic background inherent to the technologiesmentioned above. iPS are developed from a non-pluripotent cell, usually an adult somatic cell, bycausing a forced expression of several genetic sequencesand were first produced in 2006 by Takahashi &Yamanaka from mouse somatic cells. The key genesOct-4, the transcription factor Sox2, c-Myc protoonco-gene protein and Klf4 (Krueppel-like factor 4) weresufficient to reprogramme fibroblasts to cells closelyresembling ES cells (Takahashi & Yamanaka 2006;Takahashi et al. 2007).

Despite the high similarity between iPS and ES cells,tumour formation in iPS cell chimeric mice was high,presumably because of the expression of c-Myc in iPScell-derived somatic cells (Maherali et al. 2007).Thomson et al. showed that c-Myc was not necessaryas they were able to generate iPS cells using Oct-4,Sox2, Nanog and Lin28 using a lentiviral system (Yuet al. 2007). More recently, it was shown that humanfibroblasts cells can be used to generate iPS cells iftransduced with Oct-4 and Sox2 only in the presenceof valproic acid (Huangfu et al. 2008).

Terminally differentiated cells might not be the idealcandidate for iPS cell generation because a greaternumber of steps could be required to ‘reprogramme’their genome than somatic stem cells. Kim et al.(2009a,b) have shown that it is possible to generateiPS cells by transducing adult mouse neural stem cells(NSCs) and human foetal NSCs with Oct-4 only. Fur-thermore, when human adipose MSCs were used, theywere reprogrammed more efficiently than fibroblasts(Sun et al. 2009).

Few reports have also emerged on the use of cord andextra ES cells for iPS generation. Galende et al. usedterminally differentiated amniotic fluid cells as


candidates for reprogramming and found that the iPScell colonies were generated twice as fast, yieldingnearly a 200 per cent increase in number comparedwith cultured adult skin cells. However, they did notprovide absolute efficiency data (Galende et al. 2009).Li et al. reprogrammed human amniotic fluid-derivedcells (hAFDCs) with efficiencies varying from 0.059 to1.525 per cent. The iPS colonies generated expressedpluripotency markers such as Oct-4, Sox2 and SSEA 4and they maintained a normal karyotype (Li et al.2009). Wharton’s jelly cells and term placenta cellshave also been used, giving reprogramming efficienciesof 0.4 per cent and 0.1 per cent, respectively, which com-pare rather well with efficiencies reported for adultfibroblasts (less than 0.01%). The generated iPS cellcolonies expressed several pluripotency markers, formedEB and, when injected into nude mice, they generatedteratoma-containing derivates of the three germ layers(Cai et al. 2010). Giorgetti et al. selected CD133þ (hae-matopoietic stem and progenitor cell marker) cord bloodcells and managed to reprogramme them by retroviraltransduction with Oct-4 and Sox2 only without needingadditional chemical compounds. iPS cell colonies derivedfrom CD133þ cord blood cells (expressing pluripotencymarkers and capable of forming EB and teratomas)were generated in two weeks whereas no colonies wereobtained with control keratinocytes or fibroblasts usingthe two factors only (Giorgetti et al. 2009, 2010).

CD133þ cord blood cells and foetal NSCs alreadyhave a baseline level expression of Oct-4 and/or Sox2and that, along with a generally more permissive chro-matin organization, might be the key to their greaterreprogramming efficiency compared with adult cells.

5. CONCLUSION

Human FSCs can be isolated from foetal organs orextraembryonic sources. They have the advantage ofrapid proliferation, stable karyotype and low or negli-gible immunogenicity. Unlike ES cells, they do notform teratomas in vivo and have far fewer ethical con-cerns as they are mostly obtained from tissues thatwould otherwise be discarded. Many of these cellsseem to express some of the same pluipotency markersfound in ES cells, a feature largely absent from mostadult-derived stem cells. They also have the furtheradvantage over adult stem cells of senescing muchlater and being more readily amenable to geneticmodification.

All of these features make them valuable for poten-tial therapy applications. Thus far they have beenused in pre-clinical settings to treat a variety of diseasessuch as osteogenesis imperfecta, congenital diaphrag-matic hernia, Parkinson’s disease and cancer withencouraging results. Finally, their usefulness for iPSgeneration is very likely to expand their future clinicaluse even further.

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