Corso di aggiornamento in Bioetica Questioni di inizio...

Corso di aggiornamento in Bioetica

Questioni di inizio vita Roma, 22-25 settembre 2015

Caso sull’uso di linee cellulari di origine illecita Prof.ssa Elena Colombetti

Università Cattolica del Sacro Cuore, Milano

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Materiale di supporto allo studio del caso

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!!!!!"Avvertenza:"I"testi"qui"proposti"sono"di"natura"differente"e"hanno"come"unico"scopo"quello"di"offrire"alcuni"dati" scientifici" e" una" esemplificazione" delle" diverse' argomentazioni" presenti" nel" dibattito"odierno"sul"tema"in"oggetto.""!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!""

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Indice'"

*"Congregazione"per"la"Dottrina"della"Fede,"Istruzione+Dignitas+Personae"n°34@35."

*" Pontificia" Accademia" per" la" Vita," Moral+ reflections+ on+ vaccines+ prepared+ from+ cells+derived+from+aborted+human+foetuses,"Roma,"2005."

*" Augusto" Pessina," Laura" Gribaldo,' The+ key+ role+ of+ adult+ stem+ cells:+ therapeutic+perspectives,"«Current"Medical"Research"and"Opinion»,"Vol."22,"No."11"(2006),"2287–2300."

*" James" Thomson" et" al," Embryonic+ Stem+ Cell+ Lines+ Derived+ from+ Human+ Blastocysts,"«Science»,"Vol.282,"(1998),"11451@1147."

*"Scheda"di"catalogo+della+linea+cellulare+INT407"(HeLa"derivative)""

*"Angelo"Serra,"Il+figlio+di+nessuno,"tratto"da"“L’uomo"embrione”,"Cantagalli,"Siena"2003,"pag."83"e"ss.,"ripubblicato"su"«Medicina"e"Persona»,"giugno"2006.""

*"CBN,"Parere+sulle+ricerche+utilizzanti+embrioni+umani+e+cellule+staminali,'2003"

*" CNB," Risposta+ sull’utilizzo+ a+ fini+ di+ ricerca+ delle+ linee+ cellulari+ h1+ e+ h9+ derivanti+ da+embrioni+umani,"2004."

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CONGREGAZIONE PER LA DOTTRINA DELLA FEDE

ISTRUZIONE DIGNITAS PERSONAE

SU ALCUNE QUESTIONI DI BIOETICA

L’uso di “materiale biologico” umano di origine illecita

34. Per la ricerca scientifica e per la produzione di vaccini o di altri prodotti talora vengono utilizzate linee cellulari che sono il risultato di un intervento illecito contro la vita o l’integrità fisica dell’essere umano. La connessione con l’azione ingiusta può essere immediata o mediata, dato che si tratta generalmente di cellule che si riproducono facilmente e in abbondanza. Questo “materiale” talvolta viene commercializzato, talvolta è distribuito gratuitamente ai centri di ricerca da parte degli organismi statali che per legge hanno tale compito. Tutto ciò dà luogo a diversi problemi etici, in tema di cooperazione al male e di scandalo. Conviene pertanto enunciare i principi generali, a partire dai quali gli operatori di retta coscienza possono valutare e risolvere le situazioni in cui eventualmente potrebbero essere coinvolti nella loro attività professionale.

Occorre ricordare innanzitutto che la stessa valutazione morale dell’aborto «è da applicare anche alle recenti forme di intervento sugli embrioni umani che, pur mirando a scopi in sé legittimi, ne comportano inevitabilmente l’uccisione. È il caso della sperimentazione sugli embrioni, in crescente espansione nel campo della ricerca biomedica e legalmente ammessa in alcuni Stati… L’uso degli embrioni o dei feti umani come oggetto di sperimentazione costituisce un delitto nei riguardi della loro dignità di esseri umani, che hanno diritto al medesimo rispetto dovuto al bambino già nato e ad ogni persona» [54]. Queste forme di sperimentazione costituiscono sempre un disordine morale grave [55].

35. Una fattispecie diversa viene a configurarsi quando i ricercatori impiegano “materiale biologico” di origine illecita che è stato prodotto fuori dal loro centro di ricerca o che si trova in commercio. L’Istruzione Donum vitae ha formulato il principio generale che in questi casi deve essere osservato: «I cadaveri di embrioni o feti umani, volontariamente abortiti o non, devono essere rispettati come le spoglie degli altri esseri umani. In particolare non possono essere oggetto di mutilazioni o autopsie se la loro morte non è stata accertata e senza il consenso dei genitori o della madre. Inoltre va sempre fatta salva l’esigenza morale che non vi sia stata complicità alcuna con l’aborto volontario e che sia evitato il pericolo di scandalo» [56].

A tale proposito è insufficiente il criterio dell’indipendenza formulato da alcuni comitati etici, vale a dire, affermare che sarebbe eticamente lecito l’utilizzo di “materiale biologico” di illecita provenienza, sempre che esista una chiara separazione tra coloro che da una parte producono, congelano e fanno morire gli embrioni e dall’altra i ricercatori che sviluppano la sperimentazione scientifica. Il criterio di indipendenza non basta a evitare una contraddizione nell’atteggiamento di chi afferma di non approvare l’ingiustizia commessa da altri, ma nel contempo accetta per il proprio lavoro il “materiale biologico” che altri ottengono mediante tale ingiustizia. Quando l’illecito è avallato dalle leggi che regolano il sistema sanitario e scientifico, occorre prendere le distanze dagli aspetti iniqui di tale sistema, per non dare l’impressione di una certa tolleranza o accettazione tacita di azioni gravemente ingiuste [57]. Ciò infatti contribuirebbe a aumentare l’indifferenza, se non il favore con cui queste azioni sono viste in alcuni ambienti medici e politici.

Talvolta si obietta che le considerazioni precedenti sembrano presupporre che i ricercatori di buona coscienza avrebbero il dovere di opporsi attivamente a tutte le azioni illecite realizzate in ambito medico, allargando così la loro responsabilità etica in modo eccessivo. Il dovere di evitare la cooperazione al male e lo scandalo, in realtà, riguarda la loro attività professionale ordinaria, che devono impostare rettamente e mediante la quale devono testimoniare il valore della vita, opponendosi anche alle leggi gravemente ingiuste. Va pertanto precisato che il dovere di rifiutare quel “materiale biologico” – anche in assenza di una qualche connessione prossima dei ricercatori con le azioni dei tecnici della procreazione artificiale o con quella di quanti hanno procurato l’aborto, e in assenza di un previo accordo con i centri di procreazione artificiale – scaturisce dal dovere di separarsi, nell’esercizio della propria attività di ricerca, da un quadro legislativo gravemente ingiusto e di affermare con chiarezza il valore della vita umana. Perciò il sopra citato criterio di indipendenza è necessario, ma può essere eticamente insufficiente.

Naturalmente all’interno di questo quadro generale esistono responsabilità differenziate, e ragioni gravi potrebbero essere moralmente proporzionate per giustificare l’utilizzo del suddetto “materiale biologico”. Così, per esempio, il pericolo per la salute dei bambini può autorizzare i loro genitori a utilizzare un vaccino nella cui preparazione sono state utilizzate linee cellulari di origine illecita, fermo restando il dovere da parte di tutti di manifestare il proprio disaccordo al riguardo e di chiedere che i sistemi sanitari mettano a disposizione altri tipi di vaccini. D’altra parte, occorre tener presente che nelle imprese che utilizzano linee cellulari di origine illecita non è identica la responsabilità di coloro che decidono dell’orientamento della produzione rispetto a coloro che non hanno alcun potere di decisione.

Nel contesto della urgente mobilitazione delle coscienze in favore della vita, occorre ricordare agli operatori sanitari che «la loro responsabilità è oggi enormemente accresciuta e trova la sua ispirazione più profonda e il suo sostegno più forte proprio nell’intrinseca e imprescindibile dimensione etica della professione sanitaria, come già riconosceva l’antico e sempre attuale giuramento di Ippocrate, secondo il quale ad ogni medico è chiesto di impegnarsi per il rispetto assoluto della vita umana e della sua sacralità» [58].

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NOTE!

[54] Giovanni Paolo II, Lett. enc. Evangelium vitae, n. 63: AAS 87 (1995), 472-473.

[55] Cf. ibid., n. 62: l.c., 472.

[56] Congregazione per la Dottrina della Fede, Istr. Donum vitae, I, 4: AAS 80 (1988), 83.

[57] Cf. Giovanni Paolo II, Lett. enc. Evangelium vitae, n. 73: AAS 87 (1995), 486: «L’aborto e l’eutanasia sono dunque crimini che nessuna legge umana può pretendere di legittimare. Leggi di questo tipo non solo non creano nessun obbligo per la coscienza, ma sollevano piuttosto un grave e preciso obbligo di opporsi ad esse mediante obiezione di coscienza». Il diritto all’obiezione di coscienza, espressione del diritto alla libertà di coscienza, dovrebbe essere tutelato dalle legislazioni civili.

[58] Giovanni Paolo II, Lett. enc. Evangelium vitae, n. 89: AAS 87 (1995), 502.

QUI!AGGIUNGERE!PAV!IN!INGLESE!

8/7/2015 Vatican Statement on Vaccines Derived From Aborted Human Fetuses

http://www.immunize.org/concerns/vaticandocument.htm 1/7

PONTIFICIA ACADEMIA PRO VITA

Il Presidente

Prot.n.P/3431

Mrs Debra L.Vinnedge Vatican City, June 9 2005Executive Director, Children of God for Life943 Deville Drive EastLargo, Florida33771Stati Uniti

Dear Mrs Debra L.Vinnedge,

On June 4, 2003, you wrote to His Eminence Cardinal Joseph Ratzinger, with a copy of this letterforwarded to me, asking to the Sacred Congregation of the Doctrine of Faith a clarification about theliceity of vaccinating children with vaccines prepared using cell lines derived from aborted humanfetuses. Your question regarded in particular the right of the parents of these children to oppose sucha vaccination when made at school, mandated by law. As there were no formal guidelines by themagisterium concerning that topic, you said that catholic parents were often challenged by StateCourts, Health Officials and School Administrators when they filled religious exemptions for theirchildren to this type of vaccination.

This Pontifical Academy for Life, carrying out the commission entrusted to us by the Congregation forthe Doctrine of Faith, in answer to your request, has proceeded to a careful examination of thequestion of these "tainted" vaccines, and has produced as a result a study (in Italian) that has beenrealized with the help of a group of experts. This study has been approved as such by theCongregation and we send you, there enclosed, an English translation of a synthesis of this study.This synthesis can be brought to the knowledge of the interested officials and organisms.

A documented paper on the topic will be published in the journal "Medicina e Morale", edited by theCentra di Bioetica della Universita Cattolica in Rome.

The study, its synthesis, and the translation of this material took some time. We apologize for thedelay.

With my best regards,

Sincerely yours,

+E.Sgreccia

00193 Roma - Via della Concil iazione, 1 - Tel. 06 698.82423 - 06 698.81693 - Fax 06 698.82014

MORAL REFLECTIONS ON VACCINES PREPARED FROM

CELLSDERIVED FROM ABORTED HUMAN FOETUSES



The matter in question regards the lawfulness of production, distribution and use of certain vaccineswhose production is connected with acts of procured abortion. It concerns vaccines containing liveviruses which have been prepared from human cell lines of foetal origin, using tissues from abortedhuman foetuses as a source of such cells. The best known, and perhaps the most important due to itsvast distribution and its use on an almost universal level, is the vaccine against Rubella (Germanmeasles).

Rubella and its vaccine

Rubella (German measles)1 is a viral illness caused by a Togavirus of the genus Rubivirus and ischaracterized by a maculopapular rash. It consists of an infection which is common in infancy and hasno clinical manifestations in one case out of two, is self-limiting and usually benign. Nonetheless, theGerman measles virus is one of the most pathological infective agents for the embryo and foetus.When a woman catches the infection during pregnancy, especially during the first trimester, the risk offoetal infection is very high (approximately 95%). The virus replicates itself in the placenta and infectsthe foetus, causing the constellation of abnormalities denoted by the name of Congenital RubellaSyndrome. For example, the severe epidemic of German measles which affected a huge part of theUnited States in 1964 thus caused 20,000 cases of congenital rubella2, resulting in 11,250 abortions(spontaneous or surgical), 2,100 neonatal deaths, 11,600 cases of deafness, 3,580 cases ofblindness, 1,800 cases of mental retardation. It was this epidemic that pushed for the developmentand introduction on the market of an effective vaccine against rubella, thus permitting an effectiveprophylaxis against this infection.

The severity of congenital rubella and the handicaps which it causes justify systematic vaccinationagainst such a sickness. It is very difficult, perhaps even impossible, to avoid the infection of apregnant woman, even if the rubella infection of a person in contact with this woman is diagnosed fromthe first day of the eruption of the rash. Therefore, one tries to prevent transmission by suppressing thereservoir of infection among children who have not been vaccinated, by means of early immunization ofall children (universal vaccination). Universal vaccination has resulted in a considerable fall in theincidence of congenital rubella, with a general incidence reduced to less than 5 cases per 100,000livebirths. Nevertheless, this progress remains fragile. In the United States, for example, after anoverwhelming reduction in the number of cases of congenital rubella to only a few cases annually, i.e.less than 0.1 per 100,000 live births, a new epidemic wave came on in 1991, with an incidence thatrose to 0.8/100,000. Such waves of resurgence of German measles were also seen in 1997 and in theyear 2000. These periodic episodes of resurgence make it evident that there is a persistent circulationof the virus among young adults, which is the consequence of insufficient vaccination coverage. Thelatter situation allows a significant proportion of vulnerable subjects to persist, who are a source ofperiodic epidemics which put women in the fertile age group who have not been immunized at risk.Therefore, the reduction to the point of eliminating congenital rubella is considered a priority in publichealth care.

Vaccines currently produced using human cell lines that come from aborted foetuses

To date, there are two human diploid cell lines which were originally prepared from tissues of abortedfoetuses (in 1964 and 1970) and are used for the preparation of vaccines based on live attenuatedvirus: the first one is the WI-38 line (Winstar Institute 38), with human diploid lung fibroblasts, comingfrom a female foetus that was aborted because the family felt they had too many children (G. Sven etal., 1969). It was prepared and developed by Leonard Hayflick in 1964 (L. Hayflick, 1965; G. Sven etal., 1969)3 and bears the ATCC number CCL-75. WI-38 has been used for the preparation of thehistorical vaccine RA 27/3 against rubella (S.A. Plotkin et al, 1965)4. The second human cell line isMRC-5 (Medical Research Council 5) (human, lung, embryonic) (ATCC number CCL-171), with humanlung fibroblasts coming from a 14 week male foetus aborted for "psychiatric reasons" from a 27 yearold woman in the UK. MRC-5 was prepared and developed by J.P. Jacobs in 1966 (J.P. Jacobs et al,1970)5. Other human cell lines have been developed for pharmaceutical needs, but are not involved inthe vaccines actually available6.

The vaccines that are incriminated today as using human cell lines from aborted foetuses, WI-38 andMRC-5, are the following:7



A) Live vaccines against rubella8:

the monovalent vaccines against rubella Meruvax®!! (Merck) (U.S.), Rudivax® (Sanofi Pasteur,Fr.), and Ervevax® (RA 27/3) (GlaxoSmithKline, Belgium);the combined vaccine MR against rubella and measles, commercialized with the name of M-R-VAX® (Merck, US) and Rudi-Rouvax® (AVP, France);the combined vaccine against rubella and mumps marketed under the name of Biavax®!!(Merck, U.S.),the combined vaccine MMR (measles, mumps, rubella) against rubella, mumps and measles,marketed under the name of M-M-R® II (Merck, US), R.O.R.®, Trimovax® (Sanofi Pasteur, Fr.),and Priorix® (GlaxoSmithKline UK).

B) Other vaccines, also prepared using human cell lines from aborted foetuses:

two vaccines against hepatitis A, one produced by Merck (VAQTA), the other one produced byGlaxoSmithKline (HAVRIX), both of them being prepared using MRC-5;one vaccine against chicken pox, Varivax®, produced by Merck using WI-38 and MRC-5;one vaccine against poliomyelitis, the inactivated polio virus vaccine Poliovax® (Aventis-Pasteur, Fr.) using MRC-5;one vaccine against rabies, Imovax®, produced by Aventis Pasteur, harvested from infectedhuman diploid cells, MRC-5 strain;one vaccine against smallpox, AC AM 1000, prepared by Acambis using MRC-5, still on trial.

The position of the ethical problem related to these vaccines

From the point of view of prevention of viral diseases such as German measles, mumps, measles,chicken pox and hepatitis A, it is clear that the making of effective vaccines against diseases such asthese, as well as their use in the fight against these infections, up to the point of eradication, bymeans of an obligatory vaccination of all the population at risk, undoubtedly represents a "milestone"in the secular fight of man against infective and contagious diseases.

However, as the same vaccines are prepared from viruses taken from the tissues of foetuses that hadbeen infected and voluntarily aborted, and the viruses were subsequently attenuated and cultivatedfrom human cell lines which come likewise from procured abortions, they do not cease to pose ethicalproblems. The need to articulate a moral reflection on the matter in question arises mainly from theconnection which exists between the vaccines mentioned above and the procured abortions fromwhich biological material necessary for their preparation was obtained.

If someone rejects every form of voluntary abortion of human foetuses, would such a person notcontradict himself/herself by allowing the use of these vaccines of live attenuated viruses on theirchildren? Would it not be a matter of true (and illicit) cooperation in evil, even though this evil wascarried out forty years ago?

Before proceeding to consider this specific case, we need to recall briefly the principles assumed inclassical moral doctrine with regard to the problem of cooperation in evil 9, a problem which arisesevery time that a moral agent perceives the existence of a link between his own acts and a morally evilaction carried out by others.

The principle of licit cooperation in evil

The first fundamental distinction to be made is that between formal and material cooperation. Formal

cooperation is carried out when the moral agent cooperates with the immoral action of another person,sharing in the latter's evil intention. On the other hand, when a moral agent cooperates with theimmoral action of another person, without sharing his/her evil intention, it is a case of material

cooperation.

Material cooperation can be further divided into categories of immediate (direct) and mediate (indirect),depending on whether the cooperation is in the execution of the sinful action per se, or whether theagent acts by fulfilling the conditions - either by providing instruments or products - which make itpossible to commit the immoral act. Furthermore, forms of proximate cooperation and remote



cooperation can be distinguished, in relation to the "distance" (be it in terms of temporal space ormaterial connection) between the act of cooperation and the sinful act committed by someone else.Immediate material cooperation is always proximate, while mediate material cooperation can be eitherproximate or remote.

Formal cooperation is always morally illicit because it represents a form of direct and intentionalparticipation in the sinful action of another person.10 Material cooperation can sometimes be illicit(depending on the conditions of the "double effect" or "indirect voluntary" action), but when immediate

material cooperation concerns grave attacks on human life, it is always to be considered illicit, giventhe precious nature of the value in question11.

A further distinction made in classical morality is that between active (or positive) cooperation in eviland passive (or negative) cooperation in evil, the former referring to the performance of an act ofcooperation in a sinful action that is carried out by another person, while the latter refers to theomission of an act of denunciation or impediment of a sinful action carried out by another person,insomuch as there was a moral duty to do that which was omitted12.

Passive cooperation can also be formal or material, immediate or mediate, proximate or remote.Obviously, every type of formal passive cooperation is to be considered illicit, but even passive materialcooperation should generally be avoided, although it is admitted (by many authors) that there is not arigorous obligation to avoid it in a case in which it would be greatly difficult to do so.

Application to the use of vaccines prepared from cells coming from embryos or foetusesaborted voluntarily

In the specific case under examination, there are three categories of people who are involved in thecooperation in evil, evil which is obviously represented by the action of a voluntary abortion performedby others: a) those who prepare the vaccines using human cell lines coming from voluntary abortions;b) those who participate in the mass marketing of such vaccines; c) those who need to use them forhealth reasons.

Firstly, one must consider morally illicit every form of formal cooperation (sharing the evil intention) inthe action of those who have performed a voluntary abortion, which in turn has allowed the retrieval offoetal tissues, required for the preparation of vaccines. Therefore, whoever - regardless of the categoryto which he belongs — cooperates in some way, sharing its intention, to the performance of avoluntary abortion with the aim of producing the above-mentioned vaccines, participates, in actuality, inthe same moral evil as the person who has performed that abortion. Such participation would also takeplace in the case where someone, sharing the intention of the abortion, refrains from denouncing orcriticizing this illicit action, although having the moral duty to do so (passive formal cooperation).

In a case where there is no such formal sharing of the immoral intention of the person who hasperformed the abortion, any form of cooperation would be material, with the following specifications.

As regards the preparation, distribution and marketing of vaccines produced as a result of the use ofbiological material whose origin is connected with cells coming from foetuses voluntarily aborted, sucha process is stated, as a matter of principle, morally illicit, because it could contribute in encouragingthe performance of other voluntary abortions, with the purpose of the production of such vaccines.Nevertheless, it should be recognized that, within the chain of production-distribution-marketing, thevarious cooperating agents can have different moral responsibilities.

However, there is another aspect to be considered, and that is the form of passive material

cooperation which would be carried out by the producers of these vaccines, if they do not denounceand reject publicly the original immoral act (the voluntary abortion), and if they do not dedicatethemselves together to research and promote alternative ways, exempt from moral evil, for theproduction of vaccines for the same infections. Such passive material cooperation, if it should occur, isequally illicit.

As regards those who need to use such vaccines for reasons of health, it must be emphasized that,apart from every form of formal cooperation, in general, doctors or parents who resort to the use ofthese vaccines for their children, in spite of knowing their origin (voluntary abortion), carry out a form of



very remote mediate material cooperation, and thus very mild, in the performance of the original act ofabortion, and a mediate material cooperation, with regard to the marketing of cells coming fromabortions, and immediate, with regard to the marketing of vaccines produced with such cells. Thecooperation is therefore more intense on the part of the authorities and national health systems thataccept the use of the vaccines.

However, in this situation, the aspect of passive cooperation is that which stands out most. It is up tothe faithful and citizens of upright conscience (fathers of families, doctors, etc.) to oppose, even bymaking an objection of conscience, the ever more widespread attacks against life and the "culture ofdeath" which underlies them. From this point of view, the use of vaccines whose production isconnected with procured abortion constitutes at least a mediate remote passive material cooperationto the abortion, and an immediate passive material cooperation with regard to their marketing.Furthermore, on a cultural level, the use of such vaccines contributes in the creation of a generalizedsocial consensus to the operation of the pharmaceutical industries which produce them in an immoralway.

Therefore, doctors and fathers of families have a duty to take recourse to alternative vaccines13 (if theyexist), putting pressure on the political authorities and health systems so that other vaccines withoutmoral problems become available. They should take recourse, if necessary, to the use ofconscientious objection14 with regard to the use of vaccines produced by means of cell lines ofaborted human foetal origin. Equally, they should oppose by all means (in writing, through the variousassociations, mass media, etc.) the vaccines which do not yet have morally acceptable alternatives,creating pressure so that alternative vaccines are prepared, which are not connected with the abortionof a human foetus, and requesting rigorous legal control of the pharmaceutical industry producers.

As regards the diseases against which there are no alternative vaccines which are available andethically acceptable, it is right to abstain from using these vaccines if it can be done without causingchildren, and indirectly the population as a whole, to undergo significant risks to their health. However,if the latter are exposed to considerable dangers to their health, vaccines with moral problemspertaining to them may also be used on a temporary basis. The moral reason is that the duty to avoidpassive material cooperation is not obligatory if there is grave inconvenience. Moreover, we find, insuch a case, a proportional reason, in order to accept the use of these vaccines in the presence of thedanger of favouring the spread of the pathological agent, due to the lack of vaccination of children. Thisis particularly true in the case of vaccination against German measles15.

In any case, there remains a moral duty to continue to fight and to employ every lawful means in orderto make life difficult for the pharmaceutical industries which act unscrupulously and unethically.However, the burden of this important battle cannot and must not fall on innocent children and on thehealth situation of the population - especially with regard to pregnant women.

To summarize, it must be confirmed that:

there is a grave responsibility to use alternative vaccines and to make a conscientious objectionwith regard to those which have moral problems;as regards the vaccines without an alternative, the need to contest so that others may beprepared must be reaffirmed, as should be the lawfulness of using the former in the meantimeinsomuch as is necessary in order to avoid a serious risk not only for one's own children butalso, and perhaps more specifically, for the health conditions of the population as a whole -especially for pregnant women;the lawfulness of the use of these vaccines should not be misinterpreted as a declaration of thelawfulness of their production, marketing and use, but is to be understood as being a passivematerial cooperation and, in its mildest and remotest sense, also active, morally justified as anextrema ratio due to the necessity to provide for the good of one's children and of the peoplewho come in contact with the children (pregnant women);such cooperation occurs in a context of moral coercion of the conscience of parents, who areforced to choose to act against their conscience or otherwise, to put the health of their childrenand of the population as a whole at risk. This is an unjust alternative choice, which must beeliminated as soon as possible.

References



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1. J. E. Banatvala, D.W.G. Brown, Rubella, The Lancet, 3rd April 2004, vol. 363, No. 9415, pp.1127-11372. Rubella, Morbidity and Mortality Weekly Report, 1964, vol. 13, p.93. S.A. Plotkin, Virologic Assistance in the

Management of German Measles in Pregnancy, JAMA, 26th October 1964, vol.190, pp.265-2683. L. Hayflick, The Limited In Vitro Lifetime of Human Diploid Cell Strains, Experimental Cell Research, March 1965,

vol.37, no. 3, pp. 614-636.G. Sven, S. Plotkin, K. McCarthy, Gamma Globulin Prophylaxis; Inactivated Rubella Virus; Production and BiologicalControl of Live Attenuated Rubella Virus Vaccines, American journal of Diseases of Children, August 1969, vol. 118,no. 2, pp.372-381.

4. S. A. Plotkin, D. Cornfeld, Th.H. Ingalls, Studies of Immunization With Living Rubella Virus, Trials in Children With aStrain coming from an Aborted Fetus, American Journal of Diseases in children, October 1965, vol. 110, no. 4,pp.381-389.

5. J.P. Jacobs, C.M. Jones, J.P. Bailie, Characteristics of a Human Diploid Cell Designated MRC-5, Nature, 11th July1970, vol.277, pp.168-170.

6. Two other human cell l ines, that are permanent, HEK 293 aborted fetal cell l ine, from primary human embryonickidney cells transformed by sheared adenovirus type 5 (the fetal kidney material was obtained from an aborted fetus,in 1972 probably), and PER.C6, a fetal cell l ine created using retinal tissue from an 18 week gestation aborted baby,have been developed for the pharmaceutical manufacturing of adenovirus vectors (for gene therapy). They have notbeen involved in the making of any of the attenuated live viruses vaccines presently in use because of their capacityto develop tumorigenic cells in the recipient. However some vaccines, sti l l at the developmental stage, against Ebolavirus (Crucell,NV and the Vaccine Research Center of the National Institutes of Health's Allergy and InfectiousDiseases, NIAID), HIV (Merck), influenza (Medlmmune, Sanofi pasteur), Japanese encephalitis (Crucell N.V. andRhein Biotech N.V.) are prepared using PER.C6® cell l ine (Crucell N.V., Leiden, The Netherlands).

7. Against these various infectious diseases, there are some alternative vaccines that are prepared using animals' cells ortissues, and are therefore ethically acceptable. Their availabil ity depends on the country in question. Concerning theparticular case of the United States, there are no options for the time being in that country for the vaccination againstrubella, chickenpox and hepatitis A, other than the vaccines proposed by Merck, prepared using the human cell l inesWI-38 and MRC-5. There is a vaccine against smallpox prepared with the Vero cell l ine (derived from the kidney of anAfrican green monkey), ACAM2000 (Acambis-Baxter) ( a second-generation smallpox vaccine, stockpiled, notapproved in the US), which offers, therefore, an alternative to the Acambis 1000. There are alternative vaccinesagainst mumps (Mumpsvax, Merck, measles (Attenuvax, Merck), rabies (RabAvert, Chiron therapeutics), prepared fromchicken embryos. (However serious allergies have occurred with such vaccines), poliomyelitis (IPOL, Aventis-Pasteur,prepared with monkey kidney cells) and smallpox (a third-generation smallpox vaccine MVA, Modified VacciniaAnkara, Acambis-Baxter). In Europe and in Japan, there are other vaccines available against rubella and hepatitis A,produced using non-human cell l ines. The Kitasato Institute produce four vaccines against rubella, called Takahashi,TO-336 and Matuba, prepared with cells from rabbit kidney, and one (Matuura) prepared with cells from a quailembryo. The Chemo-sero-therapeutic Research Institute Kaketsuken produce one another vaccine against hepatitis A,called Ainmugen, prepared with cells from monkey kidney. The only remaining problem is with the vaccine Varivax®against chicken pox, for which there is no alternative.

8. The vaccine against rubella using the strain Wistar RA27/3 of l ive attenuated rubella virus, adapted and propagatedin WI-38 human diploid lung fibroblasts is at the centre of present controversy regarding the morality of the use ofvaccines prepared with the help of human cell l ines coming from aborted foetuses.

9. D.M. Prummer O. Pr., De cooperatione ad malum, in Manuale Theologiae Moralis secundum Principia S. ThomaeAquinatis, Tomus I, Friburgi Brisgoviae, Herder & Co., 1923, Pars I, Trat. IX, Caput III, no. 2, pp. 429-434. .K.H. Peschke, Cooperation in the sins of others, in Christian Ethics. Moral Theology in the Light of Vatican II, vol.1,General Moral Theology, C. Goodliffe Neale Ltd., Arden Forest Industrial Estate, Alcester, Warwickshire, B49 6Er,revised edition, 1986, pp. 320-324.

10. A. Fisher, Cooperation in Evil, Catholic Medical Quarterly, 1994, pp. 15-22..D. Tettamanzi, Cooperazione, in Dizionario di Bioetica, S. Leone, S. Privitera ed., Istituto Sicil iano di Bioetica,EDB-ISB, 1994, pp.194-198..L. Melina, La cooperazione con azioni moralmente cattive contra la vita umana, in Commentario Interdisciplinare alia"Evangelium Vitae", E. Sgreccia, Ramon Luca Lucas ed., Libreria Editrice Vaticana, 1997, pp.467-490..E. Sgreccia, Manuale di Bioetica, vol. I, Reprint of the third edition, Vita e Pensiero, Milan, 1999, pp.362-363.

11. Cf. John Paul II, Enc. Evangelium Vitae, no. 74.12. No. 1868 of the Catechism of the Catholic Church.13. The alternative vaccines in question are those that are prepared by means of cell l ines which are not of human

origin, for example, the Vero cell l ine (from monkeys) (D. Vinnedge), the kidney cells of rabbits or monkeys, or thecells of chicken embryos. However, it should be noted that grave forms of allergy have occurred with some of thevaccines prepared in this way. The use of recombinant DNA technology could lead to the development of newvaccines in the near future which will no longer require the use of cultures of human diploid cells for the attenuationof the virus and its growth, for such vaccines will not be prepared from a basis of attenuated virus, but from thegenome of the virus and from the antigens thus developed (G. C. Woodrow, W.M. McDonnell and F.K. Askari). Someexperimental studies have already been done using vaccines developed from DNA that has been derived from thegenome of the German measles virus. Moreover, some Asiatic researchers are trying to use the Varicella virus as avector for the insertion of genes which codify the viral antigens of Rubella. These studies are sti l l at a preliminaryphase and the refinement of vaccine preparations which can be used in clinical practice will require a lengthy periodof time and will be at high costs. .D. Vinnedge, The Smallpox Vaccine, The National Catholic Bioethics Quarterly,Spring 2000, vol.2, no. 1, p. 12. .G.C. Woodrow, An Overview of Biotechnology As Applied to Vaccine Development,



in «New Generation Vaccines)), G.C. Woodrow, M.M. Levine eds., Marcel Dekker Inc., New York and Basel, 1990, seepp.32-37. W.M. McDonnell, F.K. Askari, Immunization, JAMA, 10th December 1997, vol.278, no.22, pp.2000-2007,see pp. 2005-2006.

14. Such a duty may lead, as a consequence, to taking recourse to "objection of conscience" when the action recognizedas il l icit is an act permitted or even encouraged by the laws of the country and poses a threat to human life. TheEncyclical Letter Evangelium Vitae underlined this "obligation to oppose" the laws which permit abortion oreuthanasia "by conscientious objection" (no.73)

15. This is particularly true in the case of vaccination against German measles, because of the danger of CongenitalRubella Syndrome. This could occur, causing grave congenital malformations in the foetus, when a pregnant womanenters into contact, even if it is brief, with children who have not been immunized and are carriers of the virus. In thiscase, the parents who did not accept the vaccination of their own children become responsible for the malformationsin question, and for the subsequent abortion of foetuses, when they have been discovered to be malformed.

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REVIEW

The key role of adult stem cells: therapeutic perspectivesAugusto Pessina a and Laura Gribaldo b

a Department of Public Health, Microbiology, Virology, University of Milan, Italy

b European Centre for Validation of Alternative Methods (ECVAM), IHCP, Joint Research Centre, Ispra (Va) Italy

Address for correspondence: Augusto Pessina, Department of Public Health, Microbiology, Virology, Section of Microbiology, Cell Culture Laboratory, University of Milan, Via Pascal 36, 20133 Milan, Italy. Tel.: +39 2 50315072; email: [email protected]

Key words: Genetic engineering, therapy – Plasticity – Risk – Stem cells – Tissue engineering

0300-7995doi:10.1185/030079906X148517

All rights reserved: reproduction in whole or part not permitted

CURRENT MEDICAL RESEARCH AND OPINION®VOL. 22, NO. 11, 2006, 2287–2300

© 2006 LIBRAPHARM LIMITED

Paper 3289 2287

Background: The origin, function and physiology of totipotent embryonic cells are configured to construct organs and create cross-talk between cells for the biological and neurophysiologic development of organisms. Adult stem cells are involved in regenerating tissues for renewal and damage repair.

Findings: Adult stem cells have been isolated from adult tissue, umbilical cord blood and other non-embryonic sources, and can transform into many tissues and cell types in response to pathophysiological stimuli.

Clinical applications of adult stem cells and progenitor cells have potential in the regeneration of blood cells, skin, bone, cartilage and heart muscle, and may have potential in degenerative diseases. Multi-pluripotent adult stem cells can change their phenotype in response to trans-differentiation or fusion and their therapeutic

potential could include therapies regulated by pharmacological modulation, for example mobilising endogenous stem cells and directing them within a tissue to stimulate regeneration. Adult stem cells could also provide a vehicle for gene therapy, and genetically-engineered human adult stem cells have shown success in treatment of genetic disease.

Conclusion: Deriving embryonic stem cells from early human embryos raises ethical, legal, religious and political questions. The potential uses of stem cells for generating human tissues are the subject of ongoing public debate. Stem cells must be used in standardised and controlled conditions in order to guarantee the best safety conditions for the patients. One critical point will be to verify the risk of tumourigenicity; this issue may be more relevant to embryonic than adult stem cells.

A B S T R A C T

Introduction

Recent discoveries regarding the developmental potential of embryonic, foetal and adult stem cells have generated hope that such cells may be used for the treatment of several human diseases. Embryonic stem cells (ESC) are totipotent, retaining the capacity to generate any foetal and adult cell type both in vitro and in vivo. Adult autologous stem cells (ASC) are found in a number of tissues, and may have the ability to trans-differentiate into multiple other cell types. The derivation of ESCs from early human embryos raises

ethical, legal, religious and political questions and the potential use of stem cells for generating human tissues and perhaps organs (reparative medicine), is a subject of ongoing public debate. During the fourth National Institutes of Health Bioengineering Consortium symposium, held in Bethesda in June 2001, ‘reparative medicine’ was defined as the ‘development or growing of biological substitutes for the body in vitro and/or fostering of tissue regeneration or remodelling in vivo, with the purpose being to replace, repair, maintain or enhance tissue/organ function’. Before beginning clinical trials in humans, the issue of unregulated growth

2288 Adult stem cells and therapeutic perspectives © 2006 LIBRAPHARM LTD – Curr Med Res Opin 2006; 22(11)

potential and its relationship to stem cell differentiation must be evaluated and two essential questions must be answered, firstly, ‘are stem cells safe,’ and secondly, ‘do adult stem cells work better or worse than embryonic embryo stem cells’.

To answer the first question it is important to apply the criteria widely used in developing new drugs, with especial attention to the biological risk (e.g. immun-ological reaction, tumourigenicity, infection etc.). As haematopoietic stem cells have been largely used for the treatment of haematopoietic disorders and to help patients with malignancies undergoing intensive chemotherapy or radiation therapy, autologous stem cell transplantation is perceived as a non-harmful procedure and clinical trials are often licensed by local ethics committees without an adequate knowledge of the possible drawbacks. Furthermore, some web-sites dedicated to cytotherapy show disproportionately promising results. In fact, some of these therapies, as denounced by scientific societies, may not only be ineffective but also be considered potentially very dangerous.

Adult stem cells

The term ‘adult stem cell’ is somewhat a misnomer, because the cells are present even in infants and similar cells exist in umbilical cord and placenta. Some other terms have been proposed, such as tissue stem cells, somatic stem cells or post-natal stem cells.

Looking at the physiology of a growing organism it appears evident that the intrinsic nature of the pool of adult stem cells is very particular. At first these cells contribute to the growth of the organism, by increasing the dimension of the organs then, after growth is completed, the main role of a stem cell is homeostatic (to renew cells and/or regenerate the cells of damaged tissues). This role is unique, and takes place in tissues and organs of a healthy adult by means of depots of adult stem cells capable of responding to different tissue requests (both physiological and pathological) according to a very dynamic proliferative and differentiative plasticity. An adult organism has pluri- and multipotent stem cells that have been described to be able to trans-differentiate and to fuse with other cells and promote new genetic reprogramming processes1,2.

Embryonic stem cells and embryo totipotency

The term embryonic stem cell emphasises that these cells express the maximal degree of plasticity known so far. According to the authors of this review, however,

it may not be fully appropriate to call these cells, the cells derived from the embryo, which is totipotent, ‘stem cells’. These are actually ‘tout court’ embryo cells, because their totipotency is the expression of a completely different function and nature to adult stem cells. As reported by Thomson et al., 19983 ‘embryo stem cells are derived from totipotent cells of the early mammalian embryo ...’ and in the same article the author clarified that ‘the term ES cells was introduced to distinguish these embryo-derived pluripotent cells from embryocarcinoma cells’3. This clearly means that the cells of the inner mass can be ‘named’ ES only after their anatomical conformation has been destroyed and single cells are placed in vitro. Of course, in this new environment, these cells can no longer create a complete and functional organism, but can produce every type of tissue.

Reports in 2006 showed that this pluripotency is regulated by the interaction between Oct-4/Sox-2 and NanOg genes4. It is the opinion of the authors of this review that the intrinsic potentiality of a cell can be expressed only in relation to an appropriate environment, and that the potentiality depends on the conditions that also regulate the structural relation with other cells or with the appropriate environment. The inner mass represents the totipotent ‘developing unit’ of the blastocyst, and is able to originate tissues and organs in a three-dimensional spatial symmetry. The creation of the network of relationships amongst cells by a complex process of ‘cross-talk’ provides the neurophysiological development into a whole individual organism.

The developmental programme of these cells is determined by passages chronologically regulated by the sensitivity of specific growth factors or cytokines (or their production) that form tissues and organs according to the so called embryogenetic principles: the differential genetic expression, the tissue determination and the positional specification5. The expression of transcription factor Oct-4 is thought to be one of the decisive factors that monitors totipotency in embryonic and germ cells, both in the mouse and human6–8, and it has been proved that both the nuclei of the inner mass and trophoderm have a developmental totipotency9. A paper published in 2005 demonstrated that reconstructed embryos obtained by inter-strain inner cell mass replacement have the ability to develop to term10, confirming that nuclei of embryonic stem cells are able to reprogram somatic cells11. Tissue determination and positional specification are inter-dependent and synergise to produce polyclones having common histology.

The main characteristic that differentiates an embryo cell from a typical ‘stem cell’ is the mode of cell division. During embryogenesis, cell division mechanisms change

© 2006 LIBRAPHARM LTD – Curr Med Res Opin 2006; 22(11) Adult stem cells and therapeutic perspectives Pessina and Gribaldo 2289

rapidly and are very different. Five typologies of division can be described, but only one of these is similar to that of the stem cells (based on the mechanism of self-renewal to assure the stem cell pool)12. Generally, embryonic stem cells differentiate almost exclusively by a directional symmetric division as suggested by Zwaka et al. ‘… in some respect, embryonic stem cells more closely resemble precursor or transit amplifying cells rather than adult stem cells’13. But embryo cells can divide asymmetrically in the distribution of some genes as reported for pax 6 and Ngn-2 in neural cells14. A recent study on the mammalian gastrointestinal tract analysed the importance of its change from a mid-line structure into an asymmetric tract able to ensure a unidirectional movement of digested material15. In the heart the hedgehog gene is particularly active on the left side of the embryo, where it contributes by various mechanisms to accumulate specific growth factors influencing the development of this organ on the left hand side of the body. Thousand of genes regulated by master genes (for example Notch1, Runx1, NanOg, Sox-2, Oct –4 etc.) are probably essential in the cross-talk during embryogenesis.

Main risks related to stem cell therapy

The capacity of embryonic stem cells for virtually unlimited self-renewal and differentiation has opened up the prospect of widespread applications in biomedical and toxicological research and in reparative (or regenerative) medicine. Seven years after the first derivation of human pluripotent cell lines from pre-implantation embryos, a great deal has been learnt about their biology and how differentiation can be induced towards particular cell lineages. The ability to establish stem cell lines in vitro results in the possibility of producing large batches of allogeneic undifferenti-ated or differentiated cells. Besides the general problems associated with immunological reactions and infectious diseases, the totipotency of these cells must be considered. This could make it difficult to control the proliferative and differentiative potential of ESCs, which is wider and more dynamic than that of somatic stem cells. This risk is related to our poor knowledge of the genetic expression mechanisms and factors that regulate the complex phenomena described above.

A tumourigenic theory has been recently proposed that attributes many tumours to the stem cell compartment, where some genes playing a key-role may be dysregulated16. In 1975, it was demonstrated that cells of embryocarcinomas can perfectly integrate into normal tissues as well as the opposite17. Furthermore, it is known that many genes crucial during embryogenesis

(e.g. Oct-4, NanOg, Notch, BCR1, BCR2, hedgehog, EGF-CFC, etc.) are expressed at high levels in breast cancer, gliomas and gastrointestinal tract tumours. Supporting a theory of stem cell tumourigenesis, Tai et al. have shown that Oct-4 remains expressed in some cells of the basal layer of human epidermis16. Moreover, experiments in animals have shown that ESCs injected into the brain of syngeneic mice can generate teratocarcinomas18,19, and primary bone marrow-derived mesenchymal progenitor cells can stimulate the development of Ewing’s sarcomas20.

As stated by Andrews et al.21, ‘embryonic stem cells and embryonic carcinoma cells are the opposite side of the same coin’, and the potential risk linked to the therapeutic use of ESCs is underlined by a 2006 paper describing the transformation of early foetal cells into pre-invasive testicular carcinoma cells22. If there is evidence on the Oct-4 role to maintain embryonic cells in a pluripotent status, little is known on its potential oncogenic properties23. Furthermore, it cannot be excluded that the tumourigenesis of embryonic stem cells, as observed in animal models, could be host-dependent18. A second very important aspect, high biological risk, makes the use of ESCs of concern; it has been reported that in vitro cell lines obtained from embryo cells (even after their differentiation) show a certain degree of dysregulation in controlling the expression of the so called ‘imprinted genes’ (a group of genes involved in giving the parental imprinting)24,25. Genomic imprinting is an important genetic mech-anism influencing the transfer of nutrients to the foetus, and many human and animal growth and behavioural defects, as well as some tumours, seem to be a consequence of dysregulation of imprinted genes. Disrupted genomic imprinting appears to contribute to increased tumours, and cancer aetiology can depend on the epigenetic alterations that contribute to increased genetic modulations26.

Serious genetic alterations were found in eight of nine cell lines cultured for long periods, suggesting that if stem cells are to be used in cell therapy they must be used at early passages to avoid risks27. This observation supports the data showing that human mesenchymal stem cells (MSCs) can undergo spontaneous transform-ation during long-term culture (4–5 months)28,29. On the other hand, the observation that human mesenchymal stem cells can integrate well into gliomas after intravascular or local delivery indicate that this tropism for human gliomas could be exploited to therapeutic advantage30.

All the above reported considerations raise questions on the use of ESCs in therapy. Sapienza31 suggested that ‘ESCs might not be the best source of therapeutic material for transplantation therapy’. Regarding risk of infection, some questions have arisen recently on the


possible expression of endogenous viruses, or changes in virus susceptibility, in uncontrolled in vitro long-term manipulation.

A comparison of the main advantages and dis-advantages related to the use of adult or embryonic stem cells is reported in Table 1.

Stem cell plasticity

Unfortunately the lack of a clear definition of plasticity has made it difficult to compare the results reported in literature. This topic was covered in a review by Lakshmipathy and Verfaillie32, which suggested defining stem cell plasticity according to three main criteria:

(a) differentiation of a single cell into multiple cell lineages

(b) functionality of differentiated cells in vitro and in vivo

(c) robust and persistent engraft of transplanted cells.

It is easier to apply these criteria in some studies more than others. For instance it has been reported that bone marrow or peripheral blood contribute to the repair and genesis of cells specific for liver, cardiac and skeletal muscle, gut and brain tissue33,34 as a result of the active function of multipotent adult progenitor cells (MAPC) or mesenchymal stem cells35 that are probably the same type of cell discovered in 1970 by Friedenstein and described as CFU-F36. Studies have shown the ability of these cells to be integrated among the photoreceptors of mice with retinitis pigmentosa or to regenerate spinal cord of chicken embryos37 as

well as cardiomyocytes in the heart38. By using intra-uterine injections of bone marrow stem cells, a human embryo was recently cured for osteogenesis imperfecta syndrome (radiographically confirmed by the good bone formation observed in the baby some months after birth)39. Umbilical cord blood (UCB) contains a multipotent stem cell able to differentiate into mature blood cells, osteoblasts, chondroblasts, adipocytes, astrocytes, neurons and epatocytes40,41 as well as cells expressing markers of pancreatic transitional cells42,43. In addition to bone marrow, many other niches of multipotent stem cells have been identified, such as olfactory mucosa44 containing neural stem cells that could potentially have a regenerative role if implanted in the spinal cord in traumatic tetraplegia45,46. Very few limbic cells are needed to regenerate a cornea in vitro for transplantation into patients, and, according to some studies, the human eye could contain around 10 000 cells with characteristics of retinal stem cells47.

Important questions regarding adult stem cells include their source tissue and their ability to form other cell or tissue types. Historically only a few stem cells have been recognised in humans, such as the haematopoietic stem cell that produces all blood cells, the gastrointestinal stem cell associated with regeneration of the gastrointestinal lining, the stem cell responsible for the epidermal layer of skin, and germ cell precursors (in the adult human, the spermatogonal stem cell). These stem cells were considered to have very limited repertoires, related to replenishment of cells within their tissue of origin. These limitations were considered to be a normal part of the developmental paradigm in which cells become more and more restricted in their lineage capabilities, leading to defined and specific differentiated cells in body tissues. Thus, discovery of stem cells in other tissues, or with

Table 1. Some advantages and disadvantages of adult and embryonic stem cells

Advantages Disadvantages

Embryonic stem cells

Can make virtually any tissue (in theory) Allogeneic only (currently)

Some tissues ‘easy’ to generate (e.g. cardiac) Teratoma formation Can be propagated indefinitely Differentiation conditions to be established Amenable to genetic manipulation? Some tissues difficult to generate (blood) Ethical issues Adult stem cells Autologous Most have limited self renewal Many types and sources Differentiation outside lineage? (possibly) Some types have extensive self-renewal potential Autologous (use more cumbersome and expensive) Low or not tumourigenic Default differentiation Amenable to gene transfer Potential delivery methods attractive No ethical issues


the ability to cross typical lineage boundaries, is both exciting and thought-provoking, because such evidence challenges the canonical developmental paradigm. In any case, if these characteristics can be confirmed it could be possible in the future to regulate the SSC migration and homing by pharmacological modulation. Many studies of stem cell migration suggest that adult stem cells respond to specific and non-specific chemotactic stimuli, for example SDF-1 alpha, which attracts neuronal stem cells48 or CX3CL12, which attracts mesenchymal cells to pancreatic islets49.

The potency of adult stem cells is termed ‘plasticity’ by a wide number of authors, although the authors of this paper feel it should be the subject of debate, because many biological questions remain unanswered, such as the regulation of the proliferative mechanisms that maintain the cellular homeostasis in terms of quantity of cells rather than differentiation. Such ‘dynamic plasticity’ is not yet understood, in particular relating to the feedback control of the expansion together with the differentiation of the many transitional stem cells (or progenitors). Other intriguing problems include the so called ‘fusion mechanisms’ (having a cell reprogramming capacity) that may be mainly a function of more differentiated cells rather than that of stem cells1.

The reprogramming of cellular gene expression via hybrids is not unlike a novel method reported recently for trans-differentiation of somatic cells. In this method, fibroblasts cultured in the cytoplasm and nucleoplasm of a lysed, differentiated T-lymphocyte cell took up factors from the ‘soup’ of the cellular contents of the differentiated cell, and began expressing the functional characteristics of a T-cell50–52.

It has long been known that dedifferentiation and redifferentiation occurs in amphibians such as Urodeles, which can regenerate whole limbs. A number of studies have suggested that similar although less dramatic processes may cause dedifferentiation of somatic cells. For instance, when oligodendrocyte progenitors from the optical nerve were maintained in serum-free, low-density culture conditions, they acquired NSC characteristics53.

Other studies have suggested that cells committed to pancreatic epithelium could be switched to a hepatic phenotype, even though the functional properties of the hepatocyte lineage cells were not defined. These findings suggest that dedifferentiation and rediffer-entiation might be a third explanation for adult stem cell plasticity. In this model, adult stem or progenitor cells would be reprogrammed when removed from their usual microenvironment and introduced into a different microenvironment, which imparts signals to activate a novel genetic program needed for the new

cell’s fate54,55. Insights in the molecular mechanisms underlying nuclear reprogramming during the cloning process may, therefore, help us to better understand the phenomenon of adult stem cell plasticity and may be exploited in the future to induce lineage switch without nuclear transplantation. Likewise, insights in the molecular mechanisms underlying the de- and redifferentiation phenomena in amphibians and fish that allow regeneration of a limb, might aid in understanding adult stem cell plasticity. For instance, Msx1 is expressed in the regenerating blastema. A recent study demonstrated that over-expression of this homeobox gene in myotubes derived from the C2C12 cell line causes regression of the myotubes into multiple mononuclear myoblasts, which then proliferate and gain the ability to differentiate into osteoblasts, chondrocytes, and adipocytes56. Whether pathways that have been identified as causing de- and redifferentiation in fish and amphibians also play a role in higher mammalian stem cell plasticity will need to be defined.

Stem cell identification

Identification of cells typically relies on use of cell surface markers – cluster of differentiation (CD) antigens – that denote the expression of particular proteins associated with genomic activity related to a particular differentiation state of the cell. For bone marrow stem cells, selection of putative adult stem cells has usually excluded typical markers for haematopoietic lineages (lin-), CD45 and CD38, with inclusion or exclusion of the haematopoietic marker CD34 and inclusion of the marker c-kit (CD117). Other proposed markers for adult stem cells are AC133-2 (CD133), which are found on many stem cell populations57, and C1qRp, the receptor for complement molecule C1q58, found on a subset of CD34+/– human stem cells from bone marrow and umbilical cord blood. Attempts to determine the complete molecular signature of gene expression common to human and mouse stem cells have shown over 200 common genes between haematopoietic and neural stem cells, with some considerable overlap with mouse embryonic stem cells as well59. The function of many of these genes is as yet unknown, but may provide distinctive markers for identification of adult stem cells in different tissues. Blau et al.60 have raised the question of whether there may be a ‘universal’ adult stem cell, residing in multiple tissues and activated dependent on cellular signals, e.g., tissue injury. When recruited to a tissue, the stem cell would take its cues from the local tissue milieu in which it finds itself (including the soluble growth factors, extracellular matrix, and cell–cell


contacts). Examples of such environmental influences on choice have been noted previously61. Thus, it may not be surprising to see cell populations isolated using a common set of markers that show different patterns of maturation62–65. This can be due to the context of the isolation or experimental conditions.

As described above, in the definition of a stem cell, not only does its actual tissue of origin and differ-entiation ability have to be taken into account, but consideration has also to be given to how the concept is influenced by the experimental paradigm used66. Several possible mechanisms have been proposed for differentiation of adult stem cells into other tissues. One mechanism that has received particular attention lately is the possibility of cell fusion, whereby the stem cell fuses with a tissue cell and takes on that tissue’s characteristics. In an in vitro experiment where human mesenchymal stem cells were co-cultured with heat-shocked small airway epithelial cells, some of the stem cells differentiated directly into epithelial cells, while others formed cell fusion hybrids to repair the damage67. The ability to form cell hybrids in some tissues may be a useful mechanism for repair of certain types of tissue damage or for delivery of therapeutic genes to a tissue68.

In contrast with these results, other experiments have shown no evidence that cell fusion plays a role in differentiation of adult stem cells into other tissue types. For example, using human subjects it was shown that human bone marrow cells differentiated into buccal epithelial cells in vivo without cell fusion69, and human cord blood stem cells formed hepatocytes in mouse liver without evidence of cell fusion70. In these cases it appears that the adult stem cells underwent changes in gene expression and directly differentiated into the host tissue cell type, integrating themselves into the tissue.

Sources of adult stem cells

Adult stem cells have been isolated from numerous adult tissues, umbilical cord, and other non-embryonic sources, and have demonstrated a surprising ability for transformation into other tissue and cell types and for repair of damaged tissues.

Bone marrow stem cells

Bone marrow contains at least two, and likely more71,72, discernible stem cell populations. As well as the haematopoietic stem cell which produces blood cell progeny, a cell type termed mesenchymal or stromal stem cells also exists in marrow. This cell provides support for haematopoietic and other cells within the

marrow, and has also been a focus for possible tissue repair73.

Human mesenchymal stem cells have been shown to differentiate in vitro into various cell lineages including neuronal cells74,75, as well as cartilage, bone and fat lineages76. In vivo, human adult mesenchymal stem cells transferred in utero into foetal sheep can integrate into multiple tissues, persisting for over a year. The cells differentiated into cardiac and skeletal muscle, bone marrow stromal cells, fat cells, thymic epithelial cells and cartilage cells. Analysis of a highly purified preparation of human mesenchymal stem cells77 indicated that they could proliferate extensively in culture, constitutively expressing the telomerase enzyme, and even after extensive culture retained the ability to differentiate in vitro into bone, fat and cartilage cells.

Bone marrow-derived cells in general have shown ability to form many tissues in the body. For example, bone marrow-derived stem cells in vivo appear able to form neuronal tissues78,79 and a single adult bone marrow stem cell can contribute to tissues as diverse as marrow, liver, skin and digestive tract80. One group has now developed a method for large-scale generation of neuronal precursors from whole adult rat bone marrow81. In this procedure, treatment of unfractionated bone marrow in culture with epidermal growth factor and basic fibroblast growth factor gave rise to neurospheres with cells expressing neuronal markers. Bone marrow stem cells have also shown the ability to participate in repair of damaged retinal tissues. When bone marrow stem cells were injected into the eyes of mice, they associated with retinal astrocytes and extensively incorporated into the vascular (blood vessel) network of the eye82. Because bone marrow stem cells are of mesodermal lineage, it is not surprising that they show capabilities of forming other tissues of mesodermal origin.

Human marrow stromal cells, which have been shown to form cartilage cells, have been used in an in vitro system to define many of the molecular events associated with the formation of cartilage tissue83. Bone marrow stem cells have also shown a capability of forming kidney cells. Studies following genetically marked bone marrow stem cells in rats84 and mice85 showed that the stem cells could form mesangial cells to repopulate the glomerulus of the kidney. Liver was one of the earliest tissues recognised as showing potential contribution to differentiated cells by bone marrow stem cells. Bone marrow stem cells have been induced to form hepatocytes in culture86 and liver-specific gene expression has been induced in vitro in human bone marrow stem cells87. Heart, as a mesodermally-derived organ, is a likely candidate for regeneration with bone marrow derived stem cells.


Numerous references now document the ability of these adult stem cells to contribute to regeneration of cardiac tissue and improve performance of damaged hearts. The evidence has led numerous groups to use bone marrow derived stem cells in the treatment of patients with damaged cardiac tissue88–91. Results from these clinical trials indicate that bone marrow derived stem cells, including cells from the patients themselves, can regenerate damaged cardiac tissue and improve cardiac performance in humans.

Umbilical cord blood

Cord blood stem cells also show similarities with bone marrow stem cells in terms of their potential to differentiate into other tissue types. Human cord blood stem cells have shown expression of neural markers in vitro92, and intravenous administration of cord blood to animal models of stroke has produced functional recovery in the animals93,94. Infusion of human cord blood stem cells has also produced therapeutic benefit in rats with spinal cord injury95, and in a mouse model of ALS96. A report in 2003 noted establishment of a neural stem/progenitor cell line derived from human cord blood that has been maintained in culture over 2 years without loss of differentiation ability97. Several reports also note the production of functional liver cells from human cord blood stem cells 98–100. Additional differentiative properties of human umbilical cord blood stem cells are likely to be discovered as more investigation proceeds on this source of stem cells.

Peripheral blood

There is abundant evidence that bone marrow stem cells can leave the marrow and enter the circulation, and specific mobilization of bone marrow stem cells is used to harvest stem cells more easily for various bone marrow stem cell treatments101. Therefore, it is not surprising that adult stem cells have been isolated from peripheral blood and that monocytes have been described as cells having a role in hepatocyte fusion mechanisms102.

Neuronal stem cells

Neuronal stem cells have been isolated from various regions of the brain including the more-accessible olfactory bulb103 as well as the spinal cord104, and can even be recovered from cadavers soon after death105. Evidence now exists that neuronal stem cells can produce not only neuronal cells but also other tissues, including blood and muscle105–111. Animal studies have shown that adult neural stem cells can participate in repair of damage after stroke, either via endogenous

neuronal precursors112 or transplanted neural stem cells113. Evidence indicates that endogenous neurons and astrocytes may also secrete growth factors to induce differentiation of endogenous precursors114. In addition, two studies suggest that neural stem cells/neural progenitor cells may show low immunogenicity, being immunoprivileged on transplant115, and raising the possibility for use of donor neural stem cells to treat degenerative brain conditions.

Muscle mesenchymal stem cells

Muscle appears to contain a side population of stem cells, as seen in bone marrow and liver, with the ability to regenerate muscle tissue116. Muscle derived stem cells have been clonally isolated and used to enhance muscle and bone regeneration in animals117. An isolated population of muscle-derived stem cells has also been shown to participate in muscle regeneration in a mouse model of muscular dystrophy118. Stimulation of muscle regeneration from muscle-derived stem cells, as observed in other tissues, is greatly increased after injury of the tissue119,120. Because of the similar nature of muscle cells between skeletal muscle and heart muscle, muscle-derived stem cells have also been proposed for use in repairing cardiac damage121 with evidence that mechanical beating is necessary for full differentiation of skeletal muscle stem cells into cardiomyocytes122. At least one group has used skeletal muscle cells for clinical application to repair cardiac damage in a patient, with positive results123.

Pancreatic stem cells

Interconversion between pancreas and liver has also been demonstrated; in a study, mouse pancreatic stem cells repopulated the liver and corrected metabolic liver disease124. The possibility of repairing the pancreas, however, as a solution to the scourge of diabetes, has been a driving force in efforts to define a stem cell that could regulate insulin in a normative, glucose-dependent fashion. The pancreas itself appears to contain stem/progenitor cells that can regenerate islets in vitro and in vivo. Studies indicate that these pancreatic stem cells can functionally reverse insulin-dependent diabetes in mice125. Similar pancreatic stem cells have been isolated from humans and shown to form insulin-secreting cells in vitro126. The hormone glucagon-like peptide-1 appears to be an important inducing factor of pancreatic stem cell differentiation. Genetic engineering of rat liver cells to contain the pancreatic gene PDX-1 has also been used to generate insulin-secreting cells in vitro; the cells could also restore normal blood glucose levels when injected into mice with experimentally-induced diabetes127–130.


Corneal limbic stem cells

Corneal limbic stem cells are commonly used for replacement of corneas. Limbic cells can be maintained and cell number expanded in culture131, grown on amniotic membranes to form new corneas, and transplanted to patients with good success132–135 A recent report indicates that human corneal stem cells can also display properties of functional neuronal cells in culture136. Another report found that limbic epithelial cells or retinal cells transplanted into retina of rats could incorporate and integrate into damaged retina, but did not incorporate into normal retina137.

Mammary stem cells

Reports have indicated that mammary stem cells also exist. Isolated cells from mouse could be propagated in vitro and differentiated into all three mammary epithelial lineages138. Transcriptional profiling indicated that the mammary stem cells showed similar gene expression profiles to those of bone marrow stem cells. In that respect, there is a report that human and mouse mammary stem cells exist as a side population, as seen for bone marrow, liver and muscle stem cells139. When propagated in culture, the isolated mammary side population stem cells could form epithelial ductal structures.

Salivary gland

A 2003 report indicated that stem cells can be isolated from regenerating rat salivary gland and propagated in vitro140. Under differing culture conditions, the cells express genes typical of liver or pancreas, and when injected into rats can integrate into liver tissue.

Skin

Multipotent adult stem cells have been isolated from the dermis and hair follicle of rodents141. The cells play a role in the maintenance of epidermal and hair follicle structures, can be propagated in vitro and clonally isolated stem cells can be induced to form neurons, glia, smooth muscle and adipocytes in culture. Dermal hair follicle stem cells have also shown the ability to reform the haematopoietic system of myeloablated mice142–144.

Thymic progenitors

Bennett et al. have reported the isolation of thymic epithelial progenitor cells145. Ectopic grafting (under the kidney capsule) of the cells into mice allowed production of all thymic epithelial cell types, as well as attraction of homing T lymphocytes. In separate

experiments, Gill et al. also isolated a putative thymic progenitor cell from mice and were able to use these cells to reform miniature thymuses when the cells were transplanted under the mouse kidney capsule146.

Dental pulp stem cells

Stem cells have been isolated from human adult dental pulp that could be clonally propagated and proliferated rapidly147. Though there were some similarities with bone marrow mesenchymal stem cells, when injected into immunodeficient mice the adult dental pulp stem cells formed primarily dentin-like structures surrounded by pulpy interstitial tissue. Human baby teeth have also been identified as a source of stem cells, designated SHED cells (stem cells from human exfoliated deciduous teeth)148. In vitro, SHED cells could generate neuronal cells, adipocytes, and odontoblasts, and after injection into immunodeficient mice, the cells were indicated in the formation of bone, dentin and neural cells.

Adipose stem cells

One of the more interesting sources identified for human stem cells has been adipose (fat) tissue, in particular liposuctioned fat. While there is some debate as to whether the cells originate in the adipose tissue or are perhaps mesenchymal or peripheral blood stem cells passing through the adipose tissue, they represent a readily-available source for isolation of potentially useful stem cells. The cells can be maintained for extended periods of time in culture, have a mesenchymal-like morphology, and can be induced in vitro to form adipose, cartilage, muscle and bone tissue149–151. The cells have also shown the capability to differentiate into neuronal cells152,153.

Hepatic stem cells

The liver, strategically placed between the gut and the heart and thus exposed to a variety of xenobiotics, can call upon three distinct cell types to achieve renewal and regeneration of hepatocytes: hepatocytes, bile duct cells (oval cells) and hepatic stem cells (HSCs). All three cell types deserve the term ‘stem cells’, being capable of self-renewal and clonal expansion within the liver.

Liver cells are normally quiescent, but after cell loss, hepatocytes rapidly re-enter the cell cycle. When liver regeneration is compromised, the oval cell reaction occurs and these cells transdifferentiate into hepato-cytes. Oval cells and hepatocytes in the damaged rat liver can be derived from circulating bone marrow cells, as can hepatocytes in the undamaged murine liver.


Hepatocytes can also be derived from bone marrow cell populations in humans154.

Alison et al.155 reported that antigens traditionally associated with haematopoietic cells are also expressed by oval cells, including c-kit, flt-3, Thy-1 and CD34. In mice, the ability of bone marrow cells to cure a metabolic liver disease (hereditary tyrosinaemia) has been established. A number of animal models permit the near-total replacement of the liver parenchyma by donor cells, and all are valuable for exploring the replication and functional potential of selected populations of cells with hepatocyte lineage potential. Hepatic stem cells (hepatocytes, oval cells/cholangio-cytes or HSCs) may be therapeutically useful for treating a variety of diseases that affect the liver. This would include a number of genetic disorders that produce liver disease such as Wilson’s disease, Crigler–Najjar syndrome and tyrosinaemia, and conditions such as coagulation Factor IX deficiency.

Mobilization for tissue repair and stimulation of endogenous cells

As reported above, an important point to consider as we look ahead to utilization of adult stem cells for tissue repair is that it may be not necessary to isolate and culture stem cells before injecting them back into a patient to initiate tissue repair. Rather, it may be easier and preferable to mobilise endogenous stem cells for repair of damaged tissue. Initial results of this technique have already been seen in some animal experiments, in which bone marrow and peripheral blood stem cells were mobilised with injections of growth factors, and participated in the repair of heart and stroke damage156–158. The ability to mobilise endogenous stem cells, coupled with natural or perhaps induced targeted homing of the cells to damaged tissue, could greatly facilitate the use of adult stem cells in simplified tissue regeneration schemes159.

Such stimulation need not rely on any added stem cells. This approach would circumvent the need to isolate or grow stem cells in culture, or inject any stem cells into the body, whether the cells were derived from the patient or another source. Moreover, direct stimulation of endogenous tissue stem cells with specific growth factors might even preclude any need to mobilise stem cells to a site of tissue damage. A few experimental results suggest that this approach might be possible. One group has reported that use of glial-derived neurotrophic factor and neurotrophin-3 can stimulate regeneration of sensory axons in adult rat spinal cord160,161. Administration of transforming growth factor to the brains of mouse models of Parkinson’s disease stimulated proliferation

and differentiation of endogenous neuronal stem cells and produced therapeutic results in the mice162 and infusion of glial-derived neurotrophic factor into the brains of Parkinson’s disease patients resulted in increased dopamine production within the brain and therapeutic benefit to the patients163. Some authors163 have found that bone morphogenetic protein-7 (BMP-7) can counteract deleterious cell changes associated with tissue damage. In this latter study, a mouse model of chronic kidney damage was used. Damage to the tissue causes a transition from epithelial to mesenchymal cell types in the kidney, leading to fibrosis. The transition appears to be initiated by the action of transforming growth factor beta-1 on the tissues, and BMP-7 was shown to counteract this signalling in vitro. Systemic administration of BMP-7 in the mouse model reversed the transition in vivo and led to repair of severely damaged renal tubule epithelial cells. These experiments indicate that direct stimulation of tissues by the correct growth factors could be sufficient to prevent or repair tissue damage. The key point of treatments would be identification of the correct stimuli specific to a tissue or cell type.

Gene therapy applications with adult stem cells

Adult stem cells can provide an efficient vehicle for gene therapy applications, and engineered adult stem cells may allow increased functionality, proliferative capacity or stimulatory capability to these cells. The feasibility of genetically engineering adult stem cells has been shown, for example, in the use of bone marrow stem cells carrying stably inserted genes. The engineered stem cells when injected into mice could still participate in formation and repair of differentiated tissue, such as in the lung164. As another example, engineered stem cells containing an autoantigen to induce immune tolerance of T cells to insulin-secreting cells, were shown to prevent onset of diabetes in a mouse model of diabetes165, a strategy that may be useful for various human autoimmune diseases. Introduction of the PDX-1 gene into liver stem cells stimulated differentiation into insulin-producing cells which could normalise glucose levels when transplanted into mice with induced diabetes166.

Simply engineering cells to increase their prolifer-ative capacity can have a significant effect on their utility for tissue engineering and repair. For example, McKee et al.167 engineered human smooth muscle cells by introducing human telomerase, which greatly increased their proliferative capacity beyond the normal lifespan of smooth muscle cells in culture, while allowing retention of their normal smooth muscle characteristics. These engineered smooth


muscle cells were seeded onto biopolymer scaffolds and allowed to grow into smooth muscle layers, then seeded with human umbilical vein endothelial cells. The resulting engineered arterial vessels could be useful for transplants and bypass surgery. Similarly, human marrow stromal cells that were engineered with telomerase increased their proliferative capacity signif-icantly, but also showed enhanced ability at stimulating bone formation in experimental animals168.

Genetically-engineered human adult stem cells have already been used in the successful treatment of patients with genetic disease. Bone marrow stem cells, from infants with forms of severe combined immuno-deficiency syndrome (SCID), were removed from the patients, a functional gene inserted, and the engineered cells reintroduced to the same patients. The stem cells homed to the bone marrow, engrafted and corrected the defect169–172.

Conclusions

As a number of problems need to be addressed in order to guarantee a high level of safety in the use of stem cells for clinical applications, we suggest some important points that in our opinion should be taken into account.

• At present the methods for cell isolation and in vitro expansion or differentiation are not standardised and are not in compliance with GLP, GMP and GCCP regulations. Furthermore, there is no agreement on the characterisation of specific cell lineages and the purity of cell preparations is not defined, increasing the risk of unknown side effects.

• The traceability of the stem cell preparations needs to be regulated at a European level (see proposal of the Council of the European Community COM[2002] 319 final, August 21, 2002). Currently Europe is lagging behind the USA, where the National Institutes of Health (NIH) has already established a public online Stem Cell Registry in order to ensure that federal funds are only used to support stem cell research that is scientifically, legally and ethically sound.

• The risk of transmitting infectious agents or pyrogen contamination via the transplanted cells, either from the donor or during cell manipulation, needs careful assessment. There is also concern regarding the inadvertent transplantation of cells carrying genetic disorders.

• The use of embryonic stem cells, stem cell banking, clinical research using stem cells, and the development and marketing of stem cell products all raise a series of ethical and legal problems.

• Through use of animal models, there is a strong imperative to find a way to obtain pluripotent stem cells without producing and destroying human embryos.

• Undifferentiated embryonic stem cells are not suitable for transplantation due to the high risk of unregulated growth. If adult stem cells require specific differentiation in vitro, or if they may be capable of organ- and lineage-specific differentiation in vivo, the issue of unregulated growth potential and its relationship to stem cell differentiation must be evaluated. As our understanding of signals inducing the homing of stem cells to a specific organ or tissue is very limited, serious adverse effect must be evaluated in the case of inadequate repopulation of the target site and in the inappropriate colonisation of a non-target site.

• Therapeutic efficacy still needs to be convincingly demonstrated for several potential applications, especially when involving the possible trans-differentiation of adult stem cells.

• The immunogenicity of stem cells is still controversial and not clarified, for this reason additional studies must verify the possible rejection of allogeneic embryonic or adult stem cells by the recipient’s immune system. We do not know enough to be able to predict the likelihood and strength of the rejection and thus the need for immunosuppression, nor the susceptibility of stem cells to immunosuppression or immune modulation.

• Adequate knowledge of any possible drawbacks of autologous adult stem cell transplantation is needed.

• For routine use of stem cells in toxicity screening tests, the standardisation of culture conditions is needed. Differences in the developmental capacity of different embryonic stem or embryonic germ cell lines have to be considered, and the problem of the metabolic activation of test compounds should be addressed.

• Interaction between public institutions and private companies need to be regulated to ensure a critical mass of development, economic exploitation and equality of access to a promising technology with a potentially high impact on human health.

Acknowledgements

The authors would like to thank Mr. Gerard Bowe and Dr. Ilaria Malerba for their assistance with revising the manuscript.


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131. Meller D, Pires RT, Tseng SC. Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures. Br J Ophthalmol 2002;86:463-71

132. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. New Engl J Med 2000;343:86-93

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CrossRef links are available in the online published version of this paper:http://www.cmrojournal.com

Paper CMRO-3289_3, Accepted for publication: 25 September 2006Published Online: 16 October 2006doi:10.1185/030079906X148517

Embryonic Stem Cell LinesDerived from Human

BlastocystsJames A. Thomson,* Joseph Itskovitz-Eldor, Sander S. Shapiro,

Michelle A. Waknitz, Jennifer J. Swiergiel, Vivienne S. Marshall,Jeffrey M. Jones

Human blastocyst-derived, pluripotent cell lines are described that have normalkaryotypes, express high levels of telomerase activity, and express cell surfacemarkers that characterize primate embryonic stem cells but do not characterizeother early lineages. After undifferentiated proliferation in vitro for 4 to 5months, these cells still maintained the developmental potential to form tro-phoblast and derivatives of all three embryonic germ layers, including gutepithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle(mesoderm); and neural epithelium, embryonic ganglia, and stratified squamousepithelium (ectoderm). These cell lines should be useful in human develop-mental biology, drug discovery, and transplantation medicine.

Embryonic stem (ES) cells are derivedfrom totipotent cells of the early mamma-lian embryo and are capable of unlimited,undifferentiated proliferation in vitro (1, 2).In chimeras with intact embryos, mouse EScells contribute to a wide range of adulttissues, including germ cells, providing apowerful approach for introducing specificgenetic changes into the mouse germ line(3). The term “ES cell” was introduced todistinguish these embryo-derived pluripo-tent cells from teratocarcinoma-derivedpluripotent embryonal carcinoma (EC)cells (2). Given the historical introductionof the term “ES cell” and the properties ofmouse ES cells, we proposed that the es-sential characteristics of primate ES cellsshould include (i) derivation from the pre-implantation or periimplantation embryo,(ii) prolonged undifferentiated prolifera-tion, and (iii) stable developmental poten-tial to form derivatives of all three embry-onic germ layers even after prolonged cul-ture (4). For ethical and practical reasons,in many primate species, including humans,the ability of ES cells to contribute to thegerm line in chimeras is not a testableproperty. Nonhuman primate ES cell linesprovide an accurate in vitro model for un-derstanding the differentiation of humantissues (4, 5). We now describe human celllines that fulfill our proposed criteria to

define primate ES cells.Fresh or frozen cleavage stage human

embryos, produced by in vitro fertilization(IVF) for clinical purposes, were donatedby individuals after informed consent andafter institutional review board approval.Embryos were cultured to the blastocyststage, 14 inner cell masses were isolated,and five ES cell lines originating from fiveseparate embryos were derived, essentiallyas described for nonhuman primate ES cells(5, 6 ). The resulting cells had a high ratioof nucleus to cytoplasm, prominent nucle-oli, and a colony morphology similar to thatof rhesus monkey ES cells (Fig. 1). Threecell lines (H1, H13, and H14) had a normalXY karyotype, and two cell lines (H7 andH9) had a normal XX karyotype. Each ofthe cell lines was successfully cryopre-served and thawed. Four of the cell lineswere cryopreserved after 5 to 6 months ofcontinuous undifferentiated proliferation.The other cell line, H9, retained a normal

XX karyotype after 6 months of culture andhas now been passaged continuously formore than 8 months (32 passages). A periodof replicative crisis was not observed forany of the cell lines.

The human ES cell lines expressed highlevels of telomerase activity (Fig. 2). Telo-merase is a ribonucleoprotein that adds telo-mere repeats to chromosome ends and isinvolved in maintaining telomere length,which plays an important role in replicativelife-span (7, 8). Telomerase expression ishighly correlated with immortality in humancell lines, and reintroduction of telomeraseactivity into some diploid human somatic celllines extends replicative life-span (9). Dip-loid human somatic cells do not express tel-omerase, have shortened telomeres with age,and enter replicative senescence after a finiteproliferative life-span in tissue culture (10–13). In contrast, telomerase is present at highlevels in germ line and embryonic tissues(14). The high level of telomerase activityexpressed by the human ES cell lines there-fore suggests that their replicative life-spanwill exceed that of somatic cells.

The human ES cell lines expressed cellsurface markers that characterize undifferen-tiated nonhuman primate ES and human ECcells, including stage-specific embryonic an-tigen (SSEA)–3, SSEA-4, TRA-l-60, TRA-1-81, and alkaline phosphatase (Fig. 3) (4, 5,15, 16). The globo-series glycolipid GL7,which carries the SSEA-4 epitope, is formedby the addition of sialic acid to the globo-series glycolipid Gb5, which carries theSSEA-3 epitope (17, 18). Thus, GL7 reactswith antibodies to both SSEA-3 and SSEA-4(17, 18). Staining intensity for SSEA-4 on thehuman ES cell lines was consistently strong,but staining intensity for SSEA-3 was weakand varied both within and among colonies(Fig. 3, D and C). Because GL7 carries boththe SSEA-4 and SSEA-3 epitopes and be-cause staining for SSEA-4 was consistentlystrong, the relatively weak staining for

J. A. Thomson, M. A. Waknitz, J. J. Swiergiel, V. S.Marshall, Wisconsin Regional Primate Research Cen-ter, University of Wisconsin, Madison, WI 53715,USA. J. Itskovitz-Eldor, Department of Obstetrics andGynecology, Rambam Medical Center, Faculty ofMedicine, Technion, Haifa 31096, Israel. S. S. Shapiroand J. M. Jones, Department of Obstetrics and Gyne-cology, University of Wisconsin, Madison, WI 53715,USA.

*To whom correspondence should be addressed.

Fig. 1. Derivation of theH9 cell line. (A) Innercell mass–derived cellsattached to mouse em-bryonic fibroblast feed-er layer after 8 days ofculture, 24 hours be-fore first dissociation.Scale bar, 100 !m. (B)H9 colony. Scale bar,100 !m. (C) H9 cells.Scale bar, 50 !m. (D)Differentiated H9 cells,cultured for 5 days inthe absence of mouseembryonic fibroblasts,but in the presence ofhuman LIF (20 ng/ml;Sigma). Scale bar, 100!m.

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SSEA-3 suggests a restricted access of theantibody to the SSEA-3 epitope. In commonwith human EC cells, the undifferentiatedhuman ES cell lines did not stain for SSEA-1,but differentiated cells stained strongly forSSEA-l (15) (Fig. 3). Mouse inner cell masscells, ES cells, and EC cells express SSEA-1but do not express SSEA-3 or SSEA-4 (17,19), suggesting basic species differences be-tween early mouse and human development.

The human ES cell lines were derivedby the selection and expansion of individ-ual colonies of a uniform, undifferentiatedmorphology, but none of the ES cell lineswas derived by the clonal expansion of asingle cell. The uniform undifferentiatedmorphology that is shared by human ESand nonhuman primate ES cells and theconsistent expression by the human ES celllines of cell surface markers that uniquelycharacterize primate ES and human ECcells make it extremely unlikely that amixed population of precursor cells wasexpanded. However, because the cell lineswere not cloned from a single cell, wecannot rule out the possibility that there issome variation in developmental potentialamong the undifferentiated cells, in spite oftheir homogeneous appearance.

The human ES cell lines maintained thepotential to form derivatives of all threeembryonic germ layers. All five cell linesproduced teratomas after injection into se-vere combined immunodeficient (SCID)–beige mice. Each injected mouse formed ateratoma, and all teratomas included gutepithelium (endoderm); cartilage, bone,smooth muscle, and striated muscle (meso-derm); and neural epithelium, embryonicganglia, and stratified squamous epithelium(ectoderm) (Fig. 4). In vitro, the ES cellsdifferentiated when cultured in the absenceof mouse embryonic fibroblast feeder lay-ers, both in the presence and absence ofhuman leukemia inhibitory factor (LIF)(Fig. 1). When grown to confluence andallowed to pile up in the culture dish, theES cell lines differentiated spontaneouslyeven in the presence of fibroblasts. AfterH9 cells were allowed to differentiate for 2weeks, both !-fetoprotein (350.9 " 14.2IU/ml) and human chorionic gonadotropin(hCG, 46.7 " 5.6 mIU/ml) were detected inconditioned culture medium, indicatingendoderm and trophoblast differentiation(20).

Human ES cells should offer insightsinto developmental events that cannot bestudied directly in the intact human embryobut that have important consequences inclinical areas, including birth defects, in-fertility, and pregnancy loss. Particularly inthe early postimplantation period, knowl-edge of normal human development islargely restricted to the description of a

limited number of sectioned embryos andto analogies drawn from the experimentalembryology of other species (21). Althoughthe mouse is the mainstay of experimentalmammalian embryology, early structuresincluding the placenta, extraembryonicmembranes, and the egg cylinder all differsubstantially from the corresponding struc-ture of the human embryo. Human ES cellswill be particularly valuable for the studyof the development and function of tissuesthat differ between mice and humans.Screens based on the in vitro differentiation

of human ES cells to specific lineagescould identify gene targets for new drugs,genes that could be used for tissue regen-eration therapies, and teratogenic or toxiccompounds.

Elucidating the mechanisms that controldifferentiation will facilitate the efficient,directed differentiation of ES cells to spe-cific cell types. The standardized produc-tion of large, purified populations of eu-ploid human cells such as cardiomyocytesand neurons will provide a potentially lim-itless source of cells for drug discovery and

Fig. 2. Telomerase ex-pression by human EScell lines. MEF, irradiat-ed mouse embryonicfibroblasts used as afeeder layer for thecells in lanes 4 to 18;293, adenovirus-trans-formed kidney epithe-lial cell line 293; MDA,breast cancer cell lineMDA; TSR8, quantita-tion control template.Telomerase activitywas measured withthe TRAPEZE Telomer-ase Detection Kit (On-cor, Gaithersburg, Maryland). The ES cell lines were analyzed at passages 10 to 13. About 2000 cellswere assayed for each telomeric repeat amplification protocol assay, and 800 cell equivalents wereloaded in each well of a 12.5% nondenaturing polyacrylamide gel. Reactions were done in triplicate withthe third sample of each triplet heat inactivated for 10 to 15 min at 85°C before reaction to test fortelomerase heat sensitivity (lanes 6, 9, 12, 15, 18, 21, 24, and 27). A 36–base pair internal control foramplification efficiency and quantitative analysis was run for each reaction as indicated by thearrowhead. Data were analyzed with the Storm 840 Scanner and ImageQuant package (MolecularDynamics). Telomerase activity in the human ES cell lines ranged from 3.8 to 5.9 times that observedin the immortal human cell line MDA on a per cell basis.

A B

C D

E F

Fig. 3. Expression ofcell surface markers byH9 cells. Scale bar,100 #m. (A) Alkalinephosphatase. (B) SSEA-1. Undifferentiated cellsfailed to stain for SSEA-1 (large colony, left).Occasional coloniesconsisted of non-stained, central, undif-ferentiated cells sur-rounded by a marginof stained, differentiat-ed, epithelial cells(small colony, right).(C) SSEA-3. Somesmall colonies staineduniformly for SSEA-3(colony left of center),but most colonies con-tained a mixture ofweakly stained cellsand a majority of non-stained cells (colonyright of center). (D)SSEA-4. (E) TRA-1-60.(F) TRA-1-81. Similarresults were obtainedfor cell lines H1, H7,H13, and H14.

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transplantation therapies. Many diseases, suchas Parkinson’s disease and juvenile-onset dia-betes mellitus, result from the death or dysfunc-tion of just one or a few cell types. The replace-ment of those cells could offer lifelong treat-ment. Strategies to prevent immune rejection ofthe transplanted cells need to be developed butcould include banking ES cells with definedmajor histocompatibility complex back-grounds or genetically manipulating EScells to reduce or actively combat immunerejection. Because of the similarities to hu-mans and human ES cells, rhesus monkeysand rhesus ES cells provide an accuratemodel for developing strategies to preventimmune rejection of transplanted cells andfor demonstrating the safety and efficacy ofES cell– based therapies. Substantial ad-vances in basic developmental biology arerequired to direct ES cells efficiently tolineages of human clinical importance.However, progress has already been madein the in vitro differentiation of mouse EScells to neurons, hematopoietic cells, andcardiac muscle (22–24). Progress in basicdevelopmental biology is now extremelyrapid; human ES cells will link thisprogress even more closely to the preven-tion and treatment of human disease.

References and Notes1. M. Evans and M. Kaufman, Nature 292, 154 (1981).2. G. Martin, Proc. Natl. Acad. Sci. U.S.A. 78, 7634

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embryos produced by IVF were cultured to theblastocyst stage in G1.2 and G2.2 medium (25).Fourteen of the 20 blastocysts that developedwere selected for ES cell isolation, as described forrhesus monkey ES cells (5). The inner cell masseswere isolated by immunosurgery (26), with a rabbitantiserum to BeWO cells, and plated on irradiated(35 grays gamma irradiation) mouse embryonicfibroblasts. Culture medium consisted of 80% Dul-becco’s modified Eagle’s medium (no pyruvate,high glucose formulation; Gibco-BRL) supplement-ed with 20% fetal bovine serum (Hyclone), 1 mMglutamine, 0.1 mM !-mercaptoethanol (Sigma),and 1% nonessential amino acid stock (Gibco-BRL).After 9 to 15 days, inner cell mass– derived out-growths were dissociated into clumps either byexposure to Ca2"/Mg2"-free phosphate-bufferedsaline with 1 mM EDTA (cell line H1), by exposureto dispase (10 mg/ml; Sigma; cell line H7), or bymechanical dissociation with a micropipette (celllines H9, H13, and H14) and replated on irradiatedmouse embryonic fibroblasts in fresh medium. In-dividual colonies with a uniform undifferentiatedmorphology were individually selected by micropi-pette, mechanically dissociated into clumps, andreplated. Once established and expanded, cultures

were passaged by exposure to type IV collagenase(1 mg/ml; Gibco-BRL) or by selection of individualcolonies by micropipette. Clump sizes of about 50to 100 cells were optimal. Cell lines were initiallykaryotyped at passages 2 to 7.

7. C. B. Harley, Mutat. Res. 256, 271 (1991).8. !!!!, H. Vaziri, C. M. Counter, R. C. Allsopp, Exp.

Gerontol. 27, 375 (1992).9. A. G. Bodnar et al., Science 279, 349 (1998).

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Proc. Natl. Acad. Sci. U.S.A. 91, 2900 (1994).14. W. E. Wright, M. A. Piatyszek, W. E. Rainey, W. Byrd,

J. W. Shay, Dev. Genet. 18, 173 (1996).15. P. W. Andrews, J. Oosterhuis, I. Damjanov, in Terato-

carcinomas and Embryonic Stem Cells: A PracticalApproach, E. Robertson, Ed. (IRL, Oxford, 1987), pp.207–248.

16. Alkaline phosphatase was detected with Vector Bluesubstrate ( Vector Labs). SSEA-1, SSEA-3, SSEA-4,TRA-1-60, and TRA-1-81 were detected by immuno-cytochemistry with specific primary monoclonal an-tibodies and localized with a biotinylated secondaryantibody and then an avidin or biotinylated horse-radish peroxidase complex (Vectastain ABC system;Vector Laboratories) as previously described (5). TheES cell lines were at passages 8 to 12 at the timemarkers were analyzed.

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radioimmunoassay (double AB hCG and AFP-TC kits;Diagnostic Products, Los Angeles, CA). hCG assaysused the World Health Organization Third Interna-tional Standard 75/537. H9 cells were allowed togrow to confluence (day 0) on plates of irradiatedmouse embryonic fibroblasts. Medium was replaceddaily. After 2 weeks of differentiation, medium intriplicate wells conditioned for 24 hours was assayedfor hCG and #-fetoprotein. No hCG or #-fetoproteinwas detected in unconditioned medium.

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25. D. K. Gardner et al., Fertil. Steril. 69, 84 (1998).26. D. Solter and B. Knowles, Proc. Natl. Acad. Sci. U.S.A.

72, 5099 (1975).27. We thank the personnel of the IVF clinics at the

University of Wisconsin School of Medicine and atthe Rambam Medical Center for the initial cultureand cryopreservation of the embryos used in thisstudy; D. Gardner and M. Lane for the G1.2 andG2.2 media; P. Andrews for the NTERA2 cl.D1 cellsand the antibodies used to examine cell surfacemarkers; C. Harris for karyotype analysis; andGeron Corporation for the 293 and MDA cell pel-lets and for assistance with the telomerase TRAPassay. Supported by the University of Wisconsin(UIR grant 2060) and Geron Corporation (grant133-BU18).

5 August 1998; accepted 7 October 1998

Fig. 4. Teratomasformed by the humanES cell lines in SCID-beige mice. Human EScells after 4 to 5months of culture (pas-sages 14 to 16) fromabout 50% confluentsix-well plates were in-jected into the rear legmuscles of 4-week-oldmale SCID-beige mice(two or more mice percell line). Seven to eightweeks after injection,the resulting teratomaswere examined histo-logically. (A) Gutlikestructures. Cell line H9.Scale bar, 400 $m. (B)Rosettes of neural epi-thelium. Cell line H14.Scale bar, 200 $m. (C)Bone. Cell line H14.Scale bar, 100 $m. (D)Cartilage. Cell line H9.Scale bar, 100 $m. (E)Striated muscle. Cellline H13. Scale bar, 25$m. (F) Tubules inter-spersed with struc-tures resembling fetalglomeruli. Cell line H9.Scale bar, 100 $m.

R E P O R T S

www.sciencemag.org SCIENCE VOL 282 6 NOVEMBER 1998 1147

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General Cell Collection: INT 407(HeLa derivative)

Catalogue No.: 85051004Cell Line Name: INT 407(HeLa derivative)Keywords: Human cervix carcinomaCell Line Description: Originally derived from the jejunum and ileum of a 2 month old Caucasian

embryo. The cells have a similar virus susceptibility as HeLa but show lesssensitivity. They carry HeLa marker chromosomes This cell line has been foundto be indistinguishable from HeLa by STR PCR DNA profiling. Therefore, the cellsmust be considered as derived from HeLa. Ethnicity: Black.

Species: HumanTissue: CervixMorphology: EpithelialGrowth Mode: AdherentSubculture Routine: Split sub-confluent cultures (70-80%) 1:3 to 1:6 i.e. seeding at 2-3x10,000

cells/cm² using 0.25% trypsin or trypsin/EDTA; 5% CO2; 37°C.Culture Medium: EMEM (EBSS) + 2mM Glutamine + 1% Non Essential Amino Acids (NEAA) +

10% Foetal Bovine Serum (FBS).Karyotype: 2n = 46, HeLa markersDepositor: Dr D Newell, PHLS CAMR, Porton Down, SalisburyOriginator: NoCountry: UKReferences: J Immunol 1957;79:54Additional Bibliography: Not specifiedPatents: None specified by DepositorResearch Council Deposit: NoRelease Conditions: No

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Medicina e Persona. A proposito di staminali giugno 06

1

IL FIGLIO DI NESSUNO di Angelo Serra in “L’uomo embrione” Editore Cantagalli, marzo 2003, pag. 83 L’11 luglio 2002 era finalmente pubblicato l’atteso Rapporto del consiglio del Presidente Bush per la Bioetica, presieduto da Leon R. Kass, sul grave problema della clonazione umana. (1) Dopo sei mesi di riflessione e impegnative discussioni, il Consiglio proponeva al governo il bando definitivo della clonazione riproduttiva umana e una moratoria di 4 anni per la clonazione terapeutica a scopi biomedici. Spetterà al governo decidere. Appare, tuttavia, evidente di fronte all’unanimità contro la clonazione riproduttiva, il significato e il peso di un 47% di esperti – 7 su 17 – a favore della immediata autorizzazione alla clonazione umana a scopi terapeutici, senza alcuna ulteriore dilazione per riflettere – anche a livello pubblico – sui gravi problemi etici che questa comporta. Durante il periodo di sei mesi di attività del Consiglio era apparsa evidente la pressione soprattutto da parte degli scienziati, medici e biotecnologi. In un editoriale della stessa autorevole rivista Science, nel mese di maggio precedente, si sottolineava: “ Questa legislazione è promossa per mettere al bando la clonazione di esseri umani, ma fa qualcosa di più. Essa proibisce la clonazione riproduttiva - e ciò sta bene – ma anche mette al bando esperimenti erroneamente indicati come “clonazione terapeutica. Peggio, come l’equivalente proposta della Camera, essa criminalizza un ragionevole lavoro scientifico”. (2) E’ l’ultima conseguenza di un cammino nel campo della ricerca iniziato nel 1998. Cammino nel quale la figura che domina è ancora l’embrione umano, ma ormai ridotto a “figlio di nessuno”, mero “grumo di cellule” e prezioso strumento tecnologico. E’ impressionante il linguaggio usato da uno dei membri del Consiglio sopra ricordato, il quale votò contro la moratoria, rivolgendosi a quelli che sostenevano lo speciale rispetto dovuto all’embrione umano:”Sto parlando a gente che non ha ancora chiare le idee. Siete preoccupati di un grumo di 200 cellule o no? Molti non lo sono.” (3) ….L’embrione è dunque ridotto a strumento di lavoro. Il primo passo Il 6 novembre 1998 un gruppo di ricercatori della Wisconsin University a Madison, negli Stati Uniti, sostenuti da fondi privati offerti dalla Geron Corp. Of Menlo Park, California, pubblicavano un lavoro (4) in cui si dimostrava la possibilità di ottenere, dalle cellule dell’embrioblasto di embrioni umani allo stadio di blastociste, cellule totipoptenti non ancora differenziate, dette cellule staminali embrionali (ES). In raeltà, come dimostravano ricerche precedenti in particolare sul topo, esse avrebbero potuto dare origine, in seguito a differenziazione spontanea o indotta, a cellule dei più diversi tipi di tessuto. Sembrava di aver trovato, finalmente, una fonte inesauribile di cellule da cui derivare altre cellule – nervose, muscolari, epiteliali, ematiche ecc – che, impiantate in organi malati con le dovute attenzioni per evitarne il rigetto, ne avrebbero consentito la riparazione, ridonando così la salute a soggetti affetti da gravi patologie, quali, ad esempio il Parkinson, l’Alzheimer, il diabete. Si era aperta, così si pensava, una grande speranza per la medicina. Di fronte a questo promettente evento, non si era mai stati fermi al livello politico. Sotto le forti pressioni di scienziati, medici e pubblico in Inghilterra, il governo Blair nel giugno 1999 chiedeva al Direttore Generale della Sanità, Liam Donaldson, di formare un comitato per esaminare se avrebbero dovuto essere permesse nuove aree di ricerca su embrioni umani capaci di condurre a più ampie conoscenze su, ed eventualmente nuovi trattamenti di, tessuti o organi malati o danneggiati e di malattie mitocondriali. Il 18 agosto seguente era nominato il Gruppo di esperti; e il 14 agosto era stato reso definitivo il documento. (5) Vi si proponeva il sì per due nuovi procedimenti, che estendevano l’uso di embrioni umani precoci per ricerche che sarebbero andate molto oltre a quanto fino allora era stato concesso dalla legge del 1990, precisamente:

1) la preparazione di cellule staminali embrionali 2) la clonazione terapeutica

La “Risposta del governo alle Raccomandazioni fatte dal Gruppo di Esperti” fu immediata. Vi si diceva:”Il governo accetta in pieno le raccomandazioni del Rapporto, e preparerà una legislazione, se necessario, per renderle attive appena lo permetterà l’agenda Parlamentare” (6). Il 19 dicembre 2000 la House of Commons, con il 68% dei voti a favore (7) e il 22


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gennaio 2001 la House of Lords con il 70% dei voti a favore, approvavano il testo governativo che autorizza la derivazione delle cellule staminali da embrioni umani e la clonazione terapeutica. Era definitivamente approvato un ulteriore passo nell’aggressione dell’embrione umano: ridotto a prezioso strumento tecnologico sotto l’egida di una “buona azione” medica. Le cellule staminali embrionali (ES) Si definisce staminale una cellula che ha due caratteristiche:

• la capacità di auto-rinnovamento illimitato o prolungato, la capacità cioè di riprodursi a lungo senza differenziarsi

• la capacità di dare origine a cellule progenitrici di transito, con capacità proliferativa limitata, dalle quali discendono popolazioni di cellule altamente differenziate di circa 250 tipi (nervose, muscolari, ematiche ecc) presenti nei vari organi

Da circa 30 anni queste cellule hanno costituito un ampio campo di ricerca sia in tessuti adulti anche umani (8), sia in tessuti fetali e embrionali di animali da laboratorio. (9) Ma l’attenzione pubblica vi è stata richiamata recentemente dal nuovo traguardo sopra ricordato: la produzione in vitro di cellule staminali embrionali umane e la possibilità della loro differenziazione. Sono due gli aspetti essenziali che devono essere sottolineati. 1. La produzione delle cellule staminali embrionali a) la produzione di embrioni umani in vitro o la utilizzazione di quelli sopravanzati ai trattamenti di fecondazione in vitro nelle pratiche di riproduzione tecnicamente assistita, crioconservati o meno b) il loro sviluppo fino allo stadio di blastociste di circa 60-120 cellule c) il prelevamento, da queste, delle cellule - circa 30-40 – che ne costituiscono l’embrioblasto o massa cellulare interna (ICM): operazione che implica l’arresto dello sviluppo embrionale e la distruzione dell’embrione d) la coltura di queste cellule, con particolari accorgimenti e in adatti terreni, fino alla formazione di linee cellulari capaci di moltiplicarsi indefinitamente conservando le caratteristiche di cellule staminali embrionali (ES) per mesi e anni. J.A.Thomson e i suoi collaboratori erano riusciti a prepararne, al momento della pubblicazione del primo lavoro, cinque linee: 3 di cellule maschili, con cariotipo XY, contrassegnate H1,H13,e H14; e 2 di cellule femminili, con cariotipo XX, contrassegnate H7 e H9, che continuarono a proliferare indifferenziate per 5-8 mesi, e poi furono crioconservate a –190°C e successivamente riutilizzate per continuarne la produzione o per proseguire gli studi sulla loro differenziazione. Dinanzi a questa straordinaria conquista e ai nuovi orizzonti che si stavano aprendo alla scienza e alla tecnologia – in particolare alla medicina – con i relativi risvolti politici e commerciali (10), tutto amplificato da esaltanti e spesso frastornanti interventi massmediali, non era possibile arrestarsi. G. Keller e H.R. Snodgrass chiudevano con evidente entusiasmo un sintetico sguardo al futuro di questo nuovo promettente campo di ricerca, con queste espressioni: “E’ evidente che la tecnologia delle cellule staminali embrionali ha rivoluzionato la biologia moderna, ed offre opportunità uniche per comprendere i meccanismi che controllano processi biologici fondamentali. Lo sviluppo delle cellule staminali embrionali e germinali umane è una importante pietra miliare verso l’applicazione delle potenzialità di questa tecnologia al trattamento diretto di malattie umane [..]. Saranno necessarie ulteriori significative ricerche per capitalizzare il potenziale terapeutico totale di queste cellule, ma le nuove terapie che si otterranno rendono più che giusto lo sforzo”.(11) 2.La differenziazione delle cellule staminali embrionali Queste dovevano essere riconosciute come popolazioni di cellule omogenee e indifferenziate pluripotenti, la cui espansione – che richiede la presenza di una citokina, in particolare il fattore di inibizione della leucemia (LIF) (12) – può in poche settimane raggiungere le 10.000.000.000 – 100.000.000.000 cellule senza alcun segno di differenziazione o senescenza. E sono queste le cellule che si dovrebbero utilizzare per la preparazione di cellule differenziate desiderate., ossia di cellule dotate di ben determinate caratteristiche morfologiche e fisiologiche quali, ad esempio, cellule muscolari, nervose, epiteliali, ematiche e mesenchimatiche. In realtà, alcune osservazioni avevano già messo in evidenza la loro grande


3

potenzialità di differenziarsi. J.A. Thomson e i suoi collaboratori, infatti, avevano notato che la inoculazione delle ES umane in topi immunodeficienti seguita da sviluppo di teratomi, e la loro coltura in vitro in adatti terreni fino alla confluenza, davano spontaneamente origine a celule differenziate che deriverebbero, nello sviluppo normale, dai tre diversi foglietti embrionali: epitelio intestinale (dall’endoderma); cartilagine, osso, muscolo liscio e striato (dal mesoderma); ed epitelio neurale, epitelio squamoso (dall’esoderma); anzi tanto differenziate fino a produrre alfa-fetoproteine e gonadotropine corioniche umane, rilevabili nel terreno di coltura.(4) Si erano anche studiati a fondo i “corpi embrioidi” (embryoid bodies), che derivano dalla coltura delle cellule ES in sospensione in adatti terreni: essi erano risultati, in realtà, delle strutture multidifferenziate nelle quali è riattivato il programma di sviluppo delle cellule dell’embrioblasto, come avviene nell’embrione pre- e post- impianto, ma “in assenza completa di una organizzazione o elaborazione di un piano corporeo”.(13)

Più recentemente parziali ma promettenti risultati, relativi alle eventuali capacità terapeutiche di cellule ES differenziate si sono ottenuti in alcune mirate sperimentazioni precliniche, in generale su topo. Se ne ricordano due tra le più significative. La prima, eseguita da J.W.McDonald e coll. (14), dimostrarono che cellule ES di topo differenziate in cellule nervose, trapiantate nel midollo spinale di un ratto nove giorni dopo un evento traumatico, a 2-5 settimane dall’intervento non soltanto sopravvivevano, ma si erano differenziate in astrociti, oligodendrociti e neuroni, ed erano migrate fino a 8 mm dalla lesione, accompagnate da un buon miglioramento posturale e nel coordinamento dei movimenti. La seconda, eseguita da B. Soria (15), provò che cellule produttrici di insulina, derivate da cellule ES attraverso un processo a tre tappe che includono l’espressione di un notevole numero di fattori di trascrizione e di fattori extracellulari, trapiantate in topi diabetici portarono alla normalizzazione del livello di glucosio nel sangue, e curarono il diabete per oltre un anno. Tutte queste conoscenze hanno certamente facilitato e favorito il cammino, ancora al suo inizio, ad analoghi studi e ricerche sulle cellule staminali embrionali umane. A tre anni dalla loro scoperta, lo stesso scopritore J.A. Thomson e i suoi collaboratori scrivevano:”Le cellule staminali embrionali umane hanno un cariotipo normale, mantengono un’alta attività telomerasica e hanno un notevole potenziale proliferativo di lungo termine, offrendo la possibilità di una illimitata espansione in coltura. Inoltre esse possono differenziarsi nei derivati di tutti i tre strati germinali embrionali quando sono trasferite in un ambiente in vivo. Stanno ora emeregendo dati i quali dimostrano che le cellule ES umane possono iniziare in vitro programmi specifici di differenziazione in coltura per lo studio dei meccanismi che sottostanno ai molti aspetti dello sviluppo umano. Poiché esse hanno la doppia capacità di proliferare indefinitamente e di differenziarsi in molteplici tipi di tessuto, le cellule ES umane potrebbero provvedere una illimitata fornitura di tessuti per trapianti umani…per un notevole numero di malattie; molti ostacoli, tuttavia, rimangono ancora sulla via verso una sperimentazione clinica affidabile”.(16) Ovviamente, questi ostacoli erano da prevedere; e scienza e tecnologia si impegnarono, e si impegneranno sempre più, a superarli, facilitati dalle disposizioni legislative. Da quanto si è fatto e pubblicato fino ad oggi, nell’area umana, emergono alcuni dati e riflessioni. E’ evidente l’ampia malleabilità delle cellule staminali embrionali umane,(17) quantunque sia ancora molto limitata la comprensione del controllo della loro crescita e della loro differenziazione, complicata spesso da notevole instabilità di origine epigenetica. (18) Al fine di questo approfondimento sono, perciò, iniziate nuove linee di ricerca: si tratta, prima di tutto, di approfondire la conoscenza dei meccanismi che la controllano, ai livelli genetico, in particolare, ed epigenetico.(19) Tuttavia, i risultati sono ancora scarsi. Riferendo alcuni dati emersi a un incontro chiave tra esperti del settore, tenuto il 6-11 febbraio 2001, G. Vogel scriveva:”Sebbene le cellule staminali embrionali furono derivate più di due anni or sono, il lavoro è stato lento e frustrante; in realtà, soltanto pochi ricercatori hanno pubblicato qualche risultato su di esse. Queste cellule non solo sono esigenti per le loro condizioni di crescita, ma anche tendono a differenziarsi spontaneamente in una serie di tipi diversi da quello desiderato”.(20) Gli stessi ricercatori della Geron Co., che più di tutti gli altri hanno avuto il tempo di lavorare su queste cellule, avendone sostenuto la ricerca per la produzione e ottenuto la licenza esclusiva per il loro uso commerciale, pur ammettendo che linee cellulari derivate da una singola cellula staminale embrionale hanno continuato a replicarsi in coltura per 250 generazioni, riconoscono tuttavia, di essere ancora ben lontani dal traguardo. In


4

realtà, “molti ricercatori in questo campo – concludeva G. Vogel, riportando le affermazioni fatte al Congresso annuale della Società di Neuroscienze nel novembre 2000 – riconoscono che il prossimo passo più importante è quello di identificare i processi molecolari, che sottostanno alle impressionanti prestazioni delle cellule staminali”. (21) E’ questo il punto cui si è arrivati oggi nella ricerca sulle cellule staminali embrionali umane. Il tempo, senza dubbio, porterà – data la chiara volontà di proseguire su questa linea – a ulteriori conoscenze e progressi tecnologici e, quindi, a informazioni più attendibili sulla realizzabilità delle speranze che, oggi, stimolano la mente dei ricercatori e dei biotecnologi e affascinano il pubblico. Ma non si può non sottolineare e non riconoscere che, per tutto questo, sono centinaia di migliaia gli embrioni umani, e quindi esseri umani, che sono condannati a morte, considerati e trattati come “figli di nessuno”, come animali da esperimento. 1.Hall SS,President’s Bioethics Council delivers, Science 19 July 2002,297:322-324 2.Kennedy D, Science in the U.S. Government: Interim Report, Science 10 May 2002,296:981 3.Hall SS, President’s Bioethics....pag 323 4.Thomson JA, embryonic stem cells lines derived from human blastocysts, Science 1998,283:1145-1147 5.Department of Health Stem cell Research: Medical Progress with Responsability 14 August 2000, http://www.doh.gov.uk/cegc/stemcellreport.htm. 6.Department of Health Government Response to rhe Recommendation made in the Chief Medical Officer’s Expert Group Report. Stem cell Research. 16 August 2000, in http://www.doh.gov.uk/cegc/govres.htm. 7.Vogel C, British Parliament approves new rules, science 2001, 291:23 8.Loeffler M, Stem cfells and cellular pedigrees - a conceptual introduction, in Potten CS, (ed),Stem Cell Academic Press, london 1997, pag 1-27 9. Smith A, Embrionic stem cells, in Marshall DR, Stem Cell Biology, Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY 2001,205-230 10.Marshall E, The business of stem cells. Science, 2000,287:1419-1421 11.Keller G, Human embryonic stem cells: teh future is now. Nature Medicine 1999, 151-152 12.Williams RL, Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988,336:684-687 13.Doetschman TC, The in vitro development of blastocyst derived embrionic stem cell lines: Formation of visceral yolk sac, blood island and myocardium. J Embriol Exp Morphol 1985,87:27-45 14.MCDonald JW Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nature Medicine Dec. 1999,12:1410-1412 15.Soria B, In-vitrodifferentiation of pancreatic beta-cells. Differentiation Oct. 2001,68:205-219 16.Odorico JS, Multilineage differentiation from human embryonic stem cell lines, Ste Cells 2001,19:193-204 17.Shamblott MJ, Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proceedings of the National Academy of Sciences 2001,95,13726-13731 18.Humpheris D, Epigenetic instability in ES cells and cloned myce. Science 2001,293:95-97 19.ReiK W,Epigenetic reprogramming in mammalian development. Science 2001,293:1089-1093 20.Vogel G, The hottest stem cells are also the toughest. Science 2001, 292:429 21. ID Stem cells:New excitement, Persistent questions. Science 2000, 290:1672-1674 p.1674

Project outline: The use of human embryonic stem cell lines for the development of

an alternative methods for embryotoxicity testing in vitro Presented by the European Centre for the Validation of Alternative

Methods (ECVAM), Joint Research Centre, Ispra, Italy

SUMMARY

Le cellule staminali umane isolate in laboratorio, offrono l’opportunità di sviluppare sistemi in vitro per l’identificazione dei possibili effetti tossici di sostanze chimiche durante lo sviluppo embrionale. Il principale vantaggio derivante dall’uso di queste linee cellulari è la possibilità di creare test che rispecchiano il più possibile il sistema umano. Ciò eviterebbe i problemi derivanti dal confronto dei dati ottenuti da specie animali diverse da quella umana. Questo fatto è predominante negli studi di embrio-tossicologia, come è stato dimostrato nell’utilizzo di composti come la Talidomide tra gli anni ’50 e ’60. Inoltre la capacità delle cellule staminali umane di differenziarsi in vitro in un’ampia varietà di tessuti, permetterà l’individuazione degli effetti tossicologici delle sostanze chimiche, nei diversi tipi cellulari.

Il presente progetto di ricerca offrirà la possibilità di creare nuovi sistemi, alternativi alla sperimentazione in vivo, al fine di ridurre l’utilizzo degli animali da laboratorio. Per il seguente progetto si è pianificato l’utilizzo delle linee cellulari H1 e H9 le quali sono distribuite commercialmente da “WiCell” (USA). Entrambe le linee cellulari sono regolarmente registrate presso il “NIH”.

1. L’individuazione dei possibili effetti teratogenici e tossicologici di sostanze chimiche durante il differenziamento in specifici tipi cellulari, utilizzando tecniche immunoistochimiche, di biologia cellulare e di analisi funzionale.

2. Il progetto di ricerca contribuirà a capire i meccanismi embrio-tossicologici a livello molecolare; un’analisi a livello proteomico permetterà, inoltre, la creazione di un pattern proteico caratteristico solo di quelle cellula esposte al composto chimico.

Il presente lavoro è da considerarsi un ramo di un più ampio progetto della Comunità Europea “Reprotect Project”, il cui scopo principale è lo sviluppo di una strategia per la classificazione di sostanze potenzialmente tossiche durante il ciclo riproduttivo umano (vedere progetto allegato). Reprotect Project ha permesso la creazione di un consorzio nel quale sono coinvolti circa 30 gruppi di ricerca e sarà interamente finanziato da “DG RTD” della Comunità Europea con un contributo di 9.1 milioni di Euro. Tuttavia è necessario per ogni partecipante una valutazione, da un punto di vista etico, da parte delle competenti autorità della Stato in cui il lavoro di svolgerà.

Si richiede quindi una valutazione da parte del Comitato nazionale per la Bioetica del presente progetto al fine di garantire il regolare svolgimento di esso in Italia, nel rispetto delle norme vigenti.

Presidenza del Consiglio dei Ministri

RISPOSTA

SULL’UTILIZZO A FINI DI RICERCA DELLE LINEE CELLULARI H1 E H9 DERIVANTI DA EMBRIONI UMANI

16 luglio 2004

Risposta a un quesito sottoposto al CNB da parte dello European Centre for the Validation of Alternative Methods (ECV AM)

Joint Research Centre the European Commission, Ispra, Italy (Dr. Susanne Bremer, Key area leader: Reproductive toxicology – ECV AM)

La richiesta di parere da parte del Joint Research Centre of the European Commission, riportato in calce da presente documento, riguarda:

1. Una valutazione etica circa l’utilizzo a fini di ricerca delle linee cellulari H1 e H9 derivanti da embrioni umani, distribuite commercialmente da “WiCell” (USA) e regolarmente registrate presso l’NIH;

2. Un parere legale circa il fatto che tale utilizzo posso avvenire nel nostro Paese nel “rispetto delle norme vigenti”.

Riguardo al punto 1, il CNB rimanda al “Parere del Comitato nazionale per

la bioetica su ricerche utilizzanti embrioni umani e cellule staminali”, approvato l’11 aprile 2003, nel quale una maggioranza dei componenti ha espresso parere negativo riguardo a qualsiasi forma di sperimentazione che comporti o abbia comportato la distruzione di embrioni umani, mentre una minoranza di componenti ha espresso parere favorevole.

Fermo restando il carattere consultivo dei pareri del CNB, si fa presente che il predetto parere di maggioranza è stato assunto come posizione ufficiale del governo italiano (Vice-Ministro On. Prof. Guido Possa) nell’ambito dell’Interinstitutional Seminar on Bioethics: Human embryonic stem cells research under the 6th Fremework programme for Research svoltosi a Bruxelles il 24 aprile 2003.

Riguardo al punto 2, il CNB non è organo deputato alla formulazione di pareri legali. Si precisa tuttavia che la Legge 40/2004 (Norme in materia di procreazione medicalmente assistita), è ad oggi l’unico strumento normativo italiano che regolamenta la sperimentazione su embrioni umani a fini di ricerca. Per completezza di informazione si ricorda che la Convenzione di Oviedo, rilevante ai fini del tema in questione, è stata ratificata dal Parlamento italiano con legge n. 145 del 2001 e si è in attesa del deposito dello strumento di ratifica.

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