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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'
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*"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].
____________________________________!
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
PONTIFICIA ACADEMIA
PRO VITA
Il Presidente
Prot.n.P/3431
Mrs Debra L.Vinnedge Vatican City, June 9 2005
Executive Director, Children of God for Life
943 Deville Drive East
Largo, Florida
33771
Stati Uniti
Dear Mrs Debra L.Vinnedge,
On June 4, 2003, you wrote to His Eminence Cardinal Joseph Ratzinger, with a copy of this letter
forwarded to me, asking to the Sacred Congregation of the Doctrine of Faith a clarification about the
liceity of vaccinating children with vaccines prepared using cell lines derived from aborted human
fetuses. Your question regarded in particular the right of the parents of these children to oppose such
a vaccination when made at school, mandated by law. As there were no formal guidelines by the
magisterium concerning that topic, you said that catholic parents were often challenged by State
Courts, Health Officials and School Administrators when they filled religious exemptions for their
children to this type of vaccination.
This Pontifical Academy for Life, carrying out the commission entrusted to us by the Congregation for
the Doctrine of Faith, in answer to your request, has proceeded to a careful examination of the
question of these "tainted" vaccines, and has produced as a result a study (in Italian) that has been
realized with the help of a group of experts. This study has been approved as such by the
Congregation 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 the
Centra di Bioetica della Universita Cattolica in Rome.
The study, its synthesis, and the translation of this material took some time. We apologize for the
delay.
With my best regards,
Sincerely yours,
+E.Sgreccia
00193 Roma - Via della Conciliazione, 1 - Tel. 06 698.82423 - 06 698.81693 - Fax 06 698.82014
MORAL REFLECTIONS
ON VACCINES PREPARED FROM
CELLS
DERIVED FROM ABORTED HUMAN FOETUSES
http://www.immunize.org/concerns/vaticandocument.htm
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The matter in question regards the lawfulness of production, distribution and use of certain vaccines
whose production is connected with acts of procured abortion. It concerns vaccines containing live
viruses which have been prepared from human cell lines of foetal origin, using tissues from aborted
human foetuses as a source of such cells. The best known, and perhaps the most important due to its
vast distribution and its use on an almost universal level, is the vaccine against Rubella (German
measles).
Rubella and its vaccine
Rubella (German measles)1 is a viral illness caused by a Togavirus of the genus Rubivirus and is
characterized by a maculopapular rash. It consists of an infection which is common in infancy and has
no clinical manifestations in one case out of two, is self-limiting and usually benign. Nonetheless, the
German 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 of
foetal infection is very high (approximately 95%). The virus replicates itself in the placenta and infects
the foetus, causing the constellation of abnormalities denoted by the name of Congenital Rubella
Syndrome. For example, the severe epidemic of German measles which affected a huge part of the
United 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 of
blindness, 1,800 cases of mental retardation. It was this epidemic that pushed for the development
and introduction on the market of an effective vaccine against rubella, thus permitting an effective
prophylaxis against this infection.
The severity of congenital rubella and the handicaps which it causes justify systematic vaccination
against such a sickness. It is very difficult, perhaps even impossible, to avoid the infection of a
pregnant woman, even if the rubella infection of a person in contact with this woman is diagnosed from
the first day of the eruption of the rash. Therefore, one tries to prevent transmission by suppressing the
reservoir of infection among children who have not been vaccinated, by means of early immunization of
all children (universal vaccination). Universal vaccination has resulted in a considerable fall in the
incidence of congenital rubella, with a general incidence reduced to less than 5 cases per 100,000
livebirths. Nevertheless, this progress remains fragile. In the United States, for example, after an
overwhelming 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 that
rose to 0.8/100,000. Such waves of resurgence of German measles were also seen in 1997 and in the
year 2000. These periodic episodes of resurgence make it evident that there is a persistent circulation
of the virus among young adults, which is the consequence of insufficient vaccination coverage. The
latter situation allows a significant proportion of vulnerable subjects to persist, who are a source of
periodic 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 public
health 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 aborted
foetuses (in 1964 and 1970) and are used for the preparation of vaccines based on live attenuated
virus: the first one is the WI-38 line (Winstar Institute 38), with human diploid lung fibroblasts, coming
from a female foetus that was aborted because the family felt they had too many children (G. Sven et
al., 1969). It was prepared and developed by Leonard Hayflick in 1964 (L. Hayflick, 1965; G. Sven et
al., 1969)3 and bears the ATCC number CCL-75. WI-38 has been used for the preparation of the
historical vaccine RA 27/3 against rubella (S.A. Plotkin et al, 1965)4. The second human cell line is
MRC-5 (Medical Research Council 5) (human, lung, embryonic) (ATCC number CCL-171), with human
lung fibroblasts coming from a 14 week male foetus aborted for "psychiatric reasons" from a 27 year
old 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 in
the vaccines actually available6.
The vaccines that are incriminated today as using human cell lines from aborted foetuses, WI-38 and
MRC-5, are the following:7
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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-RVAX® (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 by
GlaxoSmithKline (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® (AventisPasteur, Fr.) using MRC-5;
one vaccine against rabies, Imovax®, produced by Aventis Pasteur, harvested from infected
human 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 as
these, as well as their use in the fight against these infections, up to the point of eradication, by
means 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 had
been infected and voluntarily aborted, and the viruses were subsequently attenuated and cultivated
from human cell lines which come likewise from procured abortions, they do not cease to pose ethical
problems. The need to articulate a moral reflection on the matter in question arises mainly from the
connection which exists between the vaccines mentioned above and the procured abortions from
which biological material necessary for their preparation was obtained.
If someone rejects every form of voluntary abortion of human foetuses, would such a person not
contradict himself/herself by allowing the use of these vaccines of live attenuated viruses on their
children? Would it not be a matter of true (and illicit) cooperation in evil, even though this evil was
carried out forty years ago?
Before proceeding to consider this specific case, we need to recall briefly the principles assumed in
classical moral doctrine with regard to the problem of cooperation in evil 9, a problem which arises
every time that a moral agent perceives the existence of a link between his own acts and a morally evil
action 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 the
immoral 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 the
agent acts by fulfilling the conditions - either by providing instruments or products - which make it
possible to commit the immoral act. Furthermore, forms of proximate cooperation and remote
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cooperation can be distinguished, in relation to the "distance" (be it in terms of temporal space or
material 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 either
proximate or remote.
Formal cooperation is always morally illicit because it represents a form of direct and intentional
participation 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, given
the precious nature of the value in question11.
A further distinction made in classical morality is that between active (or positive) cooperation in evil
and passive (or negative) cooperation in evil, the former referring to the performance of an act of
cooperation in a sinful action that is carried out by another person, while the latter refers to the
omission 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 material
cooperation should generally be avoided, although it is admitted (by many authors) that there is not a
rigorous 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 foetuses
aborted voluntarily
In the specific case under examination, there are three categories of people who are involved in the
cooperation in evil, evil which is obviously represented by the action of a voluntary abortion performed
by 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 for
health reasons.
Firstly, one must consider morally illicit every form of formal cooperation (sharing the evil intention) in
the action of those who have performed a voluntary abortion, which in turn has allowed the retrieval of
foetal tissues, required for the preparation of vaccines. Therefore, whoever - regardless of the category
to which he belongs — cooperates in some way, sharing its intention, to the performance of a
voluntary abortion with the aim of producing the above-mentioned vaccines, participates, in actuality, in
the same moral evil as the person who has performed that abortion. Such participation would also take
place in the case where someone, sharing the intention of the abortion, refrains from denouncing or
criticizing 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 has
performed 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 of
biological material whose origin is connected with cells coming from foetuses voluntarily aborted, such
a process is stated, as a matter of principle, morally illicit, because it could contribute in encouraging
the 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, the
various 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 denounce
and reject publicly the original immoral act (the voluntary abortion), and if they do not dedicate
themselves together to research and promote alternative ways, exempt from moral evil, for the
production of vaccines for the same infections. Such passive material cooperation, if it should occur, is
equally 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 of
these vaccines for their children, in spite of knowing their origin (voluntary abortion), carry out a form of
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very remote mediate material cooperation, and thus very mild, in the performance of the original act of
abortion, and a mediate material cooperation, with regard to the marketing of cells coming from
abortions, and immediate, with regard to the marketing of vaccines produced with such cells. The
cooperation is therefore more intense on the part of the authorities and national health systems that
accept the use of the vaccines.
However, in this situation, the aspect of passive cooperation is that which stands out most. It is up to
the faithful and citizens of upright conscience (fathers of families, doctors, etc.) to oppose, even by
making an objection of conscience, the ever more widespread attacks against life and the "culture of
death" which underlies them. From this point of view, the use of vaccines whose production is
connected with procured abortion constitutes at least a mediate remote passive material cooperation
to 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 generalized
social consensus to the operation of the pharmaceutical industries which produce them in an immoral
way.
Therefore, doctors and fathers of families have a duty to take recourse to alternative vaccines 13 (if they
exist), putting pressure on the political authorities and health systems so that other vaccines without
moral problems become available. They should take recourse, if necessary, to the use of
conscientious objection14 with regard to the use of vaccines produced by means of cell lines of
aborted human foetal origin. Equally, they should oppose by all means (in writing, through the various
associations, 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 abortion
of 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 and
ethically acceptable, it is right to abstain from using these vaccines if it can be done without causing
children, 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 problems
pertaining to them may also be used on a temporary basis. The moral reason is that the duty to avoid
passive material cooperation is not obligatory if there is grave inconvenience. Moreover, we find, in
such a case, a proportional reason, in order to accept the use of these vaccines in the presence of the
danger of favouring the spread of the pathological agent, due to the lack of vaccination of children. This
is particularly true in the case of vaccination against German measles 15.
In any case, there remains a moral duty to continue to fight and to employ every lawful means in order
to 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 the
health 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 objection
with regard to those which have moral problems;
as regards the vaccines without an alternative, the need to contest so that others may be
prepared must be reaffirmed, as should be the lawfulness of using the former in the meantime
insomuch as is necessary in order to avoid a serious risk not only for one's own children but
also, 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 the
lawfulness of their production, marketing and use, but is to be understood as being a passive
material cooperation and, in its mildest and remotest sense, also active, morally justified as an
extrema ratio due to the necessity to provide for the good of one's children and of the people
who come in contact with the children (pregnant women);
such cooperation occurs in a context of moral coercion of the conscience of parents, who are
forced to choose to act against their conscience or otherwise, to put the health of their children
and of the population as a whole at risk. This is an unjust alternative choice, which must be
eliminated 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.1127-1137
2. 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.265-268
3. L. Hayflick, The Limited In Vitro Lifetime of Human Diploid Cell Strains, Experimental Cell Research, March 1965,
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
vol.37, no. 3, pp. 614-636.
G. Sven, S. Plotkin, K. McCarthy, Gamma Globulin Prophylaxis; Inactivated Rubella Virus; Production and Biological
Control of Live Attenuated Rubella Virus Vaccines, American journal of Diseases of Children, August 1969, vol. 118,
no. 2, pp.372-381.
S. A. Plotkin, D. Cornfeld, Th.H. Ingalls, Studies of Immunization With Living Rubella Virus, Trials in Children With a
Strain coming from an Aborted Fetus, American Journal of Diseases in children, October 1965, vol. 110, no. 4,
pp.381-389.
J.P. Jacobs, C.M. Jones, J.P. Bailie, Characteristics of a Human Diploid Cell Designated MRC-5, Nature, 11th July
1970, vol.277, pp.168-170.
Two other human cell lines, that are permanent, HEK 293 aborted fetal cell line, from primary human embryonic
kidney 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 line 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 not
been involved in the making of any of the attenuated live viruses vaccines presently in use because of their capacity
to develop tumorigenic cells in the recipient. However some vaccines, still at the developmental stage, against Ebola
virus (Crucell,NV and the Vaccine Research Center of the National Institutes of Health's Allergy and Infectious
Diseases, NIAID), HIV (Merck), influenza (Medlmmune, Sanofi pasteur), Japanese encephalitis (Crucell N.V. and
Rhein Biotech N.V.) are prepared using PER.C6® cell line (Crucell N.V., Leiden, The Netherlands).
Against these various infectious diseases, there are some alternative vaccines that are prepared using animals' cells or
tissues, and are therefore ethically acceptable. Their availability depends on the country in question. Concerning the
particular case of the United States, there are no options for the time being in that country for the vaccination against
rubella, chickenpox and hepatitis A, other than the vaccines proposed by Merck, prepared using the human cell lines
WI-38 and MRC-5. There is a vaccine against smallpox prepared with the Vero cell line (derived from the kidney of an
African green monkey), ACAM2000 (Acambis-Baxter) ( a second-generation smallpox vaccine, stockpiled, not
approved in the US), which offers, therefore, an alternative to the Acambis 1000. There are alternative vaccines
against mumps (Mumpsvax, Merck, measles (Attenuvax, Merck), rabies (RabAvert, Chiron therapeutics), prepared from
chicken 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 Vaccinia
Ankara, Acambis-Baxter). In Europe and in Japan, there are other vaccines available against rubella and hepatitis A,
produced using non-human cell lines. 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 quail
embryo. 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.
The vaccine against rubella using the strain Wistar RA27/3 of live attenuated rubella virus, adapted and propagated
in WI-38 human diploid lung fibroblasts is at the centre of present controversy regarding the morality of the use of
vaccines prepared with the help of human cell lines coming from aborted foetuses.
D.M. Prummer O. Pr., De cooperatione ad malum, in Manuale Theologiae Moralis secundum Principia S. Thomae
Aquinatis, 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.
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 Siciliano 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.
Cf. John Paul II, Enc. Evangelium Vitae, no. 74.
No. 1868 of the Catechism of the Catholic Church.
The alternative vaccines in question are those that are prepared by means of cell lines which are not of human
origin, for example, the Vero cell line (from monkeys) (D. Vinnedge), the kidney cells of rabbits or monkeys, or the
cells of chicken embryos. However, it should be noted that grave forms of allergy have occurred with some of the
vaccines prepared in this way. The use of recombinant DNA technology could lead to the development of new
vaccines in the near future which will no longer require the use of cultures of human diploid cells for the attenuation
of the virus and its growth, for such vaccines will not be prepared from a basis of attenuated virus, but from the
genome of the virus and from the antigens thus developed (G. C. Woodrow, W.M. McDonnell and F.K. Askari). Some
experimental studies have already been done using vaccines developed from DNA that has been derived from the
genome of the German measles virus. Moreover, some Asiatic researchers are trying to use the Varicella virus as a
vector for the insertion of genes which codify the viral antigens of Rubella. These studies are still at a preliminary
phase and the refinement of vaccine preparations which can be used in clinical practice will require a lengthy period
of 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,
6/7
8/7/2015
in «New Generation Vaccines)), G.C. Woodrow, M.M. Levine eds., Marcel Dekker Inc., New York and Basel, 1990, see
pp.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 recognized
as illicit is an act permitted or even encouraged by the laws of the country and poses a threat to human life. The
Encyclical Letter Evangelium Vitae underlined this "obligation to oppose" the laws which permit abortion or
euthanasia "by conscientious objection" (no.73)
15. This is particularly true in the case of vaccination against German measles, because of the danger of Congenital
Rubella Syndrome. This could occur, causing grave congenital malformations in the foetus, when a pregnant woman
enters into contact, even if it is brief, with children who have not been immunized and are carriers of the virus. In this
case, the parents who did not accept the vaccination of their own children become responsible for the malformations
in question, and for the subsequent abortion of foetuses, when they have been discovered to be malformed.
Top of page
Immunization Action Coalition 1573 Selby Avenue St. Paul MN 55104
E-mail: [email protected] Web: http://www.immunize.org/
Tel: (651) 647-9009 Fax: (651) 647-9131
7/7
0300-7995
doi:10.1185/030079906X148517
CURRENT MEDICAL RESEARCH AND OPINION®
VOL. 22, NO. 11, 2006, 2287–2300
© 2006 LIBRAPHARM LIMITED
All rights reserved: reproduction in whole or part not permitted
REVIEW
The key role of adult stem cells:
therapeutic perspectives
Augusto 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
ABSTRACT
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 transdifferentiation or fusion and their therapeutic
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
Paper 3289
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.
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
2287
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. immunological 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 pluriand 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,
2288 Adult stem cells and therapeutic perspectives
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/Sox2 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 cells 11. Tissue
determination and positional specification are interdependent 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)
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 crosstalk 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 preimplantation 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 undifferentiated 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 epidermis 16.
Moreover, experiments in animals have shown that
ESCs injected into the brain of syngeneic mice can
generate teratocarcinomas 18,19, 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 cells 22. If there is
evidence on the Oct-4 role to maintain embryonic cells
in a pluripotent status, little is known on its potential
oncogenic properties 23. Furthermore, it cannot be
excluded that the tumourigenesis of embryonic stem
cells, as observed in animal models, could be hostdependent 18. 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 mechanism 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 transformation 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. Sapienza 31 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
Adult stem cells and therapeutic perspectives Pessina and Gribaldo
2289
possible expression of endogenous viruses, or changes
in virus susceptibility, in uncontrolled in vitro longterm manipulation.
A comparison of the main advantages and disadvantages 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 Verfaillie 32, 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 cells 35 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 intrauterine 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 tetraplegia 45,46. 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
Embryonic stem
cells
Adult stem cells
Disadvantages
Can make virtually any tissue (in theory)
Allogeneic only (currently)
Some tissues ‘easy’ to generate (e.g. cardiac)
Can be propagated indefinitely
Amenable to genetic manipulation?
Teratoma formation
Differentiation conditions to be established
Some tissues difficult to generate (blood)
Ethical issues
Autologous
Many types and sources
Some types have extensive self-renewal potential
Low or not tumourigenic
Default differentiation
Amenable to gene transfer
Potential delivery methods attractive
No ethical issues
Most have limited self renewal
Differentiation outside lineage? (possibly)
Autologous (use more cumbersome and expensive)
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 redifferentiation 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
2291
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 differentiation 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 heatshocked small airway epithelial cells, some of the
stem cells differentiated directly into epithelial cells,
while others formed cell fusion hybrids to repair the
damage 67. 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 tissues 78,79 and a single adult bone
marrow stem cell can contribute to tissues as diverse
as marrow, liver, skin and digestive tract 80. One
group has now developed a method for large-scale
generation of neuronal precursors from whole adult
rat bone marrow 81. 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 tissue 83.
Bone marrow stem cells have also shown a capability
of forming kidney cells. Studies following genetically
marked bone marrow stem cells in rats 84 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 culture 86 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.
neuronal precursors 112 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
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 animals 93,94. 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.
Muscle appears to contain a side population of stem
cells, as seen in bone marrow and liver, with the ability
to regenerate muscle tissue 116. 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.
Peripheral blood
Pancreatic stem cells
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.
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 insulindependent 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.
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
2293
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.
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.
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 cells 145. 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
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 hepatocytes. 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/cholangiocytes 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 BMP7 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 proliferative 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
2295
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 significantly, 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 immunodeficiency 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 transdifferentiation 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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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 2006
Published Online: 16 October 2006
doi:10.1185/030079906X148517
Embryonic Stem Cell Lines
Derived from Human
Blastocysts
James 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 normal
karyotypes, express high levels of telomerase activity, and express cell surface
markers that characterize primate embryonic stem cells but do not characterize
other early lineages. After undifferentiated proliferation in vitro for 4 to 5
months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut
epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle
(mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous
epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.
Embryonic stem (ES) cells are derived
from totipotent cells of the early mammalian embryo and are capable of unlimited,
undifferentiated proliferation in vitro (1, 2).
In chimeras with intact embryos, mouse ES
cells contribute to a wide range of adult
tissues, including germ cells, providing a
powerful approach for introducing specific
genetic changes into the mouse germ line
(3). The term “ES cell” was introduced to
distinguish these embryo-derived pluripotent cells from teratocarcinoma-derived
pluripotent embryonal carcinoma (EC)
cells (2). Given the historical introduction
of the term “ES cell” and the properties of
mouse ES cells, we proposed that the essential characteristics of primate ES cells
should include (i) derivation from the preimplantation or periimplantation embryo,
(ii) prolonged undifferentiated proliferation, and (iii) stable developmental potential to form derivatives of all three embryonic germ layers even after prolonged culture (4). For ethical and practical reasons,
in many primate species, including humans,
the ability of ES cells to contribute to the
germ line in chimeras is not a testable
property. Nonhuman primate ES cell lines
provide an accurate in vitro model for understanding the differentiation of human
tissues (4, 5). We now describe human cell
lines that fulfill our proposed criteria to
J. A. Thomson, M. A. Waknitz, J. J. Swiergiel, V. S.
Marshall, Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, WI 53715,
USA. J. Itskovitz-Eldor, Department of Obstetrics and
Gynecology, Rambam Medical Center, Faculty of
Medicine, Technion, Haifa 31096, Israel. S. S. Shapiro
and J. M. Jones, Department of Obstetrics and Gynecology, University of Wisconsin, Madison, WI 53715,
USA.
*To whom correspondence should be addressed.
define primate ES cells.
Fresh or frozen cleavage stage human
embryos, produced by in vitro fertilization
(IVF) for clinical purposes, were donated
by individuals after informed consent and
after institutional review board approval.
Embryos were cultured to the blastocyst
stage, 14 inner cell masses were isolated,
and five ES cell lines originating from five
separate embryos were derived, essentially
as described for nonhuman primate ES cells
(5, 6 ). The resulting cells had a high ratio
of nucleus to cytoplasm, prominent nucleoli, and a colony morphology similar to that
of rhesus monkey ES cells (Fig. 1). Three
cell lines (H1, H13, and H14) had a normal
XY karyotype, and two cell lines (H7 and
H9) had a normal XX karyotype. Each of
the cell lines was successfully cryopreserved and thawed. Four of the cell lines
were cryopreserved after 5 to 6 months of
continuous undifferentiated proliferation.
The other cell line, H9, retained a normal
XX karyotype after 6 months of culture and
has now been passaged continuously for
more than 8 months (32 passages). A period
of replicative crisis was not observed for
any of the cell lines.
The human ES cell lines expressed high
levels of telomerase activity (Fig. 2). Telomerase is a ribonucleoprotein that adds telomere repeats to chromosome ends and is
involved in maintaining telomere length,
which plays an important role in replicative
life-span (7, 8). Telomerase expression is
highly correlated with immortality in human
cell lines, and reintroduction of telomerase
activity into some diploid human somatic cell
lines extends replicative life-span (9). Diploid human somatic cells do not express telomerase, have shortened telomeres with age,
and enter replicative senescence after a finite
proliferative life-span in tissue culture (10–
13). In contrast, telomerase is present at high
levels in germ line and embryonic tissues
(14). The high level of telomerase activity
expressed by the human ES cell lines therefore suggests that their replicative life-span
will exceed that of somatic cells.
The human ES cell lines expressed cell
surface markers that characterize undifferentiated nonhuman primate ES and human EC
cells, including stage-specific embryonic antigen (SSEA)–3, SSEA-4, TRA-l-60, TRA-181, and alkaline phosphatase (Fig. 3) (4, 5,
15, 16). The globo-series glycolipid GL7,
which carries the SSEA-4 epitope, is formed
by the addition of sialic acid to the globoseries glycolipid Gb5, which carries the
SSEA-3 epitope (17, 18). Thus, GL7 reacts
with antibodies to both SSEA-3 and SSEA-4
(17, 18). Staining intensity for SSEA-4 on the
human ES cell lines was consistently strong,
but staining intensity for SSEA-3 was weak
and varied both within and among colonies
(Fig. 3, D and C). Because GL7 carries both
the SSEA-4 and SSEA-3 epitopes and because staining for SSEA-4 was consistently
strong, the relatively weak staining for
Downloaded from www.sciencemag.org on July 8, 2015
REPORTS
Fig. 1. Derivation of the
H9 cell line. (A) Inner
cell mass–derived cells
attached to mouse embryonic fibroblast feeder layer after 8 days of
culture, 24 hours before 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 in
the absence of mouse
embryonic fibroblasts,
but in the presence of
human LIF (20 ng/ml;
Sigma). Scale bar, 100
!m.
www.sciencemag.org SCIENCE VOL 282 6 NOVEMBER 1998
1145
REPORTS
SSEA-3 suggests a restricted access of the
antibody to the SSEA-3 epitope. In common
with human EC cells, the undifferentiated
human ES cell lines did not stain for SSEA-1,
but differentiated cells stained strongly for
SSEA-l (15) (Fig. 3). Mouse inner cell mass
cells, ES cells, and EC cells express SSEA-1
but do not express SSEA-3 or SSEA-4 (17,
19), suggesting basic species differences between early mouse and human development.
The human ES cell lines were derived
by the selection and expansion of individual colonies of a uniform, undifferentiated
morphology, but none of the ES cell lines
was derived by the clonal expansion of a
single cell. The uniform undifferentiated
morphology that is shared by human ES
and nonhuman primate ES cells and the
consistent expression by the human ES cell
lines of cell surface markers that uniquely
characterize primate ES and human EC
cells make it extremely unlikely that a
mixed population of precursor cells was
expanded. However, because the cell lines
were not cloned from a single cell, we
cannot rule out the possibility that there is
some variation in developmental potential
among the undifferentiated cells, in spite of
their homogeneous appearance.
The human ES cell lines maintained the
potential to form derivatives of all three
embryonic germ layers. All five cell lines
produced teratomas after injection into severe combined immunodeficient (SCID)–
beige mice. Each injected mouse formed a
teratoma, and all teratomas included gut
epithelium (endoderm); cartilage, bone,
smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic
ganglia, and stratified squamous epithelium
(ectoderm) (Fig. 4). In vitro, the ES cells
differentiated when cultured in the absence
of mouse embryonic fibroblast feeder layers, both in the presence and absence of
human leukemia inhibitory factor (LIF)
(Fig. 1). When grown to confluence and
allowed to pile up in the culture dish, the
ES cell lines differentiated spontaneously
even in the presence of fibroblasts. After
H9 cells were allowed to differentiate for 2
weeks, both !-fetoprotein (350.9 " 14.2
IU/ml) and human chorionic gonadotropin
(hCG, 46.7 " 5.6 mIU/ml) were detected in
conditioned culture medium, indicating
endoderm and trophoblast differentiation
(20).
Human ES cells should offer insights
into developmental events that cannot be
studied directly in the intact human embryo
but that have important consequences in
clinical areas, including birth defects, infertility, and pregnancy loss. Particularly in
the early postimplantation period, knowledge of normal human development is
largely restricted to the description of a
1146
Fig. 2. Telomerase expression by human ES
cell lines. MEF, irradiated mouse embryonic
fibroblasts used as a
feeder layer for the
cells in lanes 4 to 18;
293, adenovirus-transformed kidney epithelial cell line 293; MDA,
breast cancer cell line
MDA; TSR8, quantitation control template.
Telomerase
activity
was measured with
the TRAPEZE Telomerase Detection Kit (Oncor, Gaithersburg, Maryland). The ES cell lines were analyzed at passages 10 to 13. About 2000 cells
were assayed for each telomeric repeat amplification protocol assay, and 800 cell equivalents were
loaded in each well of a 12.5% nondenaturing polyacrylamide gel. Reactions were done in triplicate with
the third sample of each triplet heat inactivated for 10 to 15 min at 85°C before reaction to test for
telomerase heat sensitivity (lanes 6, 9, 12, 15, 18, 21, 24, and 27). A 36–base pair internal control for
amplification efficiency and quantitative analysis was run for each reaction as indicated by the
arrowhead. Data were analyzed with the Storm 840 Scanner and ImageQuant package (Molecular
Dynamics). Telomerase activity in the human ES cell lines ranged from 3.8 to 5.9 times that observed
in the immortal human cell line MDA on a per cell basis.
Fig. 3. Expression of
cell surface markers by
H9 cells. Scale bar,
100 #m. (A) Alkaline
phosphatase. (B) SSEA1. Undifferentiated cells
failed to stain for SSEA1 (large colony, left).
Occasional colonies
consisted of nonstained, central, undifferentiated cells surrounded by a margin
of stained, differentiated, epithelial cells
(small colony, right).
(C) SSEA-3. Some
small colonies stained
uniformly for SSEA-3
(colony left of center),
but most colonies contained a mixture of
weakly stained cells
and a majority of nonstained cells (colony
right of center). (D)
SSEA-4. (E) TRA-1-60.
(F) TRA-1-81. Similar
results were obtained
for cell lines H1, H7,
H13, and H14.
A
B
C
D
E
F
limited number of sectioned embryos and
to analogies drawn from the experimental
embryology of other species (21). Although
the mouse is the mainstay of experimental
mammalian embryology, early structures
including the placenta, extraembryonic
membranes, and the egg cylinder all differ
substantially from the corresponding structure of the human embryo. Human ES cells
will be particularly valuable for the study
of the development and function of tissues
that differ between mice and humans.
Screens based on the in vitro differentiation
of human ES cells to specific lineages
could identify gene targets for new drugs,
genes that could be used for tissue regeneration therapies, and teratogenic or toxic
compounds.
Elucidating the mechanisms that control
differentiation will facilitate the efficient,
directed differentiation of ES cells to specific cell types. The standardized production of large, purified populations of euploid human cells such as cardiomyocytes
and neurons will provide a potentially limitless source of cells for drug discovery and
6 NOVEMBER 1998 VOL 282 SCIENCE www.sciencemag.org
REPORTS
Fig. 4. Teratomas
formed by the human
ES cell lines in SCIDbeige mice. Human ES
cells after 4 to 5
months of culture (passages 14 to 16) from
about 50% confluent
six-well plates were injected into the rear leg
muscles of 4-week-old
male SCID-beige mice
(two or more mice per
cell line). Seven to eight
weeks after injection,
the resulting teratomas
were examined histologically. (A) Gutlike
structures. Cell line H9.
Scale bar, 400 $m. (B)
Rosettes of neural epithelium. 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. Cell
line H13. Scale bar, 25
$m. (F) Tubules interspersed with structures resembling fetal
glomeruli. Cell line H9.
Scale bar, 100 $m.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
transplantation therapies. Many diseases, such
as Parkinson’s disease and juvenile-onset diabetes mellitus, result from the death or dysfunction of just one or a few cell types. The replacement of those cells could offer lifelong treatment. Strategies to prevent immune rejection of
the transplanted cells need to be developed but
could include banking ES cells with defined
major histocompatibility complex backgrounds or genetically manipulating ES
cells to reduce or actively combat immune
rejection. Because of the similarities to humans and human ES cells, rhesus monkeys
and rhesus ES cells provide an accurate
model for developing strategies to prevent
immune rejection of transplanted cells and
for demonstrating the safety and efficacy of
ES cell– based therapies. Substantial advances in basic developmental biology are
required to direct ES cells efficiently to
lineages of human clinical importance.
However, progress has already been made
in the in vitro differentiation of mouse ES
cells to neurons, hematopoietic cells, and
cardiac muscle (22–24). Progress in basic
developmental biology is now extremely
rapid; human ES cells will link this
progress even more closely to the prevention and treatment of human disease.
References and Notes
1. M. Evans and M. Kaufman, Nature 292, 154 (1981).
2. G. Martin, Proc. Natl. Acad. Sci. U.S.A. 78, 7634
(1981).
3. A. Bradley, M. Evans, M. Kaufman, E. Robertson, Nature 309, 255 (1984).
4. J. A. Thomson and V. S. Marshall, Curr. Top. Dev. Biol.
38, 133 (1998).
5. J. A. Thomson et al., Proc. Natl. Acad. Sci. U.S.A. 92,
7844 (1995).
6. Thirty-six fresh or frozen-thawed donated human
embryos produced by IVF were cultured to the
blastocyst stage in G1.2 and G2.2 medium (25).
Fourteen of the 20 blastocysts that developed
were selected for ES cell isolation, as described for
rhesus monkey ES cells (5). The inner cell masses
were isolated by immunosurgery (26), with a rabbit
antiserum to BeWO cells, and plated on irradiated
(35 grays gamma irradiation) mouse embryonic
fibroblasts. Culture medium consisted of 80% Dulbecco’s modified Eagle’s medium (no pyruvate,
high glucose formulation; Gibco-BRL) supplemented with 20% fetal bovine serum (Hyclone), 1 mM
glutamine, 0.1 mM !-mercaptoethanol (Sigma),
and 1% nonessential amino acid stock (Gibco-BRL).
After 9 to 15 days, inner cell mass– derived outgrowths were dissociated into clumps either by
exposure to Ca2"/Mg2"-free phosphate-buffered
saline with 1 mM EDTA (cell line H1), by exposure
to dispase (10 mg/ml; Sigma; cell line H7), or by
mechanical dissociation with a micropipette (cell
lines H9, H13, and H14) and replated on irradiated
mouse embryonic fibroblasts in fresh medium. Individual colonies with a uniform undifferentiated
morphology were individually selected by micropipette, mechanically dissociated into clumps, and
replated. Once established and expanded, cultures
21.
22.
23.
24.
25.
26.
27.
were passaged by exposure to type IV collagenase
(1 mg/ml; Gibco-BRL) or by selection of individual
colonies by micropipette. Clump sizes of about 50
to 100 cells were optimal. Cell lines were initially
karyotyped at passages 2 to 7.
C. B. Harley, Mutat. Res. 256, 271 (1991).
, H. Vaziri, C. M. Counter, R. C. Allsopp, Exp.
Gerontol. 27, 375 (1992).
A. G. Bodnar et al., Science 279, 349 (1998).
L. Hayflick and P. S. Moorhead, Exp. Cell Res. 25, 581
(1961).
R. C. Allsopp et al., Proc. Natl. Acad. Sci. U.S.A. 89,
10114 (1992).
C. M. Counter et al., EMBO J. 11, 1921 (1992).
C. M. Counter, H. W. Hirte, S. Bacchetti, C. B. Harley,
Proc. Natl. Acad. Sci. U.S.A. 91, 2900 (1994).
W. E. Wright, M. A. Piatyszek, W. E. Rainey, W. Byrd,
J. W. Shay, Dev. Genet. 18, 173 (1996).
P. W. Andrews, J. Oosterhuis, I. Damjanov, in Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, E. Robertson, Ed. (IRL, Oxford, 1987), pp.
207–248.
Alkaline phosphatase was detected with Vector Blue
substrate ( Vector Labs). SSEA-1, SSEA-3, SSEA-4,
TRA-1-60, and TRA-1-81 were detected by immunocytochemistry with specific primary monoclonal antibodies and localized with a biotinylated secondary
antibody and then an avidin or biotinylated horseradish peroxidase complex (Vectastain ABC system;
Vector Laboratories) as previously described (5). The
ES cell lines were at passages 8 to 12 at the time
markers were analyzed.
R. Kannagi et al., EMBO J. 2, 2355 (1983).
R. Kannagi et al., J. Biol. Chem. 258, 8934 (1983).
D. Solter and B. B. Knowles, Proc. Natl. Acad. Sci.
U.S.A. 75, 5565 (1978).
hCG and #-fetoprotein were measured by specific
radioimmunoassay (double AB hCG and AFP-TC kits;
Diagnostic Products, Los Angeles, CA). hCG assays
used the World Health Organization Third International Standard 75/537. H9 cells were allowed to
grow to confluence (day 0) on plates of irradiated
mouse embryonic fibroblasts. Medium was replaced
daily. After 2 weeks of differentiation, medium in
triplicate wells conditioned for 24 hours was assayed
for hCG and #-fetoprotein. No hCG or #-fetoprotein
was detected in unconditioned medium.
R. O’Rahilly and F. Müller, Developmental Stages in
Human Embryos (Carnegie Institution of Washington,
Washington, DC, 1987).
G. Bain, D. Kitchens, M. Yao, J. E. Huettner, D. I.
Gottlieb, Dev. Biol. 168, 342 (1995).
M. V. Wiles and G. Keller, Development 111, 259
(1991).
M. G. Klung, M. H. Soonpaa, G. Y. Koh, L. J. Field,
J. Clin. Invest. 98, 216 (1996).
D. K. Gardner et al., Fertil. Steril. 69, 84 (1998).
D. Solter and B. Knowles, Proc. Natl. Acad. Sci. U.S.A.
72, 5099 (1975).
We thank the personnel of the IVF clinics at the
University of Wisconsin School of Medicine and at
the Rambam Medical Center for the initial culture
and cryopreservation of the embryos used in this
study; D. Gardner and M. Lane for the G1.2 and
G2.2 media; P. Andrews for the NTERA2 cl.D1 cells
and the antibodies used to examine cell surface
markers; C. Harris for karyotype analysis; and
Geron Corporation for the 293 and MDA cell pellets and for assistance with the telomerase TRAP
assay. Supported by the University of Wisconsin
(UIR grant 2060) and Geron Corporation (grant
133-BU18).
!!!!
5 August 1998; accepted 7 October 1998
www.sciencemag.org SCIENCE VOL 282 6 NOVEMBER 1998
1147
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Medicina e Persona. A proposito di staminali giugno 06
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
1
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
2
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
3
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. 2001,68:205219
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 2001,293:10891093
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
4
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 embriotossicologici 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.

materiale per PUSC - Pontificia Università della Santa Croce

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