In utero stem cell transplantation: two steps forward but one step back?

JonesIn utero stem cell transplantation

Emerging Biotherapeutic Technologies

In utero stem cell transplantation: twosteps forward but one step back?

DRE Jones

Department of Immunology, Queen’s Medical Centre, Nottingham, NG7 2UH,UK

The concept of in utero stem cell transplantation has provided hope that atleast some of the genetic disorders which can be diagnosed prenatally canbe treated before the pathological sequelae become manifested. Ourincreasing knowledge of haematopoietic stem cell biology has providedfurther possibilities for optimising this transplant procedure. However, therelatively poor success rate achieved using this procedure has promptedsome to suggest that there may be fetal rejection of donor cells via animmune mechanism. However, the prevailing evidence from both animalmodels and from human cases, suggest that there may be othermechanisms which are preventing successful engraftment in somedisorders and these must be addressed if the procedure is to receive furtherattention. The ability to achieve sustained engraftment after in uterotransplantation will have relevance for those seeking to use gene therapy asan alternative therapeutic stratagem.

Keywords: fetal haematopoiesis, in utero transplantation, stem cells, tolerance

Exp. Opin. Biol. Ther. (2001) 1(2):205-212

1. Introduction

’A paradigm is a commonly held belief among scientists. It need not becorrect, merely accepted. Thus, science moves forward by changingparadigms. All experiments are designed to prove the paradigms. Whenenough data are generated so that the paradigm begins to be less univer-sally accepted, a new paradigm takes its place.’Thomas S Kuhn: The Structure of Scientific Revolutions(The University of Chicago Press, 1970)

The evolutionary success of the human immune system relies on the ability,at the cellular and at the molecular level, to recognise and to eliminate thatwhich is ‘foreign’. To achieve this, the ability to recognise self and todiscriminate that which is non-self must be ‘learned’ before birth. Thus, theparadigm of learned self tolerance in fetal life [1] has become one of thebasic tenets of immunology and has served to underpin the concept of inutero transplantation (IUT). Indeed, ‘experiments of nature’ have beenrecorded which indicate the efficacy of this mode of tolerance induction,both in animals [2] and in man [3]; where blood group chimerism throughvascular anastomoses has occurred in utero and is subsequently toleratedinto postnatal life. The IUT procedure builds on these observations by usingdonor haematopoietic stem cells (HSC) for transplant into a recipient fetusat a developmental stage when (or before) the process of self tolerance isbeing undertaken. Indeed, if performed sufficiently early in gestation it is

2052001 © Ashley Publications Ltd. 1471-2598

Review

1. Introduction

2. Is lack of engraftmentindicative ofimmune-mediatedrejection?

3. Is IUT in human patientssubject to a differentparadigm?

4. Stem cell plasticity

5. Whither gene therapy?

6. Conclusions

7. Expert opinion

Bibliography

http://www.ashley-pub.com

Expert Opinion on Biological Therapy

www.ashley-pub.com

theoretically possible to use mismatched cells for thetransplant procedure, because the process of immunedevelopment should include even these cells as ‘self’.Animal studies have been carried out, where HSCfrom a different species have been transplanted inutero and long-term engraftment achieved. These arethe experiments which serve to reinforce theparadigm of learned self tolerance, which develops inutero. Against this background, human IUTprocedures have been performed and there are nowestimated to be in excess of 35 cases worldwide.There have been notable successes after IUT forimmunodeficiency disorders (in particular, forX-linked severe combined immunodeficiency(X-SCID); but for other types of disorder, transplanta-tion in utero has generally not been effective. This hasprompted some workers to suggest that it is immunerecognition of the foreign (donor) cells which has ledto a lack of engraftment in, for example, thehaemoglobinopathies and the successful outcomeafter IUT for immunodeficiency disorders has beencited as evidence. Thus, the paradigm of learned selftolerance is becoming less universally accepted andhas generated a renewed interest in fetal haemato-poiesis, tolerance induction and the question ofimmune competence in fetal life.

2. Is lack of engraftment indicative ofimmune-mediated rejection?

Susceptibility to tolerance is greatest during immunedevelopment, which in humans and other animalswith a long gestation, occurs in utero [4]. The firsttrimester human fetal liver is predominantlycommitted to the erythroid lineage [5] and bygestational week 15 there is already active migrationof cells to seed the bone marrow [6] and inevitably, thethymus and spleen. We know with certainty that, ataround this time the lineage commitment of the stemcells within the fetal liver changes and multilineagecommitment becomes evident [6]. During this phaseof fetal haematopoietic development, CD3+T-lymphocytes can be detected within the liver [7] butthere is some controversy over the function of thesecells. It has recently been suggested that the humanfetus can react against foreign transplantationantigens perhaps as early as 11 - 12 weeks gestation[8], although the onset of reactivity appeared to differconsiderably between fetal samples studied. It hasalso been shown that T-cell receptor Vβ2 rearrange-ments can be detected in RNA extracted from early

gestation fetal liver cells [9] and taking these data inconjunction with in vitro proliferation assays, it wasconcluded that the first trimester fetal liver containsfunctional T-cells which are capable of reacting toforeign tissue. However, these (and similar) studiesare subject to many confounding variables which aredifficult to reconcile. For example, the number ofT-cells identified was very low and it was impossibleto determine which (fetal liver-derived) cell popula-tion was responding in the proliferation assay [8]. Theresponsiveness to proliferative stimuli, taken toindicate a functional T-cell repertoire, could simplyhave been a response to IL-2: a known feature of fetalT-cells, which are predominantly CD45RO+ (i.e., theyare memory T-cells) and which express the IL-2receptor (CD25) [10]. It is known that non-reactive(i.e., anergic) T-cells can be stimulated to proliferatein an in vitro assay simply by addition of exogenousIL-2 [11]. Indeed, it has been suggested that theCD45RO+ (memory) T-cells present in the earlygestation human fetus may actually represent apopulation of self-reactive T-cells ‘leaking’ from thedeveloping thymus and that they will ultimately bedeleted or be rendered anergic [10,12]; a conceptwhich lends further currency to the paradigm oflearned self tolerance in utero.

The most compelling examples of successful IUThave occurred in a non-conditioned large animal(sheep) model, where the success of xenotransplanta-tion is testament to the immunologically receptiveenvironment which must be present in the earlygestation fetus [13,14] and which can result inevidence of postnatal tolerance to the xenograft [13].The sheep model of IUT is a very important additionto the tools used for the study of ways to improveengraftment after IUT: it has a relatively long gestationperiod (∼ 145 days) within which there is, initially,liver-dependent haematopoiesis and subsequentmigration of haematopoietic cells to seed otherlymphohaematopoietic sites; in a manner analogousto that in the human fetus [6]. No conditioning orgenetic manipulation is required to achieve engraft-ment of, for example, human fetal liver-derived HSCafter IUT in the fetal sheep and the transplanted cellshave been shown to be functional in postnatal life[15]. Thus, there is tantalising evidence to suggest thatearly in gestation, transplanted (donor) cells will berecognised as part of the self repertoire. It would beinteresting to seek CD45RO+ cells in the fetal sheep,post IUT and to determine whether any of thispopulation can react to autologous donor cells in

© Ashley Publications Ltd. All rights reserved. Exp. Opin. Biol. Ther. (2001) 1(2)

206 In utero stem cell transplantation

vitro under the relatively non-specific conditions in aproliferation assay: an important experiment requiredfor furthering our understanding of self tolerance inthe context of an in utero transplant.

In the fetal sheep recipient of an IUT using humanfetal liver-derived HSC, engraftment levels of 1 - 2%can be achieved and sustained long-term [15]. This is aremarkable achievement, considering that thetransplant is not just across MHC barriers but alsoacross a species barrier. However, this level of engraft-ment would be insufficient to ameliorate the seriouspathological sequelae in, for example, a human fetusdiagnosed with a life-threatening thalassaemia. Theavailable evidence, from bone marrow transplanta-tion (BMTx) experience in adults, shows that levels ofperhaps 20% donor cells are required for ameliorationof the pathology in such cases [16]. It is clear that iflevels of up to 2% (mismatched) donor cells can betolerated after an IUT procedure in a mismatchedanimal model (and persist into adult life), it could beanticipated that higher (i.e., clinically relevant) levelsof engraftment would also fail to provoke immuno-logical sequelae. Indeed, dogma informs us thathigher doses of antigen are more likely to provokeimmunological tolerance. This is borne out by theobservation that persistent and clinically relevantlevels of chimerism have been recorded in adultsfollowing BMTx for thalassaemia [17]. Thus, the sheepmodel can serve as a useful tool in the search formethods of increasing the levels of donor cell engraft-ment following an IUT procedure. To this end, thereare a number of similarities between the haemato-poietic system in the fetal sheep and that in athalassaemic (human) fetus: both have an indigenoushaematopoietic output tuned to their specific physio-logical requirements and in neither scenario does atransplanted donor cell population offer any selectiveadvantage over the status quo.

3. Is IUT in human patients subject to adifferent paradigm?

The number of cases of IUT performed in humanmedicine is variously estimated to be in excess of 35,worldwide. It is difficult to provide an exact figurebecause not all procedures have been reportedwidely [18] particularly when there has been a lack ofengraftment, no clinical improvement or subsequentdeath in utero. However, it is clear from those caseswhere a diagnosis of haemoglobinopathy was the

reason for choosing IUT as a therapeutic option, thatresults have been very poor. To some groups, this hasreinforced the paradigm that it is immune-mediatedrejection which provoked the lack of engraftment;which is tantamount to accepting that immunedevelopment in human fetal life differs profoundlyfrom that in large animal models described in theliterature. It is pertinent to study the haemoglobino-pathy IUT cases more closely and to seek alternativeanswers for the apparent lack of engraftment. Firstly,in a serious haemoglobinopathy (such as α thalas-saemia) oxygen delivery to the developing fetaltissues is radically diminished. The developing fetusrequires rapid and efficient oxygen delivery and forthis reason the haematopoietic output is predomi-nantly erythroid during the first trimester [7]. Clearly,any defect in this system will be catastrophic andwhere the haemoglobin molecule is ineffective orunstable, the haematopoietic output will be increasedin order to compensate for the defect. Inevitably, thiswill result in haematopoiesis in sites other than thefetal liver or fetal bone marrow. Thus, in such afundamental defect all potential sites for haemato-poiesis are occupied and there is no space forengraftment by incoming donor cells. To achieveeven a 1% level of engraftment in the normal fetalsheep, there must be steady-state haematopoiesis (forthat stage of fetal development). Where the defect iswithin the erythroid compartment and where thiscompromises oxygen delivery, there can be a marked,compensatory, increase in erythropoiesis. Underthese circumstances, it is not realistic to anticipatedonor cell engraftment at levels sufficient toovercome the indigenous defect in haemoglobinsynthesis (Table 1).

Thus, if the paradigm of learned self tolerance in uterois to remain tenable, is there evidence in cases thus fartransplanted, to support the ‘telephone networkhypothesis’ (Table 1)? In many cases, the lack ofengraftment was assessed with reference to theclinical outcome. Thus, where IUT had beenperformed for a thalassaemia and where the infantwas subsequently born with the disorder and with noevidence of donor cells in the circulation, the IUTwould be deemed unsuccessful [19]. However, someworkers have pursued this issue in more detail andhave shown that, despite the apparent lack of engraft-ment in such cases (no amelioration of the disorder,subsequent to IUT), there is indeed evidence thattolerance to the donor cell type is present [20]. In oneparticular case (a fetus with α thalassaemia) there was


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evidence of microchimerism at birth but this wasclearly insufficient per se to have had any clinicalimpact. As in the sheep model of IUT, where thedonor cells have no selective advantage over theindigenous haematopoietic cells, then levels ofengraftment will be low or will be undetectable. It hasbeen shown, in a mouse model of inherited anaemia,that where the defect is within the stem cell compart-ment then IUT will correct the disorder [21]. In(human) thalassaemia, the defect is within thematuring erythroid lineage, so the likelihood ofsignificant engraftment is diminished [21]. Neverthe-less, tolerance to the donor cell type can still beachieved and as has been shown in a mouse model ofIUT (without a haematological defect), could possiblybe exploited postnatally to boost the levels ofcirculating donor cells (to perhaps clinically relevantnumbers?) [22,23]. However, this strategy has, thus far,been unsuccessful in human recipients of an IUT [20].Long-term transfusion and further BMTx in similarpost-IUT patients have also failed to influence theinitially poor outcome [19].

In some IUT procedures, the cell preparation whichwas infused had been derived from more than onedonor [19]. This has been deemed necessary whenusing fetal liver-derived HSC, because it may not bepossible to obtain sufficient cell numbers fortransplant from a single donor. Interestingly, it hasbeen shown in a mouse model of IUT, that the use ofcells from multiple donors can reduce the level ofengraftment: it appears then that tolerance is restrictedto only one disparate tissue type [24]. The use of fetalliver versus bone marrow as the source of HSC hasproved to be another contentious issue. Fetal liver-derived HSC have a significantly greater develop-mental potential than do adult BM-derived HSC [25]and their proliferative capacity is enhanced whencompared to either BM- or cord blood-derived HSC[26]. Fetal liver-derived HSC are able to sustainlong-term engraftment after transplantation far more

readily than do their adult counterparts [27] probablyowing to their comparatively longer telomeric repeats[28]. However, it is likely that HSC obtained fromdifferent sources might be appropriate for differentdisorders. Early gestation fetal liver is a source of HSCcommitted to erythroid production [7] and as suchmight be suitable for IUT in dyseythropoieticdisorders. We know also that before fetal bonemarrow-derived haematopoiesis has developed theseHSC, when transplanted, home to the recipient fetalliver in the first trimester [29] and this is the site whereengraftment must initially occur if clinically relevantlevels of haematological chimerism are to be achievedin a serious thalassaemia. After development of thefetal bone marrow and subsequent to the start ofmigration of HSC from the liver, the likelihood ofsignificant levels of engraftment are diminished asmore of the donor cells home to the marrow [6], wherethey will not contribute to the circulating blood in fetallife [29].

Thus, there is a significant body of evidencedeveloping to suggest that it may not be immunefunction that impairs the engraftment potential ofdonor cells in an IUT procedure in man. The ‘learnedself tolerance’ paradigm remains intact but with sofew human IUT cases thus far, it is being sustained bylimited data gleaned from individual patients.

4. Stem cell plasticity

Future directions for in utero intervention with stemcell transplantation have, in recent years, taken on anew significance with the finding that not all stemcells are the same. As the name implies, stem cells arecapable of producing progenitors of all blood celllineages and thus, are capable of repopulating acytoablated recipient marrow. However, the humanbone marrow also harbours a population ofmesenchymal stem cells [30] with the capacity todifferentiate into cells of the adipocyte, chondrocyte,



Table 1: Successful in utero stem cell transplantation in a fetus diagnosed with a haemoglobinopathy can only be achieved byovercoming the indigenous microenvironmental advantages for haematopoiesis in the recipient. In an immunodeficiency disorder,the donor cells will predominate because they possess an intact molecular apparatus for cell-cell communication. The donor cellsestablish communication and proliferation proceeds: the ‘telephone network hypothesis.’

Haemoglobinopathy Immunodeficiency

Cell development is normal

Compensatory overproduction

All haematopoietic space is occupied

No donor cell advantage

Cell development is defective

Cells do not communicate

Lack of proliferation to occupy niches

Donor cells have a selective advantage

or osteocyte lineages [30]. Studies in the fetal sheepmodel have shown that IUT using humanmesenchymal stem cells results in human cellsappearing in multiple tissues for long periods after thetransplant [31]. Also, in this IUT model mesenchymalstem cells appear to have unique immunologicalcharacteristics, which allow engraftment at andbeyond, a gestational age when immunologicalcompetence has been shown to have developed [31].Thus, there is a population of stem cells which appearto be able to evade the usual immune surveillancemechanisms by being themselves ‘invisible’ toimmune detection. Another paradigm. The prospectof using an in utero stem cell transplant to correctdefects other than those diagnosed in the lymphohae-matopoietic system is tantalising and already, studieshave been designed to investigate this exciting area[32]. Mesenchymal stem cells may hold the key to thecurrent poor success rate after IUT for metabolicdisorders.

5. Whither gene therapy?

Prenatal gene therapy is rapidly becoming lauded as anew concept for treating pre- and postnatal manifes-tations of genetic diseases and developmentaldisorders but there are numerous obstacles to beovercome before this therapy can be used in theclinic. Clearly, the prospect for gene therapy in uteroholds great promise because very similar criteria arerequired for use of such therapy as are proposed forIUT with unmodified stem cells (Table 2). Themajority of gene therapy protocols thus far approvedare intended for paediatric and adult patients [33] anda recently reported success in the treatment of X-SCIDhas generated hope that the methodology is ready for

more general use [34]. Transduction of ‘self’ cells willovercome any postnatal immune-mediated recogni-tion, provided the cells do not secrete a new ormodified protein; in which case in utero transfer ofthe transduced cells would be preferred. Again, thisstrategy relies on the paradigm of ‘learned selftolerance’ being de rigueur in the early gestation fetus.There is therefore, a remarkable similarity in therationale underlining IUT using HSC and that for genetherapy in utero (Table 2). It can be argued that if theIUT procedure using HSC can be manipulated toachieve clinically relevant levels of engraftment thenin utero gene therapy is not needed. A number ofgene transfer strategies have been undertaken inanimal models [35] but a big hurdle is the need totransduce a long-lived quiescent cell which is not incell cycle: the haematopoietic stem cell. In thiscontext, fetal liver-derived HSC may be better targetsfor gene transduction because there are increasednumbers of actively proliferating multipotent stemcells in this tissue [36].

6. Conclusions

The concept of transplanting HSC into a fetus early inpregnancy, to offer therapy for a prenatally diagnosedgenetic disorder is an attractive therapeutic option.Transplantation in utero offers the chance of alteringthe course of a potentially fatal, or at least severelydisabling, disorder before the pathological sequelaebecome apparent. However, the initial enthusiasm forthe procedure has been tempered by the poor successrate in those disorders where alternative therapycarries either an uncertain outcome or is entirelylacking. Disorders such as X-SCID have proved to bevery successful targets for IUT and this has prompted


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Table 2: A number of selection criteria which are applicable to gene therapy could also be used for choosing a haematopoieticstem cell transplant.

Criteria for use of in utero therapy Via stem cell transplantation Via gene therapy

The disease should be life-threatening in utero ü ü

The defective gene should be isolated and cloned. û ü

The pathology of the disease should be understood ü ü

No reasonable alternative therapy should be available§. ? ü

Prenatal diagnosis should be available. ü ü

Fine regulation of the transgene should be unnecessary. û ü

Overexpression of the transgene should be non-toxic. û ü

The target organ should be accessible. û ü

§This may be contentious for IUT using stem cells because disorders targeted may include those for which no intervention can resultin death in utero. (Modified from Senut & Gage) [35].

some groups to widen the repertoire of immunedefects amenable to the procedure [37]. In somequarters this has reinforced the concept that it is fetalimmune competence which determines the success ofIUT. Thus, one of the paradigms which is a basic tenetof immunology is being challenged. The issue ofimmune competence in fetal life must urgently beaddressed, otherwise not only will the therapeutic useof IUT with stem cells cease but the prospect of genetherapy in utero will be called into question.

7. Expert opinion

Further work on issues relevant to IUT is underwayand as the various groups return to fundamentalquestions of fetal development then many of thecurrent dilemmas will be addressed. The issues whichrequire attention are:

• Choice of HSC for therapy in utero. There are cleardifferences between HSC obtained from differentsources and it is likely that these differences can beexploited to achieve an optimal therapeuticstrategy in the various disorders deemed to beamenable to IUT.

• Can tolerance be achieved in utero ? This is the areawhere basic research into fetal immune develop-ment is needed. Where tolerance to donor cells hasbeen demonstrated, either in utero and/or inpostnatal life there is a real prospect for finding themeans to increase the levels of donor cellchimerism. If tolerance is possible, there must be areceptive haematopoietic microenvironment in thefetal recipient. The next task must therefore be toexploit this state of tolerance to seek ways to obtainengraftment at levels which are clinically relevant.

• With the current state of knowledge, it is pertinentthat workers in the field use IUT for only thoseprocedures which have been proven to give goodengraftment levels. In this way, the pool of datarelevant to human IUT can be increased andmeaningful extrapolations made. Animal models ofIUT must be used more rigorously to address theproblems of lack of engraftment: useful data hasbeen provided in this area and now is the time tocapitalise on the studies thus far performed.

Bibliography

Papers of special note have been highlighted as:• of interest•• of considerable interest

1. BILLINGHAM R, BRENT EL, MEDAWAR PB: ‘Activelyacquired tolerance’ of foreign cells. Nature (Lond.).(1953) 172:603-606.

2. OWEN RD: Immunogenetic consequences of vascularanastomoses between bovine twins. Science (1945)102:400-401.

3. VAN DIJK BA, BOOMSMA DI. DE MAN AJM: Blood-groupchimerism in human multiple births is not rare. Am. J.Med. Genet. (1996) 61:264-268.

• Reinforces the original ‘experiments of nature’ by showingthat in utero tolerance is not uncommon.

4. WEST L, MORRIS JPJ, WOOD KJ: Fetal liver haemato-poietic cells and tolerance to organ allografts. Lancet(1994) 343:148-149.

5. ANDERSON EM, JONES DRE, LIU DTY, EVANS AA:Gestational age and cell viability determine the effectof frozen storage on human fetal hematopoieticprogenitor cell preparations. Fetal. Diagn. Ther. (1996)11:427-432.

6. JONES DRE: Why do in utero stem cell transplantssometimes fail? Mol Med. Today. (1999) 5:288-291.

7. JONES DRE, ANDERSON EM, EVANS AA, LIU DTY:Long-term storage of human fetal hematopoieticprogenitor cells and their subsequent reconstitution -implications for in utero transplantation. Bone MarrowTransplant. (1995) 16:297-301.

8. LINDTON B, MARKLING L, RINGDÉN O, KJAELDGAARD A,GUSTAFSON O, WESTGREN M: Mixed lymphocyteculture of human fetal liver cells. Fetal Diagn. Ther.(2000) 15:71-78.

9. RENDA MC, FECAROTTA E, DIELI F et al.: Evidence ofalloreactive T lymphocytes in fetal liver: implicationsfor fetal hematopoietic stem cell transplantation. BoneMarrow Transplant. (2000) 25:135-141.

• Investigations which suggest immune function in the fetalliver.

10. BYRNE JA, STANKOVIC AK, COOPER MD: A novelsubpopulation of primed T cells in the human fetus. J.Immunol. (1994) 152:3098-3106.

• An alternative view of the significance of immune cells in thefetus.

11. KIM HB, SHAABAN AF, MILNER R, FICHTER C, FLAKE AW:In utero bone marrow transplantation inducesdonor-specific tolerance by a combination of clonaldeletion and clonal anergy. J. Pediatr. Surg. (1999)34:726-730.

•• A thorough study of the mechanisms by which tolerance canbe achieved in utero.

12. FLAKE AW, ZANJANI ED: Role of the alloimmuneresponse after in utero hematopoietic stem celltransplantation. Blood (2000)96:1608-1609.



13. JONES DRE, ANDERSON EM, LIU DTY, WALKER RM:Tolerance induction following in utero stem celltransplantation. In: Stem Cells from Cord Blood, in uteroStem cell Development and Transplantation-Inclusive GeneTherapy, Workshop 33. Holzgreve W, Lessl M (Eds.),Springer-Verlag, Berlin Heidelberg (2000):187-196.

14. ZANJANI ED, ALMEIDA-PORADA G, FLAKE AW: Thehuman/sheep xenograft model: A large animal modelof human hematopoiesis. Int. J. Hematol. (1996)63:179-192.

15. JONES DRE, LIU DTY, ANDERSON EM, LAMMING GETransplantation of human fetal liver-derivedhaematopoietic stem cells into sheep in utero. InCorrection of Genetic Disorders by Transplantation, vol. IV.Ringdén O, Hobbs JR, Steward CG (Eds.): COGENT Trust,London (1997):130-136.

16. ANDREANI M, MANNA M, LUCARELLI G et al.: Persistenceof mixed chimerism in patients transplanted for thetreatment of thalassemia. Blood (1996) 87:3494-3499.

• Evidence from adult thalassaemia patients which hasrelevance to IUT for this condition.

17. ANDREANI M, NESCI S, LUCARELLI G et al.: Long-termsurvival of ex-thalassemic patients with persistentmixed chimerism after bone marrow transplantation.Bone Marrow Transplant. (2000) 25:401-404.

18. WESTGREN M, SHIELDS LE: In utero stem cell transplan-tation in humans. In: Stem Cells from Cord Blood, in uteroStem cell Development and Transplantation-Inclusive GeneTherapy, Workshop 33. Holzgreve W, Lessl M (Eds.),Springer-Verlag, Berlin Heidelberg (2000):197-221.

19. WESTGREN M, RINGDÉN O, EIK-NES S et al.: Lack ofevidence of permanent engraftment after in utero fetalstem cell transplantation in congenital hemoglobino-pathies. Transplantation (1996) 61:1176-1179.

20. HAYWARD A, AMBRUSO D, BATTAGLIA F et al.:Microchimerism and tolerance following intrauterinetransplantation and transfusion for alpha-thalassemia-1. Fetal Diagn. Ther. (1998) 13:8-14.

•• A good workup of the consequences of an IUT which hasapparently failed.

21. FLEISCHMAN RA, CUSTER RP MINTZ B: Totipotenthematopoietic stem cells: normal self-renewal anddifferentiation after transplantation between mousefetuses. Cell (1982) 30:351-359.

22. CARRIER E, LEE TH, BUSCH MP, COWAN MJ: Induction oftolerance in nondefective mice after in uterotransplantation of major histocompatibility complex-mismatched fetal hematopoietic stem-cells. Blood(1995) 86:4681-4690

• An important paper because the transplant recipients didnot have a haematological defect.

23. KIM HB, SHAABAN AF, YANG EY, LIECHTY KW, FLAKEAW: Microchimerism and tolerance after in utero bonemarrow transplantation in mice. J. Surg. Res. (1998)77:1-5.

24. MINTZ B, ANTHONY K, LITWIN S: Monoclonal deriva-tion of mouse myeloid and lymphoid lineages fromtotipotent hematopoietic stem cells experimentally

engrafted in fetal hosts. Proc. Natl. Acad. Sci. USA (1984)81:7835-7839.

25. UCHIDA N, FLEMING WH, ALPERN EJ, WEISSMAN IL:Heterogeneity of hematopoietic stem cells. Curr. Opin.Immunol. (1993) 5:177-184.

26. WEEKX SF, VAN BOCKSTAELE DR, PLUM J et al.: CD34+CD38- and CD34+ CD38+ human hematopoieticprogenitors from fetal liver, cord blood and adult bonemarrow respond differently to hematopoieticcytokines depending on the ontogenic source. Exp.Hematol. (1998) 26:1034-1042.

•• A seminal paper outlining the differences between stemcells from various sources.

27. HARRISON DE, ZHONG RK, JORDAN CT, LEMISCHKA IR,ASTLE CM: Relative to adult marrow, fetal liver repopu-lates nearly five times more effectively long-term thanshort-term. Exp. Hematol. (1997) 25:293-297.

• A useful reference to the functional differences betweenstem cells.

28. LANSDORP PM: Telomere length and proliferationpotential of hematopoietic stem cells. J. Cell Sci. (1995)108:1-6.

29. ZANJANI ED, ASCENSAO JL, TAVASSOLI M: Liver-derivedfetal hematopoietic stem cells selectively and prefer-entially home to the fetal bone marrow. Blood (1993)81:399-404.

30. PITTENGER MF, MACKAY AM, BECK SC et al. :Multilineage potential of adult human mesenchymalstem cells. Science (1999) 284:143-147.

31. LIECHTY KW, MACKENZIE TC, SHAABAN AF et al.:Human mesenchymal stem cells engraft anddemonstrate site-specific differentiation after in uterotransplantation in sheep. Nature Med . (2000)6:1282-1286.

•• A milestone in our understanding of stem cell function andpotential.

32. PARTRIDGE T: The ‘fantastic voyage’ of muscleprogenitor cells. Nature Med. (1998) 4:554-555.

33. ZANJANI ED, ANDERSON WF: Prospects for in uterohuman gene therapy. Science (1999) 285:2084-2088.

• A good overview of the state of gene therapy at thebeginning of the new millennium.

34. CAVAZZANA-CALVO M, HACEIN-BEY S, DE SAINT-BASILEG et al.: Gene therapy of human severe combinedimmunodeficiency (SCID)-X1 disease. Science (2000)288:669-672.

•• A seminal paper with the first convincing series of genetherapy cases. Future developments from this group areeagerly awaited.

35. SENUT MC, GAGE FH: Prenatal gene therapy: can thetechnical hurdles be overcome? Mol. Med. Today (1999)5:152-156.

• A good overview.

36. CLAPP DW, DUMENCO LL, HATZOGLOU M, GERSON SL:Fetal liver hematopoietic stem cells as a target for inutero retroviral gene transfer. Blood (1991)78:1132-1139.


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37. LANFRANCHI A, NEVA A, TETTONI K et al.: In uterotransplantations (IUT) of parental CD34+ cells in threepatients affected by primary immunodeficiencies.Bone Marrow Transplant. (1998) 21:127.

D Rhodri E JonesDepartment of Immunology, Queen’s Medical Centre, Nottingham,NG7 2UH, UKTel.:+44 (0)115 970 9058; Fax :+44 (0)115 970 9125;E-mail: [email protected]



In utero stem cell transplantation: two steps forward but one step back?

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