Use Of Hematopoietic Stem Cells Research Articles

Selective Activation of STAT5 Unveils Its Role in the Maintenance of Hematopoietic Stem Cells and the Development of Myeloproliferative Disorder.

Recent studies have implicated the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the maintenance of stem cells, such as mouse embryonic stem cells and Drosophila germ cells. We have previously reported that thrombopoietin (TPO) can support in vitro self-renewal division of murine hematopoietic stem cells (HSCs) (CD34−/lowc-Kit+Sca-1+lineage marker-negative; CD34−KSL cells). Signal transducers and activators of transcription 5 (STAT5) is one of the major signaling molecules that mediate TPO signals. All these findings suggest that STAT5 could be an attractive candidate for therapeutic manipulation of HSCs. Cytokines activate JAK/STAT5 pathway along with other signaling pathways, causing difficulty to dissect STAT5-specific functions in hematopoietic stem cells (HSCs). Here we took advantage of constitutively active STAT5 mutants to selectively activate STAT5 signaling pathway in HSCs. The mutants used are STAT5A 1*6 that harbors two amino acid mutations S710F and H298R in the effecter domain, and STAT5A #2 that harbors a point mutation N642H in the SH2 domain. Retroviral transduction of either STAT5 1*6 or STAT5#2 mutant into purified CD34−KSL HSCs caused a drastic expansion of multipotential progenitors in vitro and promoted multi-lineage differentiation in vitro. During 7 days of culture supplemented with SCF and TPO, the number of high proliferative potential colonies (HPPC) increased ten-fold compared with the GFP control and half of them were derived from multipotential progenitor cells. Notably, even in the culture supplemented with SCF only, expression of STAT5 mutants in HSCs supported a similar mode of expansion of progenitors cells and multi-lineage differentiation, indicating that activation of STAT5 can substitute major biological effects of TPO in HSCs. In all in vitro experiments, STAT5 1*6 showed stronger effects than STAT5#2. To evaluate the effect of STAT5A mutants in the maintenance of long-term bone marrow repopulating HSC ex vivo, cultured transduced cells corresponding to 30 initial CD34−KSL HSCs were transplanted into lethally irradiated mice 7 to 10 days after transduction. Although rapid hamatopoietic repopulation was observed with HSCs expressing STAT5A 1*6, mice developed myeloproliferative disease (MPD) and succumbed to death within two months. In contrast, HSCs expressing STAT5A #2 presented significantly higher long-term repopulating capacity than the GFP control. These data indicate that selective activation of STAT5 maintains long-term repopulating ability of HSCs ex vivo. Oncostatin M, a well known STAT5 target gene, has been postulated to be involved in the development of MPD and was actually induced STAT5A 1*6-expressing cells. However, transplantation of OSM−/− HSCs expressing STAT5A 1*6 similarly caused a lethal MPD in wild-type mice, indicating that Oncostatin is not the main target for STAT5 in MPD development. Taken together, our findings establish a role for STAT5 in the self-renewal of HSCs and provide STAT5 as novel target for therapeutic manipulation of HSCs ex vivo.

Genetic manipulation of hematopoietic stem cells

Over the past two decades, the ability to transfer genes into hematopoietic stem cells (HSCs) has provided new insights into the behavior of individual stem cells and offered a novel approach for the treatment of various inherited or acquired disorders. At present, gene transfer into HSCs has been achieved mainly using modified retroviruses. While retrovirus-based vectors could efficiently transduce murine HSCs, extrapolation of these methods to large mammals and human clinical trials resulted in very low numbers of gene-marked engrafted cells. In addition, in vitro progenitor assays used to optimize gene transfer procedures were found to poorly predict the outcome of stem cell gene transfer. The focus rapidly turned to the development of superior and more relevant preclinical assays in human stem cell gene transfer research. Xenogeneic transplant models and large animal transplantation system have been invaluable. The development of better assays for evaluating human gene therapy protocols and a better understanding of stem cell and vector biology has culminated over the past decade in multiple strategies to improve gene transfer efficiency into HSCs. Improved gene transfer vectors, optimization of cytokine combination, and incorporation of a recombinant fragment of fibronectin during transduction are examples of novel successful additions to the early gene transfer protocols that have contributed to the first unequivocal clinical benefits resulting from genetic manipulation of HSC.

Identification of hematopoietic stem/progenitor cells: strength and drawbacks of functional assays.

A major challenge in hematopoiesis is to conceive assays that could bring useful insights into experimental and clinical hematology. This means identifying separately the various classes of hematopoietic progenitors that are produced sequentially during the progression from stem cells to differentiated functional cells. Standardized short-term colony assays easily quantify lineage-committed myeloid precursors, but identification of primitive cells, which have both the ability to repopulate durably myeloid and lymphoid lineages and perhaps to self-renew, still depends on in vivo assays. Whatever the assay, two important requisites have to be solved: one is the definition of appropriate read-outs that will depend solely on the function of these cells, and the second is to evaluate precisely their numbers and proliferative potential in quantitative assays. When evaluating hematopoiesis, three parameters have to be taken into account: (1) the lack of reliable correlation between the phenotype of a given cell and its function. This is especially problematic in post-transplantation situations where cells from transplanted animals are analysed; (2) functionally heterogeneous cells are identified in a single assay; and (3) ontogeny-related changes in hematopoietic cell proliferation and self-renewal that, in human beings, hampers the exploration of adult stem cells. Nevertheless, years of progress in the manipulation of hematopoietic stem cells have recently resulted in the purification of a cell subset that repopulates irradiated recipients with absolute efficiency.

185. Systemic Protein Delivery by Muscle-Directed Gene Transfer Is Limited by a Local Immune Response

Hemophilia A is an inherited bleeding disorder caused by the lack of functional coagulation factor VIII (fVIII). Although fVIII protein replacement therapy is effective, it is inconvenient, very expensive, and has disadvantages such as unpredictable product shortage and possible transmission of viral blood-borne diseases in the case of plasma-derived product. Considering these limitations, a gene-based treatment for hemophilia A with the potential to cure the disease is very attractive. Hemophilia A is an excellent candidate for gene therapy because it is a monogenic disorder, modest elevation of fVIII levels to 1.5–2% of normal is sufficient to significantly improve the clinical symptoms, and tissue-specific expression is not required. Although much progress has been made in hemophilia A gene therapy, major hurdles that remain are low frequencies of transgene delivery, suboptimal expression of fVIII mRNA and inefficient secretion of fVIII protein, plus the development of inhibitory antibodies to fVIII neoantigen. We have taken several approaches to overcome these difficulties: use of an oncoretroviral vector backbone containing strong transcriptional control elements such as the MSCV LTR; inclusion of introns for more efficient processing and accumulation of fVIII mRNA; construction of a secretion-enhanced variant of fVIII (sfVIII) and use of hematopoietic stem cells (HSCs) as a target population. HSCs are a promising target cell population because they allow long-term expression of an integrating transgene from circulating blood cells. More importantly, their use as the target population may prevent inhibitor formation to recombinant fVIII by inducing immune tolerance during lymphoid reconstitution. To evaluate the potential of HSCs for hemophilia A gene therapy, primary bone marrow cells from immunocompetent (E16; fVIII exon 16 disrupted) and immunocompromised (E12; fVIII exon 16 and B7-2 disrupted) hemophilic mice were transduced with an optimized MSCV-based splice-gag oncoretroviral vector containing an sfVIII-IRES-EGFP cassette. Transplantation of 2–3.5 106 EGFP-expressing bone marrow cells into sublethally irradiated syngeneic hemophilic recipients resulted in persistant expression of EGFP in peripheral blood cells for at least 21 weeks. Therapeutic levels of sfVIII (2 to 101 ng/ml) corresponding to the percentage of peripheral blood EGFP-expressing cells were detected in the plasma of all E12 recipients and led to correction of their hemophilic phenotype as observed by survival of tail-clipping. Transplantation of bone marrow cells obtained from sfVIII-expressing E12 mice (wk 15 post-transplant) into sublethally irradiated secondary E12 recipients has indicated that hematopoietic stem cells are the target population responsible for maintenance of sfVIII production in vivo. Ongoing studies with immunocompetent E16 mice are encouraging, with 30 12 ng/ml of sfVIII detected in the best example for up to 20 wks. Further investigations are focused on strategies to reproducibly induce immune tolerance to curative levels of fVIII neoantigen.

916. Systematic Analysis of Retroviral Vector Integrations in Human Marrow-Repopulating Cells by the Aid of a Data Warehouse

Increasing use of hematopoietic stem cells for retroviral vector-mediated gene therapy and recent reports on insertional mutagenesis in mice and humans have created intense interest to characterize vector integrations on the genomic level. We have studied retroviral integration sites in the genome of human marrow-repopulating cells. Therefore, we transduced peripheral blood progenitor cells (PBPC) from 5 healthy donors with retroviral supernatant of the SF-vector (based on the Friend mink cell-forming/murine embryonic stem-cell virus; transduction efficiency: 7%–41%) and transplanted them into immunodeficient NOD/SCID mice. After 8 weeks of engraftment chimeric mouse bone marrow DNA was analyzed with ligation-mediated PCR (LM-PCR) followed by blast analysis.

Review article: a medicine based on cell transplantation -- is there a future for treating liver diseases?

Cell-based therapies, comprising isolated hepatocyte transplantation, ex vivo gene therapy and bioartificial liver devices, may actually design a new scenario in the treatment of patients with liver failure. Recent advances in the liver repopulation field and the considerable excitement surrounding the use of haematopoietic stem cells for a 'regenerative medicine', allow us to see under a new light the alternative cell sources actually under investigation. In particular, the major drawbacks and the most important advantages of xenogenic primary cells, tumour-derived cell lines, immortalised hepatocytes and stem cells in the wide range of experimental and clinical applications are presented. Even if up to now none of them represent a 'gold standard' in the clinical practice, the intersecting advances in the development of mechanical components of artificial devices and in the liver biology and bioengineering will open tantalising possibilities to treat patients with liver failure, by tailoring the therapeutic choice on the basis of the aetiology and the stage of liver disease.

Donor-derived hematopoietic stem cells in organ transplantation: technical aspects and hurdles yet to be cleared.

The use of hematopoietic stem-cell (HSC) therapy in organ transplantation is a challenge to promote chimerism with the aim of enhancing organ tolerance. Several HSC sources are available, including bone marrow (most of the time), peripheral blood after stem-cell mobilization, and placental blood. HSC collection techniques from vertebral bodies or iliac crests require a number of complex manipulations. The best yield of HSC is obtained from vertebral bodies. HSC harvesting by cytapheresis after cell mobilization with a cytokine such as granulocyte colony-stimulating factor should be preferred with a live donor. The number of CD3+ T cells is more than 10-fold higher in peripheral blood than in bone marrow. Cell separation by the immunoselection technique (positive selection of the CD34+ cell population) eliminates erythrocytes, granulocytes, and T cells, thus preventing the possible occurrence of acute graft-versus-host disease. In the future, an accreditation will be required for HSC collection and processing. In Europe, the reference tool is the Joint Accreditation Committee of Ishage-Europe or the Foundation for the Accreditation of Haematopoietic Cell Therapy manual, which provides standards for every technical step of these procedures.

Retroviral vector integration occurs in preferred genomic targets of human bone marrow–repopulating cells

Increasing use of hematopoietic stem cells for retroviral vector-mediated gene therapy and recent reports on insertional mutagenesis in mice and humans have created intense interest to characterize vector integrations on a genomic level. We studied retrovirally transduced human peripheral blood progenitor cells with bone marrow-repopulating ability in immune-deficient mice. By using a highly sensitive and specific ligation-mediated polymerase chain reaction (PCR) followed by sequencing of vector integration sites, we found a multitude of simultaneously active human stem cell clones 8 weeks after transplantation. Vector integrations occurred with significantly increased frequency into chromosomes 17 and 19 and into specific regions of chromosomes 6, 13, and 16, although most of the chromosomes were targeted. Preferred genomic target sites have previously only been reported for wild-type retroviruses. Our findings reveal for the first time that retroviral vector integration into human marrow-repopulating cells can be nonrandom (P =.000 37).

Development of improved factor VIII molecules and new gene transfer approaches for hemophilia A.

Hemophilia A, the most common inherited bleeding disorder, is caused by deficiency or functional defects in coagulation factor VIII (fVIII). Conventional treatment for this disease involves intravenous infusions of plasma-derived or recombinant fVIII products. Although replacement therapy effectively stops the bleeding episodes, it has a risk of transmission of viral blood-borne diseases and development of neutralizing antibodies that inactivate the administered fVIII protein. Hemophilia A is an attractive candidate for application of gene therapy approaches because the therapeutic window is wide and even modest elevation of fVIII levels will correct the hemophilic phenotype. Ongoing preclinical investigations utilize animal models of hemophilia A, including genetically fVIII-deficient mice and naturally fVIII-deficient dogs, to optimize vectors, transgenes and target cell populations for Phase I clinical trials. In this review, we outline the progress in understanding the mechanisms of fVIII turnover, which provides a basis for development of improved fVIII molecules with prolonged half-life in the circulation. We discuss the possibility of incorporating these improved fVIII molecules as transgenes into self-inactivating lentiviral vectors carrying chromatin insulator sequences, representing a new generation of gene delivery vehicle, to target hematopoietic stem cells and endothelial cells. The use of hematopoietic stem cells as the target cell population may prevent inhibitor formation to transduced fVIII by induction of immune tolerance. Alternatively, endothelial cells may support optimal synthesis of fVIII and myeloablative conditioning of patients with radiation or chemotherapy may not be required for efficient engraftment of the engineered cells. Collectively, these proposed advances represent promising prophylactic strategies toward long-term correction of the coagulation defect in this progressively debilitating, life-threatening disease.

Allogeneic hematopoietic stem cell transplantation--yesterday, today, and tomorrow.

During the past 50 years, intensive studies into the use of hematopoietic stem cell transplantation (HSCT) for therapy of cancer and nonmalignant hematologic diseases have changed this treatment modality from one that was thought to be plagued by insurmountable complications to one that is now standard therapy for some diseases. More-recent research has opened up the way to include elderly patients with a wide variety of hematologic malignant diseases. Continued research by transplant teams worldwide is likely to allow continued progress toward developing novel and improved treatment modalities and even wider application of the use of pluripotent hematopoietic stem cells in the treatment of human diseases.

New concepts about graft-versus-host and graft-versus-leukaemia-reactions. A summary of the 5th International Symposium held in Munich, 21 and 22 March 2002.

The Fifth International Symposium on Graft-versus-Host and Graft-versus-Leukemia Reactions was held on 21 and 22 March 2002 in the University Hospital (Klinikum Grosshadern) of the University of Munich. As in previous years, it was dedicated to the encounter of scientists and clinicians involved in hematopoietic cell transplantation. This year's symposium focused on the characterization of stem cells potentially expanding the use of hematopoietic stem cells and on gene therapy. The immunology section dealt with mechanisms of tolerance, and the characterization of minor histocompatibility antigens presented by major histocompatibility molecules. Further important topics were cytokines and dendritic cells. In 1 and 1/2 days of intense work, the invited speakers, chairmen, authors and an active audience experienced an exciting exchange of ideas and collaboration. Again, new impulses were given for basic research and clinical transplantation. The authors would like to express their deep appreciation and thanks to all participants of this symposium.

Adult versus embryonic stem cells: it's still a tie.

The field of gene therapy has always had a special interest in stem cell biology because the ex vivo approach to gene transfer involved the genetic manipulation of cells followed by retransplantation [1]. Much of the early work in this area was specifically directed at genetic manipulation of hematopoietic stem cells (HSCs) and the first clinical success in our field has come from this approach [2]. Thus, for many investigators in gene therapy, research required knowledge in transplantation biology, as well as an understanding of the gene transfer technology itself.

Arylsulfatase-A in umbilical cord blood: gestational age and mode of delivery do not influence enzyme activity.

The possibility of using umbilical cord blood for transplantation in several enzyme deficiencies has received increasing attention because of the availability of cord blood, the reduced incidence of post-transplantation complications, such as graft-versus-host disease and the possible accomplishment of good corrective results following transplantation, even in cases of greater HLA disparity. The use of hematopoietic stem cells from unrelated donors is even more highly recommended for the treatment of inherited enzyme deficiencies, because it might reduce the risk of the transplanted cells originating from a carrier of the defect, which might have an inadequate corrective ability. Our study was designed to elucidate whether the gestational age and mode of delivery influences the arylsulfatase-A activity in the umbilical cord blood. Enzyme activities proved to be similar in the four populations studied (full-term normal spontaneous vaginal delivery, full-term caesarean section, preterm normal spontaneous vaginal delivery and preterm caesarean section). Therefore, umbilical cord blood samples seem to be suitable for transplantation in metachromatic leukodystrophy, regardless of gestational age and mode of delivery. Moreover, our results are the first published data on normal values for arylsulfatase-A activity in human umbilical cord blood.

Role of STAT3 for hematopoietic stem cells

Self-renewal of hematopoietic stem cells (HSCs) is a major factor determining the yield of ex-vivo manipulation of HSC or regeneration of bone marrow after transplantation. Despite the many interests, molecular mechanisms regulating the self-renewing process of HSC remains largely uncertain. Based upon previous finding that activating gp-130 signal enhance self-renewal of HSC, we speculated that STAT3, one of down stream molecule of gp-130, could be a effector molecule. In the expression analysis, higher level of STAT3 transcripts were detected in primitive human bone marrow cells (CD34+CD38−) cells compared to their in-vitro differentiated products or uncultured CD34+CD38+ cells. Similar quantitative difference was also observed in protein level of STAT3 in 5-FU treated murine bone marrow cells. To test the effect of STAT3 activity in various stages of hematopoiesis, murine fetal liver cells (TER119 depleted) were transduced with retrovirus (MSCV-IRES-GFP) encoding either wild type (wt) or dominant negative form (dn) of STAT3. Profound suppression of lympho-myeloid repopulation was observed in the cells transduced with do form of STAT3, but no changes in further down stage of hematopoietic activities as defined by spleen colony formation (CFU-S) or in-vitro colony formation (CFC). Similar results were observed murine bone marrow cells stimulated with 5-FU, suggesting that HSCs of more broad spectrum requires STAT3 activity for their repopulating activities. In contrast, overexpression of wild type STAT3 did not affect any of those hematopoietic activities. However, when similar study was performed using another mutant form of STAT3 (STAT3-C) that can dimerize without being phosphorylated, significant increases in the repopulating activities of transduced cells were observed. Our study suggests that the STAT3 plays a direct regulatory role for gp-130 mediated induction of self-renewal in HSCs.

Hematopoietic stem cells

The adequate production of blood cells is maintained by a set of immature hematopoietic stem cells (HSC) located in the bone marrow after birth. HSC are able to reconstitute the hematopoietic system in disease-related bone marrow failure and bone marrow aplasia. Nowadays, HSC cells can be mobilized from the bone marrow into the peripheral blood using hematopoietic cytokines, allowing a convenient harvest of these cells for clinical transplantation. This review outlines the development of the hematopoietic system in the embryo and in adults and the characterization, enumeration, purification and ex vivo expansion of HSC for clinical use. Future directions include the genetic manipulation of HSC and the identification/expansion of bone marrow-derived stem cells capable of generating non-hematopoietic tissues.

Liver transplantation of hepatic stem cells: potential use for treating liver diseases.

Hepatic stem cells are an alternative means for repopulating the liver after various injuries instead of liver transplantation. The first step before use is to select stem cells that will be good candidates. This review discusses the different candidates including fetal progenitor bipotential hepatic stem cells; adult hepatocytes, which can be considered as unipotential committed stem cells; and oval cells, a type of nonparenchymal pluripotential hepatic stem cell. The advantages and disadvantages of each type of cell are discussed and several other possible alternatives, such as the use of hematopoietic stem cells are analyzed.

A ligand-receptor signaling threshold model of stem cell differentiation control: a biologically conserved mechanism applicable to hematopoiesis

A major limitation to the widespread use of hematopoietic stem cells (HSC) is the relatively crude level of our knowledge of how to maintain these cells in vitro without loss of the long-term multilineage growth and differentiation properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of HSC stimulation by exogenous cytokines would thus be useful. An emerging theme from recent HSC expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by extracellular matrix- or cell-bound cytokine presentation. According to such a model, when the relevant ligand-receptor interaction falls below a critical level, the probability of a differentiation response is increased; otherwise, self-renewal is favored. Thus, in addition to the identity of a particular receptor-ligand interaction being important to the regulation of stem cell responses, the quantitative nature of this interaction, as well as the dynamics of receptor expression, internalization, and signaling, may have a significant influence on stem cell fate decisions. This review uses examples from hematopoiesis and other tissue systems to examine existing evidence for a role of receptor activation thresholds in regulating hematopoietic stem cell self-renewal versus differentiation events.

Docetaxel-induced mobilization of hematopoietic stem cells in a murine model: Kinetics, dose titration, and toxicity

Objective Docetaxel (DXT) is an anticancer agent that has demonstrated therapeutic efficacy against solid tumors, particularly breast cancer. Based on the use of hematopoietic stem cell (HSC) transplantation to restore hematopoietic reconstitution after myeloablative therapy, this study was performed to determine if DXT could mobilize HSCs in vivo. Materials and Methods C57Bl/6 mice were injected intraperitoneally with varying doses of DXT (equivalent to human doses of 40 to 120 mg/m 2). Spleens were harvested on days 2, 4, 6, 8, 10, and 12 after DXT administration for recovery of mononuclear cells (MNCs). The number of HSCs present within the MNCs was determined by clonogenic assay for colony-forming units in culture (CFU-C) and by FACS analysis for CD34 + cells. Peripheral blood samples were obtained at the time of spleen harvest to determine the hematologic profile. Liver and renal function tests were performed to monitor toxicity. Results DXT mobilized HSCs in a dose- and time-dependent manner. When measured by the CFU-C assay, maximal mobilization of HSC (>10-fold increase over control; p < 0.01) was observed at a dose of 30 mg/kg (equivalent to human dose of 75 mg/m 2) on day 7. The number of mobilized HSCs peaked on days 6 to 8 at all doses of DXT tested. There was no evidence of weight loss, liver, or renal toxicity at any of the DXT doses tested. Conclusion These results indicate that DXT efficiently mobilizes HSCs in a murine model and provide the rationale for similar studies in a clinical trial.

Expansion of human cord blood CD34+CD38−cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function

Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin− CD34+CD38−cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin−CD34+ CD38− cells and SRC, CD34+–enriched lineage–depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both CD34+CD38− cells (20.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP+ human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34+CD38− phenotype (n = 20). However, there was no increase in SRC numbers, indicating dissociation between the CD34+CD38− phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34+CD38+ cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. 2000;95:102-110)

Evidence of alloreactive T lymphocytes in fetal liver: implications for fetal hematopoietic stem cell transplantation.

The use of hematopoietic stem cells for in utero transplantation to create permanent hematochimerism represents a new concept in fetal therapy, although this approach has provided heterogeneous results. In this paper we have undertaken molecular, phenotypic and functional studies aimed at identifying the presence of fully competent T lymphocytes in samples of fetal livers and cord blood. We found mature VDJ TCR beta chain transcripts in fetal liver cells taken from 7 to 16 weeks of gestation and a similar pattern was detected in cord blood cells sampled from 13.5 to 20.5 weeks of gestation. A Vbeta8 gene sequence comparable to that detected in adult PBMC was found in fetal liver samples at 9 or 17 weeks gestation. PreTalpha message was detected in all samples and its expression decreased in fetal blood samples with increasing gestational age while Calpha message appeared at 9.4 weeks and its expression increased during gestational age. T cell clones obtained from fetal liver cells showed a mature TCR alphabeta+, CD8+ phenotype and displayed strong alloreactivity against allo-MHC class I molecules. The presence of alloreactive T lymphocytes may explain the failure to engraft in fetuses older than 13 to 16 weeks and may provide insights into fetal liver transplantation. Bone Marrow Transplantation (2000) 25, 135-141.