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In view of the ever-increasing demand for human stem cells for transplantation, we initiated in vitro and in vivo studies of human fetal bone marrow stem/progenitor cells derived from lost pregnancies at 16–20 weeks. Utilizing non-human primates as models, we demonstrated that fetal tissue has distinctive biological and therapeutic properties that are optimal for transplantation. Subsequently, we tested and compared the phenotypic and functional characteristics of fetal bone marrow (FBM), adult bone marrow (ABM), and cord blood (CB) and peripheral blood (PB) sources of the most primitive stem/progenitor cells. A striking ontogenic difference in the proportion of CD34+ cells in FBM, ABM, PB and CB was observed (24.6 vs. 2.1 vs. 0.5 vs. 2%). The clonogenic potential, as measured by the CFU-c assay, was also higher in FBM when compared with ABM, PB and CB (202.5 vs. 73.5 vs. 40.8 vs. 65.5 colonies/105 cells). Moreover, there was a significant decrease in proliferative responsiveness in the mixed lymphocyte reaction (MLR) assay of FBM and CB as compared to ABM and PB. The cytokinetic profiles of the cells from the four sources were also analyzed. This study revealed that both FBM and ABM had a higher proportion of S-phase (21.7 and 11.5%, respectively), compared to PB and CB cells (1.2 and 2.8%, respectively). FBM and ABM also showed a higher proportion of cells in the G2-M phase (6.4 and 2.6%, respectively) compared with PB and CB (1.7 and 1.2%, respectively). These data show that FBM has the highest number of proliferating cells. We have also investigated the ontogenic differences in stromal cells derived from FBM, ABM and CB, with a special focus on the expression of selected cytokines, such as CSF, GM-CSF, G-CSF, M-CSF, IL-3, IL-6, IL-10 and IL-11. FBM showed the highest levels of expression of CSF, IL-6 and IL-11 when compared to the other sources. These cytokines may have an important role in engraftment and homing of stem cells. The levels of expression of the other cytokines were similar in all sources of stromal cells, with the exception of G-CSF, which was not detected in CB. Moreover, the number of colonies FBM and ABM cells was higher when inoculated with fetal stromal cells. These results suggested an important regulatory role of cytokines in ontogeny of hematopoiesis. In summary, the foregoing observations indicate that each source of hematopoietic and stromal cells has different intrinsic properties, closely correlated with ontogenetic age, which is a vital determinant for phenotypic characteristics, lineage commitments, immunogenicity as well as proliferative potentials. Our data clearly indicate that FBM is the best source of stem cells for engraftment and therapeutic reconstitution due to its very high proliferative capacity, low immunogenicity and highest number of primitive stem/progenitor cells. It should also be stressed that FBM stem cells retrieved at their optimal stage of hematopoiesis (16–20 weeks) may be the cells of choice for both therapeutic cellular reconstitution and gene targeting.

A detailed review of various methods of bone marrow transplantation is presented. Special emphasis is placed on the newest reports of fetal bone marrow transplantation in utero and stem cell reconstitution.

Accessible online at: www.karger.com/fdt Paper presented at the meeting ‘In Utero Stem Cell Transplantation and Gene Therapy’ organized by the Merieux Foundation, Lyon (France), 13–14 March, 2003. The rapid advances in biological sciences have allowed a better understanding and management of various diseases. Cellular therapies, cell engineering and gene therapies may improve and/or replace many conventional treatments. Unfortunately, the progress in the achievement of those goals is frequently marred by overly enthusiastic and poorly supported claims by some of the scientists involved in this rapidly developing cutting-edge research. There are a number of areas that should be of special concern. Above all, it is crucial to protect patients involved in experimental therapies and to preserve high standards of inquiry and scientific integrity. The increasing demand for stem cells for transplantation has resulted in the exploitation of new sources of human stem cells, including embryonic stem cells. New methods of human cell cloning have opened an area of embryonic research that is associated with major ethical problems and controversies. As a result, opponents of research on human embryos have raised politically powerful arguments against embryonic stem cell research, suggesting that adult stem cells may offer the same therapeutic promise. Unfortunately, there is accumulating evidence that adult stem cells exhibit time-dependent changes, which affect long-term engraftment and curative cell reconstitution. Moreover, recent studies show that adult stem cells have limited plasticity and extensive heterogeneity. These studies also suggest that enriched but impure cell populations may lead to a misinterpretation of in vitro results. Thus, it is impossible to draw a distinction between plasticity of adult stem cells and heterogeneity of stem cell types that may pre-exist within tissue. Consequently, the great excitement concerning trans-differentiation of adult stem cells has to be tempered by the new, conflicting findings. The scientific ‘dogma’ that adult stem cells can morph into many types of cells may be based on misinterpretation of somewhat uncritical interpretation of data. What was initially claimed to be cell re-programming in vitro has currently been recognized by many as simple cell fusion with enlarged nuclei and double chromosomes, typical of hybrid cells. It is also recognized that the number of adult stem cells is limited and that the engraftment as well as reconstitution is impaired due to telomere shortening and DNA replicative exhaustion. It is clear that new discoveries continue to refine the understanding of what adult stem cells are capable of doing and more relevant animal studies are needed to establish the suitability of these cells for therapy. Embryonic stem cells have been suggested as a virtually inexhaustible source of stem cells for therapeutic uses because they can be propagated easily in culture. Unfortu

We hypothesized that maternal serum levels of the isoenzyme creatine kinase (CK)-BB, which is highly expressed in the placenta, may be elevated during the early second trimester in gestations destined to deliver prematurely or of a small-for-gestational-age infant (birthweight below 10th percentile). To test this hypothesis, we compared maternal serum CK-BB levels and percentage of CK-BB over total CK, in 69 normal pregnancies (delivering at term of appropriate-for-gestational-age infants) with those of 25 cases complicated by preterm delivery at < or = 34 weeks (n = 14), of a small-for-gestational-age infant (n = 8), or both (n = 3). No differences were present in maternal serum CK BB levels between normal and complicated pregnancies. Moreover, no correlation was found between gestational age at delivery and CK BB levels (r = 0.03; p = 0.7).

Hemopoietic and immune capacities of fetal bone marrow (FBM) obtained from 2nd-trimester lost pregnancies and adult bone marrow (ABM) were compared. Progenitor cell assays for both sources were also enumerated. Out data showed ontogeny-related functional differences between hemopoietic cells, particularly in the ability to produce CD34+ cells (24.6% in FBM, 3.1% in ABM). The phenotypic composition of FBM and ABM were quite different. The clonogenic/proliferative potentials, as measured by CFU-C assays, were significantly higher in FBM when compared to ABM (202.5 vs. 73.5/10(5) cells). Moreover, FBM had a lower percentage of CD3+ T lymphocytes as compared to ABM (1.47 vs. 7.58), and there was a significantly decreased proliferative responsiveness in mixed lymphocyte reactions of FBM as compared to ABM. Thus, our data clearly showed distinct advantages of FBM over ABM, which include a higher number of stem cells, lower immunological reactivity, and higher clonogenic/proliferative potential. These characteristics provide optimal conditions for successful engraftment without graft-versus-host disease. These data support the possible advantages of FBM from these sources for hemopoietic stem cell reconstitution and gene therapy.

The current status of the intrauterine treatment of fetal hydrocephalus is presented. The new data from the International Fetal Surgery Registry at the University of Manitoba in Winnipeg are discussed. Technical problems of antenatal shunting, clinical trials and experimental treatment are assessed. The outcome of the antenatal diagnosis and treatment of fetal hydrocephalus is evaluated.

Teratogen-induced herniation of the occipital lobes (encephalocele) associated with cranium bifidum in monkey fetuses (Macaca mulatta) were repaired by amputation and cranioplasty in utero or postnatally. Fetal allogeneic cranial bone which had been fresh frozen was used in conjunction with a bone paste to provide a protective covering of the neural tissue following repair of the encephalocele. The bone graft in those animals treated in utero or 1–2 weeks post-partum developed a strong union with the calvarium, provided good protection for the underlying dura and neural tissue, had a smooth surface, and provided good cosmetic-reconstructive results. In contrast, when the cranioplasty procedure was performed on infant monkeys at 2–3 months of age the bone graft was rejected.

While transplantation of fetal tissues may alleviate a great deal of suffering, current policy bars federal funding of research using tissue from elective abortions. Using fetal tissue from spontaneous abortions would obviate the moral concerns. If existing studies of small samples are extrapolated to the US population, then each year about 750,000 fetuses are aborted spontaneously by week 28 (500,000 in the first trimester and 250,000 in the second trimester). The consensus among researchers is that spontaneously aborted fetuses are few in number, and inappropriate for transplantation because they are born dead or have defects. This intuition may not be supported by the evidence, and there is a compelling need to focus research on these issues.

Pathogenesis and intrauterine treatment of central nervous system defects are described. Current and future developments in fetal therapies and/or surgical correction of malformations and stem cell engraftments are discussed.