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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.

The epidemiology and biologic characteristics of fetal tissue harvested from elective and spontaneous abortions are reviewed. The use of fetal bone marrow obtained from second trimester lost pregnancies is discussed. Allogeneic fetal tissue transplantation carried out in utero is reviewed. Data on intrauterine transplantation of human fetal bone marrow obtained from second trimester lost pregnancies into baboon fetuses are presented. The viability of this tissue, its clonogenic efficiency, engraftment, use in the future, and banking are discussed.

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

Hematopoietic stem cell transplantation has been increasingly used to replace a defective hematopoietic system and to treat various genetic defects as well as malignant diseases. However, the limitations of conventional bone marrow transplantation have stimulated an intense interest in exploring the use of alternative sources of hematopoietic stem cells, including peripheral blood mononuclear cells (PBMC) and cord blood (CB). A major investigative effort of our laboratory has been focused on evaluating fetal bone marrow (FBM) for transplantation. The current study compares and characterizes the functional and phenotypic characteristics of FBM, CB, adult bone marrow (ABM), and PBMC by clonogenicity assays, immunogenicity, and the quantification of progenitor cells. There was a striking difference in the proportion of CD34+ cells in FBM, ABM, PBMC, and CB (24.6%, 2.1%, 0.5%, and 2.0%, respectively). The clonogenic potential, as measured by colony forming unit in culture (CFU-C) assay, was significantly higher in FBM when compared with ABM, PBMC, and CB (202.5, 73.5, 40.8, and 65.5 colonies/10(5) cells, respectively). There was a significant decrease in proliferative responsiveness in mixed lymphocyte reaction (MLR) assay of FBM and CB compared with ABM and PBMC. These observations indicate that each source of hematopoietic stem cells has different intrinsic properties closely correlated with ontogenetic age that is a vital determinant for phenotypic characteristics, lineage commitments, immunogenicity, and proliferative potentials.

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.

We have previously reported that fetal bone marrow (FBM) has higher number of CD34+ cells, greater clonogenic capacity, and lowered immunologic reactivity compared to adult bone marrow (ABM), cord blood (CB), and peripheral blood lymphocytes (PBL). The present studies were designed to investigate whether similar ontogenetic differences exist in the expression of hematopoietic-related cytokines by stromal cells from FBM, ABM, and CB using RT-PCR. Stromal cells were prepared in long-term cultures supplemented with hydrocortisone to 80% confluency from specimens obtained from 8 FBM (n=8), collected from spontaneously aborted fetuses at gestational age of 18-20 weeks; 8 ABM (n=8) from allogeneic donors; 7 CB (n=7) samples. RNA was extracted from the stromal cells and reverse-transcribed into cDNA which was then subjected to PCR using specific primers for stem cell factor (SCF), GM-CSF, G-CSF, M-CSF, IL-3, IL-6, IL-10, IL-11, respectively. Quantitation of products was measured by densitometry using a Howtech Scanner. A strong expression of SCF and IL-11 was observed in the stromal cells from FBM compared to ABM and CB. Although M-CSF was not detected in any of 8 FBM specimens, it was highly expressed in all ABM and CB samples. IL-6 was detected in 6/8 ABM but in only 1/8 FBM and 0/7 CB. Although G-CSF was not observed in CB, it was expressed in all FBM and ABM specimens. In contrast, expression of GM-CSF, IL-3, and IL-10 from different sources of stromal cells was similar. These results strongly indicate that there are age-related changes in cytokine expression in stromal cells at different maturational stages. Further, these results suggest an important role for these cytokines in their capacity to regulate differentiation and proliferation of stem cells during ontogentic development, and these may provide better predictive information for treatment of hematologic diseases as well as selection of stem cells for transplantation.

Stem Cell Transplantation: Superiority of Fetal Bone Marrow Over Cord Blood and Adult Bone Marrow. • 1290

To the Editor. —The 6-month study of the suitability for transplantation 1 of fetal tissues derived from spontaneous abortions (SAbs) and ectopic pregnancies (EPs) was conceived and delivered in a very political climate. The question of the use of fetal tissue(s) from spontaneous vs elective abortions has been caught up in the political debate of pro-life vs prochoice issues, which also directly affects the scientific community. Unfortunately, the emotions and the lack of objectivity, as well as the lack of true scientific inquiry in certain quarters of the scientific community, have jeopardized the scientific integrity of the study even before it was initiated. The results reported in the aforementioned article represent data from five independent studies supported by National Institute of Child Health and Human Development and National Institutes of Health funds. The results reported in the article by Dr Branch and colleagues 1 on EPs confirmed our earlier prediction

Fetal therapy has now become a reality and many centres specializing in this field have been established in several countries. The in utero treatment of fetal hydrocephalus is bedevilled by many problems, including poor patient selection, problems with shunt design and general lack of understanding of the pathogenesis of hydrocephalus. Implantation of a ventriculo-amniotic shunt employing a one-way valve into the fetal skull allows continuous evacuation of CSF. One such model has been developed by us and should be ready for clinical trials in the near future. There is strong evidence that the fetal brain has a high capacity for rapid growth and regeneration. However, the process is poorly understood and there is great need for further study. No attempts have yet been made to treat spina bifida in utero. However, the advantages demonstrated in our earlier studies using allogeneic bone transplantation in utero resulted in the development of an allogenic bone paste. This substance can be moulded to any desired shape and is ideal for the restoration of bone loss resulting from cranial injuries or the correction of skull defects. The allogeneic bone paste has been also applied in utero for the repair of surgically induced spina bifida-like lesions in the animal model.

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.

The flexion of the cranial base and ontogenic migration of the foramen magnum in Macaca mulatta were investigated. Longitudinal cephalometric studies were carried out in infant and juvenile groups. Significant ontogenic changes of the cranial base were observed only in juvenile age group. The infant group did not exhibit any changes of the cranial base angle or migration of the foramen magnum. The diameter of the latter in infants exhibited small but statistically significant changes.

An experimental study of functional and adaptive growth changes of the gonial region associated with removal of all anterior teeth is being carried out through the life span of the Macaca mulatta monkeys. Radiographic cephalometry, using metallic implants as consistent points of reference and histological methods using in vivo tetracycline bone labeling, are the principal experimental techniques in the study. The present paper covers the results obtained from the longitudinal observations of the infant group from two to 24 months of age. The angular measurements of the gonial angle, linear measurements of the horizontal and vertical rates of growth in the gonial region were made in experimental and control groups. The data obtained from the angular measurements showed negative regression trends in both groups. The data from linear measurements of horizontal and vertical rate of growth exhibited positive linear regression trends with age. The histological findings, however, indicate differences between the two groups. Sections from the control group show some bone apposition in the gonial region, while experimental animals show extensive resorption. The results of this study show the inadequacy of standard anthropometric methods in the determination of adaptive growth changes of the gonial region at that early age.

Cross-sectional studies of the degree of the cranial base flexion were carried out in infant, juvenile and adult skulls in four genera of nonhuman primates (P. paniscus, H. lar, P. urinus, and M. mullatta). The cephalometric observations of the cranial base included linear and angular measurements of each specimen. The data obtained in this study showed that the anterior portion of the cranial base exhibits a significant shortening trend as the mammalian evolutionary scale ascends. Moreover, the growth pattern of the anterior portion of the skull base follows that of the facial bony structures. The ontogenic growth changes of the posterior portion of the skull base follows the growth pattern of the endocranial cavity. The significant trend of elongation in this area directly contributes to the posterior migration of the foramen magnum. The magnitude of these growth changes decreases as the evolutionary scale ascends. The angular measurements of the cranial flexion showed a less obtuse cranial base angle in young specimens and the ones higher on the mammalian scale. The skull kyphosis was less pronounced in these specimens and the anatomical features of the cranial base were more humanlike, including the balance of the head expressed by the position of the foramen magnum.

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.

Teratogen-induced herniation of the occipital lobes (encephalocele) in monkey fetuses was repaired by amputation and cranioplasty in utero or postnatally. The in utero-treated animals had normal vision, CT scans indicated normal brain density, and autoradiographic visualization of the striate cortex by the [14C]-2-deoxyglucose mapping revealed almost complete functional and anatomic recovery. The animals treated postnatally were blind, and exhibited significant structural deficit in the operated area.

At a time when the intrauterine diagnosis of hydrocephalus is common and pioneering efforts of antenatal therapy are evolving, an assessment of intrauterine treatment of this disorder becomes pertinent. Consequently, 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. The technical problems of antenatal shunting, the clinical trials, and experimental treatment are assessed. The prognostication and possible new approaches in intrauterine treatment of hydrocephalus are presented as well as the new diagnostic and surgical techniques. The outcome of the antenatal diagnosis and treatment of fetal hydrocephalus is evaluated.

Intrauterine allogeneic bone transplantation was performed on 25 monkey fetuses at 120 to 135 days of gestation. Two surgical techniques for orthotopic transplantation of the humeral midshaft were utilized: (1) A segment (5 to 7 mm) of fetal humerus was transplanted from fetus (donor) to fetus (recipient). An intramedullary pin (Kirschner orthopedic wire) was used for fixation of the severed humerus. (2) Without severing the humerus, a midshaft area (5 to 7 mm X 5 to 6 mm) was surgically ablated. A bone paste composed of crushed bone particles mixed with an agar-enriched culture medium was sculpted to fill this lesion. We used fresh frozen or fresh allograft tissue with equal success. Postsurgical observations included serial roentgenographic and histological evaluation of humeral osteogenesis, as well as postnatal assessment of limb use. The contralateral arm served as a control. Our findings indicate that the immune surveillance system of fetal monkeys may be tolerant of these bone allografts; alternatively, healing by substitution may also occur. The roentgenographic and histological results demonstrate that both of the transplantation procedures used here achieved restoration of the long bone. The use of bone paste allowed us to sculpt the allograft to the desired conformation. These results from laboratory primate models encourage continued investigation of fetal allogeneic bone transplantation, because of its ultimate potential for intrauterine repair of skeletal anomalies in man.

The postnatal course of a form of infantile porencephaly under pressure in monkeys was evaluated by comparing brain development between animals that had received the hydrocephalic antenatal vent for intrauterine treatment and those that had not been treated antenatally. Brain development was assessed by computed tomographic scans. The studies showed asymmetric malformations and associated porencephalic lesions in infant monkeys not treated in utero, despite installation of a conventional postnatal ventriculoperitoneal shunt. In contrast, apparently normal development of brain tissue in infants given intrauterine therapy was observed. (JAMA1984;251:2548-2552)

A spina-bifida-like condition was induced in 8 Macaca mulatta fetuses by intra-uterine lumbar laminectomy (L3-L5) followed by displacement of the spinal cord from the central canal. This condition was repaired in utero in 5 animals. A key feature of the repaired in utero in 5 animals. A key feature of the repair method was the use of allogeneic bone paste made from fetal bone particles suspended in an agar-thickened modified Dulbecco's medium. The bone paste was used to correct the bone deficit produced by the laminectomy. All of the monkey babies were delivered by cesarean section at near term (160-164 days of gestation). Neurological test on the neonates and subsequent morphological studies indicated that the in utero treated animals developed normally. In contrast, 3 control animals, which had the induced spinal dysraphism but were left untreated, showed severe spina-bifida-like abnormalities, including paraplegia, incontinence and somatosensory loss.

The contribution of the basicranial synchondroses in the growth of neurocranial length and ontogenetic development of the cranial base were investigated. The study concentrated on the midsphenoidal synchondrosis and its delayed fusion in nonhuman primates when compared to man, and on the spheno-occipital synchondrosis. The mode and time of fusion of both growth centers were observed, and their role in the ontogenetic growth changes (flattening processes) of the cranial base were established. The chondrogenic ossification of midsphenoidal and spheno-occipital synchondroses was studied on 20 skulls of Macaca mulatta females, ranging in age from newborn specimens to those 24 months old. The technique of in vivo tetracycline bone labeling was used for histologic evaluation of the material. Different chondrogenic growth patterns were observed in both synchondroses. The endochondral activity of the spheno-occipital synchondrosis increased with age, from a nonactive narrow cartilaginous column in the neonatal specimen to a broad band with high chondrogenic ossification in the 24-month-old specimens. This growth center contributes to elongation of the posterior portion of the cranial base and is a secondary factor in its flexion. The midsphenoidal synchondrosis seems to be the primary factor in the mode of flexure of the cranial base in Macaques. This growth center is very active in the first ten months of life but later exhibits cessation of chondrogenic activity and long remains unfused. The first signs of fusion were observed as late as 72 months of age. At the same time, the continuous process of cranial base flattening showed the first signs of tapering off.

We describe the antenatal diagnosis and intrauterine treatment of hydrocephalus induced (corticosteroid teratism) in fetal rhesus monkeys. Diagnostic techniques included (1) maternal serum α-fetoprotein (AFP) measurement, (2) ultrasonography of the fetal ventricular system, (3) roentgenography, and (4) fetoscopy. After measuring excessive intracranial CSF pressures in hydrocephalic fetuses, an indwelling prosthetic valve was devised—the hydrocephalic antenatal vent for intrauterine treatment (HAVIT). The HAVIT was surgically implanted in the fetal skull so that when CSF pressures exceeded 60 mm H 2 O, the ports opened to vent CSF from the fetal ventricular system into the amniotic fluid. Whereas unaided hydrocephalic neonates seldom survived more than ten to 14 days, manifesting progressive muscular weakness and frequent seizures, fetal monkeys receiving the HAVIT demonstrated markedly superior postnatal development of motor skills and weight gain. Although additional laboratory studies are indicated, ultimately, clinical investigations combining early diagnosis and in utero insertion of the HAVIT or similar prosthesis may significantly enhance the prognosis of children in whom severe antenatal hydrocephalus develops. ( JAMA 1981;246:1093-1097)