In utero bone marrow transplantation of fetal baboons with mismatched adult baboon marrow.

In utero bone marrow transplantation to fetuses offers the potential advantage of ameliorating the effects of genetic disorders by transplanting allogeneic hematopoietic stem cells into recipients who are immunoincompetent and require no preparative regimen. Therefore, we undertook studies to examine the feasibility of in utero bone marrow transplantation of unrelated allogeneic adult bone marrow into fetal baboons. Thirty-one baboon fetuses were transplanted between the ages of 60 and 160 days gestation (normal gestation, 182 days) with unrelated allogeneic adult bone marrow containing a different isozyme of glucose-phosphate isomerase (GPI). Approximately one third of the 80-day fetuses demonstrated engraftment 1 month after transplantation. Three of three of the initial chimeras died in utero 45 to 80 days after transplantation and the remaining chimeras lost their graft. Furthermore, 80-day fetal baboons were able to recognize donor cells, maternal cells, and other adult baboon peripheral blood cells in a mixed lymphocyte culture (MLC) reaction but still could engraft with allogeneic bone marrow. In contrast all nonchimeric animals survived to term. These data suggest that fetal transplantation of primates is feasible using techniques employed in these studies and that transplantation of younger fetuses who are immunocompetent should be attempted.     

同种异体宫内移植小鼠嵌合模型的建立

干细胞宫内移植是一种很有前途的产前治疗方式。为深入研究干细胞移植后的细胞行为,采用宫内移植的方法建立同种异体的嵌合小鼠模型。将雄鼠骨髓单核细胞宫内注射到胎鼠腹腔,在受体鼠出生后检测雌性受体鼠外周血细胞。应用PCR检测外周血是否存在雄性鼠的DNA,并采用定量PCR技术确定其嵌合量;同时用荧光原位杂交（FISH）技术直观观察外周血中雄性来源的细胞。结果表明：共获得4只阳性外周血嵌合小鼠,其中3只稳定嵌合达到6个月以上。应用宫内移植成功建立了外周血中存在异源细胞的小鼠嵌合模型。     

Ethnically mismatched cord blood transplants in African Americans: the Saint Louis Cord Blood Bank experience

BACKGROUND: For ethnic minority patients where a suitably matched BM or peripheral blood donor is frequently unavailable, cord blood offers an opportunity for hematopoietic stem cell transplantation. Focused recruitment of ethnic minorities for cord blood donation has been proposed as the preferred strategy to improve access for minority recipients to cord blood for transplantation. The aim of this study was to evaluate cord blood characteristics for Caucasian and African American donors and the success of ethnically mismatched UC blood transplantation in African American recipients. METHODS: Retrospective data analysis was performed comparing the characteristics of 556 cord blood units from African American and Caucasian donors. The outcomes of 18 African American ethnically mismatched transplant recipients were compared with a paired sample of 18 ethnically matched Caucasian recipients. RESULTS: The fraction of collected units meeting acceptability criteria from African Americans was significantly lower compared with Caucasians (P = <0.0001). Additionally, African Americans had a significantly lower post-processing total nucleated cell count (TNC) compared with Caucasians (P=0.007) but there were no other significant differences in conventionally measured product characteristics. In the transplant analysis, there was no difference in overall survival at 1 year (P=0.85) or time to neutrophil engraftment (P=0.92) between the two patient populations. DISCUSSION: At comparable levels of TNC dose and HLA matching, the use of ethnically mismatched UC blood units as a source for allogeneic unrelated transplant can result in successful transplant outcomes for African American patients.     

Long-term engraftment of p18INK4C-deficient hematopoietic stem cells is enhanced in the sublethally-irradiated recipients

Hematopoietic stem cell transplantation (HSCT) has been widely used for the treatment of hematologi-cal malignancies and congenital deficiencies. In recent years, non-myeloablative and reduced-intensity condi-tioning regimens have significantly expanded t…     

The use of CD 34(+) mobilized peripheral blood as a donor cell source does not improve chimerism after in utero hematopoietic stem cell transplantation in non-human primates

In utero hematopoietic stem cell transplantation is a therapeutic procedure that could potentially cure many developmental diseases affecting the immune and hematopoietic systems. In most clinical and experimental settings of fetal hematopoietic transplantation the level of donor cell engraftment has been low, suggesting that even in the fetus there are significant barriers to donor cell engraftment. In postnatal hematopoietic transplantation donor cells obtained from mobilized peripheral blood engraft more rapidly than cells derived from marrow. We tested the hypothesis that use of donor hematopoietic/stem cells obtained from mobilized peripheral blood would improve engraftment and the level of chimerism after in utero transplantation in non-human primates. Despite the potential competitive advantage from the use of CD 34(+) from mobilized peripheral blood, the level of chimerism was not appreciably different from a group of animals receiving marrow-derived CD 34(+) donor cells. Based on these results, it is unlikely that this single change in cell source will influence the clinical outcome of fetal hematopoietic transplantation.     

Hematopoietic chimerism in sheep and nonhuman primates by in utero transplantation of fetal hematopoietic stem cells.

Bone marrow transplantation to reconstitute defective hematopoietic cell lines in children with congenital defects is limited by donor availability, graft rejection, and graft-versus-host disease (GVHD). These problems can be eliminated by transplanting normal preimmune fetal hematopoietic stem cells (HSC) into an unrelated preimmune fetal recipient. We show here that injections of allogeneic fetal stem cells into preimmune fetal lambs and monkeys result in long-term stable hematopoietic chimerism. HSCs harvested from the livers of preimmune fetal sheep and monkeys when injected into the peritoneal cavity of young unrelated fetal sheep and monkey recipients results in stable, long-term postnatal hematopoietic chimerism involving lymphoid, erythroid, and myeloid cells of donor origin. Donor cell engraftment was achieved without the use of cytoablative procedures and without the development of GVHD.     

Long-term engraftment of p18 INK4C-deficient hematopoietic stem cells is enhanced in the sublethally-irradiated recipients

Non-myeloablative regimens for host conditioning have been widely used in clinical hematopoietic stem cell transplantation due to their reduced toxicity on the recipients. But a milder conditioning regimen may require a higher engrafting ability of donor stem cells in competing with endogenous stem cells. Thus, new strategies for enhancing the competitiveness of donor stem cells in non-myeloablative recipients would have important implications for current clinical stem cell transplantation. It is known that the absence of p18 ^INK4C (p18) gene can enhance the self-renewal potential of hematopoietic stem cells (HSCs). We applied the approach of competitive bone marrow transplantation to evaluate the impact of p18 gene deletion on long-term engraftment of HSCs in sublethally irradiated hosts. We found that p18 ^−/− HSCs had a significant advantage over wild-type HSCs during long-term engraftment in the mouse recipients that received a sub-lethal irradiation (5-Gy). The engraftment efficiency of p18 ^−/− HSCs in the sub-lethally irradiated recipients was similar to that in the lethally irradiated (10-Gy) recipients. Our current study demonstrates that enhanced engraftment of donor HSCs in the absence of p18 does not strictly depend on the dose of irradiation used for host conditioning. Therefore, p18 might serve as a potential drug target for increasing the efficacy of stem cell transplant in the patients that are preconditioned with either a myeloablative or non-myeloablative regimen.     

Long-term engraftment of p18INK4C-deficient hematopoietic stem cells is enhanced in the sublethally-irradiated recipients

Non-myeloablative regimens for host conditioning have been widely used in clinical hematopoietic stem cell transplantation due to their reduced toxicity on the recipients. But a milder conditioning regimen may require a higher engrafting ability of donor stem cells in competing with endogenous stem cells. Thus, new strategies for enhancing the competitiveness of donor stem cells in non-myeloablative recipients would have important implications for current clinical stem cell transplantation. It is known that the absence of p18 ^INK4C (p18) gene can enhance the self-renewal potential of hematopoietic stem cells (HSCs). We applied the approach of competitive bone marrow transplantation to evaluate the impact of p18 gene deletion on long-term engraftment of HSCs in sublethally irradiated hosts. We found that p18 ^−/− HSCs had a significant advantage over wild-type HSCs during long-term engraftment in the mouse recipients that received a sub-lethal irradiation (5-Gy). The engraftment efficiency of p18 ^−/− HSCs in the sub-lethally irradiated recipients was similar to that in the lethally irradiated (10-Gy) recipients. Our current study demonstrates that enhanced engraftment of donor HSCs in the absence of p18 does not strictly depend on the dose of irradiation used for host conditioning. Therefore, p18 might serve as a potential drug target for increasing the efficacy of stem cell transplant in the patients that are preconditioned with either a myeloablative or non-myeloablative regimen.     

Totipotent hematopoietic stem cells: normal self-renewal and differentiation after transplantation between mouse fetuses

Successful engraftment of mouse fetal liver cells in early fetal recipients, after microinjection via the placental circulation, is attributable to seeding of the recipient's liver by a cell type that is ancestral to both the myeloid and lymphoid definitive lineages and is capable of sustained self-renewal and differentiation for more than 2 years. This primitive cell is therefore the normal totipotent hematopoietic stem cell (THSC). The use of a large series of mutant anemic recipients with decreasing severity of an endogenous stem-cell defect (W/W, Wv/Wv, Wf/Wf, Wv/+), and therefore of graded selective advantage to normal donor cells, has revealed that engraftment entails marginal numbers of cells--probably individual ones--in the least afflicted hosts. Thus the observed progressive and coordinate shift toward donor-strain erythrocytes, granulocytes and B and T lymphocytes, over time, indicates THSC expansion to form a larger stem-cell pool and normally regulated differentiation of cells from the pool. This transplant system allows allogeneic combinations with impunity and therefore provides many novel experimental possibilities for investigating THSC normal development, genetic abnormalities or neoplastic potential in relation to the intact developmental succession of hematopoietic tissue environments in vivo.     

Hematopoietic stem cell transplantation for thalassemia

Hematopoietic stem cell transplantation (HSCT) represents the only cure for patients with thalassemia. At present HSCT in younger patients from an HLA- matched sibling donor offers 80% to 87% probability of cure according to risk classes. However, results HSCT in adult patients continue to be inferior due to advanced of disease. High-resolution tissue typing techniques have enabled transplant centres to offer allogeneic HSCT from unrelated donors to patients with thalassemia who could not benefit from matched sibling donor transplantation with results comparable to those obtained using sibling donors. Advances in transplantation biology have made it possible to perform haploidentical HSCT in patients with thalassemia who lack a related or unrelated matched donor. Although limited number of patients, results of unrelated cord blood transplantation for thalassemia are encouraging. Patients with graft failure could now benefit from second transplantation using the same donor with a high disease-free survival rate. Most ex-thalassemics continue to have disease and treatment-related complications acquired before transplantation which require adequate treatment following BMT.     

Recent advances in bone marrow transplantation in hemoglobinopathies

Allogeneic hematopoietic cell transplantation (HCT) is currently the only treatment with curative potential for sickle cell disease (SCD) and beta-thalassemia. HCT was first used to treat SCD and thalassemia more than two decades ago, and with increasing experience this treatment modality has shifted from being an experimental intervention to one in which selected patient populations are targeted for treatment. Recent multicenter clinical studies show an event-free survival (EFS) of 85% after human leukocyte antigen (HLA)-identical sibling transplantation for SCD, using conventional myeloablative conditioning with a backbone of busulfan (BU) and cyclophosphamide (CY) [1-3]. Results of HCT for thalassemia show very similar outcomes, with EFS probabilities that range from 81%-87% [4,5]. However, the risk of graft failure, recurrent disease, graft-versus-host-disease (GVHD), infections, and long-term sequelae of chronic GVHD and endocrinopathies related to Fe overload and myeloablative BU limit broader application of this therapy. Non-myeloablative conditioning regimens may offer a lower risk of toxicity, and investigations to identify a regimen that is sufficiently immunosuppressive to ensure stable engraftment of donor cells are ongoing. Alternative sources of donor hematopoietic cells that include HLA-matched unrelated donor (URD) and umbilical cord blood (UCB), are being pursued for hemoglobinopathies, with promising initial results. This review discusses the successes, challenges and future direction of HCT for SCD and thalassemia.     

Selective Enhancement of Donor Hematopoietic Cell Engraftment by the CXCR4 Antagonist AMD3100 in a Mouse Transplantation Model

The interaction between stromal cell-derived factor-1 (SDF-1) with CXCR4 chemokine receptors plays an important role in hematopoiesis following hematopoietic stem cell transplantation. We examined the efficacy of post transplant administration of a specific CXCR4 antagonist (AMD3100) in improving animal survival and in enhancing donor hematopoietic cell engraftment using a congeneic mouse transplantation model. AMD3100 was administered subcutaneously at 5 mg/kg body weight 3 times a week beginning at day +2 post-transplant. Post-transplant administration of AMD3100 significantly improves animal survival. AMD3100 reduces pro-inflammatory cytokine/chemokine production. Furthermore, post transplant administration of AMD3100 selectively enhances donor cell engraftment and promotes recovery of all donor cell lineages (myeloid cells, T and B lymphocytes, erythrocytes and platelets). This enhancement results from a combined effect of increased marrow niche availability and greater cell division induced by AMD3100. Our studies shed new lights into the biological roles of SDF-1/CXCR4 interaction in hematopoietic stem cell engraftment following transplantation and in transplant-related mortality. Our results indicate that AMD3100 provides a novel approach for enhancing hematological recovery following transplantation, and will likely benefit patients undergoing transplantation.     

New approach to organ transplantation based on the fetal allograft.

A new approach to organ transplantation that may be particularly applicable to kidney transplantation is suggested by analogy with the immunological mechanism responsible for the survival of the fetal allograft. The method concerns identifying donor-recipient tissue compatibility by use of the two-way mixed lymphocyte reaction (MLR), in which reacting cells from patients awaiting transplants are primed with phytohaemagglutinin (PHA) and stored. When a donor becomes available, these PHA-primed cells may then be tested against donor lymphocytes, possibly giving a result within 36 hours. Immunosuppressive agents occurring naturally in pregnancy, such as alpha-fetoprotein and chorionic gonadotrophin, may eventually replace standard immunosuppressive treatment with potentially toxic regimens in transplant recipients. If the results of the two-way MLR using PHA-primed cells are shown to be comparable to those of the standard two-way MLR graft survival may be successful in 80% of cases.     

Hematopoietic stem cell transplantation from alternative donors for high-risk acute leukemia: the haploidentical option

Much progress has been made in the clinical, biological and technical aspects of the T-cell-depleted full-haplotype mismatched transplants for acute leukemia. Our experience demonstrates that infusing a megadose of extensively T-cell-depleted hematopoietic peripheral blood stem cells after an immuno-myeloablative conditioning regimen in acute leukemia patients ensures sustained engraftment with minimal graft-vs-host disease (GvHD) without the need of any post-transplant immunosuppressive treatment. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in more than 200 high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype mismatched donor, who is immediately available, a T-cell depleted mismatched transplant should be offered, not as a last resort, but as a viable option to high risk acute leukemia patients who do not have, or cannot find, a matched donor.     

Cardiac injury protection from mouse bone marrow stromal cells with in utero transplantation followed by secondary postnatal boost

Limited donor-cell engraftment to the injured tissue restricts therapeutic efficacy of stem cell transplantation. Herein, we proposed an alternative strategy by using in utero transplantation (IUT) to create mixed-chimerism environment in recipients and to facilitate donor-cell engraftment followed by postnatal secondary boost with the same cells. Mouse bone marrow stromal cells (BMSCs) were used as the xenogenic donor cells and given into rat fetus as an early exposure of IUT treatment. The engraftment potential was analyzed for the presence of BMSCs by flow cytometry or PCR in recipient tissues. The function of a second boost of mouse BMSCs, in terms of cardioprotection, was tested by given 1×10? cells to rat IUT hearts with ischemia/reperfusion (IR) injury that was induced by a 45 min of left coronary ligation and released for 72 h. Mouse BMSCs demonstrated an immunosuppressive effect when mixed with mouse or rat lymphocytes. IUT treatment only caused few BMSCs engrafted to fetal (embryonic day 20) and adult (4 weeks after birth) rat organs including heart, but engraftment was increased in hearts of the IUT rats after second boost. This was coincided with attenuation of cardiac injury caused by IR. Interestingly, an up-regulation of CXC chemokine receptor type 4 (CXCR4) was seen when BMSCs were exposed to hypoxia. This indicates that enhanced engraftment of mouse BMSCs to post-ischemic rat hearts possibly is dependent on CXCR4. Moreover, results of flow cytometry demonstrated that the presence of CD34? cells in rat IUT hearts with IR injury was increased. These observations suggest that enhanced engraftment of donor BMSCs to rat IR hearts by CXCR4 may recruit endogenous CD34? cells of recipients which in turn protects heart against IR. This also supports the notion of fetal preconditioning with BMSC enhances the efficiency of progenitor cell-mediated organ protection after a postnatal second boost in xeno-transplantation.     

Successful bone marrow transplantation from an unrelated donor in a child with AML in second remission

In February 1986 we transplanted a 10-year-old girl with AML in second remission with the bone marrow of an unrelated donor. HLA-types were different for one A- and one B-antigen between patient and donor. Conditioning regimen consisted of 14 Gy total body irradiation with lung shielding, 8 X 3 g/m2 cytosin arabinoside and 90 mg/kg cyclophosphamide. GVHD-prophylaxis was performed with cyclosporin A, methotrexate and prednisolone. Only mild GVHD I of the skin could be observed after rapid engraftment. 100 days after transplantation the patient was in good clinical condition and GVHD-prophylaxis was discontinued without any reactivation of acute or chronic GVHD. Engraftment was documented by sex chromosome and blood group typing. 120 days after transplantation leukemic blasts were detected in the peripheral blood and the child died 130 days after BMT from relapse of the leukemia. Despite the negative outcome, this was the first successful bone marrow transplantation from a unrelated donor in Germany.     

A novel HLA-A*3303-restricted minor histocompatibility antigen encoded by an unconventional open reading frame of human TMSB4Y gene

Female-to-male hemopoietic stem cell transplantation (HSCT) elicits T cell responses against male-specific minor histocompatibility (H-Y) Ags encoded by the Y chromosome. All previously identified H-Y Ags are encoded by conventional open reading frames, but we report in this study the identification of a novel H-Y Ag encoded in the 5'-untranslated region of the TMSB4Y gene. An HLA-A*3303-restricted CD8(+) CTL clone was isolated from a male patient after an HSCT from his HLA-identical sister. Using a panel of cell lines carrying Y chromosome terminal deletions, a narrow region controlling the susceptibility of these target cells to CTL recognition was localized. Minigene transfection and epitope reconstitution assays identified an 11-mer peptide, EVLLRPGLHFR, designated TMSB4Y/A33, whose first amino acid was located 405 bp upstream of the TMSB4Y initiation codon. Analysis of the precursor frequency of CTL specific for recipient minor histocompatibility Ags in post-HSCT peripheral blood T cells revealed that a significant fraction of the total donor CTL response in this patient was directed against the TMSB4Y epitope. Tetramer analysis continued to detect TMSB4Y/A33-specific CD8(+) T cells at least up to 700 days post-HSCT. This finding underscores the in vivo immunological relevance of minor histocompatibility Ags derived from unconventional open reading frame products.     

Cytomegalovirus Inhibits the Engraftment of Donor Bone Marrow Cells by Downregulation of Hemopoietin Gene Expression in Recipient Stroma

Cytomegalovirus (CMV) disease after bone marrow (BM) transplantation is often associated with BM graft failure. There are two possible reasons for such a correlation. First, a poor hematopoietic reconstitution of unrelated etiology could promote the progression of CMV infection by the lack of immune control. Alternatively, CMV infection could interfere with the engraftment of donor BM cells in recipient BM stroma. Evidence for a causative role of CMV in BM aplasia came from studies in long-term BM cultures and from the murine in vivo model of CMV-induced aplastic anemia. A deficiency in the expression of essential stromal hemopoietins, such as stem cell factor (SCF), has indicated a functional insufficiency of the stromal microenvironment. It remained open to question whether CMV mediates a negative regulation of hemopoietin gene expression (the downregulation model) or whether it causes the default of a positive regulator (the lack-of-induction model). Further, even though implicitly assumed, it has never been formally documented that CMV directly interferes with the engraftment of a BM cell transplant. We addressed these problems in a murine model of CMV infection after experimental male-into-female BM transplantation. The data indicate that the downregulation model applies. Quantitation of the male-sex-determining gene tdy demonstrated an impaired engraftment of donor BM cells in the BM stroma of the female recipients. This graft failure was reflected by a diminished population of SCF-receptor-expressing hematopoietic progenitor cells and correlated with a reduced level of stromal SCF gene expression. Interestingly, high doses of BM cells protected against stromal insufficiency by a mechanism unrelated to control of infection.     

Prolonged human/sheep cellular chimerism following transplantation of human hemopoietic stem cells into the ewe celomic cavity

We evaluated the possibility of prolonged chimerism formation in fetus and lamb, following human cord blood-selected CD133+ hemopoietic stem cell (HSC) transplantation into the celomic cavity of ewes at a pre-immune fetal age (44-45 days of pregnancy). Nineteen ewes were injected with HSC and 5 controls with a saline solution. By PCR, HLA-DQ alpha 1 and 6 human microsatellites (CODIS) were used for HSC traceability. FISH analysis was performed with 8 human DNA probes from different chromosomes, to confirm chromosomal integrity, nuclear DNA localization and donor DNA identification. Immunological staining for revealing HLA-DQ alpha 1 expression demonstrated multilineage engraftment. Both HLA-DQ alpha 1 and microsatellites were detected in different tissues of 3 available aborted fetuses, to a lesser extent in 11 lambs tested at 2-months, but not 12-months after birth. Although only 1 fetus of siblings of each sheep was injected, all siblings revealed positive engraftments. Microsatellite analysis showed evidence of human allele segregation in different tissues of individual fetuses and lambs. FISH analysis confirmed chimerism and the presence of human chromosomes. Non-detection of some human gene sequences in different chromosomes and random finding of allele segregation for some human heterozygous microsatellites were found in different tissues of individual animals. Controls born from un-transplanted ewes never revealed any human DNA sequences nor HLADQ alpha 1 expression.