Fetal hematopoietic stem cell transplantation into beta-thalassemic mice

The transplantation of fetal-derived hematopoietic stem cells (HSCs) may potentially be used to treat hemoglobinopathies, immunodeficiencies, and storage diseases. The levels of donor cell engraftment needed to reconstitute the recipient's hematopoietic system are disease-dependent and remain unknown for most deficiencies. We have explored the application of fetal hematopoietic stem cell transplants for the amelioration of hemolytic disease in a murine model of beta-thalassemia. Nonirradiated neonatal homozygous beta-thalassemic mice were transplanted intraperitoneally (IP) with 10(6) fetal liver cells from syngeneic nonthalassemic murine fetal donors (14 to 16 days gestation). Donor hemoglobin was demonstrated in the peripheral blood of 9 of 14 transplant recipients at levels ranging from 8.8% to 27.1% at 30 days. The levels of engraftment in 6 of these 9 transplant chimeras remained stable or increased up to 150 days after transplantation, with levels ranging from 13.6% to 54.6% at 280 days. Three chimeras have demonstrated gradually decreasing engraftment after 200 days. The degree of engraftment correlated with clinically relevant improvement: decreased reticulocyte counts (8.4% to 15.7% in chimeras [n = 9] v 17.1% to 19.1% in controls [n = 8], P = .01), increased mean RBC deformability, and the significant reduction in extramedullary hematopoiesis and iron deposits seen on histological examination of chimeric liver and spleen. These data demonstrate that fetal HSC transplants results in significant long-term chimerism with favorable alterations in red cell characteristics, and decreased hemolytic anemia in beta-thalassemia.     

Mature human hematopoietic cells in donor bone marrow complicate interpretation of stem/progenitor cell assays in xenogeneic hematopoietic chimeras

Xenogeneic hematopoietic chimeras have been used to assay the growth and differentiation of human stem/progenitor cells. The presence of human hematopoietic cells in immunodeficient mice transplanted with human marrow cells may be caused by proliferation and differentiation of early stem/progenitor cells and/or proliferation of mature cells. Unpurified human marrow mononuclear cells, T cell-depleted, or stem/progenitor cell-enriched (CD34+ or CD34+CD38-) populations were injected into sublethally irradiated NOD/LtSz scid/scid (NOD/SCID) mice. High levels of human cells were detected in mice (hu/mu chimeras) transplanted with each of the above human marrow populations. Large numbers of mature human T lymphocytes were found in marrow, spleens, and thymuses from hu/mu chimeras that had been transplanted with unpurified human mononuclear marrow cells. Human immunoglobulin was detected in sera from these chimeras, and some exhibited a clinical syndrome suggestive of graft-versus-host disease. In contrast, in hu/mu chimeras that had received T cell-depleted or stem/progenitor cell-enriched populations, multilineage hematopoiesis (myeloid, B lymphoid, and progenitor cells by immunophenotype) was detected but T lymphocytes and human immunoglobulin were not; in addition, no human cells were detected in the thymuses. Thus, injection of adult human marrow cells into immunodeficient mice can result in hematopoietic chimerism for at least 3 months after transplant. However, the types of cells present in hu/mu chimeras differ depending on the human cell population transplanted. This should be taken into account when hematopoietic chimeras are used to assess human stem/progenitor cell function.     

Human mini-chromosomes in mouse embryonal stem cells

We have introduced human mini-chromosomes of 4 Mb and approximately 15 Mb in size into mouse embryonal stem cells. Although these human mini-chromosomes are stable in hamster and chicken cells, they re-arrange or segregate aberrantly in the embryonal stem cells and are rapidly lost in the absence of selection. However, one of the mini-chromosomes re-arranged, acquired mouse centromeric sequences and was then stably maintained for at least 60 population doublings in culture. This mini-chromosome, which is 4 Mb in size, is a candidate for a mouse germ line chromosome vector.     

Human apolipoproteins A-I and A-II in cell cholesterol efflux: studies with transgenic mice

The first step in reverse cholesterol transport is the movement of cholesterol out of cells onto lipoprotein acceptors in the interstitial fluid. The contribution of specific lipoprotein components to this process remains to be established. In this study, the role of human apolipoproteins (apo) A-I and A-II in the efflux of cellular cholesterol was investigated in transgenic mouse models in which the expression of murine apoA-I was abolished due to gene targeting (A-IKO). Serum from A-IKO mice and from mice expressing human apoA-I and/or human apoA-II was incubated with [3H]cholesterol-labeled Fu5AH rat hepatoma cells for 4 hours at 37 degrees C. The cholesterol efflux to the serum of A-IKO mice was markedly lower than that to the serum of mice transgenic for human apoA-I (5.0 +/- 1.5% versus 25.0 +/- 4.0%). Expression of human apoA-II alone did not modify the cholesterol efflux capacity of A-IKO mouse serum. Cholesterol efflux to serum of mice expressing human apoA-II together with human apoA-I was significantly lower than that to human apoA-I mouse serum (20.0 +/- 2.3% versus 25.0 +/- 4.0%). Regression analysis of cholesterol efflux versus the lipid/apolipoprotein concentrations of mouse serum suggested that 3 independent factors contribute to determine the cholesterol efflux potential of serum: the apolipoprotein composition of HDL, the serum concentration of HDL phospholipids, and the presence of a small fraction of particles containing apoA-I.     

Late-onset chronic inflammatory encephalopathy in immune-competent and severe combined immune-deficient (SCID) mice with astrocyte-targeted expression of tumor necrosis factor

To examine the role of tumor necrosis factor (TNF)-alpha in the pathogenesis of degenerative disorders of the central nervous system (CNS), transgenic mice were developed in which expression of murine TNF-alpha was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF-alpha fusion gene. In two independent GFAP-TNFalpha transgenic lines (termed GT-8 or GT-2) adult (>4 months of age) animals developed a progressive ataxia (GT-8) or total paralysis affecting the lower body (GT-2). Symptomatic mice had prominent meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large numbers of B cells and CD4+ and CD8+ T cells accumulated at predominantly perivascular sites. The majority of these lymphocytes displayed a memory cell phenotype (CD44high, CD62Llow, CD25-) and expressed an early activation marker (CD69). Parenchymal lesions contained mostly CD45+ high, MHC class II+, and Mac-1+ cells of the macrophage microglial lineage with lower numbers of neutrophils and few CD4+ and CD8+ T cells. Cerebral expression of the cellular adhesion molecules ICAM-1, VCAM-1, and MAdCAM as well as a number of alpha- and beta-chemokines was induced or upregulated and preceded the development of inflammation, suggesting an important signaling role for these molecules in the CNS leukocyte migration. Degenerative changes in the CNS of the GFAP-TNFalpha mice paralleled the development of the inflammatory lesions and included primary and secondary demyelination and neurodegeneration. Disease exacerbation with more extensive inflammatory lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFalpha mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-alpha in the CNS induces a late-onset chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury.     

Human growth factor-enhanced regeneration of transplantable human hematopoietic stem cells in nonobese diabetic/severe combined immunodeficient mice

Self-renewal is considered to be the essential defining property of a stem cell. Retroviral marking, in vitro amplification, and serial transplantation of human cells that can sustain long-term lymphomyelopoiesis in vivo have provided evidence that human hematopoietic stem cell self-renewal occurs both in vitro and in vivo. To investigate whether this process can be manipulated by cytokines, we administered two different combinations of human growth factors to sublethally irradiated nonobese diabetic/severe combined immunodeficient (SCID) mice transplanted with 10(7) light-density human cord blood cells and then performed secondary transplants to compare the number of transplantable human lymphomyeloid reconstituting cells present 4 to 6 weeks post-transplant. A 2-week course of Steel factor + interleukin (IL)-3 + granulocyte-macrophage colony-stimulating factor + erythropoietin (3 times per week just before sacrifice) specifically and significantly enhanced the numbers of transplantable human lymphomyeloid stem cells detectable in the primary mice (by a factor of 10). Steel factor + Flt3-ligand + IL-6 (using either the same schedule or administered daily until sacrifice 4 weeks post-transplant) gave a threefold enhancement of this population. These effects were obtained at a time when the regenerating human progenitor populations in such primary mice are known to be maximally cycling even in the absence of growth factor administration suggesting that the underlying mechanism may reflect an ability of these growth factors to alter the probability of differentiation of stem cells stimulated to proliferate in vivo.     

Engraftment and retroviral marking of CD34+ and CD34+CD38- human hematopoietic progenitors assessed in immune-deficient mice

Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+ progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38- cells. Comparable extents of human engraftment and lineage development were obtained from 5 x 10(5) CD34+ cells and 2,000 CD34+CD38- cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38- cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38- cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.     

间充质干细胞在造血干细胞移植中的应用

间充质干细胞是一种能够从各种人成体组织分离出来的非造血多能干细胞,近年来,许多研究表明间充质干细胞具有免疫调节能力及促进组织重建等功能.就其在造血干细胞移植中的应用,如急慢性移植物抗宿主病(GVHD)、GVHD造成的移植失败、纯红细胞再生障碍性贫血及免疫性血小板减少性紫癜、出血性膀胱炎作以综述.     

Ethical issues in stem cell transplantation

Citrate transport into the vacuoles of acid lime juice cells was investigated using isolated tonoplast vesicles. ATP stimulated citrate uptake in the presence or in the absence of a Delta mu H+. Energization of the vesicles only by an artificial K+ gradient (establishing an inside-positive Delta psi) also resulted in citrate uptake as was the case of a Delta pH dominated Delta mu H+. Addition of inhibitors to endomembrane ATPases showed no direct correlation between the inhibition to the tonoplast bound H+/ATPase and citrate uptake. The data indicated that, although some citrate uptake can be accounted for by Delta psi and by a direct primary active transport mechanism involving ATP, under in vivo conditions of vacuolar pH of 2.0, citrate uptake is driven by Delta pH.     

Embryonic stem cells and hematopoietic stem cell biology

Two advances in murine embryonic stem (ES) cell technology and their applications for the study of hematopoietic stem cells (HSCs) are discussed in this article. First, ES cells induced to differentiate in vitro form hematopoietic lineages in a fashion that recapitulates the ontogeny of blood formation in the embryo. This system offers a unique opportunity to isolate, examine, and manipulate the most primitive hematopoietic progenitors. Second, targeted gene ablation (knockout) studies in ES cells have identified several genes that are required for normal hematopoiesis and may function in the formation, maintenance, and differentiation of HSCs. Insights into murine hematopoiesis gained through the study of ES cells generally should be applicable to other vertebrates, including humans.     

Embryonic stem cell lines from MRL mice allow genetic modification in a murine model of autoimmune disease

The MRL/MpJ-Fas(lpr) (MRL-lpr/lpr) mouse spontaneously develops a generalized autoimmune disease with features similar to those of systemic lupus erythematosus. This mouse strain provides a valuable system for identifying and characterizing the multiple genetic factors that influence the pathogenesis of autoimmune diseases. One of the most powerful means of examining the role of a specific gene product in vivo is by inactivating the gene in mouse embryonic stem (ES) cells by homologous recombination and using these cells to derive mouse lines carrying the inactivated gene. The successful application of this approach, however, requires an ES cell line that will remain stable in culture during the processes of genetic manipulation and selection. To date, ES cell lines that meet this criterion have been derived from only a few mouse strains. Here we describe the production and characterization of stable ES cell lines from the MRL mouse strain. Approximately 7% of the blastocysts derived from the MRL/MpJ+ (MRL-+/+) strain gave rise to ES cell lines, and both of the male MRL-+/+ ES cell lines tested were shown to be germline competent. We show that the MRL-+/+ ES cell lines undergo gene targeting by homologous recombination at high frequency by inactivating the gene encoding the EP2 prostaglandin receptor. These Ep2-targeted MRL ES cell lines were used to generate MRL mouse lines heterozygous for the disrupted Ep2 gene, thus demonstrating the feasibility of using a genetic approach to dissect the pathobiology of the autoimmune disease in the MRL mouse.     

Quantitative methods for scoring cell migration and invasion in filter-based assays

The ability of cells to traverse pores in a biocompatible filter provides means for examining cell chemoattraction. Filter-based assays also permit rapid, quantitative assessment of the in vitro migratory and invasive potential of tumor cells. Scoring migration has relied on visual counting of stained cells which appear on the underside of the filter and determining a true percentage score involves arduous counting of cells on both filter surfaces. Visual counting of random fields may be unreliable, and counting all fields is laborious. In the present study we developed and compared two alternative methods for scoring cell numbers in filter-based assays, a colorimetric assay of toluidine blue binding, and a radioassay of cells prelabeled with [3H]thymidine. Each method was evaluated for sensitivity, variability, ease of use and efficiency, and suitability for use in assays of cell migration and invasion. The radiolabeling method proved to be sensitive and reliable and was the most efficient technique. Although less sensitive and specific, the colorimetric dye method offered a rapid and reliable, nonradioactive alternative with the distinct advantage of preserving intact cultures for follow-up visual assessments. We conclude that colorimetric and radiolabel scoring of filter-based assays are reliable and efficient semiautomated methods which provide means to obtain more complete assessments of cell migration and invasion.     

New developments in hematopoietic stem cell expansion

In the present study we investigated the effects of various doses of gamma-irradiation, followed by induction of granulocytic differentiation with all-trans-retinoic acid (ATRA), on proliferative rate, differentiation capability and oxidative metabolism of leukaemic cells from two different myeloid leukaemia cell lines, HL-60 and PLB-985. Regarding the effects of such combined treatment on the proliferative capabilities of HL-60 and PLB-985 cell lines, we showed that their growth kinetics were similar after 2 Gy gamma-irradiation combined with ATRA. However, with doses >2 Gy, the behaviour of the cell lines differed largely. Indeed, HL-60 appeared to be more radiosensitive than PLB-985 regarding cell viability and proliferation. Besides, whatever dose of irradiation (2, 5 or 10 Gy) was applied, ATRA was still able to induce differentiation of HL-60 and PLB-985 into granulocytes that retained the capacity to produce superoxide anion. The results of these in vitro studies suggest that leukaemia cell lines retain their ability to respond to ATRA, a granulocytic-differentiating inducer following high doses of irradiation. This may have implications for the use of radiation therapy in combination with ATRA for the treatment of extramedullary infiltrations of myeloid leukaemias in humans.