Increased reactive oxygen species and exhaustion of quiescent CD34-positive bone marrow cells may contribute to poor graft function after allotransplants.

Poor graft function (PGF) is a fatal complication following allogeneic haematopoietic stem cell transplantation. However, the underlying mechanism is unclear. Effective cross-talk between haematopoietic stem cells (HSCs) and bone marrow microenvironment is important for normal haematopoiesis. Normal HSCs reside in a hypoxic bone marrow microenvironment that protects them from oxidative stress that would otherwise inhibit their self-renewal and results in bone marrow failure. Whether an increased level of reactive oxygen species (ROS) causes PGF following allotransplant is unclear. Using a prospective case-pair study, we identified increased levels of ROS in CD34+ bone marrow cells in subjects with PGF. Elevated ROS levels was associated with an increased frequency of DNA strand breaks, apoptosis, exhaustion of quiescent CD34+ cells and defective colony-forming unit plating efficiency, particularly in the CD34+CD38- fraction. Up-regulated intracellular p53, p21, caspase-3 and caspase-9 levels (but not p38) were detected in CD34+ cells, particularly in the CD34+CD38- fraction. To further study the potential role of ROS levels in post-transplant haematopoiesis, CD34+ bone marrow cells from subjects with good graft function were treated with H2O2. This increased ROS levels resulting in defective CD34+ cells, an effect partially reversed by N-acetyl-L-cysteine. Moreover, CD34+ bone marrow cells from the donors to subjects with poor or good graft function exhibited comparable haematopoietic reconstitution capacities in the xeno-transplanted NOD-PrkdcscidIL2rgnull mice. Thus, even if the transplanted donors' bone marrow CD34+ cells are functionally normal pre-transplant, ROS-induced apoptosis may contribute to the exhaustion of CD34+ bone marrow cells in subjects with PGF following allotransplant.

Evaluation of islets derived from human fetal pancreatic progenitor cells in diabetes treatment.

INTRODUCTION: With the shortage of donor organs for islet transplantation, insulin-producing cells have been generated from different types of stem cell. Human fetal pancreatic stem cells have a better self-renewal capacity than adult stem cells and can readily differentiate into pancreatic endocrine cells, making them a potential source for islets in diabetes treatment. In the present study, the functions of pancreatic islets derived from human fetal pancreatic progenitor cells were evaluated in vitro and in vivo. METHODS: Human pancreatic progenitor cells isolated from the fetal pancreas were expanded and differentiated into islet endocrine cells in culture. Markers for endocrine and exocrine functions as well as those for alpha and beta cells were analyzed by immunofluorescent staining and enzyme-linked immunosorbent assay (ELISA). To evaluate the functions of these islets in vivo, the islet-like structures were transplanted into renal capsules of diabetic nude mice. Immunohistochemical staining for human C-peptide and human mitochondrion antigen was applied to confirm the human origin and the survival of grafted islets. RESULTS: Human fetal pancreatic progenitor cells were able to expand in medium containing basic fibroblast growth factor (bFGF) and leukemia inhibitor factor (LIF), and to differentiate into pancreatic endocrine cells with high efficiency upon the actions of glucagon-like peptide-1 and activin-A. The differentiated cells expressed insulin, glucagon, glucose transporter-1 (GLUT1), GLUT2 and voltage-dependent calcium channel (VDCC), and were able to aggregate into islet-like structures containing alpha and beta cells upon suspension. These structures expressed and released a higher level of insulin than adhesion cultured cells, and helped to maintain normoglycemia in diabetic nude mice after transplantation. CONCLUSIONS: Human fetal pancreatic progenitor cells have good capacity for generating insulin producing cells and provide a promising potential source for diabetes treatment.

Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability.

BACKGROUND: Amplification of 3q26 is one of the most frequent genetic alterations in many human malignancies. Recently, we isolated a novel oncogene eIF-5A2 within the 3q26 region. Functional study has demonstrated the oncogenic role of eIF-5A2 in the initiation and progression of human cancers. In the present study, we aim to investigate the physiological and pathological effect of eIF-5A2 in an eIF-5A2 transgenic mouse model. METHODS: An eIF-5A2 transgenic mouse model was generated using human eIF-5A2 cDNA. The eIF-5A2 transgenic mice were characterized by histological and immunohistochemistry analyses. The aging phenotypes were further characterized by wound healing, bone X-ray imaging and calcification analysis. Mouse embryo fibroblasts (MEF) were isolated to further investigate molecular mechanism of eIF-5A2 in aging. RESULTS: Instead of resulting in spontaneous tumor formation, overexpression of eIF-5A2 accelerated the aging process in adult transgenic mice. This included decreased growth rate and body weight, shortened life span, kyphosis, osteoporosis, delay of wound healing and ossification. Investigation of the correlation between cellular senescence and aging showed that cellular senescence is not required for the aging phenotypes in eIF-5A2 mice. Interestingly, we found that activation of eIF-5A2 repressed p19 level and therefore destabilized p53 in transgenic mouse embryo fibroblast (MEF) cells. This subsequently allowed for the accumulation of chromosomal instability, such as errors in cell dividing during metaphase and anaphase. Additionally, a significantly increase in number of aneuploidy cells (p < 0.05) resulted from an increase in the incidences of misaligned and lagging chromosomal materials, anaphase bridges, and micronuclei in the transgenic mice. CONCLUSION: These observations suggest that eIF-5A2 mouse models could accelerate organismal aging by increasing chromosome instability.

Mice transgenic for human angiotensin-converting enzyme 2 provide a model for SARS coronavirus infection.

To establish a small animal model of severe acute respiratory syndrome (SARS), we developed a mouse model of human severe acute respiratory syndrome coronavirus (SARS-CoV) infection by introducing the human gene for angiotensin-converting enzyme 2 (hACE2) (the cellular receptor of SARS-CoV), driven by the mouse ACE2 promoter, into the mouse genome. The hACE2 gene was expressed in lung, heart, kidney, and intestine. We also evaluated the responses of wild-type and transgenic mice to SARS-CoV inoculation. At days 3 and 7 postinoculation, SARS-CoV replicated more efficiently in the lungs of transgenic mice than in those of wild-type mice. In addition, transgenic mice had more severe pulmonary lesions, including interstitial hyperemia and hemorrhage, monocytic and lymphocytic infiltration, protein exudation, and alveolar epithelial cell proliferation and desquamation. Other pathologic changes, including vasculitis, degeneration, and necrosis, were found in the extrapulmonary organs of transgenic mice, and viral antigen was found in brain. Therefore, transgenic mice were more susceptible to SARS-CoV than were wild-type mice, and susceptibility was associated with severe pathologic changes that resembled human SARS infection. These mice will be valuable for testing potential vaccine and antiviral drug therapies and for furthering our understanding of SARS pathogenesis.

Identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a recently identified human coronavirus. The extremely high homology of the viral genomic sequences between the viruses isolated from human (huSARS-CoV) and those of palm civet origin (pcSARS-CoV) suggested possible palm civet-to-human transmission. Genetic analysis revealed that the spike (S) protein of pcSARS-CoV and huSARS-CoV was subjected to the strongest positive selection pressure during transmission, and there were six amino acid residues within the receptor-binding domain of the S protein being potentially important for SARS progression and tropism. Using the single-round infection assay, we found that a two-amino acid substitution (N479K/T487S) of a huSARS-CoV for those of pcSARS-CoV almost abolished its infection of human cells expressing the SARS-CoV receptor ACE2 but no effect upon the infection of mouse ACE2 cells. Although single substitution of these two residues had no effects on the infectivity of huSARS-CoV, these recombinant S proteins bound to human ACE2 with different levels of reduced affinity, and the two-amino acid-substituted S protein showed extremely low affinity. On the contrary, substitution of these two amino acid residues of pcSARS-CoV for those of huSRAS-CoV made pcSARS-CoV capable of infecting human ACE2-expressing cells. These results suggest that amino acid residues at position 479 and 487 of the S protein are important determinants for SARS-CoV tropism and animal-to-human transmission.

Functional cloning of IGFBP-3 from human microvascular endothelial cells reveals its novel role in promoting proliferation of primitive CD34+CD38- hematopoietic cells in vitro.

Ex vivo expansion of hematopoietic stem cells (HSCs) has been investigated as a means of enhancing engraftment of transplantation therapies, but current ex vivo expansion methods typically result in a loss of functional stem cell activity. Factors that can selectively expand human HSCs remain elusive. Recently we have isolated three functionally distinct human brain microvascular endothelial cells (HBMVECs) that differ greatly in their ability to support in vitro proliferation of human umbilical cord blood (UBC) CD34+CD38-cells. Using these distinct HBMVEC populations, we have devised a cell-based functional cloning assay to identify a molecule(s) capable of facilitating expansion of HSCs in vitro. A gene encoded for IGFBP-3 (insulin-like growth factor binding protein-3) has been identified. IGFBP-3 mRNA and protein are differentially expressed in distinct HBMVEC populations. In vitro cell proliferation assay and CD34+CD38- immunophenotype analysis showed that the addition of an exogenous IGFBP-3 to cultures of purified CD34+/-CD38-Lin- cells (CD2/CD3/CD14/CD16/CD19/CD24/CD56/CD66b/GlyA depleted) enhanced proliferation of primitive hematopoietic cells with CD34+CD38- phenotype, suggesting that IGFBP-3 is capable of expanding primitive human blood cells. These expanded primitive blood cells were illustrated to maintain ability to generate functional progenitors. IGFBP-3 belongs to a family of high-affinity IGFBPs, which binds to IGFs and modulates their actions. IGFBP-3 appears to have intrinsic bioactivity that is independent of IGF binding. We are currently exploring the underlying mechanism by which IGFBP-3 modulates proliferation of primitive hematopoietic cells, and the potential of IGFBP-3 to expand pluripotent human repopulating cells capable of hematopoietic reconstitution of irradiated NOD/SCID recipients.