The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.

The low level of amphotropic retrovirus-mediated gene transfer into human hematopoietic stem cells (HSC) has been a major impediment to gene therapy for hematopoietic diseases. In the present study, we have examined amphotropic retrovirus receptor (amphoR) and ecotropic retrovirus receptor mRNA expression in highly purified populations of mouse and human HSC. Murine HSC with low to undetectable levels of amphoR mRNA and relatively high levels of ecotropic retrovirus receptor mRNA were studied. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, ecotropic provirus sequences were detected in 10 of 13 long-term repopulated animals, while amphotropic proviral sequences were detected in only one recipient. A second distinct population of murine HSC were isolated that express 3-fold higher levels of amphoR mRNA. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, 11 of 11 repopulated mice contained ecotropic provirus and 6 of 11 contained amphotropic provirus sequences, a significant increase in the amphotropic retrovirus transduction (P = 0.018). These results indicate that, among the heterogeneous populations of HSC present in adult mouse bone marrow, the subpopulation with the highest level of amphoR mRNA is more efficiently transduced by amphotropic retrovirus. In a related study, we found low levels of human amphoR mRNA in purified populations of human HSC (CD34+ CD38-) and higher levels in committed progenitor cells (CD34+ CD38+). We conclude that the amphoR mRNA level in HSC correlates with amphotropic retrovirus transduction efficiency.

Thrombopoietic potential and serial repopulating ability of murine hematopoietic stem cells constitutively expressing interleukin 11.

Based on transplantation studies with bone marrow cultured under various conditions, a role of interleukin 11 (IL-11) in the self-renewal and/or the differentiation commitment of hematopoietic stem cells has been indicated. To better evaluate the in vivo effects of IL-11 on stem/progenitor cell biology, lethally irradiated mice were serially transplanted with bone marrow cells transduced with a defective retrovirus, termed MSCV-mIL-11, carrying the murine IL-11 (mIL-11) cDNA and the bacterial neomycin phosphotransferase (neo) gene. High serum levels (i.e., > 1 ng/ml) of mIL-11 in all (20/20) primary and 86% (12/14) of secondary long-term reconstituted mice, as well as 86% (12/14) of tertiary recipients examined at 6 weeks posttransplant, demonstrated persistence of vector expression subsequent to transduction of bone marrow precursors functionally definable as totipotent hematopoietic stem cells. In agreement with results obtained with human IL-11 in other myeloablation models, ectopic mIL-11 expression accelerated recovery of platelets, neutrophils, and, to some extent, total leukocytes while preferentially increasing peripheral platelet counts in fully reconstituted mice. When analyzed 5 months posttransplant, tertiary MSCV-mIL-11 recipients had a significantly greater percentage of G418-resistant colony-forming cells in their bone marrow compared with control MSCV animals. Collectively, these data show that persistent stimulation of platelet production by IL-11 is not detrimental to stem cell repopulating ability; rather, they suggest that IL-11 expression in vivo may have resulted in enhanced maintenance of the most primitive hematopoietic stem cell compartment. The prolonged expression achieved by the MSCV retroviral vector, despite the presence of a selectable marker, contrasts with the frequent transcriptional extinction observed with other retroviral vectors carrying two genes. These findings have potentially important implications for clinical bone marrow transplantation and gene therapy of the hematopoietic system.

Interleukin 7 independent development of human B cells.

Mammalian hematopoietic stem cell (HSC) commitment and differentiation into lymphoid lineage cells proceed through a series of developmentally restricted progenitor compartments. A complete understanding of this process, and how it differs from HSC commitment and differentiation into cells of the myeloid/erythroid lineages, requires the development of model systems that support HSC commitment to the lymphoid lineages. We now describe a human bone marrow stromal cell culture that preferentially supports commitment and differentiation of human HSC to CD19+ B-lineage cells. Fluorescence activated cell sorterpurified CD34++/lineage-cells were isolated from fetal bone marrow and cultured on human fetal bone marrow stromal cells in serum-free conditions containing no exogenous cytokines. Over a period of 3 weeks, CD34++/lineage- cells underwent commitment, differentiation, and expansion into the B lineage. Progressive changes included: loss of CD34, acquisition of and graded increases in the level of cell surface CD19, and appearance of immature B cells expressing mu/kappa or mu/lambda cell surface Ig receptors. The tempo and phenotype of B-cell development was not influenced by the addition of IL-7 (10 ng/ml), or by the addition of goat anti-IL-7 neutralizing antibody. These results indicate a profound difference between mouse and human in the requirement for IL-7 in normal B-cell development, and provide an experimental system to identify and characterize human bone marrow stromal cell-derived molecules crucial for human B lymphopoiesis.

The genesis of avian neural crest cells: a classic embryonic induction.

Neural crest cells arise from the ectoderm and are first recognizable as discrete cells in the chicken embryo when they emerge from the neural tube. Despite the classical view that neural crest precursors are a distinct population lying between epidermis and neuroepithelium, our results demonstrate that they are not a segregated population. Cell lineage analyses have demonstrated that individual precursor cells within the neural folds can give rise to epidermal, neural crest, and neural tube derivatives. Interactions between the neural plate and epidermis can generate neural crest cells, since juxtaposition of these tissues at early stages results in the formation of neural crest cells at the interface. Inductive interactions between the epidermis and neural plate can also result in "dorsalization" of the neural plate, as assayed by the expression of the Wnt transcripts characteristic of the dorsal neural tube. The competence of the neural plate changes with time, however, such that interaction of early neural plate with epidermis generates only neural crest cells, whereas interaction of slightly older neural plate with epidermis generates neural crest cells and Wnt-expressing cells. At cranial levels, neuroepithelial cells can regulate to generate neural crest cells when the endogenous neural folds are removed, probably via interaction of the remaining neural tube with the epidermis. Taken together, these experiments demonstrate that: (i) progenitor cells in the neural folds are multipotent, having the ability to form multiple ectodermal derivatives, including epidermal, neural crest, and neural tube cells; (ii) the neural crest is an induced population that arises by interactions between the neural plate and the epidermis; and (iii) the competence of the neural plate to respond to inductive interactions changes as a function of embryonic age.

Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5.

Signaling through the erythropoietin receptor (EPO-R) is crucial for proliferation, differentiation, and survival of erythroid progenitor cells. EPO induces homodimerization of the EPO-R, triggering activation of the receptor-associated kinase JAK2 and activation of STAT5. By mutating the eight tyrosine residues in the cytosolic domain of the EPO-R, we show that either Y343 or Y401 is sufficient to mediate maximal activation of STAT5; tyrosine residues Y429 and Y431 can partially activate STAT5. Comparison of the sequences surrounding these tyrosines reveals YXXL as the probable motif specifying recruitment of STAT5 to the EPO-R. Expression of a mutant EPO-R lacking all eight tyrosine residues in the cytosolic domain supported a low but detectable level of EPO-induced STAT5 activation, indicating the existence of an alternative pathway for STAT5 activation independent of any tyrosine in the EPO-R. The kinetics of STAT5 activation and inactivation were the same, regardless of which tyrosine residue in the EPO-R mediated its activation or whether the alternative pathway was used. The ability of mutant EPO-Rs to activate STAT5 did not directly correlate with their mitogenic potential.

Conserved motifs in prokaryotic and eukaryotic polypeptide release factors: tRNA-protein mimicry hypothesis.

Translation termination requires two codon-specific polypeptide release factors in prokaryotes and one omnipotent factor in eukaryotes. Sequences of 17 different polypeptide release factors from prokaryotes and eukaryotes were compared. The prokaryotic release factors share residues split into seven motifs. Conservation of many discrete, perhaps critical, amino acids is observed in eukaryotic release factors, as well as in the C-terminal portion of elongation factor (EF) G. Given that the C-terminal domains of EF-G interacts with ribosomes by mimicry of a tRNA structure, the pattern of conservation of residues in release factors may reflect requirements for a tRNA-mimicry for binding to the A site of the ribosome. This mimicry would explain why release factors recognize stop codons and suggests that all prokaryotic and eukaryotic release factors evolved from the progenitor of EF-G.

Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells.

Because of their known myelopoietic activities, both interleukin (IL)-3 and IL-1 are often used in combination with other cytokines for in vitro (ex vivo) expansion of stem cells. We have investigated the effects of IL-3 and IL-1 on in vitro expansion of murine hematopoietic stem cells with long-term engraftment capabilities, using a highly purified progenitor population. Lineage-negative, Ly-6A/E+, c-kit+ bone marrow cells from male mice were cultured in suspension in the presence of stem cell factor, IL-6, IL-11, and erythropoietin with or without IL-3 or IL-1. Kinetic studies revealed an exponential increase in total nucleated cells and about 10-fold enhancement of nucleated cells by IL-3 during the initial 10 days. Addition of IL-3 hastened the development but significantly suppressed the peak production of colony-forming cells. Addition of IL-1 also significantly suppressed the numbers of colony-forming cells. The reconstituting ability of the cultured cells was tested by transplanting the expanded male cells into lethally irradiated female mice. The cells expanded from enriched cells in the absence of IL-3 and IL-1 revealed engraftment at 2, 4, 5, and 6 months, whereas addition of IL-3 or IL-1 to the cultures significantly reduced the reconstituting ability. The results suggest that these cytokines may have a modulatory role on the self-renewal of stem cells and further indicate that the use of IL-3 and IL-1 for in vitro expansion of human stem cells needs to be cautiously evaluated.

The brain-specific activator p35 allows Cdk5 to escape inhibition by p27Kip1 in neurons.

Cell cycle withdrawal in postmitotic cells involves cyclin-dependent kinase (Cdk) inhibitors that repress cell cycle Cdk activity. During mouse neurogenesis, cortical postmitotic neurons are shown here to accumulate high levels of the p27 Cdk inhibitor compared with their progenitor neuroblasts. Elevated p27 levels in staged embryo brain extracts correlate with p27 binding to Cdk2, and Cdk inactivation. Yet, Cdk5, which is associated with the noncyclin activator p35 in neurons, remains active in the presence of high p27 levels. Both in vitro and in vivo, p27 and related inhibitors can recognize a cyclin D-Cdk5 complex but not a p35-Cdk5 complex. The results indicate that the choice of activator determines the susceptibility of Cdk5 to p27 and related Cdk inhibitors, and thus its ability to act in postmitotic cells.

BEN/SC1/DM-GRASP, a homophilic adhesion molecule, is required for in vitro myeloid colony formation by avian hemopoietic progenitors.

BEN/SC1/DM-GRASP is a membrane glycoprotein of the immunoglobulin superfamily isolated in the chick by several groups, including ours. Its expression is strictly developmentally regulated in several cell types of the nervous and hemopoietic systems and in certain epithelia. Each of these cell types expresses isoforms of BEN which differ by their level of N-glycosylation and by the presence or absence of the HNK-1 carbohydrate epitope. In the present work, the influence of glycosylation on BEN homophilic binding properties was investigated by two in vitro assays. First, each BEN isoform was covalently coupled to microspheres carrying different fluorescent dyes and an aggregation test was performed. We found that homophilic aggregates form indifferently between the same or different BEN isoforms, showing that glycosylation does not affect BEN homophilic binding properties. This was confirmed in the second test, where the BEN-coated microspheres bound to the neurites of BEN- expressing neurons, irrespective of the isoform considered. The transient expression of the BEN antigen on hemopoietic progenitors prompted us to see whether it might play a role in their proliferation and differentiation. When added to hemopoietic progenitor cells in an in vitro colony formation assay anti-BEN immunoglobulin strongly inhibited myeloid, but not erythroid, colony formation although both types of precursors express the molecule.

Genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa.

The gastric mucosa of mammalian stomach contains several differentiated cell types specialized for the secretion of acid, digestive enzymes, mucus, and hormones. Understanding whether each of these cell lineages is derived from a common stem cell has been a challenging problem. We have used a genetic approach to analyze the ontogeny of progenitor cells within mouse stomach. Herpes simplex virus 1 thymidine kinase was targeted to parietal cells within the gastric mucosa of transgenic mice, and parietal cells were ablated by treatment of animals with the antiherpetic drug ganciclovir. Ganciclovir treatment produced complete ablation of parietal cells, dissolution of gastric glands, and loss of chief and mucus-producing cells. Termination of drug treatment led to the reemergence of all major gastric epithelial cell types and restoration of glandular architecture. Our results imply the existence of a pluripotent stem cell for the gastric mucosa. Parietal cell ablation should provide a model for analyzing cell lineage relationships within the stomach as well as mechanisms underlying gastric injury and repair.

DANA elements: a family of composite, tRNA-derived short interspersed DNA elements associated with mutational activities in zebrafish (Danio rerio).

DNA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of these tRNA-derived elements. DANA is unique in its clearly defined substructure of distinct cassettes. In contrast to generic SINE elements, DANA appears to have been assembled by insertions of short sequences into a progenitor, tRNA-derived element. Once associated with each other, these subunits were amplified as a new transposable element with such a remarkable success that DANA-related sequences comprise approximately 10% of the modern zebrafish genome. At least some of the sequences comprised by the full-length element were capable of movement, forming a new group of mobile, composite transposons, one of which caused an insertional mutation in the zebrafish no tail gene. Being present only in the genus Danio, and estimated to be as old as the genus itself, DANA may have played a role in Danio speciation by massive amplification and genome-wide dispersion. There are extensive DNA polymorphisms between zebrafish populations and strains detected by PCR amplification using primers specific to DANA, suggesting that the DANA element will be useful as a molecular tool for genetic and phylogenetic analyses.

Cell fate determination in the vertebrate retina.

In the vertebrate central nervous system, the retina has been a useful model for studies of cell fate determination. Recent results from studies conducted in vitro and in vivo suggest a model of retinal development in which both the progenitor cells and the environment change over time. The model is based upon the notion that the mitotic cells within the retina change in their response properties, or "competence", during development. These changes presage the ordered appearance of distinct cell types during development and appear to be necessary for the production of the distinct cell types. As the response properties of the cells change, so too do the environmental signals that the cells encounter. Together, intrinsic properties and extrinsic cues direct the choice of cell fate.

The stem cell antigen CD34 functions as a regulator of hemopoietic cell adhesion.

Although the CD34 antigen is widely used in the identification and purification of hemopoietic stem and progenitor cells, its function within hemopoiesis is unknown. We have investigated this issue by ectopically expressing human (hu) CD34 on the surface of murine hemopoietic cells. Forced expression of hu-CD34 in the thymocytes of transgenic mice did not appear to affect the development, maturation, or distribution of murine T cells but did significantly increase their ability to adhere to bone marrow stromal layers of human but not mouse origin. Ectopic expression of hu-CD34 on murine 416B cells, a multipotential progenitor that expresses murine CD34, yielded similar results. In both cases hu-CD34-dependent adhesion was enhanced by molecular engagement of the hu-CD34 protein using anti-CD34 antibodies. These results provide evidence that CD34 promotes the adhesive interactions of hemopoietic cells with the stromal microenvironment of the bone marrow thereby implicating CD34 in regulation and compartmentalization of stem cells. We propose that CD34 regulates these processes in part via an indirect mechanism, signaling changes in cellular adhesion in response to molecular recognition of an as yet unidentified stromal CD34 counterreceptor or ligand.

A recombinant bcl-x s adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.

Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.

Clinical evidence of hematopoietic restoration with placental/umbilical cord blood (PCB) grafts indicates that PCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. In the unrelated setting, human leukocyte antigen (HLA)-matched donors must be obtained for candidate patients and, hence, large panels of frozen HLA-typed PCB units must be established. The large volume of unprocessed units, consisting mostly of red blood cells, plasma, and cryopreservation medium, poses a serious difficulty in this effort because storage space in liquid nitrogen is limited and costly. We report here that almost all the hematopoietic colony-forming cells present in PCB units can be recovered in a uniform volume of 20 ml by using rouleaux formation induced by hydroxyethyl starch and centrifugation to reduce the bulk of erythrocytes and plasma and, thus, concentrate leukocytes. This method multiples the number of units that can be stored in the same freezer space as much as 10-fold depending on the format of the storage system. We have also investigated the proportion of functional stem/progenitor cells initially present that are actually available to the recipient when thawed cryopreserved PCB units are infused. Progenitor cell viability is measurably decreased when thawed cells, still suspended in hypertonic cryopreservative solutions, are rapidly mixed with large volumes of isotonic solutions or plasma. The osmotic damage inflicted by the severe solute concentration gradient, however, can be averted by a simple 2-fold dilution after thawing, providing almost total recovery of viable hematopoietic progenitor cells.