Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.

Stem cell homing and repopulation are not well understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 were found to be critical for murine bone marrow engraftment by human severe combined immunodeficient (SCID) repopulating stem cells. Treatment of human cells with antibodies to CXCR4 prevented engraftment. In vitro CXCR4-dependent migration to SDF-1 of CD34+CD38-/low cells correlated with in vivo engraftment and stem cell function. Stem cell factor and interleukin-6 induced CXCR4 expression on CD34+ cells, which potentiated migration to SDF-1 and engraftment in primary and secondary transplanted mice. Thus, up-regulation of CXCR4 expression may be useful for improving engraftment of repopulating stem cells in clinical transplantation.

Lipofectamine and related cationic lipids strongly improve adenoviral infection efficiency of primitive human hematopoietic cells.

Adenoviral vectors have the potential to infect a large number of cell types including quiescent cells. Their use in hematopoietic cells is limited by the episomal form of their DNA, leading to transgene loss in the progeny cells. However, the use of this vector may be interesting for short-term in vitro modifications of primitive human hematopoietic cells. Therefore, we have investigated the ability of adenovirus to transduce cord blood CD34+ cells. Several promoters were tested using the lacZ reporter gene. The PGK and CMV promoters induced transgene expression in 18-25% of the cells, whereas the HTLV-I and especially the RSV promoter were almost inactive. To improve infection efficiency, adenovirus was complexed with cationic lipids. Lipofectamine, Cellfectin, and RPR120535b, but not Lipofectin, Lipofectace, or DOTAP, markedly improved transgene expression in CD34+ cells (from 19 to 35%). Lipofectamine strongly enhanced infection efficiency of the poorly infectable primitive CD34+CD38low cells (from 11 to 28%) whereas the more mature CD34+CD38+ cells were only slightly affected (from 24 to 31%). Lipofectamine tripled the infection of CFU-GMs and LTC-ICs derived from the CD34+CD38low cell fraction (from 4 to 12% and from 5 to 16%, respectively) and doubled that of BFU-Es (from 13 to 26%). We conclude that cationic lipids can markedly increase the efficiency of adenovirus-mediated gene transfer into primitive hematopoietic cells.

SCLIP: a novel SCG10-like protein of the stathmin family expressed in the nervous system.

Stathmin is a cytosolic phosphoprotein previously described as a ubiquitous relay integrating various signaling pathways. It is the generic element of a protein family in mammals as in Xenopus, including SCG10, a neuron-specific, growth-associated protein, and RB3/XB3, related to the expression of differentiated functions of mature cells of the nervous system. In an extensive search for other members of the stathmin family, we identified cDNAs coding for two novel stathmin-related proteins: (a) a cDNA from a rat striatum cDNA library codes for RB3", a splice variant of RB3, with an additional basic domain in its N-terminal region; and (b) another cDNA identified through a systematic search in EST databases codes for a novel protein, SCLIP, for "SCG10-like protein," displaying 70% identity with SCG10 and sharing the same domain organization, with an N-terminal domain likely involved in membrane attachment and a C-terminal stathmin-like domain. Northern blot analysis as well as in situ hybridization on 14-day rat embryos showed that SCLIP mRNA is expressed only in neural structures. SCLIP mRNA is expressed at comparable levels in neonatal and adult rat brain, suggesting a potential role not only in the acquisition, but also in the expression of differentiated neuronal functions.

The Ulip family phosphoproteins--common and specific properties.

The search for intracellular phosphoproteins implicated in the regulation of neuronal differentiation led to the identification of Ulip1, a mammalian protein related to the Caenorhabditis elegans unc-33 gene product [Byk, T., Dobransky, T., Cifuentes-Diaz, C. & Sobel, A. (1996) J. Neurosc. 16, 688-701]. The expression level and phosphorylation pattern of Ulip1 were shown to be strongly regulated during development and neuronal differentiation. We have isolated three additional complete coding sequences for members of the Ulip family in the mouse, Ulips 2-4, all preferentially expressed in the nervous system. Furthermore, two Ulip sequences, Ulips A and Ulips B, could be identified in C. elegans. The Ulip family is highly conserved throughout evolution (more than 96 % for Ulips 1-3 and 92.5 % for Ulip4 between mouse and human) and the various members of the family within a single species display about 75% similarity. Sequence comparisons further reveal several highly similar domains and subdomains, including a 32-amino-acid region highly conserved from a bacterial hydantoinase to human Ulips. Two-dimensional immunoblot analysis of in vitro translated Ulips 1-4 demonstrates the existence, for each Ulip protein, of several, most probably differentially phosphorylated forms, in agreement with the presence of conserved phosphorylation consensus sites within their sequences. The expression of Ulips 1-4 mRNAs is differentially regulated during development and nerve-growth-factor-induced neuronal differentiation of PC12 cells. Our results indicate a differential, possibly complementary role of phosphoproteins of the highly conserved Ulip family in the control of neuronal differentiation, in relation with the development and plasticity of the nervous system.

Identification and molecular characterization of Unc-33-like phosphoprotein (Ulip), a putative mammalian homolog of the axonal guidance-associated unc-33 gene product.

The control of neuritic extension and guidance is critical for the development, maturation, and regeneration of functional neuronal circuits. We identified a neuronal 64-85 kDa phosphoprotein, the expression of which in mouse brain is regulated during development, reaching a peak at approximately 5 d postnatal, when maturation of neurons and synaptic connections is highly active. The amino acid sequence of the mouse protein deduced from its cloned cDNA reveals similarities with that of the neuritic outgrowth- and guidance-related product of the unc-33 gene in Caenorhabditis elegans. The regulation of its phosphorylation in response to nerve growth factor, as well as its localization in neurites and growth cones and at the neuromuscular junction, further indicates that Ulip (for Unc-33-like phosphoprotein) is not only a structural but likely is also a functional mammalian homolog of Unc-33, potentially involved in the control of neuritic outgrowth and axonal guidance.

Regulatory arrestin cycle secures the fidelity and maintenance of the fly photoreceptor cell.

Excitation of fly photoreceptor cells is initiated by photoisomerization of rhodopsin to the active form of metarhodopsin. Fly metarhodopsin is thermostable, does not bleach, and does not regenerate spontaneously to rhodopsin. For this reason, the activity of metarhodopsin must be stopped by an effective termination reaction. On the other hand, there is also a need to restore the inactivated photopigment to an excitable state in order to keep a sufficient number of photopigment molecules available for excitation. The following findings reveal how these demands are met. The photopigment undergoes rapid phosphorylation upon photoconversion of rhodopsin to metarhodopsin and an efficient Ca2+ dependent dephosphorylation upon regeneration of metarhodopsin to rhodopsin. Phosphorylation decreases the ability of metarhodopsin to activate the guanine nucleotide-binding protein. Binding of 49-kDa arrestin further quenches the activity of metarhodopsin and protects it from dephosphorylation. Light-dependent binding and release of 49-kDa arrestin from metarhodopsin- and rhodopsin-containing membranes, respectively, directs the dephosphorylation reaction toward rhodopsin. This ensures the return of phosphorylated metarhodopsin to the rhodopsin pool without initiating transduction in the dark. Assays of rhodopsin dephosphorylation in the Drosophila retinal degeneration C (rdgC) mutant, a mutant in a gene previously cloned and predicted to encode a serine/threonine protein phosphatase, reveal that phosphorylated rhodopsin is a major substrate for the rdgC phosphatase. We propose that mutations resulting in either a decrease or an improper regulation of rhodopsin phosphatase activity bring about degeneration of the fly photoreceptor cells.

beta2 microglobulin-deficient (B2m(null)) NOD/SCID mice are excellent recipients for studying human stem cell function.

Human SCID repopulating cells (SRC) are defined based on their functional ability to repopulate the bone marrow of NOD/SCID mice with both myeloid and lymphoid cell populations. The frequency of SRC in umbilical cord blood cells is 1 in 9.3 x 10(5) mononuclear cells. We report that as few as 8 x 10(4) human cord blood mononuclear cells transplanted into NOD/SCID/B2m(null )mice resulted in multilineage differentiation in the murine bone marrow, revealing a more than 11-fold higher SRC frequency than in NOD/SCID mice. Moreover, as few as 2 to 5 x 10(3) CD34(+) cells recovered from the bone marrow of primary transplanted NOD/SCID mice were sufficient for engrafting secondary NOD/SCID/B2m(null )mice with SRC, suggesting SRC self-renewal. Thus, by using NOD/SCID/B2m(null )mice as recipients, we established a functional assay for human stem cells capable of engrafting the bone marrow of primary and secondary transplanted immune-deficient mice with SRC, providing a model that better resembles autologous stem cell transplantation.

Calcium channel blockers inhibit retinal degeneration in the retinal-degeneration-B mutant of Drosophila.

Light accelerates degeneration of photoreceptor cells of the retinal degeneration B (rdgB) mutant of Drosophila. During early stages of degeneration, light stimuli evoke spikes from photoreceptors of the mutant fly; no spikes can be recorded from photoreceptors of the wild-type fly. Production of spike potentials from mutant photoreceptors was blocked by diltiazem, verapamil hydrochloride, and cadmium. Little, if any, effect of the (-)-cis isomer or (+)-cis isomer of diltiazem on the light response was seen. Further, the (+)-cis isomer was approximately 50 times more effective than the (-)-cis isomer in blocking the Ca2+ spikes, indicating that diltiazem action on the rdgB eye is mediated by means of blocking voltage-sensitive Ca2+ channels, rather than by blocking the light-sensitive channels. Application of the Ca(2+)-channel blockers (+)-cis-diltiazem and verapamil hydrochloride to the eyes of rdgB flies over a 7-day period largely inhibited light-dependent degeneration of the photoreceptor cells. Pulse labeling with [32P]phosphate showed much greater incorporation into eye proteins of [32P]phosphate in rdgB flies than in wild-type flies. Retarding the light-induced photoreceptor degeneration in the mutant by Ca(2+)-channel blockers, thus, suggests that toxic increase in intracellular Ca2+ by means of voltage-gated Ca2+ channels, possibly secondary to excessive phosphorylation, leads to photoreceptor degeneration in the rdgB mutant.

Rapid and efficient homing of human CD34(+)CD38(-/low)CXCR4(+) stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2m(null) mice.

Stem cell homing into the bone microenvironment is the first step in the initiation of marrow-derived blood cells. It is reported that human severe combined immunodeficient (SCID) repopulating cells home and accumulate rapidly, within a few hours, in the bone marrow and spleen of immunodeficient mice previously conditioned with total body irradiation. Primitive CD34(+)CD38(-/low)CXCR4(+) cells capable of engrafting primary and secondary recipient mice selectively homed to the bone marrow and spleen, whereas CD34(-)CD38(-/low)Lin(-) cells were not detected. Moreover, whereas freshly isolated CD34(+)CD38(+/high) cells did not home, in vivo stimulation with granulocyte colony-stimulating factor as part of the mobilization process, or in vitro stem cell factor stimulation for 2 to 4 days, potentiated the homing capabilities of cytokine-stimulated CD34(+)CD38(+) cells. Homing of enriched human CD34(+) cells was inhibited by pretreatment with anti-CXCR4 antibodies. Moreover, primitive CD34(+)CD38(-/low)CXCR4(+) cells also homed in response to a gradient of human stromal cell-derived factor 1 (SDF-1), directly injected into the bone marrow or spleen of nonirradiated NOD/SCID mice. Homing was also inhibited by pretreatment of CD34(+) cells with antibodies for the major integrins VLA-4, VLA-5, and LFA-1. Pertussis toxin, an inhibitor of signals mediated by Galpha(i) proteins, inhibited SDF-1-mediated in vitro transwell migration but not adhesion or in vivo homing of CD34(+) cells. Homing of human CD34(+) cells was also blocked by chelerythrine chloride, a broad-range protein kinase C inhibitor. This study reveals rapid and efficient homing to the murine bone marrow by primitive human CD34(+)CD38(-/low)CXCR4(+) cells that is integrin mediated and depends on activation of the protein kinase C signal transduction pathway by SDF-1.

Ulip/CRMP proteins are recognized by autoantibodies in paraneoplastic neurological syndromes.

Anti-CV2 autoantibodies have recently been discovered in patients with paraneoplastic neurological diseases (PND). These disorders are associated with neuronal degeneration, mediated by autoimmune processes, in patients with systemic cancer. Anti-CV2 autoantibodies recognize a brain protein of 66 kDa developmentally regulated and specifically expressed by a subpopulation of oligodendrocytes in the adult brain. Here, we demonstrate that anti-CV2 sera recognize several post-translationally modified forms of Ulip4/CRMP3, a member of a protein family related to the axonal guidance and homologous to the Unc-33 gene product in Caenorhabditis elegans. The sequence of the human Ulip4/CRMP3 was determined and the gene localized to chromosome 10q25.2-q26, a region mutated in glioblastomas and containing tumour suppressor genes. The identification of the Ulip/CRMP proteins as recognized by anti-CV2 sera should provide new insights into the role of Ulip/CRMPs in oligodendrocytes and into pathophysiology of PND.