BM cells giving rise to MSC in culture have a heterogeneous CD34 and CD45 phenotype.

BACKGROUND: Mesenchymal stromal cells (MSC) isolated from adult human BM are characterized by their fibroblast-like morphology, adherent growth and capacity to differentiate into adipocytes, osteocytes, chondrocytes, cardiomyocytes and neuroprogenitors. After culturing these cells in vitro, they express the cell-surface molecules CD44, CD90, SH2 and SH3, and are negative for CD34 and the hematopoietic marker CD45. The aim of this study was to characterize the in vivo phenotype of MSC relative to the expression of CD34 and CD45. METHODS: BM mononuclear cells were stained with Ab against both molecules and separated into the CD34(+), CD34(-), CD45(+) CD34(+), CD45(high+) CD34(-), CD45(med,low+) CD34(-) and CD45(-) CD34(-) subpopulations, which were then cultured under the same conditions and analyzed for growth of MSC. RESULTS: A small population of MSC arose from the CD45(+) CD34(+) fraction, although the majority was obtained from the CD45(-) CD34(-) subpopulation. MSC from all fractions could be differentiated into adipocytes and osteocytes. In addition, MSC from the CD34(+) and CD34(-) fractions were shown to differentiate into chondrocytes. After in vitro culture, MSC from both fractions possessed the same phenotype, which was negative for CD34 and CD45. DISCUSSION: MSC from the CD45(+) CD34(+) fraction change their phenotype under in vitro conditions.

Perforin-independent rejection of transplanted human stem cells.

The NOD/SCID mouse model is one of the most established model systems for the analysis of human stem cells in vivo. The lack of mature B and T cells renders NOD/SCID mice susceptible to transplantable human stem and progenitor cells. One remaining functional component of the immune system in NOD/SCID mice is natural killer (NK) cells. We rationalized that by eliminating NK cell-mediated cytotoxicity in this model system engraftment of human haematopoietic stem cells could be improved. Thus perforin-deficient NOD/SCID mice (PNOD/SCID) were generated, which display a complete lack of NK cell-mediated cytotoxicity. To test the engraftment potential of human stem cells in PNOD/SCID mice, we compared the repopulating potential of human haematopoietic stem cells in these mice with the repopulating potential in NOD/SCID mice. Upon injection with varying numbers of mononuclear cells from human cord blood, the number of engrafted PNOD/SCID mice was lower (34.8%) than the number of engrafted NOD/SCID mice (64.7%). Similarly, injection of purified CD34(+) human cord blood cells led to engraftment in 32.3% PNOD/SCID versus 60% in NOD/SCID mice. Surprisingly, these results show that the inactivation of cytotoxic activity of NK cells in PNOD/SCID mice did not result in better engraftment with human haematopoietic stem cells. A potential reason for this observation could be that compensatory activation of NK cells in PNOD/SCID mice induces high levels of soluble factors resulting in an environment unfavourable for human stem cell engraftment.

pH dependent Ni(II) binding and aggregation of Escherichia coli and Helicobacter pylori NikR.

NikR proteins are bacterial metallo-regulatory transcription factors that control the expression of the nickel uptake system and/or nickel containing enzymes such as urease, and are involved in the acid stress response. Here, a comparative study is reported on NikR from Helicobacter pylori (HpNikR) and Escherichia coli (EcNikR), as well as the Q2E mutant of EcNikR. Most attention was focused on the Ni(II) binding properties of these proteins, as a function of pH. The influence of the pH on the Ni(II) binding and aggregation properties was studied using gel filtration analysis and UV-visible absorption spectroscopy in the presence of an increasing concentration of nickel. Q2E and wt EcNikR are identical in Ni(II) binding but the Q2E mutant is impaired to some extent in DNA-binding. For EcNikR it is shown that between pH 6 and 8, addition of Ni(II) above 1 equiv. induces mass aggregation and precipitation, concomitant with binding of Ni(II) up to a maximum of 5-8 Ni(II) ions per monomer. The Ni(II) site with highest affinity is the well-described square planar site with three histidines and one cysteine ligands. Aggregation is complete in the presence of less than 1 extra equiv. of Ni(II) and aggregation is fully reversible and precipitates are rapidly solubilized by addition of EDTA. The sensitivity of EcNikR to aggregation decreases with decreasing pH, concurrent with histidines being the main ligands of the site responsible for aggregation. HpNikR does not display aggregation except at alkaline pH, where 3 Ni(II) equiv. are needed. The participation of a cluster consisting of surface-exposed histidines present in EcNikR but not in HpNikR, is proposed to be involved in aggregation. Our results on HpNikR are compatible with the crystallographic data and with the ability of this protein to bind more than one nickel.

Lack of telomerase activity in human mesenchymal stem cells.

Telomerase activity and telomere maintenance have been associated with immortality in tumor and embryonic stem cells. Whereas most normal somatic cells are telomerase negative, low levels of this enzyme have been found in adult stem cells from the skin, gut and the hematopoietic system. Here, we show that telomerase activity is not detectable in human mesenchymal stem cells (hMSCs), which have the phenotype SH2+, SH3+, SH4+, CD29+, CD44+, CD14-, CD34- and CD45-, and have the capacity to differentiate into adipocytes, chondrocytes and osteoblasts. These data suggest that hMSCs have a different telomere biology compared to other adult stem cells. Alternatively, true mesenchymal stem cells might be a very rare subpopulation that have a detection level that is below the sensitivity of the TRAP assay.

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle.

The mammalian signal recognition particle (SRP) catalytically promotes cotranslational translocation of signal sequence containing proteins across the endoplasmic reticulum membrane. While the S-domain of SRP binds the N-terminal signal sequence on the nascent polypeptide, the Alu domain of SRP temporarily interferes with the ribosomal elongation cycle until the translocation pore in the membrane is correctly engaged. Here we present biochemical and biophysical evidence for a hierarchical assembly pathway of the SRP Alu domain. The proteins SRP9 and SRP14 first heterodimerize and then initially bind to the Alu RNA 5' domain. This creates the binding site for the Alu RNA 3' domain. Alu RNA then undergoes a large conformational change with the flexibly linked 3' domain folding back by 180 degrees onto the 5' domain complex to form the final compact Alu ribonucleoprotein particle (Alu RNP). We discuss the possible mechanistic consequences of the likely reversibility of this final step with reference to translational regulation by the SRP Alu domain and with reference to the structurally similar Alu RNP retroposition intermediates derived from Alu elements in genomic DNA.

Interleukin 13 and interleukin 13 receptor are frequently expressed by Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma.

Hodgkin lymphoma (HL) is characterized by the abnormal expression of multiple cytokines, accounting for its unique clinicopathologic features. We have previously shown that interleukin-13 (IL-13) is secreted by HL cell lines and may serve as an autocrine growth factor. To determine the frequency of IL-13 expression in lymphoma patients, tissue sections from 36 patients with classical HL, 5 patients with nodular lymphocyte predominance HL (NLPHL), and 23 patients with non-Hodgkin lymphoma (NHL) were subjected to in situ hybridization. In 31 of 36 cases (86%) of classical HL patients of all histologic subtypes, between 25% to almost 100% of Hodgkin and Reed Sternberg (HRS) cells were positive for IL-13 expression. In contrast, in no case of NLPHL and in only 4 of 23 NHL cases (1 of 5 T-cell-rich B-cell lymphomas, 2 of 5 anaplastic large cell lymphomas, and 1 of 5 peripheral T-cell lymphomas) did the neoplastic cells express IL-13. The expression of the IL-13 receptor chain alpha1 (IL-13Ralpha1) was also analyzed by in situ hybridization. In 24 of 27 (89%) cases of classical HL, between 25% to 75% of HRS cells, as well as a high frequency of lymphocytes and histiocytes, were positive for IL-13Ralpha1 expression. These results were confirmed by the construction of complementary DNA libraries from single HRS cells, followed by polymerase chain reaction analysis, in which IL-13Ralpha1 transcripts were found to be present in all 6 cases of HL. These data indicate that expression of IL-13 and IL-13Ralpha1 is a common feature of HRS cells in HL, consistent with the hypothesis that IL-13 may play a role in autocrine growth in classical HL.

Interleukin 13: a growth factor in hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) is a malignant disorder of lymph nodes with distinctive clinical and pathologic features. These features are thought to be primarily due to the abnormal production of multiple cytokines by the malignant cell population of HL, the Reed-Sternberg (RS) cells. We have previously demonstrated that interleukin (IL)-13 expression is a common feature of HL and have studied its role as an autocrine growth factor for RS cells. IL-13 and IL-13R(alpha)1, the IL-13-specific receptor chain, are frequently expressed by HL-derived cell lines and by RS cells from biopsy material of tissues involved by HL. Neutralization of IL-13 in cultures of the HL-derived cell lines HDLM-2 and L-1236 leads to a dose-dependent inhibition of proliferation, and is associated with increased apoptosis in L-1236 cells. Signal transducer and activator of transcription (STAT) 6 is an important mediator of IL-13 signaling. STAT6 is constitutively activated in HL cell lines due to autocrine secretion of IL-13. STAT6 is also phosphorylated (P-STAT6) in RS cells from many primary HL samples, supporting the hypothesis that IL-13 signaling occurs in these malignant cells in vivo. Coexpression of IL-13, IL-13R(alpha)1 and P-STAT6 is uncommon in non-Hodgkin lymphomas. Following a description of the clinical and pathologic features of HL, this review will discuss the function of IL-13 as an autocrine growth factor for RS cells in HL and its potential role in mediating other features of this disease.

Interleukin 13 is secreted by and stimulates the growth of Hodgkin and Reed-Sternberg cells.

Gene expression patterns can provide vital clues to the pathogenesis of neoplastic diseases. We investigated the expression of 950 genes in Hodgkin's disease (HD) by analyzing differential mRNA expression using microarrays. In two independent microarray experiments, the HD-derived cell lines L428 and KMH2 were compared with an Epstein-Barr virus (EBV)-immortalized lymphoblastoid B cell line, LCL-GK. Interleukin (IL)-13 and IL-5 were found to be highly expressed in the HD-derived cell lines. Examination of IL-13 and IL-5 expression by Northern blot analysis and enzyme-linked immunosorbent assay confirmed these results and revealed the expression of IL-13 in a third HD-derived cell line, HDLM2. Control LCL and EBV-negative non-Hodgkin lymphoma-derived cell lines did not express IL-13. In situ hybridization of lymph node tissue from HD patients showed that elevated levels of IL-13 were specifically expressed by Hodgkin/Reed-Sternberg (H/RS) tumor cells. Treatment of a HD-derived cell line with a neutralizing antibody to IL-13 resulted in a dose-dependent inhibition of H/RS cell proliferation. These data suggest that H/RS cells produce IL-13 and that IL-13 plays an important role in the stimulation of H/RS cell growth, possibly by an autocrine mechanism. Modulation of the IL-13 signaling pathway may be a logical objective for future therapeutic strategies.

Cytokine treatment or accessory cells are required to initiate engraftment of purified primitive human hematopoietic cells transplanted at limiting doses into NOD/SCID mice.

Little is known about the cell types or mechanisms that underlie the engraftment process. Here, we have examined parameters affecting the engraftment of purified human Lin-CD34+CD38- normal and AML cells transplanted at limiting doses into NOD/SCID recipients. Mice transplanted with 500 to 1000 Lin-CD34+CD38- cord blood (CB) or AML cells required the co-transplantation of accessory cells (ACs) or short-term in vivo cytokine treatment for engraftment, whereas transplantation of higher doses (>5000 Lin-CD34+CD38- cells) did not show these requirements suggesting that ACs are effective for both normal and leukemic stem cell engraftment in this model. Mature Lin+CD34- and primitive Lin-CD34+CD38+ cells were capable of acting as ACs even though no repopulating cells are present. Cytokine treatment of NOD/SCID mice could partially replace the requirement for co-transplantation of AC. Furthermore, no difference was seen between the percentage of engrafted mice treated with cytokines for only the first 10 days after transplant compared to those receiving cytokines for the entire time of repopulation. Surprisingly, no engraftment was detected in mice when cytokine treatment was delayed until 10 days posttransplant. Together, these studies suggest that the engraftment process requires pluripotent stem cells plus accessory cells or cytokine treatment which act early after transplantation. The NOD/SCID xenotransplant system provides the means to further clarify the processes underlying human stem cell engraftment.

Treatment of non-obese diabetic (NOD)/Severe-combined immunodeficient mice (SCID) with flt3 ligand and interleukin-7 impairs the B-lineage commitment of repopulating cells after transplantation of human hematopoietic cells.

Until recently, the identification of cellular factors that govern the developmental program of human stem cells has been difficult due to the absence of repopulation assays that detect human stem cells. The transplantation of human bone marrow (BM) or cord blood (CB) into non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice has enabled identification of primitive human cells capable of multilineage repopulation of NOD/SCID mice (termed the SCID-repopulating cell [SRC]). Here, we examined the effect of long-term in vivo treatment with various combinations of human cytokines on the developmental program of SRC. Detailed flow cytometric analysis of engrafted mice indicated that the vast majority of the human graft of untreated mice was comprised of B lymphocytes at various stages of development as well as myeloid and primitive cells; T cells were not reproducibly detected. Many studies, including murine in vitro and in vivo data and human in vitro experiments, have suggested that flt3 ligand (FL) and/or Interleukin-7 (IL-7) promotes T- and B-cell development. Unexpectedly, we found that treatment of engrafted mice with the FL/IL-7 combination did not induce human T- or B-cell development, but instead markedly reduced B-cell development with a concomitant shift in the lineage distribution towards the myeloid lineage. Effects on lineage distribution were similar in engrafted mice transplanted with highly purified cells indicating that the action of the cytokines was not via cotransplanted mature cells from CB or BM cells. These data show that the lineage development of the human graft in NOD/SCID mice can be modulated by administration of human cytokines providing a valuable tool to evaluate the in vivo action of human cytokines on human repopulating cells.

Identification of a minimal Alu RNA folding domain that specifically binds SRP9/14.

We have identified functionally and analyzed a minimal Alu RNA folding domain that is recognized by SRPphi14-9. Recombinant SRPphi14-9 is a fusion protein containing on a single polypeptide chain the sequences of both the SRP14 and SRP9 proteins that are part of the Alu domain of the signal recognition particle (SRP). SRPphi14-9 has been shown to bind to the 7SL RNA of SRP and it confers elongation arrest activity to reconstituted SRP in vitro. Alu RNA variants with homogeneous 3' ends were produced in vitro using ribozyme technology and tested for specific SRPphi14-9 binding in a quantitative equilibrium competition assay. This enabled identification of an Alu RNA of 86 nt (SA86) that competes efficiently with 7SL RNA for SRPphi14-9 binding, whereas smaller RNAs did not. The secondary structure of SA86 includes two stem-loops that are connected by a highly conserved bulge and, in addition, a part of the central adaptor stem that contains the sequence at the very 3' end of 7SL RNA. Circularly permuted variants of SA86 competed only if the 5' and 3' ends were joined with an extended linker of four nucleotides. SA86 can thus be defined as an autonomous RNA folding unit that does not require its 5' and 3' ends for folding or for specific recognition by SRPphi14-9. These results suggest that Alu RNA identity is determined by a characteristic tertiary structure, which might consist of two flexibly linked domains.

Salt effects on the structure and internal dynamics of superhelical DNAs studied by light scattering and Brownian dynamics.

Using laser light scattering, we have measured the static and dynamic structure factor of two different superhelical DNAs, p1868 (1868 bp) and simian virus 40 (SV40) (5243 bp), in dilute aqueous solution at salt concentrations between 1 mM and 3 M NaCl. For both DNA molecules, Brownian dynamics (BD) simulations were also performed, using a previously described model. A Fourier mode decomposition procedure was used to compute theoretical light scattering autocorrelation functions (ACFs) from the BD trajectories. Both measured and computed autocorrelation functions were then subjected to the same multiexponential decomposition procedure. Simulated and measured relaxation times as a function of scattering angle were in very good agreement. Similarly, computed and measured static structure factors and radii of gyration agreed within experimental error. One main result of this study is that the amplitudes of the fast-relaxing component in the ACF show a peak at 1 M salt concentration. This nonmonotonic behavior might be caused by an initial increase in the amplitudes of internal motions due to diminishing long-range electrostatic repulsions, followed by a decrease at higher salt concentration due to a compaction of the structure.

Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays, knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38- cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38- cells and colony-forming cells, respectively, as well as a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were lost, despite further increases in total cells, CFC content, and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells.

The crystal structure of the signal recognition particle Alu RNA binding heterodimer, SRP9/14.

The mammalian signal recognition particle (SRP) is an 11S cytoplasmic ribonucleoprotein that plays an essential role in protein sorting. SRP recognizes the signal sequence of the nascent polypeptide chain emerging from the ribosome, and targets the ribosome-nascent chain-SRP complex to the rough endoplasmic reticulum. The SRP consists of six polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72) and a single 300 nucleotide RNA molecule. SRP9 and SRP14 proteins form a heterodimer that binds to the Alu domain of SRP RNA which is responsible for translation arrest. We report the first crystal structure of a mammalian SRP protein, that of the mouse SRP9/14 heterodimer, determined at 2.5 A resolution. SRP9 and SRP14 are found to be structurally homologous, containing the same alpha-beta-beta-beta-alpha fold. This we designate the Alu binding module (Alu bm), an additional member of the family of small alpha/beta RNA binding domains. The heterodimer has pseudo 2-fold symmetry and is saddle like, comprising a strongly curved six-stranded amphipathic beta-sheet with the four helices packed on the convex side and the exposed concave surface being lined with positively charged residues.

Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice.

The purification of primitive human hematopoietic stem cells has been impaired by the absence of repopulation assays. By using a stringent two-step strategy involving depletion of lineage-positive cells followed by fluorescence-activated cell sorting, we have purified a cell population that is highly enriched for cells capable of multilineage repopulation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) recipients. These SCID-repopulating cells (SRCs) were exclusively found in a cell fraction that expressed high levels of CD34 and no CD38. Through limiting dilution analysis using Poisson statistics, we calculated a frequency of 1 SRC in 617 CD34(+) CD38(-) cells. The highly purified SRC were capable of extensive proliferation in NOD/SCID mice. Mice transplanted with 1 SRC (at limiting cell doses) were able to produce approximately 400, 000 progeny 6 weeks after the transplant. Detailed flow cytometric analysis of the marrow of highly engrafted mice demonstrated both lymphoid and myeloid differentiation, as well as the retention of a significant fraction of CD34(+) CD38(-) cells. These highly purified fractions should be useful for identification of the cellular and molecular mechanisms that regulate primitive human hematopoietic cells. Moreover, the ability to detect and purify primitive cells provides a means to develop conditions for maintaining and/or expanding these cells during in vitro culture.

Assay of human stem cells by repopulation of NOD/SCID mice.

The only conclusive method to assay stem cells is to follow their ability to repopulate conditioned recipients, making it difficult to study human stem cells. The development of systems to transplant human hematopoietic cells into immune-deficient mice lays the foundation for such an experimental repopulation assay for primitive human cells. Cell purification and gene marking studies have shown that the repopulating cells, termed severe-combined immunodeficiency (SCID) mouse-repopulating cells (SRC), are primitive and distinct from most of the progenitors that are detected using short and long-term in vitro culture assays. The SRC are exclusively CD34+CD38- and poorly infected with retrovirus vectors. These gene marking data are reminiscent of the human clinical trials establishing that the SRC assay is a good surrogate to develop improved transduction methods. Limiting dilution analysis has been used to establish a quantitative assay for SRC that can be used to precisely determine the effect of various cytokine cocktails on the proliferation and differentiation of SRC during in vitro culture.

Crystallization and preliminary crystallographic analysis of the signal recognition particle SRPphi14-9 fusion protein.

The SRPphi14-9 fusion protein, which can functionally replace the SRP9/14 heterodimer in the mammalian signal recognition particle (SRP), has been crystallized using the vapor diffusion method. Four different crystal forms were grown. SRPphi14-9 form IV crystals belong to the space group P4(1)22/ P4(3)22 with cell parameters a = b = 69.7 Angstroms, c = 95.7 Angstroms, alpha = beta = gamma = 90 degrees. A complete data set to 2.8 Angstroms resolution with an Rsym on intensities of 7.0% was collected on a single flash-frozen crystal.

Crystallization and preliminary X-ray analysis of the 9 kDa protein of the mouse signal recognition particle and the selenomethionyl-SRP9.

Two different crystal forms of the 9 kDa protein of the signal recognition particle (SRP9) have been prepared by the hanging drop vapor diffusion technique using 28% (w/v) PEG8000 or 28% saturated ammonium sulphate as precipitant. The crystals are hexagonal bipyramids with average dimensions of 0.2 X 0.1 X 0.1 mm(3) and they diffract to a resolution of 2.3 Angstroms. They belong to the space groups P6(2)22/P6(4)22 or P3(1)21/P3(2)21 with cell dimensions a = b = 63.0 Angstroms, and c = 111.5 Angstroms. Crystals have also been grown from the selenomethionyl protein and multiwavelength data sets have been collected.

Peripheral blood mononuclear cells of a patient with advanced Hodgkin's lymphoma give rise to permanently growing Hodgkin-Reed Sternberg cells.

A novel Hodgkin's disease (HD) derived cell line, L1236, was established from the peripheral blood of a patient with advanced Hodgkin's disease. Analysis of immunoglobulin (Ig) gene rearrangements revealed a biallelic Ig heavy chain and a monoallelic Ig kappa light chain gene rearrangement, pointing to a B-lymphoid origin of these cells. No DNA of Epstein-Barr virus was detected in L1236. The cells expressed the HD-associated surface antigens CD30 and CD15 as well as the transferrin receptor (CD71). Cytogenetic analysis of early passages of L1236 cells revealed a grossly disordered karyotype including cytogenetic aberrations described previously in other HD-derived cell lines. The Hodgkin/Reed-Sternberg (H-RS) cell origin of L1236 cells is further confirmed by Kanzler et al (Blood 87:3429, 1996), who found identical Ig gene rearrangement sequences in L1236 cells and H-RS cells of the same patient's bone marrow. L1236 cells expressed antigens necessary for efficient antigen presentation to T cells including HLA class I and II, B7.1 and B7.2, as well as adhesion molecules ICAM 1 and LFA 3. The cells secreted the interleukins (IL)-6, -8, -10, tumor necrosis factor (TNF) alpha, interferon (IFN) gamma, transforming growth factor (TGF) beta, and the granulocyte-macrophage colony stimulating factor (GM-CSF). After subcutaneous inoculation into SCID mice, a necrotic regression of initially growing tumors at the injection site was followed by disseminated intralymphatic growth. Our findings, together with the results of Kanzler et al, demonstrate that H-RS cells of B-lymphoid origin were present in the peripheral blood of a patient with advanced HD. These cells exerted a malignant phenotype with regard to their in vitro and in vivo characteristics.