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Strategies for transplanting primitive hematopoietic progenitor (PHP) cells are under development that require in vitro manipulation of cells for several hours to several days prior to transplantation. This applies to gene-therapy protocols involving transduction with adenoviral or lentiviral vectors (typically 1 day of ex vivo culture) or retroviral vectors (up to 3 days of culture).Human mobilized peripheral blood (MPB) CD34(+) cells were cultured with the cytokines thrombopoietin mimetic peptide (mTPO), flt3 ligand (FL), and c-kit ligand (KL). Equal numbers of CD34(+) cells, either uncultured or cultured for various time periods up to 5 days, were tested for engraftment in sublethally irradiated 8-10 week-old NOD/SCID mice. Cells were also compared for expression and function of several key surface molecules.At a limiting dose of 1 million cells, mice receiving uncultured cells had a mean of 20% CD45(+) (human) cells in their BM 6 weeks post-transplantation, versus 3% for mice receiving 3-5 day cultured cells. Analysis of 10 surface molecules, CD11a, CD18, CD29, CD49d, CD49e, CXCR-4, CD62L, CD31, CD43, and CD44 over a 5-day culture period showed that their expression levels were either maintained or up-regulated on CD34(+) cells and the primitive Thy-1(+) subset. Similar percentages of uncultured and 3-day cultured MPB CD34(+) cells bound to plates coated with vascular cell adhesion molecule-1 (VCAM-1) under both static and physiological flow conditions, and chemotaxis of cultured cells towards stromal-derived factor-1 (SDF-1) was not impaired, suggesting that VLA-4 and CXCR-4 were functional on cultured cells. CD34(+) Thy-1(+) MPB cells cultured with cytokines expressed increasing levels of Fas receptor beginning at 20 h in culture, with peak expression levels after 3 days (mean Day 0 expression, 39%; mean Day 3 expression, 86%), without increased apoptosis. Including inhibitors of caspases in the media of cells cultured for 24-48 h significantly improved their engraftment in a SCID-hu bone-engraftment model.Increased susceptibility to apoptosis upon in vivo injection may contribute to impaired engraftment of in vitro manipulated cells. Inhibitors of apoptosis may increase their engrafting capacity in clinical settings.

The sequence of a novel hemopoietic cytokine was discovered in a computational screen of genomic databases, and its homology to mouse thymic stromal lymphopoietin (TSLP) suggests that it is the human orthologue. Human TSLP is proposed to signal through a heterodimeric receptor complex that consists of a new member of the hemopoietin family termed human TSLP receptor and the IL-7R α-chain. Cells transfected with both receptor subunits proliferated in response to purified, recombinant human TSLP, with induced phosphorylation of Stat3 and Stat5. Human TSLPR and IL-7Rα are principally coexpressed on monocytes and dendritic cell populations and to a much lesser extent on various lymphoid cells. In accord, we find that human TSLP functions mainly on myeloid cells; it induces the release of T cell-attracting chemokines from monocytes and, in particular, enhances the maturation of CD11c+ dendritic cells, as evidenced by the strong induction of the costimulatory molecules CD40 and CD80 and the enhanced capacity to elicit proliferation of naive T cells.

Absence of durable high-level expression of transgenes from Moloney murine leukemia (Mo-MuLV) retroviral vectors has been a hurdle in bringing effective gene therapy to the clinic. In this study we have analyzed transgene expression among the progeny of mobilized hematopoietic stem cells (HSCs), comparing Mo-MuLV and mouse stem cell virus (MSCV) vectors, with or without addition of a scaffold attachment region (SAR) from the human interferon beta gene. Retroviral (RV) vector supernatant quality was assessed by comparing NGFR transgene expression by HEL cells, and transgene delivery and expression by CD34(+) cells 72 hr after transduction, using real-time PCR and FACS analysis. This is the first description of the effect of SAR within both Mo-MuLV and MSCV vector backbones on long-term RV transgene expression among in vivo HSC progeny in HSC repopulation assays (SCID-hu bone and NOD/SCID). After transduction of mobilized CD34(+) cells with MSCV-SAR vector, transgene expression was observed among a mean of 10% of donor HSC progeny in the SCID-hu bone (range, 0.6-43%). The predominant effect of SAR was to increase the mean fluorescence intensity (MFI) of transgene expression among HSC progeny in both in vivo bone repopulation models (three- to fourfold), and after long-term stromal cultures (twofold).

CD34+Thy-1+Lin− cells are enriched for primitive hematopoietic progenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic stem cells (HSC), as defined by the in vivo SCID-hu bone assay. We evaluated the effects of different cytokine combinations on BM-derived PKH26-labeled CD34+Thy-1+Lin− cells in 6-day stroma-free cultures. Nearly all (>95%) of the CD34+Thy-1+Lin− cells divided by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34hiPKHlo (postdivision) cell population retained a high CAFC frequency, a mean 3.2-fold increase of CAFC numbers, as well as a capacity for in vivo marrow repopulation similar to freshly isolated CD34+Thy-1+Lin− cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal loss of CD34 and Thy-1 expression. In contrast, cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory factor contained a mean of 75% of undivided cells at day 6. These CD34hi PKHhi cells retained a high frequency of CAFC, whereas the small population of CD34hiPKHlo postdivision cells contained a decreased frequency of CAFC. These data suggest that use of a combination of TPO, KL, and FL for short-term culture of CD34+Thy-1+Lin− cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity for in vivo engraftment.

The CD34 antigen is expressed on most, if not all, human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and its use for the enrichment of HSCs with repopulating potential is well established. However, despite homology between human and murine CD34, its expression on subsets of primitive murine hematopoietic cells has not been examined in full detail. To address this issue, we used a novel monoclonal antibody against murine CD34 (RAM34) to fractionate bone marrow (BM) cells that were then assayed in vitro and in vivo with respect to differing functional properties. A total of 4% to 17% of murine BM cells expressed CD34 at intermediate to high levels, representing a marked improvement over the resolution obtained with previously described polyclonal anti-CD34 antibodies. Sixty percent of CD34+ BM cells lacked lineage (Lin) markers expressed on mature lymphoid or myeloid cells. Eighty-five percent of Sca-1+Thy-1(10)Lin- /10 cells that are highly enriched in HSCs expressed intermediate, but not high, levels of CD34 antigen. The remainder of these phenotypically defined stem cells were CD34-. In vitro colony-forming cells, day-8 and -12 spleen colony-forming units (CFU-S), primitive progenitors able to differentiate into B lymphocytes in vitro or into T lymphocytes in SCID mice, and stem cells with radioprotective and competitive long-term repopulating activity were all markedly enriched in the CD34+ fraction after single-parameter cell sorting. In contrast, CD34-BM cells were depleted of such activities at the cell doses tested and were capable of only short-term B-cell production in vitro. The results indicate that a significant proportion of murine HSCs and multilineage progenitor cells express detectable levels of CD34, and that the RAM34 monoclonal antibody is a useful tool to subset primitive murine hematopoietic cells. These findings should facilitate more direct comparisons of the biology of CD34+ murine and human stem and progenitor cells.

Mammalian skeletal muscle is generated by two waves of fiber formation, resulting in primary and secondary fibers. These fibers mature to give rise to several classes of adult muscle fibers with distinct contractile properties. Here we describe fast myosin heavy chain (MyHC) isoforms that are expressed in nascent secondary, but not primary, fibers in the early development of rat and human muscle. These fast MyHCs are distinct from previously described embryonic and neonatal fast MyHCs. To identify these MyHCs, monoclonal antibodies were used whose specificity was determined in western blots of MyHCs on denaturing gels and reactivity with muscle tissue at various stages of development. To facilitate a comparison of our results with those of others obtained using different antibodies or species, we have identified cDNAs that encode the epitopes recognized by our antibodies wherever possible. The results suggest that epitopes characteristic of adult fast MyHCs are expressed very early in muscle fiber development and distinguish newly formed secondary fibers from primary fibers. This marker of secondary fibers, which is detectable at the time of their inception, should prove useful in future studies of the derivation of primary and secondary fibers in mammalian muscle development.

Background Increasing use of phenotypically-enriched stem cell populations for clinical hematopoietic transplants has led to an urgent demand for a reliable, rapid and simple functional assay which would provide an estimation of the reconstituting potential of cells prior to transplantation. Methods We have developed a 2-week quantitative, stroma-free assay to measure the frequency of primitive progenitors within hematopoietic cell samples. This relatively short-term assay provides frequency information which correlates with that measured by a 5-week stroma-dependent CAFC assay. Cells with the phenotype CD34 + Thy-1 + were purified by fluorescenceactivated cell sorting from peripheral blood apheresis products of multiple myeloma patients mobilized with cytoxan and GM-CSF. CD34 + Thy-1 + cells were plated at limiting dilution into microtiter wells and cultured in an Iscove's based serumdeprived culture medium, supplemented with the cytokines, interleukin (IL)-3, IL-6, G-CSF, Flk2/Flt3 ligand (FL) and Kit ligand (KL). After 2 weeks, cell proliferation in individual wells was quantified by microscopy and bright-field imaging, or by using a fluorescent nucleic acid-binding dye and fluorimetry. Poisson statistics were used to calculate the frequency of wells containing cells with high proliferative potential (wells containing ≥500 cells). Results Progenitor cell frequencies generated using this assay were compared by linear regression analysis to those generated from 32 parallel CAFC and CFU-C assays performed on the same patient samples. Correlations were r=0.80, r 2 =0.65, and r=0.76, r 2 =0.58, respectively; these correlations were highly significant (p –7 ). Discussion This limiting dilution assay should more directly quantitate the potential of primitive hematopoietic cells than a CFU-C assay. It also has advantages over both the CAFC and the CFU-C assay, in that scoring has been automated, making it simple, rapid, and objective compared with manual cobblestone area or colony counting. The described limiting dilution assay may provide a useful alternative to assays currently used to evaluate the viability and proliferative potential of purified hematopoietic cells intended for transplant.

A recombinant gene encoding human growth hormone (hGH) was stably introduced into cultured myoblasts with a retroviral vector. After injection of genetically engineered myoblasts into mouse muscle, hGH could be detected in serum for 3 months. The fate of injected myoblasts was assessed by coinfecting the cells with two retroviral vectors, one encoding hGH and the other encoding beta-galactosidase from Escherichia coli. These results provide evidence that myoblasts, which can fuse into preexisting multinucleated myofibers that are vascularized and innervated, may be advantageous as vehicles for systemic delivery of recombinant proteins.

IL-23 is a heterodimeric cytokine composed of the IL-12p40 “soluble receptor” subunit and a novel cytokine-like subunit related to IL-12p35, termed p19. Human and mouse IL-23 exhibit some activities similar to IL-12, but differ in their capacities to stimulate particular populations of memory T cells. Like IL-12, IL-23 binds to the IL-12R subunit IL-12Rβ1. However, it does not use IL-12Rβ2. In this study, we identify a novel member of the hemopoietin receptor family as a subunit of the receptor for IL-23, “IL-23R.” IL-23R pairs with IL-12Rβ1 to confer IL-23 responsiveness on cells expressing both subunits. Human IL-23, but not IL-12, exhibits detectable affinity for human IL-23R. Anti-IL-12Rβ1 and anti-IL-23R Abs block IL-23 responses of an NK cell line and Ba/F3 cells expressing the two receptor chains. IL-23 activates the same Jak-stat signaling molecules as IL-12: Jak2, Tyk2, and stat1, -3, -4, and -5, but stat4 activation is substantially weaker and different DNA-binding stat complexes form in response to IL-23 compared with IL-12. IL-23R associates constitutively with Jak2 and in a ligand-dependent manner with stat3. The ability of cells to respond to IL-23 or IL-12 correlates with expression of IL-23R or IL-12Rβ2, respectively. The human IL-23R gene is on human chromosome 1 within 150 kb of IL-12Rβ2.

Ex vivo cell cycling of hematopoietic stem cells (HSC), a subset of primitive hematopoietic progenitors (PHP) with engrafting capacity, is required for transduction with retroviral vectors and to increase transplantable HSC numbers. However, induction of division of HSC ex vivo also may lead to differentiation and loss of in vivo marrow repopulating potential. We evaluated mobilized peripheral blood (MPB) PHP for maintenance of stem cell function after ex vivo culture under conditions that we show can induce cycling of a majority of PHP with minimal differentiation. The following methods were combined: cell labeling with the division tracking dye carboxyfluorescein-diacetate succinimidylester (CFSE), analysis of primitive cell surface marker expression, an ex vivo PHP assay, and an in vivo marrow repopulating assay. MPB-purified CD34+ Thy-1+ cells were labeled with CFSE dye and cultured for 112 hours in serum-deprived medium in the presence of the cytokine combinations of thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL), or TPO, FL, and interleukin 6 (IL-6). Both cytokine combinations supported division of greater than 95% of cells within 112 hours with an average 2.1-fold (TPO, FL, KL) or 1.3-fold (TPO, FL, IL-6) increase in total cell numbers. An average of 21.6% (TPO, FL, KL) and 27.4% (TPO, FL, IL-6) of the divided cells still expressed the Thy-1 marker after 112 hours. Functional assays were performed to compare cultured and uncultured cells. CD34+ Thy-1+ CFSElo (post division) cells showed maintenance of cobblestone area-forming cell (CAFC) frequency (a mean of 1/9.0) relative to the starting population of uncultured CD34+ Thy-1+ cells (a mean of 1/8.4). In contrast, CD34+ cells that had lost Thy-1 expression during culture (CD34+ Thy-1 CFSElo) showed a mean 5.8-fold reduction in CAFC frequency (a mean of 1/52.5). Only the Thy-1-expressing fraction of cells post culture could engraft in vivo in the SCID-hu bone assay. Because the majority of HSC functional activity post culture was found in the CD34+ Thy-1+ fraction, we focused on this fraction for subsequent analysis. CFSE labeling allows segregation and purification by flow cytometry of cells having undergone discrete numbers of divisions during culture. Very few cells that divided more than four times in culture still expressed Thy-1. Cells that retained expression of Thy-1 during culture retained CAFC activity relative to fresh CD34+ Thy-1+ cells, after undergoing at least two divisions. CAFC frequency decreased after four divisions in culture with TPO, FL, and KL or after three divisions in TPO, FL, and IL-6. We then compared populations of Thy-1+ cells that had undergone sequential numbers of divisions in culture for their ability to engraft in the SCID-hu bone assay. Engrafting ability was retained throughout four divisions in both cytokine combinations. These data demonstrate that primitive MPB CD34+ cells maintain HSC function coincident with Thy-1 expression while undergoing two to four divisions under these culture conditions. Essentially all CD34+ Thy-1+ cells divided under the conditions tested, promoting susceptibility to retroviral transduction.

The early stages of lymphold cell formation were studied by testing the differentiative potential of phenotypically defined subsets of CD34+ bone marrow cells. A subpopulation of CD34+ Lin− CD45RA+ cells expressing CD10 was isolated by flow cytometry. Such cells are CD38+ , HLA-DR+, do not express significant levels of Thy-1 and c-kit, lack erythroid, myeloid, megakaryocytic potential, and give rise only to lymphoid T, B, natural killer (NK), and dendritic cells (DC) in kinetics and titration experiments. Limiting dilution analysis demonstrates the existence of multipotential B/NK/DC progenitor clones in the CD34hi Lin−CD10+ adult bone marrow cell population. Thus, nonprimitive progenitors for lymphoid cells and for DCs can be distinct from those of myeloid, megakaryocytic, and erythroid cells, implying that the DC lineage is developmentally more closely related to the lymphoid lineage than to the myeloid lineage. This study provides new insights into the organization and development of the human lympho-hematopoietic system.

Myosin heavy chain (MyHC) isoforms show a striking diversity of expression patterns during mammalian development. Using a set of monoclonal antibodies that recognize different epitopes on myosin heavy chain isoforms we show that there exist in human and rat skeletal muscle at least three isoforms of slow twitch myosin heavy chain. To facilitate a comparison of our results to others obtained using different antibodies or species, we have identified cDNAs encoding the epitopes recognized by the three slow antibodies. Using these reagents, we show that the onset of expression of three slow MyHC isoforms is temporally distinct during early gestation. This result suggests that a sequence of MyHC transitions plays an important role in determining muscle fiber function at fetal, neonatal, and adult stages.

Some investigators have proposed myoblast transfer as a potential therapy for the treatment of Duchenne muscular dystrophy. Little is known about the immunobiology of myoblast transplantation. Transplantation rejection is mediated to a large extent by CD8+ T cells, which recognize alloantigens encoded by class I HLA genes, and by CD4+ T cells, which recognize alloantigens encoded by class II HLA genes. Gamma interferon (IFN-gamma) is a potent inducer of HLA class II molecules as well as beta 2-microglobulin, which is co-expressed with HLA class I. IFN-gamma may be a critical cytokine involved in graft rejection. We purified human myoblasts by flow cytometry and incubated them in vitro for varying time periods with recombinant human IFN-gamma. The inducibility of HLA-DR and -DP molecules raises a note of caution concerning possible rejection phenomenon which might occur following myoblast transplantation.

Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. We show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. Our results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also to a direct effect on muscle. We propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.

Myosin heavy chains are encoded by distinct members of a multigene family at different stages of muscle development. Study of the underlying regulatory mechanisms has been hindered because transitions in myosin expression have not been readily attained in tissue culture. Here we show a transition from early (fetal) to late (perinatal/adult) myosins defined by two monoclonal antibodies, F1.652 and N3.36, in the myotubes of mouse C2C12 cells. On day 1 of differentiation, essentially all myosin was early myosin. By day 8, early myosin dropped to 25% of its day 1 value and was replaced by late myosin. The transition occurred without neural contact, connective tissue components, or complex substrates, suggesting that its regulation may be intrinsic to the muscle cell. Our results demonstrate that a developmental progression in myosin gene expression, which occurs rapidly, with high frequency, and under relatively simple conditions, is now amenable to molecular analysis in cultured muscle cells.

Vertebrate myosin heavy chains (MHC) are represented by multiple genes that are expressed in a spatially and temporally distinct pattern during development. In order to obtain molecular probes for developmentally regulated human MHC isoforms, we used monoclonal antibodies to screen an expression cDNA library constructed from primary human myotube cultures. A 3.4 kb cDNA was isolated that encodes one of the first MHCs to be transcribed in human skeletal muscle development. A portion of the corresponding gene encoding this isoform has also been isolated. Expression of this embryonic MHC is a hallmark of muscle regeneration after birth and is a characteristic marker of human muscular dystrophies. During normal human development, expression is restricted to the embryonic period of development prior to birth. In primary human muscle cell cultures, devoid of other cell types, mRNA accumulation begins as myotubes form, reaches a peak 2 days later and declines to undetectable levels within 10 days. The expression of the protein encoded by the embryonic skeletal MHC gene follows a similar time course, lagging behind the mRNA by approximately two days. Thus, expression of the human embryonic gene is efficiently induced and then repressed in cultured muscle cells, as it is in muscle tissue. The study of the regulation of a human MHC isoform with a central role in muscle development and in muscle regeneration in disease states is therefore amendable to analysis at a molecular level.

An efficient Th1-driven adaptive immune response requires activation of the T cell receptor and secretion of the T cell stimulatory cytokine IL-12 by activated antigen-presenting cells. IL-12 triggers Th1 polarization of naive CD4(+) T cells and secretion of IFN-gamma. We describe a new heterodimeric cytokine termed IL-27 that consists of EBI3, an IL-12p40-related protein, and p28, a newly discovered IL-12p35-related polypeptide. IL-27 is an early product of activated antigen-presenting cells and drives rapid clonal expansion of naive but not memory CD4(+) T cells. It also strongly synergizes with IL-12 to trigger IFN-gamma production of naive CD4(+) T cells. IL-27 mediates its biologic effects through the orphan cytokine receptor WSX-1/TCCR.

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage-- (Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin-ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA-cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.