Efficient and durable gene marking of hematopoietic progenitor cells in nonhuman primates after nonablative conditioning.

Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 microgram/kg) and G-CSF (20 microgram/kg) for 7 days and autologous CD34(+) peripheral blood stem cells harvested by leukapheresis. CD34(+) cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% +/- 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% +/- 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naïve (CD45RA(+) and CD62L(+)) CD4(+) and CD8(+) cells were the predominant phenotype of the marked CD3(+) T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naïve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.

Early genes induced in hepatic stellate cells during wound healing.

Activation of mesenchymal cells is a central event in the wound healing response of most tissues. In liver, the mesenchymal element responsible for organ fibrosis is the hepatic stellate cell (HSC) (formerly known as lipocyte or Ito cell). The phenotypic cascade of stellate cell activation in liver fibrosis has been well documented and involves both marked morphologic changes and upregulation of several functional components including extracellular matrix, cytokine receptors, contractile filaments and metalloproteinases. However, the genetic regulation of stellate cell activation is poorly understood. In an attempt to clone genes that are involved in the regulation of HSC activation we have combined cDNA library amplification by PCR with subtraction hybridization/differential screening, and have successfully identified genes induced in vivo during early stellate cell activation in a rat model of liver fibrosis. The subtracted cDNA library comprised less than 100 unique sequences. Of these, 13 clones with sizes ranging from 322 to 745 were sequenced and characterized. Gene induction in HSCs was monitored by RNAse protection assay during early liver injury induced by the hepatotoxin CCl4. The sequenced cDNAs corresponding to the known genes included type II transforming growth factor beta receptor, glutathione peroxidase I, transferrin and several clones encoding cellular retrotransposons, whose expression was not previously identified in non-parenchymal liver cells. In addition, one partial cDNA predicted a zinc-finger motif, suggesting a possible role of a novel transcriptional regulator. Our approach represents a valuable strategy for clarifying in vivo regulatory mechanisms of mesenchymal cell activation in wound healing.

High-resolution cell cycle analysis of defined phenotypic subsets within primitive human hematopoietic cell populations.

We have developed a novel protocol for analysis of cell cycle status within specific subsets of primitive human hematopoietic cells. The technique, referred to as SID (surface, intracellular, and DNA) analysis, allows for the simultaneous characterization of cell surface and intracellular antigens, as well as quantitation of DNA content. To evaluate the technique, early human hematopoietic cells were examined using surface staining for the CD34 and CD38 antigens to identify primitive cells. Relative cell cycle status within defined phenotypic subsets (CD34+ and CD34+/CD38-) was determined by simultaneous two-parameter analysis using DNA content vs. antibody staining for the Ki-67 antigen. Ki-67 is not expressed in quiescent cells, but is quickly up-regulated as cells are induced to cycle. Consequently, expression of Ki-67, in combination with DNA content can be used to delineate all phases of the cell cycle (G0, G1, S, and GZ/M). We demonstrate that cycle induction of CD34+ cells, using IL-3, IL-6, and stem cell factor (SCF), does not correlate with activation of the CD34+CD38- subpopulation during ex vivo culture. Rather, CD34+/CD38- cells are much more refractory to cycle activation, requiring at least 72 hours to show significant levels of induction. In addition, primitive cells derived from bone marrow (BM) vs. mobilized peripheral blood (PB) show differing degrees of responsiveness to conventional ex vivo culture conditions. Finally, the effect of IL-3, IL-6, SCF, and Flt3 ligand (FL) on cycle induction was examined. It was observed that IL-3 synergized strongly with IL6+SCF to activate quiescent CD34+/CD38- cells. Moreover, when FL was combined with IL-3+IL-6+SCF, there was a small but reproducible increase in activation of CD34+/CD38- cells from G0 to G1. These data suggest that ex vivo behavior of primitive human stem cell populations is amenable to comprehensive flow cytometric analysis, and that such studies can provide detailed information on the biological response of stem cells to ex vivo culture and manipulation.

Leukemic burden in subpopulations of CD34+ cells isolated from the mobilized peripheral blood of alpha-interferon-resistant or -intolerant patients with chronic myeloid leukemia.

We attempted to determine the frequency of normal hematopoietic stem cells (HSC) and contaminating leukemic cells in mobilized peripheral blood (MPB) collected from chronic myeloid leukemia (CML) patients, intolerant of alpha-interferon or with interferon-resistant disease. A total of 14 MPB samples, six from patients in chronic phase (CP) and eight from patients in accelerated phase or blast crisis (AP/BC) were studied. Cytogenetic analysis of MPB collected from AP/BC patients showed that 100% of the cells were Ph+, whereas cells from four of five CP MPB were Ph-. By contrast, fluorescence in situ hybridization (FISH) analysis of CP MPB showed a mean frequency of 14.7% Ph+ cells, while AP/BC MPB contained 39.2% Ph+ cells. In an attempt to purify normal HSC, subpopulations of the MPB CD34+ cells were isolated based on expression of the Thy-1 antigen (CDw90). The mean Ph+ cell frequency as determined by FISH within the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- populations from CP patients was 19.2% and 33.9%, respectively. In the AP/BC patients, levels of residual leukemic cells were significantly greater with mean Ph+ cell frequencies of 59.2% and 72.7% for the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- fractions, respectively. The frequency of cobblestone area forming cells (CAFC) was used as a means of quantitating the numbers of functional HSC within these cell subpopulations. The mean CAFC frequency was 1 of 19 for the CD34+Thy-1+Lin- cells as compared with 1 of 133 for the Thy-1-fraction indicating a higher frequency of primitive progenitor cells in the Thy-1+ subpopulation. CD34+ cell subsets from two patients were also injected into SCID-hu bone assays to determine the in vivo behavior of these cell populations. After 8 weeks, multilineage donor engraftment was observed in these grafts. FISH analysis of the donor cells within the grafts showed that 55.3% and 60.0% of the cells were Ph+. We conclude that unfractionated MPB from this patient population is not leukemia-free and that the CD34+Thy-1+Lin- cell subpopulation, although predominantly enriched for normal HSC, still contains substantial numbers of residual leukemic cells.

Induction of beta-platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture.

A consistent response to liver injury is the activation of resident mesenchymal cells known as lipocytes (Ito, fat-storing cells) into a proliferating cell type. In cultured lipocytes, platelet-derived growth factor (PDGF) is the most potent proliferative cytokine, but requires the activation-dependent expression of its receptor protein (Friedman, S. L., and M. J. P. Arthur. 1989. J. Clin. Invest. 84:1780-1785); the role of PDGF receptor (PDGFR) in liver injury is unknown. We have examined PDGFR gene expression in freshly isolated lipocytes during liver injury and correlated these findings with a culture model of cellular activation. Whereas lipocytes from normal rats had no detectable transcript for the beta-PDGFR subunit, this mRNA was induced within 1 h after a dose of carbon tetrachloride (CCl4). In contrast, alpha subunit mRNA was detected in normal cells, but was unchanged after liver injury. Similar results were observed in lipocytes from bile duct-obstructed rats, although beta-PDGFR induction was less marked. By immunoblot, induction of beta-PDGFR protein in lipocytes isolated from CCl4-treated animals correlated with mRNA increases. In contrast to lipocytes, endothelial cells from normal liver expressed low levels of alpha- and beta-receptor subunit mRNA, which did not increase with injury. Using a beta-PDGFR antibody, receptor protein could be identified within fibrotic septa in CCl4-treated animals in regions where cells expressed proliferating cell nuclear antigen (PCNA). In cultured lipocytes activated by growth on uncoated plastic, beta-PDGFR transcripts appeared within 3 d after plating, which coincided with the onset of cellular proliferation. In contrast, quiescent cells in suspension culture had no detectable beta-PDGFR mRNA. These results indicate that beta-PDGF receptor induction by lipocytes is an early event during hepatic injury in vivo and in primary culture.

Modulation of transforming growth factor beta receptors of rat lipocytes during the hepatic wound healing response. Enhanced binding and reduced gene expression accompany cellular activation in culture and in vivo.

Activation of lipocytes, characterized by increased proliferation and fibrogenesis, is a central feature of the hepatic wound healing response. We have examined whether modulation of receptors for transforming growth factor beta (TGF-beta) contributes to the fibrogenic behavior of activated lipocytes. Isolated lipocytes were maintained in a quiescent state by culturing the cells in suspension, where they displayed minimal specific binding for TGF-beta 1 and only a small amount of type III (betaglycan) receptor by affinity labeling. In contrast, lipocytes activated by growth on uncoated plastic displayed saturable binding of TGF-beta 1 (Kd = 28 pM, 7,730 receptors/cell), and receptors types I, II, and III. Binding activity in quiescent and activated cells correlated with responsiveness to TGF-beta 1; TGF-beta 1 induced cellular fibronectin mRNA expression only in activated and not quiescent cells. Despite the absence of binding in quiescent cells, type II receptor was detectable by immunoblot. By RNase protection assay, mRNAs for receptor types II and III were greater in quiescent than activated cells. In freshly isolated lipocytes from animals with liver injury caused by the administration of carbon tetrachloride, a rapid but transient increase in mRNA for receptor types I (approximately 3.2-fold), II (approximately 1.5-fold), and III (approximately 3-fold) was observed with peaks at 12 h for type I receptor, 1 h for type II receptor, and 6 h for type III receptor; mRNA induction was followed by down-regulation for all receptors. The modest changes in mRNAs compared with marked alterations in binding activity during mesenchymal cell activation suggest that TGF-beta receptors may be regulated in vivo in part by a post-translational mechanism.

Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture.

The development of techniques for isolating hepatic lipocytes (Ito, stellate or fat-storing cells) from rodents has been instrumental in defining their role in hepatic vitamin A storage and fibrogenesis. In this study, we developed a method for the purification of lipocytes and Kupffer cell from wedge sections of normal human liver and examined their properties in primary culture. Sections of donor liver (400 to 600 gm) harvested but not used for transplantation were perfused in situ with University of Wisconsin solution and used for lipocyte isolation within 48 hr. Cells were isolated by catheter perfusion of the wedge through several large vessels with L-15 salts, Pronase and collagenase, followed by Larex density gradient centrifugation. Lipocytes were plated on either uncoated plastic or a basement membrane-like gel. Lipocyte and Kupffer cell yields were 2.3 +/- 0.6 x 10(5) and 8.6 +/- 1.4 x 10(5) cells, respectively, per gram of liver (n = 5). Lipocyte purity was 91% as assessed by vitamin A autofluorescence, and Kupffer cell purity was 83% as determined by uptake of fluorescinated staphylococci. Lipocytes cultured on the plastic spread within 48 to 72 hr, displaying slightly more heterogeneous retinoid droplet size than comparable rat cells; on a basement-membrane gel, the cells remained aggregated and spherical with occasional spindlelike extensions. Lipocytes on plastic expressed procollagens I and III, collagen IV and laminin by immunocytochemistry, and types I, III and IV procollagen messenger RNAs by RNAse protection. Northern blot and polymerase chain reaction, respectively. Transmission electron microscopy of lipocytes at 7 days demonstrated a prominent rough endoplasmic reticulum and contractile filaments. Scanning electron microscopy revealed a smooth cell surface with perinuclear droplets beneath the cell membrane. With continued primary culture on plastic (more than 7 days), cells appeared "activated" (i.e., increased spreading and diminished retinoid droplets) and began proliferating as assessed by nuclear autoradiography and [3H]thymidine incorporation. Kupffer cells observed by scanning electron microscopy in early primary culture displayed prominent membrane ruffling and lamellipodia. In summary, we have established a reproducible method for the isolation and primary culture of human lipocytes and Kupffer cells.

Hybrid bioartificial liver in hepatic failure: preliminary clinical report.

A hybrid bioartificial liver has been developed on the principle of hemodialysis against a suspension of functioning hepatocytes. The new chimeric device of synthetics and living parts was used successfully to treat a patient in hepatic failure due to an inoperable bile duct carcinoma that involved the bifurcation of the common duct.

Insulin-releasing activity and successful transplantation of pancreatic islets preserved by tissue culture.

Preserving pancreatic islets for 7 days by a making use of the organ culture, we studied the insulin-releasing activity at the time of administration of glucose and various digestive tract hormones for the purpose of clarifying the function of preserved pancreatic islets. Furthermore, we transplanted pancreatic islets preserved for 3 to 5 days into the portal vein of rats with streptozotocin-induced diabetes and reached the following conclusions: (1). The islets of Langerhans of the pancreas responded well to glucose up to the seventh day of preservation and showed patterns similar to those of fresh pancreatic islets with respect to both the dose response and the time response. (2). Preserved pancreatic islets of the pancreas had insulin-releasing activity almost equal to that of fresh pancreatic islets against stimulation by glucagon, tolbutamide, and various digestive tract hormones. (3). Rats with streptozotocin-induced diabetes showed a marked improvement in blood glucose and urine glucose following transplantation of preserved pancreatic islets into the portal vein, and this effectiveness persisted for 6 to 8 weeks.

Compact far ultraviolet emission source with rich spectral emission 1150-3100 A.

A new compact far uv emission source suitable for laboratory or space applications is described. The source is small, rugged, lightweight, spectrally rich, and bright in the 1150-3200-A region. It has met rigid spacecraft environmental tests and also is a very useful transfer standard for absolute sensitivity calibrations of spectrometric instruments.