Punam Malik

Pediatric Tumor Cells Express Erythropoietin and a Functional Erythropoietin Receptor that Promotes Angiogenesis and Tumor Cell Survival

Erythropoietin was traditionally considered an erythroid-restricted cytokine, but recent evidence indicates a broader role for it in nonhematopoietic tissues, specifically in neural development. Pediatric solid tumors are mostly developmental in origin, and more than 50% of the solid tumors are neural in origin. We found erythropoietin receptor and erythropoietin expression in common pediatric tumor cells: neuroblastomas, Ewings sarcoma family of tumors, pediatric brain tumors (medulloblastoma, astrocytoma, and ependymoma), Wilms tumors, rhabdomyosarcomas, and hepatoblastomas (n = 24), and in cell lines derived from some of these tumors (n = 25). Expression of erythropoietin in tumor cell lines was hypoxia-inducible. Addition of exogenous erythropoietin to tumor cell lines expressing erythropoietin receptor increased nuclear DNA binding activity of nuclear factor kappa B and increased the expression of the antiapoptotic genes bcl-1, bcl-xL, and mcl-1. Additionally, exogenous erythropoietin increased production and secretion of angiogenic growth factors, vascular endothelial growth factor, or placenta growth factor from the tumor cell lines, which promoted endothelial cell proliferation and chemotaxis. Erythropoietin receptor expression that promotes tumor cell survival and releases angiogenic growth factors in pediatric tumors has not been previously described. Therefore, a careful evaluation of the impact of erythropoietin is warranted in vivo, in xenograft models of pediatric tumors, followed by evaluation in pediatric patients with cancer.

Ineffective erythropoiesis in β-thalassemia major is due to apoptosis at the polychromatophilic normoblast stage

Beta-thalassemia major is characterized by ineffective erythropoiesis, although it is difficult to define the dynamics of this process from the static information revealed by analysis of bone marrow (BM) aspirates. We aimed to study the kinetics of sequential erythroid differentiation in beta-thalassemia major. We isolated the progenitor cells (CD34(+) and CD34(+)CD38(-) cells) from BM of thalassemia major patients and studied in vitro erythropoiesis. This is the first report of an in vitro study in human beta-thalassemia major from purified BM CD34(+) progenitor cells, using erythroid culture conditions, which allow unilineage differentiation to mature enucleated red blood cells. In contrast to normal donors, a high proportion of BM CD34(+) and CD34(+)CD38(-) progenitors from beta-thalassemia major coexpressed the late erythroid lineage-specific protein glycophorin A and generated a higher proportion of erythroid colonies. However, despite the marked increase in erythroid clonogenicity of the progenitor population, erythroid cultures initiated from beta-thalassemia major BM CD34(+) cells expanded 10- to 20-fold less than from normal BM. There were less viable cells during differentiation, specifically after the polychromatophilic normoblast stage. There was a progressive increase in the apoptotic erythroid progeny with differentiation, and apoptosis occurred predominantly at the polychromatophilic normoblast stage. In thalassemia major, BM progenitor cells show increased erythroid clonogenicity, increased expression of late erythroid lineage-specific proteins, and accelerated erythroid differentiation. However, despite the apparent increased erythroid commitment, ineffective erythropoiesis occurs due to apoptosis at the polychromatophil stage. Identification of the differentiation stage at which apoptosis occurs will permit further studies of the underlying mechanisms and target therapeutic strategies to improve red cell production.

Retinal Pigment Epithelium Cells Promote the Maturation of Monocytes to Macrophages in vitro

Proliferative vitreoretinopathy is characterized by excessive cell proliferation within the eye; retinal pigment epithelial (RPE) cells form the majority of proliferating cells and interact with infiltrating leukocytes including monocytes. The purpose of this study was to determine the effect of RPE cells on the maturation of monocytes to macrophages. The enriched monocyte fraction of peripheral blood mononuclear cells was either cultured with or without RPE cells. The expression of the maturation-associated antigen CD16 on monocytes was assessed by flow cytometry, and the concentration of bioactive transforming growth factor-beta (TGF-beta) in the culture supernatant measured by mink lung epithelial cell (Mv1Lu) bioassay. The cellular density of CD16 in terms of mean fluorescence intensity was significantly higher on monocytes in coculture with RPE cells (p = 0.0153) than on monocytes in monoculture. The CD16 expression was significantly (p = 0.0093) reduced when antibodies to TGF-beta were added to the culture medium. RPE cells did not express CD16. Supernatants from cocultures also contained active TGF-beta (76.7 +/- 23.8 pg/ml), while in those of cell monocultures TGF-beta was close to the detection limit. We conclude that RPE cells stimulate and modulate the differentiation of monocytes to macrophages. Bioactive TGF-beta generated in the coculture was in part responsible for this effect. It seems likely that RPE cells or interactions between RPE cells and monocytes could be an important factor in inflammatory/immune processes and wound healing in the eye, which are probably involved in proliferative vitreoretinopathy.

Monocyte-Macrophage Differentiation Induced by Coculture of Retinal Pigment Epithelium Cells with Monocytes

Macrophages play an important role in proliferative vitreoretinopathy (PVR). Since macrophage maturation may be modulated by the local microenvironment, we determined the effect of retinal pigment epithelium (RPE) cells interacting with monocytes on macrophage maturation. The enriched monocyte fraction of peripheral blood mononuclear cells was cocultured with RPE cells. Cell-free supernatants conditioned during the culture periods 0-4 days (growing RPE cells) and 4-8 days (confluent RPE cells) were tested for their capacity to induce monocyte/macrophage differentiation of the promyelocytic cell line HL-60, which was measured by the expression of CD11c and CD14 and flow cytometry. RPE cells released factors that increased the CD14 expression on HL-60 cells in terms of percentage of positive cells (22.7% vs. control 10.4%). RPE-cell-conditioned supernatants had no effect on the CD11c expression. Monocytes secreted substances that increased the expression of CD11c (20.5% vs. control 9.1%; p = 0.003) and CD14 (31.6% vs. control 10.4%; p < 0.0001). Supernatants from cocultures increased the CD11c (19.8%) and CD14 expression (40.8%) to values that were similar to the sum of those of cell monocultures. Supernatants conditioned during the later culture had no effect on CD14 and CD11c expression. We conclude that invading monocytes and RPE cells could create an intraocular microenvironment that supports macrophage maturation during the initial stage of PVR.

Report on the Workshop “New Technologies in Stem Cell Research,” Society for Pediatric Research, San Francisco, California, April 29, 2006

RNA interference (RNAi) is a powerful tool with which to study gene function, especially in stem cells. Small interfering RNAs (siRNAs) can effectively be introduced either with a vehicle or through viral vectors to transiently or stably inhibit the expression of a particular gene target. Much is known about the optimization of siRNAs and method of delivery in mammalian cells. In this review, we discuss design considerations for siRNAs, methods of delivery, optimization of siRNAs, applications to study genes in stem cells, therapeutic applications, and remaining hurdles. With recent advances in RNAi, it is likely that application of this technology will increase in the future. Correspondence: Kathleen M. Sakamoto, M.D., Ph.D., Division of Hematology-Oncology, Mattel Children’s Hospital at UCLA, 10833 Le Conte Avenue, Los Angeles, California, 90095-1752. Telephone: 310-794-7007; Fax: 310-206-8089; e-mail: kms@ucla.edu Received June 28, 2006; accepted for publication January 5, 2007; first published online in STEM CELLS EXPRESS January 25, 2007. ©AlphaMed Press 1066-5099/2007/

467. Lentiviral Vector (LV) Non-Coding Elements That Impart High Titers and Expression to β-Globin (hβ) Cassettes

30.00/0 doi: 10.1634/stemcells.2006-0397 MEETING REPORT STEM CELLS 2007;25:1070–1088 www.StemCells.com

Engineered cytokine secretion from human mesenchymal stem cells influences in vivo hematopoiesis

Top of pageAbstract nThalassemias are the most common monogenic defects that result from absent/reduced h|[beta]| in RBC. However, the complexities of the h|[beta]| gene and its regulatory elements have been significant obstacles for optimization of gene therapy approaches using |[gamma]|-oncoretroviral vectors. Recently, LV have been shown to stably transfer and express h|[beta]| at high levels. The goal of our study was to identify non-coding elements in LV required for this effect. A series of LV carrying different elements from the gag and env fragments were made. All vectors encoded h|[beta]| gene driven by |[beta]|-regulatory elements (|[beta]|-promoter, HS234 and |[beta]| 3 enhancer), contained the same 5 and 3 SIN-LTR: sBG-0 was a gutted LV vector containing only the packaging (|[Psi]| region including 40bp of the 5 gag).In sBG-1 the central polypurine tract (cPPT) element was added to sBG-0.In sBG-2, the rev response element (RRE) was added to sBG-1. In sBG-3 and sBG-4, the wt or the mutated splice acceptor (SA) in the env sequence was added to sBG-2, respectively. In sBG-5, |[sim]|600bp of gag and RRE was added to sBG-1. In sBG-6, the |[sim]|400bp of gag was added to sBG-3. All vectors were packaged using the standard transient co-transfection system in 293T cells, concentrated 1400-fold and assayed in differentiated mouse erythroleukemia (MEL) cells. The level of h|[beta]| expression in MEL cells was analyzed by RNase protection assay (RPA) and FACS analysis with 3 independent viral lots. sBG-6 had the highest titers (6|[times]|10E8 TU/mL) while the gutted LV vector sBG-0 and sBG-1 had very poor titers (Fig. 1A). Addition of RRE (sBG-2) increased the titers very significantly. Addition of the env fragment (sBG-3) further increased titers nearly 2-3 fold. However, when the SA in the env gene was mutated (sBG-4), the titers of sBG-4 vectors dropped to levels lower than sBG-2, despite sBG-3 and sBG-4 containing RRE, suggesting that some contribution of titers by the env fragment in sBG-3 reside in the SA, and its mutation has a negative effect on RRE. Replacement of a |[sim]|600bp of gag in place of the env (+SA) in sBG-5 was sufficient to improve titers to 84% of sBG-6. These results were confirmed at the RNA level (RPA in Fig. 1B). When probed with an LCR fragment, Northern blot analysis showed stable transmission of full-length LV mRNA in 293T cells, with varying ratio of spliced and unspliced LTR transcripts in different vectors. The mean fluorescence intensity of h|[beta]| expression in differentiated MEL cells was similar with all vectors, suggesting that upon integration, viral cis elements probably play a minimal role in improving expression of the h|[beta]| transcripts, although this is under investigation; as is RNA export and stable transmission from these vectors. In conclusion, while the rev/RRE in HIV is likely the most important element imparting high titers to h|[beta]| cassettes, the env SA and regions of gag appear to contribute to the titers from SIN-LV.

Gene therapy of a mouse model of protoporphyria with a self-inactivating erythroid-specific lentiviral vector without preselection.

Abstract In the current studies, we examined the duration and effects of cytokine cDNA introduction and protein secretion from human mesenchymal stem cells (HuMSC), in beige/nude/xid (bnx) mice. HuMSC were engineered to secrete human Erythropoietin, Interleukin (IL-)3, or IL-7, using MoMuLV-based retroviral vectors. Engineered MSC were introduced via neo-organoid implantation, biochamber implantation, or intravenous injection. The latter route resulted in the highest sustained levels of human cytokines in the murine serum. Sequentially measured systemic levels of huEpo were 1.1±0.4 U/ml serum at 1 month post-transplantation and 0.6±0.1 U/ml at 2 months (N=8). HuIL-7 levels were 281±29 pg/ml at 1 month and 45.7±6 at 6 months (N=7). HuIL-3 levels were 209.4±15.5 pg/ml at 1 month and 35.5±5 at 6 months (N=7). To determine whether the decrease was due to cell loss or to reduction in expression, we are now implanting the engineered MSC in a dissolvable polymer, to allow precise recovery of the vascularized cell mass. There were significant influences from the MSC on the development of co-transplanted human hematopoietic stem cells (HSC). HuIL-3 allowed huHSC survival in bnx mice, with none in control animals. HuEpo significantly increased the hematocrit and production of BFU-E and mature RBC. Human RBC were blocked at the BFU-E stage in mice that did not receive huEpo. HuIL-7 significantly increased the levels of human CD3+CD4+ T cells that developed from purified HSC in the murine marrow. Retroviral-mediated engineering of human MSC is an effective model to study the impact of sustained, supraphysiological levels of specific cytokines on co-transplanted human hematopoietic stem cells.