Judson Ratliff

Blastula-stage stem cells can differentiate into dopaminergic and serotonergic neurons after transplantation.

In order to assess the potential of embryonic stem cells to undergo neuronal differentiation in vivo, totipotent stem cells from mouse blastocysts (D3 and E14TG2a; previously expanded in the presence of leukemia inhibitory factor) were transplanted, with or without retinoic acid pretreatment, into adult mouse brain, adult lesioned rat brain, and into the mouse kidney capsule. Intracerebral grafts survived in 61% of cyclosporine immunosuppressed rats and 100% of mouse hosts, exhibited variable size and morphology, and both intracerebral and kidney capsule grafts developed large numbers of cells exhibiting neuronal morphology and immunoreactivity for neurofilament, neuron-specific enolase, tyrosine hydroxylase (TH), 5-hydroxytryptamine (5-HT), and cells immunoreactive for glial fibrillary acidic protein. Though graft size and histology were variable, typical grafts of 5-10 mm3 contained 10-20,000 TH+ neurons, whereas dopamine-beta-hydroxylase+ cells were rare. Most grafts also included nonneuronal regions. In intracerebral grafts, large numbers of astrocytes immunoreactive for glial fibrillary acidic protein were present. Both TH+ and 5-HT+ axons from intracerebral grafts grew into regions of the dopamine-lesioned host striatum. TH+ axons grew preferentially into striatal gray matter, while 5-HT+ axons showed no white/gray matter preference. These findings demonstrate that transplantation to the brain or kidney capsule can induce a significant fraction of totipotent embryonic stem cells to become putative dopaminergic or serotonergic neurons and that when transplanted to the brain these neurons are capable of innervating the adult host striatum.

Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation

The controlled differentiation of mouse embryonic stem (ES) cells into near homogeneous populations of both neurons and skeletal muscle cells that can survive and function in vivo after transplantation is reported. We show that treatment of pluripotent ES cells with retinoic acid (RA) and dimethylsulfoxide (DMSO) induce differentiation of these cells into highly enriched populations of gamma-aminobutyric acid (GABA) expressing neurons and skeletal myoblasts, respectively. For neuronal differentiation, RA alone is sufficient to induce ES cells to differentiate into neuronal cells that show properties of postmitotic neurons both in vitro and in vivo. In vivo function of RA-induced neuronal cells was demonstrated by transplantation into the quinolinic acid lesioned striatum of rats (a rat model for Huntingtons disease), where cells integrated and survived for up to 6 wk. The response of embryonic stem cells to DMSO to form muscle was less dramatic than that observed for RA. DMSO-induced ES cells formed mixed populations of muscle cells composed of cardiac, smooth, and skeletal muscle instead of homogeneous populations of a single muscle cell type. To determine whether the response of ES cells to DMSO induction could be further controlled, ES cells were stably transfected with a gene coding for the muscle-specific regulatory factor, MyoD. When induced with DMSO, ES cells constitutively expressing high levels of MyoD differentiated exclusively into skeletal myoblasts (no cardiac or smooth muscle cells) that fused to form myotubes capable of spontaneous contraction. Thus, the specific muscle cell type formed was controlled by the expression of MyoD. These results provided evidence that the specific cell type formed (whether it be muscle, neuronal, or other cell types) can be controlled in vitro. Further, these results demonstrated that ES cells can provide a source of multiple differentiated cell types that can be used for transplantation.

Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

TM601 is a synthetic form of chlorotoxin, a 36-amino acid peptide derived from the venom of the Israeli scorpion, Leirius quinquestriatus, initially found to specifically bind and inhibit the migration of glioma cells in culture. Subsequent studies demonstrated specific in vitro binding to additional tumor cell lines. Recently, we demonstrated that proliferating human vascular endothelial cells are the only normal cell line tested that exhibits specific binding to TM601. Here, we identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and human umbilical vein endothelial cell (HUVEC). We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2. Identification of annexin A2 as a binding partner for TM601 is also consistent with the anti-angiogenic effects of TM601. Annexin A2 functions in angiogenesis by binding to tissue plasminogen activator and regulating plasminogen activation on vascular endothelial cells. We demonstrate that in HUVECs, TM601 inhibits both vascular endothelial growth factor- and basic fibroblast growth factor-induced tissue plasminogen activator activation, which is required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits platelet-derived growth factor-C induced trans-well migration of both HUVEC and U373-MG glioma cells.

Long-term survival of fetal porcine lateral ganglionic eminence cells in the hippocampus of rats

Embryonic porcine brain tissue from the lateral ganglionic eminence was transplanted into the adult rat hippocampus to determine whether fetal striatal cells could survive, differentiate, and integrate in a heterotopic site. The hippocampus, a common site of epileptic seizure activity, was chosen to determine if fetal striatal cells could supply inhibitory GABAergic neurons that may serve to block seizures. Cells were either implanted with a single deposit using a standard metal cannula or by five smaller disseminated deposits with a glass micropipette. At 20–24 weeks, animals immunosuppressed with cyclosporin showed long‐term survival of porcine cells in the adult hippocampus. Analysis by immunohistochemistry and in situ hybridization showed that the grafts contained glial and neuronal cell types, including GABAergic neurons within graft core and networks of porcine neuronal fibers extending from the graft into the host parenchyma. In addition, a marker of porcine presynaptic terminals, synaptobrevin, was abundant within the grafts and was found associated with hippocampal structures and cell layers suggesting functional integration of grafted cells within the host. The survival of xenografts in the hippocampus and potential integration of inhibitory components provides evidence that these grafts may serve as an internal negative feedback mechanism to quench epileptiform activity. J. Neurosci. Res. 56:581–594, 1999. 

Regulated expression of the homeobox gene, rPtx2, in the developing rat

Using degenerate primers designed to amplify genes containing homeodomains, we have used reverse transcription and polymerase chain reaction to amplify and clone a rat homeobox gene. Based on the nucleotide and predicted amino acid sequences, the rat cDNA clone contains a high degree of sequence similarity to murine genes which are members of the paired-like class of homeobox genes (Ptx2, Otlx2, solurshin and Ptx1). Considering the high degree of sequence similarity and similar restricted expression patterns, we have named the cloned rat gene rPtx2 (rat Ptx2 homolog). Northern analysis revealed two rPtx2 transcripts expressed in the developing rat brain. Yet, only a single gene was detected by Southern blot hybridization, suggesting that multiple messages are the result of alternative transcriptional initiation, splicing or processing of a common message. The expression pattern of rPtx2 was further delineated by in situ hybridization to rat embryos. Within the brain, tissue specific expression was observed in the differentiating neural cells of the posterior hypothalamus, tegmentum, and rhombomere r1. Expression was also observed in the developing pituitary, maxilla, mandible, tongue and umbilical cord. To further study the control of Ptx2 gene expression, we used an in vitro model for neural differentiation by treating mouse embryonic stem cells with retinoic acid. Within 24 h and prior to detection of a neural phenotype in the culture, murine Ptx transcripts were induced and remained elevated for at least 6 days. This suggests that retinoic acid may be an important inductive signal which regulates the developmental and tissue-specific expression of Ptx2.

Comparison of fresh and cryopreserved porcine ventral mesencephalon cells transplanted in A rat model of Parkinson's disease

To evaluate whether cryopreservation of porcine ventral mesencephalon cells influences graft survival and function in vivo, we have transplanted either freshly prepared or cryopreserved cells into the striatum of 6‐hydroxydopamine‐lesioned rats. A single cell suspension of porcine ventral mesencephalon cells from the same isolation either was stored at 4°C and transplanted the next day or was cryopreserved for 4 weeks in liquid nitrogen vapor. The cryopreserved cells were then rapidly thawed, rinsed, and transplanted in the same manner as the fresh cells, with the same dose of viable cells. All animals received daily injections of cyclosporin A to prevent xenograft rejection. To monitor graft function, amphetamine‐induced rotation was measured every 3 weeks between 6 and 15 weeks posttransplantation. After sacrifice at 15 weeks posttransplantation, histological methods were used to compare fresh cell and cryopreserved cell transplants with respect to graft survival, differentiation and integration, and host immune response. Cryopreserved cells were found to be either equivalent or in some cases superior to fresh cells with respect to rotational correction, graft survival, graft volume, numbers of graft‐derived dopaminergic neurons, and host immune responses. In conclusion, the results indicate that it is feasible to cryopreserve porcine ventral mesencephalon cells for long‐term storage of cells prior to transplantation in an animal model of Parkinsons disease.

Abstract B217: Annexin A2 is a novel molecular target for TM601, a peptide with specific tumor targeting and antiangiogenic properties

In this study we sought to identify the cell surface target for TM601, a peptide with tumor‐targeting and anti‐angiogenic effects. The 131I‐radiolabeled form of TM601 is currently in clinical trials for having been shown to specifically bind several human tumor types including malignant glioblastoma, colorectal, pancreatic, prostate and non‐small cell lung carcinomas. We used a biochemical approach of surface cross‐linking followed by affinity‐binding assays in conjunction with mass spectrometry and siRNA knock‐down to identify and validate the target for TM601 on tumor and vascular endothelial cell. We identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and HUVEC vascular endothelial cells. We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line, Panc‐1 is dependent on the expression of annexin A2. Annexin A2 is over‐expressed in several human cancers and has functions in angiogenesis by binding to tissue plasminogen activator (tPA) and regulating plasminogen activation on vascular endothelial cells. In HUVEC cells, TM601 inhibits both VEGF and bFGF induced tPA activation which is in turn required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits PDGF‐CC induced trans‐well migration of both HUVEC vascular endothelial and U373‐MG glioma cells. In conclusion, we have identified annexin A2 as a novel cell surface target for TM601 that might mediate both the tumor‐targeting and anti‐angiogenic effects of the peptide. Importantly, our results also support a role annexin A2 as a molecular target in cancer therapeutics. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B217.