Our goal is to develop a cellular strategy for repairing the damage seen in children's myelin disease, Multiple Sclerosis and other neurological diseases.

 

Research Progress Report

Seung U. Kim, MD, PhD
Division of Neurology, Department of Medicine
UBC Hospital, University of British Columbia
2211 Wesbrook Mall, Vancouver, BC V6T 2B5, Canada
Tel: 604 822-7145 FAX: 604 822-7897
E-mail: sukim@interchange.ubc.ca

Human neural stem cell lines - Stable, continuously growing cell lines of human neural stem cells (hNSC) were generated via a retroviral vector encoding v-myc. These hNSC cell lines carry normal human karyotype pf 46XX or 46 XY and express nestin, vimentin and ABCG2, cell type-specific markers for NSCs (Cho et al., 2003; Ryu et al., 2003). One of the hNSC cell line, HB1.F3 (other lines are F5, A4 and A7), has been successfully utilized in experimental animal models of Parkinson disease, Huntington disease (Ryu et al., 2004), stroke (Chu et al., 2003; Jeong et al., 2003) and pediatric storage disease (Sly disease/ mucopolysaccharidosis VII, Meng et al., 2003). Presently we have in storage of following 16 sublines of F3 including F3.LacZ, F3.eGFP, F3.TH, F3.GTPCH, F3.TH.GTPCH, F3.BDNF, F3.GDNF, F3.Nurr1, F3.Bcl-XL, F3.Olig2, F3.Mash1, F3.Neurogenin1, F3.NeuroD, F3.beta-glucuronidase, F3.CD and F3.FUC. Last two sublines are for brain tumor therapy.

New human neural stem cell line suitable for human clinical trials - Since our immortalized human neural stem cell (HB1.F3) line was generated using v-myc oncogene, institutional review boards of Universities/Hospitals are cautious and slow to grant permission to use these v-myc encoded hNSC cell lines for the clinical trials in human patients. To circumvent this issue, we have recently generated a new hNSC cell line using tet-on-v-myc system (HB2.G2). In this system, v-myc is activated in the presence of tetracycline antibiotic, thus in the absence of tetracycline the immortalized hNSCs do not multiply and differentiate into neurons. When HB2.G2 hNSCs were grafted into rat brain they survive well and develop into neurons. HB2.G2 hNSC line appears most suitable hNSCs for cell source for the cell therapy for patients with neurological disorders. Recently we have generated sublines of G2 including G2.eGFP and G2.GDNF.

Huntington disease (HD) - We have proactively grafted F3 hNSC cells in the brain of rat HD model and the grafted F3 hNSCs protected host neurons from cell death (Ryu et al., 2004). Dr. B. Leavitt of UBC has previously generated a transgenic HD mouse model, and the animals were given grafts of F3 hNSCs over-expressing BDNF (F3.BDNF) and final results will be available in September 2004.

Parkinson disease (PD) - We have transduced F3 hNSC cell line to carry two enzyme genes required for dopamine production, TH and GTPCH genes. There was a 2000-fold increase in L-DOPA (800 ng/106 cells/ 24 h) as compared to non-transduced parental F3 cells. L-DOPA is a dopamine precursor that is deficient in PD brain. We initiated animal studies in rat PD models at the UBC and preliminary results indicate that the cell line is effective in repairing behavioral deficits in PD animal models. Dr. Evan Snyder of the Burnham Institute has recently generated primate models of PD and asked me to provide F3.TH.GTP and F3.GDNF cell lines for brain transplantation in these animals. We will send him the cell lines under a collaborative arrangement.

Stroke - My collaborators at the Seoul University Hospital has published two papers in animal models of stroke (there are two forms of stroke, focal ischemia and haemorrhage) showing that intravenously introduced hNSCs migrate to brain and restore fuctional deficits (focal ischemia: Chu et al., 2003; cerebral hemorrhage: Jeong et al., 2003). F3 hNSCs injected into tail vein of rat stroke model, migrate into the lesion sites and induce improvement in functional parameters such as corner test or rotarod test. We have recently generated F3.BDNF and F3.GDNF sub lines carrying neurotrophic factor genes. These modified hNSCs are currently grafted into rat models of focal/global ischemia and cerebral haemorrhage to see if these hNSCs should provide neuroprotection of host neurons from cell death.

Paediatric lysosomal storage diseases - F3 hNSCs carrying beta-glucuronidase gene correct clinical course and pathology of Sly disease (mucopolysaccharidosis VII) animals (Meng et al., 2003; this work was performed in collaboration with Prof. Y. Eto of Jikei Medical University). Prof. Y. Eto is currently working in the area of Krabbe and Gauchet diseases using F3 hNSCs. I am interested in collaborating with Kyushu University investigators who carry adrenoleukodystrophy (ALD) transgenic mice (Prof. J. Kira of Neurology Department) and we will generate F3/G2 hNSCs over expressing ALDP gene and transplant into ALD mice.

Brain tumour/ glioblastoma – Our collaborator, Dr. Karen Aboody (formerly at the Harvard Neurosurgery and currently at the City of Hope Medical Center), has generated most exciting findings for F3 hNSCs to be used for treatment of brain tumour. F3 hNSCs could selectively and specifically migrate into brain tumour loci when grafted into the tumor carrying animals. In collaboration with her, we have produced F3 hNSCs carrying suicide genes such cytosine deaminase (CD) or CD-TK (hybrid gene of CD and herpes virus thymidine kinase). When F3.CD or F3.CD-TK hNSCs are introduced intracerebrally or intravenously, they migrate selectively into the brain tumour loci and when prodrugs such as fluorocytosine or gancyclovil are applied, hNSCs commit suicide and release anti-cancer drugs and kill the tumor cells by “by-stander-effect”. Since F3 hNSCs carry suicide gene, the question of v-myc oncogene becomes non-issue here. I am certain that the clinical trials in brain tumour/gliobastoma could be the first cell therapy trial utilizing the hNSCs we generated. We have already started animal experiments using F3.CD cell line at the Ajou University (Suwon, Korea), City of Hope Medical Center (Dr. K. Aboody), Harvard Neurosutrgery (Dr. P. Black) and Nagoya University Neurosurgery (Prof. J. Yoshida).

More recently Dr. Mary Danks (St. Jude Children’s Medical Center) has informed us that F3 NSCs transduced with therapeutic transgene carboxylesterase (CE) via adenovirus selectively migrated into metastatic neuroblastoma loci in mice and killed tumor cells. Human neuroblastoma cells are injected into the tail veins of mice to produce metastatic tumor in bone marrow, liver and spleen, and intravenously introduced F3.CE cells selectively infiltrate metastatic neuroblastoma loci, and product-turned anti-cancer drug induces tumour cell death. We have recently generated a F3.CE sub line using a retroviral vector and the line will be provided to Dr. Danks shortly for animal studies.

ALS - When F3 hNSCs were injected into the tail vein of mouse ALS model (carrying a mutant superoxide dismutase/ SOD gene), LacZ-labeled F3 hNSCs were found in hippocampus 3-4 weeks later. Only small number of F3.LacZ cells was found in spinal cord. We are currently performing long-term follow up of the animals to see if F3 cells arrive at spinal cord lesions. This project is carried out in Suwon, Korea. If this work produces positive results, we expect to initiate clinical trials in Korea.

The new sub line F3.Olig2 (see section 9) is demonstrated to express motor neuron marker HB9. We are continuing our research effort to develop motor neuron from the F3.Olig2 cell line.

Generation of neurons and oligodendrocytes from neural stem cells -Recently we have introduced neurogenic master genes into HB1.F3 hNSCs to induce neurons or oligodendrocytes from the NSCs. These master genes (basic helix loop helix transcription factors) include NeuroD, Neurogenin1 and Mash1 for induction of neurons and Olig2 for oligodendrocytes, were introduced in the F3 hNSCs and push these cells to develop into neurons or oligodendrocytes (F3.Neurogenin1, F3.NeuroD, F3.Olig2 have been generated previously). By introduction of NeuroD gene in F3 hNSCs, we have demonstrated that the F3 cNSCs differentiate into neurons as determined by generation of sodium current, a specific marker for neurons (Cho et al., 2003). When and if we generate a large number of olgiodendrocytes, then we will implant them in animal models of demyelination.

Human bone marrow mesenchymal stem cell line -We generated immortalized cell lines of human bone marrow stem cells (from fetal human bone marrow) using retroviral vectors carrying v-myc (as in F3 hNSC line, BM3.B10) or teromerase gene (BM4.C3). The BM4.C3 cell line is quite unique since it carries teromerase catalytic unit gene and unlike hNSC line carrying v-myc this cell line does not pose risk for tumour formation. These hBMSC cell lines we produced are pluripotent and they could transdifferentiate into bone, cartilage, fat cells and neurons (these results are most recent). It appears that hBMSCs has endless potential for clinical application much more than ES cells that gets world-wide attention these days.

Human neural crest stem cell line -We have previously generated immortalized cell lines of human neural crest stem cells (HNC10.K1, K10) detailed account on the generation and characterization of the cell line has been published previously (Kim et al., 2002; Nakagawa et al., 2002).

Immortalized human microglia lines -We have generated an immortalized human microglia cell line (HMO6.N2, Nagai et al., 2003) for investigation in new drug discovery in the field of neuroinflammation. Neuroinflammation is known to be cause of neurological disorders such as Alzheimer disease, stroke and spinal cord injury. In addition micrroglia is a good candidate as a therapeutic vehicle to carry transgene or drug into the brain since we have evidence that the intravenously injected human microglia cells (HMO6) migrated successfully into the brain parenchyma. This line was provisionally licensed to the Astra-Zeneca and Amgen.

We have produced immortalized cell lines of human mygenic progenitors (HM4), human osteocyte progenitors (HO5), human chondrocyte progenitors (three cell lines, each from cartilage tissues of ear, nose and knee joint), human liver hepatocyte progenitors (HL7) and human heart myocardial progenitors (HH8). Most of these cell lines have been generated via retroviral vector encoding v-myc and carry cell type-specific markers of their normal counterparts.

Recently we have published a paper in which we reported that human umbilical cord blood-derived hematopoietic cells (CD-133-positive cells selectively purified by magnetic cell sorting and FACS) could be induced to differentiate into neurons and glial cells by treatment with retinoic acid (Jang et al., 2004).

References:

Cho T, Bae JH, Choi HB, Min CG, Kim SU. Human neural stem cells: Electrophysi-ological properties of voltage-gated ion channels. Neuroreport 13, 1447-1452, 2002.

Ryu JK, Choi HB, Hatori K, Heisel R, Pelech S, McLarnon JG, Kim SU. Adenosine triphosphate induces proliferation of human neural stem cells: Role of calcium and p70 ribosomal protein S6 kinase. J Neurosci Res 72, 352-362, 2003.
Ryu JK, Kim J, Hong SH, Choi HB, Lee MC, Kim SU. Proactive transplantation of human neural stem cells blocks neuronal cell death in rat model of Huntington disease. Neurobiol Disease 16, 68-77, 2004

Meng X, Shen J, Ohashi T, Sly WS, Kim SU, Eto Y. Brain transplantation of genetically engineered human neural stem cells globally corrects brain lesions in mucopolysaccharidosis VII mouse. J Neurosci Res 74, 266-277, 2003.

Chu K, Kim M, Jeong SW, Kim SU, Yoon BW. Human neural stem cells can migrate, differentiate and integrate after intravenous transplantation in adult rats with transient forbrain ischemia. Neuroci Lett 343, 637-643, 2003.
Jeong SW, Chu K, Jung KH, Kim MH, Kim SU, Roh JK. Human neural stem cell transplantation in experimental intracerebral hemorrhage. Stroke 34, 2258-2263, 2003.

Kim SU, Nakagawa E, Hatori K, Nagai A, Lee MA, Bang JH. Production of immortalized human neural crest stem cells. In: Neural Stem Cells: Methods and Protocols, Eds: T. Zigova, J. Sanches, Humana Press, Totowa, NJ, pp 55-65, 2002.

Nakagawa E, Hatori K, Nagai A, Choi HB, Lee MA, Bang JH, Kim J, Ryu JK, Lee MC, Snyder EY, Kim SU. Generation, characterization and transplantation of immortalized human neural crest stem cells. In: Neural Stem Cells for Brain and Spinal Cord Repair, Eds: T. Zigova, EY Snyder, P Sanberg, Humana Press, Totowa, NJ, pp 89-106, 2002.

Nagai A, Nakagawa E, Hatori K, Choi HB, McLarnon JG, Lee MA, Kim SU. Genration and characterization of immortalized human microglial cell lines: Expression of cytokines and chemokines. Neurobiol Disease 8, 1057-1068, 2001.

Jang YK, Park JJ, Lee MC, Yoon BH, Yang YS, Yang SE, Kim SU. Retinoic acid-mediated induction of neurons and glial cells from human umbilical cord derived hematopoietic stem cells. J Neurosci Res 74, 573-584, 2004.

St. Kitts Biomedical Research Foundation celebrates 20th Anniversary

BASSETERRE, ST. KITTS (JULY 26TH 2002) –

A major announcement is expected this weekend that the St. Kitts Biomedical Research Foundation will get funding from the United States Public Health Service to allow the world-class primate facility here to embark on a series of experiments to determine whether human neural stem cells can cure Parkinson’s disease in monkeys.

Dr. Redmond will announce funding of a new grant to the Foundation by the United States Public Health Service to embark on a series of experiments to determine whether human neural stem cells can cure Parkinson’s diease in monkeys.

Human Parkinson’s results from unknown processes which kill dopamine cells, causing muscle rigidity, lack of coordination, difficulty moving and tremors. Human neural stem cells, which are primordial and uncommitted, can be propagated in large numbers and then safely differentiated into the necessary dopamine-producing neurons after they are injected into the brain. "The human neural stem cells migrate to populate developing or degenerating brain regions, perhaps allowing a functionally correct and effective reconstruction,' Dr. Redmond said.

Retinoic acid-mediated induction of neurons and glial cells from human umbilical cord-derived hematopoietic stem cells.

Jang YK, Park JJ, Lee MC, Yoon BH, Yang YS, Yang SE, Kim SU.

Brain Disease Research Center, Ajou University School of Medicine, Suwon, Korea.

Recent studies reporting trans-differentiation of mononucleated cells derived from human umbilical cord blood into neuronal cells aroused interest among investigators for their clinical implication and significance in regenerative medicine. In the present study, purified populations of hematopoietic stem cells were isolated via magnetic bead sorting and fluorescence-activated cell sorter (FACS) using a specific CD133 antibody, a cell type-specific marker for hematopoietic stem cells, and grown in culture in the presence of retinoic acid (RA). CD133+ hematopoietic stem cells expressed neuronal and glial phenotypes after RA treatment. RT-PCR analysis indicated that the RA treated CD133+ cells expressed mRNA transcripts for ATP-binding cassettes transporter ABCG2 (a universal stem cell marker), nestin (a specific cell type marker for neural stem cells), Musashi1 (a specific marker for neural stem cells) and RA receptors (RAR) including RAR-alpha, RAR-beta, and retinoid X receptor (RXR)-gamma. RA-treated CD133+ cells expressed mRNA transcripts for neuron-specific markers neurofilament proteins (NF-L, -M, -H) and synaptophysin as determined by RT-PCR, structural proteins characteristic of neurons including tubulin beta III and neuron specific enolase (NSE) by Western blot, and neuron-specific markers NeuN and microtubule-associated protein-2 (MAP2) by immunocytochemistry. RA-treated CD133+ cells also expressed the astrocyte-specific marker glial fibrillary acidic protein (GFAP), as demonstrated by RT-PCR, Western blot, and immunocytochemistry. In addition, RA-treated CD133+ cells expressed cell type-specific markers for oligodendrocytes including myelin basic protein (MBP) as shown by RT-PCR, proteolipid protein (PLP) by Western blot analysis, and cyclic nucleotide phosphodiesterase (CNPase) by immunostaining. Upregulated expression of several basic helix-loop-helix (bHLH) transcription factors important for early neurogenesis, including Otx2, Pax6, Wnt1, Olig2, Hash1 and NeuroD1, was also demonstrated in CD133+ cells after RA treatment. These results indicate that human cord blood-derived CD133+ hematopoietic stem cells could trans-differentiate into neural cell types of neuron-like cells, astrocytes, and oligodendrocytes by RA treatment. Copyright 2003 Wiley-Liss, Inc.




posted by Canadian Myelin Research Initiative at 10:37 PM