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

 
Dear Wayne,

I am writing this note to inform you of recent development in my research activity.

(1) I agreed to provide the HB1.F3 human neural stem cell line and its sub lines to Evan Snyder to be implanted in the brain of monkey Parkinson disease (PD) models. Dr. Eugene Redmond of Yale and Dr. Ted Teng of Harvard at the Primate Center in St Kitts will conduct the brain transplantation. In addition to F3 parental cell line, F3.LacZ and F3.eGFP (both cell lines carry cell-specific tags so that one can identify grafted cells in the animal brain), F3.TH.GTP (this cell line makes L-DOPA 2000 times over parental F3 cells; L-DOPA is a precursor molecule of dopamine which is short supply in PD patients' brain), F3.GDNF (GDNF is a neurotrophic factor well known to protect host neurons in PD patient's brain) and F3.Nurr1 (Nurr1 is a master gene to induce dopaminergic neurons). I have already sent F3, F3.LacZ and F3.eGFP cell lines to Snyder. In the month of October, I will provide him with F3.TH.GTP, F3.GDNF and F3.Nurr1 cell lines to be propagated and implanted in PD monkeys.

(2) Dr. Karen Aboody (City of Hope Medical Center -One of the most active cancer hospital in the world located outside of Los Angeles) and Dr. Peter Black (Chief of Neurosurgery at the Brigham and Women’s Hospital-Harvard and one of the top brain tumour specialists) are currently using F3.CD (cytosine deaminase, a suicide gene) cell line we previously developed for brain tumour treatment. F3 human NSC showed extensive migratory and tumour-tropic properties, and could be used for treatment of invasive brain tumours. Intracranial injection of human F3.CD Neural Stem Cells (NSCs) carrying a suicide gene cytosine deaminase (CD) resulted in a significant anti-tumour response in rat glioma models. Moreover, intravascularly delivered human NSCs used to target human primary and metastatic tumours in animal models of neuroblastoma and breast cancer show promise. Dr. Mary Danks of St Jude Children's Medical Center in Memphis USA, is conducting a neuroblastoma study and Dr. Carlotta Glackin of the City of Hope Medical Center is performing a breast cancer.

(3) We will concentrate our research effort during the next 6 months to develop sub lines of the new human NSC cell line, HB2.G2. We generated this new cell line by the use of Tet-on-v-myc system. We required a v-myc oncogene to produce our human neural stem cell line F3, and so there is a risk of these cells developing a brain tumour once transplanted into a patients' brain. For that reason, we used the Tet-on-v-myc system by which activates v-myc only in the presence of tetracycline. In the absence of tetracycline, NSCs will not increase in number but differentiate into neurons. We have already generated sub lines of G2 including G2.eGFP, G2.LacZ and G2.BDNF (G2.BDNF was grafted into rat model of stroke and improved clinical outcome markedly).

(4) We have generated a new human bone marrow mesenchymal stem cell lines using a retoroviral vector carrying v-myc, and we have successfully produced bone, cartilage, fat cells and neurons from these bone marrow stem cells. We will collaborate with other investigators to develop pancreatic insulin producers, heart muscle cells and liver parenchymal cells from these human bone marrow stem cells.

The following is an abstract of my presentation at the Annual meeting of the Japanese Neuropathology Society in Nagoya in May 2004:

IMMORTALIZED HUMAN NEURAL STEM CELL LINE GENETICALLY MODIFIED FOR BRAIN REPAIR

Seung Up Kim, MD, PhD
Brain Disease Research Center, Ajou University School of Medicine, Suwon, Korea; Department of Neurology, University of British Columbia, Vancouver, Canada

Cell replacement and gene transfer to the diseased or injured CNS have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases including Parkinson disease (PD), Huntington disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD), stroke and spinal cord injury. In PD and HD, previous studies have documented improvements in motor and cognition performance in patients following fetal human brain cell transplantation. However, ethical, religious and logistic difficulties associated with the use of fetal tissues limit wide adoption of this approach. An ideal source of cell therapy in neurological diseases is human neural stem cells (NSCs) that could integrate into host brain tissue and differentiate into neurons or glial cells in response to environmental cues. Successful application of in vivo gene transfer to the CNS will depend on the identification of suitable cells that can serve as carriers for a wide range of potentially therapeutic transgenes and provide platforms for functionally efficient expression and secretion of transgene products. Immortalized NSCs have recently been introduced as potentially interesting candidates for this purpose.

We have previously generated stable cell lines of human NSCs from primary cultures of human fetal telencephalon via transfection with a retroviral vector encoding v-myc oncogene. One of the cell lines, HB1.F3, expresses nestin and ABCG2, both cell type-specific markers for neural stem cells and normal human karyotype of 46XX without any chromosomal abnormality. F3 NSC cell line has the ability to self-renew, differentiate into cells of neuronal and glail lineage (Cho et al., Neuroreport 13, 1447, 01), and integrate into the damaged CNS loci upon transplantation into the brain of animal models of stroke (Jeong et al., Stroke 34, 2258, 03; Chu et al., Brain Res 1016, 145, 04), HD (Ryu et al., Neurobiol Disease 16, 145, 04) and lysosomal storage disease (Meng et al., J Neurosci Res 74, 266, 03).

In animal models of PD, F3 NSC cell line was transduced to carry tyrosine hydroxylase (TH) and GTP cyclohydrolase I (GTPCHI), via retrovirally mediated procedures. HPLC analysis indicated that the level of L-DOPA produced by the F3.TH.GTP is 62 ng/hr/106 cells, a 2000-fold increase over parental F3 cell line. L-DOPA producing NSCs were transplanted into the striatum of PD rats to replace missing dopaminergic neurons, and the results indicated that there was a marked improvement in amphetamine-induced turning behavior and a good survival of TH-positive cells in the PD animals.

Human NSCs generated by us showed extensive migratory and tumor-tropic properties, and was used for the treatment of invasive tumors. NSCs meet two major challenges facing current gene therapy strategies: effective delivery and distribution of a therapeutic agent throughout the tumor masses. Recently we have demonstrated that the intracranial injection of human F3.CD NSCs carrying a suicide gene, cytosine deaminase (CD) resulted in a significant anti-tumor response in rat glioma models. In addition, intravascularly delivered human NSCs targeting human primary and metastatic tumors in animal models of neuroblastoma and breast cancer showed promise.

The results of the present study have particular relevance to cell therapy in neurodegenerative diseases including PD, HD, ALS and AD and also for stroke and spinal cord injury. NSC transplantation has the great potential to prevent or restore anatomic or functional deficits associated with CNS injury or disease via cell replacement, release of specific neurotransmitters, and production of molecules that promote neuronal growth and regeneration.

Grants from KOSEF/ BDRC-Ajou University and Canadian Myelin Research Initiative supported this research.

Regards, Seung Kim, MD
posted by Canadian Myelin Research Initiative at 10:26 PM

 

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

 
First Cloned Embryo Yields Stem Cells

This is the achievement reported on Thursday by a team of researchers led by Woo Suk Hwang of Seoul National University.

"Because these cells carry the nuclear genome of the individual, after differentiation they could be expected to be transplanted without immune rejection for treatment of degenerative disorders," Woo said. "Our approach opens the door for the use of these specially developed cells in transplantation medicine."

The study was published online by the journal Science ahead of a meeting in Seattle of the American Association for the Advancement of Science.

Until now, only mouse cells have yielded embryonic stem cells, and there have been many failures to do the same using human and monkey embryos.

The South Korean team used the classic cloning technique, pioneered in Dolly the sheep, which is to take an egg and remove the nucleus, which contains virtually all of the DNA code for programming the egg into a human being. They then replaced the nucleus with one taken from an adult non-reproductive cell, fused them together and then cultured the egg in a warm nutrient bath so that it divided and developed into an early embryo.

Where they improved on this technique, though, was on several fronts. They had access to an extraordinarily large batch of fresh eggs — 242 donated by 16 unpaid volunteers — which enabled them to finetune their work as they went alone.

They also had stringent programs for timing the way the eggs were handled, and used a new procedure that gently extruded the nucleus rather than suctioned it out, thus reducing the risk of DNA damage to the egg.

Out of 242 eggs, the team cultured 30 blastocysts, or fertilized eggs, of which 20 developed into embryonic cell clusters. Only one of these 20 developed into a stem cell line; the scientists suspect that some of the cloned blastocysts may have suffered chromosomal abnormalities seen in other primate cloning attempts.


posted by Canadian Myelin Research Initiative at 8:01 PM

 
Scientists Restore Crucial Myelin In Brains Of Mice



Scientists for the first time have restored a crucial substance known as myelin in a widespread area of an animal's brain, opening the door toward new ways to improve treatment of an assortment of "demyelinating" diseases as well as the side effects of such common conditions as high blood pressure and heart disease. The research by a team led by Steven Goldman, M.D., Ph.D., of the University of Rochester Medical Center, is in the January issue of Nature Medicine.

The work has implications for a wide variety of children's diseases known as pediatric leukodystrophies, where the myelin is damaged or doesn't work correctly, such as Canavan disease, Krabbe disease, or Tay-Sachs disease.

The team remyelinated the mice – restored the "insulation" to the brain cells– by injecting into the mice highly purified human "progenitor" cells, which ultimately evolve into the cells that make myelin. These cells are known as oligodendrocytes: While these and other types of glial cells aren't as well known as information-processing brain cells called neurons, they are vital to the brain's health.

Goldman says that while scientists have used other methods during the past two decades to remyelinate neurons in small portions of the brains of mice, the remyelination seen in the Nature Medicine paper is much more extensive. He estimates that about 10 percent of the axons in the mouse brains were remyelinated, compared to a tiny fraction of 1 percent in previous studies.

In addition to MS, many diseases affecting tens of millions of people in the United States involve myelin problems, Goldman says. These include widespread diseases like diabetes, heart disease and high blood pressure, where decreased blood flow can damage myelin and hurt brain cells, as well as strokes, which often destroy brain cells in part by knocking out the cells that pump out myelin. In addition, cerebral palsy is largely caused by a myelin problem in infants born prematurely.

The team found that adult human cells were much more adept at settling into the brain, becoming oligodendrocytes and producing myelin than the fetal cells. After just four weeks, adult cells but not fetal cells were producing myelin. After 12 weeks, four times as many oligodendrocytes derived from adult cells were producing myelin – 40 percent, compared to 10 percent of the cells from fetal cells. In addition, adult cells were likely to take root and form oligodendrocytes, not other brain cells such as neurons or astrocytes, which are not necessary for myelin production. On average, each oligodendrocyte from an adult cell successfully remyelinated five axons, compared to just one axon for fetal cells.


posted by Canadian Myelin Research Initiative at 11:56 AM

 
Dear Wayne and Julie,

Margaret and I wish you and your family a happy holiday season.

Following items are intended for the newsletter of the Canadian Myelin Research Initiative:

I trust that the copies of our recent publications I sent you last week are in your hand now.

1.One of the papers on cell/gene therapy in mucopolysaccharidosis VII (Sly disease) mice must be interest to you. This work has been a collaboration between my self and Prof. Eto of Tokyo, Japan (he is an outstanding medical geneticist/ paediatrician specializing lysosomal storage diseases such as Krabbe, Gauchet and ALD) and we wish to continue our collaboration in same lines of work in paediatric storage diseases using human neural stem cells I generated in Vancouver. Next target diseases we are planning are Krabbe and ALD; mouse models of these diseases are available now.

2. As I informed you earlier, we have generated a new human neural stem cell line using a tetracyclin-regulatory gene expression system. This new human cell line multiplies only in the presence of tetracyclin (Tet), and in the absence of Tet most of these cells differentiate into neurons. We could grow them in large numbers in culture in the presence of Tet, modify them to carry a gene of our interest, and then transplant them into the brain of animal models or even in human patients.

We will explore more into the cell/gene therapy using this new human neural stem cell system.

3. Recently we have generated immortalized human mesenchymal stem cell lines (from human foetal bone marrow). These human mesenchymal stem cells become bone cartilage; muscle or neurons depend on their culture environment (a manuscript is in preparation). We will study further their ability to develop into neurons but also into insulin producing beta islet cells, heart muscle cells or liver cells.

4. More recently we have generated oligodendrocytes/oligo progenitors from our human neural stem cells using a master gene called Olig2 transcription factor. Until recently we could not generate oligodendrocytes from our immortalized human neural stem cells probably because of the absence of an oligo-inducing signal in the system/environment. Now we found the signal that tells neural stem cells to become oligodendrocytes. We will pursue this line of work further in New Year by transplanting neural stem cell-derived human oligos in an animal model of MS.

5. We have collaborative works at UBC including transplantation of human neural stem cells carrying brain-derived growth factor gene (these cells produce a large amount of BDNF/neurotrophic factor that keeps host neurons and grafted neurons survive well) into rat model of spinal cord injury (Dr. Wolf Tetzlaff has a spinal cord injury model) and also mouse model of Huntington disease (Dr. Bruce Levitte of UBC has a mutant mouse model of HD).

6. We have a mouse model of ALS in Suwon, Korea and we have transplanted LacZ-labelled human neural stem cells via tail vein and found survival of grafted cells in the neocortex, hippocampus and spinal cord (only small number of cells in spinal cord) and the animal behaviour was visibly improved. We are continuing further experiments in ALS animals by grafting neural stem cells directly into spinal cord.

7. As I sent you copies of two papers earlier in which rat models of stroke were given human neural stem cell transplantation via tail vein injection. LacZ-labelled neural stem cells found their way to the stroke lesions, integrated into the host brain and improved behaviour. Further study is planned to use neural stem cells carrying BDNF gene (see above).

8. In collaboration with Dr. Karen Aboody (formerly of Harvard Neurosurgery and fellow of Evan Snyder, and now at the City of Hope Medical Center near LA), we generated new cell line by modifying our human neural stem cells to carry a suicide gene called cytosine deaminase (F3.CD cell line). Our neural stem cell line was found to track down brain tumour cells selectively and penetrate into the tumour mass. Brain tumour bearing animals are grafted with F3.CD cells, then non-toxic drug fluorocytosine is injected intraperitoneal route, drug reaches to the tumour site and the CD enzyme in F3 cells turn the drug into fluorouracil, a potent anticancer drug. Fluorouracil kills the F3 neural stem cells but the drug diffuses out into wide surrounding area and kills target brain tumour cells via by-stander effect. Earlier studies using mouse neural stem cells showed 90% reduction in tumour size.

I wish to tell you that Margaret and I are most grateful to you for your support, financial as well as moral, during the past several years for our research effort at UBC. If I can be of help in any way to support your good work with Canadian Myelin Research Initiative, I would do my utmost to help you.

With kindest regards, Seung Kim


posted by Canadian Myelin Research Initiative at 3:03 PM

 
Dr. Seung U. Kim updates progress with his stem cell research -

As I informed you earlier, we are conducting several interesting projects here at UBC and also in Suwon, Korea:


1. New human neural stem cell lines - Since our immortalized human neural stem cell (hNSC, F3) line was generated using v-myc oncogene, institutional review boards including one at UBC are reluctant to grant permission to use the cell line for the clinical trials in neurological diseases. To circumvent this issue, we have recently generated a new hNSC cell line using tet-on-vmyc system (G12). In this system, v-myc is only activated in the presence of tetracycline antibiotic, thus in the absence of tetracycline, the immortalized hNSCs do not multiply. We will develop this G12 hNSC cell line further and utilize it for animal studies and clinical trials.

2. Huntington disease (HD) - We have proactively grafted F3 hNSC cells in rat model of HD and grafted F3 cells protected host neurons from cell death (a paper submitted for publication). Dr. B. Levitte of UBC has generated a transgenic HD mouse model, and he kindly offered his animals for hNSC transplantation. The first experiment will take place next week.

3. Parkinson disease (PD) - We have generated subline of F3 hNSC carrying TH and GTPCH genes so that this subline produces L-DOPA 2000 times over the parental cells.

L-DOPA is the precursor of dopamine which is deficient in PD brain. We initiated animal studies in rat PD models at UBC in the collaboration with Dr. C. Lee who is a specialist of PD here.

4. ALS - When F3 hNSCs were injected into the tail vein of mouse ALS model (carrying mutant superoxide dismutase/ SOD gene), F3-turned-neurons were found in hippocampus 2-3 weeks later. We are currently doing long-term follow up 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 a clinical trials in Korea.

5. Stroke - My collaborators at the Seoul University Hospital (my alma mater) has published two papers (one in focal ischemia model and the other in cerebral hemorrhage model). F3 hNSCs were injected into tail vein of rat stroke models and later they migrated into the perilesion sites and then induced improvement in functional parameters such as corner test or rotarod tests. We have recently generated F3.BDNF and F3.GDNF sublines carrying neurotrophic factor genes. These modified hNSCs should provide protection of neurons from cell death. We will provide these cell lines to the investigators to graft into the stroke model animals.

6. Pediatirc storage diseases - Prof. Eto of Tokyo has completed a work in which F3 hNSCs carrying beta-glucuronidase gene coreect clinical course and pathology of Sly disease (mucopolysaccharidosis VII) animals. His group is currently working in the area of Krabbe and Gauchet diseases using F3 hNSCs. I am interested in collaborating again with Kyushu University investigators who carry adrenoleukodystrophy (ALD) transgenic mice (Yamada who came to Canada several years ago is no longer with the group) and we will generate G12 hNSCs overexpressing ALDP gene and transplant into ALD mice (we obtained ALDP cDNA recently).

7. Brain tumor/ glioma � Dr. Karen Aboody (former fellow of Evan Snyder and currently at the City of Hope Medical Center) has generated most exciting findings for F3 hNSCs to be used for treatment of brain tumor. F3 hNSCs could selectively and specifically migrate into brain tumor loci when grafted into the tumor carrying animals. In collaboration with her, we are currently producing F3 hNSCs carrying suicidal genes such as cytosine deaminase (CD) or HSV-TK (herpes virus thymidine kinase). F3.CD or F3.TK cells when they are grafted into the brain or intravenously injected, should migrate into the brain tumor loci and when drugs such as fluorocytosine or gancyclovile are applied, hNSCs commit suicide and release anticancer drug and kill the tumor cells by ?�by-stander-effect?�. Since F3 cells kill themselves later, the question of V-myc oncogene is non-issue here. I am certain clinical trials in brain tumor/gliobastoma could be the first one for the hNSCs we generated. We are determined to generate F3.CD and F3.HSVTK cell lines within a month or two.

8. We have generated immortalized cell lines of human bone marrow stem cells (from fetal bone marrow) using retroviral vectors carrying v-myc (as in hNSC F3 line) or teromerase gene. The hBMSC cell line generated is quite unique since it carrys teromerase which is physiological and does not cause tumor formation. 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 everybody are talking about.

posted by Canadian Myelin Research Initiative at 3:31 PM

 
From Suffolk Life, Wednesday July 2nd, 2003 (via Professor Hersh Chadha)

A study of a new drug called Campath, at Stoney Brook University, in Suffolk England, is accepting participants from the region.

"To date, every patient has shown a predictable response to Campath through suppressed disease activity" said Professor Alistair Compton.

It is hoped the drug will slow or stop the progression of MS.
posted by Canadian Myelin Research Initiative at 11:36 AM