CytoTherapeutics Researchers Demonstrate Potential for Human Neural Stem Cells to Repair or Replace CNS Tissue.

LINCOLN, R.I.--(BUSINESS WIRE)--Nov. 6, 1998--CytoTherapeutics, Inc. (Nasdaq: CTII) and its collaborators will present four abstracts based on the use of the Company's proprietary neural stem/progenitor cell cultures to repair or replace central nervous system (CNS) cells at the 28th Annual Meeting of the Society for Neuroscience in Los Angeles on Monday, November 9th.

The abstracts describe results of studies which demonstrate CytoTherapeutics' proprietary neural stem cell technology may be used to:

--isolate and differentiate human neural stem cells in vitro ; --maintain and exponentially expand human neural stem cells in vitro; --successfully engraft, migrate and differentiate human neural stem cells into the appropriate neuronal phenotypes in an animal model.

"The results from these studies demonstrate the potential for the neural stem/progenitor cells to repair or replace damaged cells of the central nervous system, including the brain, the eye and the spinal cord," stated Richard M. Rose, President and Chief Executive Officer.

In March 1994, the Company obtained an exclusive, worldwide license from NeuroSpheres, Ltd. for the commercial development and use of its proprietary neural stem cells for transplantation to treat human disease. In 1997, through the acquisition of StemCells, Inc., a company focused on the discovery and development of stem cells, CytoTherapeutics acquired additional intellectual property and licenses related to neural stem/progenitor cells.

CytoTherapeutics, Inc. is a leader in the development of gene and cell-based therapeutics. The Company's encapsulated-cell technology is designed to treat a number of serious diseases and disorders by delivering therapeutic substances directly to the site in the body where they are needed. The Company's stem cell programs seek to repair or repopulate tissue that has been damaged or lost as a result of disease or injury. For additional information, visit the company's Internet web site at http://www.cyto.com.

Certain statements in this press release include forward-looking statements regarding, among other things, intellectual property rights, products, and product development. CytoTherapeutics actual results may vary materially from those forward looking statements due to risks and uncertainties, such as delays in obtaining regulatory and other approvals or delays in product development, the need for additional intellectual property, the possible loss of existing intellectual property, the failure to achieve anticipated partnering agreements and other risks to which the company is subject and which are described in Exhibit 99 to the Company's Annual Report on Form 10-K entitled "Cautionary Factors Relevant to Forward Looking Statements."

Abstracts Follow

IN VITRO PROLIFERATION OF HUMAN NEURAL PROGENITOR CELLS THAT MAINTAIN

GROWTH FACTOR REGULATION

M.K. Carpenter*1, X. Cui2 , J. Jackson1, S. Sherman1, Z. Hu1 , S. Bruhn1, R. Fricker4, A. Seiger3, L. Wahlberg1

1Cell and Molecular Neurobiology, CytoTherapeutics, Inc., Lincoln, RI 02865; 2Dept. Clin. Neurosci., Karolinska Inst., 3Dept. of Geriatric Med., Huddinje Univ. Hospital, Stockholm, 4Wallenberg Neurosci. Center, Lund, Sweden.

A population of human neural progenitor cells from the human embryonic forebrain (gestational weeks 5-11) have proliferated in vitro as non-adherent spheres for at least one year (30-40 passages) using defined serum-free media culture conditions. To date, we have cultured at least 8 different "cell lines" which all proliferate at similar rates and have similar differentiative capacity. Upon immunocytochemical staining for a variety of markers, all the spheres show positive immunoreactivity for nestin. A small number of GFAP and (beta)-tubulin-positive cells were identified within the spheres indicating the heterogencity of this population.

When plated on appropriate substrate, this population of cells can differentiate into all three major neural phenotypes, neurons, astrocytes and oligodendrocytes. Depending on the "cell line" and culture conditions, 20-40% of the cells form (beta)-tubulin-positive neurons; 15-50% GFAP-positive astrocytes; and 0-15% GalC-positive oligodendrocytes. Immunocytochemistry and/or RT-PCR analysis demonstrates that this population of differentiated cells contains specific neuronal subtypes (e.g. GABAergic) and reveals expression of the neurotrophin receptor, trkB. Exposure to BDNF during differentiation can increase the number of neurons. Furthermore, pre-exposure to specific growth factors before differentiation can influence the percentage of cells which form neurons. For example, the presence or absence of basic fibroblast growth factor (bFGF) and/or leukemia inhibitory factor (LIF) appears to modulate the quantity of neurons. This study demonstrates that exposure to growth factors and/or cytokines prior to or during the differentiation process can influence the fate of these cells. The in vitro proliferation of populations of neural progenitors whose differentiation can be regulated in vitro may provide a source of cells for transplantation to treat some CNS disorders.

SITE-SPECIFIC DIFFERENTIATION OF HUMAN NEURAL PROGENITOR CELLS AFTER

TRANSPLANTATION TO THE DEVELOPING RAT CNS.

U.Englund1, R.A. Fricker1,2 , M.K. Carpenter2, J.Jackson2, S. Sherman2, K. Wictorin1* and A. Bjorklund1

1Lund University, Wallenberg Neuroscience Center, Dept. of Physiology and Neuroscience, Lund, Sweden. 2CytoTherapeutics, Inc. Lincoln, RI, 02865, USA

Expandable human neural progenitor cells have been generated from the embryonic forebrain, and cultured in serum-free growth factor-containing medium, for over one year, with repeated passaging every 7-14 days. This population is able to form several phenotypes characteristic of the mature central nervous system (CNS): neurons, astrocytes and oligodendrocytes in vitro, even after long periods in culture. In this study we compared different cultures of human neural progenitors in their ability to differentiate after transplantation to the neonatal rat brain.

Cell suspensions of small spheres from either 6.5wk, 7 wk, 8.5wk or 9wk forebrain, at passage 4-28, labelled with Bromo-deoxy-uridine (1uM, 48 hours), were transplanted to the postnatal day 1 (P1) rat CNS, in either the hippocampus, striatum, midbrain, cerebellum or spinal cord. The neonates received no immunosuppression and grafts were analysed at 2-15 wks. There was good graft survival, with extensive migration of BrdU-positive cells either specifically within the subgranular layer of the dentate gyrus (DG); or throughout the striatum, in particular toward the globus pallidus. Cells were double stained by immunohistochemistry with antibodies for BrdU and neuronal markers, or single-labelled with a human-specific Tau antibody, and revealed that grafted cells had undergone site-specific neuronal differentiation, both in the DG and striatum, extending axons from the striatal transplants in to the fibres of the internal capsule. Further analysis of both neuronal and glial differentiation in other transplant sites is currently underway. These results indicate that human growth factor-dependent progenitor cells are able to differentiate appropriately after transplantation to different regions of the developing CNS. Supported by the Wellcome Trust, the Wenner Gren Foundation, the Segerfalk Foundation and Swedish MRC.

SITE-SPECIFIC DIFFERENTIATION OF HUMAN NEURAL PROGENITOR CELLS

AFTER TRANSPLANTATION TO THE ADULT RAT BRAIN.

R.A. Fricker1,2*, M.K. Carpenter2, C. Winkler1, M.A. Gates1, and A. Bjorklund1

1Lund University, Wallenberg Neuroscience Center, Dept. of Physiology and Neuroscience, Lund, Sweden. 2CytoTherapeutics, Inc. Lincoln, RI, 02865, USA

Human neural progenitor cells can be generated from the embryonic forebrain, and cultured in growth factor-containing medium, for over one year, with repeated passaging every 7-14 days. These cells retain differentiative capacity, and form several characteristic phenotypes of the mature central nervous system (CNS): neurons, astrocytes and oligodendrocytes in vitro, even after long-term culture. In this study we compared two different cultures of human progenitors after transplantation to the adult rat brain.

Cell suspensions of small spheres from either 6.5wk or 9wk forebrain, at passage 9-21, labelled with Bromo-deoxy-uridine (BrdU, 1uM, 48 hours), were transplanted to the adult rat brain, in either the anterior subventricular zone, rostral migratory pathway (RMP), hippocampus or striatum. The adult rats were maintained under cyclosporin immunosuppression during the timecourse of the experiment, and the brains were assessed either 2 or 6 weeks after transplantation. BrdU-labelled grafted cells exhibited extensive migration along the RMP to the olfactory bulb (OB); within the subgranular layer of the dentate gyrus (DG); and nondirectionally throughout the striatum. Double-labelling for BrdU and other phenotypic markers, revealed that grafted cells had undergone site-specific neuronal differentiation; forming tyrosine hydroxylase-positive neurons in the periglomerular layer of the OB; calbindin-positive neurons in the subgranular and granular layers of the DG; and calbindin and DARPP-32-positive cells in the striatum. Single-labelling using a human-specific Tau antibody, showed Tau-positive neuronal projections along the internal capsule to the globus pallidus. These results indicate that human growth factor-dependent progenitor cells are able to respond to local environmental signals in the adust brain, undergoing site-specific neuronal differentiation. Supported by the Wellcome Trust, the Wenner Gren Foundation and Swedish MRC.

PROPAGATION AND CHARACTERIZATION OF NEURAL PROGENITOR CELLS DERIVED FROM THE EMBRYONIC HUMAN SPINAL CORD, BRAINSTEM, AND FOREBRAIN

X. Cui3, M.K. Carpenter1 , A. Seiger2 and L. Wahlberg 1, 3 *

1CytoTherapeutics, Inc., Cell and Molecular Neurobiology, Lincoln, RI 02865; 2Karolinska Institute, Department of Geriatric Medicine, Huddinge University Hospital, Stockholm, Sweden; 3Karolinska Institute, Department of Clinical Neuroscience, Section of Neurosurgery, Stockholm, Sweden.

There is a growing body of data showing that rodent neural progenitor/stem cells from different anatomical regions of the embryonic central nervous system (CNS) can be cultured for long periods (months) while maintaining multipotency. We asked whether neural progenitor cells from human embryonic anatomical counterparts could be isolated and yield progenitor cell cultures that could be propagated. First trimester (6-6.5 weeks gestation) human embryonic tissues were collected and grossly divided into forebrain, brainstem and spinal cord. The tissues were mechanically dissociated and kept in serum-free, defined media in the presence of growth factors including epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).

The results show that progenitor cells from all three regions can be expanded for at least 4 months. The forebrain and spinal cord cultures expanded the fastest and could be passaged every 7-14 days. The mesencephalic cultures showed a similar rate initially but after about 60 days in vitro, slowed their overall rate of expansion. The undifferentiated progenitor cells incorporated bromodeoxyuridine (BrdU) and showed nestin immunoreactivity. Upon differentiation, the progenitor cells from all three regions gave rise to the three major cell types of the CNS: neurons, astrocytes and oligodendrocytes. Regional differences in the relative number of phenotypes formed were observed but, because of the single embryonic sources, need to be repeated before any major conclusions can be drawn. In conclusion, these data show that neural progenitor cells can be derived and propagated from three major regions of the developing human CNS and may provide a source of transplantable cells for the treatment of CNS diseases. This study was funded by CytoTherapeutics, Inc.