WO2001076507A2

WO2001076507A2 - Use of oxygen carriers to improve grafted cell survival in neural transplantation

Info

Publication number: WO2001076507A2
Application number: PCT/US2001/011564
Authority: WO
Inventors: Jacqueline Sagen; Elizabeth Potter
Original assignee: The University Of Miami
Priority date: 2000-04-11
Filing date: 2001-04-11
Publication date: 2001-10-18
Also published as: WO2001076507A3

Abstract

A stem cell transplantation medium containing a serum-free physiologically suitable aqueous carrier; about 1 % to about 20 % of an oxygen carrier; and optionally one or more growth factors or differentiation factors is disclosed. Also disclosed is the use of an oxygen carrier in a stem cell transplantation medium.

Description

USE OF OXYGEN CARRIERS TO IMPROVE GRAFTED CELL SURVIVAL IN NEURAL TRANSPLANTATION

This application claims priority to U.S. Serial No. 60/196,094, the entire contents of which is hereby incorporated herein by reference.

The invention relates to a medium and its method of use in improving stem cell survival during transplantation into host brain tissue for treatment of neurodegenerative diseases.

BACKGROUND OF THE INVENTION

Stem cells have been the subject of transplantation research in the search for improved therapies for human disease and injury. Progenitor cells, which may be considered a subset of stem cells, are pluripotent, self-renewing cells that are lineage restricted. Unlike omnipotent stem cells that, theoretically, can differentiate into any cell type, progenitor cells differentiate into a restricted set of cell types. Stem cells and progenitor cells, residing in the subependymal zone of adult and fetal brain can migrate and differentiate into neurons, oligodendrocytes, and glia. During development, local environment factors including agents released from neighboring floor plate cells can induce, for example, dopamine neuron differentiation in mesencephalic subependymal cells. Thus, it may be possible to produce enriched cultures of stem cells, such as fetal mesencephalic cells, capable of differentiating into functional neurons using the appropriate combination of environmental cues.

By screening numerous combinations of cytokines and trophic factors, interleukin-1 (IL- 1) has been identified as a dopamine neuronal differentiation factor in mesencephalic progenitor cells. Bromo-deoxyuridine-immunoreactivity (BrdU-ir), which permits visualization of labeled progenitor cells via immunocytochemical techniques, demonstrated that rat fetal (El 5) mesencephalic progenitor cells are mitotically active after expansion in epidermal growth factor (EGF)-supplemented media for at least 21 days. Treatment with IL- 1 resulted in a marked conversion of these cells into tyrosine hydroxylase (TH)- immunoreactive (ir) neurons in vitro compared with control cultures incubated in complete media (CM). Complete media contained DMEM/F-12 (1:1) 10% fetal calf serum (FCS), without IL-1. IL-1 treated cultures were also enriched over CM-incubated controls in Hu- immunoreactivity, (Hu-ir), Hu being the earliest marker for neuronal differentiation, and GAP-43, a marker for neuronal process extension. In addition to neuronal phenotypes, both CM and IL-1 treated cultures contained a substantial population of GFAP-positive astrocytic cells, a common feature of differentiated neural progenitor cultures.

Previously, embryonic mesencephalic progenitor cells were found to be substantially converted into dopamine neurons in vitro using interleukin-1 and FCS-supplemented media. However, while FCS nourishes mature cells, it inhibits growth of source progenitors, and may even decrease their conversion rate into dopaminergic neurons. Since serum antibodies and cytokines could be responsible for these effects, serum-free media may be required for preparing progenitor cells for transplantation.

Accordingly, a need exists to develop a medium and method of its use for transplanting stem cells into neural tissue in a fashion that promotes survival of the stem cell transplants into active, functional cells capable of replacing the functions lost by aberrant or missing neural cells involved in disease and trauma.

SUMMARY OF THE INVENTION

The invention provides a transplantation medium containing oxygen carriers and its use in a method of enhancing cell viability, thereby enabling successful grafting of neural stem cells transplanted into recipient host central nervous system tissue.

Thus, a preferred embodiment of the invention is a stem cell transplantation medium comprising a serum-free physiologically suitable aqueous carrier and about 1% to about 20% of an oxygen carrier, more preferably 7.5% to about 10%. The aqueous carrier may any suitable pharmaceutically useful aqueous carrier, such as saline. Optionally, the transplantation medium may contain at least one member selected from the group consisting of a growth factor and a differentiation factor. The stem cells may be progenitor cells.

Another preferred embodiment of the invention is a method of transplanting stem cells into vertebrate central nervous system tissue, wherein the cultured stem cells are in a transplantation medium containing at least one oxygen carrier. The method comprises isolating stem cells from central nervous system tissue of a donor animal, growing the isolated stem cells in a culture medium, and introducing the cultured stem cells into central nervous system tissue of a recipient vertebrate host. The transplantation medium may further comprise at least one member selected from the group consisting of a growth factor and a differentiation factor.

Another preferred embodiment is a method of treating a neurological disorder in vertebrate central nervous system tissue comprising isolating stem cells from a predetermined region of a vertebrate nervous system; growing the isolated stem cells in a culture medium; introducing the cultured stem cells in a transplantation medium containing at least one oxygen carrier into central nervous system tissue of a recipient vertebrate host at a site predetermined to be associated with the neurological disorder; and allowing the stem cells to graft into the central nervous system tissue of the host, thereby providing neural cells capable of functioning in lieu of host neural cells associated with the neurological disorder.

The at least one oxygen carrier may comprise a member selected from the group consisting of hemoglobin-based oxygen carriers and perfluorocarbons. The at least one growth factor may comprise bFGF. The differentiation factor may comprise interleukin-1.

The cultured stem cells introduced to the host may be present in a transplantation medium containing at least one oxygen carrier at least one member selected from the group consisting of a growth factor and a differentiation factor. Such stem cells may be fetal progenitor cells isolated from fetal brain mesencephalon.

In another preferred embodiment, the method of treating a neurological disorder in vertebrate central nervous system tissue may be used to treat such maladies as stroke, traumatic brain injury, spinal cord injury, cerebral palsy, Huntington's disease, Alzheimer's disease, amyotropic lateral sclerosis, epilepsy, multiple sclerosis, Parkinson's disease and chronic pain.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated in the Figures in which:

Figure la shows fetal mesencephalic progenitor cells incubated for three days in culture in complete medium supplemented with 10% bovine hemoglobin-based oxygen carrier;

Figure lb shows fetal mesencephalic progenitor cells incubated in N2 serum- free medium supplemented with 10 ng/ml FGF and 10% bovine hemoglobin-based oxygen carrier;

Figure 2a shows BrdU-labeled fetal mesencephalic progenitor cells (black) 7 days after being transplanted into rat striatum (control),

Figure 2b shows BrdU labeled mesencephalic progenitor cells (black) after 7 days of co- transplantation into rat striatum with 10% hemoglobin-based oxygen carrier contained in HBSS transplantation medium,

Figure 3 graphically depicts enhanced survival of grafted progenitor cells in the presence of a hemoglobin-based oxygen carrier (UPH),

Figure 4a shows photomicrographs at 20X magnification of mesencephalic progenitor cell transplants in untreated regions;

Figure 4b shows mesencephalic progenitor cell transplants treated with 7.5% UPH;

Figure 4c shows mesencephalic progenitor cell transplants treated with 10% UPH; and Figure 4d shows mesencephalic progenitor cell transplants treated with 20% UPH.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Because serum interferes with progenitor cell growth and differentiation in vitro, N2 medium, a serum-free medium supporting dopaminergic neuron viability, was supplemented with bFGF and used to determine progenitor cell viability and differentiation under various conditions. Specifically, N2 is a defined serum-free media that contains DMEM (Dulbecco's

minimal essential medium, Gibco), 25 μg/ml insulin, 100 μg/ml human transferring, 100 μM

putrescine, 30 nM sodium selenite and 20 nM progesterone. These studies showed, inter alia, that while mesencephalic progenitor cells thrive in N2/bFGF, neuronal differentiation is somewhat inhibited by the mitotic factor bFGF. By contrast, differentiated dopaminergic neurons cultivated in N2 without bFGF required additional factors for long term viability. Thus, viability of stem cells in vitro is highly influenced by the type of medium and supplements used to culture these cells. Supplements in addition to those typically used to maintain cells in culture were sought to tailor transplantation media capable of improving viability of stem cells during transplantation procedures.

It was determined that factors suitable for additional media supplementation include hemoglobin-based oxygen carriers and perfluorocarbons (PFCs), which are cytokine-free oxygen carriers that support cell viability. An ultrapurified bovine hemoglobin-based oxygen carrier was tested as a media supplement, and was found to support growth of mesencephalic progenitor cells in both complete media and N2 media containing bFGF (Figs, la and lb). Thus, oxygen carriers may be used as a media supplement during transplantation procedures to improve cell survival and enable transplant cell growth and differentiation.

Pertinent to the issue of neural cell graft survival, hemoglobin and PFC blood substitutes can improve in vivo cell viability through oxygen delivery during hypoxic conditions. Thus, an object of the invention is to enhance survival of dopaminergic progenitor cells transplanted into host brains afflicted with neurodegenerative diseases, such as Parkinson's disease, via use of transplantation media containing oxygen carriers.

To test this concept, UPH (Biopure) was included in transplantation media and the effect of this oxygen carrier on grafted cell viability was evaluated. Improved survival of fetal mesencephalic progenitor cells transplanted into rat striatum was achieved when transplantation media supplemented 5%-10% UPH was employed (Fig. 2a and 2b). Conditions resulting in maximal survival of grafted dopaminergic neurons will be followed for longer periods to assess behavioral recovery of treated animal models of Parkinson's disease, such as 6-hydroxy-dopamine lesion models.

Fetal neocortical stem cells have produced similar results upon implantation into adult animal host spinal cord tissue. Thus, the benefit of oxygen carriers on stem cell transplant survival is not limited to a single cell type.

Effective increases in transplant stem cell survival have been observed employing 2.5% to 10% UPH, and stem cells from the rat CNS have optimal responses at about 7.5% concentrations of this oxygen carrier. The hemoglobin-based oxygen carriers have been found to be suitable for use in humans for conditions such as ischemia, and therefore may be used in the effective concentration range of 2.5% to 10% for improving human cell transplantation survival. Descriptions of such hemoglobin-based oxygen carriers can be found in U.S. Patent Nos. 6,150,507; 5,895,810; and 5,952,470, the contents of which are incorporated herein by reference. Optimal concentrations of oxygen carriers will vary depending upon the stem cells used and the types of tissue transplantation undertaken. Much of this variation is driven by the sensitivity of a given cell population to hypoxic stresses. A skilled artisan will be able to optimize the most effective concentrations of oxygen carrier for a particular transplantation procedure based upon the general knowledge in the art in combination with the information disclosed herein.

The beneficial effects of oxygen carriers such as UPH are expected to enhance survival of transplanted stem cells from non-fetal sources as well as fetal sources. Stem cells derived from adult sources are known to be difficult to cultivate, and therefore their survival should be enhanced by inclusion of oxygen carriers in transplantation media.

The oxygen carriers noted herein and their use to enhance transplantation survival of brain-derived progenitor cells may be used to treat numerous neurological disorders requiring replacement of lost or dysfunctional cells in the central nervous system of a recipient host. The central nervous system is defined herein to comprise neural components of the brain and spinal cord. Cellular transplantation in the central nervous system may be used for the treatment of such disorders as stroke, traumatic brain injury, spinal cord injury, cerebral palsy, Huntington's disease, Alzheimer's disease, epilepsy, ALS, chronic pain, multiple sclerosis, Parkinson's disease, and the like.

Regions of the central nervous system that may be targeted for stem cell transplantation include areas negatively affected by trauma as well as areas associated with genetically inherited or disease-mediated neurological disorders. Examples of specific CNS areas known to be associated with neurological diseases and the types of neural cell functions impaired in each include: Parkinson's disease: nigrostriatal dopamine cells; Huntington's disease: striatal interneurons; Alzheimer's disease: nucleus basalis of Meynert, cortical and hippocampal cholinergic neurons; ALS: lower and upper motor neurons; MS: oligodendrocytes in multiple areas of brain and spinal cord; stroke, traumatic brain injury, cerebral palsy: common sites include neocortical areas, hippocampus, and basal ganglia; spinal cord injury: oligodendrocytes in white matter tracts, motor neurons and interneurons in gray matter; chronic pain: inhibitory interneurons in spinal cord dorsal horn. All of these maladies may be improved or overcome by employing stems cells grafted according to the invention. Ideally, the progenitor cells and stem cells can be differentiated into specific neuronal cell types and grafted into the areas affected by neuronal cell death as a replacement therapy for the various CNS diseases.

Recipient hosts that may be treated by transplantation of stem cells according to the invention include vertebrate animals, particularly mammals such as companion animals and humans. For example, the replacement of nigrostriatal dopamine neurons in Parkinson's disease may be an effective treatment using the improved stem cell transplantation technology of the invention.

Human stem cell transplants have been conducted in cancer patients for hematopoiesis (e.g. Alvamas, J.C., Negrin, R.S., Horning, S.J., Hu, W.W., Long, G.D., Schriber, J.R., Stockerl-Goldstein, K., Tierney, K., Wong, R., Blume, K.G., Chao, N.J., Biol. Bone Marrow Transplant. 6: 352-358, 2000; Lehman, S., Isberg, B., Ljungman, P., Paul, C, Bone Marrow Transplant. 26: 187-192, 2000). In addition, transplants using a stem cell-like teratoma cell line (the hNT cells) have been conducted in stroke patients (Kondziolka, D., Wechsler, L., Goldstein, S., Meltzer, C, Thulborn, K.R., Gebel, J., Jannetta, P., DeCesare, S., Elder, E.M., McGrogan, M., Reitman, M.A., Bynum, L., Neurology 55: 565-569, 2000). The contents of all of these citations are hereby incorporated herein by reference.

Roughly 300 transplantation cases have been documented using fetal mesencephalic tissue or cell suspensions to treat Parkinson's patients. These transplantations were not limited to use of neural stem cells per se, although the cell suspensions likely contain some stem cells and progenitors in the mix with committed and differentiated neurons and glia. Fetal tissue transplants have also been used in trials for Huntington's disease, spinal cord injury, and stroke. Therefore, a skilled artisan would know from these previous human therapies how to implement the use of oxygen carriers to improve stem cell transplantation in human subjects.

The transplantation media of the invention provides an environment that stimulates the growth and differentiation of transplanted stem cells. Thus, fetal progenitor cells of neural origin transplanted into the central nervous system of a recipient host are able may be able to grow and differentiate into functional neural cells in the host. By functional is meant that these differentiated graft cells are capable of acting in lieu of endogenous neural cells that no longer act or respond to stimuli in a normal fashion. For example, neurons that no longer secrete neurotransmitters in a normal fashion often give rise to pathologies that may be treated by replacement with functional cells that do secrete neurotransmitters upon stimulation.

Accordingly, the transplantation medium comprises a physiologically suitable aqueous solution supplemented with at least one oxygen carrier to counteract effects of hypoxia during the transplantation process. Cell survival two weeks following transplantation increases in the presence of oxygen carriers in a dose-dependent fashion, as is depicted in Figure 3. In this study, very poor survival of untreated mesencephalic progenitor cells was noted at the two-week stage, while improved survival was observed correlating with increasing UPH concentrations in transplantation media up to 7.5% to 10%. Figure 4 shows photomicrographs of transplanted mesencephalic progenitor cells that were not exposed to UPH (control, Figure 4a), in comparison with cells exposed to 7.5 % (Figure 4b), 10% (Figure 4c) and 20% (Figure 4d) UPH. The greatest amount of cell labeling was detected in cells exposed to 7.5% and 10% UPH, indicating that these levels of UPH are optimal for promoting mesencephalic progenitor cell division post transplantation.

Suitable oxygen carriers include perfluorocarbons and hemoglobin-based oxygen carriers. The oxygen carrier may be present in a transplantation medium in an amount ranging from about 1% to about 20% by volume. A range of about 7.5% to about 10% oxygen carrier is preferred.

Stem cell transplantation medium according to the invention may include a differentiation factor and a growth factor. Thus, a transplantation medium may prepared to contain one or more growth factors, one or differentiation factors, or a combination thereof, to facilitate proper grafting of the transplanted cells. For example, fibroblast growth factor (bFGF) may be included in the transplantation medium to promote growth of transplanted cells in vivo. Likewise, a differentiation cytokine and a neurotrophic factor may be included in such medium to induce conversion of grafted mesencephalic progenitors in situ into dopaminergic neurons.

A balance must be achieved between the advantages of pre-differentiating stem cells in order to direct them towards the desired phenotype before transplantation, and allowing them to differentiate too much, which would result in process shearing and trauma to the cells, reducing their viability. One way to achieve this balance may be initiating differentiation of stem cells using a brief pulse with the cytokine IL-1 (e.g. overnight) prior to transplantation in order to induce the dopaminergic phenotype. Another approach is infusing FGF-2 (bFGF) in order to expand the population of transplanted stem cells when the number of cells needed is likely to be low compared to the number of cells lost, for example in traumatic brain injury or stroke. The optimal methods for administering growth factors or differentiation factors may be determined for particular cell populations. For example, in neuronal differentiation, BDNF or NT-3 may be useful, whereas T3 or PDGF may be useful for oligodendrocytes.

The invention is now further described in the following non-limiting examples. EXAMPLES

Example 1 : In vitro Analysis and Testing

Progenitor Cell Culture Preparation: Fetal rat mesencephalic cells (El 4) were prepared as previously described. In particular, cytokine-induced conversion of mesencephalic-derived progenitor cells into dopamine neurons was conducted as described by Potter et al. in Cell Tissue Res. 296: 235-246, 1999, the entire contents of which is hereby incorporated herein by reference.

Specifically, fetal rat mesencephalic cells are microdissected from El 4- 15 rat brains into Hank's balanced salt solution (HBSS; Gibco) and a cell suspension created via mechanical trituration. The cells are plated (400,000 cells/ml) in 25 cm flasks, and incubated in expansion media (Svenson et al., 1995) consisting of F12/DMEM (1:3), B27 (Gibco; 1:50), and epidermal growth factor (EGF, 20ng/ml). EGF stimulates progenitor cells to form 50-100 proliferation spheres/cm².

For initial viability studies, progenitor cell cultures were transferred to poly-L-lysine (PLL)-coated wells of multiwell tissue culture plates and incubated with DMEM/F-12 (1:2) supplemented by fetal bovine serum (FBS) at a concentration of 10%, or N2 serum-free media supplemented with bFGF and UPH (Biopure) for 7 days. Control cultures were grown in N2 serum-free media supplemented with bFGF but were not exposed to UPH. Cell viability was determined by observation of the cultures using light microscopy.

Preparation of Cortical Stem Cells: To prepare neocortical stem cells, E14-15 fetal rat neocortical tissue is microdissected in Hank's balanced salt solution (HBSS; Gibco) and a cell suspension created via mechanical trituration. Cells are plated (1.5xl0⁶) onto poly-L- ornithine (PLO; Sigma; 15 μg/ml)/fιbronectin (Sigma; 1 μg/ml) treated 10 cm² tissue culture plates and grown in DMEM F12 containing N2 and 10 ng/ml of human recombinant basic fibroblast growth factor (bFGF or FGF-2; Sigma) (D/F-N2-bFGF) for stem cell growth. bFGF (10 ng/ml) is added daily and the culture media changed every two days. Cells are grown at 37° C, 5% C0₂ and passaged at 60-70% confluence by brief incubation in HBSS and dislodging with a cell lifter.

Example 2: In vivo analysis

Mesencephalic Progenitor Cell Transplantation into the Striatum: Adult host rats receiving the progenitor cell transplant were anesthetized and prepared for stereotactic surgery under sterile conditions. Bilateral transplants were performed with the animal receiving "untreated" mesencephalic progenitor cells in HBSS vehicle on the right side, and "treated" mesencephalic progenitor cells in HBSS supplemented with 1%-10% UPH (Biopure) on the left side. The cells were pre-labeled with bromo-deoxyuridine (BrdU; Amersham) for identification in the transplanted graft.

Five microliters of mesencephalic progenitor cells (50,000 cells/μl in Hank's Buffered Saline Solution (HBSS)) were stereotactically injected through a stainless steel needle into the striatum using methods previously described by Carvey et al. (Carvey et al. The injection of biologically active substances into the brain. In: Methods in Neuroscience, Vol. 21, Flanagan TR, Emerich DF, Winn SR, eds. Academic Press, Orlando, FL (1994):214-234), the contents of which are hereby incorporated herein by reference. Graft survival and differentiation were assessed immuocytochemically at 1 and 2 weeks following transplantation using BrdU-ir, TH-ir, neuronal and glial markers as previously described. Once optimal dopamine neuron survival is established, behavioral studies assessing the effects of the transplanted neural cells in animal models may be conducted, employing standard amphetamine and apomorphine rotational models. Cortical Stem Cell Transplantation: Cortical cells were obtained as described in Example 1. Twenty- four hours prior to transplantation, cells were incubated overnight in D/F-N2- FGF-2 containing lOμM bromodeoxyuridine (BrdU; Amersham) to label the cells for later identification. The adult host rats receiving the cortical cell transplants were anesthetized and prepared for stereotactic surgery under sterile conditions.

For transplantation in the spinal cord, a laminectomy was performed at L1-L2 and cortical stem cells were implanted bilaterally as described above for mesencephalic cell transplantation into the striatum. Injections were made using a 10 μl Hamilton syringe attached to a pulled glass pipet (diameter ~50 μm). A small slit was made in the dura and 1.5 μl of cell suspension (~ 10⁵ cells) was stereotactically placed into the ventral horn at each graft site (0.5 mm lateral; 1.3 mm ventral from the surface of the dorsal vein). The needle was held in place for about one min and then gradually withdrawn. Similar approaches were used to transplant cortical cells into the striatum and cortex to evaluate the effects the inventive stem cell transplantation process in animal models of stroke and traumatic injury, respectively.

The invention as described herein provides for enhancement of stem cell viability immediately post-transplantation, when oxygen deprivation is critical. Stem cells are one type of cell which benefit from exposure to oxygen carriers during the transplantation process. Mesencephlaic progenitor cells are particularly vulnerable to this early stage oxygen deprivation, although survival of cortical stem cells in the spinal cord, while more hearty, is enhanced as well.

While the invention has been described above with respect to certain embodiments thereof, it will be appreciated by one skilled in the art that variation and modifications may be made without departing from the spirit and scope of the invention.

Claims

What is claimed is:

1. Use of at least one oxygen carrier in a medium for transplantation of stem cells into vertebrate central nervous system tissue.

2. The use of an oxygen carrier according to claim 1, wherein the at least one oxygen carrier comprises a member selected from the group consisting of hemoglobin-based oxygen carriers and perfluorocarbons.

3. The use of an oxygen carrier according to claim 1 or claim 2, wherein the medium further comprises at least one member selected from the group consisting of a growth factor and a differentiation factor.

4. The use of an oxygen carrier according to any of claims 1-3, wherein the stem cells comprise progenitor cells.

5. The use of an oxygen carrier according to any of claims 1-5, wherein the at least one oxygen carrier is present in a concentration of 1% to 20%, preferably 7.5% to 10%.

6. A stem cell transplantation medium comprising: a serum-free physiologically suitable aqueous carrier; about 1% to about 20% of an oxygen carrier; and optionally at least one member selected from the group consisting of a growth factor and a differentiation factor.

7. The transplantation medium according to claim 6, wherein the oxygen carrier comprises a member selected from the group consisting of hemoglobin-based oxygen carriers and perfluorocarbons.

8. The transplantation medium according to claim 6 or claim 7, wherein the at least one growth factor comprises bFGF.

9. The transplantation medium according to any of claims 6-8, wherein the at least one differentiation factor comprises interleukin-1.

10. The transplantation medium according to any of claims 6-9, wherein the oxygen carrier is present in an amount ranging from 7.5% to 10%.