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Title:  Schwann cells originating in myeloid interstitial cells
United States Patent: 
6,989,271
Issued: 
January 24, 2006
Inventors:
 Dezawa; Mari (Yokosuka, JP); Sawada; Hajime (Yokohama, JP); Takano; Masahiko (Yokohama, JP)
Assignee:
 Sanbio, Inc. (Mountain View, CA)
Appl. No.: 
481603
Filed: 
June 21, 2002
PCT Filed: 
June 21, 2002
PCT NO: 
PCT/JP02/06249
371 Date: 
December 22, 2003
102(e) Date: 
December 22, 2003
PCT PUB.NO.: 
WO03/000871
PCT PUB. Date: 
January 3, 2003


 

George Washington University's Healthcare MBA


Abstract

There is provided a method of inducing bone marrow stromal cells to differentiate into bone marrow stromal cell-derived Schwann cells in vitro, comprising the steps of: collecting bone marrow stromal cells from bone marrow and culturing the cells in a standard essential culture medium supplemented with a serum; adding a reducing agent to the culture medium and further culturing the cells; adding a differentiation inducing agent to the culture medium and further culturing the cells; and adding a cyclic AMP-augmenting agent or a cyclic AMP analogue and/or a glial cell differentiation and survival stimulating factor to the culture medium, and further culturing the cells to obtain the bone marrow stromal cell-derived Schwann cells. There are also provided bone marrow stromal cell-derived Schwann cells obtained thereby and a pharmaceutical composition for neural regeneration that comprises them.

DISCLOSURE OF THE INVENTION

As a result of repeated experimentation, the present inventors are the first to have succeeded in inducing differentiation of bone marrow stromal cells into Schwann cells with a high degree of efficiency by a multistage operation. Moreover, it was confirmed that actual regeneration and elongation of nerves occurred upon transplanting the bone marrow stromal cell-derived Schwann cells obtained by the differentiation inducing method into damaged optic nerves (central nervous system), and the present invention was thus completed.

The present invention therefore provides a method of inducing bone marrow stromal cells to differentiate into bone marrow stromal cell-derived Schwann cells in vitro, comprising the steps of:

(1) collecting bone marrow stromal cells from bone marrow and culturing the cells in a standard essential culture medium supplemented with a serum;

(2) adding a reducing agent to the culture medium and further culturing the cells;

(3) adding a differentiation inducing agent to the culture medium and further culturing the cells; and

(4) adding a cyclic AMP-augmenting agent or a cyclic AMP analogue and/or a glial cell differentiation and survival stimulating factor to the culture medium, and further culturing the cells to obtain the bone marrow stromal cell-derived Schwann cells.

The invention further provides bone marrow stromal cell-derived Schwann cells obtained by the aforementioned method and a pharmaceutical composition for neural regeneration comprising the bone marrow stromal cell-derived Schwann cells. The invention still further provides a method of treating neural disease by transplanting the aforementioned bone marrow stromal cell-derived Schwann cells or a pharmaceutical composition for neural regeneration comprising them into a patient with neural disease to promote regeneration of the neural cells with which the disease is associated.

DETAILED DESCRIPTION OF THE INVENTION

According to one mode of the present invention there is provided a method of inducing bone marrow stromal cells to differentiate into bone marrow stromal cell-derived Schwann cells in vitro, comprising the steps of:

(1) collecting bone marrow stromal cells from bone marrow and culturing the cells in a standard essential culture medium supplemented with a serum;

(2) adding a reducing agent to the culture medium and further culturing the cells;

(3) adding a differentiation inducing agent to the culture medium and further culturing the cells; and

(4) adding a cyclic AMP-augmenting agent or a cyclic AMP analogue and/or a glial cell differentiation and survival stimulating factor to the culture medium, and further culturing the cells to obtain the bone marrow stromal cell-derived Schwann cells.

The density of the cells in step (1) may be 50% confluency, and the cells are preferably subcultured to four generations.

The standard essential culture medium may be Minimum Essential Medium Eagle Alpha Modification (M4526, Sigma) and the serum may be fetal calf serum (14-501F, Lot #61-1012, BioWhittaker Co.). The serum may be added to a concentration of 20%. The reducing agent is an SH reagent, and the SH reagent is preferably β-mercaptoethanol (214-18, Lot# MOM7582, Nacalai Tesque). The concentration of the reducing agent may be 1 nM to 10 mM, preferably 10 nM to 5 mM and more preferably 100 μM to 2 mM. The culturing time in step (2) may be 1 hour to 5 days, preferably 12-48 hours and more preferably 18-30 hours. The aforementioned reagent concentration is the concentration in the culture medium with which the cells are in direct contact (same for reagents referred to hereunder).

The differentiation inducing agent may be retinoic acid (all-trans) (R-2625, Sigma). The differentiation inducing agent concentration may be 0.001 ng/ml to 1 μg/ml, preferably 1 ng/ml to 200 ng/ml and more preferably 10 ng/ml to 60 ng/ml. In step (3), the culture medium used in step (2) may be exchanged with fresh differentiation inducing agent-containing medium after step (2) has been completed. The fresh culture medium is identical to the culture medium used in step (1) except that it contains the differentiation inducing agent. The culturing time for step (3) may be 1 hour to 30 days, preferably 12 hours to 7 days and more preferably 2-4 days.

The cyclic AMP-augmenting agent or cyclic AMP analogue may be forskolin (344273, Calbiochem). The concentration of the cyclic AMP-augmenting agent or cyclic AMP analogue may be 0.001 ng/ml to 100 μg/ml, preferably 100 ng/ml to 50 μg/ml and more preferably 1 μg/ml to 10 μg/ml.

The glial cell differentiation and survival stimulating factor may be one selected from the group consisting of neuregulin, platelet-derived growth factor-AA (396-HB, Peprotech EC, Ltd.), basic fibroblast growth factor (100-18B, Peprotech EC, Ltd.) or mixtures thereof. Neuregulin is available as Heregulin™ (396-HB, R&D Corp.) The concentration of the glial cell differentiation and survival stimulating factor may be 0.001 ng/ml to 100 μg/ml, with a concentration of preferably 0.1 ng/ml to 100 ng/ml and more preferably 1 ng/ml to 10 ng/ml for platelet-derived growth factor-AA, and a concentration of preferably 10 ng/ml to 1 μg/ml and more preferably 100 ng/ml to 300 ng/ml for basic fibroblast growth factor. The culturing time in step (4) may be 1 hour to 30 days, and preferably 4 to 10 days.

According to another mode of the invention there are provided bone marrow stromal cell-derived Schwann cells obtained by the aforementioned differentiation inducing method.

According to yet another mode of the invention there is provided a pharmaceutical composition for neural regeneration comprising bone marrow stromal cell-derived Schwann cells.

According to yet another mode of the invention there is provided a method of treating neural disease by transplanting the aforementioned bone marrow stromal cell-derived Schwann cells or a pharmaceutical composition for neural regeneration comprising them into a patient with neural disease to cause regeneration of the neural cells with which the disease is associated.

Throughout the present specification, the term "bone marrow stromal cells" refers to cells in the bone marrow which are not of the hemopoietic system and are considered capable of differentiating to cells of the bone, cartilage, etc. Bone marrow stromal cells are positive for Thy1.2 and (β1-integrin) and negative for CD34, as shown in the immunofluorescent photographs of FIG. 7 (see Original Patent). They may be positive or negative for S-100 (calcium-binding protein). Antibodies for Thy1.2, β1-integrin and CD34 were used.

Throughout the present specification, the term "natural Schwann cells" refers to Schwann cells collected from the peripheral nerves of living bodies, namely dorsal root ganglions. As seen in the upper immunofluorescent photograph of FIG. 8 (see Original Patent), they are positive for S-100.

Throughout the present specification, the term "bone marrow stromal cell-derived Schwann cells" refers to Schwann cells which (1) closely resemble natural Schwann cells in morphology and do not revert to the form of bone marrow stromal cells by subculturing, (2) exhibit the same reaction as Schwann cells with respect to P75 (nerve growth factor (NGF) receptor, low affinity), S-100, GFAP (glial fibrillary acidic protein, a type of intermediate filament), nestin (a type of intermediate filament) and O4 (a marker for myelin-producing cells such as Schwann cells and oligodendrocytes) based on immunostaining as shown in the lower immunofluorescent photographs of FIG. 8, and (3) have features similar to natural Schwann cells in their neurogenic ability, but can be distinguished from natural Schwann cells due to their distinct differentiation histories. Antibodies for P75, S-100, GFAP, nestin and O4 were obtained from the following sources: Anti-nerve growth factor-receptor, Boehringer Mannheim, 1198645; Anti-S-100, z-311, Dako Corp.; Anti-glial fibrillary acidic protein, L-1812, Dako Corp.; Anti-nestin, BMS4353, Bioproducts; Anti-O4, 1518925, Boehringer Mannheim.

Bone marrow was treated in multistages according to the invention, as shown in the micrograph of FIG. 9 (see Original Patent).

Stromal cells (right upper and lower photographs in FIG. 9) exhibit the same morphology as natural Schwann cells (left photograph in FIG. 9).

FIG. 10 (see Original Patent) shows the morphology of bone marrow stromal cells obtained by the method of inducing differentiation of bone marrow stromal cells according to the invention, without steps (2) to (4) or some of the reagents used therein. The micrograph at the upper left in FIG. 10 shows natural Schwann cells. The micrograph at the lower right shows bone marrow stromal cells before treatment. The top center micrograph shows results by the method of inducing differentiation of bone marrow stromal cells according to the invention conducted without omission of steps (2) to (4), and it is seen that the method of the invention produced cells exhibiting a morphology similar to natural Schwann cells. The micrograph at the upper right was obtained with omission of step (4), and the micrographs at the lower left and bottom center were taken without the differentiation inducing agent retinoic acid and without forskolin in step (3) and step (4), respectively.

It was thus demonstrated that the multistage treatment described in steps (2) to (4) above induces differentiation of bone marrow stromal cells into bone marrow stromal cell-derived Schwann cells with high efficiency.

The term "high efficiency" as used throughout the present specification means that the differentiation inducing method of the invention converts a high proportion of the original bone marrow stromal cells into the final bone marrow stromal cell-derived Schwann cells. The high efficiency of the differentiation inducing method of the invention is 50% or greater, preferably 75% or greater, more preferably 90% or greater and most preferably 95% or greater. Although each of the individual steps described above have been known, the selection and optimum combination of the steps as according to the present invention were first discovered by the present inventors, and the discovery is highly significant. Specifically, while it was known that bone marrow stromal cells are mesenchymal stem cells or precursor cells that are capable of being induced to differentiate into osteoblasts, vascular endothelial cells, skeletal muscle cells, adipocytes, smooth muscle cells and the like, as explained above, it was not known whether bone marrow stromal cells could actually be differentiated into neural crest cell-derived Schwann cells, and no party had successfully achieved this despite a strong desire to do so. The present inventors, though not wishing to be constrained by any theory, conjecture that the treatment with a reducing agent in step (2) produces a shock on the cells while the treatment with retinoic acid in step (3) resets the cells, after which the treatment with a cyclic AMP-augmenting agent or cyclic AMP analogue and a glial cell differentiation and survival stimulating factor in step (4) induces differentiation of the cells.

Bone marrow stromal cells may be collected and subjected to treatment involving multiple steps according to the present invention, to induce their differentiation into cells having the same features as natural Schwann cells with respect to neurogenic ability, with high efficiency. By transplanting the bone marrow stromal cell-derived Schwann cells into the peripheral and central nervous system, it has become possible to induce regeneration and elongation of injured nerves.

As explained above, the fact that natural Schwann cells must be collected from peripheral nerves presents a difficulty for application to humans. Bone marrow stromal cells, on the other hand, are easy to obtain without damaging the human body. Moreover, since the cells have a high rate of growth and can therefore be supplied rapidly in large amounts, the present invention makes possible a wider application of bone marrow stromal cells for a variety of nervous system disorders.

Another major advantage afforded by bone marrow stromal cells is their suitability for autologous transplantation. Collecting one's own bone marrow stromal cells, inducing them to differentiate and transplanting the differentiated cells into nerves produces no rejection reaction and therefore requires no immunosuppressants or the like, which should allow regeneration to be achieved in a more stable manner. Since bone marrow stromal cells can also be obtained from bone marrow banks, this method is also practical from the standpoint of supply.

As will be apparent by the examples provided below, the bone marrow stromal cell-derived Schwann cells of the present invention are considered widely applicable for regeneration of peripheral nerves or central nerves. According to one mode, therefore, the invention provides the bone marrow stromal cell-derived Schwann cells themselves. Due to their different induced differentiation histories as mentioned above, they are artificially modified cells which are distinct from natural Schwann cells. According to another mode, the invention provides the bone marrow stromal cell-derived Schwann cells in the form of a pharmaceutical composition for neural regeneration. The bone marrow stromal cell-derived Schwann cells of the invention are suitable for autologous transplantation as explained above, but they may also be allogenically transplanted. This is because the cells of the nervous system are not as susceptible to immune system attack, and rejection reaction can therefore be avoided by using donor cells with matching histocompatibility antigens from a bone marrow bank. The pharmaceutical composition may also contain common pharmaceutically acceptable carriers, buffers, salts, excipients and the like. The composition may be injected into the affected site directly or it may be filled into a hollow tube for transplantation at the site of a severed central or peripheral nerve.

Although peripheral nerves intrinsically are capable of regeneration, it is known that they cannot regenerate over gaps of several centimeters; such cases are also considered to be included among the practical applications to peripheral nerves.

Central nerve conditions wherein reconstruction is considered impossible encompass a wide gamut of different conditions, including injury-related spinal cord damage or cerebrovascular damage and diseases ranging from blinding glaucoma to degenerative diseases such as Parkinson's, which have a high estimated incidence rate among the population. The pharmaceutical composition of the invention may be used for regeneration of many and various types of central nerves. Research on methods of neural regeneration for the aforementioned conditions is an urgent social need, and the present invention is believed to have direct application for the human body.
 

Claim 1 of 14 Claims

1. A method of inducing bone marrow stromal cells to differentiate into bone marrow stromal cell-derived Schwann cells in vitro, comprising: (1) collecting bone marrow stromal cells from bone marrow, and culturing said cells in a standard essential culture medium supplemented with a serum (2) adding a reducing agent to said culture medium, and further culturing said cells; (3) adding retinoic acid to said culture medium, and further culturing said cells; and (4) adding forskolin, and/or a differentiation, survival and growth stimulating factor which acts on nerves and glial cells to said culture medium, and further culturing said cells to obtain said bone marrow stromal cell-derived Schwann cells.
 

 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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