JP3244696B2 - Isolation, proliferation and differentiation of human muscle cells - Google Patents

Description

Description: FIELD OF THE INVENTION The field of the invention relates to the generation of myogenic cells for use in the treatment of neuromuscular diseases and for the transformation of cells of diverse etiology to treat cells of disease.

(Background) Myogenic cells are mesodermal precursor cells used for myogenesis. Certain myogenic cells recognize other myogenic cells that produce differentiated myotubes and can naturally fuse with them. Multinucleated myotubes no longer divide and do not synthesize DNA, but produce large amounts of muscle protein. These include the components of the contractile device and certain cell surface components required for neuromuscular transmission. The terminally differentiated myocytes show characteristic striations and rhythmic contractions. The next step in this pathway is maturity. Contractile devices and muscles at different stages of development contain other types of muscle proteins, such as myosin and actin, encoded by different members of a multigene group.

Methods for producing myogenic cells from fetal and adult tissues have been developed. These methods show that large amounts of myogenic cells can be produced from adult muscle tissue substantially free of other cells. Myogenic cells have a variety of uses.
First, myogenic cells can be used to treat a variety of genetic diseases involving muscle tissue. Myogenic cells are also useful as vehicles for cell therapy where one or more genes are introduced to provide the desired product.

(Related technology) Blau and Webster, Pro
Acad. Sci., USA (1981) 78 : 5623-5627, reports the isolation and cloning of myoblasts for the purpose of growing and differentiating individual clones. Blow (Bla
u) et al., Proc. Natl. Acad. Sci., USA (1983) 80: 4856-486.
0, myogenic cells (satellite cells) in clonal analysis of cultured cells from patients with Dachen's muscular dystrophy,
We report a lack of proliferative capacity of mononuclear precursors in mature muscle fibers. Blau et al., Exp. Cell. Res. (1983) 14
4 : 495-503, report the production and analysis of a pure myogenic cell clone from a biopsy of a patient with Dachen's muscular dystrophy. Blau et al., Science (1985) 2
30 : 758-766 report the fusion of non-muscle cells with activated muscle genes to muscle cells. Webster et al., Exp. Cell. Res. (1988) 174 : 252-265,
Report purification of human myogenic cells using a fluorescence activated cell sorter. In Vitro Cel, such as Ham
l. & Dev. Biology (1988) 24 : 833-844, report a serum-free medium for clonal expansion of human muscle satellite cells. Cel, such as Webster
1 (1988) 52 : 503-513, showed that Dachen's muscular dystrophy selectively affects a group of skeletal muscle fibers specialized for fast contraction (see references cited therein).

SUMMARY OF THE INVENTION Myogenic cells are prepared in serum-free or low-serum media for cell therapy. Myoblasts can migrate, fuse with pre-existing fibers, and are also used as carriers for genes that are introduced as a result of transformation. The migration of myogenic cells through the basal layer can reduce infusion for the purpose of treating various diseases.

DESCRIPTION OF SPECIFIC EMBODIMENTS Methods and cells for use in cellular therapy for the treatment of disease are provided. Methods for preparing large quantities of clonally pure or substantially enriched myoblasts in the absence or substantially absence of serum in a nutrient medium are described. The resulting cells are used in the treatment of various diseases involving muscle tissue or other tissues in which soluble factors are involved.

The cells used are myogenic cells obtained from a tissue sample containing tissue from a fetal, neonatal or more mature human. These cells may be fresh, frozen immediately after being obtained from the patient, or clone cultures that have been grown about 5 to 30-fold and stored frozen until use. The cells are grown under optimal conditions using a cell culture incubator (37 ° C., 5% CO ₂ containing air, saturation humidity). Use other conditions if necessary. The medium used grows without significant differentiation. Thus, this medium maintains the level of maturation of the myoblasts, but can be introduced into an environment where they differentiate and mature as needed.

This medium contains essential amino acids, inorganic salts, trace elements and vitamins, and other organic components. The following table shows that, as is well known in the art, each component is changed so as not to have a detrimental effect on the proliferation of myoblasts, but is generally considered optimal. .

Supplement the following factors in addition to the basal medium called MCDB120. The first factor is 0.3-0.5 μg / ml, preferably about 0.39
/0.40 μg / ml dexamethasone. 0.25-0.75μg / m
1, preferably about 0.50 mg / ml of serum albumin, especially bovine serum albumin, is also used. About 5 to 15 ng, preferably about 10 ng, of epidermal growth factor is also used. About 0.25-0.75mg / m
1, preferably 0.5 mg / ml fetuin is also used. Finally, about 150-200 μg / ml, preferably about 180 μg / ml of insulin, conveniently bovine insulin, is also used.

For the growth of myoblasts, the inoculum is introduced into the medium described above and the cells are grown under the conditions described above. After addition of the inoculum, the medium is gently agitated to maintain a uniform distribution of cells for the purpose of increasing confluency.

Cells are harvested in a convenient manner. The tissue is dissociated by performing 2 to 3 continuous treatments for a total of 40 to 60 minutes using 0.05% trypsin-EDTA in a Wheatin trypsinization flask with constant stirring.

The cells recovered from the supernatant after each trypsin treatment are harvested and cooled to 4 ° C. on ice. Equine serum is added to a final 10% (v / v) to stop protease activity. Centrifuge the dissociated cells (2 minutes, 25 ° C). The cell pellet is suspended in conditioned medium and plated on the medium or frozen in liquid nitrogen to a density of about 0.1 cm ³ of tissue per ml.

In the case of culture, the myoblasts are transformed by various methods including fusion, transfection, infection, electroporation, particle bombardment, and the like.
The method of introducing the foreign DNA is not critical to the invention. Depending on the purpose of the DNA transfer, it is of interest to perform homologous or orthologous integration specifically by recombination or by heterologous recombination. Smithies, etc. (1985)
Nature 317 : 230-235; Thomas and Capecchi (1987) Cell 51 : 503-512 and Mansour et al. (1988) Nature 336 : 348-352. When performing specific integration, the gene of interest is flanked by at least 50 bp, usually 100 bp, on each side of the DNA homologous to the integration site. It is preferable that the size of this homologous DNA is about 10 kbp, usually about 5 kbp at most, and that the adjacent regions have substantially the same size.

The integration region contains a DNA sequence associated with a particular muscle defect. Thus, the host myogenic cells are removed from the host and transformed by homologous recombination. The cells are then cloned and screened for homologous recombination at the defective site. Alternatively, for natural inducible genes that are normally repressed in myoblasts or mature muscle tissue, transcription initiation regulatory sequences, such as promoters, with or without enhancers, may be modified and induced or constitutively modified. Homologous recombination can occur that provides the basis for transcription. Thus, the myogenic cells can be used to express endogenous (to the host) or heterologous genes not normally expressed in muscle tissue. For example, expression of cytokines, growth factors, colony stimulating factors, interferons, surface membrane receptors, insulin and the like is possible. By modifying the transcription initiation regulatory region, myogenic cells can achieve constitutive production of the expression product, and
Separately or simultaneously, a receptor for the soluble product can be introduced, which provides for inducible transcription of cellular proteins, such as the cytoplasm and nucleus. By activating this receptor, myogenic cells are induced to produce an expression product under conditions that induce the related ligand.

Various vectors are used for transformation of myoblasts. Of particular interest for transfection and infection are replication defective viral vectors, DNA virus or retroviral vectors that can be introduced into cells. Usually, these vectors contain prokaryotic DNA.
Is not included, and various functional sequences are included.

As previously discussed, functional sequences include DNA regions that traverse transcriptional and translational start and stop regulatory sequences, open reading frames encoding the protein of interest, and additional flanking regions for site-specific integration. May include. As already indicated, in some situations, the 5 'flanking region alters the nature of the transcription initiation region by homologous recombination. For example, the presence or absence of an enhancer causes a difference in whether or not transcription can be induced or the level of transcription increases or decreases. Similarly, promoter regions may be modified to alter induction sensitivity and transcription levels.

Structural genes to be used include those that are maintained in the cell, such as organelles such as the cytoplasm and nucleus, or that produce intracellular membranes and intracellular products that are transported to the cell membrane, and the natural signal sequences that are naturally present in the structural genes Some are secreted by providing a signal sequence that does not exist originally. If the soluble protein of interest is a large protein fragment, it is necessary to provide such a protein with a signal sequence. As a result, the protein desired for processing at the secretory or processing site will be of the native sequence.

Markers exist to select for cells containing the vehicle construct. Usually this marker allows for a positive selection that provides one or more cytotoxic drug resistances. For example, kanamycin resistance, which selects cells with G418, or dihydrofolate reductase, which exhibits resistance to methotrexate, is used. Since these markers use inducible or non-inducible genes, selection is performed under inducing or non-inducing conditions.

The vector also contains an origin of replication and other genes required for replication in the host. An origin-containing replication system and replication-related proteins encoded by the particular virus are included as part of the construct. Care must be taken in choosing a replication system so that the gene encoding replication does not cause myogenic cell transformation. An exemplary replication system is the Epstein-Barr virus. In addition, replication-defective vehicles, especially replication-defective retroviral vectors, are used. Price for these vectors
Proc. Natl. Acad. Sci., USA (1987) 84 : 156-160 and Sales et al., EMBO J. (1986) 5 : 3133-3142.
There is a report by. The final vehicle construct contains one or more genes of interest. Either cDNA or chromosomal genes can be used. It is of particular interest to provide at least one intron in the 5 'non-coding or coding region. Messenger RN
A has been found to increase stability.

In addition, cells can be transformed in vivo by injection of an infectious replication-defective viral vector. The vector is introduced into retroviral producer cells for ecotropic packing. The cells are then harvested, filtered and concentrated by centrifugation. The virus stock is then injected at one site in vivo. Because myogenic cells have been shown to migrate and spread to adjacent areas, relatively little injection is required for muscle fibers.

For administration of the vector, it is convenient to inject using a physiologically acceptable medium such as water, saline, or phosphate buffer. Generally, the virus concentration is about 10 ⁵ ffu or more. Other additives include polybrene. Usually, about 10 ⁵ cells per cm ³ of muscle tissue are used for injection. Damage to the tissue is minimized by reducing the number of injections to the target area. It is clear that it is desirable to reduce the number of injections to a particular area, especially if the patient requires extensive treatment.

The following examples are for illustrative purposes and do not limit the invention.

EXPERIMENTAL (Vectors) Two β-galactosidase vectors called BAG and pMMuLVSVnlsLacZ were used, each of which contains a β-galactosidase coding sequence under the transcriptional control of the MMULV promoter / enhancer or the SV40 early promoter. Furthermore, these vectors are ne
o Has ^γ and LacZ genes, and in some cases has seven amino acid codons for the SV40 large T nuclear localization sequence.

[Psi _CRE or [psi ₂ ecotropic each BAG or pMMuLVSVnlsLacZ each CREBAG2 or P produced by transfection of the packaging cell line of a recombinant resistant
A12Ψ ₂ 12-C2 were collected retrovirus producer cell-derived supernatants were concentrated by filtration after centrifugation (Price (Pr
ice) et al., 1987; Sanes et al., 1986). The virus stock (50-200μ) was mixed with activated charcoal and 10μM polybrene and injected with a 26-gauge needle into the dorsal and dorsal hind paw of anesthetized Wistar rats. After about two weeks, the animals were fixed with cardiac perfusion using 4% paraformaldehyde, 0.5% glutaraldehyde, 100 mM PIPES (pH 7.4). After 30-60 minutes, the hind paw skin was removed and immersed in the same fixative overnight at 4 ° C, followed by immersion in 30% sucrose phosphate buffer (PBS) at 4 ° C for 24 hours. This was frozen in freezing isopentane and sliced continuously using a cryostat to a thickness of 30 μm. After fixing these fragments with 2% paraformaldehyde, and washing, 1 mg /
mlX-gal, 35 mM ferri and potassium ferrocyanide,
Stain overnight at 30 ° C. with 1 mM MgCl ₂ phosphate buffer, place in glycerin: PBS (9: 1), then check for the presence of the blue X-gal reaction product on a Zeiss Axiophoto brightfield microscope. Examined. Muscle fibers stained blue were scattered on the hind legs. Activated carbon particles were generally present between the heel and lateral gastrocnemius muscle and were used to identify the site of infection. Differences in the distribution or size of the labeled myocyte clusters were BAG or p, respectively.
It was observed with the MMuLVSVnlsLacZ vector. BAG vector is P
At 9 (P = postnatal), analysis was performed on injection P23 and pMMuLVSVnlsLac
Z was injected at P16 and analyzed at P29. In addition, labeled cell clusters close to activated carbon particles were indistinguishable from those a few millimeters away, indicating that the injection did not destroy nearby tissue.

Rats injected on day 9 and analyzed on day 23 had a high fiber count and 16/25 clusters, whereas rats injected on day 19 and analyzed on day 35 in the case of,
The number of clusters spanning many fibers was found to be one in one.

The cluster of stained muscle fibers in the lateral gastrocnemius muscle indicates that the myogenic cells can be infected even after fusion to polynuclear fibers and can express β-galactosidase. Many of the observed clusters represent single cell-derived clones, some of whose progeny migrate through the basal layer of a given muscle fiber and fuse with neighboring muscle fibers. As each myogenic cell associates with a single fiber upon infection, the data indicate that myogenic cells can migrate through the basal layer from one fiber to another. Furthermore, since most infections result in clones that span many muscle fibers, their migration is likely to occur relatively frequently.

To show that each cluster of β-galactosidase positive cells present in infected rat paws was derived from a single retrovirus-infected myogenic cell, a mixture of two vectors showing different β-galactosidase staining patterns was injected. . To maximize the number and size of clones, the two vector mixtures were injected into the hind paws of P0 rats, where proliferation and fibrosis were active. Fibers adjacent to cytoplasmic stained fibers contained nuclear staining in only two cases in two paws well infected with 87 β-galactosidase positive clusters, including 23 with cytoplasmic β-galactosidase. Therefore, the frequency of independent infection of adjacent myogenic cells is 40-50 per hindpaw.
Less than 15% in animals infected at the clonal level. Therefore, in the case of mildly infected hind legs, virtually 1
It is unlikely that one or more or two clones originate from two or more separate infections. Most are clones from a single infection.

Whether the labeling of each fiber was from a separate infection was determined by using two viral techniques to determine whether the cytoplasmic and nuclear staining in all labeled fibers was randomly distributed. The cytoplasmic and nuclear staining patterns were segregated into distinct regions, the opposite being observed in that each contained a cluster of several fibers.

Since the number of fibers is clearly kept constant, it is unlikely that new fibers were formed during the experiment. The average diameter of the fibers in the clone was the same as the total average fiber diameter of the muscle, indicating that the multi-fiber clones were not selectively containing small newly formed fibers.
The generation of multiple fiber clones at a high frequency compared to the highest rate of new fibrogenesis indicates that myogenic cells destined to form new fibrils are much more susceptible to vector infection than a dividing myogenic cell population. It indicates that selectivity must be high. These data strongly support the migration of myogenic cells through the basal layer as a mechanism by which transformed myogenic cells are contained in different fibers.

It is clear from the above results that the present invention allows the use of muscle forming cells for the treatment of muscle fiber related diseases or for the production of soluble or other proteins in a host. Myogenic cells can form new muscle tissue and participate in fibrosis, where these cells can provide useful properties or correct defects. In addition, modifying these cells with a marker can allow for the selection of transformed cells among naturally occurring cells. All publications and patent applications cited herein are each incorporated by reference as though they were incorporated by reference.

Although the present invention has been described in detail and by way of example for a clearer understanding, it will be apparent to those skilled in the art that modifications may be made without departing from the spirit and scope of the appended claims. Would.

Continuation of the front page (72) Inventor Hughes Simon M. United States of America 94301 Palo Alto Addison Street 733 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 48/00 ABJ C12N 5/10 C12N 15 / 09 BIOSIS (DIALOG) MEDLINE (STN)

Claims (19)

(57) [Claims] 1. A population of clonally pure or substantially enriched myogenic cells grown in primary serum cells in a substantially serum-free medium that provides for expansion of the myogenic cells without significant differentiation. A myoblast composition obtainable by a method comprising the step of obtaining exogenous DNA into said primary myoblasts or said clonally pure or substantially enriched population of myoblasts in vitro. The myogenic cell composition contains foreign DNA as a result of the transformation;
Contains a gene encoding a protein, wherein the myogenic cells of the myogenic cell composition are incorporated into pre-existing myofibers and the foreign DN
A myogenic cell composition capable of expressing A. 2. The myogenic cell composition of claim 1, wherein said exogenous DNA has been introduced by transfection into primary myogenic cells or a clonally pure or substantially enriched population of myogenic cells. 3. The myogenic cell composition according to claim 2, wherein said transfection comprises a replication-defective retrovirus. 4. The myogenic cell composition of claim 1, wherein said gene is integrated into the genome of a primary myogenic cell or a population of clonally pure or substantially enriched myogenic cells by homologous recombination. . 5. The transcription initiation region wherein the exogenous DNA functions in a primary myoblast or a population of clonally pure or substantially enriched myoblasts, wherein the gene is transcribed from the transcription initiation region. 5. The myoblast composition according to any one of claims 1 to 4, which is under control. 6. The myoblast composition of claim 5, wherein said transcription initiation region provides for constitutive expression of said gene. 7. The myoblast composition of claim 5, wherein said transcription initiation region provides for inducible expression of said gene. 8. The myoblast composition according to claim 1, wherein the foreign DNA further contains a signal sequence. 9. The exogenous DNA of claim 1, wherein said exogenous DNA contains a gene expressed in a primary myoblast or a population of clonally pure or substantially enriched myoblasts.
Item 8. The myogenic cell composition according to Item. 10. The myoblast composition according to claim 1, wherein the foreign DNA contains a gene encoding a secreted protein. 11. The secretory protein is a cytokine, a growth factor,
11. The myogenic cell composition according to claim 10, comprising a colony stimulating factor, an interferon, a surface membrane receptor, or insulin. 12. The myogenic cell composition according to claim 1, wherein the primary myogenic cells are mature myogenic cells. 13. The myogenic cell composition according to claim 12, wherein the primary myogenic cells are human myogenic cells. 14. A pharmaceutical composition for treating a disease associated with muscle tissue, comprising the myogenic cell composition according to any one of claims 1 to 13. 15. A pharmaceutical composition comprising the myogenic cell composition according to any one of claims 1 to 13 for treating a disease relating to a tissue other than muscle tissue associated with a soluble factor. 16. The method for producing a myogenic cell composition according to claim 1, wherein a DNA construct containing a gene that can be expressed in primary myogenic cells is used. 17. The method for producing a pharmaceutical composition according to claim 14, wherein a replication-defective virus containing a DNA construct that can be expressed in primary myogenic cells is used. 18. The method for producing a pharmaceutical composition according to claim 15, wherein a replication-defective virus containing a DNA construct that can be expressed in primary myogenic cells is used. 19. The method according to claim 17, wherein the replication-defective virus is a retrovirus.