Patent Application
20220184138
The present invention relates to transcription factors (nucleus pulposus cell master regulator transcription factors) that enable direct cell reconstruction (direct reprogramming) from terminally differentiated cells or cells with differentiation capacity to an active nucleus pulposus cell phenotype, that is, transdifferentiation or differentiation induction from undifferentiated cells to nucleus pulposus cells. The present invention further relates to an active nucleus pulposus cell phenotype (induced nucleus pulposus cells) obtained by transdifferentiation or differentiation induction, and to use thereof.
Low back pain and neck pain are common health problems that affect 632 million people all over the world and a major cause of disability. The two disorders place significant social and economic burdens due to work disability and medical costs. Intervertebral disc degeneration, which is estimated to develop 20% of all low back pain cases, destructs biomechanics along the spinal column and leads to intervertebral hernia, spinal canal stenosis, spondylolisthesis, and other spinal disorders. Intervertebral disc degeneration is an intervertebral disc disorder that is particularly characterized by irreversible degeneration of the extracellular matrix composition in the core of an intervertebral disc.
At present, there is no clinically effective treatment that enables recovery from such a degenerative state or can stop the underlying pathogenesis. Therefore, there is a strong demand for the development of new treatments. Nevertheless, general understanding of intervertebral disc homeostasis, cell phenotype, and development and progression of the pathogenesis is poor, particularly as compared with knowledge insight in the field of bone and articular cartilage.
An intervertebral disc is the fibrocartilage structure between each two vertebrae along the spine. The intervertebral disc is involved in distributing mechanical forces along the spinal column while imparting flexibility. This feature arises from the hydrostatic pressure established within a sealed nucleus pulposus. The nucleus pulposus (NP) is composed mainly of proteoglycans and type II collagen fibers that are gently arranged and absorbs a relatively large amount of water. The nucleus pulposus is laterally surrounded by a fibrocartilage layer called annulus fibrosus (AF). Finally, the intervertebral disc is covered by a thin layer of hyaline cartilage on the boundary with each vertebra, that is, an endplate.
It is suggested that native nucleus pulposus cells change their phenotype from vacuolated notochord cells to active nucleus pulposus cells, senescent cells, and fibrous nucleus pulposus cells, to form a lot of heterogeneous cell populations in the nucleus pulposus. Such changes to different cell types seem to play a decisive role in the progression of intervertebral disc degeneration. Due to aging and biological and mechanical stress (wear), the nucleus pulposus shows a gradual decrease in Tie2/GD2-positive progenitor cells (Non Patent Literature 1: Sakai et al., 2012), and the cells are aged to be switched to fibrous cells. These changes result in a change in a proteoglycan-rich nucleus pulposus extracellular matrix into a fibrous structure, thereby deteriorating the hydrostatic pressure and other biomechanical characteristics of the intervertebral disc. A cascade of these events potentially causes low back pain and other spinal disorders. Conversely, intervertebral disc degeneration and related disorders are rarely seen in other animal models (such as pigs, mice, and rats). This is likely due to the facts that these animals maintain the original notochordal cell phenotype, and as such are able to maintain healthy intervertebral discs with an active phenotype of nucleus pulposus cells, which play a decisive role in preventing the intervertebral disc disorders. Accordingly, for treating human intervertebral disc degeneration or the like or alleviating pains and disorders, a strong research emphasis is placed on establishing strategies to repopulate or induce proliferation of endemic cell populations in the intervertebral disc tissue to actively produce and regenerative intervertebral disc matrix.
As part of such strategies, various studies show the advantage of transplanting active nucleus pulposus cells or chondrocytes into affected areas by intervertebral disc degeneration or the like (as reviewed in, Non Patent Literature 2: Schol and Sakai, 2018), and it is regarded as a promising option for treatment of intervertebral disc degeneration or the like. Nevertheless, supply of active nucleus pulposus cells or the like for cell transplantation is insufficient both clinically and scientifically. Conventionally, the possibility of regenerating intervertebral discs by transplanting autologous or allogeneic donor nucleus pulposus cells that are isolated from an intervertebral disc surgically removed during surgery or chondrocytes that essentially have the ability to produce an extracellular matrix like the nucleus pulposus cells in the intervertebral disc environment has been explored. However, since the intervertebral discs or cartilage collected from donors by surgery or the like are often damaged due to diseases, trauma, aging, or the like, there is a possibility that nucleus pulposus cells or chondrocytes contained therein do not have sufficient effectiveness as transplantation regeneration materials (Non Patent Literature 1). Further, in the treatment method of transplanting nucleus pulposus cells, donor cells are reactivated and generated by in-vitro culture to prepare the final purified product of active nucleus pulposus cell populations, but the traits of nucleus pulposus cells are known to be lost (dedifferentiated) by culture, and it is a technical problem to amplify nucleus pulposus cells without dedifferentiation (Non Patent Literature 1).
As cells to be transplanted into the intervertebral disc, it is also conceivable to use stem cells capable of differentiating into nucleus pulposus cells, such as mesenchymal stem cells (MSCs) that are derived from bone marrow, placenta, fat, or the like, comparatively easily accessible, and available in a large amount. However, the hypoglycemia, hyperosmolarity, and hypoxic environment caused by avascular and progressive degeneration of an intervertebral disc is a significant obstacle to the survival and thriving of MSCs. Therefore, it is unknown to what extent the transplanted MSCs can actively produce and secrete matrix proteins or cytokines for reactivating surrounding cells to regenerate the intervertebral disc tissue for long-term effects, and thus clinical application of MSCs to intervertebral disc disorders is limited (Non Patent Literature 3: Fang, Z., et al., 2013). There are similar problems in pluripotent stem cells other than mesenchymal stem cells, such as hematopoietic stem cells.
Therefore, a technique for artificially maintaining or inducing the traits (phenotype) of active nucleus pulposus cells is required, but information on transcription factors and their control, which is the key of the technique, is limited, and induction of the active nucleus pulposus cell phenotype itself in vitro has not been realized so far. Conventionally, the possibility of obtaining a nucleus pulposus cell-like phenotype as an alternative to actual nucleus pulposus cells by applying various stimuli to cells that seem promising among cells other than nucleus pulposus cells has been studied. As such candidate cells, induced pluripotent stem cells (iPS cells) are used other than MSCs, although less frequently. It is known that various cells can be obtained from such pluripotent or multipotent stem cells by differentiation induction under appropriate conditions. In order to obtain a nucleus pulposus cell-like phenotype from pluripotent or multipotent stem cells, stimulation by extracellular matrix compositions, oxygen partial pressure, mechanical or osmotic pressure, application of scaffold such as hydrogel, stimulation by growth factors or cytokines, stimulation by co-culture with other cells or culture supernatant, or combinations thereof are attempted. These studies have generally reported that the expression level of nucleus pulposus cell-related genes such as type II collagen (COL2), aggrecan (ACAN), and differentiation cluster 24 (CD24) can be enhanced, or the production of those proteins can be improved. Further, it has been also reported that cell encapsulation, aggregation, or direct transplantation in vivo causes deposition of an extracellular matrix similar to an NP-cell like extracellular matrix rich in proteoglycans and COL2, which is generally reported as regeneration of an intervertebral disc.
In general, there is a concern about the reproducibility in the method using growth factors. In order to elicit a desired response by applying growth factors, cells need to present the correct cell membrane-bound receptors that correspond to the growth factors. The presentation of appropriate receptors is highly specific to the cell type and donor, thus potentially causing problems in reproducibility of the induction procedure. Further, it is still unknown whether there is a possibility that cell transplantation in an environment with no or different growth factors (that is, in an environment in the original nucleus pulposus tissue or the like), can negatively alter the transplanted cell phenotype. Finally, continuous supplementation of growth factors accounts for a relatively expensive part of a culture process for producing or using induced nucleus pulposus cells, thereby further limiting the clinical applicability.
Only a few studies have explored the possibility of fabricating an active nucleus pulposus cell phenotype from pluripotent or multipotent cells, such as MSCs and iPSCs, or terminally differentiated cells other than nucleus pulposus cells by more direct manipulation to the gene expression profile, instead of adjusting the culture conditions as above, and information on transcription factors and their control, which is the key of such gene operation, has been exceptionally limited.
For example, Non Patent Literature 4 (Xu et al., 2016) discloses that bone morphogenetic protein 7 (BMP7) was overexpressed in rabbit bone marrow-derived MSCs, in order to stimulate differentiation into NP cells by enhancing secretion of BMP7, as a result of which, the expressions of COL2, ACAN, SOX9, keratin (KRT)-8, and KRT19 were enhanced at the mRNA level by monolayer culture two to three weeks after transfection. However, when a cell product obtained in this method was transplanted into a rabbit model with intervertebral disc degeneration, a beneficial effect was observed in both the sham control and the BMP7 transfection group 6 weeks and 12 weeks after transplantation, but the glycosaminoglycan/DNA ratio in the BMP7 overexpression group at the 12th week were merely slightly increased as compared with the sham control.
Non Patent Literature 5 (Chen et al., 2017) discloses that, by the same approach, an established factor WNT11 was overexpressed by lentivirus transduction in rat fat-derived MSCs, and an in-vitro evaluation thereof showed a slight but significant increase in COL2, ACAN, and SOX9 at both the mRNA and protein levels, as compared with the sham control was transfected to overexpress green fluorescent protein (GFP).
As another approach, Non Patent Literature 6 (Outani et al., 2013) discloses that introduction of SOX9, known as a master regulator transcription factor for forming cartilage into fibroblasts (Non Patent Literature 7: Wright et al., 1995, and Non Patent Literature 8: Bi et al., 1999), enabled direct reprogramming of their cells into a chondrocyte phenotype.
Non Patent Literature 9 (Yang et al, 2011) discloses that a cell line overexpressing SOX9 was established in rat adipose tissue-derived MSCs by a leukemia virus-derived viral vector pseudotyped with vesicular stomatitis virus G-envelope glycoprotein. Further, it also discloses that, as a result of culturing the established cell line with or without addition of transforming growth factor β (TGFβ)-3, the ratios GAG/DNA and COL2/DNA changed, and such a result showed that the overexpression of SOX9 could induce a relative increase in the ratios of GAG and COL2 with supplementation of TGFβ3, conversely, manipulation by only any one of supplementation of TGFβ3 to the culture medium or transduction with SOX9 would not result in a significant improvement. However, the literature does not present any further evaluation regarding a nucleus pulposus cell phenotype.
Likewise, Non Patent Literature 10 (Sun et al. 2014) also discloses that rabbit bone marrow-derived MSCs were transduced with SOX9 through an adenoviral vector. In an in-vitro monolayer evaluation, the transduced cells clearly showed an obviously strong increase in expressions of ACAN, COL2, and SOX9 at the mRNA level but showed only a slight decrease in type I collagen (COL1), as compared with the GFP-control group. The same results were obtained from transduced cells cultured using chitosan-glycerophosphate gel. Finally, when transduced cells were transplanted into a rabbit model with induced intervertebral disc degeneration, both a GFP-expressing MSC-administered group and a SOX9-overexpressing MSC-administered group showed improved T2 intensity, which showed the advantage of MSC transplantation again, but the SOX9-overexpressing MSC-administered group showed a small but significant improvement as compared with the GFP-expressing cells. Likewise, the histological results also revealed a benefit by MSC transplantation with a slight increase due to the SOX9 overexpression, as shown by safranin-O staining (representing the content of proteoglycan), and COL2 targeted staining, in contrast to the GFP expression.
Non Patent Literature 11 (Hiramatsu et al., 2011) discloses that mouse adult dermal fibroblasts were differentiated using combinations of reprogramming factor c-MYC with transcription factors KLF4 and SOX9, and as a result of overexpression of their genes, expressions of ACAN and COL2 specific to chondrocytes could be stimulated, despite the mature differentiated state of starting cell populations.
Non Patent Literature 12 (Outani et al, 2013, by the same group as Non Patent Literature 11) also discloses evidence of successful reprogramming from fibroblasts to a chondrocyte phenotype by overexpression of KLF4, c-MYC and SOX9. This literature discloses that expressions of ACAN and COL2 were enhanced by application to dermal fibroblasts derived from humans. Further, it is also revealed that transplantation of the reprogrammed fibroblasts enhanced deposition of a chondrogenic matrix in a mouse model with any one of subcutaneous and chondrogenic defect indications.
The aforementioned conventional art literatures generally indicate that expression of transcription factor SOX9 can stimulate the chondrogenic phenotype. Accordingly, overexpression of SOX9 highly possibly results in a more common chondrocyte phenotype rather than a specific nucleus pulposus cell phenotype. The conventional art literatures did not examine markers or morphological characteristics specifically associated with nucleus pulposus cells, as a comparison with other cells exhibiting cartilage traits.
In addition, as markers associated with the traits of nucleus pulposus cells, hypoxia-inducible factor 1 sub-unit α (HIF1α) (Non Patent Literature 13: Risbud et al., 2006), Brachyury (T) (Non Patent Literature 14: Sheyn et al., 2017), Paired Box 1 (PAX1) (Non Patent Literature 15: Risbud et al., 2015), and the like have been reported, but their gene expression profiles have not been sufficiently evaluated so far.
[Non Patent Literature 1] Sakai, D. et al. Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nature communications 3, 1264, doi: 10.1038/ncomms 2226 (2012).
[Non Patent Literature 2] Schol J, Sakai D. Cell therapy for intervertebral disc herniation and degenerative disc disease: clinical trials. International orthopaedics. (2018) doi: https://doi.org/10.1007/s00264-018-4223-1.
[Non Patent Literature 3] Fang, Z., et al., Differentiation of GFP-Bcl-2-engineered mesenchymal stem cells towards a nucleus pulposus-like phenotype under hypoxia in vitro. Biochem Biophys Res Commun, 2013. 432 (3): p. 444-50.
[Non Patent Literature 4] Xu, J., et al., BMP7 enhances the effect of BMSCs on extracellular matrix remodeling in a rabbit model of intervertebral disc degeneration. FEBS J, 2016. 283 (9): p. 1689-700.
[Non Patent Literature 5] Chen, H. T., et al., Wnt11 overexpression promote adipose-derived stem cells differentiating to the nucleus pulposus-like phenotype. Eur Rev Med Pharmacol Sci, 2017. 21 (7): p. 1462-1470.
[Non Patent Literature 6] Outani, H. et al. Direct induction of chondrogenic cells from human dermal fibroblasts cultured by defined factors (PloS one, 2013).
[Non Patent Literature 7] Wright, E., et al., The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet, 1995. 9 (1): p, 15-20.
[Non Patent Literature 8] Bi, W., et al., Sox9 is required for cartilage formation. Nat Genet, 1999. 22 (1): p. 85-9.
[Non Patent Literature 9] Yang, Z., et al., Sox-9 facilitates differentiation of adipose tissue-derived stem cells into a chondrocyte-like phenotype in vitro. J Orthop Res, 2011. 29 (8): p. 1291-7.
[Non Patent Literature 10] Sun, W., et al., Sox9 gene transfer enhanced regenerative effect of bone marrow mesenchymal stem cells on the degenerated intervertebral disc in a rabbit model. PLoS One, 2014. 9 (4): p. e93570.
[Non Patent Literature 11] Hiramatsu, K., et al., Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors. J Clin Invest, 2011. 121 (2): p, 640-57.
[Non Patent Literature 12] Outani, H., et al., Direct induction of chondrogenic cells from human dermal fibroblast culture by defined factors. PLoS One, 2013. 8 (10): p. e 77365.
[Non Patent Literature 13] Risbud, M. V., et al., Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment. JC ell Biochem, 2006.98 (1): p. 152-9.
[Non Patent Literature 14] Sheyn et al (2017) Human iPS-derived notochordal cells survive and retain their phenotype in degenerated porcine IVD-ORS 2017 Annual Meeting Paper No. 0051.
[Non Patent Literature 15] Risbud, M. V., et al., Defining the phenotype of young healthy nucleus pulposus cells: recommendations of the Spine Research Interest Group at the 2014 annual ORS meeting. J Orthop Res, 2015. 33 (3): p. 283-93.
As described above, there are various problems in transplanting intervertebral disc (nucleus pulposus) cells or chondrocytes collected from donors, and also in transplanting stem cells capable of differentiating into nucleus pulposus cells such as mesenchymal stem cells and hematopoietic stem cells, in treating intervertebral disc degeneration or the like. Further, there are also problems of reproducibility and cost in the method of differentiation induction of stem cells such as MSCs into an active nucleus pulposus cell phenotype using specific growth factors, and there is no evidence for achieving the safety or therapeutic effects that counteract such problems.
Therefore, it seems ideal to perform differentiation induction into an active nucleus pulposus cell phenotype by allowing specific transcription factors to act on stem cells or the like (that is, by changing transcription factor profiles), and to use the active nucleus pulposus cell phenotype thus obtained for transplantation. However, “nucleus pulposus cell master regulator transcription factors” that enable such differentiation induction, keep exerting sufficient functions as an active nucleus pulposus cell phenotype also after transplantation of the active nucleus pulposus cell phenotype obtained (such as producing a sufficient amount of extracellular matrix), and thus can be clinically effective have not been established so far. Further, potent transcription factors (master regulator transcription factors) that enable an active nucleus pulposus cell phenotype to be obtained by transdifferentiation from terminally differentiated cells other than nucleus pulposus cells, not from stem cells such as MSCs, have still not been found. SOX9, which has been established as a master regulator transcription factor for cartilage formation, has not been demonstrated to have sufficient usefulness as a master regulator transcription factor for an active nucleus pulposus cell phenotype.
It is an object of the present invention to provide reproducible means that enables the production of an active nucleus pulposus cell phenotype from desired cells such as terminally differentiated cells or pluripotent or multipotent stem cells.
The inventors screened transcription factors specific to nucleus pulposus cells by microarray assay, iPS cell interference assay, siRNA assay, or the like as described in Examples below, instead of using the transcription factor SOX9 (chondrogenic master regulator) associated with cartilage formation as a starting point as in conventional arts. Further, the inventors have found that a combination of specific transcription factors selected from the candidates, typically, a combination of at least two selected from the group consisting of Brachyury (T), SRY-box6 (SOX6), and Forkhead Box Q1 (FOXQ1) serve as a set of potent transcription factors which should be called “nucleus pulposus cell master regulator transcription factors” that enable induction into an active nucleus pulposus cell phenotype not only by differentiation induction from stem cells such as MSCs but also transdifferentiation, for example, from terminally differentiated cells such as fibroblasts, thereby accomplishing the present invention.
That is, the present invention provides at least the following items.
(1) A differentiation inducer that is an agent comprising an effective amount of a gene of transcription factors for differentiation induction of nucleated cells other than active nucleus pulposus cell phenotypes into an active nucleus pulposus cell phenotype (hereinafter referred to as “nucleus pulposus cell master regulator transcription factors”) or a product thereof (hereinafter referred to as “differentiation inducer”), wherein the master regulator transcription factors comprise at least two selected from the group consisting of Brachyury (T), SRY-box6 (SOX6), and Forkhead Box Q1 (FOXQ1), or homologs thereof.
(2) The differentiation inducer according to item (1), wherein the nucleus pulposus cell master regulator transcription factors further comprise at least one selected from the group consisting of Paired Like Homeodomain1 (PITX1) and Paired Box 1 (PAX1), or a homolog thereof.
(3) The differentiation inducer according to item (1) or (2), wherein the nucleus pulposus cell master regulator transcription factors further comprise at least one selected from the group consisting of Hypoxia inducible factor 3 alpha (HIF3α), SRY-box9 (SOX9), Runt-related Transcription Factor 1 (RUNX1), hypoxia Inducible Factor 1 alpha (HIF1α), and Forehead Box A2 (FOXA2), or a homolog thereof.
(4) The differentiation inducer according to any one of items (1) to (3), wherein the nucleus pulposus cell master regulator transcription factors are in the form of a gene inserted into an expression vector.
(5) A pharmaceutical composition for treating or preventing an intervertebral disc disorder in a vertebrate animal, comprising the differentiation inducer according to any one of items (1) to (4).
(6) A method for producing induced nucleus pulposus cells, comprising the steps of: introducing the differentiation inducer according to any one of items (1) to (4) in vitro into nucleated cells other than active nucleus pulposus cell phenotypes (hereinafter referred to as “introduction step”); and performing transdifferentiation or differentiation induction into an active nucleus pulposus cell phenotype through culturing the transcription factor introduction cells obtained by the introduction step (hereinafter referred to as “differentiation induction step”).
(7) The method for producing induced nucleus pulposus cells according to item (6), further comprising a step of checking an expression status of at least one selected from the group consisting of CD24, aggrecan, and type II collagen in the cells during culture or after culture in the differentiation induction step.
(8) The method for producing induced nucleus pulposus cells according to item (7), wherein the differentiation induction step comprises culturing the transcription factor introduction cells in a culture medium supplemented with transforming growth factor β1 (TGFβ1) and growth differentiation factor 5 (GDF5).
(9) The method for producing induced nucleus pulposus cells according to item (7) or (8), wherein the differentiation induction step comprises culturing the transcription factor introduction cells under at least one condition selected from the group consisting of a hypoxic environment, an acidic environment, and a low-glucose environment.
(10) Transcription factor introduction cells that are cells comprising an effective amount of the nucleus pulposus cell master regulator transcription factors defined in any one of items (1) to (4).
(11) Induced nucleus pulposus cells that are cells having an active nucleus pulposus cell phenotype obtained through culturing the transcription factor introduction cells according to item (10).
(12) The induced nucleus pulposus cells according to item (11), wherein the induced nucleus pulposus cells are expressing at least one selected from the group consisting of CD24, aggrecan, and type II collagen.
(13) The induced nucleus pulposus cells according to item (11) or (12), wherein the induced nucleus pulposus cells are viable under at least one condition selected from the group consisting of a hypoxic environment, an acidic environment, and a low-glucose environment.
(14) The induced nucleus pulposus cells according to any one of items (11) to (13), wherein the induced nucleus pulposus cells have intercellular vacuoles.
(15) A cell population comprising the transcription factor introduction cells according to item (10) and/or the induced nucleus pulposus cells according to any one of items (11) to (14).
(16) A cell preparation for treating or preventing an intervertebral disc disorder in a vertebrate animal, comprising the induced nucleus pulposus cells according to any one of items (11) to (14) or the cell population according to item (15).
(17) A method for treating or preventing an intervertebral disc disorder in a vertebrate animal (excluding human), comprising transplanting or administering the induced nucleus pulposus cells according to any one of items (11) to (14), the cell population according to item (15), or the cell preparation according to item (16) in vivo so as to act on the intervertebral disc nucleus pulposus tissue.
(18) A method for treating or preventing an intervertebral disc disorder in a vertebrate animal (excluding human), comprising administering the differentiation inducer according to any one of items (1) to (4) or the pharmaceutical composition according to item (5) in vivo so as to act on nucleus pulposus cells in an intervertebral disc.
(19) A method for screening for a medicine or a method for treating or preventing an intervertebral disc disorder in a vertebrate animal, comprising a step of testing effectiveness and safety in a subject using the transcription factor introduction cells according to item (10), the induced nucleus pulposus cells according to any one of items (11) to (14), or the cell population according to item (15).
(20) A method for obtaining an indicator associated with an aging, degenerating or disease state of nucleus pulposus cells, comprising measuring expression levels of the nucleus pulposus cell master regulator transcription factors defined in any one of items (1) to (4) in isolated nucleus pulposus cells.
In the aforementioned items, the provision that the application target of the invention “excludes humans” is made only in view of industrial applicability, and the target can “include human” from a technical point of view.
The present invention enables cell transcription profiles to be directly changed, that is, cell transcription factor profiles to be induced into the same profiles as those of nucleus pulposus cells by specifically stimulating the master regulator of an active nucleus pulposus cell phenotype using specific transcription factors. This approach enables cells to be directly modified without the need for specific cell membrane receptors or related signaling proteins and therefore can be implemented relatively inexpensively with reliability (reproducibility). Further, even disseminated vacuolar presenting cells have a morphology similar to that of nucleus pulposus cells.
Further, the nucleus pulposus cell master regulator transcription factors specified by the present invention are potent and thus expand the range of cells to which direct reprogramming can be applied. In the conventional art, pluripotent or multipotent cell types (such as MSCs) were necessary, but the present invention also enables transdifferentiation of mature differentiated cells such as human dermal fibroblasts that have been terminally differentiated. A clear up-regulation can be given to extracellular matrix components such as type II collagen and aggrecan only by using the nucleus pulposus cell master regulator transcription factors specified by the present invention without using SOX9 that is a master regulator for cartilage formation. Accordingly, induced nucleus pulposus cells to be obtained by the present invention can survive under a severe nucleus pulposus microenvironment and can actively produce a nucleus pulposus-related extracellular matrix such as proteoglycans and type II collagen. The present invention can be further implemented by an embodiment of introducing the nucleus pulposus cell master regulator transcription factors into senescent cells or fibrous nucleus pulposus cells contained in an intervertebral disc or dedifferentiated nucleus pulposus cells during culture, to “regenerate” the cells into an active nucleus pulposus cell phenotype.
Currently, effective treatment for chronic low back pain or the like is limited to symptomatic therapy, but application of the present invention enables mass supply of superior cell products for intervertebral disc regenerative medicine, thereby solving the problem of the number and quality of donors and the problem of production cost. The situation in which nucleus pulposus cells to be transplanted are collected from a tissue removed from affected areas of intervertebral disc degeneration such as scoliosis and intervertebral disc hernia or other organs, as before, can be overcome, and functional nucleus pulposus cells that are more healthy and closer to the original traits can be fabricated from various somatic cell types such as cells contained in the skin or adipose tissue. Further, nucleus pulposus cells to be fabricated by the present invention can also be autologous, and therefore the risk of using non-autologous cells can be avoided.
According to another aspect, induced nucleus pulposus cells to be obtained by the present invention can be fabricated from healthy individual donor cells and therefore can also be used for studying the health state of nucleus pulposus of young individuals under in-vitro conditions. Further, the present invention enables nucleus pulposus cells derived from patients presenting specific gene mutations that affect the spinal or general cell biology to be fabricated. That is, the present invention enables induced nucleus pulposus cells to be fabricated from cells contained in easy-to-access tissue sources such as the skin or adipose tissue of patients, and enables how gene mutations in the original cells affect the behavior of nucleus pulposus cells in vitro to be evaluated. Finally, use of induced nucleus pulposus cells to be obtained by the present invention in a personalized medical care strategy enables how medicine that becomes a treatment candidate and its dose affect induced nucleus pulposus cell populations specific to the patient to be determined, thereby revealing patient-specific potential negative effects before actual administration to the patient, which can be useful for knowing an appropriate treatment method.
The differentiation inducer of the present invention is an agent containing a gene of transcription factors (nucleus pulposus cell master regulator transcription factors) or a product thereof in an effective amount for differentiating cells into an active nucleus pulposus cell phenotype. In the present invention, “inducing cells into an active nucleus pulposus cell phenotype” includes embodiments such as (i) maintaining “transdifferentiation” into nucleus pulposus cells from terminally differentiated cells other than nucleus pulposus cells or cells committed to differentiation into cells other than nucleus pulposus cells and inducing the cells into an active nucleus pulposus cell phenotype (which may be referred to as “first embodiment” in this description), (ii) differentiation induction into an active nucleus pulposus cell phenotype from stem cells or the like capable of differentiating into nucleus pulposus cells and other cells (having pluripotency or multipotency) (which may be referred to as “second embodiment” in this description), and (iii) reactivating dedifferentiated or otherwise compromised nucleus pulposus cells into an active nucleus pulposus cell phenotype (which may be referred to as “third embodiment” in this description). The differentiation inducer of the present invention can be used in any form of the first, second, and third embodiments, and the term “differentiation inducer” is used as a general term, but it is also possible to use the term “transdifferentiation agent” particularly for use in the first embodiment, the term “differentiation inducer” particularly for use in the second embodiment, and the term “reactivation agent” particularly for use in the third embodiment.
Examples of the nucleus pulposus cell master regulator transcription factors as the subject of the present invention, that is, the master regulator transcription factors that are important for maintaining an active nucleus pulposus cell phenotype include Brachyury (T), SRY-box 6 (SOX6), Forkhead Box Q1 (FOXQ1), Paired Like Homeodomain 1 (PITX1), Paired Box Protein1 (PAX1), Hypoxia inducible factor 3 alpha (HIF3α), SRY-box9 (SOX9), Krueppel-like factor 6 (KLF6), Runt-related Transcription Factor 1 (RUNX1), hypoxia Inducible Factor 1 alpha (HIF1α), and Forehead Box A2 (FOXA2), and homologs thereof (which may be referred to as “transcription factor group of the present invention” in this description). The homolog of each master regulator transcription factor is known to those skilled in the art and can be searched by databases such as Japan DNA data bank (DDBJ), NCBI BenBank, and EMBL. In the present invention, any one of these nucleus pulposus cell master regulator transcription factors may be used alone, or two or more, three or more, four or more, or more types of them may be optionally combined for use. Which (combination of) nucleus pulposus cell master regulator transcription factors are to be selected can be appropriately adjusted corresponding to whether the differentiation inducer of the present invention is to be used as a transdifferentiation agent (for terminally differentiated cells or the like other than nucleus pulposus cells) or as a differentiation inducer (for stem cells or the like capable of differentiating into nucleus pulposus cells and other cells) or as a reactivation agent (for dedifferentiated or damaged nucleus pulposus cells).
In the present invention, the “active nucleus pulposus cell phenotype” means to have one or more traits selected from (i) producing a large amount of nucleus pulposus-related matrix proteins such as proteoglycans (for example, aggrecan) and type II collagen or expressing at least one of cell markers specific to nucleus pulposus cells, such as CD24, KRT8, and KRT18, (ii) being capable of coping with hypoglycemia, acidity, hypoxia, and/or hyperosmolarity conditions imitating a healthy or moderately degenerated intervertebral disc, and (iii) having the same morphological characteristics as nucleus pulposus cells, such as cytoskeletal deposition and comparatively large intercellular vacuoles, preferably, to have traits of a plurality of groups (i), (ii), or (iii). Further, not all the cells belonging to the cell population necessarily have the same traits uniformly, and trait (iii) above may be seen only in a part of the cell population, for example.
In a typical embodiment of the present invention, the nucleus pulposus cell master regulator transcription factors include at least two selected from the group consisting of T, SOX6, and FOXQ1, or homologs thereof (which may be referred to as “first transcription factor group” in this description). In this typical embodiment, the nucleus pulposus cell master regulator transcription factors preferably include T (or a homolog thereof) and SOX6 (or a homolog thereof), or T (or a homolog thereof) and FOXQ1 (or a homolog thereof), more preferably all of T, SOX6, and FOXQ1.
According to one embodiment of the present invention, the master regulator transcription factors preferably include at least one selected from the group consisting of PITX1 and PAX1, or a homolog thereof (herein referred to as “second transcription factor group”) in addition to the aforementioned first transcription factor group. For example, in view of the above (iii) relating to the active nucleus pulposus cell phenotype, PITX1 is preferably added to the master regulator transcription factors in the case of fabricating the induced nucleus pulposus cells by transdifferentiation from fibroblasts or the like in the first embodiment of the present invention (see
According to one embodiment of the present invention, the master regulator transcription factors preferably include at least one selected from the group consisting of HIF3α, SOX9, RUNX1, HIF1α, and FOXA2, or a homolog thereof (herein referred to as “third transcription factor group”) in addition to the first transcription factor group and/or the second transcription factor group.
According to still another embodiment of the present invention, the master regulator transcription factors preferably include:
T or a homolog thereof; and
at least one selected from the aforementioned transcription factor groups of the present invention excluding T, SOX6, and FOXQ1, or a homolog thereof.
The nucleus pulposus cell master regulator transcription factors of the present invention may be introduced into target cells in the form of a gene (nucleic acid) or in the form of a protein that is a product of the gene. A variety of means for introducing the gene (nucleic acid) or protein into the target cells are known to those skilled in the art, and suitable means and conditions corresponding to the mean can be used in the present invention. The gene (nucleic acid) of the nucleus pulposus cell master regulator transcription factors may be, for example, in the form of DNA such as plasmid or in the form of RNA such as mRNA, and the gene can be introduced into the target cells in each form, for example, by transfection using a composite with a liposome, lipid particles, a polymer, or the like; electroporation; or a viral vector using retrovirus, lentivirus, adeno-associated virus, adenovirus, Sendai virus, or the like. Further, a protein of the nucleus pulposus cell master regulator transcription factors can be introduced into the target cells, for example, by coupling cell penetrating peptides with the protein.
According to one embodiment of the present invention, the nucleus pulposus cell master regulator transcription factors are introduced into the target cells in the form of an expression vector (such as a viral vector plasmid and an expression plasmid) into which a gene encoding at least one of the nucleus pulposus cell master regulator transcription factors has been inserted. The gene on the vector is expressed within the target cells, thereby producing a protein of the nucleus pulposus cell master regulator transcription factor, as a result of which, gene expression specific to nucleus pulposus cells such as aggrecan, type II collagen, and CD24 is directly or indirectly induced. The vector may be an embodiment of existing in the nucleus or cytoplasm either temporarily or continuously while being replicated, or may be an embodiment of permanently existing in the genomic DNA while being incorporated therein. In the case where a plurality of nucleus pulposus cell master regulator transcription factors are expressed, one of the nucleus pulposus cell master regulator transcription factors may be expressed by one expression vector (using a plurality of such expression vectors in combination), or the plurality of nucleus pulposus cell master regulator transcription factors may be expressed by one expression vector.
For example, plasmid DNAs containing genes encoding T, SOX6, and FOXQ1 may be introduced into human newborn dermal fibroblasts by transfection using lentivirus, and the factors can be expressed by a 35M cauliflower mosaic virus promoter (pCMV) disposed in the upstream of the genes. This causes overexpression of the T, SOX6, and FOXQ1 genes in the human newborn dermal fibroblasts having the three nucleus pulposus cell master regulator transcription factors introduced thereinto, so that the cells can be induced into nucleus pulposus cells. The induced nucleus pulposus cells thus obtained are characterized by expression and production of nucleus pulposus cell-specific markers such as aggrecan, type II collagen, and CD24.
According to one embodiment of the present invention, the nucleus pulposus cell master regulator transcription factors of the present invention function as markers of a mature active nucleus pulposus cell phenotype. The nucleus pulposus cell master regulator transcription factors are rarely expressed in the cell population mainly composed of nucleus pulposus cells that have aged and differentiated into a fibrous cell type, such as a degenerated intervertebral disc. In contrast, the expression levels in the cell population mainly composed of healthy nucleus pulposus cells are high. Therefore, the expression levels of the nucleus pulposus cell master regulator transcription factors in a cell population that contains isolated nucleus pulposus cells or nucleus pulposus cells collected from a tissue such as a nucleus pulposus and an intervertebral disc are measured, thereby enabling indices of the nucleus pulposus cell maturity, the cell activity, and the health of the cell population or the tissue to be obtained as indices associated with the aging, degenerating or disease state of the nucleus pulposus cells or the cell population.
The method for producing induced nucleus pulposus cells of the present invention includes at least the steps of introducing the differentiation inducer of the present invention (an effective amount of a gene of nucleus pulposus cell master regulator transcription factors or a product thereof) into cells in vitro (introduction step); and differentiating the cells into an active nucleus pulposus cell phenotype through culturing the cells (differentiation induction step), and preferably further includes the step of testing whether the cells during the differentiation induction step or after the differentiation induction step express at least one selected from the group consisting of CD24, aggrecan, and type II collagen (test step).
The introduction step is a step of introducing the differentiation inducer of the present invention (an effective amount of a gene of nucleus pulposus cell master regulator transcription factors or a product thereof) into cells.
The cells as targets for introduction of the differentiation inducer (herein referred to as “introduction target cells”) are not specifically limited, as long as they are nucleated cells other than active nucleus pulposus cell phenotypes, preferably, somatic cells (cells other than germ cells), and include various cell types. The introduction target cells may be an established cell line or may be primary cultured cells (autologous cells or allogeneic cells) collected from an individual or their passage cells.
In the first embodiment of the present invention, the introduction target cells are various somatic cells that have terminally differentiated into cells other than nucleus pulposus cells, or cells that have already been committed to differentiation into cells other than nucleus pulposus cells (progenitor cells of a particular lineage other than nucleus pulposus cells) and are, for example, skin tissue cells that are relatively easy to obtain and culture, typically preferably, fibroblasts.
In the second embodiment of the present invention, the introduction target cells are stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) possessing pluripotency, various stem cells possessing multipotency into nucleus pulposus cells and other cells, or other cells that have not been committed to differentiation into nucleus pulposus cells and are, for example, preferably mesenchymal stem cells (MSC), which are adult stem cells that can be collected from tissues such as fat, umbilical cord, synovium, and bone marrow.
The introduction target cells are generally derived from vertebrate animals, typically, mammals and may be derived from humans or non-human mammals. Examples of mammals can include mice, rats, dogs, cats, sheep, and bovines, in addition to humans. In the case where the introduction target somatic cells are primary cultured cells collected from an individual or their passage cells, the individual may be an individual into which induced nucleus pulposus cells are to be transplanted (such as patients of intervertebral disc disorders) or may be an individual different from the aforementioned individual (a healthy individual or a donor).
The introduction target cells may be derived from a vertebrate animal with genetic variation. For example, nucleus pulposus cell master regulator transcription factors are introduced into skin fibroblasts derived from a target (a human or a non-human vertebrate) with genetic variation to produce induced nucleus pulposus cells, thereby enabling analysis of the influence of genetic variation (the role of the gene) on the behavior of nucleus pulposus cells, intervertebral disc development, and other phenomena.
The differentiation inducer used in the introduction step may be used in an effective (necessary) amount for (i) maintaining and inducing transdifferentiation, in the case where the introduction target cells are terminally differentiated cells other than nucleus pulposus cells or the like, from the cells to an active nucleus pulposus cell phenotype, or an effective (necessary) amount for (ii) differentiation induction, in the case where the introduction target cells are undifferentiated cells or the like, from the cells into an active nucleus pulposus cell phenotype. The amount to be used as the transdifferentiation agent (i) and the amount to be used as the differentiation inducer (ii) each vary depending on the embodiment of the present invention, such as the type of introduction target cells, the types of nucleus pulposus cell master regulator transcription factors and selection of whether they are introduced into the cells in the form of a gene (such as expression plasmid and mRNA) or in the form of a protein, further the means (such as a viral vector, electroporation, microinjection, lipofection, coupling of cell penetrating peptides, and other transfection reagents) for introducing them into the cells, and the cell culture conditions. The numerical range thereof cannot be generally determined. Those skilled in the art would be able to adjust and set the amount to be used, for example, using the ratio of the number of induced nucleus pulposus cells with respect to the total number of cells as an indicator, so that an expected purpose can be achieved.
As an indicator, it is conceivable to set the number of viral vectors per cell to a suitable range according to the concept of the multiplicity of viral infection (MOI), when nucleus pulposus cell master regulator transcription factors are introduced into the introduction target cells in the form of genes using viral vectors. As an example, it is conceivable to use a viral vector solution so that about 8 viral vectors are introduced into each cell (equivalent to MOI=8). Since the number of “about 8” above is mentioned as an example, a larger or smaller number may be employed, and the number can be appropriately set and adjusted by those skilled in the art. Likewise, also in the embodiment in which genes of nucleus pulposus cell master regulator transcription factors are introduced into the cells in a form other than viral vectors, such adjustment can be performed so that an appropriate number of genes of nucleus pulposus cell master regulator transcription factors are introduced into each introduction target cell.
Accordingly, the differentiation inducer of the present invention can be configured as a solution containing genes of nucleus pulposus cell master regulator transcription factors prepared so as to have an appropriate “multiplicity of infection” (such as expression vectors introduced into each cell) or a kit therefor, corresponding to the type of introduction target somatic cells, the number of cells, and other culture conditions.
The introduction step may be performed under conditions suitable for introducing an effective amount of a gene of nucleus pulposus cell master regulator transcription factors or a product thereof into introduction target cells, and culturing the cells after introduction. The culture conditions such as components in the culture medium used therefor (such as the basal culture medium, growth factors, other additive components, vectors, and transfection reagents), the culture period, and the atmosphere can be appropriately set by those skilled in the art.
The differentiation induction step is a step of differentiating the cells into an active nucleus pulposus cell phenotype by culturing cells having the differentiation inducer of the present invention (an effective amount of a gene of nucleus pulposus cell master regulator transcription factors or a product thereof) introduced thereinto by the introduction step.
The differentiation induction step may be performed under conditions suitable for culturing until the cells having the nucleus pulposus cell master regulator transcription factors introduced thereinto become an active nucleus pulposus cell phenotype. The culture conditions such as components in the culture medium used therefor (such as the basal culture medium, growth factors, and other additive components), the culture period, and the atmosphere can be appropriately set by those skilled in the art. Further, the culture in the differentiation induction step may be two-dimensional culture (for example, monolayer culture) or may be three-dimensional culture (for example, 3D pellet culture).
According to one embodiment of the present invention, the culture medium (cell cultures) in the differentiation induction step can contain at least one selected from the group consisting of transforming growth factor β1 (TGFβ1), transforming growth factor β2 (TGFβ2), and transforming growth factor β3 (TGFβ3), and at least one selected from the group consisting of growth differentiation factor 5 (GDF5) and growth differentiation factor 6 (GDF6). In some embodiments of the present invention, it may be preferable, for example, to culture the cells having the differentiation inducer introduced thereinto within a culture medium supplemented with transforming growth factor β1 (TGFβ1) and growth differentiation factor 5 (GDF5), for maintaining and inducing transdifferentiation into an active nucleus pulposus cell phenotype. The concentration of each of TGFβ1 and GDF5 in the culture medium can be appropriately adjusted, but the concentration of TGFβ1 is generally 1 to 10,000 ng/mL, preferably 10 to 100 ng/mL, for example, about 10 ng/mL, and the concentration of GDF5 is generally 1 to 100,000 ng/mL, preferably 10 to 500 ng/mL, for example, about 100 ng/mL.
Further, the culture medium in the differentiation induction step can contain dexamethasone at a concentration of generally 1 to 1,000 ng/mL, preferably 4 to 500 ng/mL, for example, about 10 ng/mL. The culture medium in the differentiation induction step can contain L-ascorbic acid at a concentration of generally 1 to 1,000 μM, preferably 5 to 500 μM, for example, about 50 μM.
According to one embodiment of the present invention, the differentiation induction step can include culturing cells under at least one condition selected from the group consisting of a hypoxic environment, an acidic environment, and a low-glucose environment, more preferably under all these conditions. Induced nucleus pulposus cells which are viable in a hypoglycemic, acidic, and hypoxic (and hyperosmolar state) environment that is the environment in a healthy or moderately degenerated intervertebral disc can be fabricated and recovered by culturing cells having nucleus pulposus cell master regulator transcription factors introduced thereinto in such an environment. The hypoxic environment generally refers to an environment in which the oxygen concentration in the atmosphere of the culture medium is 1 to 10%, preferably 1 to 5%, for example, about 2%. The acidic environment generally refers to an environment in which the pH of the culture medium at room temperature (for example, 25° C.) is in the range of 6.5 to 7.4, for example, about 6.8. The low-glucose environment generally refers to an environment in which the glucose concentration in the culture medium is 4.5 g/L or less, for example, about 1 g/L. The culture period under such an environment can be appropriately adjusted but is generally 2 to 90 days (3 months), for example, 14 days (two weeks). For example, it is preferable to culture cells having with nucleus pulposus cell master regulator transcription factors introduced thereinto under a hypoxic environment with an oxygen concentration of 2% for two weeks.
In the checking step, the expression status of a gene or a protein that characterizes induced nucleus pulposus cells, that is, a nucleus pulposus cell marker in the cells during culture or after culture in the differentiation induction step is checked. As such a nucleus pulposus cell marker, either a marker that is positive in an active nucleus pulposus cell phenotype (positive marker) or a marker that is negative therein (negative marker) can be used. The expression status of a gene and/or a protein serving as a nucleus pulposus cell marker can be checked quantitatively or qualitatively, for example, by a general approach such as real time polymerase chain reaction (real time PCR), immunohistological staining (IHC), Western blotting, and flow cytometry. Whether the expression is positive or negative can be determined based on the results.
Examples of the positive marker in induced nucleus pulposus cells include CD24, aggrecan, type II collagen, keratin 8, and keratin 18. According to one embodiment of the present invention, the cells during culture or after culture in the differentiation induction step, that is, (the cells estimated to be) induced nucleus pulposus cells preferably express (being positive in) at least one selected from the group consisting of CD24, aggrecan, and type II collagen, more preferably express (being positive in) all these three types.
Examples of the negative marker in induced nucleus pulposus cells include type I collagen. According to one embodiment of the present invention, the cells during culture or after culture in the differentiation induction step, that is, (the cells estimated to be) induced nucleus pulposus cells preferably do not express (being negative in) or weakly express type I collagen.
Transdifferentiation or differentiation induction from the cells into an active nucleus pulposus cell phenotype is paired with a decrease in proliferative capacity, thereby achieving differentiation and maturity of the cells having master regulator transcription factors introduced thereinto. When expression of such a nucleus pulposus cell-specific marker in the growth factor introduction cells (the growth factor introduction cells contained in a cell population in a cell culture at a desired ratio) during culture is confirmed in the test step, the production of induced nucleus pulposus cells can be ended.
The method for producing induced nucleus pulposus cells of the present invention as described above enables an active nucleus pulposus cell phenotype to be supplied substantially infinitely (inexhaustibly). Use of induced nucleus pulposus cells to be obtained by the present invention is not limited, but the cells can be used typically for administration to an intervertebral disc in methods for treating and preventing the intervertebral disc disorders described below, particularly, for preparing cell preparations used in such applications.
Both the transcription factor introduction cells and induced nucleus pulposus cells of the present invention are cells produced by the method for producing induced nucleus pulposus cells of the present invention. In this description, cells containing an effective amount of a gene of nucleus pulposus cell master regulator transcription factors or a product thereof, that is, cells just having master regulator transcription factors introduced thereinto, and cells that overexpress nucleus pulposus cell master regulator transcription factors due to culture after the introduction but are not yet induced into an active nucleus pulposus cell phenotype are referred to as “transcription factor introduction cells”, whereas cells that are obtained by culturing the transcription factor introduction cells and induced into an active nucleus pulposus cell phenotype are referred to as “induced nucleus pulposus cells”, so as to distinguish the two. The embodiment of a cell population containing transcription factor introduction cells and/or induced nucleus pulposus cells, and the embodiment of a cell culture containing such a cell population can be adjusted corresponding to their applications. For example, in the case where a cell population containing induced nucleus pulposus cells is used as a raw material for producing a cell preparation for treating or preventing an intervertebral disc disorder, it is desirable to set the ratio of induced nucleus pulposus cells in the cell population as high as possible (conversely, to set the ratio of transcription factor introduction cells that have not transformed to induced nucleus pulposus cells as low as possible).
Further, according to one embodiment of the present invention, inducer introduction cells, cell cultures, induced nucleus pulposus cells, or cell populations obtained by the present invention can be used in a method for screening for a medicine or a method for treating or preventing an intervertebral disc disorder in a vertebrate animal including a step of testing the effectiveness and safety in a subject, that is, can be used as an in-vitro test model serving as a scientific, diagnostic, or prognostic tool for evaluating the reaction of nucleus pulposus cells or patient-specific nucleus pulposus cells to dosing, factors, or other (environmental) conditions. Further, a methodology established by the present invention can be used, for example, for fabricating nucleus pulposus cells from patients with genetic defects, in order to evaluate the influence of the genetic defects on nucleus pulposus cell phenotypes, homeostasis, development and pathology of an intervertebral disc, and reactions to drugs. The embodiment based on such applications, for example, enables the effectiveness of specific drugs on individual patients to be evaluated before administration, or the effectiveness of a treatment for preventing toxic side effects in administration of induced nucleus pulposus cells to patients or suppressing medical cost to be evaluated, in an individualized medical approach.
The pharmaceutical composition of the present invention contains the differentiation inducer of the present invention, that is, an effective amount of a gene (nucleic acid) of nucleus pulposus cell master regulator transcription factors or a product thereof (protein). Further, the cell preparation of the present invention contains induced nucleus pulposus cells obtained by introducing an effective amount of nucleus pulposus cell master regulator transcription factors into cells outside the body (in vitro or ex vivo). The pharmaceutical composition and the cell preparation can be used for treating and preventing spine-related diseases of humans and non-human vertebrates. The pharmaceutical composition of the present invention can be used in the form of so-called gene treatment for transforming cells present in the nucleus pulposus into an active nucleus pulposus cell phenotype inside the body (in vivo or in situ). Meanwhile, the cell preparation of the present invention can be used as an effective supply source, which potentially recovers biomechanical characteristics of the spine, for reconstituting and recovering the structure of an intervertebral disc by being transplanted into a nucleus pulposus with a degenerated intervertebral disc through allogeneic transplantation, xenotransplantation, or autologous cell transplantation.
The dosage forms of the pharmaceutical composition and the cell preparation of the present invention need only to enable delivery to a nucleus pulposus of an intervertebral disc serving as a target but can be, for example, injections, preferably, injections for local administration to the intervertebral disc (nucleus pulposus). The pharmaceutical composition and the cell preparation, for example, when prepared as injections, can contain pharmaceutically acceptable substances such as injection solvents, normal saline, culture liquids, other suitable solvents/dispersion media, additives, and the like, as required. Further, the pharmaceutical composition and the cell preparation each can be produced also as a kit including a syringe and a pharmaceutical agent to be used in combination, as required.
A first embodiment (which may be referred to as “first treatment and prevention method” in this description) of the method for treating and preventing an intervertebral disc disorder of the present invention includes transplanting or administering (a cell population containing) the induced nucleus pulposus cells of the present invention or the cell preparation of the present invention containing the induced nucleus pulposus cells (cell population) in vivo so as to act on the intervertebral disc nucleus pulposus tissue. The phrase “so as to act on the intervertebral disc nucleus pulposus tissue” means that the embodiment of transplantation or administration is not specifically limited, as long as the induced nucleus pulposus cells or the like that have been transplanted or administered can reach the intervertebral disc nucleus pulposus tissue that is an affected area, so that the effects of treatment or prevention can be exerted. The phrase includes transplanting the induced nucleus pulposus cells or the like into the intervertebral disc nucleus pulposus tissue or the vicinity thereof or injecting the induced nucleus pulposus cells or the like so as to reach the affected area through blood vessels, for example.
A second embodiment (which may be referred to as “second treatment and prevention method” in this description) of the method for treating and preventing an intervertebral disc disorder of the present invention includes administering the differentiation inducer of the present invention or the pharmaceutical composition of the present invention containing the differentiation inducer in vivo so as to act on nucleus pulposus cells in an intervertebral disc. The phrase “so as to act on nucleus pulposus cells in an intervertebral disc” means that the embodiment of administration is not specifically limited, as long as the administered differentiation inducer or the like can be incorporated into the nucleus pulposus cells in the intervertebral disc to reactivate the cells, so that the effects of treatment or prevention can be exerted. The phrase includes administering the differentiation inducer or the like into the intervertebral disc nucleus pulposus tissue or the vicinity thereof in situ or injecting the differentiation inducer or the like so as to reach the affected area through blood vessels, for example.
The intervertebral disc (nucleus pulposus tissue) that is subjected to the action of the cell preparation of the first treatment and prevention method of the present invention or the like and the pharmaceutical composition or the like of the second treatment and prevention method is an intervertebral disc with degeneration, aging, other symptoms. In such an intervertebral disc with degeneration or the like, healthy nucleus pulposus cells decrease, and senescent cells and fibrous nucleus pulposus cells increase. The induced nucleus pulposus cells transplanted or administered by the first treatment and prevention method and the induced nucleus pulposus cells generated (reactivated) by the second treatment and prevention method can survive in an intervertebral disc microenvironment, which is acidic, hyperosmotic and hypoglycemic, thereby increasing the amount of aggrecan, type II collagen, and the like to be produced in the extracellular matrix, so that the intervertebral disc disorder can be treated or prevented. In the second treatment and prevention method of the present invention, nucleus pulposus cell master regulator transcription factors are introduced into senescent cells and fibrous nucleus pulposus cells contained in the intervertebral disc, so that those cells can be transdifferentiated and induced into an active nucleus pulposus cell phenotype.
The cell preparation or the like of the first treatment and prevention method of the present invention and the pharmaceutical composition or the like of the second treatment and prevention method may be administered in an effective amount for exerting desired treatment or prevention effects. Such an effective amount can be appropriately adjusted depending on the dose per administration, the number of doses, and the administration interval (the number of doses within a certain period), in consideration of the embodiments of the cell preparation and the pharmaceutical composition or the like, the administration subject, the administration route, and the like. Both the first and second treatment and prevention methods can be performed on humans and non-human vertebrates.
In conducting this study, collection and use of human tissue samples were approved by the institutional ethics review committee of Tokai University Hospital. In addition, surgically excised tissue materials were obtained only from patients which have provided their informed consent.
A human intervertebral disc tissue collected was macroscopically examined to separate the nucleus pulposus tissue from fibrous tissue and other tissue structures. Subsequently, the nucleus pulposus tissue was incised into about 1 cm3 sections. The human nucleus pulposus tissue was further digested with TrypLE express (Gibco, USA) at 37° C. for 1 hour. Thereafter, the first digested tissue was transferred to 0.25 mg/mL collagenase-P (F. Hoffmann-La Roche, Ltd., Switzerland) for 2 hours. The suspension obtained was filtered with a 100 μm cell strainer, washed twice with 10% FBS-added αMem (Dulbecco, USA), and inoculated at a cell density of 3,000 to 5,000 cells/cm2. The seeded cells were cultured at 37° C. under 5% CO2, and 5% O2 until further use.
The nucleus pulposus cells were seeded at a density of 3,000 to 5,000 cells/cm2 and grown with 10% (vol/vol) FBS and αMEM (Gibco) in a wet chamber at 37° C. under 25% O2. For cell purification, the nucleus pulposus cells were incubated with anti-human disialoganglioside GD2 (GD2) (BD Pharmingen; 14; G2a) mAb for 30 minutes and then with FITC-conjugated anti-mouse Ig goat (BD Biosciences) at 4° C. for 30 minutes. After washing, the cells were stained with allophycocyanin-conjugated anti-human Tie2 (R&D Systems, Inc., clone 83715) mAb and PE-conjugated or biotinylated anti-human CD24 (BD Biosciences; clone ML5) mAb for 1 hour. The cell suspension was centrifuged at 4° C. and 1200 rpm for 5 minutes and washed. Subsequently, the nucleus pulposus cells were divided into Tie2+/GD2+/CD24−, Tie2−/GD2+/CD24−/+, and Tie2−/GD2−/CD24+ populations by FACS Vantage cells (BD Biosciences). At the same time, the Tie2−/GD2−/CD24+ population was also separated from spheroids formed by 3D cell culture, using a methylcellulose culture medium, MethoCult H4230 (STEMCELL Technologies Inc.). Subsequently, the fractionated nucleus pulposus cell population was dissolved in RNA buffer of RNeasy Mini Kit (QIAGEN). Total RNA was isolated. Finally, Cy3-labeled cRNA was prepared from each of the four populations using a low-input Quick Amp Labeling Kit, one color (Agilent Technology, Inc.), analyzed using SurePrint G3 Human GE 8x60K v2 Microarray (Agilent Technology, Inc.) and Gene Expression Hybridization Kit (Agilent Technology, Inc.), and analyzed by Agilent DNA Microarray Scanner (G2565CA) using a software, Feature Extraction 7 (Agilent Technology, Inc.). The expression values obtained were compared with the expression levels in neural progenitor cells (ID GSM1608144, GSM1608145), fibroblasts (ID GSM1191059, GSM1191060, GSM1191061), iPS cells (ID GSM1598135, GSM1598136, GSM1598137), and lung cells (ID GSM1700910, GSM1700913) obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/gds/) by subtraction.
A plasmid with transgenes for all nucleus pulposus cell master regulator transcription factor candidates inserted into the pMXs-GW skeleton, and a plasmid with green fluorescent protein (GFP), additionally Brachyury (T) was also inserted into the pMX-IRES-GFP skeleton, were used. The plasmids were provided by courtesy of the iPS cell research and use center of Kyoto University in Japan. Plasmid amplification was established by cloning each plasmid into MAX Efficiency Stbl2 chemically competent cells (Invitrogen, USA) through heat shock at 42° C. for 45 seconds and subsequent overnight incubation in LB-broth (Miller) liquid microbial growth medium (Sigma-Aldrich Co. LLC, USA). On the next day, plasmid DNA was recovered using the NucleoBond Xtra Midi kit (Takara Bio Inc., Japan) according to the manufacturer's instructions.
Virus particles were produced by separate transfection of the plasmid DNA obtained according to research previously published by Kitazawa et al. (Kitazawa, K. et al. OVOL2 Maintains the Transcriptional Program of Human Corneal Epithelium by Suppressing Epithelial-to-Mesenchymal Transition. Cell reports 15, 1359-1368, doi:10.1016/j.celrep.2016.04.020 (2016)). In summary, platinum-GP retroviral packaging (PLAT-GP) cells in a DMEM high glucose culture medium supplemented with 10% FBS, 1% pyruvate, and 50 U/mL (50 μg/mL) penicillin/streptomycin were seeded at a density of 55×103 cells/cm2 on a plastic coated with 0.1% gelatin (Sigma-Aldrich Co. LLC, USA) derived from pig skin. On the next day, 5.4 μL of FuGENE (registered trademark, Promega KK., USA), 600 ng (0.6 μL) of pLP/VSVG (registered trademark, Invitrogen, USA) (expression plasmid of vesicular stomatitis virus G protein as an alternative envelope), 1200 ng (1.2 μL) of pMXs-GW plasmid or pMX-IRES-GFP vector DNA encoding a singular transgene, and 60 μL of Opti-MEM (registered trademark, Thermo Fisher Scientific, Inc., USA), which account for a total of 69 μL, were added per 2 mL of PLAT-GP culture medium. PLAT-GP cells were incubated in a wet chamber at 37° C. under 21% O2 for 24 hours, and thereafter the culture medium was refreshed. After further culture for 24 hours, the culture medium was recovered and filtered with a 0.45 μm pore size filter unit (Sigma-Aldrich Co. LLC, USA). The virus-containing culture medium was directly applied.
According to research by Takahashi et al. (Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126 (4): p. 663-76.), mouse SNL feeder cells for iPS cell culture were seeded on 0.1% gelatin-coated wells and grown to 70 to 80% confluence.
Meanwhile, the nucleus pulposus cells were seeded at 555 cells/cm2 on 6-well plate wells. Next, the culture medium was replaced with DMEM high glucose (Thermo Fisher Scientific, Inc.) supplemented with 10 ng/mL of polybrene (Santa Cruz Biotechnology, Inc., USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Inc., USA). Subsequently, 2 mL of a mixed culture medium rich in VSVG particles containing the plasmid DNA encoding the transgene was added thereto at maximum. Viral transduction was promoted by centrifuging the nucleus pulposus cultures at 800 G and 35° C. for 30 minutes. Subsequently, the culture medium was aspirated and was replaced with 2 mL of DMEM high glucose culture medium supplemented with 10% FBS and 1% penicillin/streptomycin. Transduction was performed using a viral vector (pMX-GW) containing genes for Yamanaka factors (OCT3/4, SOX2, KLF4, and CMYC) and additional master regulator transcription factor candidates (see the microarray data above). The nucleus pulposus cells were recovered under 2% O2 hypoxia and tension for 48 hours with the culture medium replaced everyday.
Two days later, the SNL feeder cells were treated with 13 μg/mL of mitomycin C (FUJIFILM Wako Pure Chemical Corporation, Japan) for 1 hour. Subsequently, the culture medium was replaced with 10% FBS (Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin and refreshed. The nucleus pulposus cells were trypsinized and seeded on the mitomycin C-treated SNL feeder cells. Further, the nucleus pulposus cultures were switched to 21% O2 every other day, and replacement of the culture medium was also conducted.
Six days after transduction, the culture medium was replaced with ReproCELL primate ES cell culture medium (ReproCell Inc., Japan) supplemented with 10 ng/mL of fibroblast growth factor (FGF) and 1% (v/v%) penicillin/streptomycin. After iPS cell induction was continued for a total of two weeks, the cultures were fixed and stained with alkaline phosphatase staining kit (MUTO pure chemicals Co., Ltd., Japan) according to the manufacturer's instructions. Positively stained colonies were manually counted as an indicator of iPSC induction efficiency.
In order to examine how each master regulator transcription factor candidate identified by the microarray assay (the results will be described later) affects nucleus pulposus cell marker expression by RNA interference (RNA i), nucleus pulposus cells seeded at 15,000 cells/cm2 were transduced with DharmaFECT and custom-made siRNA molecules (GE Healthcare, USA) targeting the mRNA of each master regulator transcription factor candidate, according to the manufacturer's instructions. After transduction with siRNA, the cells were cultured at 37° C. under 2% O2 in αMem supplemented with 10% FBS for 48 hours.
The cells after culture were collected, and the total RNA was recovered according to the manufacturer's protocol using SVTotal RNA Isolation System (Promega KK., USA). The total RNA recovered was converted into cDNA according to the manufacturer's protocol using High Capacity cDNA Reverse Transcription kit (Applied Biosystem, USA). The mRNA expression level was evaluated from cDNA by 7300RT-PCR system (Applied Biosystems, USA) using SYBR Green PCR master mix (Applied Biosystems) and a primer manually designed for each nucleus pulposus cell marker. CT values obtained by such a system were normalized to the CT value of glyceraldehyde 3-phosphate dehydrogenase (GAPDH; housekeeping gene) and were then compared with the CT value of nucleus pulposus cell sham control (SHAM control) normalized in the same manner, thereby calculating relative values of the expression level.
Human newborn skin fibroblasts (Lonza, Switzerland) were grown in DMEM containing 10% (v/v) FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The day before transduction, human fibroblasts were separated by incubation in 0.25% (w/v) trypsin, 0.001% (w/v) ethylenediaminetetraacetic acid (EDTA), and PBS for 5 minutes and then seeded at a density of 5.5×103 cells/cm2 in a well plate sufficiently covered with DMEM containing 6100 μg/mL penicillin and 100 μg/mL streptomycin.
To each well, 500 μL of viral culture medium transduced with each transgene was added in a maximum volume of 2 ml. As the SHAM control, a GFP transgene vector was added. DMEM containing 100 U/mL of penicillin and 100 μg/mL of streptomycin was added in a total capacity per well of 4 ml. Further, the culture medium was supplemented with 40 μg of polybrene (Santa Cruz Biotechnology, Inc.). The cultures were spun down at 30° C. and about 800 G for 30 minutes. Subsequently, the virus-containing culture medium was removed, and the cells were washed with excess PBS. Finally, the cells were cultured at 37° C. under 2% O2 for two weeks in DMEM containing 0.1% (v/v) insulin-transferrin-selenium-ethanolamine solution (ITS-X; Thermo Fisher Scientific, Inc.), 1% (v/v) FBS, 50 μM L-magnesium ascorbyl phosphate n-hydrate (FUJIFILM Wako Pure Chemical Corporation, Japan), 100 U/mL penicillin and 100 μg/mL streptomycin, with or without supplementation with 10 ng/mL of TGFβ1 (PeproTech, Inc., Japan) and 100 ng/mL of growth differentiation factor 5 (GDF5; PeproTech, Inc.), and a fresh culture medium was given every 3 to 4 days. The cell morphology was captured by an optical microscope, and temporal changes were revealed depending on the combinations of transcription factors used for transduction.
Finally, cells of a group transduced with transcription factors in combination sporadically presented cells with intercellular vacuoles similar to vacuoles observed in notochord cells in vitro (
The transduced cells were collected with 0.25% trypsin and 0.001% EDTA and were subjected to further evaluation. For samples used for qPCR evaluation, the total RNA was isolated as described above (see (8) siRNA-mediated interference assay). The isolated RNA was thereafter converted into cDNA using a high-volume RNA-to-cDNA kit (Thermo Fisher Scientific, Inc.) according to the accompanying instructions. Subsequently, about 10 to 100 ng of cDNA was used for SYBRGREEN (Thermo Fisher Scientific, Inc.)-mediated qPCR analysis, and a custom-designed primer set for nucleus pulposus markers and notochord markers was applied. Each expression level obtained was calculated as a value of 2−ΔΔCT comparing the gene expression level with that of GAPDH and subsequent SHAM control.
Further, two weeks after monolayer differentiation culture, transduced fibroblasts were counted as above at a density of 250,000 cells put into a 15 ml polypropylene conical tube (BD Biosciences) in 0.5 ml of the aforementioned differentiation culture medium. The cell suspension was spun down at 1500 rpm for 5 minutes at room temperature. After the cell pellets obtained were cultured for one day, the pellets were gently tapped from the bottom of the conical tube, and the spherical cell aggregates were further cultured under 2% O2 for three weeks. Finally, the pellets were fixed with 4% (v/v) paraformaldehyde, supplemented with Tissue-TEK O.C.T. compound (Sakura Finetek Japan Co., Ltd., Japan), and rapidly frozen in liquid nitrogen. The sample obtained was cryosectioned into an 8 μm section on a silane-coated slide (MUTO pure chemical substance). Subsequently, production of ECM within a pellet was visualized by staining the section with a 1 g/L Safranin-O (Merck, USA) and 800 mg/L Fast Green FCF staining (Merck) solution or hematoxylin eosin staining.
In the same manner as in the protocols of (8) to (10) above, the master regulator transcription factors were used also for MSCs to induce differentiation into a nucleus pulposus cell phenotype, and the differentiation process was investigated from cell morphological changes, mRNA expression changes, and protein production tested by pellet culture based on histology.
Transduced MSCs cultured for three weeks in pellet cultures were similar to those identified in fibroblast cultures but showed more exaggerated characteristics. The SHAM control or T-transduced MSC pellet did not bring about identifiable Safranin-O-stained proteoglycans in the pellet, and vacuolated cells could not be identified therein. Transduction with T+SOX6+PITX1 or T+SOX6+PITX1+FOXQ1 showed some notochord cell-like cells, vacuoles, and a sporadic but small Safranin-O positive regions. T+SOX6-transduced cells showed a pellet filled with vacuolated cells together with potent but localized proteoglycan deposition. Finally, the combination of T+SOX6+FOXQ1 showed proteoglycan deposition distributed throughout the pellet. It was similar to extracellular matrix deposition derived from nucleus pulposus cells cultured in a pellet. Sporadically, vacuole-forming cells could be distinguished, but most part of cells showed a mature nucleus pulposus cell-like phenotype.
Finally, the expression levels of proteins of nucleus pulposus cell markers and notochord cell markers within MSC pellet cultures were checked by immunohistochemistry. A cryosection slide was rehydrated and blocked with 3% (v/v) BSA/PBS for 30 minutes. An intercellular protein section was blocked with 0.1% (w/v) Triton-X (FUJIFILM Wako Pure Chemical Corporation)/3% (v/v) BSA/PBS. Rabbit anti-COL2 antibody was used as a primary antibody for staining COL2. Likewise, mouse anti-CD24 antibody (Funakoshi Co., Ltd., Japan), rabbit anti-ACAN antibody (Millipore, USA), mouse anti-KRT18 antibody (Abcam plc., UK), goat anti-PAX1 antibody (Abcam plc.), and rabbit anti-PITX1 antibody were used. Finally, each section was stained with PE labeling antibody corresponding to the primary antibody and was mounted with VECTASHIELD mounting medium (Funakoshi Co., Ltd.) containing DAPI. Images were taken with a ZeissLSM 510 Meta Confocal Microscope (Zeiss, Germany).
In the case where fibroblasts were transduced with T alone, no obvious increase was observed in expression levels of nucleus pulposus cell markers, but only the expression level of ACAN increased, as compared with the SHAM control. In contrast, in the case where fibroblasts were transduced with a combination of T and other transcription factors, a stronger and complete increase was observed in the expression levels of nucleus pulposus cell markers. For all three types of nucleus pulposus cell markers (ACAN, COL2, and CD24) tested, the expression levels with FOXQ1 combined with T were particularly excellent, and the expression levels strongly increased in the following order of SOX6, SOX9, HIF1α, FOXA2, PITX1, HIF3α, and RUNX1 when combined with T.
Human bone marrow-derived MSCs (Lonza, Switzerland) were cultured in nucleus pulposus cell induction medium (NPIM) composed of DMEM high glucose (Dulbecco, USA) supplemented with 50 μM magnesium L-ascorbyl phosphate n-hydrate (FUJIFILM Wako Pure Chemical Corporation, Japan), 6.25 μg/mL insulin-transferrin-selenium-x (Thermo Fisher Scientific, Inc., USA) and 1% penicillin/streptomycin for two weeks. Further, 10 nM dexamethasone was added thereto. Concurrently, MSCs were separately suspended in 5 mL of NPIM and were centrifuged in a 15 mL propylene centrifugation tube for 5 minutes at 250 g. The cell cultures were incubated in a wet chamber at 37° C. under 21% O2. Two days later, monolayer and cell pellet culture media containing TGFβ1, TGFβ2, or TGFβ3 (10 ng/mL) were further supplemented with GDF5 or GDF6 (100 ng/mL), and the cultures were transferred to a 5% O2 wet chamber. The culture media were refreshed every two days. The monolayer cultures were harvested two weeks later by trypsinization with 0.25% EDTA/trypsin and lysed with a lysis solution (Ambion, USA) for real-time PCR analysis. The pellet cultures were harvested in the third week, supplemented with a Tissue-TEK O.C.T. compound (Sakura Finetek USA, Inc., USA), and snap-frozen in liquid nitrogen. Subsequently, fixed pellets were sliced into a 8 μm section using a cryostat. Each pellet was stained with a 1 g/L safranin-O and 800 mg/L fast green FCF staining solution.
Regarding the MSC pellet culture (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-066535 | Mar 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/004449 | 2/6/2020 | WO | 00 |
