Patent Application
20210386790
Uterine scar development after Caesarean Delivery and other uterine surgeries can cause complications such as uterine rupture and abnormal placentation. These complications can result in a very serious prognosis for both the mother and the fetus. A clinical study reported that Caesarean Delivery should be performed very quickly (e.g. within 18 minutes) after diagnosis of a fetal disorder due to uterine rupture for preventing a higher incidence of perinatal mortality and morbidity. See Leung et al., 1993, Am J Obstet Gynecol 169: 945-950. The risk of these complications can increase depending on the number of times a Caesarean Delivery is performed. In particular, women having multiple Caesarean Deliveries could have higher risk. Therefore, a need exists to prevent potential complications resulting from Caesarean Deliveries and other uterine surgeries.
In certain aspects, the present disclosure relates to a method of reducing formation of fibrotic tissue in a uterus of a subject in need thereof, comprising applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, wherein the MSC sheet comprises one or more layers of aggregated confluent mesenchymal stem cells (MSCs), and wherein applying the MSC sheet to the uterus reduces the formation of fibrotic tissue in the uterus relative to a uterus in which the MSC sheet is not applied.
In certain aspects, the present disclosure relates to a method of increasing myometrial regeneration in a uterus of a subject in need thereof, comprising applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, wherein the MSC sheet comprises one or more layers of aggregated confluent mesenchymal stem cells (MSCs), and wherein applying the MSC sheet to the uterus increases myometrial regeneration relative to a uterus in which the MSC sheet is not applied.
In certain aspects, the present disclosure relates to a method of preventing or reducing rupture of a uterine incision and abnormal placentation in a subject in need thereof, comprising applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, wherein the MSC sheet comprises one or more layers of aggregated confluent mesenchymal stem cells (MSCs), and wherein applying the MSC sheet to the uterus prevents or reduces rupture of the uterine incision and abnormal placentation relative to an incision in a uterus to which the MSC sheet is not applied.
In some embodiments, the MSC sheet is applied to an incision site in the uterus. In some embodiments, applying the MSC sheet to the uterus reduces fibrotic surface area of the uterus by at least 20% relative to a uterus in which the MSC sheet is not applied. In some embodiments, the MSC sheet consists essentially of MSCs. In some embodiments, the cell sheet comprises an extracellular matrix. In some embodiments, the extracellular matrix comprises one or more proteins selected from the group consisting of fibronectin, laminin and collagen. In some embodiments, the cell sheet comprises cell adhesion proteins and cell junction proteins. In some embodiments, the cell junction proteins are selected from the group consisting of Vinculin, Integrin-β1, Connexin 43, β-catenin, Integrin-linked kinase and N-cadherin. In some embodiments, the MSCs are isolated from the subepithelial layer of human umbilical cord tissue. In some embodiments, the MSCs express a protein selected from CD44 and CD90. In some embodiments, the MSCs express a cytokine selected from the group consisting of hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and interleukin-10 (IL-10). In some embodiments, expression of the cytokine in the cell sheet is increased relative to a suspension of cultured, isolated MSCs containing an equivalent number of cells. In some embodiments, the cell sheet expresses the cytokine for at least 10 days after transplantation to a tissue in a host organism.
In some embodiments, the cell sheet expresses extracellular matrix proteins and cell junction proteins for at least 10 days after transplantation to a tissue in a host organism. In some embodiments, the extracellular matrix proteins are selected from the group consisting of fibronectin, laminin and collagen. In some embodiments, the cell junction proteins are selected from the group consisting of Vinculin, Integrin-β1, Connexin 43, β-catenin, Integrin-linked kinase and N-cadherin. In some embodiments, initial cell density of the MSCs in a cell culture support used to prepare the cell sheet is from 0.5×104/cm2 to 9×105/cm2. In some embodiments, the MSCs do not express Human Leukocyte Antigen-DR isotype (HLA-DR), Human Leukocyte Antigen-DP isotype (HLA-DP), or Human Leukocyte Antigen-DQ isotype (HLA-DQ). In some embodiments, the MSCs comprise microvilli and filopodia.
In some embodiments, the cell sheet remains attached to a tissue in a host organism for at least 10 days after transplantation to the tissue.
In some embodiments, the MSCs in the cell sheet are allogeneic to the subject. In some embodiments, the subject is a human. In some embodiments, the subject has had at least one Caesarean Delivery. In some embodiments, the subject has had at least two Caesarean Deliveries. In some embodiments, the subject has had at least one uterine surgery. In some embodiments, the MSC is a human umbilical cord mesenchymal stem cell (hUC-MSC).
This disclosure describes decreased uterine scar formation and increased myometrial regeneration resulting from transplantation of a mesenchymal stem cell (MSC) sheet. For example, this disclosure describes decreased formation of fibrotic tissue and increased myometrial regeneration following transplantation of a human umbilical cord mesenchymal stem cell (hUC-MSC) sheet to a sutured incision site of a uterus, indicating that applying the hUC-MSC sheet reduced uterine scar formation. In the control group, a large fibrotic area was present between the host myometrium areas as a result of wound healing. In contrast, the fibrotic area in the hUC-MSC sheet transplantation group was significantly smaller than in the control group. Specifically, application of the hUC-MSC sheet to the uterus reduced the fibrotic surface area of the uterus by approximately 27% relative to the control group, and reduced the ratio of fibrotic-to-normal myometrial surface by greater than 33% relative to the control group. Thus transplantation of the hUC-MSC sheet significantly reduced fibrosis on the myometrial surface. Accordingly, the hUC-MSC sheets described herein improve healing of the uterine scar and have the potential to decrease morbidities related to abnormal uterine scar formation.
This disclosure also describes methods of preparing MSC sheets for use in reducing uterine scar formation. For example, hUC-MSCs were used to prepare cell sheets in vitro using temperature-responsive cell culture dishes (TRCDs) coated with a temperature-responsive polymer. Confluent cell sheets formed at 4-6 days after seeding and were detached from the TRCD by cooling the cultures to room temperature. Various culture conditions were identified that allow for successful production of robust, uniform hUC-MSC sheets containing one or more layers of aggregated, confluent cells. These culture conditions included optimization of subculture (passage) number before adding cells to the TRCD, initial cell density in the TRCD, addition of cell growth factors such as human platelet lysate (hPL) to the cell culture solution, and culture time in the TRCD before detachment from the temperature-responsive polymer.
I. Mesenchymal Stem Cells (MSCs)
Mesenchymal stem cells (MSCs) suitable for use in the methods described herein include, but are not limited to MSCs from umbilical cord, cord blood, limb bud, bone marrow, dental tissue (e.g. molars), adipose tissue, muscle and amniotic fluid.
In a particular embodiment, the mesenchymal stem cell is a human umbilical cord mesenchymal stem cell. The term “human umbilical cord mesenchymal stem cell” or “hUC-MSC” as used herein refers to a mesenchymal stem cell that has been isolated from a human umbilical cord.
Mesenchymal stem cells (MSCs) have a remarkable clinical potential to treat a wide range of debilitating diseases, mainly due to their unique immunomodulatory role and regenerative capacity (Caplan and Sorrell, 2015, Immunol Lett 168(2): 136-139). A convenient source for human MSCs is the umbilical cord, which is discarded after birth and provides an easily accessible and non-controversial source of stem cells for therapy (El Omar et al., 2014, Tissue Eng Part B Rev 20(5): 523-544). hUC-MSCs have been validated for safety and efficacy in human clinical trials as suspensions (Bartolucci et al., 2017, Circ Res, 121(10), 1192-1204). Moreover, hUC-MSCs have been successfully used in experimental animal disease models (Zhang et al., 2017, Cytotherapy 19(2): 194-199).
Methods for isolating MSCs from umbilical cords are known in the art and are described, for example, in U.S. Pat. No. 9,903,176, which is incorporated by reference herein in its entirety. The human umbilical cord comprises the umbilical artery, the umbilical veins, Wharton's Jelly, and the subepithelial layer. In some embodiments, the hUC-MSCs are isolated from the subepithelial layer of the human umbilical cord. In some embodiments, the hUC-MSCs are isolated from Wharton's Jelly of the human umbilical cord. Various cellular markers may be used to identify hUC-MSCs isolated from the subepithelial layer. For example, in some embodiments, the hUC-MSCs isolated from the subepithelial layer express one or more cell markers selected from CD29, CD73, CD90, CD146, CD166, SSEA4, CD9, CD44, CD146, and CD105. In a particular embodiment, the hUC-MSCs express CD73. In some embodiments, the hUC-MSCs isolated from the subepithelial layer do not express one or more cell markers selected from CD45, CD34, CD14, CD79, CD106, CD86, CD80, CD19, CD117, Stro-1, HLA-DR, HLA-DP and HLA-DQ. In a particular embodiment, the hUC-MSCs do not express HLA-DR, HLA-DP or HLA-DQ. In some embodiments, the cell sheets described herein are prepared with mesenchymal stem cells (MSCs) with low HLA expression, e.g. less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the MSCs in the cell sheet express HLA (e.g. HLA-DR, HLA-DP and/or HLA-DQ).
hUC-MSCs in the umbilical cord are surrounded by extracellular matrix (ECM) and connected with other types of umbilical cord cells (e.g. endothelial cells, epithelial cells, muscle cells, and fibroblasts) through cell-cell junction structures. In contrast to endogenous hUC-MSCs in the umbilical cord, the hUC-MSC sheets described herein comprise one or more layers of aggregated confluent hUC-MSCs in which the hUC-MSCs are connected to other hUC-MSCs, not to other types of umbilical cord cells. The hUC-MSC sheets described herein also differ from hUC-MSC suspension cultures in several ways. Suspension cultures of hUC-MSCs comprise single cells lacking an ECM or cell-cell junctions because these cell adhesive proteins in these cell-cell junctions must be removed (e.g. by proteolytic trypsin treatment) to harvest and suspend cells from culture surfaces commonly used for preparation of the cell suspension culture. In contrast to singe cell suspensions of hUC-MSCs, the hUC-MSC sheets described herein contain both an endogenous cell-produced ECM and intact cell-cell junctions among the hUC-MSCs that are generated during formation of the cell sheet. The endogenous ECM and intrinsic cell-cell junctions retained during cell sheet formation, fabrication and handling facilitate retention of important properties for their phenotypic preservation, cell functions and adhesion of the hUC-MSC sheet to target tissue during transplantation to a host organism.
II. Cell Sheets Produced from MSCs
In certain aspects the present disclosure relates to a mesenchymal stem cell sheet comprising one or more layers of confluent mesenchymal stem cells (MSCs). The term “mesenchymal stem cell sheet” or “MSC sheet” as used herein refers to a cell sheet obtained by growing mesenchymal stem cells on a cell culture support in vitro. In some embodiments, the MSCs in the MSC sheet are aggregated or physically contiguous. In some embodiments, the mesenchymal stem cell sheet is a human umbilical cord mesenchymal stem cell (hUC-MSC) sheet. The MSC sheets described herein are harvested as an intact sheet by temperature shift using a temperature-responsive culture dish (TRCD) without any enzyme treatment. The MSC sheets maintain their integrity and shape by retaining tissue-like structures, actin filaments, extracellular matrix, intercellular proteins, and high cell viability, all of which are related to improved cell survival and cellular function. Accordingly, the cell sheets described herein may comprise structural features that improve cell survival and cell function, including native extracellular matrix, cell adhesion proteins and cell junction proteins. Thus, the MSC sheets prepared by the methods described herein have several beneficial characteristics compared to MSCs produced by other methods. For example, the chemical disruption method is unable to maintain tissue-like structures of cells as well as cell-cell communication, since enzyme treatment disrupts the extracellular and intracellular proteins (cell-cell and cell-ECM junctions). Accordingly, protein cleavage by enzymes reduces cell viability and cellular functions. Physical disruption (i.e., by rubber policeman or media aspiration) produces disruption of cell-cell junctions and disintegration of the cultured adherent sheet into cell aggregates.
In some embodiments, the extracellular matrix comprises one or more proteins selected from the group consisting of fibronectin, laminin and collagen. In some embodiments, the cell junction proteins are selected from the group consisting of Vinculin, Integrin-β1, Connexin 43, β-catenin, Integrin-linked kinase and N-cadherin.
The MSCs in the cell sheets may also maintain additional structural features, such as microvilli and filopodia. Microvilli are cell membrane protrusions involved in a wide variety of cell functions, including absorption, secretion, and cellular adhesion. Filopodia are cytoplasmic projections that play a role in cell-cell interactions. Thus maintenance of these structural features may also help to maintain cell function and signaling useful for their application.
In some embodiments, the cell sheet consists of MSCs. In some embodiments, the cell sheet consists essentially of MSCs. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of cells in the cell sheet are MSCs. In some embodiments, 100% of the cells in the cell sheet are MSCs.
The MSCs may be added to the culture solution on the temperature-responsive polymer in the cell culture support at various cell densities to optimize formation of the cell sheet or its characteristics. For example, cytokine expression levels in the MSC may be optimized by controlling the initial cell density of the MSCs in the cell culture support (e.g. TRCD). In some embodiments, increasing the initial cell density of the MSCs in the cell culture support increases cytokine expression (e.g. HGF). In some embodiments, increasing the initial cell density of the MSCs in the cell culture support decreases cytokine expression. In some embodiments the initial cell density of the MSCs in the cell culture support used for preparation of the cell sheet is from 0.5×104/cm2 to 9×105/cm2. In some embodiments, the initial cell density of the MSCs in the cell culture support is at least 0.5×104, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, or 9×105 cells/cm2. Any of these values may be used to define a range for the initial cell density of the MSCs in the cell culture support. For example, in some embodiments, the initial cell density in the cell culture support is from 2×104 to 1×105 cells/cm2, 4×104 to 1×105 cells/cm2, or 1×104 to 5×104 cells/cm2.
The MSC sheets described herein may be transplanted to a target tissue in a host organism (e.g. a human) for therapeutic uses. Transplantation of the MSC sheets to the target tissue may prompt formation of new blood capillaries (angiogenesis) in the host tissue, as well as blood vessel formation between the transplanted cell sheet and the host tissue. This neocapillary formation is an important capability for engraftment, viability and tissue regeneration. In addition, this new blood vessel recruitment into sheets on the target tissue suggests that implanted MSC sheets continually secret paracrine factors to modulate this engraftment.
In some embodiments, the MSC sheets express one or more cytokines, for example, one or more anti-inflammatory cytokines and/or one or more inflammatory cytokines. In some embodiments the anti-inflammatory cytokine is derived from hepatocyte growth factor (HGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and interleukin-10 (IL-10). In some embodiments, the inflammatory cytokine is tumor necrosis factor-α (TNF-α). In some embodiments, cytokine expression (e.g. an anti-inflammatory cytokine or an inflammatory cytokine) in the cell sheet is increased relative to a suspension of MSCs containing an equivalent number of cells. In some embodiments, expression of the cytokine (e.g. an anti-inflammatory cytokine or an inflammatory cytokine) is decreased relative to a suspension of MSCs containing an equivalent number of cells. For some therapeutic uses, reducing secretion of inflammatory cytokines by the cell sheet would be beneficial. For example, in a particular embodiment, the cell sheet secretes tumor necrosis factor-α (TNF-α) into a culture solution in vitro at a rate of less than 100, 90, 80, 70, 60, 50, 40 or 30 pg/mL of culture solution/24 hours.
The MSC sheets described herein may continue to express cytokines after transplantation to a target tissue in a host organism. In some embodiments, the cell sheet expresses the cytokine for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 days after transplantation to a tissue in a host organism. In some embodiments, the cell sheet expresses the cytokine for at least 1, 2, 3, 4, 5 or 6 months after transplantation to a tissue in a host organism.
The MSC sheets described herein may also continue to express extracellular matrix proteins and cell junction proteins after transplantation to a target tissue in a host organism. For example, in some embodiments the cell sheet expresses extracellular matrix proteins and/or cell junction proteins for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 days after transplantation to a tissue in a host organism. In some embodiments, the cell sheet expresses the extracellular matrix proteins and/or cell junction proteins for at least 1, 2, 3, 4, 5 or 6 months after transplantation to a tissue of a host organism. In some embodiments the extracellular matrix proteins expressed in the cell sheet after transplantation are selected from fibronectin, laminin and collagen. In some embodiments the cell junction proteins expressed in the cell sheet after transplantation are selected from Vinculin, Integrin-β1, Connexin 43, β-catenin, Integrin-linked kinase and N-cadherin.
Current stem cell therapies often use cultured stem cells isolated from biopsies as injectable cell suspensions (Bayoussef et al., 2012, J Tissue Eng Regen Med, 6(10)). Injected cell suspensions typically exhibit lower engraftment into and retention within diseased organs or tissues (Devine et al., 2003, Blood, 101(8), 2999-3001). Loss of intact ECM and cell-cell junctions (i.e., communication) in stem cell suspensions through enzymatic disruption at harvest compromises stem cell function, engraftment and survival in vivo, and can limit therapeutic efficacy in vivo. In contrast, the methods of preparing MSC sheets described herein preserve intrinsic cell functional structures, improving attachment of the cell sheet to the target tissue after transplantation. For example, in some embodiments, the cell sheet remains attached to the target tissue in the host organism for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 days after transplantation to a tissue in a host organism. In some embodiments, the cell sheet remains attached to the target tissue in the host organism for at least 1, 2, 3, 4, 5 or 6 months after transplantation to a tissue of a host organism.
Human leukocyte antigens (HLAs) are cell-surface proteins that make up the major histocompatibility complex (MHC) proteins in humans and are responsible for regulation of the immune system. HLA markers are important to control for tissue transplantation and host acceptance. HLAs corresponding to MHC class II (DP, DM, DO, DQ, and DR) present antigens from the cell surface to host T-lymphocytes to modulate host recognition as “self”. These antigens stimulate the multiplication of T-helper cells (CD4+ T cells), which in turn stimulate antibody-producing B-cells to produce antibodies to that specific antigen. Thus minimizing expression of HLAs is beneficial for minimizing a host immune response to transplanted hUC-MSC sheets in a host organism. In some embodiments, the hUC-MSC sheets described herein do not express one or more of Human Leukocyte Antigen-DR isotype (HLA-DR), Human Leukocyte Antigen-DP isotype (HLA-DP), or Human Leukocyte Antigen-DQ isotype (HLA-DQ). In some embodiments, less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the hUC-MSCs in the cell sheet express HLA (e.g. HLA-DR, HLA-DP and/or HLA-DQ).
III. Methods for Producing MSC Sheets In Vitro
In certain aspects, the present disclosure relates to a method for producing a cell sheet comprising one or more layers of aggregated confluent mesenchymal stem cells (MSCs), the method comprising:
In some embodiments, the MSCs are human umbilical cord mesenchymal stem cells (hUC-MSCs). Methods for isolating hUC-MSCs are known in the art and are described, for example, in U.S. Pat. No. 9,803,176, which is incorporated by reference herein in its entirety. For example, hUC-MSCs may be isolated from the subepithelial layer of an umbilical by washing the umbilical cord to remove blood, Wharton's Jelly, and any other material, and dissecting the subepithelial layer (SL) from the umbilical cord. The cord tissue may be washed multiple times in a solution of Phosphate-Buffered Saline (PBS) such as Dulbecco's Phosphate-Buffered Saline (DPBS). The PBS can include a platelet lysate (i.e. 10% PRP lysate of platelet lysate). The SL can then be placed interior side down on a substrate. An entire dissected umbilical cord with the Wharton's Jelly removed can be placed directly onto the substrate, or the dissected umbilical cord can be cut into smaller sections (e.g. 1-3 mm) and these sections can be placed directly onto the substrate. The substrate can be a solid polymeric material such as a cell culture dish. The SL can be placed upon the substrate of the cell culture dish without any additional pretreatment to the cell culture treated plastic, or on a semi-solid culture medium such as agar. Following placement of the SL on the substrate, the SL is cultured in a suitable medium (e.g. Dulbecco's Modified Eagle Medium (DMEM) glucose (500-6000 mg/mL) without phenol red, 1× glutamine, 1×NEAA, and 0.1-20% PRP lysate or platelet lysate). The culture can then be cultured under either normoxic or hypoxic culture conditions for a period of time sufficient to establish primary cell cultures (e.g. 3-7 days). After primary cell cultures have been established, the SL tissue is removed and discarded. Cells or stem cells are further cultured and expanded in larger culture flasks in either a normoxic or hypoxic culture conditions.
General methods for preparing cell sheets are known in the art and are described, for example, in U.S. Pat. Nos. 8,642,338; 8,889,417; 9,981,064; and 9,114,192, each of which is incorporated by reference herein in its entirety.
The temperature-responsive polymer used to coat the substrate of the cell culture support has an upper or lower critical solution temperature in aqueous solution which is generally in the range of 0° C. to 80° C., for example, 10° C. to 50° C., 0° C. to 50° C., or 20° C. to 45° C.
The temperature-responsive polymer may be a homopolymer or a copolymer. Exemplary polymers are described, for example, in Japanese Patent Laid-Open No. 211865/1990. Specifically, they may be obtained by homo- or co-polymerization of monomers such as, for example, (meth)acrylamide compounds ((meth)acrylamide refers to both acrylamide and methacrylamide), N-(or N,N-di)alkyl-substituted (meth)acrylamide derivatives, and vinyl ether derivatives. In the case of copolymers, any two or more monomers, such as the monomers described above, may be employed. Further, those monomers may be copolymerized with other monomers, one polymer may be grafted to another, two polymers may be copolymerized, or a mixture of polymer and copolymer may be employed. If desired, polymers may be crosslinked to an extent that will not impair their inherent properties.
The substrate which is coated with the polymer may be of any types including those which are commonly used in cell culture, such as glass, modified glass, silicon oxide, polystyrene, poly(methyl methacrylate), polyester, polycarbonate, and ceramics.
Methods of coating the support with the temperature-responsive polymer are known in the art and are described, for example, in Japanese Patent Laid-Open No. 211865/1990. Specifically, such coating can be achieved by subjecting the substrate and the above-mentioned monomer or polymer to, for example, electron beam (EB) exposure, irradiation with γ-rays, irradiation with UV rays, plasma treatment, corona treatment, or organic polymerization reaction.
The coverage of the temperature responsive polymer may be in the range of 0.4-3.0m/cm2, for example, 0.7-2.8 μg/cm2, or 0.9-2.5 μg/cm2. The morphology of the cell culture support may be, for example, a dish, a multi-plate, a flask or a cell insert.
The cultured cells may be detached and recovered from the cell culture support by adjusting the temperature of the support material to the temperature at which the polymer on the support substrate hydrates, whereupon the cells can be detached. Smooth detachment can be realized by applying a water stream to the gap between the cell sheet and the support. Detachment of the cell sheet may be affected within the culture solution in which the cells have been cultivated or in other isotonic fluids, whichever is suitable.
In a particular embodiment, the temperature-responsive polymer is poly(N-isopropyl acrylamide) Poly(N-isopropyl acrylamide) has a lower critical solution temperature in water of 31° C. If it is in a free state, it undergoes dehydration in water at temperatures above 31° C. and the polymer chains aggregate to cause turbidity. Conversely, at temperatures of 31° C. and below, the polymer chains hydrate to become dissolved in water, thereby causing release of the cell sheet from the polymer. In a particular embodiment, this polymer covers the surface of a substrate such as a Petri dish and is immobilized on it. Therefore, at temperatures above 31° C., the polymer on the substrate surface also dehydrates but since the polymer chains cover the substrate surface and are immobilized on it, the substrate surface becomes hydrophobic. Conversely, at temperatures of 31° C. and below, the polymer on the substrate surface hydrates but since the polymer chains cover the substrate surface and are immobilized on it, the substrate surface becomes hydrophilic. The hydrophobic surface is an appropriate surface for the adhesion and growth of cells, whereas the hydrophilic surface inhibits the adhesion of cells and the cells are detached simply by cooling the culture solution.
Culture solutions for mesenchymal stem cells are known in the art and are described, for example, in U.S. Pat. Nos. 9,803,176 and 9,782,439, each of which is incorporated by reference herein in its entirety. In some embodiments, the cell culture solution comprises human platelet lysate (hPL). In some embodiments, the culture solution comprises fetal bovine serum (FBS). In some embodiments, the culture solution comprises ascorbic acid. In some embodiments, the culture solution is a xeno-free medium, i.e. a medium that may contain products obtained from humans but does not contain products obtained from non-human animals. In some embodiments, the culture solution contains at least one product obtained from a non-human animal (e.g. FBS). In some embodiments, the culture solution does not contain a product obtained from a human.
In a particular embodiment, the culture solution comprises one or more of Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies, CA, USA), human platelet lysate (hPL, iBiologics, Phoenix, USA), Glutamax (Life Technologies), MEM Non-Essential Amino Acids Solution (NEAA) (Life Technologies) and an antibiotic, e.g. penicillin streptomycin.
The MSCs (e.g. hUC-MSCs) may be passed through one or more subcultures (i.e. passages) prior to culturing the cells in culture solution on a temperature-responsive polymer which has been coated onto a substrate surface of a cell culture support. In some embodiments, the MSCs (e.g. hUC-MSCs) are passed through 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 subcultures prior to culturing the cells in culture solution on a temperature-responsive polymer which has been coated onto a substrate surface of a cell culture support. Any of these values may be used to define a range for the number of subcultures. For example, in some embodiments, the MSCs (e.g. hUC-MSCs) are passed through 2 to 10, 4 to 8, or 1 to 12 subcultures prior to culturing the cells on a temperature-responsive polymer. In some embodiments the number of subcultures is less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, the number of subcultures is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
The MSC sheet may be prepared in a range of different sizes depending on the application. In some embodiments, the MSC sheet has a diameter of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 cm. Any of these values may be used to define a range for the size of the MSC sheet. For example, in some embodiments, the MSC sheet has a diameter from 1 to 20 cm, from 1 to 10 cm or from 2 to 10 cm. In some embodiments, the MSC sheet has an area of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 cm2. Any of these values may be used to define a range for the size of the MSC sheet. For example, in some embodiments, the MSC sheet has an area from 1 to 100 cm2, 3 to 70 cm2, or 1 to 300 cm2. The methods described herein result in an hUC-MSC sheet in which the surface area of the hUC-MSC sheet is much greater than its thickness. For example, in some embodiments the ratio of the surface area of the hUC-MSC sheet to its thickness is at least 10:1, 100:1, 1000:1, or 10,000:1. The hUC-MSC sheets described herein comprise one or more layers of confluent human umbilical cord mesenchymal stem cells (hUC-MSCs), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 layers of hUC-MSCs. In some embodiments, the hUC-MSC sheet comprises fewer than 2, 3, 4, 5, 6, 7, 8, 9 or 10 layers of hUC-MSCs. In some embodiments, the hUC-MSC sheet comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 layers of hUC-MSCs.
IV. Methods for Using MSC Sheets to Reduce Formation of Fibrotic Tissue and Increase Myometrial Regeneration in the Uterus
In some aspects, the present disclosure relates to a method of reducing formation of fibrotic tissue in a uterus of a subject in need thereof, comprising applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, wherein the SC sheet comprises one or more layers of aggregated confluent mesenchymal stem cells (MSCs), and wherein applying the −MSC sheet to the uterus reduces the formation of fibrotic tissue in the uterus relative to a uterus in which the MSC sheet is not applied.
In some aspects, the present disclosure relates to a method of increasing myometrial regeneration in a uterus of a subject in need thereof, comprising applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, wherein the MSC sheet comprises one or more layers of aggregated confluent mesenchymal stem cells (MSCs), and wherein applying the MSC sheet to the uterus increases myometrial regeneration relative to a uterus in which the MSC sheet is not applied.
The uterus comprises four layers, the endometrium epithelium, endometrium stroma, myometrium and perimetrium. The endometrium comprises epithelial and stromal layers as inner layers. It has a basal layer and a functional layer; the functional layer thickens and then is sloughed during the menstrual cycle or estrous cycle. The myometrium is the middle layer of the uterine wall, consisting mainly of uterine smooth muscle cells (also called uterine myocytes), but also of supporting stromal and vascular tissue. The main function of the myometrium is to induce uterine contractions. The outer layer of the uterus is the perimetrium.
For Caesarean delivery, an incision of about 15 cm is typically made through the mother's lower abdomen and the uterus is then opened with a second incision and the baby delivered. The incisions are then stitched closed in multiple tissue layers.
For other uterine surgeries such as myomectomy which is removed myoma nodules, an incision of any length and location depended on size of lesions are made through the patient's lower abdomen or abdominal hole for endoscopic surgeries and the lesions are removed with an incision in border line between normal tissue and abnormal tissue. The incisions are then stitched closed in single or multiple tissue layers. After uterine surgeries, the initial scar is typically fibrous tissue; and that fibrotic scar is weak, prone to rupture and other problems, and in need of mitigation with normal scar formation, remodeling and myometrial regeneration.
MSC sheets may also be used after non-surgical procedures. For example, in some embodiments, the MSC sheet is applied to the uterus after dilation and curettage (D&C) (e.g. after miscarriage) or after removal of uterine fibroids. The MSC sheets could be applied with an endoscope deploying cell sheets vaginally.
One advantage of the MSC sheets described herein is that the extracellular matrix of the applied cell sheet acts as a natural adhesive to bind the cell sheet to the uterine tissue of the subject, such that suturing or stitching is not required to adhere the cell sheet to the tissue. A support membrane or other devices may be used to transfer the MSC sheet to the uterine tissue of the subject and then removed after sheet transfer. The supports can be, for example, poly(vinylidene difluoride) (PVDF), cellulose acetate, cellulose esters, plastic and metal. The MSC sheets readily adhere to target tissue, self-stabilizing without suturing after being placed directly onto the target tissue for a short period of time. For example, in some embodiments, the MSC sheet adheres to the target tissue within 5, 10, 15, 20, 25, or 30 minutes after contact with the tissue. Once the MSC sheet has adhered to the uterine tissue, the support membrane may be excised. In certain embodiments, the MSCs in the cell sheet are allogeneic to the subject, i.e. are isolated from a different individual from the same species as the subject, such that the genes at one or more loci are not identical. In certain reported cases, MSCs seemingly avoid allogeneic rejection in humans and in animal models (Jiang et al., 2005, Blood, 105(10), 4120-4126). Thus, the MSC sheets described herein may be used in allogeneic cell therapies as an off-the-shelf product.
Allogeneic cell sources must be capable of eliciting meaningful therapies under standard immunologic competence in host patient allogeneic tissues. This includes reliable cell homing to and fractional dose engraftment or retention for sufficient duration at the tissue site of therapeutic interest (Leor et al., 2000, Circulation, 102(19 Suppl 3), III 56-61). Current estimates are that when stem cell suspensions are administered to a subject, less than 3% of injected stem cells are retained in damaged myocardium 3 days post-injection following ischemic injury (Devine et al., 2003, Blood, 101(8), 2999-3001). Additionally, most administered cells from a cell suspension that engraft into target tissue will die within the first few weeks (Reinecke & Murry, 2002, J Mol Cell Cardiol, 34(3), 251-253). In contrast, the MSC cell sheets described herein stably engraft at high fractional retention to host tissue 7 days after transplantation. Thus the MSC sheets described herein provide distinct advantages over injected or administered mesenchymal stem cell suspensions.
In some embodiments, the MSC sheet is applied to an incision site in the uterus. In some embodiments, the incision is sutured closed before the MSC sheet is applied to the uterus (e.g. to the top incision site of the myometrium). In some embodiments, applying the MSC sheet to the uterus reduces fibrotic area of the uterus by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a uterus in which the MSC sheet is not applied. In some embodiments applying the MSC sheet to the uterus reduces the thickness of the uterine scar after Caesarean Delivery by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a uterus in which the MSC sheet is not applied.
The risk of complications for Caesarean Delivery often increases if the subject has had one or move previous Caesarean Deliveries or at least one previous uterine surgery. In some embodiments, the subject has not had a previous Caesarean Delivery or any uterine surgery. In some embodiments, the subject has had at least 1, 2, 3, 4, 5, 6, 7 or 8 previous Caesarean Deliveries and at least one previous uterine surgery.
More than one MSC sheet may be applied to the uterus in the methods described herein. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more MSC sheets may be applied to the uterus. Any of these values may be used to define a range for the number of MSC sheets applied to the uterus. For example, in some embodiments, 2-4, 3-5 or 1-10 MSC sheets are applied to the uterus.
1.1 Human Umbilical Cord Stem Cell (hUC-MSC) Culture
Banked human umbilical cord mesenchymal stem cells isolated from the subepithelial layer of human umbilical cord tissue (Jadi Cell LLC, Miami, USA IRB-35242) (Patel et al., 2013, Cell Transplant, 22(3), 513-519) were cultured in xeno-free cell culture media with Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies, CA, USA) supplemented with 10% human platelet lysate (hPL, iBiologics, Phoenix, USA), 1% Glutamax (Life Technologies), 1% MEM NEAA (Life Technologies), 1% penicillin streptomycin (Life Technologies), at 37° C. in a humidified atmosphere with 5% CO2 for 5 days. Subculture was performed from passage 4 until passage 12. Cell culture media was changed every two days.
1.2 hUC-MSC Proliferation Rate
hUC-MSCs were seeded on 35-mm tissue culture plates (TCP) (Corning, N.Y.) at cell numbers of 5×104, 1×105 and 2×105 cells/dish (i.e. initial cell densities of 5×103/cm2, 1×104/cm2, and 2×104/cm2, respectively) in xeno-free cell culture media. Cells on TCP were dissociated with trypsin and cell number counted using a hemocytometer at 1, 2, 3, 4, 5 and 6 days. hUC-MSCs were seeded at a cell density of 3.5×103/cm2 on 175 cm2 tissue culture flasks (Corning, N.Y.) and passaged at 5 days with TrypLE (life technologies) after culturing from passage 4 until 12. Cell number was counted each passage using a hemocytometer.
1.3 hUC-MSC Characterization in Differentiation Potential
hUC-MSCs were cultured in xeno-free cell culture media for two passages on TCP. At passages 4, 6, 8, 10, and 12, cells were prepared and induced for osteogenic and adipogenic differentiation. For osteogenic differentiation, cells were plated at 5×103 cells/cm2 in 35 mm TCP dishes in xeno-free cell culture media. When 60% confluent, cells were induced with osteogenic differentiation media containing αMEM, 10 nM dexamethasone, 82 μg/mL ascorbic acid 2-phosphate, 10 mM β-glycerolphosphate (Sigma-Aldrich). Cells were cultured in osteogenic media at 37° C. for 21 days with media changed every 3 days. To detect positive differentiation, cells were fixed with cold 4% paraformaldehyde for 12 minutes and stained with Alizarin Red S-(Sigma-Aldrich) using standard protocols. For adipogenic differentiation, cells were plated at 1×104 cells/cm2 in 35 mm TCP dishes in xeno-free cell culture media. When 80% confluent, cells were induced with adipogenic differentiation media containing high-glucose DMEM, 100 nM dexamethasone, 0.5 mM IBMX, and 50 μM IND (all Sigma-Aldrich). Cells were cultured in adipogenic media at 37° C. for 21 days and media changed every 3 days. To detect positive differentiation, cells were fixed with cold 4% paraformaldehyde for 12 minutes and stained with Oil Red 0 (Sigma-Aldrich) using standard protocols.
1.4 hUC-MSC Surface Phenotype Assay
hUC-MSCs were cultured in xeno-free cell culture media on TCP. Cell suspensions were prepared of P6, P8, P10, and P12 HPL and FBS cultured cells. Cells were then detached enzymatically and washed once with PBS. To minimize non-specific binding of antibodies, cells were incubated with 2% w/v Bovine Serum Albumin (BSA) in PBS for 30 minutes. Cells were then aliquoted at concentrations of 3-5×105/100 μL. One aliquot was reserved as an unstained control and those remaining were stained with the following antibodies: CD44, CD90 and HLA-DR, DP, DQ (Biolegend, San Diego, Calif.). Primary antibody was added to each aliquot to achieve a ratio of about 20:1 of cells in buffer to antibody. About 3-5×105 cells were stained with saturating concentrations of (fluorophore)-conjugated antibodies. Cells were incubated in the dark on ice for 30 minutes. After incubation, cells were washed three times and then re-suspended in PBS. The cells were immediately analyzed by flow cytometry. Flow cytometry was performed on a Becton, Dickinson FACS Canto (BD Biosciences, Sparks, MD). Flow cytometer instruments were set using unstained cells. Cells were gated by forward versus side scatter to eliminate doublets. A minimum of 10,000 events was counted for each analysis.
1.5 hUC-MSC Sheet Preparation Using Different Initial Cell Numbers and Passage Numbers
hUC-MSC sheets were prepared on temperature-responsive cell culture dishes (TRCDs) in various conditions including different initial cell density and passage numbers (
1.6 Immunohistochemical Staining
Cultured cell sheets were removed from TRCD at room temperature and fixed with 4% paraformaldehyde for 30 min and then embedded in paraffin. Embedded specimens were sectioned into 4 μm slices and stained with H&E, stem cell surface markers, ECMs (fibronectin; FN and laminin; LM) and cell-cell junctions (integrin-linked kinase; β-catenin). For fluorescence staining (FN, LM, and β-catenin), slides were immersed in antigen retriever solution (Sigma-Aldrich) for 20 min at 100° C. and washed with PBS 1×. Non-specific binding was blocked in PBS 1× containing 10% goat serum (Vector Laboratories, Burlingame, USA), for 1 h at room temperature. Primary antibody labeling (Abcam, Cambridge, USA) (1:100) at 4° C. proceeded overnight and then washed with PBS 1×. These specimens were treated with Alexa Fluor 594-conjugated secondary antibodies (Life Technologies) (1:200) for 1 h and mounted with ProLong Gold Antifade Reagent (Life Technologies). Immunofluorescence images were obtained using an AX 10 microscope (Carl Zeiss Microimaging) and analyzed with Axiovision software (Carl Zeiss Microimaging). For H&E stain, specimens were treated with hematoxylin solution (Sigma-Aldrich) for 3 min and subsequently with eosin solution (Thermo Fisher Scientific, Kalamazoo, USA) for 5 min. The H&E stained specimens were dehydrated and mounted with Permount™ (Thermo Fisher Scientific). H&E images were obtained using a BX 41 microscope (Olympus, Hamburg, Germany).
1.7 Cell Sheet Microstructure Observed Using Transmission Electron Microscopy
hUC-MSC sheets were fixed with a mixture of 2% paraformaldehyde, 2% glutaraldehyde, 0.1 M sodium phosphate buffer, and 2% osmium tetroxide (OsO4) in sodium phosphate buffer and dehydrated in a grade series of ethanol. Samples were then embedded in epoxy resin. Ultrathin sections (70 nm thickness) were observed with a transmission electron microscope (JEOL JEM1200EX) (JEOL USA, Peabody, USA).
1.8 Determination of Hepatocyte Growth Factor (HGF) and Tumor Necrosis Factor Alpha (TNF-α) Secretion from hUC-MSC Sheets
hUC-MSC cell sheets were fabricated on TRCDs. Supernatant media over adherent cultured cells for 24 hours was collected just prior to cell sheet detachment from TRCD at room temperature (RT). HGF and TNF-α amounts secreted from hUC-MSCs were measured by human HGF Quantikine ELISA and human TNF-α Quantikine ELISA kits, respectively (R&D Systems, Minneapolis, USA).
1.9 Cell Sheet Placement into Immune-Deficient Mice Subcutaneous Tissue
hUC-MSC (passage 6) cell sheets were detached from TRCD at RT after 4 days of culture and transplanted into subcutaneous dorsal tissues of 6-week old immune-deficient mice (NOD.CB17-Prkdcscid/NCrCrl) (Charles River, San Diego, USA). Sterilized non-cytotoxic silicone membrane (Invitrogen) was placed between the cell sheet and subcutaneous dorsal tissues to prohibit tissue adhesion. Implanted mice were sacrificed 10 days after cell sheet transplantation. The cell sheet-transplanted subcutaneous tissue was fixed with 10% paraformaldehyde (Sigma-Aldrich) for 1 day for histological analysis. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) (protocol #16-12017) at The University of Utah and conducted in accordance with national guidelines.
1.10 Statistical Analyses
All quantitative values are expressed as mean and standard error (SE, mean±SE). Significant differences between groups were tested by one-way Analysis of Variance using origin 2017 software (OriginLab, Northampton, USA). A probability value of less than 0.05 (p<0.05) was considered statistically significant.
1.11 Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis
hUC-MSC cell sheets were collected after detachment from TRCD at RT. Total RNA from cell sheets was extracted using Trizol and PureLink RNA Mini Kit (Life Technologies) according to manufacturer's protocols. cDNA was prepared from 1 μg of total RNA using high capacity cDNA reverse transcription kits (Life Technologies). RT-PCR analysis was performed with TapMan Universal PCR Master Mix using an Applied Biosystems Step One instrument (Applied Biosystems™, Foster City, USA). Gene expression levels were assessed for the following genes: 1) glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs02786624_g1) as a housekeeping gene, 2) integrin-linked kinase (ILK, Hs00177914_m1), 3) N-cadherin (N-cad, Hs00983056_m1). All primers were manufactured by Applied Biosystems. Relative gene expression levels were quantified by the comparative CT method (Schmittgen & Livak, 2008). Gene expression levels were normalized to GAPDH expression levels. Gene expression levels are relative to the level at passage 6 cell group.
Results
hUC-MSC Sheet Preparation with Different Initial Cell Numbers and Passage Numbers
hUC-MSCs were cultured on flasks and sub-cultured using trypsin every 5 days from passages 4 to 12 (Table 1). Cells were proliferated 16-20 times from initial cell seeding numbers between passages 4-8 during sub-culture. However, cell proliferation rate dramatically decreases from passage 9. Cell numbers were 14, 10.9, 7.5, and 3.1-fold increased from initial cell seeding numbers at passage 9, 10, 11, and 12, respectively. Cells in passage 10 required one day more to reach confluence and yield cell sheets than cells in passages 4-8 at the same seeding density (
Initially seeded cell numbers of 5×104, 1×105, and 2×105 cells/dish reached confluence at 6, 5, and 4 days, respectively (
hUC-MSC Surface Marker Characterization
CD44 and CD90 expression was measured in hUC-MSC suspension cultures and in hUC-MSC sheets in vitro. As shown in
Structural Analysis of hUC-MSC Sheets
Passage 6 cells were cultured on TRCD for 4 days and resulting cell sheets were recovered from TRCD with temperature reduction to RT. The cell sheet was stained with fibronectin, laminin, and β-catenin to verify hUC-MSC sheet retention of functional structures during culture and after sheet detachment. Fibronectin and laminin, important ECM components that promote cell and tissue attachment (Yue, 2014, J Glaucoma 23: S20-S23; Kim et al., 2016, Int Neurourol J.: S23-S29), were strongly expressed across the entire cell sheet surface (
Inter-cellular structures within cell sheets were observed by TEM. Horizontal sectioning showed ECM structures within cell sheets (
Secretion of Hepatocyte Growth Factor (HGF) and Tumor Necrosis Factor-Alpha (TNF-α)
Human anti-inflammatory cytokine HGF (Gong, Rifai, & Dworkin, 2006; J Am Soc Nephrol, 17(9), 2464-2473), and pro-inflammatory cytokine TNF-α (Ertel et al., 1995, J Cell Sci, 123(Pt 24), 4195-4200) (REF) secreted from hUC-MSCs in culture supernatant were measured to support paracrine effects of the fabricated hUC-MSC sheets in vitro. No significant differences in amounts of hHGF were seen in 2×105, 1×105, and 5×104 cells/dish groups at passage 6 (
hUC-MSC sheets fabricated using passage 4 cells secreted significantly higher concentrations of hHGF (633 pg/mL), compared to hUC-MSC sheets fabricated using passage 6, 8, 10, and 12 cells. Amounts of hHGF secreted from hUC-MSC sheets dramatically decreased as passage number increased (
Cell Sheet Implantation into Immune-Deficient Mice
hUC-MSC sheets were implanted into dorsal subcutaneous pockets in immune-deficient mice for 10 days to demonstrate stability and engraftment in vivo. At 10 days after transplantation, formation of capillaries (angiogenesis) was observed in cell sheet-transplanted tissue, while subcutaneous tissue without cell sheet transplantation showed only a few fine blood vessels (
Discussion
Xeno-free hUC-MSC sheet fabrication was demonstrated from cultures using temperature responsive culture dishes (TRCD). These hUC-MSC sheets exhibit: 1) retention of native functional inter-cellular structures essential to cell-cell communication, act as a natural matrix adhesive when implanted onto target organs (
To fabricate potent MSC cell sheets reproducibly, hUC-MSCs from passages 4 to 12 were expanded and transformed to sheets in cell culture media supplemented with hPL. Cell proliferation rates for hUC-MSCs were remarkably reduced after passage 10, affecting the cell sheet creation process and timelines to harvest (
Rapid growth of hUC-MSCs cultured in cell culture media including hPL could be beneficial in reducing time required to fabricate cell sheets. Conversely, it may also introduce some processing difficulties because sheet cultures reach confluence quickly and are prone to spontaneous detachment upon reaching confluence. Therefore, judicious use of appropriate initial cell seeding numbers is important for the hUC-MSC sheet fabrication process. Initial cell seeding density higher than 2×105 cells/dish does not yield a monolayer sheet: such high density induces spontaneous cell detachment from TRCDs within 2 days of cell culture (data not shown). In this study, initial densities of 2×105, 1×105, and 5×104 cells were used and all successfully yielded hUC-MSC sheets at day 4, 5, and 6, respectively, when each culture reached confluence (
Central to these results is the reliable capability to produce a stable, robust layer of hUC-MSCs using a commercial TRCD grafted with temperature-responsive polymer coating that facilitates cell harvest without destructive enzymes using temperature reduction (Okano et al., 1995, Biomaterials, 16(4), 297-303; Okano et al., 1993, J Biomed Mater Res, 27(10), 1243-1251). This cell sheet technology produces cultured cell recovery with intact native cell-cell organization, cell-cell communication, intact ECMs, and tissue-like phenotypes. Cell sheets recovered from TRCDs by small changes in culture temperature preserve cell surface-associated ECMs such as fibronectin and laminin, and cell-cell junction proteins such as β-catenin (
Overall, hUC-MSC sheets display several beneficial properties for improving allogeneic MSC cell therapy. Results here have determined (1) specific conditions for reliable xeno-free hUC-MSC sheet fabrication; (2) intact features of hUC-MSC sheets that preserve important cell functional structures and paracrine effects after cell harvest from TRCDs; (3) intact hUC-MSC sheet retention in implant target tissue sites for 10 days; and (4) new blood vessel recruitment into sheets on the target tissue, suggesting that implanted hUC-MSC sheets continually secret paracrine factors to modulate engraftment.
Conclusions
hUC-MSC cell sheet technology represents a unique cellular delivery method aimed to improve MSC therapy over current injected cell suspensions. The simple fabrication method on TRCDs in hPL allows rapid xeno-free production of robust uniform hUC-MSC sheets, harvested with small changes of temperature instead of destructive proteolytic enzymes. Cell production depends on several controlled culture variables, including cell seeding density, passage number, media (hPL), and culture time and TRCDs. When cultured homogeneously under optimized conditions, hUC-MSC cell sheet reproducibility is enhanced and the hUC-MSC cell sheet production process is simplified to a routine amenable to scaling. This enables future production of hUC-MSC sheets having higher cell numbers to increase paracrine action and therapeutic benefits. Given their paracrine effects and low HLA profile, fabricated xeno-free hUC-MSC sheets represent promising tissue regeneration potential both structurally and functionally in vitro and in vivo. With reliable topical tissue site placement, high engraftment efficiency, long-term retention and survival in vivo, the hUC-MSC sheet has a potential to improve therapeutic value of allogeneic cell therapy over injected stem cells used currently.
Materials and Methods
2.1 Antibodies
The following antibodies were used in this study; actin (ab8226) (Abcam, Cambridge, USA), vinculin (ab129002) (Abcam), fibronectin (ab6328) (Abcam), laminin (ab11575) (Abcam), integrin β-1 (ab179471) (Abcam), connexin 43/GJA1 (ab11370) (Abcam), YAP (#140794) (Cell Signaling Technology (CST), Massachusetts, USA), phospho-YAP (Ser127, #4911)) (CST), FAK (ab40794) (Abcam), Phospho-FAK (Tyr397, #8556) (CST), GAPDH (ab9484) (Abcam). Alexa flour 568 goat anti-rabbit, 568 goat anti-mouse, 488 goat anti-rabbit, and 488 goat anti-mouse (life technologies) were used as secondary antibodies.
2.2 Human Umbilical Cord Stem Cell (hUC-MSC) Culture
Banked human umbilical cord mesenchymal stem cells (hUC-MSCs) were isolated from the subepithelial layer of human umbilical cord tissue (Jadi Cell LLC, Miami, USA IRB-35242) and were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% GlutaMAX (Gibco), 1% MEM non-essential amino acids (NEAA) (Gibco), 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco). hUC-MSC was incubated at 37° C. with 5% CO2 in a humidified chamber and passaged when cells reached confluent. hUC-MSC was passaged with TrypLE (Gibco) treatment for 5 minutes and subculture at 3000 cells/cm2 between passages 4 and 6.
2.3 Preparation of hUC-MSC Sheet
hUC-MSCs were seeded on a 35 mm temperature responsive culture dish (TRCD) (CellSeed, Tokyo, Japan). hUC-MSC was seeded at the density of 2×105 cells/dish (Day 0) and cultured to confluence (Day 5). Cell culture media including 16.4 μg/mL of ascorbic acid (Wako, Osaka, Japan) was replaced at 1 day after seeding. hUC-MSC was harvested as a mono-layer sheet from TRCD within 60 minutes by reducing the temperature to 20° C. Total cell number of hUC-MSC sheet was counted with trypan blue (Gibco) exclusion test using hemocytometer.
2.4 Hematoxylin and Eosin (H&E) Staining of hUC-MSC Sheet
Samples were fixed with 4% buffered paraformaldehyde (PFA) and embedded in paraffin. Then, the samples were cut into 4 μm-thick sections. The sections were stained with Mayer's hematoxylin and 1% eosin alcohol solution. Then, it was mounted with Permount™ (Thermo Fisher Scientific). The stained samples were visualized using a BX53 microscope (Olympus, Tokyo).
2.5 Morphological Observation of hUC-MSCs Using Scanning Electron Microscopy and Transmission Electron Microscope
For scanning electron microscopy (SEM) analysis, samples were rinsed in wash buffer (0.1M sodium cacodylate buffer with 2.4% sucrose and 8 mM calcium chloride) for 5 minutes and then fixed with 2% osmium tetroxide (OsO4) in wash buffer for 1 hour at room temperature. Samples were rinsed with DI water to remove unbound osmium, then dehydrated through grade series of ethanol. Subsequently, ethanol was replaced with hexamethyldisilazane (HMDS) and dried at −30° C. The samples were observed with scanning electron microscope (FEI Quanta 600 FEG, FEI, Oregon). For transmission electron microscope (TEM) analysis, samples were fixed with a mixture of 2% paraformaldehyde, 2% glutaraldehyde, 2% OsO4 in 0.1M sodium phosphate buffer and dehydrated in grade series of ethanol. The samples were then embedded in epoxy resin to cut into 70 nm thickness. The ultrathin sections were observed with a transmission electron microscope (JEOL JEM-1400 Plus, JEOL, Tokyo).
2.6 Cell Viability Assay
Cell viability was measured with live and dead viability/cytotoxicity assay (Thermo Fisher Scientific, Massachusetts). Cell sheet and trypsin treated cell groups were washed twice and incubated with Live/Dead working solution (4 mM ethidium homodimer-1 and 2 mM calcein AM) for 30 minutes at 37° C. in the dark. The samples were washed with and visualized using an AX 10 microscope (Carl Zeiss Microimaging, Gottingen, Germany) and analyzed with Axiovision software (Carl Zeiss Microimaging) (Ex/Em 495/635 ethidium homodimer-1; Ex/Em 495/515 calcein). The number of live and dead cells in single suspension group were counted using image J (National Institutes of Health, Bethesda, Md., USA). The number of dead cells in cell sheet was also counted using image J (National Institutes of Health), whereas live cells in cell sheet was calculated based on the following;
As shown in
2.7 Qualitative Analysis of Proteins Related to Cell Functions
hUC-MSCs (2×105 cells/dish) were cultured for 5 days and harvested by temperature change (cell sheet technology), trypsin treatment (chemical disruption), or cell scraper (physical disruption) (
2.8 Immunocytochemistry Staining of Proteins Related to Cell Functions
Samples were fixed in 4% buffered PFA and then permeabilized with 0.1% triton X-100 (Thermo Fisher Scientific). The samples were blocked with 1% BSA in 10% goat serum for 15 minutes and then incubated in primary antibodies overnight at 4° C.; actin (5 μg/ml), vinculin (1:50 dilution), fibronectin (1:100 dilution), laminin (1:50 dilution), collagen-1 (1:100 dilution), integrin β-1 (1:200 dilution), connexin 43 (1:100 dilution) in the presence of 1% BSA with 10% goat serum. The samples were treated with secondary antibodies for 1 hour. Finally, it was mounted with mounting solution (ProLong Gold Antifade Mountant with DAPI) (Thermo Fisher Scientific) and inspected using IX73 fluorescence microscope (Olympus).
2. 9 Statistical Analysis
All values are expressed as the mean±SEM. Two-way analysis of variance followed by the Tukey's test was used to evaluate differences between more than two groups. Probabilities (p<0.1, 0.05) were considered significant.
Results
Human Umbilical Cord Stem Cell (hUC-MSC) Sheet Preparation
To verify morphologies and growth rates of hUC-MSCs cultured on temperature responsive cell culture dishes (TRCD), hUC-MSCs were seeded at a density of 2×105 cells on conventional tissue culture plates (TCP) or on 35-mm diameter TRCD and were cultured for 5 days. Cells cultured on TRCD have changed its morphology from rounded shape to spindle shape when cells attached to the bottom surface of TRCD. This morphological change was also observed in cells cultured with TCP (
Morphological Observation of hUC-MSC Sheet
The surface and intercellular structures of hUC-MSC sheets were observed by scanning electron microscopy (SEM) (
In TEM analysis, hUC-MSC sheets maintained ECMs (white dotted line) and cell-cell junctions (white solid arrow), which are related to cell adhesion and cell-cell communication (Gattazzo et al., 2014, Biochim Biophys Acta 1840(8): 2506-19). (
hUC-MSC Maintains Actin Filament Proteins Relating with Cell Dynamics
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein expression was detected as a loading control to normalize protein amounts for western blotting assay. GAPDH protein expression level was similar in all groups. Cells treated with 0.50% trypsin for 20 and 60 minutes expressed lower actin than that in cell sheet, 0.05% trypsin, and cell scraper groups (
Vinculin is a membrane cytoskeletal protein that forms focal adhesion by linking integrin family and actin, associated with cell movement (Peng, 2011, Int Rev Cell Mol Biol 287: 191-231). Vinculin expression was observed in both cell sheet and 0.05% trypsin treated groups when stained with immunohistochemistry (
hUC-MSC Sheet Maintains Extracellular Proteins Related with Cell Adhesion
Fibronectin and laminin are important proteins in cell- and tissue-adhesion. Cell sheet, 0.05% or 0.50% trypsin treatment for 5 minutes, and cell scraper groups in western blot assay expressed fibronectin. However, 0.05% and 0.50% trypsin treatment for 20 minutes, and 60 minutes groups had no detectable expression of fibronectin. Laminin expression was observed in cell sheets, 0.05% trypsin treatment, 0.50% trypsin treatment for 5 minutes, and cell scraper groups. However, 0.50% trypsin treatment groups for 20 minutes and 60 minutes showed no detectable laminin expression.
Cells were stained using fibronectin and laminin antibodies to observe the ECM protein structures of ECM proteins (
hUC-MSC Sheet Maintains Cell Junction Proteins Associated with Cell Communication
Integrin β-1 is a major protein of the integrin family of transmembrane membrane proteins that forms cell-ECM junction. It is known that integrin links to the cell's actin cytoskeleton through adapter proteins (e.g. vinculin, talin) and is involved in cell phenotypic preservation, survival, cell adhesion and tissue repair (Moreno-Layseca, 2014, Matrix Biol 34: 144-53. Cell sheet, 0.05% trypsin treatment for 5 minutes and cell scraper groups showed similar Integrin β-1 expression. Integrin β-1 was cleaved gradually as with trypsin concentration and treatment time. Connexin 43 is a transmembrane protein that in gap junctions that facilitates cell-cell communication. Connexin 43 plays essential role in maintaining homeostasis and function of cells and tissues by exchange of biological information (Ribeiro-Rodrigues, 2017, J Cell Sci 130(21): 3619-3630). Connexin 43 was expressed in the cell sheet, 0.05% trypsin treated (5, 20, 60 minutes) and 0.5% trypsin treated (5 minutes) group. However, 0.50% trypsin treatment for 20 and 60 minutes had no expression of Connexin 43. This suggests that Connexin 43 protein was cleaved by 0.50% trypsin when treated for 20 and 60 minutes.
Structural observation of cell junction proteins was performed with Integrin β-1 and Connexin 43 protein was observed by immunostaining. Cell sheet group showed positive expression of Integrin β-1 all uniformly over the cell sheet, whereas Integrin β-1 was expressed slightly on the cell surface in 0.05% and 0.50% trypsin treated groups (
Chemical Disruption Method Induces Cell Death
Cells were stained using calcein and ethidium homodimer-1 immediately after cell detachment by trypsin treatment (chemical disruption) or temperature changes (cell sheet technology). Green color shows live cells and red color shows dead cells in
Apoptotic Cell Death is Activated by Chemical Disruption
Mechanosensor controls for cellular homeostasis convert extracellular physical stimuli to intracellular chemical stimuli (Humphrey, 2014, Nat Rev Mol Cell Biol 15(12): 802-12). Yes-associated protein (YAP) is a major mechanosensor protein localized at cell nuclei to regulate cell survival and proliferation (Jaalouk, 2009, Nat Rev Mol Cell Biol 10(1): 63-73). YAP is inhibited via phosphorylation of Ser127 (phosphor-YAP, pYAP), which results in cytoplasmic retention and induction of cell apoptosis. When cells lose cell-ECM junctions, apoptotic cell death, namely anoikis, is induced subsequently to phosphorylation of YAP (Halder et al. 2012, Nat Rev Mol Cell Biol 13(9): 591-600). YAP and phospho-YAP (pYAP) expression of cell sheets, 0.05% and 0.50% trypsin treatments for 5, 20 and 60 minutes and in the cell scraper group were determined with western blotting (
Discussion
Chemical disruption methods are commonly used to harvest cells from cell culture dishes through disruption of extracellular (Huang et al., 2010, J Biomed Sci 17: 36) and intercellular (Besingi, 2015, Nat Protoc 10(12): 2074-80) proteins associated with the cytoskeleton, cell junctions, cell metabolism, and cell growth. Hence, cells harvested by chemical disruption methods (e.g., trypsin-treated cells) had insufficient ECM which are necessary to adhere to target tissues, and insufficient cell junctions to maintain their normal cellular functions through graft-host communication (
TEM results showed that extracellular protein cleavage was observed in cells treated with 0.05% trypsin for 5 minutes in the chemical disruption group. Cytoplasm cleavage was observed in after 20 minutes of 0.05% trypsin treated cells; and cell nucleus has degraded at 60 minutes of 0.05% trypsin treatment. In addition, endoplasmic reticulum changes related to cell death were observed at 60 minutes of 0.05% trypsin treatment (
Yes-associated protein (YAP) has an important role in regulating cell adhesion, proliferation and survival. It is known that apoptotic cell death is induced through inhibition of YAP and subsequent pYAP induction. Similarly, breakdown of cell-ECM junction induces apoptotic cell death through inhibition of YAP (Codelia, 2012, Cell 150(4): 669-70). When cells were treated with trypsin (chemical disruption), integrin β-1 was cleaved (
This study demonstrates that ECM, cell-cell junction and cell-ECM junction proteins are important in retaining higher cell survival rates. In conventional stem cell therapies that use chemical disruption harvesting methods, it is not possible to avoid low engraftment rate efficiencies and low cell survival rates, since chemical disruption (e.g. trypsin treatment) cleaves cell-cell junctions, cell-ECM junctions and cell adhesion proteins. Cell sheet technology enables cells to be harvested in a viable sheet form without any structural disruption. Furthermore, cell sheet technology maintains important structures for cells (ECMs, cell-ECM junctions, cell-cell junctions, cytoskeleton and mechanosensors) enhancing cell survival rates, engraftment efficiencies and maintaining various critical cellular functions. As a result, cell survival rates in hUC-MSC sheets are significantly higher than for cells harvested with chemical disruption methods.
Conclusion
We demonstrate that retaining tissue-like structure such as ECMs cell-cell junction and cell-ECM junctions are associated with enhanced cell survival rates for of transplanted cells. Cell sheet technology allows harvest of cells in sheet form without using any damaging proteolytic enzymes (chemical disruption). Harvested hUC-MSC sheets that retain tissue-like cell structures, ECMs, cell-cell junctions and cell-ECM junctions had higher cell survival rates, compared to conventional chemical disruption methods (trypsin treatment). This technology will provide not only a higher therapeutic effect of stem cell therapy, but also new concepts for improving cell functions in regenerative medicine research since cell sheets mimic native tissue-like structures.
Cell sheets were prepared from hUC-MSCs by the methods described in Example 1 above, except that the cell culture medium contained either 20% hPL or 20% FBS. The hUC-MSC sheets are shown in
hUC-MSC sheets were cultured in medium containing 20% hPL and implanted within the subcutaneous tissue of immuno-deficient mice as described in Example 1 above, and the hUC-MSC sheets were harvested from the subcutaneous tissue sites for histological observation at 1 day and 10 days after implantation. After harvest, the samples were stained with human growth factor (HGF) antibody for detection of HGF expression, and cell nuclei were stained with DAPI. As shown in
The effect of initial cell density on HGF expression in hUC-MSC sheets was also determined. Cell sheets were prepared from hUC-MSCs in TRCD with an initial cell density of 2×104, 4×104, 6×104, 8×104 or 10×104 cells/cm2 in cell culture medium containing 20% FBS. As shown in
HLA DR, DP, DQ expression was determined in hUC-MSCs in suspension cultures from passage 4 to 12, and in cell sheets prepared from human adipose-derived mesenchymal stem cells (hADSC), human bone marrow-derived mesenchymal stem cells (hBMSC), or hUC-MSCs. Cells were grown in culture media containing 20% hPL. HLA expression was determined as described above in Example 1. As shown in
Human umbilical cord mesenchymal stem cells (hUC-MSCs) were used for making cell sheets as described in Example 1 above using an initial cell density of 3.0×105 in a thermo-responsive cell culture dish (TRCD) (UpCell®, Tokyo, Japan). Images of the cell sheets are provided in
After harvesting, the cell sheet was labeled with a fluorescent marker, carboxyfluorescein succinimidyl ester (CFSE), to allow the cell sheet to be identified after transplantation. Uterine incision was performed to induce scar formation using non-pregnant female nude rats (
Longitudinal incisions were made on each horn of the didelphic rat uterus. After suture repair of both uterine incisions, a hUC-MSC sheet was transplanted to the surface of the hysterotomy repair of one horn of the uterus, while the contralateral horn of the uterus served as a control. In the transplantation group, a hUC-MSC sheet approximately 1 cm2 in surface area was transplanted to the uterus, and the position of the transplanted cell sheet was confirmed by fluorescent microscopy as a green colored area (
Proper positioning and location of the cell sheet was confirmed by fluorescent microscopy and evaluated on days one, three, and seven after surgery. At 1, 3, 7 and 14 days post-surgery, uteri were harvested for macroscopic analysis and histological evaluation by hematoxylin and eosin (HE) staining and Masson-Trichrome staining. The fibrotic (blue) and normal (purple/red) myometrium surface was analyzed using AmScope® software, and the ratio of fibrotic-to-normal myometrial surface area was calculated for each specimen.
Expression of human hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGFA) was measured by quantitative real-time PCR. Scars in both control and cell sheet-transplanted horns were harvested and frozen in liquid nitrogen on day 1, 3, 7 and 14 (n=3 horns per group). RNA was then extracted from each sample using Rneasy® Fibrous Tissue Mini Kit (74704, Qiagen, Germany) after mincing each horn with a mechanical homogenizer. Using a High Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher Scientific), cDNA was synthesized and subjected to PCR analysis (StepOnePlus™ Real-Time PCR System, 4376600, Thermo Fisher Scientific)) using TaqMan® Gene Expression Assays (4331182, Thermo Fisher Scientific) and PCR probes specific to each target gene.
Results
Eighteen uterine specimens from six rats were included in this analysis, 2 or 4 from each horn of the uterus. At 1, 3, and 7 days after transplantation, the presence of the hUC-MSC sheet transplanted to the uterine wound was confirmed by fluorescent microscopy (
In the control group, a large fibrotic area was present between the host myometrium areas as a result of wound healing. In contrast, the fibrotic area in the hUC-MSC sheet transplantation group was significantly smaller than in the control group (
The thickness in incision areas in both control (n=18) and the hUC-MSC sheet transplant group (n=18) were 191.5 μm (range 90.0-296.4 μm) and 274.3 μm (range 143.7-448.4 μm) respectively (p<0.01) (
Gene expression of human hepatocyte growth factor (HGF) and human vascular endothelial growth factor (VEGF) from cell sheet-transplanted uteruses was measured at 1, 3, 7 and 14 days after transplantation. The gene expression of human HGF message and human VEGF message from scarred control uterus were not detected on all days. Human gene expression differences between the cell sheet transplantation and control groups are significant at days 1 and 3 (*p<0.01). Scarred control uterine gene expression was not detectable, and not shown.
Numbers of fibroblast cells in myometrial tissues were detected using immunostaining for S100A4 protein, specific for fibroblasts (Kong, et al., 2013, Am J Physiol Heart Circ Physiol. 305(9): p. H1363-72). The border of the myometrial (dotted line), endometrial lining, and transplanted cell sheet areas (arrows) were identified with HE-stained specimens. S100A4-positive cells were counted in myometrial areas using fibroblast specific protein-specific immunostaining. The mean number of fibroblasts in cell sheet transplanted horns was 483/mm2 (SD: 137/mm2) compared to 716/mm2 (SD: 194/mm2) in control horns (
Gene expression of human hepatocyte growth factor (HGF) and human vascular endothelial growth factor A (VEGFA) were detected on days 1 and 3 and were higher in cell sheet-transplanted horns compared to control horns. However, these genes were not expressed on days 7 and day 14 (
Discussion In an immune-deficient rat hysterotomy model, we demonstrated that the transplantation of human umbilical cord mesenchymal stem cell sheets onto the hysterotomy at the time of surgical repair is feasible, as measured by the development of appropriately sized sheets with natural adhesion and by retention of the stem cells in the target area by immunohistochemistry after several days. We further demonstrated that stem cell sheet transplantation stimulates improved wound healing, as demonstrated by reduced fibroblast recruitment, decreased fibrosis formation, increased postoperative myometrial thickness, and improved ratio of normal myometrium to fibrotic tissue in the area of the hysterotomy.
This study used human umbilical cord-derived stem cells that have been previously used for treating human heart failure as clinically injected cell suspensions. Previous work has shown that these human umbilical cord mesenchymal stem cell sheets cultured in fetal bovine serum (FBS) express low major histocompatibility complex (MHC) class II antigens and maintain low MHC class II antigen expression during cell sheet preparation. These human umbilical cord mesenchymal stem cells express not only normal MHC class I genes, but also non-canonical class I MHC genes (Human Leukocyte Antigen [HLA]-E, HLA-F, and HLA-G) (La Rocca, et al., 2009, Histochem Cell Biol, 131(2): p. 267-82). MHC class I antigens can serve to protect cells from some natural killer cell-induced killing processes. All three non-canonical class I MHC proteins have been reported to be expressed by extravillous trophoblasts, and are associated with maternal tolerance to the semi-allogeneic embryo.
Clinical stem cell preparations are most often administered as injected suspensions. Injected stem cell suspensions exhibit weak, transient short-term cytokine secretion (i.e., less than 3 days) (Elman, et al., 2014, PLoS One, 9(2): p. e89882), and very low tissue engraftment efficiency, decreasing their local effects. However, the cell sheet transplants described herein engrafted locally, spontaneously, rapidly, and efficiently, promoting maximal therapeutic effects at the transplant site without migration. Hence, these human umbilical cord mesenchymal stem cell sheets exhibit reliable phenotypic traits, tissue engagement, and structural features deemed important for human umbilical cord mesenchymal stem cell for host immune histocompatibility, optimal therapeutic and reliable engraftment processes.
This study expands the use of human umbilical cord mesenchymal stem cell sheets to uterine surgical applications, demonstrating feasibility. Cell sheets constructed from these stem cells, in contrast to infused or injected stem cells, retain critical cell adhesive proteins related to therapeutic and engraftment efficacy, including cell-cell junction proteins associated with cell-cell communication and extracellular matrix proteins related to cell adhesion, and cytokine hepatocyte growth factor (HGF) (Kim, et al., 2019, Sci Rep, 2019. 9(1): p. 14415). These cell sheets could be optically tracked for up to 7 days post-transplant (c.f.,
The precise mechanisms for how transplanted stem cell sheets promote myometrial regeneration are uncertain. However, an essential stem cell function is their paracrine effects, through secretion of cytokines and chemokines, including plasminogen activator inhibitor-1 (PAI-1), macrophage migration inhibitory factor (MIF), and interleukin-6 (IL-6), which decrease inflammation, HGF and others to promote regeneration and proliferation of host tissue, and with VEGF, promote angiogenesis. Previous studies have shown that transformation of transplanted stem cells into myometrial tissue is unlikely to be a significant contributor to repair, at least not in the acute phase (Ho, C. H., et al., 2018, J Chin Med Assoc, 81(3): p. 268-276). Further, transplantation of allogenic myometrial cells did not improve hysterotomy wound healing in an animal uterine repair model (Ho et al., 2018, ibid).
Noted reductions in both wound site fibrotic surface area as well fibroblast abundance in this model can be attributed to intrinsic human umbilical cord mesenchymal stem cell sheet anti-inflammatory effects Inflammatory cell mediators (monocytes and macrophages) are attenuated with human umbilical cord mesenchymal stem cell sheet transplantation (La Rocca, et al., 2009, Histochem Cell Biol, 131(2): p. 267-82).
Conclusion
These results demonstrate decreased formation of fibrotic tissue and increased myometrial regeneration following hUC-MSC sheet transplantation onto repaired hysterotomies of rat uteri relative to a control without hUC-MSC sheet transplantation. Accordingly, the hUC-MSC sheets described herein improve healing of the uterine scar and have the potential to decrease morbidities related to abnormal uterine scar formation.
This application claims priority to U.S. Provisional Patent Application No. 62/793,195 filed on Jan. 16, 2019, the contents of which are incorporated herein in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/013622 | 1/15/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62793195 | Jan 2019 | US |
