Use of mesenchymal stem cell sheets for preventing uterine scar formation

Abstract

To provide a method of reducing the formation of fibrotic tissue in a uterus of a subject in need thereof, and a method of increasing myometrial regeneration in a uterus of a subject in need thereof.SOLUTION: A method of reducing the formation of fibrotic tissue in a uterus comprises applying a mesenchymal stem cell (MSC) sheet to a uterus of a subject, where the MSC sheet comprises one or more layers of aggregated confluent mesenchymal stem cells (MSCs), and where applying the MSC sheet to the uterus reduces the formation of fibrotic tissue in the uterus relative to a uterus to which the MSC sheet is not applied.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 793,195, filed January 16, 2019, the contents of which are incorporated herein in their entirety. [Background technology]

[0002] The development of uterine scars after cesarean delivery and other uterine surgery can lead to complications such as uterine rupture and abnormal placentation. These complications can have very serious prognosis for both the mother and the fetus. Clinical studies have reported that cesarean delivery should be performed very promptly (e.g., within 18 minutes) after diagnosis of fetal damage due to uterine rupture to prevent higher incidence of perinatal mortality and morbidity. See Leung et al., 1993, Am J Obstet Gynecol 169: 945-950. The risk of these complications may increase with the number of times cesarean deliveries are performed. In particular, women who have had multiple cesarean deliveries may be at higher risk. Thus, there is a need to prevent potential complications resulting from cesarean delivery and other uterine surgery. Summary of the Invention

[0003] In certain aspects, the present disclosure relates to a method of reducing fibrotic tissue formation in a uterus of a subject in need thereof, comprising applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, the MSC sheet comprising one or more layers of aggregated, confluent mesenchymal stem cells (MSCs), wherein applying the MSC sheet to the uterus reduces fibrotic tissue formation in the uterus compared to a uterus to which the MSC sheet is not applied.

[0004] 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, the MSC sheet comprising one or more layers of aggregated, confluent mesenchymal stem cells (MSCs), wherein application of the MSC sheet to the uterus increases myometrial regeneration as compared to a uterus to which the MSC sheet is not applied.

[0005] In certain aspects, the present disclosure relates to a method of preventing or reducing uterine incision rupture and abnormal placentation in a subject in need thereof, comprising applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, the MSC sheet comprising one or more layers of aggregated confluent mesenchymal stem cells (MSCs), wherein application of the MSC sheet to the uterus prevents or reduces uterine incision rupture and abnormal placentation as compared to a uterine incision to which the MSC sheet is not applied.

[0006] In some embodiments, an MSC sheet is applied to the incision site of the uterus. In some embodiments, applying an MSC sheet to the uterus reduces the fibrotic surface area of ​​the uterus by at least 20% compared to a uterus to which an 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 compared to a cultured, isolated MSC suspension containing a comparable number of cells. In some embodiments, the cell sheet expresses the cytokine for at least 10 days following implantation into a tissue of a host organism.

[0007] In some embodiments, the cell sheet expresses extracellular matrix proteins and cell junction proteins for at least 10 days after implantation into a tissue of 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, the initial cell density of the MSCs in the cell culture support used to prepare the cell sheet is 0.5×10 4 / cm2 From 9×10 5 / cm 2 In some embodiments, the MSCs do not express the 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 the tissue of the host organism for at least 10 days following transplantation into the tissue.

[0008] In some embodiments, the MSCs in the cell sheet are allogeneic to the subject. In some embodiments, the subject is 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 MSCs are human umbilical cord mesenchymal stem cells (hUC-MSCs). [Brief description of the drawings]

[0009] [Figure 1] Figure 1 shows the cell sheet experimental protocol. Human umbilical cord stem cells (hUC-MSCs) were seeded on a temperature-responsive cell culture dish (TRCD) and cultured in a cell culture incubator at 37°C until confluent. The cultured cells were detached from the TRCD as an intact cell sheet within 20 min at room temperature (RT) without the use of proteolytic enzyme treatment, preserving essential features including the extracellular matrix (ECM) and cell-cell junctions. [Figure 2A-B]Figures 2A-B show morphological observations of hUC-MSC sheets using cell passages 4, 6, 8, 10 and 12 seeded at 2 x 104 cells / cm2. (a) Morphology of passage 4, 6, 8, 10 and 12 cells observed using phase contrast microscopy before sheet detachment. (b) Successful hUC-MSC sheet fabrication using passage 4, 6, 8 and 10 cells. In contrast, passage 12 cells detached as non-contiguous disconnected cell structures. Scale bar = 100 μm. [Figure 3A-C] Figures 3A-C show morphological observations, cell proliferation rates and cell sheet fabrication for hUC-MSC seeded at initial cell numbers of 2x105, 1x105 and 5x104 on a 35 mm diameter TRCD with a surface area of ​​9.6 cm2. (a) Cells cultured on a TRCD and observed prior to sheet detachment. (b) Cell numbers counted using a hemocytometer after cells were seeded on a TRCD until a hUC-MSC sheet was observed. (c) Cells detached as disconnected fragments one day before confluence, on days 3, 4 and 5 for the 2x105, 1x105 and 5x104 initial cell seeding groups, respectively. Intact cell sheets were successfully fabricated on days 4, 5 and 6 for the seeding densities of 2x105, 1x105 and 5x104 initial cell numbers groups, respectively. One day after confluence, the cultured cells spontaneously detach as aggregated fragments without TRCD temperature change on days 5, 6, and 7 for initial cell seeding groups of 2 x 105, 1 x 105, and 5 x 104, respectively. The scale bar indicates 100 μm in (a). The scale bar indicates 1 cm in (c). [Figure 4A-D] 4A to 4D show positive expression of CD44 and CD90 in hUC-MSCs in cell suspension cultures (A and B) and in vitro hUC-MSC sheets (C and D). [Figure 5A-E]Figures 5A-5E show cell-cell structure analysis using immunohistochemistry (IHC) and transmission electron microscopy (TEM). The cultured cells were successfully detached as sheets from the TRCD by temperature shift on the 4th day after seeding. The cell sheets were stained with antibodies for ECM ((a) fibronectin and (b) laminin) and (c) cell junction β-catenin, respectively (bright areas, 5A-5C), to confirm that the cell sheets preserved their functional structures after detachment. In the TEM images, the hUC-MSC sheets preserved their (d) ECM and (e) cell-cell junction structures after detachment. In the hUC-MSC sheets, 5D arrow = ECM; 5E arrow = cell junction. The scale bars indicate 100 μm in (a-c). The scale bars (d) and (e) indicate 5 μm and 1 μm, respectively. [Figure 6A-D] Figures 6A-6D show cytokine analysis of human hepatocyte growth factor (HGF) and tumor necrosis factor-alpha (TNF-α) secreted from hUC-MSC sheets. hHGF (anti-inflammatory cytokine) and hTNF-α (pro-inflammatory cytokine) were detected in the culture supernatant of cells cultured for 24 hours. (a) No significant difference in hHGF secretion in the initial cell seeding groups of 2×105, 1×105 and 5×104. (b) hTNF-α was barely detectable and was not significantly different in the initial cell seeding groups of 2×105, 1×105 and 5×104. (c) Significant reduction in hHGF secreted from hUC-MSC sheets with increasing passage. (d) hUC-MSC sheets fabricated using passage 4 cells secreted significantly less hTNF-α compared to hUC-MSC sheets fabricated using passage 6, 8, 10 and 12 cells. *p<0.05. [Figure 7A-E]7A-7E show the retention of implanted hUC-MSC sheets in vivo. hUC-MSC sheets implanted in the subcutaneous tissue of immunodeficient mice. (c and d) On the 10th day after implantation, the subcutaneous tissue sites implanted with hUC-MSCs were harvested for histological observation. In the H&E staining images, (b) the hUC-MSC cell sheet was clearly confirmed in the subcutaneous tissue implantation site compared with (a) the normal subcutaneous tissue (control). Moreover, abundant vascular structures were observed in (e) the cell sheet implantation group. Arrows in (b) = implanted cell sheet; arrows in (e) = blood vessels. Scale bars (a and b) and (e) indicate 100 μm and 50 μm, respectively. Scale bars (c and d) indicate 0.5 cm. [Figure 8A-B] 8A to 8B show the expression levels of intercellular junction-related genes in hUC-MSC sheets. The gene expression levels of (a) integrin-linked protein kinase (ILK) and (b) N-cadherin (Ncad), which are related to cell junctions, at passage 12 were lower than those at passage 6. [Figure 9A-C] Figures 9A-C show a schematic of the cell harvesting process. Human umbilical cord mesenchymal stem cells (hUC-MSCs) were seeded on 35 mm temperature-responsive cell culture dishes (TRCDs) or tissue culture plates (TCPs) and cultured for 5 days until confluence was reached. hUC-MSCs were harvested using three different methods representing cell sheet technology, chemical disruption, and physical disruption: A) cell sheets were harvested in a seamless and intact state by temperature shift, B) cells were treated with enzymatic proteolysis (trypsin) to produce single cell suspensions and cell clumps, and C) cells were physically harvested using a cell scraper to produce heterogeneous multicellular fragments and clumps. [Figure 10A-D]10A-10D show the preparation of human umbilical cord mesenchymal stem cell (hUC-MSC) sheets. (A) Cells were cultured in conventional tissue culture plates (TCP) or temperature-responsive cell culture dishes (TRCD) for 5 days. A phase contrast microscope was used to observe the cell morphology cultured in TCP and TRCD. (B) The cell numbers when cultured in TCP or TRCD for 100 hours were counted using a hemocytometer. (C) The cells cultured in TRCD were detached in the form of a sheet by temperature reduction. (D) Histological analysis of the harvested cell sheets was performed by H&E staining to show individual cell bodies and nuclei in the form of a sheet. The scale bar indicates 200 μm in A and D and 10 mm in C. [Figure 11A-H] Figure 11A-H distinguish different morphological observations of hUC-MSC cells harvested by trypsin proteolysis and hUC-MSC sheets harvested using temperature shift without enzymes. (A) Morphology of trypsinized MSC surface observed using scanning electron microscopy (SEM). (B) Microstructure of temperature harvested hUC-MSC sheets and trypsinized hUC-MSC analyzed using transmission electron microscopy (TEM). White arrows in (E) point to intact MSC sheet cell junctions and dark grey arrows in (H) point to intact hUC-MSC sheet ECM. Scale bars in SEM and TEM = 5 μm. [Figure 12A-C]12A-C show cell kinetics-related protein expression analysis using Western blot and immunohistochemistry for temperature-harvested hUC-MSC cell sheets compared to trypsinized hUC-MSCs. (A) Western blot (whole cell lysate, 10 mg protein / lane) of F-actin, vinculin and GAPDH in intact cell sheets (left lane) compared to trypsinized cells. (B) Immunostaining fluorescent imaging comparison of intact hUC-MSC cell sheets (left image) compared to trypsinized hUC-MSC harvested (right lane) for F-actin cytoskeleton, (C) vinculin and nuclear DAPI (bright punctate spots). Scale bar = 10 μm. [Figure 13A-C] Figures 13A-C show ECM protein expression comparison using Western blot and immunohistochemical fluorescence imaging of intact hUC-MSC cell sheets (left image) compared to trypsinized hUC-MSC harvests (right image). (A) Western blot of fibronectin, laminin and GAPDH in whole cell lysates (10 mg protein / lane). (B) Immunostaining of fibronectin, (C) laminin and nucelar DAPI (bright spots). Scale bar = 10 μm. [Figure 14A-C] Figure 14A-C show cell-ECM and cell-cell junction protein expression comparison using Western blot and immunohistochemical fluorescent imaging comparison of intact hUC-MSC cell sheets (left image) compared to trypsinized hUC-MSC harvest (right lane). (A) Western blot of integrin β-1, connexin 43 and GAPDH in whole cell lysates (10 mg protein / lane). (B) Immunostained images of integrin β-1, (C) connexin 43 and DAPI (bright spots). Scale bar = 10 μm. [Figure 15A-C]Figures 15A-C compare live and dead cells in intact hUC-MSC cell sheets and trypsinized hUC-MSC suspensions using a dye-based assay: (A) Microscopic images of live and dead staining of cell sheets (left lane) and trypsinized cell suspensions (right lane). Cells were stained with calcein and ethidium homodimer-1 immediately after cell detachment. Scale bar = 100 μm; (B) Quantification of live and dead cells for cell sheets compared to trypsinized hUC-MSCs from image analysis; (C) Live / dead cell quantification data for the graph shown in (B). [Figure 16] Figure 16 compares protein mechanosensor expression analysis using Western blot of hUC-MSC cell sheets and trypsinized hUC-MSC suspensions. Western blot of Yes-associated protein (YAP), phosphorylated YAP and GAPDH in whole cell lysates (10 μg protein / lane). [Figure 17] Figure 17 shows hUC-MSC sheets prepared in culture medium containing human platelet lysate (hPL) (left) or fetal bovine serum (FBS) (right). Rulers shown are in cm. [Figure 18A-B] 18A-18B show immunostaining fluorescence imaging of in vivo HGF expression in a hUC-MSC sheet implanted in the subcutaneous tissue of an immunodeficient mouse. On day 1 (A) and day 10 (B) after implantation, the subcutaneous tissue site where the MSC sheet was implanted was harvested for histological observation. To detect human HGF expression from the hUC-MSC sheet, the samples were stained with an anti-human HGF antibody, and the cell nuclei were stained with DAPI. [Figure 19A-B] 19A-B show hUC-MSC sheets generated in TRCD at initial cell densities of 2×104, 4×104, 6×104, 8×104, or 10×104 cells / cm2 in cell culture medium containing 20% ​​FBS (A). Increasing initial cell density increased HGF gene expression in a concentration-dependent manner (B). [Figure 20A-B]Figures 20A-B show HLA DR, DP, and DQ expression in hUC-MSC single cell suspension cultures (A) and in harvested MSC cell sheets (B) as a function of passage number. HLA expression was measured in single cell suspension cultures from passage 4 to 12 (A). The percentages in (A) represent the percentage of cells expressing HLA. HLA-DR gene expression was undetectable in hUC-MSC sheets, while cell sheets similarly prepared from human adipose-derived stem cells (hADSCs) or human bone marrow-derived mesenchymal stem cells (hBMSCs) showed relatively high levels of HLA-DR gene expression in comparison (B). [Figure 21] FIG. 21 shows the sutured uterus in a nude rat model of uterine scar development prior to transplantation of hUC-MSC sheets. [Figure 22] Figure 22 shows the sutured uterus in a nude rat model of uterine scar development before and after implantation of a fluorescently labeled hUC-MSC sheet (top right image) into the rat uterus. A bright in vivo microscopy image (bottom right image) indicates the fluorescent cell sheet in situ in the rat uterus after suturing and sheet implantation. [Figure 23] Figure 23 shows nude rat uterus harvested 1, 3, 7 or 14 days after fluorescently labeled hUC-MSC sheet implantation. Bright images in / around the uterus indicate signal from the retained hUC-MSC fluorescent cell sheet. [Figure 24] Figure 24 shows a histological comparison of a control (no cell treatment, suture only) uterine section and a uterine section 14 days after hUC-MSC sheet implantation. The control uterine horn cross section displays increased fibrotic areas by dye staining compared to the cell sheet uterine horn implantation group. [Diagram 25] Figure 25 compares fibrotic areas to myometrial areas assessed between control and hUC-MSC-implanted corneas of nude rat uterus 14 days after sheet implantation. Six rats and a total of 18 histological specimens were assessed for fibrotic scar area from histological dye staining (following Figure 24). [Figure 26]Figure 26 compares the fibrous to myometrial area ratios assessed between control and hUC-MSC-implanted corneas of nude rat uterus 14 days after sheet implantation. Six rats and a total of 18 histological specimens were assessed for fibrous scar area from histological dye staining (following Figures 24 and 25). [Figure 27] Figure 27 compares scar (control) and thickness (microns) in the cell sheet implanted area between control and hUC-MSC sheet implanted corners of nude rat uterus 14 days after implantation. Six rats and a total of 18 histological specimens were assessed for fibrous thickness from histological dye stained samples. [Fig. 28A-B] Figure 28A-B shows the nude rat uterine scar model and the hUC-MSC cell sheet transplantation procedure after sheet fluorescent dye staining. (a) Schematic diagram of the cell sheet transplantation process (see i-vi): i) surgically expose the uterine horns, ii) open both uterine cavities, iii) close the wound, iv) cartoon of harvested dye-labeled human stem cell sheet and corresponding actual bright field sheet micrograph image below, v) cartoon of cell sheet stained by green fluorescent dye and corresponding actual fluorescent sheet micrograph image below, vi) transplantation of the labeled cell sheet into only the rat left uterine horn (right horn control). (b) Macroscopic in vivo photographs of the hUC-MSC cell sheet transplantation process in situ in nude rats. ii) (corresponding to element ii in the schematic diagram) Surgically opened uterine cavity, dashed line indicates the endometrial surface; iii) Uterine wound closure, black arrow indicates the sutured uterine site; vi) Implantation of fluorescently stained hUC-MSC sheet, white arrow indicates the implanted cell sheet at the uterine suture line. [Figure 29A-C]29A-C show hUC-MSC cell sheet fabrication. (a) Top-down microscopy images morphology of hUC-MSC in culture. Left column: 1 day after seeding; right column: 5 days after seeding, cells are confluent on the culture surface. Scale bar = 200 μm in upper microscopy image and 500 μm in lower microscopy image, (b) Macroscopic observation of harvested cell sheet. After reducing the culture temperature to 20°C, the cell sheet harvests spontaneously and naturally. Scale = 1 mm. (c) Histologically stained cross-sectional images of released hUC-MSC cell sheet. Scale bar = 200 μm (left) and 50 μm (right); right micrograph points to the inset box enlarged from the left image to show individual stem cell nuclei (dark) and connected cell bodies with expressed cell-cell junctions and extracellular matrix. [Fig. 30A-B] Figure 30A-B show the tracking of fluorescently stained hUC-MSC sheets. (a) Macroscopic observation of nude rat uterus imaged using in situ fluorescence microscopy and bright field microscopy (top and second rows, respectively). Horizontal cross-sectional fluorescence image of uterine horn (third row). Histological imaging of cell sheet fluorescence (bottom row). Scale bar = 100 μm. Bright fluorescent signals (arrows) from cell sheets confirmed in situ 1 and 3 days after rodent horn implantation. Small fluorescently stained fragments (arrows) were confirmed 7 days after implantation. No stained cells were observed 14 days after implantation. (b) Gene expression of human hepatocyte growth factor (HGF) and human vascular endothelial growth factor (VEGF) from cell sheet-implanted uterus on days 1, 3, 7, and 14 after implantation. Gene expression of human HGF message and human VEGF message from scar control uterus (without cell sheet) was not detected on all days. The difference in human gene expression between cell sheet implanted and control groups is significant (*p<0.01) on days 1 and 3. Scar control uterine gene expression was undetectable and data not shown. [Fig. 31A-E]Figure 31A-E show the evaluation of fibroblast numbers in rat uterine horn tissue samples 3 days after transplantation (a-d). Cross-sectional HE stained histological microscopic images (a, b) and fibroblast-specific immunohistochemically stained S100A4 protein images (c, d) of rat uterine horns. Scale bar = 500 μm. The dark dashed lines in the histological images indicate the myometrium of the rat uterus. The black arrows indicate the transplanted human umbilical cord mesenchymal stem cell sheets. The S100A4 positive stained cells (fibroblasts) in the uterine scar control group show a higher abundance than the cell sheet transplant group. (e) Graph comparing the number of S100A4 positive cells in rat uterine scar control and hUC-MSC sheet uterus transplant groups. The S100A4 positive cells in the scar control group are significantly more abundant than the hUC-MSC sheet transplant group (*p<0.05, n=18). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] This disclosure describes the reduction of uterine scar formation and the increase of myometrial regeneration resulting from the transplantation of mesenchymal stem cell (MSC) sheets. For example, this disclosure describes the reduction of fibrous tissue formation and the increase of myometrial regeneration after transplantation of human umbilical cord mesenchymal stem cell (hUC-MSC) sheets into sutured incision sites of the uterus, showing that application of hUC-MSC sheets reduced uterine scar formation. In the control group, large fibrous areas were present between host myometrial areas as a result of wound healing. In contrast, the fibrous areas in the hUC-MSC sheet transplantation group were significantly smaller than in the control group. Specifically, application of hUC-MSC sheets to the uterus reduced the fibrous surface area of ​​the uterus by approximately 27% compared to the control group, and reduced the ratio of fibrous myometrial surface to normal myometrial surface by more than 33% compared to the control group. Thus, transplantation of hUC-MSC sheets significantly reduced fibrosis of the myometrial surface. Thus, the hUC-MSC sheets described herein have the potential to improve uterine scar healing and reduce morbidity associated with abnormal uterine scar formation.

[0011] This disclosure also describes methods for 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 were formed 4-6 days after seeding and detached from the TRCD by cooling the culture to room temperature. Various culture conditions were identified that allowed for successful generation of robust, uniform hUC-MSC sheets containing one or more layers of aggregated, confluent cells. These culture conditions included optimization of the number of subcultures (passages) before adding cells to the TRCD, the initial cell density in the TRCD, the addition of cell growth factors such as human platelet lysate (hPL) to the cell culture solution, and the culture time in the TRCD before detachment from the temperature-responsive polymer.

[0012] 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, umbilical cord blood, limb bud, bone marrow, dental tissue (e.g., molars), adipose tissue, muscle, and amniotic fluid.

[0013] In certain embodiments, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells. As used herein, the term "human umbilical cord mesenchymal stem cells" or "hUC-MSCs" refers to mesenchymal stem cells isolated from human umbilical cord.

[0014] Mesenchymal stem cells (MSCs) have remarkable clinical potential to treat a wide range of debilitating diseases, primarily 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 thus provides an easily accessible and uncontroversial source of stem cells for therapy (El Omar et al., 2014, Tissue Eng Part B Rev 20(5): 523-544). hUC-MSCs have been tested for safety and efficacy as suspensions in human clinical trials (Bartolucci et al., 2017, Circ Res, 121(10), 1192-1204). Furthermore, hUC-MSCs have been used successfully in experimental animal disease models (Zhang et al., 2017, Cytotherapy 19(2): 194-199).

[0015] Methods for isolating MSCs from umbilical cord are known in the art and described, for example, in U.S. Patent No. 9,903,176, which is incorporated herein by reference in its entirety. Human umbilical cord includes umbilical artery, umbilical vein, Wharton's jelly, and subepithelial layer. In some embodiments, hUC-MSCs are isolated from the subepithelial layer of human umbilical cord. In some embodiments, hUC-MSCs are isolated from the Wharton's jelly of human umbilical cord. Various cell markers can be used to identify hUC-MSCs isolated from the subepithelial layer. For example, in some embodiments, 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 certain embodiments, 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 certain embodiments, 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).

[0016] hUC-MSCs in the umbilical cord are surrounded by an extracellular matrix (ECM) and are connected to other types of umbilical cord cells (e.g., endothelial cells, epithelial cells, muscle cells, and fibroblasts) by intercellular 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 but not to other types of umbilical cord cells. The hUC-MSC sheets described herein also differ in several ways from hUC-MSC suspension cultures. Suspension cultures of hUC-MSCs comprise single cells that lack ECM or intercellular junctions, since their cell adhesion proteins in their intercellular junctions must be removed (e.g., by proteolytic trypsinization) from culture surfaces commonly used to prepare cell suspension cultures in order to harvest and suspend the cells. In contrast to single cell suspensions of hUC-MSCs, the hUC-MSC sheets described herein contain endogenous ECM produced by the cells and intact cell-cell junctions between the hUC-MSCs that are generated during the formation of the cell sheet. The endogenous ECM and intrinsic cell-cell junctions that are maintained during the formation, fabrication and handling of the cell sheet facilitate the preservation of their phenotype during transplantation into the host organism, important properties for cell function and adhesion of the hUC-MSC sheet to the target tissue.

[0017] II. Cell sheets generated 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). As used herein, the term "mesenchymal stem cell sheet" or "MSC sheet" 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 continuous. 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 intact sheets by temperature shift using a temperature-responsive culture dish (TRCD) without any enzymatic treatment. 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 cell function. Thus, the cell sheets described herein can include structural features that improve cell survival and cell function, including natural extracellular matrix, cell adhesion proteins, and cell junction proteins. Thus, the MSC sheets prepared by the methods described herein have several beneficial features compared to MSCs generated by other methods. For example, chemical disruption methods cannot maintain the tissue-like structure of cells and cell-cell communication because enzymatic treatment destroys extracellular and intracellular proteins (junctions between cells and between cells and ECM). Thus, protein cleavage by enzymes reduces cell viability and cell function. Physical disruption (i.e., by rubber policeman or medium aspiration) results in the destruction of cell-cell junctions and the collapse of the cultured adherent sheets into cell aggregates.

[0018] In some embodiments, the extracellular matrix comprises one or more proteins selected from the group consisting of fibronectin, laminin, and collagen, hi some embodiments, the cell junction protein is selected from the group consisting of vinculin, integrin-β1, connexin 43, β-catenin, integrin-linked kinase, and N-cadherin.

[0019] MSCs in cell sheets can also maintain additional structural features, such as microvilli and filopodia. Microvilli are cell membrane protrusions that are involved in a wide variety of cell functions, including absorption, secretion, and cell adhesion. Filopodia are cytoplasmic protrusions that play a role in cell-cell interactions. Thus, the maintenance of these structural features can also help maintain cell functions and signaling that are beneficial for their application.

[0020] 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 the cells in the cell sheet are MSCs. In some embodiments, 100% of the cells in the cell sheet are MSCs.

[0021] To optimize the formation or characteristics of the cell sheet, MSCs can be added to the culture medium on the temperature-responsive polymer in the cell culture support at various cell densities. For example, cytokine expression levels in MSCs can be optimized by controlling the initial cell density of MSCs in the cell culture support (e.g., TRCD). In some embodiments, increasing the initial cell density of MSCs in the cell culture support increases cytokine expression (e.g., HGF). In some embodiments, increasing the initial cell density of MSCs in the cell culture support decreases cytokine expression. In some embodiments, the initial cell density of MSCs in the cell culture support used for the preparation of cell sheets is 0.5×10 4 / cm 2 From 9×10 5 / cm 2 In some embodiments, the initial cell density of the MSCs in the cell culture support is at least 0.5×10 4 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×104 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 Or 9 x 10 5 Cells / cm 2 Any of these values ​​can be used to define a range of initial cell densities of MSCs in the cell culture support. For example, in some embodiments, the initial cell density in the cell culture support is 2×10 4 From 1×10 5 Cells / cm 2 , 4×10 4 From 1×10 5 Cells / cm 2 , or 1 × 10 4 From 5×10 4 Cells / cm 2 It is.

[0022] The MSC sheet described herein can be transplanted into a target tissue of a host organism (e.g., human) for therapeutic use. The transplantation of the MSC sheet into the target tissue can promote the formation of new capillaries in the host tissue (angiogenesis) as well as blood vessel formation between the transplanted cell sheet and the host tissue. This new capillary formation is a key function for engraftment, viability, and tissue regeneration. Furthermore, this new blood vessel recruitment to the sheet on the target tissue suggests that the implanted MSC sheet continuously secretes paracrine factors to modulate this engraftment.

[0023] In some embodiments, the MSC sheet expresses one or more cytokines, such as one or more anti-inflammatory cytokines and / or one or more inflammatory cytokines. In some embodiments, the anti-inflammatory cytokines are 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., anti-inflammatory cytokines or inflammatory cytokines) in the cell sheet is increased compared to an MSC suspension containing a comparable number of cells. In some embodiments, cytokine expression (e.g., anti-inflammatory cytokines or inflammatory cytokines) is decreased compared to an MSC suspension containing a comparable number of cells. For some therapeutic uses, it may be beneficial to reduce secretion of inflammatory cytokines by the cell sheet. For example, in certain embodiments, the cell sheets secrete tumor necrosis factor-α (TNF-α) into culture medium at a rate of less than 100, 90, 80, 70, 60, 50, 40, or 30 pg / mL of culture medium per 24 hours in vitro.

[0024] The MSC sheet described herein can continuously express cytokines after transplantation into target tissue of a host organism. In some embodiments, the cell sheet expresses cytokines for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 days after transplantation into tissue of a host organism. In some embodiments, the cell sheet expresses cytokines for at least 1, 2, 3, 4, 5 or 6 months after transplantation into tissue of a host organism.

[0025] The MSC sheets described herein can also continuously express extracellular matrix proteins and cell junction proteins after transplantation into a target tissue of a host organism. For example, in some embodiments, the cell sheets express 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 into a tissue of a host organism. In some embodiments, the cell sheets express extracellular matrix proteins and / or cell junction proteins for at least 1, 2, 3, 4, 5, or 6 months after transplantation into a tissue of a host organism. In some embodiments, the extracellular matrix proteins expressed in the cell sheets after transplantation are selected from fibronectin, laminin, and collagen. In some embodiments, the cell junction proteins expressed in the cell sheets after transplantation are selected from vinculin, integrin-β1, connexin 43, β-catenin, integrin-linked kinase, and N-cadherin.

[0026] 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 generally show poorer engraftment and retention in 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 due to enzymatic destruction during harvesting may impair stem cell function, engraftment, and survival in vivo, limiting therapeutic efficacy in vivo. In contrast, the method for preparing MSC sheets described herein preserves the intrinsic cell functional structure and improves the attachment of cell sheets to target tissues after transplantation. For example, in some embodiments, the cell sheet remains attached to the target tissue of the host organism for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days after implantation into the tissue of the host organism. In some embodiments, the cell sheet remains attached to the target tissue of the host organism for at least 1, 2, 3, 4, 5, or 6 months after implantation into the tissue of the host organism.

[0027] Human leukocyte antigens (HLA) are cell surface proteins that constitute the major histocompatibility complex (MHC) proteins in humans and play a role in regulating the immune system. HLA markers are important for controlling tissue transplantation and host acceptance. HLA (DP, DM, DO, DQ and DR) corresponding to MHC class II present antigens from the cell surface to host T lymphocytes to modulate host recognition as "self". These antigens are expressed by helper T cells (CD4 +HLA stimulates proliferation of HLA-specific T cells, which in turn stimulates antibody-producing B cells to generate antibodies against their specific antigens. Therefore, minimizing the expression of HLA is beneficial to minimize the host immune response to the transplanted hUC-MSC sheet in the 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).

[0028] III. Methods for generating MSC sheets in vitro In certain aspects, the present disclosure provides a method for producing a cell sheet comprising one or more layers of aggregated, confluent mesenchymal stem cells (MSCs), comprising: a) culturing MSCs in a culture medium on a temperature-responsive polymer surface, which is typically coated as a thin film on the substrate surface of a cell culture support, the temperature-responsive polymer having a lower critical solution temperature in water of 0 to 80°C; b) adjusting the temperature of the culture medium below the lower critical solution temperature of the polymer, thereby rendering the substrate surface hydrophilic and weakening the adhesion of the cell sheet to the surface; and c) Detaching the cell sheet from the culture support The present invention relates to a method comprising the steps of:

[0029] 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. Patent No. 9,803,176, which is incorporated herein by reference in its entirety. For example, hUC-MSCs can be isolated from the subepithelial layer of the umbilical cord by washing the cord to remove blood, Wharton's jelly, and any other material, and dissecting the subepithelial layer (SL) from the cord. The umbilical 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 may contain platelet lysate (i.e., 10% PRP lysate of platelet lysate). The SLs may then be placed medially down on the substrate. The entire dissected umbilical cord from which the Wharton's jelly has been removed may be placed directly on the substrate, or the dissected cord may be cut into smaller pieces (e.g., 1-3 mm) and these pieces may be placed directly on the substrate. The substrate may be a solid polymeric material such as a cell culture dish. The SLs may be placed on top of the substrate in a cell culture dish without additional pretreatment to the cell culture treated plastic, or may be placed on top of a semi-solid culture medium such as agar. After placing the SLs on the substrate, the SLs are cultured in a suitable medium (e.g., Dulbecco's Modified Eagle Medium (DMEM) without phenol red, glucose (500-6000 mg / mL), 1x glutamine, 1x NEAA, and 0.1-20% PRP lysate or platelet lysate)). The culture may then be cultured under normoxic or hypoxic culture conditions for a sufficient time (e.g., 3-7 days) to establish a primary cell culture. After the primary cell culture is established, the SL tissue is removed and discarded. The cells or stem cells are further cultured and expanded in larger culture flasks under normoxic or hypoxic culture conditions.

[0030] General methods for preparing cell sheets are known in the art and are described, for example, in U.S. Patent Nos. 8,642,338, 8,889,417, 9,981,064, and 9,114,192, each of which is incorporated herein by reference in its entirety.

[0031] The temperature responsive polymers used to coat the substrate of the cell culture support have a higher or lower critical solution temperature in aqueous solution, which is generally within the range of 0°C to 80°C, e.g., 10°C to 50°C, 0°C to 50°C, or 20°C to 45°C.

[0032] The temperature-responsive polymers may be homopolymers or copolymers. Exemplary polymers are described, for example, in JP 211865 / 1990. Specifically, they can be obtained by homopolymerization or copolymerization of monomers, such as (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 above-mentioned monomers, can be used. Furthermore, the monomers can be copolymerized with other monomers, one polymer can be grafted to another, two polymers can be copolymerized, or a mixture of polymers and copolymers can be used. If desired, the polymers can be crosslinked to an extent that does not impair their inherent properties.

[0033] The substrate to be coated with the polymer can be of any type commonly used in cell culture, including, for example, glass, modified glass, silicon oxide, polystyrene, poly(methyl methacrylate), polyester, polycarbonate, and ceramic.

[0034] Methods for coating a support with a temperature-responsive polymer are known in the art and are described, for example, in JP 211865 / 1990 A. 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 gamma rays, irradiation with ultraviolet rays, plasma treatment, corona treatment, or organic polymerization reaction.

[0035] Temperature-responsive polymer coverage is 0.4-3.0μg / cm 2, for example, 0.7 to 2.8 μg / cm 2 or 0.9 to 2.5 μg / cm 2 The form of the cell culture support may be, for example, a dish, a multiplate, a flask or a cell insert.

[0036] The cultured cells can be detached and harvested from the cell culture support by adjusting the temperature of the support to a temperature at which the polymer on the support substrate is hydrated, thereby allowing the cells to detach. A smooth detachment can be achieved by applying a water flow to the gap between the cell sheet and the support. The detachment of the cell sheet can be effected in the culture medium in which the cells were cultured or in another isotonic liquid, whichever is suitable.

[0037] In a particular embodiment, the temperature-responsive polymer is poly(N-isopropylacrylamide). Poly(N-isopropylacrylamide) has a lower critical solution temperature in water at 31°C. When it is in a free state, it will dehydrate in water at a temperature above 31°C, and the polymer chains will aggregate and cause turbidity. Conversely, at temperatures below 31°C, the polymer chains will hydrate and dissolve in water, thereby causing the release of the cell sheet from the polymer. In a particular embodiment, the polymer covers the surface of a substrate, such as a Petri dish, and is immobilized thereon. Thus, at temperatures above 31°C, the polymer on the substrate surface will also dehydrate, but the polymer chains will cover and immobilize the substrate surface, making the substrate surface hydrophobic. Conversely, at temperatures below 31°C, the polymer on the substrate surface will hydrate, but the polymer chains will cover and immobilize the substrate surface, making the substrate surface hydrophilic. Hydrophobic surfaces are suitable surfaces for cell attachment and proliferation, whereas hydrophilic surfaces inhibit cell attachment and cells are detached simply by cooling the culture medium.

[0038] Culture media for mesenchymal stem cells are known in the art and are described, for example, in U.S. Patent Nos. 9,803,176 and 9,782,439, each of which is incorporated herein by reference in its entirety. In some embodiments, the cell culture medium comprises human platelet lysate (hPL). In some embodiments, the culture medium comprises fetal bovine serum (FBS). In some embodiments, the culture medium comprises ascorbic acid. In some embodiments, the culture medium is a xeno-free medium, i.e., a medium that may contain products derived from humans but does not contain products derived from non-human animals. In some embodiments, the culture medium contains at least one product derived from a non-human animal (e.g., FBS). In some embodiments, the culture medium does not contain products derived from humans. In certain embodiments, the culture medium 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 acid solution (NEAA) (Life Technologies) and antibiotics, such as penicillin, streptomycin.

[0039] MSCs (e.g., hUC-MSCs) can be subcultured (i.e., passaged) one or more times before culturing the cells in a culture medium on the temperature-responsive polymer coated on the substrate surface of the cell culture support. In some embodiments, MSCs (e.g., hUC-MSCs) are subcultured 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times before culturing the cells in a culture medium on the temperature-responsive polymer coated on the substrate surface of the cell culture support. Any of these values ​​can be used to define the range of the number of subcultures. For example, in some embodiments, MSCs (e.g., hUC-MSCs) are subcultured 2 to 10 times, 4 to 8 times, or 1 to 12 times before culturing the cells on the 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 times. 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 times.

[0040] MSC sheets can be prepared in a range of different sizes depending on the application. In some embodiments, the MSC sheets have a diameter of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 cm. Any of these values ​​can be used to define a range of sizes for the MSC sheets. For example, in some embodiments, the MSC sheets have a diameter of 1 to 20 cm, 1 to 10 cm, or 2 to 10 cm. In some embodiments, the MSC sheets have a diameter 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 cm. 2 Any of these values ​​can be used to define a range of sizes for the MSC sheet. For example, in some embodiments, the MSC sheet has an area of ​​1 to 100 cm 2 , 3 to 70 cm 2 Or 1 to 300 cm 2The methods described herein result in hUC-MSC sheets 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), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 layers of hUC-MSCs. In some embodiments, the hUC-MSC sheets comprise less 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.

[0041] IV. Methods of Using MSC Sheets to Reduce Fibrous Tissue Formation and Increase Myometrial Regeneration in the Uterus In some aspects, the present disclosure relates to a method of reducing fibrotic tissue formation in a uterus of a subject in need thereof, comprising applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, the SC sheet comprising one or more layers of aggregated confluent mesenchymal stem cells (MSCs), wherein applying the MSC sheet to the uterus reduces the formation of fibrotic tissue in the uterus compared to a uterus to which the MSC sheet is not applied.

[0042] 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, the MSC sheet comprising one or more layers of aggregated, confluent mesenchymal stem cells (MSCs), wherein applying the MSC sheet to the uterus increases myometrial regeneration as compared to a uterus to which the MSC sheet is not applied.

[0043] The uterus contains four layers, endometrial epithelium, endometrial stroma, myometrium and perimetrium. The endometrium contains the epithelial and stromal layers as its inner layers. It has a basal layer and a functional layer. The functional layer thickens and is then shed during the menstrual or estrous cycle. The myometrium is the middle layer of the uterine wall, composed primarily of uterine smooth muscle cells (also known as myometrial cells), but also of supporting stromal and vascular tissue. The primary function of the myometrium is to induce uterine contractions. The outer layer of the uterus is the perimetrium.

[0044] In a Caesarean section delivery, a 15cm incision is typically made through the mother's lower abdomen, followed by a second incision to expose the uterus and deliver the baby. The incision is then sewn shut with multiple layers of tissue.

[0045] For other uterine surgeries such as myomectomy, where fibroid nodules are removed, an incision of any length and location depending on the size of the lesion is made through the patient's lower abdomen or through an abdominal opening for endoscopic surgery, and the lesion is removed with an incision at the border between normal and abnormal tissue. The incision is then sutured closed with single or multiple layers of tissue. After uterine surgery, the initial scar is generally fibrous tissue, which is weak and prone to tearing and other problems, requiring relief through normal scar formation, remodeling, and myometrial regeneration.

[0046] MSC sheets can also be used after non-surgical procedures. For example, in some embodiments, MSC sheets are applied to the uterus after dilation and curettage (D&C) (e.g., after a miscarriage) or after removal of uterine fibroids. MSC sheets can be applied with an endoscope to apply the cell sheet transvaginally.

[0047] One advantage of the MSC sheet 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, so that no sutures or stitches are required to attach the cell sheet to the tissue. A support membrane or other device can be used to transfer the MSC sheet to the uterine tissue of the subject, and then removed after sheet transfer. The support can be, for example, poly(vinylidene difluoride) (PVDF), cellulose acetate, cellulose ester, plastic, and metal. The MSC sheet easily adheres to the target tissue and self-stabilizes without sutures after being placed directly on 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 of contact with the tissue. Once the MSC sheet adheres to the uterine tissue, the support membrane can be excise. In certain embodiments, the MSCs in the cell sheet are allogeneic to the subject, i.e., isolated from a different individual of the same species as the subject, and therefore the genes at one or more loci are not identical.In certain reported cases, MSCs appear to avoid allogeneic rejection in humans and animal models (Jiang et al., 2005, Blood, 105(10), 4120-4126).Therefore, the MSC sheet described herein can be used in allogeneic cell therapy as a ready-made product.

[0048] The allogeneic cell source must be capable of eliciting meaningful therapy under standard immunocompetence in the host patient allogeneic tissue. This includes reliable cell homing to the tissue site of therapeutic interest and engraftment or retention there in fractional doses for a sufficient period of time (Leor et al., 2000, Circulation, 102(19 Suppl 3), III 56-61). Current expectations are that when a stem cell suspension is administered to a subject, less than 3% of the injected stem cells will be retained in the injured myocardium 3 days after injection following ischemic injury (Devine et al., 2003, Blood, 101(8), 2999-3001). Furthermore, most administered cells from the cell suspension that engraft in the target tissue 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 are stably engrafted into host tissues with high cell division retention 7 days after transplantation. Thus, the MSC sheets described herein offer distinct advantages over injected or administered mesenchymal stem cell suspensions.

[0049] In some embodiments, the MSC sheet is applied to the incision site of the uterus. In some embodiments, the incision is sutured closed before the MSC sheet is applied to the uterus (e.g., to the upper incision site of the myometrium). In some embodiments, application of the MSC sheet to the uterus reduces the fibrotic area of ​​the uterus by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a uterus to which the MSC sheet is not applied. In some embodiments, application of 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% compared to a uterus to which the MSC sheet is not applied.

[0050] If the subject has had one or more previous cesarean deliveries or at least one previous uterine surgery, the risk of complications from cesarean delivery is often increased. In some embodiments, the subject has not had a previous cesarean delivery or any previous uterine surgery. In some embodiments, the subject has had at least 1, 2, 3, 4, 5, 6, 7, or 8 previous cesarean deliveries and at least one previous uterine surgery.

[0051] In the methods described herein, multiple MSC sheets can be applied to the uterus. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more MSC sheets can be applied to the uterus. Any of these values ​​can be used to define a range of 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.

[0052] [Example] [Example 1] Characteristics of umbilical cord mesenchymal stem cell sheets prepared in xeno-free medium Materials and Methods 1.1 Human umbilical cord stem cell (hUC-MSC) culture Human umbilical cord mesenchymal stem cells (Jadi Cell LLC, Miami, USA IRB-35242) isolated from the subepithelial layer of human umbilical cord tissue stored in a bank (Patel et al., 2013, Cell Transplant, 22(3), 513-519) were cultured in xeno-free cell culture medium containing 10% human platelet lysate (hPL, iBiologics, Phoenix, USA), 1% Glutamax (Life Technologies), 1% MEM NEAA (Life Technologies), and 1% penicillin-streptomycin (Life Technologies) in a humidified atmosphere of 5% CO2 at 37°C for 5 days. Subculture was performed from passage 4 to passage 12. The cell culture medium was changed every 2 days.

[0053] 1.2 hUC-MSC proliferation rate 5 × 10 4 , 1×10 5 and 2 x 10 5 Number of cells / dish (i.e., 5 x 10 cells / dish) 3 / cm 2 , 1×10 4 / cm 2 and 2 x 10 4 / cm 2 hUC-MSCs were seeded in xeno-free cell culture medium at an initial cell density of 3.5 × 10. The cells in TCP were dissociated using trypsin, and the cell numbers were counted on days 1, 2, 3, 4, 5, and 6 using a hemocytometer. 3 / cm 2 At a cell density of 175 cm 2 hUC-MSCs were seeded in tissue culture flasks (Corning, NY) and cultured from passage 4 to 12, then passaged by TrypLE (life technologies) on day 5. Cell numbers were counted at each passage using a hemocytometer.

[0054] 1.3 hUC-MSC characterization in differentiation potential hUC-MSCs were cultured in xeno-free cell culture medium for two passages in TCP. Cells were prepared and induced for osteogenic and adipogenic differentiation at passages 4, 6, 8, 10, and 12. For osteogenic differentiation, 5 × 10 3 Cells / cm 2Cells were plated in xeno-free cell culture medium in 35 mm TCP dishes at 4 °C. When 60% confluent, cells were induced with osteogenic differentiation medium containing αMEM, 10 nM dexamethasone, 82 μg / mL ascorbic acid 2-phosphate, and 10 mM β-glycerolphosphate (Sigma-Aldrich). Cells were cultured in osteogenic medium at 37 °C for 21 days, and the medium was changed every 3 days. To detect positive differentiation, cells were fixed with cold 4% paraformaldehyde for 12 min and stained with Alizarin Red S- (Sigma-Aldrich) using standard protocols. For adipogenic differentiation, 1 × 10 4 Cells / cm 2 Cells were plated in xeno-free cell culture medium in 35 mm TCP dishes at 20 °C. When 80% confluent, cells were induced with adipogenic differentiation medium containing high glucose DMEM, 100 nM dexamethasone, 0.5 mM IBMX, and 50 μM IND (all Sigma-Aldrich). Cells were cultured in adipogenic medium at 37 °C for 21 days, and medium was changed every 3 days. To detect positive differentiation, cells were fixed with cold 4% paraformaldehyde for 12 min and stained with Oil Red O (Sigma-Aldrich) using standard protocols.

[0055] 1.4 hUC-MSC surface phenotype assay hUC-MSCs were cultured in xeno-free cell culture medium with TCP. Cell suspensions of P6, P8, P10 and P12 cells cultured with HPL and FBS were prepared. The cells were then enzymatically detached and washed once with PBS. To minimize non-specific binding of the antibodies, the cells were incubated with 2% w / v bovine serum albumin (BSA) in PBS for 30 min. Then, 3–5 × 10 5 Cells were divided into aliquots at a concentration of 1000 x 10 cells / 100 μL. One aliquot was reserved as an unstained control and the rest were stained with the following antibodies: CD44, CD90 and HLA-DR, DP, DQ (Biolegend, San Diego, CA). Primary antibodies were added to each aliquot to achieve a cell to antibody ratio of approximately 20:1 in buffer. Approximately 3–5 × 105 Cells were stained with saturating concentrations of (fluorophore)-conjugated antibodies. Cells were incubated on ice for 30 min in the dark. After incubation, cells were washed three times and then resuspended in PBS. Cells were immediately analyzed by flow cytometry. Flow cytometry was performed on a Becton, Dickinson FACS Canto (BD Biosciences, Sparks, MD). Unstained cells were used to set up the flow cytometer instrument. Cells were gated by forward vs. side scatter to exclude doublets. A minimum of 10,000 events were counted per analysis.

[0056] 1.5 hUC-MSC sheet preparation using different initial cell numbers and passage numbers hUC-MSC sheets were prepared under various conditions, including different initial cell densities and passage numbers, in temperature-responsive cell culture dishes (TRCDs) (Figure 2). 4 Cells / dish, 1 x 10 5 cells / dish and 2 x 10 5 Passage 6 cells were seeded at a cell number of 2 x 10 cells / dish. 5 Number of cells / dish (i.e., 2 x 10 4 / cm 2 Passage 4–12 cells were seeded at an initial cell density of 100 μg / mL. Fresh xeno-free cell culture medium containing 16.4 μg / mL ascorbic acid (Sigma-Aldrich, St. Louis, USA) for producing cell sheets was added 1 day after seeding. Confluent cell sheets formed 4–6 days after seeding were detached from the TRCD at room temperature. Before cell sheet detachment, cell morphology was monitored using an AX 10 microscope (Carl Zeiss Microimaging, Göttingen, Germany) by Axio Vision software (Carl Zeiss Microimaging).

[0057] 1.6 Immunohistochemical staining Cultured cell sheets were removed from the TRCD at room temperature, fixed in 4% paraformaldehyde for 30 min, and then embedded in paraffin. Embedded specimens were sectioned into 4 μm slices and stained for H&E, stem cell surface markers, ECM (fibronectin; FN and laminin; LM) and cell-cell junctions (integrin-linked kinase; β-catenin). For fluorescent staining (FN, LM and β-catenin), slides were immersed in antigen retrieval 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) was proceeded overnight at 4°C and then washed with PBS 1×. The specimens were treated with Alexa Fluor 594-conjugated secondary antibody (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 staining, specimens were treated with hematoxylin solution (Sigma-Aldrich) for 3 min, followed by eosin solution (Thermo Fisher Scientific, Kalamazoo, USA) for 5 min. 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).

[0058] 1.7 Cell sheet ultrastructure observed using a transmission electron microscope hUC-MSC sheets were fixed in 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. The samples were then embedded in epoxy resin. Ultrathin sections (70 nm thick) were observed using a transmission electron microscope (JEOL JEM1200EX) (JEOL USA, Peabody, USA).

[0059] 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 in the TRCD. The supernatant medium above the adherent cultured cells for 24 h was collected immediately before cell sheet detachment from the TRCD at room temperature (RT). The amount of HGF and TNF-α secreted from hUC-MSC was measured by human HGF Quantikine ELISA and human TNF-α Quantikine ELISA kits (R&D Systems, Minneapolis, USA), respectively.

[0060] 1.9 Cell sheet placement into subcutaneous tissue of immunodeficient mice After 4 days of culture, the hUC-MSC (passage 6) cell sheets were detached from the TRCD at RT and transferred to 6-week-old immunodeficient mice (NOD.CB17-Prkdc scidCell sheets were implanted into the subcutaneous dorsal tissue of mice (Neurology, California, USA) using the 100% 100% 100% 15 ...50% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150% 150%

[0061] 1.10 Statistical analysis All quantitative values ​​are expressed as the 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). Probability values ​​less than 0.05 (p < 0.05) were considered statistically significant.

[0062] 1.11 Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis hUC-MSC cell sheets were harvested after detachment from TRCD at RT. Total RNA was extracted from cell sheets using Trizol and PureLink RNA Mini kit (Life Technologies) according to the manufacturer's protocol. cDNA was prepared from 1 μg of total RNA using high capacity cDNA reverse transcription kit (Life Technologies). RT-PCR analysis was performed using 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 housekeeping gene, 2) integrin-linked kinase (ILK, Hs00177914_m1), 3) N-cadherin (N-cad, Hs00983056_m1). All primers were manufactured by Applied Biosystems. Comparative C T Relative gene expression levels were quantified by the method (Schmittgen & Livak, 2008). Gene expression levels were normalized to GAPDH expression levels. Gene expression levels are relative to the levels of passage 6 cells.

[0063] result Preparation of hUC-MSC sheets using different initial cell numbers and passage numbers hUC-MSCs were cultured in flasks and subcultured every 5 days using trypsin from passage 4 to 12 (Table 1). Cells expanded 16-20 fold from the initial cell seeding number during passages 4-8 in subculture. However, the cell proliferation rate dramatically decreased from passage 9. The cell number increased 14, 10.9, 7.5 and 3.1 fold from the initial cell seeding number at passages 9, 10, 11 and 12, respectively. Cells at passage 10 required one more day to reach confluence and obtain cell sheets than cells at passages 4-8 at the same seeding density (Figures 2a and 2b). Cells at passage 12 showed heterogeneous culture morphology, lost contact inhibition and clumped into multi-layered aggregates instead of a consistent monolayer (Figure 2a). When the culture temperature was reduced to room temperature (RT), cells at passage 12 were detached from the TRCD but were not as sheet-like as those harvested from passages 4 to 10 (Figure 2b). Cells should therefore be used at passages 4 to 8 to produce consistent cell sheet quality.

[0064] [Table 1] Table 1. Proliferation rate of hUC-MSCs during passages 4–12 JPEG2025024004000002.jpg81167

[0065] 5×10 4 , 1×10 5 and 2 x 10 5 Initial cell seeding numbers of 1 × 10 cells / dish reached confluence on days 6, 5, and 4, respectively (Figures 3a and 3b), and reliably produced hUC-MSC sheets (Figure 3c). Cell sheets derived from all different initial cell number groups were grown at 2 × 10 cells / dish once the cells became over-confluent. 5 , 1×10 5 and 5 x 10 4In the seeded cells / dish group, cells spontaneously detached from the TRCD without temperature change (i.e., at 37 °C) on days 5, 6, and 7, respectively (Figure 3c). One day before cell confluence, the adherent cells were less than 70-80% confluent and detached as partially broken sheet fragments when the temperature was reduced to RT due to insufficient cell density. Recovery of hUC-MSC sheets either one day before or one day after the cells reached confluence in the TRCD was not possible (Figure 3c). These results indicate that in order for cells to detach as thermally harvested sheets, the cell sheets must be carefully prepared and harvested at a precise time point when the cells are neither sparsely nor overcrowded.

[0066] hUC-MSC surface marker characterization CD44 and CD90 expression was measured in vitro in hUC-MSC suspension cultures and hUC-MSC sheets. As shown in FIG. 4, hUC-MSC expressed CD44 and CD90 in suspension cultures (FIG. 4A and FIG. 4B) and cell sheets (FIG. 4C and FIG. 4D) in vitro. CD44 and CD90 are known to be expressed in hUC-MSC. Therefore, these results indicate that the hUC-MSC sheets maintained the hUC-MSC-specific phenotype. In particular, the results in FIG. 4C and FIG. 4D indicate that the cell sheets contained hUC-MSCs and did not contain other cell types and differentiated cells derived from the umbilical cord.

[0067] Structural analysis of hUC-MSC sheets Passage 6 cells were cultured in the TRCD for 4 days, and the resulting cell sheets were harvested from the TRCD with temperature reduction to RT. The cell sheets were stained for fibronectin, laminin, and β-catenin to verify the retention of functional structure during culture and after sheet detachment of the hUC-MSC sheets. Fibronectin and laminin, key 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 throughout the cell sheet surface (Figure 5a and Figure 5b). β-catenin, part of a protein complex that forms cell adhesive junctions (Nelson & Nusse, 2004, Science, 303(5663), 1483-1487), shows prominent staining between cells (Figure 5c). Retention of ECM and cell junction proteins indicates that functional proteins produced during culture are preserved after cell sheet harvest.

[0068] The intercellular structures within the cell sheets were observed by TEM. Horizontal sectioning revealed ECM structures within the cell sheets (FIG. 5d), which contained numerous intercellular junctions (FIG. 5e). These results suggest that hUC-MSC sheets structurally retain functional proteins related to native cellular functions, such as cell communication and cell adhesion.

[0069] Secretion of hepatocyte growth factor (HGF) and tumor necrosis factor-alpha (TNF-α) To support the paracrine effect of the fabricated hUC-MSC sheets in vitro, 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 the culture supernatant were measured. A significant difference in the amount of hHGF was observed between 2 × 10 5 , 1×10 5 and 5 x 104 The proinflammatory cytokine (hTNF-α) was not observed in the 2 × 10 cells / dish group (Fig. 6a). 5 , 1×10 5 and 5 x 10 4 was barely detectable in the 10 cells / dish group (Figure 6b).

[0070] hUC-MSC sheets fabricated using passage 4 cells secreted a significantly higher concentration of hHGF (633 pg / mL) compared to hUC-MSC sheets fabricated using passage 6, 8, 10, and 12 cells. The amount of hHGF secreted from hUC-MSC sheets dramatically decreased as the passage number increased (Figure 6c). hTNF-α was barely secreted from hUC-MSC sheets (16-35 pg / mL) (Figure 6d), and hUC-MSC sheets fabricated using passage 4 had a significantly lower concentration of hTNF-α compared to hUC-MSC sheets fabricated using passage 6, 8, 10, and 12 cells. The results therefore demonstrate that the passage number is an important factor in the hUC-MSC sheet cytokine profile.

[0071] Cell sheet implantation into immunodeficient mice hUC-MSC sheets were implanted in dorsal subcutaneous pockets in immunodeficient mice for 10 days to demonstrate stability and engraftment in vivo. On the 10th day after implantation, capillary formation (angiogenesis) was observed in the tissues with cell sheets implanted, while the subcutaneous tissues without cell sheets showed very few microvessels (Figures 7c and 7d). H&E staining data demonstrated that the cell sheets maintained their localization in the implanted area for 10 days after implantation (Figures 7a and 7b). In the cell sheet implantation group, numerous vascular structures were observed between the implanted cell sheets and the host tissue (Figure 7e). This indicates that the cell sheets are implantable and can engraft and preserve the cell sheet structure for 10 days in vivo. Furthermore, the cell sheets induce neocapillary formation as a key ability for engraftment, viability, and tissue regeneration.

[0072] Consideration Fabrication of xeno-free hUC-MSC sheets was demonstrated from cultures using temperature-responsive culture dishes (TRCDs). Such hUC-MSC sheets (1) retained native functional intercellular structures essential for cell-cell communication, acting as a natural matrix adhesive when implanted into target organs (Figure 5); (2) secreted hepatocyte growth factor (HGF) to induce angiogenesis and anti-fibrotic effects (Figure 6); (3) retained cells in vivo for 10 days after implantation; and (4) showed in vivo angiogenesis supporting sheet-tissue engraftment (Figure 7).

[0073] To fabricate reproducibly strong MSC cell sheets, hUC-MSCs from passages 4 to 12 were expanded and converted into sheets in cell culture medium supplemented with hPL. The cell proliferation rate of hUC-MSCs was significantly reduced after passage 10, affecting the cell sheet fabrication process and harvesting timeline (Figure 2). Furthermore, passage 12 cells could not form stable sheets due to reduced cell proliferation rate and inadequate cell-cell junction formation after increasing passages (Table 1 and Figure 8). In addition, microscopic phase contrast images (Figure 2) showed cells stacked on top of each other and the formation of cell aggregates at higher passage numbers. This feature tends to increase as the passage number increases, especially for passage 12 cells. Cell aggregation using bone marrow-derived (BMSC) and adipose-derived (ADSC) stem cells is reported to occur when cultured in medium containing 5% hPL (Hemeda, Giebel, & Wagner, 2014, Cytotherapy, 16(2), 170-180). Activated coagulation factors in hPL may be involved in inducing such aggregation. This cell aggregation hinders uniform cell growth and cell sheet fabrication. To prepare reproducible hUC-MSC sheets, a passage number below passage 10 would be preferred.

[0074] The rapid proliferation of hUC-MSCs cultured in cell culture medium containing hPL can be beneficial in reducing the time required for cell sheet fabrication. Conversely, it may also introduce some processing difficulties, as the sheet cultures tend to reach confluence quickly and spontaneously detach upon reaching confluence. Therefore, the judicious use of an appropriate initial cell seeding number is critical to the hUC-MSC sheet fabrication process. 2×10 5 Initial cell seeding densities higher than 2 × 10 cells / dish do not result in monolayer sheets: such high densities induce spontaneous cell detachment from the TRCD within 2 days of cell culture (data not shown). 5 , 1×10 5 and 5 x 10 4 Initial densities of cells were used, all of which successfully produced hUC-MSC sheets on days 4, 5, and 6, respectively, when each culture reached confluence (Figure 3). Poor cell sheet quality was observed when cells were detached one day before or one day after reaching confluence (Figure 3c). Therefore, for optimal hUC-MSC sheet fabrication, the harvest time for TRCD cell sheet recovery depends on both the passage number and seeding cell density.

[0075] Central to these results is the reliable ability to use temperature reduction to produce stable, robust layers of hUC-MSCs using commercially available TRCDs grafted with a temperature-responsive polymer coating that facilitates cell harvesting without destructive enzymes (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 intact native cell-cell organization, cell-cell communication, intact ECM, and the recovery of cultured cells with tissue-like phenotypes. Cell sheets retrieved from TRCD by slight changes in culture temperature preserve cell surface-associated ECM, such as fibronectin and laminin, and cell-cell junction proteins, such as β-catenin, which play important roles in promoting cell adhesion and paracrine signaling (Figure 5) (Brownlee, 2002, Curr Opin Plant Biol, 5(5), 396-401). Cell sheets with native morphology, confluent phenotype and organization, cell-cell communication, intact extracellular matrix (ECM), and tissue-like behavior can be easily transplanted into target tissues (Miyahara et al., 2006, Nat Med, 12(4), 459-465). hUC-MSC sheets implanted at subcutaneous tissue sites in immunodeficient mice rapidly and spontaneously attached to the subcutaneous tissue surface within 10 minutes. After 10 days in vivo, the implanted cell sheets remained as intact sheets (Figure 7).

[0076] Overall, hUC-MSC sheets display several beneficial properties for improving allogeneic MSC cell therapy. The results here determined (1) specific conditions for reliable xeno-free hUC-MSC sheet fabrication; (2) intact features of hUC-MSC sheets that preserve structure and paracrine effects on important cell functions after cell harvesting from the TRCD; (3) intact hUC-MSC sheets maintained at the implanted target tissue site for 10 days; and (4) new blood vessel recruitment to the sheets in the target tissue, suggesting that implanted hUC-MSC sheets continuously secrete paracrine factors to modulate engraftment.

[0077] conclusion The hUC-MSC cell sheet technology represents a unique cell delivery method that aims to improve MSC therapy beyond current injected cell suspensions. The simple fabrication method in hPL and TRCD allows rapid xeno-free production of robust and homogenous hUC-MSC sheets that are harvested by slight changes in temperature instead of destructive proteolytic enzymes. Cell production depends on several controlled culture variables, including cell seeding density, passage number, medium (hPL) and culture time and TRCD. When cultured uniformly under optimized conditions, hUC-MSC cell sheet reproducibility is enhanced and the hUC-MSC cell sheet production process is simplified into a routine that is amenable to scaling. This allows future production of hUC-MSC sheets with higher cell numbers for increased paracrine effects and therapeutic benefits. Considering their paracrine effects and low HLA profile, the fabricated xeno-free hUC-MSC sheets exhibit promising tissue regeneration potential both structurally and functionally in vitro and in vivo. With reliable local tissue site placement, high engraftment efficiency, and long-term retention and survival in vivo, hUC-MSC sheets have the potential to improve the therapeutic value of allogeneic cell therapy over currently used injected stem cells.

[0078] [Example 2] Comparison of human umbilical cord mesenchymal stem cells (hUC-MSCs) harvested by temperature shift, trypsinization, and cell scraper 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), and 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.

[0079] 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 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-MSCs were incubated at 37°C in a humidified chamber with 5% CO2 and passaged when cells reached confluence. hUC-MSCs were passaged by treatment with TrypLE (Gibco) for 5 min and maintained at 3000 cells / cm between passages 4 and 6. 2 and subcultured.

[0080] 2.3 Preparation of hUC-MSC sheets hUC-MSCs were seeded on a 35 mm temperature-responsive culture dish (TRCD) (CellSeed, Tokyo, Japan). 5 The cells were seeded at a density of 10 cells / dish (day 0) and cultured until confluent (day 5). The cell culture medium containing 16.4 μg / mL ascorbic acid (Wako, Osaka, Japan) was replaced 1 day after seeding. hUC-MSCs were harvested as monolayer sheets from the TRCD within 60 min by reducing the temperature to 20 °C. The total cell number of the hUC-MSC sheets was counted by trypan blue (Gibco) exclusion test using a hemocytometer.

[0081] 2.4 Hematoxylin and eosin (H&E) staining of hUC-MSC sheets The samples were fixed in 4% buffered paraformaldehyde (PFA) and embedded in paraffin. The samples were then cut into 4 μm thick sections. The sections were stained with Mayer's hematoxylin and 1% eosin alcohol solution. They were then mounted with permount™ (Thermo Fisher Scientific). The stained samples were visualized using a BX53 microscope (Olympus, Tokyo).

[0082] 2.5 Morphological observation of hUC-MSCs using scanning electron microscope and transmission electron microscope For scanning electron microscopy (SEM) analysis, samples were rinsed in wash buffer (0.1 M sodium cacodylate buffer containing 2.4% sucrose and 8 mM calcium chloride) for 5 min and then fixed in 2% osmium tetroxide (OsO4) in wash buffer for 1 h at room temperature. Samples were rinsed with DI water to remove unbound osmium and then dehydrated through a graded series of ethanol. Ethanol was then exchanged for hexamethyldisilazane (HMDS) and dried at -30°C. Samples were observed under a scanning electron microscope (FEI Quanta 600 FEG, FEI, Oregon). For transmission electron microscopy (TEM) analysis, samples were fixed in a mixture of 2% paraformaldehyde, 2% glutaraldehyde, 2% OsO4 in 0.1 M sodium phosphate buffer and dehydrated in a graded series of ethanol. The samples were then embedded in epoxy resin and cut to a thickness of 70 nm. Ultrathin sections were observed under a transmission electron microscope (JEOL JEM-1400 Plus, JEOL, Tokyo).

[0083] 2.6 Cell viability assay Cell viability was measured by live and dead viability / cytotoxicity assay (Thermo Fisher Scientific, Massachusetts). Cell sheets and trypsinized cell populations were washed twice and incubated with live / dead working solution (4 mM ethidium homodimer-1 and 2 mM calcein AM) for 30 min at 37°C in the dark. Samples were washed and visualized using an AX 10 microscope (Carl Zeiss Microimaging, Göttingen, Germany) and analyzed by 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 the single suspension population was counted using image J (National Institutes of Health, Bethesda, Maryland, USA). The number of dead cells in the cell sheets was also counted using Image J (National Institutes of Health), while the viable cells in the cell sheets were calculated based on the following formula:

[0084]

number

[0085] The percentage of dead cells was calculated to compare cell viability in each sample, as shown in FIG. 15B.

[0086] 2.7 Qualitative analysis of proteins related to cellular functions hUC-MSCs (2 × 10 5Cells (1000 cells / dish) were cultured for 5 days and harvested by temperature shift (cell sheet technique), trypsinization (chemical disruption) or cell scraper (physical disruption) (Figure 9). Protein extracts were isolated by lysing cells with cell lysis buffer (RIPA buffer, proteinase inhibitors and phosphatase inhibitors) (Thermo Fisher Scientific) for 15 min at 4 °C. Samples were then sonicated three times for 9 s. Protein concentration of each sample was determined by the Bradford method (Galipeau et al., 2018, Cell Stem Cell 22(6): 824-833). Samples containing the same amount of protein (10 μg) were denatured at 70 °C for 10 min, loaded onto SDS-PAGE gels (3-8% Tris-acetate gels or 4-12% Tris-glycine gels (Thermo Fisher Scientific)) and electrophoretically transferred to a PVDF membrane (LC2002) (Thermo Fisher Scientific). The membranes were treated with blocking solution 5% bovine serum albumin (BSA) for 1 h at room temperature and incubated overnight at 4° C. with primary antibodies: actin (1:1000 dilution), vinculin (1:10000 dilution), fibronectin (1:2000 dilution), laminin (1:1000 dilution), integrin β-1 (1:2000 dilution), connexin 43 (1:8000 dilution), YAP (1:1000 dilution), phosphor-YAP (Ser127) (1:1000 dilution), FAK (1:1000 dilution), phospho-FAK (Tyr397) (1:1000 dilution), GAPDH (1:5000 dilution). The incubated membranes were treated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Membranes were visualized by using enhanced chemiluminescence (FluorChem HD2, ProteinSimple, California, USA). Expression levels were normalized to GAPDH.

[0087] 2.8 Immunocytochemical staining of proteins related to cell function Samples were fixed in 4% buffered PFA and then permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific). Samples were blocked with 1% BSA in 10% goat serum for 15 min and then incubated overnight at 4°C in primary antibodies; 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 and 10% goat serum. Samples were treated with secondary antibodies for 1 h. Finally, they were mounted with mounting solution (ProLong Gold antifade mounting medium containing DAPI) (Thermo Fisher Scientific) and examined using an IX73 fluorescent microscope (Olympus).

[0088] 2.9 Statistical analysis All values ​​are expressed as mean±SEM. Two-way analysis of variance followed by Tukey's test was used to assess differences between groups greater than 2. Probabilities (p<0.1, 0.05) were considered significant.

[0089] result Preparation of human umbilical cord stem cell (hUC-MSC) sheets To verify the morphology and proliferation rate of hUC-MSCs cultured on temperature-responsive cell culture dishes (TRCDs), 2 × 10 hUC-MSCs were cultured on conventional tissue culture plates (TCPs) or 35 mm diameter TRCDs. 5Cells were seeded at a density of 100 μg / cm2 and cultured for 5 days. Cells cultured on the TRCD changed their morphology from round to spindle shape when the cells attached to the bottom surface of the TRCD. This morphological change was also observed in cells cultured on TCP (Figure 10A). Furthermore, the proliferation rate of hUC-MSCs cultured on the TRCD showed the same proliferation curve as that of TCP (Figure 10B). This indicates that the cell culture dish surface coated with the temperature-responsive polymer did not affect the cultured cell proliferation and cell morphology. Furthermore, as a result of the temperature reduction from 37°C to 20°C, the cells were successfully detached from the TRCD and the sheet was maintained (Figure 10C). The fabricated cell sheet formed contained an intact and continuous cell layer and maintained cell-associated proteins similar to the native structure (Figure 10d).

[0090] Morphological observation of hUC-MSC sheets The surface and intercellular structures of hUC-MSC sheets were observed by scanning electron microscopy (SEM) (Figure 11A-D) and transmission electron microscopy (TEM) (Figure 11E-F). In SEM analysis, hUC-MSC sheets showed connected cell membrane structures at the cell surface. It means that even after cell detachment, hUC-MSC sheets preserved the retention of the native structures formed when they are cultured on cell culture dishes. Native cell membrane structures are composed of cell surface proteins and membrane proteins, which are involved in cell adhesion and function. This finding suggests that hUC-MSC sheets retain cell surface proteins and membrane proteins, and this retention can improve cell adhesion and cell function (Albuschies et al., 2013, Sci Rep 3: 1658). In contrast, hUC-MSCs treated with 0.05% trypsin showed dissociated single-cell shapes without connected tissue structures (Figure 11B-D). Furthermore, the cell surfaces in the 0.05% trypsin treatment groups (5 min, 20 min, and 60 min) lost their microvilli-like structures in a trypsin treatment time-dependent manner (Figures 11B-D). As a result, the hUC-MSC sheets maintained the tissue-like junction structure and the microvilli-like structure, and the proteins on the cell surfaces in the 0.05% trypsin treatment group were cleaved.

[0091] TEM analysis showed that hUC-MSC sheets maintained ECM (white dotted line) and cell-cell junctions (white solid arrows), which are involved in cell adhesion and cell-cell communication (Gattazzo et al., 2014, Biochim Biophys Acta 1840(8): 2506-19) (Figure 11E). However, hUC-MSCs treated with 0.05% trypsin for 5 min showed disrupted cell-cell junctions and ECM compared to the cell sheet group (Figure 11F). Furthermore, when hUC-MSCs were treated with 0.05% trypsin for 20 and 60 min, hUC-MSCs lost their natural filopodia on their cell surface and had unclear nuclear shapes of nuclei (Figures 11G and H). hUC-MSCs treated with 0.05% trypsin for 60 min showed endoplasmic reticulum (dark gray arrows), which are known to be associated with cell death (Figure 11H). SEM and TEM results show that hUC-MSC sheets maintained cell surface and intercellular proteins, such as microvilli-like structures, filopodia, ECM, and cell-cell junctions, even after the cells were detached from the cell culture dish. In contrast, the hUC-MSC group treated with 0.05% trypsin showed severed microvilli, ECM, and cell-cell junctions, and the nuclei were damaged. These findings suggest that trypsin treatment (chemical destruction) causes damage in cell and tissue structures (i.e., junction proteins, ECM, nuclei, and endoplasmic reticulum).

[0092] hUC-MSCs maintain actin filament proteins involved in cell kinetic dynamics Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein expression was detected as a loading control to normalize protein amount for Western blot assay. The expression level of GAPDH protein was similar in all groups. Cells treated with 0.50% trypsin for 20 and 60 min expressed lower actin than that in cell sheet, 0.05% trypsin, and cell scraper groups (Figure 12A). This indicates that 0.50% trypsin treatment destroys actin in the cytoplasm. To observe the cytoskeleton structure, hUC-MSCs were stained with actin. When cells are attached to the culture dish, actin forms a stress fiber structure that plays an important role in cell survival (Bachir et al., 2017, Cold Spring Harb Perspect Biol 9(7)). Even after the cell sheet was detached from the cell culture dish, the cell sheet group showed actin stress fiber structure of actin. In contrast, the 0.05% trypsin treatment groups for 5, 20, and 60 min showed actin-positive areas, but no stress fiber structures were observed (Figure 12B). The amount of F-actin protein was similar in the cell sheet and 0.05% trypsin treatment groups. However, only the cell sheet group maintained the actin stress fiber structure, which is characteristic of cellular actin.

[0093] Vinculin is a membrane cytoskeleton protein that forms focal adhesions by linking the integrin family and actin, which are associated with cell motility (Peng, 2011, Int Rev Cell Mol Biol 287: 191-231). When stained by immunohistochemistry, vinculin expression was observed in both the cell sheet and 0.05% trypsin-treated groups (Figure 12C). Western blot analysis of vinculin expression showed multiple bands with lower molecular weights in the chemically disrupted group (Figure 12A), indicating that vinculin protein was cleaved in the chemically disrupted group. Trypsin-treated (chemically disrupted) cells revealed delocalized actin fiber structures, reduced actin protein, and cleaved vinculin protein, suggesting that the chemical disruption method cleaved proteins related to cell shape and cell kinetics. This cleavage increased when the trypsin concentration was increased.

[0094] hUC-MSC sheets maintain extracellular proteins involved in cell adhesion Fibronectin and laminin are important proteins in cell adhesion and tissue adhesion. In Western blot assay, cell sheet, 0.05% or 0.50% trypsin treatment for 5 min, and cell scraper group expressed fibronectin. However, 0.05% and 0.50% trypsin treatment for 20 min and 60 min groups had no detectable expression of fibronectin. Laminin expression was observed in cell sheet, 0.05% trypsin treatment for 5 min, 0.50% trypsin treatment, and cell scraper group. However, 0.50% trypsin treatment for 20 min and 60 min groups showed no detectable laminin expression.

[0095] To observe the ECM protein structure, cells were stained using fibronectin and laminin antibodies (Figure 13B). Higher expression of fibronectin was observed in the cell sheet group compared to cells treated with 0.05% trypsin. Similar to the native tissue structure (fibrous structure of ECM), the cell sheet group showed higher expression of fibronectin and laminin across all cells of the cell sheet. These results suggest that the cell sheet group was able to detach cells without disruption of the ECM. In contrast, ECM proteins were cleaved by trypsin treatment (chemical disruption) after cell detachment from the cell culture dish.

[0096] hUC-MSC sheets maintain cell junction proteins associated with cell communication Integrin β-1 is a major member of the integrin family of transmembrane proteins that form cell-ECM junctions. Integrins are linked to the actin cytoskeleton of cells through adaptor proteins (e.g., vinculin, talin) and are known to be involved in cell phenotype preservation, survival, cell adhesion and tissue repair (Moreno-Layseca, 2014, Matrix Biol 34: 144-53). Cell sheet, 0.05% trypsin treatment for 5 min and cell scraper groups showed similar integrin β-1 expression. Integrin β-1 was gradually cleaved with trypsin concentration and treatment time. Connexin 43 is a transmembrane protein that facilitates cell-cell communication at gap junctions. Connexin 43 plays an essential role in maintaining cell and tissue homeostasis and function by exchanging biological information (Ribeiro-Rodrigues, 2017, J Cell Sci 130(21): 3619-3630). Connexin 43 was expressed in the cell sheet, 0.05% trypsin treatment (5, 20, 60 min) and 0.5% trypsin treatment (5 min) groups. However, 0.50% trypsin treatment for 20 and 60 min did not result in the expression of connexin 43. This suggests that connexin 43 protein was cleaved by 0.50% trypsin when treated for 20 and 60 min.

[0097] Structural observation of cell junction proteins was performed with integrin β-1, and connexin 43 protein was observed by immunostaining. The cell sheet group showed positive expression of integrin β-1 uniformly all over the cell sheet, while in the 0.05% and 0.50% trypsin treatment groups, integrin β-1 was slightly expressed on the cell surface (Figure 14B). Connexin 43 expression was observed in all groups (Figure 14C). Notably, the cell sheet group revealed connexin 43 expression throughout the visualization area. This demonstrates that the cell sheet has a connected tissue cell-cell structure and maintains cell-junction proteins. In contrast, trypsin treatment (chemical disruption) cleaved these important junction proteins.

[0098] Chemical disruption methods induce cell death Immediately after cell detachment by trypsin treatment (chemical destruction) or temperature change (cell sheet technique), cells were stained using calcein and ethidium homodimer-1. In FIG. 15, green indicates live cells and red indicates dead cells. As for the results, the ratios of dead cells to live cells in the 0.05% trypsin treatment groups for 5 and 20 minutes were similar. Furthermore, the ratios of dead cells to live cells in the 0.05% trypsin treatment groups for 60 minutes were significantly increased compared to cells treated with 0.05% trypsin for 5 and 20 minutes (FIG. 15B). This result suggests that cell death was induced by trypsin treatment (chemical destruction).

[0099] Apoptotic cell death is activated by chemical destruction Mechanosensor control for cell homeostasis transduces extracellular physical stimuli into intracellular chemical stimuli (Humphrey, 2014, Nat Rev Mol Cell Biol 15(12): 802-12). Yes-associated protein (YAP) is a major mechanosensor protein localized in the cell nucleus for regulating cell survival and proliferation (Jaalouk, 2009, Nat Rev Mol Cell Biol 10(1): 63-73). YAP is inhibited through phosphorylation of Ser127 (phosphoro-YAP, pYAP), which leads to cytoplasmic retention and induction of cell apoptosis. When cells lose cell-ECM junctions, apoptotic cell death, i.e., anoikis, is induced following phosphorylation of YAP (Halder et al. 2012, Nat Rev Mol Cell Biol 13(9): 591-600). The expression of YAP and phospho-YAP (pYAP) in cell sheet, 0.05% and 0.50% trypsin treatment for 5, 20 and 60 min, and cell scraper groups was determined by Western blotting (Figure 16). All groups showed similar YAP protein expression, but the expression of pYAP was increased in 0.05% and 0.50% trypsin-treated cells compared to cell sheet and cell scraper groups (Figure 16A). This demonstrates that trypsin treatment (chemical disruption) inhibited YAP activity and induced phosphorylation of YAP. Furthermore, induction of pYAP is known to shift cellular responses toward apoptosis.

[0100] Consideration Chemical disruption methods are commonly used to harvest cells from cell culture dishes through the disruption of extracellular (Huang et al., 2010, J Biomed Sci 17: 36) and intercellular (Besingi, 2015, Nat Protoc 10(12): 2074-80) proteins related to cytoskeleton, cell junctions, cell metabolism, and cell proliferation. Thus, cells harvested by chemical disruption methods (e.g., trypsin-treated cells) had insufficient ECM, which is necessary to adhere to the target tissue, and insufficient cell junctions to maintain their normal cell functions through graft-host communication (Figures 13 and 14). On the other hand, hUC-MSC sheets harvested by cell sheet technology using TRCD maintained tissue-like structures, including smooth surfaces, microvilli, ECM, and intercellular junctions of connected cells (Figures 10, 13, and 14).

[0101] TEM results showed that cleavage of extracellular proteins was observed in cells treated with 0.05% trypsin for 5 min in the chemical disruption group. Cytoplasmic cleavage was observed in cells after 20 min of 0.05% trypsin treatment, and cell nuclei were disassembled after 60 min of 0.05% trypsin treatment. Furthermore, endoplasmic reticulum changes related to cell death were observed after 60 min of 0.05% trypsin treatment (Figure 11). Integrins are key proteins involved in the interaction between cell membranes and ECM, and also link ECM to the cytoskeleton (actin) to form focal adhesions (Kim et al., 2011, J Endocrinol 209(2): 139-51). Harvesting by chemical disruption induced cleavage of integrin β-1, as well as cytoskeleton (F-actin), focal adhesion proteins (vinculin), and ECM (fibronectin and laminin) (Figures 12, 13, and 14). On the other hand, hUC-MSC sheets maintained all of their integrin β-1, cytoskeleton, focal adhesion proteins (vinculin), and ECM (fibronectin and laminin) even after detachment from the cell culture dish (Figures 12, 13, and 14). These findings suggested that chemical disruption methods (e.g., proteolytic trypsinization) could result in a harsh environment that compromises cell survival through enzymatic proteolytic cleavage of membrane receptors and disruption of cell-ECM junctions.

[0102] 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, disassembly of cell-ECM junctions 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 (Figure 14), and cleavage of integrin β-1 inactivated YAP and induced pYAP (Figure 16). Finally, apoptotic cell death occurred in the chemical disruption group (Figures 11, 15 and 16). In contrast, hUC-MSC sheets maintained integrin β-1 and reduced the expression of pYAP (Figures 14 and 16), and it showed significantly higher cell viability (Figures 15 and 16). It has been reported that pYAP can be induced not only by integrin β-1 cleavage but also by inhibition of F-actin polymerization. hUC-MSC sheets showed cytoskeletal fiber structures of F-actin, indicating active actin polymerization, even after cell detachment from cell culture dishes (Figure 12). This suggests that hUC-MSC sheets retain integrin β-1 (cell-ECM junctions) and the fiber structures of F-actin fibers, allowing cell sheets to maintain higher cell viability compared to trypsin treatment (the traditional chemical disruption cell harvesting method). These findings suggest that cell-ECM junctions and actin fiber-maintaining structures are important factors for maintaining cell survival. High cell viability is difficult when using chemical disruption methods.

[0103] This study demonstrates that proteins of ECM, cell-cell junctions and cell-ECM junctions are important to maintain higher cell viability. Since chemical disruption (e.g. trypsinization) cuts cell-cell junctions, cell-ECM junctions and cell adhesion proteins, it is not possible to avoid low engraftment efficiency rates and low cell viability in conventional stem cell therapy using chemical disruption harvesting methods. Cell sheet technology allows cells to be harvested in the form of viable sheets without any structural disruption. Furthermore, cell sheet technology maintains important structures for cells (ECM, cell-ECM junctions, cell-cell junctions, cytoskeleton and mechanosensors), enhancing cell viability, engraftment efficiency and maintaining various critical cell functions. As a result, cell viability in hUC-MSC sheets is significantly higher than cells harvested by chemical disruption methods.

[0104] conclusion We demonstrate that preserving tissue-like structures such as ECM cell-cell junctions and cell-ECM junctions is associated with enhanced cell viability of transplanted cells. The cell sheet technology allows for harvesting cells in the form of sheets without using any harmful proteolytic enzymes (chemical disruption). The harvested hUC-MSC sheets, which retained tissue-like cell structures, ECM, cell-cell junctions and cell-ECM junctions, had higher cell viability compared to traditional chemical disruption methods (trypsinization). Since the cell sheet mimics the natural tissue-like structure, this technology provides not only a higher therapeutic effect of stem cell therapy but also a new concept for improving cell function in regenerative medicine research.

[0105] [Example 3] Gene expression in human umbilical cord mesenchymal stem cell (hUC-MSC) sheets Cell sheets were prepared from hUC-MSCs by the method described in Example 1 above, except that the cell culture medium contained 20% hPL or 20% FBS. The hUC-MSC sheets are shown in Figure 17. Single cell suspension cultures of hUC-MSCs were prepared by culturing hUC-MSCs on cell culture dishes and treating the cells with trypsin (TryLE, Gibco) when they were confluent. Trypsinized single cell suspensions of hUC-MSCs were analyzed by flow cytometry.

[0106] hUC-MSC sheets were cultured in medium containing 20% ​​hPL and implanted into the subcutaneous tissue of immunodeficient mice as described in Example 1 above, and the hUC-MSC sheets were harvested from the subcutaneous tissue site for histological observation 1 and 10 days after implantation. After harvesting, the samples were stained with human growth factor (HGF) antibody to detect HGF expression, and cell nuclei were stained with DAPI. As shown in Figure 18, the hUC-MSC sheets expressed HGF 1 day after implantation and still maintained significant HGF expression 10 days after implantation. These results suggest that the hUC-MSC sheets maintain continuous HGF expression in the tissues of the host organism for at least 10 days after implantation.

[0107] The effect of initial cell density on HGF expression in hUC-MSC sheets was also determined. Cell sheets were grown at 2 × 10 4 , 4×10 4 , 6×10 4 , 8×10 4 Or 10 x 10 4 Cells / cm 2 HGF expression was increased in a cell density-dependent manner by increasing the initial cell density. For example, at an initial cell density of 1 × 10 5 Cells / cm 2 The cell sheet generated in 4 , 4×10 4 , 6×10 4 , 8×10 4 Or 1×10 5 Cells / cm2 The hUC-MSC sheets had higher HGF gene expression than the cell sheets produced with the 100% 15 ...

[0108] HLA DR, DP, DQ expression was determined in hUC-MSCs in suspension culture at passages 4 to 12, and in cell sheets prepared from human adipose-derived mesenchymal stem cells (hADSCs), human bone marrow-derived mesenchymal stem cells (hBMSCs), or hUC-MSCs. Cells were grown in culture medium containing 20% ​​hPL. HLA expression was determined as described in Example 1 above. As shown in FIG. 20A, hUC-MSCs maintained low cell surface expression of HLA DR, DP, DQ at passages 4 to 12 in cell suspension culture. As shown in FIG. 20B, HLA-DR gene expression was undetectable in hUC-MSC sheets, while cell sheets prepared from hADSCs or hBMSCs showed relatively high levels of HLA-DR gene expression. Low HLA expression is desirable for reducing host immune responses to huC-MSC cell sheets transplanted into host organs. Therefore, these results suggest that hUC-MSC sheets are less likely to induce an immune response in the host organism after transplantation, compared with cell sheets generated from hADSCs or hBMSCs.

[0109] [Example 4] Transplantation of human umbilical cord mesenchymal stem cell (hUC-MSC) sheets prevents uterine scarring in a nude rat model 3.0 × 10 cells in a thermoresponsive cell culture dish (TRCD) (UpCell®, Tokyo, Japan). 5Human umbilical cord mesenchymal stem cells (hUC-MSCs) were used to generate cell sheets as described in Example 1 above using an initial cell density of 1000 μg / ml. Images of the cell sheets are provided in Figures 29A-29C. Commercially available human umbilical cord mesenchymal stem cells used in a previous human clinical trial were cultured in a commercially available temperature-responsive cell culture dish and grown to confluence on day 5 (Figure 29A). The cell sheets were naturally harvested at reduced culture temperature (20°C) without destructive proteolytic enzymes for 30 minutes (Figure 29B). HE staining clearly showed a continuous cell sheet with regular tight cell-cell junctions and single bilayered cuboidal cells comprising the cell sheet (Figure 29C). All seeded temperature-responsive cell culture dishes yielded cell sheets, confirming the feasibility of the method used. The resulting human umbilical cord mesenchymal stem cell sheets could then be transferred to the rat uterus implantation site using a fine plastic spatula (Figure 29B).

[0110] After harvesting, the cell sheets were labeled with a fluorescent marker, carboxyfluorescein succinimidyl ester (CFSE), to allow identification of the cell sheets after transplantation. Non-pregnant female nude rats were used and a uterine incision was performed to induce scar formation (FIG. 21). Schematic diagrams and photographs of the cell transplantation process are provided in FIGS. 28A-28B.

[0111] A longitudinal incision was made in each horn of the uterus of a diploic rat. After suture repair of both uterine incisions, a hUC-MSC sheet was transplanted onto the surface of the uterine incision repair in one horn of the uterus, while the contralateral horn of the uterus served as a control. In the transplantation group, the surface area was approximately 1 cm 2hUC-MSC sheets were transplanted into the uterus, and the location of the transplanted cell sheet was confirmed as a green area by fluorescence microscopy (Figure 22). For transplantation, the cell sheet was suspended in medium in a cell culture dish and then transferred to a rectangular plastic sheet with a width of approximately 1 cm. The cell sheet was transferred to the uterus on the plastic sheet. The cell sheet was transferred to the uterine wound by sliding it off the plastic sheet with forceps (Figure 22). The shape and adhesive extracellular matrix of the cell sheet allowed it to be directly fixed to the uterine wound without any scaffold or sutures.

[0112] Proper placement and positioning of the cell sheets was confirmed by fluorescence microscopy and evaluated on days 1, 3, and 7 after surgery. Uteri were harvested for macroscopic analysis and histological evaluation with hematoxylin-eosin (HE) and Masson's trichrome staining on days 1, 3, 7, and 14 after surgery. The fibrous (blue) and normal (purple / red) myometrial surfaces were analyzed using AmScope® software, and the ratio of fibrous to normal myometrial surface area was calculated for each specimen.

[0113] Expression of human hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGFA) was measured by quantitative real-time PCR. Scars from control and cell sheet-implanted horns were harvested on days 1, 3, 7 and 14 and frozen in liquid nitrogen (n=3 horns per group). RNA was then extracted from each sample using the Rneasy® Fibrous Tissue Mini Kit (74704, Qiagen, Germany) after each horn was minced with a mechanical homogenizer. cDNA was synthesized using the High Performance cDNA Reverse Transcription Kit (4368814, Thermo Fisher Scientific) and subjected to TaqMan® Gene Expression Assays (4331182, Thermo Fisher Scientific) and PCR analysis (StepOnePlus™ Real-Time PCR System, 4376600, Thermo Fisher Scientific) using PCR probes specific for each target gene.

[0114] result Eighteen uterine specimens, two or four from each horn of the uterus, from six rats were included in this analysis. The presence of transplanted hUC-MSC sheets in the uterine wound was confirmed by fluorescence microscopy at 1, 3, and 7 days after transplantation (Figures 23 and 28A). The cell sheets spontaneously and stably attached to the horn wound site surface after 30 minutes without suturing. A group of uteri were harvested at 1, 3, 7, and 14 days after surgery. The transplanted cell sheets were easily detected in situ by direct fluorescence microscopy observation at 1 and 3 days. However, fewer fluorescent cell sheet fragments were noted at 7 days. By 14 days, no cells were observable (Figure 30A, top row). The boundary between the transplanted cell sheet and the uterine surface was well defined at 1 and 3 days, but became unclear at 7 and 14 days (Figure 30A, second and third rows). Green stained cells were present in the histological samples on days 1, 3, and 7, but absent on day 14. The presence of stained cells progressively decreased and was no longer evident on day 14 (FIG. 23 and FIG. 30A, bottom row).

[0115] In the control group, large fibrous areas were present between the host myometrial regions as a result of wound healing. In contrast, the fibrous areas in the hUC-MSC sheet transplantation group were significantly smaller than those in the control group (Figure 24). Specifically, the average fibrous surface area in the control group (n = 18) was 129185.7 μm 2 (range 60838.1~245836.1μm 2 ), whereas in the hUC-MSC sheet transplant group (n=18), it was 95861.6μm 2 (range 47090.5~154446.7μm 2) (Figure 25). Thus, application of the hUC-MSC sheet to the uterus reduced the fibrotic surface area of ​​the uterus by approximately 27% compared to the control group in which the hUC-MSC sheet was not applied. The average calculated ratios of fibrotic myometrium to normal myometrium surface in the control and hUC-MSC sheet transplantation groups were 0.28 and 0.19, respectively (p<0.01) (Figure 26). Thus, application of the hUC-MSC sheet to the uterus reduced the ratio of fibrotic myometrium to normal myometrium surface by more than 33% compared to the control group in which the hUC-MSC sheet was not applied. Thus, transplantation of the hUC-MSC sheet significantly reduced fibrosis of the myometrium surface.

[0116] The thickness of the incision area in both the control (n=18) and hUC-MSC sheet transplantation groups (n=18) was 191.5 μm (range 90.0-296.4 μm) and 274.3 μm (range 143.7-448.8 μm), respectively (p<0.01) (Figure 27).

[0117] Gene expression of human hepatocyte growth factor (HGF) and human vascular endothelial growth factor (VEGF) from cell sheet implanted uterus was measured on days 1, 3, 7 and 14 after implantation. Gene expression of human HGF message and human VEGF message from scarred control uterus was not detectable on all days. Differences in human gene expression between cell sheet implanted group and control group are significant on days 1 and 3 (*p<0.01). Gene expression from scarred control uterus was not detectable and is not shown.

[0118] The number of fibroblasts in the myometrial tissue was detected using immunostaining for fibroblast-specific S100A4 protein (Kong, et al., 2013, Am J Physiol Heart Circ Physiol. 305(9): p. H1363-72). The borders of the myometrium (dotted line), endometrium, and implanted cell sheet areas (arrows) were identified in HE-stained specimens. Using fibroblast-specific protein-specific immunostaining, S100A4-positive cells were counted in the myometrial area. 716 / mm in the control angle. 2 (SD:194 / mm2 ), the average number of fibroblasts in the cell sheet-implanted horns was 483 / mm 2 (SD:137 / mm 2 ) (FIG. 31B). The number of S100A4-positive cells in the control horn was significantly higher than that in the cell sheet transplantation group (p=0.0002).

[0119] Gene expression of human hepatocyte growth factor (HGF) and human vascular endothelial growth factor A (VEGFA) was detected on days 1 and 3 and was higher in the cell sheet-implanted corneas compared to the control corneas, but these genes were not expressed on days 7 and 14 (Figure 30B).

[0120] Consideration In an immunodeficient rat uterine incision model, we demonstrated that transplantation of human umbilical cord mesenchymal stem cell sheets into the uterine incision at the time of surgical repair is feasible, as judged by the development of appropriately sized sheets with natural adhesion and by retention of stem cells in the target area by immunohistochemistry several days later.Furthermore, we demonstrated that stem cell sheet transplantation stimulated improved wound healing, as demonstrated by reduced fibroblast recruitment in the uterine incision area, reduced fibrosis formation, increased myometrial thickness after surgery, and improved ratio of normal myometrial to fibrotic tissue.

[0121] This study used stem cells derived from human umbilical cord, which were previously used as a clinically injected cell suspension to treat human heart failure. Previous studies have 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 may serve to protect cells from some natural killer cell-induced death processes. All three non-canonical class I MHC proteins are expressed by supernumerary chorionic trophoblast cells and have been reported to be associated with maternal tolerance to semi-allogeneic embryos.

[0122] Most clinical stem cell preparations are administered as injection suspensions. Injected stem cell suspensions exhibit weak and 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, reducing their local effects. However, the cell sheet transplants described herein engrafted locally, spontaneously, rapidly, and efficiently, promoting maximum therapeutic effects at the transplantation site without migration. Thus, these human umbilical cord mesenchymal stem cell sheets exhibit host immune tissue compatibility, structural features believed to be important for human umbilical cord mesenchymal stem cells for optimal therapeutic and reliable engraftment process, reliable phenotypic traits, and tissue engagement.

[0123] This study extends the use of human umbilical cord mesenchymal stem cell sheets to uterine surgical applications and demonstrates feasibility. Cell sheets constructed from these stem cells, in contrast to infused or injected stem cells, retain critical cell adhesion proteins, including intercellular junction proteins associated with cell-cell communication and extracellular matrix proteins associated with cell adhesion, as well as the cytokine hepatocyte growth factor (HGF), which are related to therapeutic and engraftment efficacy (Kim, et al., 2019, Sci Rep, 2019. 9(1): p. 14415). These cell sheets could be optically tracked for up to 7 days after transplantation (see Figure 30). Human umbilical cord mesenchymal stem cell sheet transplantation was associated with increased uterine wall thickness, reduced wound site fibrosis, and reduced presence of wound fibroblasts. Human hepatocyte growth factor (HGF) and human vascular endothelial growth factor (VEGF), both of which are important for wound healing and tissue regeneration, were detected in human umbilical cord mesenchymal stem cell sheet-implanted horns during the early healing phase compared to control horns (Figure 30). These findings suggest that human umbilical cord mesenchymal stem cell sheet transplantation improves wound healing in nude rat uterine incision.Our data suggest that human umbilical cord mesenchymal stem cells applied as permanent living sheets are promising for uterine incision repair.

[0124] The exact mechanism of how transplanted stem cell sheets promote myometrial regeneration remains uncertain. However, the essential stem cell function is their paracrine effect through secretion of cytokines and chemokines, including plasminogen activator inhibitor-1 (PAI-1), macrophage migration inhibitory factor (MIF), and interleukin-6 (IL-6), which reduce inflammation, promote host tissue regeneration and proliferation, and VEGF and HGF, among others, which promote angiogenesis. Previous studies have shown that conversion of transplanted stem cells into myometrial tissue is unlikely to be a significant contributor to repair, at least outside the acute phase (Ho, CH, et al., 2018, J Chin Med Assoc, 81(3): p. 268-276). Furthermore, transplantation of allogeneic myometrial cells did not improve uterine incision wound healing in an animal uterine repair model (Ho et al., 2018, ibid).

[0125] The significant reduction in both fibrotic surface area and fibroblast abundance at the wound site in this model can be attributed to the inherent anti-inflammatory effect of the human umbilical cord mesenchymal stem cell sheet. Inflammatory cellular mediators (monocytes and macrophages) are attenuated upon human umbilical cord mesenchymal stem cell sheet transplantation (La Rocca, et al., 2009, Histochem Cell Biol, 131(2): p. 267-82).

[0126] conclusion These results demonstrate a reduction in fibrous tissue formation and an increase in myometrial regeneration following hUC-MSC sheet implantation into repaired uterine incisions of rat uteruses compared to controls without hUC-MSC sheet implantation. Thus, the hUC-MSC sheets described herein have the potential to improve uterine scar healing and reduce morbidity associated with abnormal uterine scar formation. The present invention includes, for example, the following embodiments: [Embodiment 1] A method for reducing the formation of fibrous tissue in the uterus of a subject in need thereof, comprising the step of applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, the MSC sheet comprising one or more layers of aggregated, confluent mesenchymal stem cells (MSCs), and wherein application of the MSC sheet to the uterus reduces the formation of fibrous tissue in the uterus compared to a uterus to which the MSC sheet is not applied. [Embodiment 2] A method for increasing myometrial regeneration in the uterus of a subject in need thereof, comprising the step of applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, the MSC sheet comprising one or more layers of aggregated, confluent mesenchymal stem cells (MSCs), and application of the MSC sheet to the uterus results in increased myometrial regeneration compared to a uterus to which the MSC sheet is not applied. [Embodiment 3] The method described in embodiment 1 or 2, wherein the MSC sheet is applied to the incision site of the uterus. [Embodiment 4] The method described in embodiment 1, wherein application of the MSC sheet to the uterus reduces the fibrous surface area of ​​the uterus by at least 20% compared to a uterus to which the MSC sheet is not applied. [Embodiment 5] The method described in embodiment 1 or 2, wherein the MSC sheet essentially consists of MSCs. [Embodiment 6] The method of embodiment 1 or 2, wherein the cell sheet comprises an extracellular matrix. [Embodiment 7] The method described in embodiment 6, wherein the extracellular matrix comprises one or more proteins selected from the group consisting of fibronectin, laminin, and collagen. [Embodiment 8] The method described in embodiment 1 or 2, wherein the cell sheet contains cell adhesion proteins and cell junction proteins. [Embodiment 9] The method of embodiment 8, wherein the cell junction protein is selected from the group consisting of vinculin, integrin-β1, connexin 43, β-catenin, integrin-linked kinase, and N-cadherin. [Embodiment 10] The method according to any one of embodiments 1 to 9, wherein the MSCs are isolated from the subepithelial layer of human umbilical cord tissue. [Embodiment 11] The method described in any one of embodiments 1 to 10, wherein the MSCs express a protein selected from CD44 and CD90. [Embodiment 12] The method described in any one of embodiments 1 to 11, wherein 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). [Embodiment 13] The method described in embodiment 12, wherein expression of the cytokine in the cell sheet is increased compared to an MSC suspension containing a comparable number of cells. [Embodiment 14] The method described in embodiment 13, wherein the cell sheet expresses the cytokine for at least 10 days after transplantation into the tissue of a host organism. [Embodiment 15] The method described in any one of embodiments 1 to 14, wherein the cell sheet expresses extracellular matrix proteins and cell junction proteins for at least 10 days after transplantation into the tissue of a host organism. [Embodiment 16] The method described in embodiment 15, wherein the extracellular matrix protein is selected from the group consisting of fibronectin, laminin and collagen. [Embodiment 17] The method of embodiment 15, wherein the cell junction protein is selected from the group consisting of vinculin, integrin-β1, connexin 43, β-catenin, integrin-linked kinase, and N-cadherin. [Embodiment 18] The initial cell density of the MSCs in the cell culture support used to prepare the cell sheet is 0.5 × 10 4 / cm 2 From 9×10 5 / cm 2 18. The method of any preceding embodiment, wherein [Embodiment 19] The method described in any one of embodiments 1 to 18, wherein 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). [Embodiment 20] The method of any one of embodiments 1 to 19, wherein the MSCs comprise microvilli and filopodia. [Embodiment 21] The method of any one of embodiments 1 to 20, wherein the cell sheet remains attached to the tissue of the host organism for at least 10 days after transplantation into the tissue. [Embodiment 22] The method of any one of embodiments 1 to 21, wherein the MSCs in the cell sheet are allogeneic to the subject. [Embodiment 23] The method of any one of embodiments 1 to 22, wherein the subject is a human. [Embodiment 24] The method of any one of embodiments 1 to 23, wherein the subject has had at least one cesarean section delivery. [Embodiment 25] The method of any one of embodiments 1 to 23, wherein the subject has had at least two cesarean deliveries. [Embodiment 26] The method described in any one of embodiments 1 to 25, wherein the subject has had at least one uterine surgery. [Embodiment 27] A method for preventing or reducing uterine incision tearing in a subject in need thereof, comprising the step of applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, the MSC sheet comprising one or more layers of aggregated confluent mesenchymal stem cells (MSCs), and wherein application of the MSC sheet to the uterus prevents or reduces tearing of the uterine incision compared to a uterine incision to which the MSC sheet is not applied. [Embodiment 28] The method described in any one of embodiments 1 to 27, wherein the MSCs are human umbilical cord mesenchymal stem cells (hUC-MSCs).

Claims

1. 1. A method for reducing fibrous tissue formation in a uterus of a subject in need thereof, comprising the step of applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, the MSC sheet comprising one or more layers of aggregated, confluent mesenchymal stem cells (MSCs), wherein application of the MSC sheet to the uterus reduces fibrous tissue formation in the uterus compared to a uterus to which the MSC sheet is not applied.

2. 1. A method for 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, the MSC sheet comprising one or more layers of aggregated, confluent mesenchymal stem cells (MSCs), wherein application of the MSC sheet to the uterus increases myometrial regeneration compared to a uterus to which the MSC sheet is not applied.

3. 3. The method of claim 1 or 2, wherein the MSC sheet is applied to the incision site of the uterus.

4. The method of claim 1, wherein application of the MSC sheet to the uterus reduces the fibrous surface area of ​​the uterus by at least 20% compared to a uterus to which the MSC sheet is not applied.

5. 3. The method of claim 1 or 2, wherein the MSC sheet consists essentially of MSCs.

6. The method of claim 1 or 2, wherein the cell sheet comprises an extracellular matrix.

7. The method of claim 6, wherein the extracellular matrix comprises one or more proteins selected from the group consisting of fibronectin, laminin, and collagen.

8. The method of claim 1 or 2, wherein the cell sheet comprises cell adhesion proteins and cell junction proteins.

9. 9. The method of claim 8, wherein the cell junction protein is selected from the group consisting of vinculin, integrin-β1, connexin 43, β-catenin, integrin-linked kinase, and N-cadherin.

10. 10. The method of any one of claims 1 to 9, wherein the MSCs are isolated from the subepithelial layer of human umbilical cord tissue.

11. 11. The method of any one of claims 1 to 10, wherein the MSCs express a protein selected from CD44 and CD90.

12. 12. The method of any one of claims 1 to 11, wherein 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).

13. 13. The method of claim 12, wherein expression of said cytokines in said cell sheet is increased compared to an MSC suspension containing a comparable number of cells.

14. The method of claim 13, wherein the cell sheet expresses the cytokine for at least 10 days following transplantation into a tissue of a host organism.

15. 15. The method of any one of claims 1 to 14, wherein the cell sheet expresses extracellular matrix proteins and cell junction proteins for at least 10 days after transplantation into a tissue of a host organism.

16. 16. The method of claim 15, wherein the extracellular matrix protein is selected from the group consisting of fibronectin, laminin, and collagen.

17. 16. The method of claim 15, wherein the cell junction protein is selected from the group consisting of vinculin, integrin-β1, connexin 43, β-catenin, integrin-linked kinase, and N-cadherin.

18. The initial cell density of the MSCs in the cell culture support used to prepare the cell sheet was 0.5×10 4 / cm 2 From 9×10 5 / cm 2 18. The method according to any one of claims 1 to 17, wherein

19. 19. The method of any one of claims 1 to 18, wherein 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).

20. 20. The method of any one of claims 1 to 19, wherein the MSCs comprise microvilli and filopodia.

21. 21. The method of any one of claims 1 to 20, wherein the cell sheet remains attached to the tissue of the host organism for at least 10 days following transplantation into the tissue.

22. 22. The method of any one of claims 1 to 21, wherein the MSCs in the cell sheet are allogeneic to the subject.

23. 23. The method of any one of claims 1 to 22, wherein the subject is a human.

24. 24. The method of any one of claims 1 to 23, wherein the subject has had at least one cesarean delivery.

25. 24. The method of any one of claims 1 to 23, wherein the subject has had at least two cesarean deliveries.

26. 26. The method of any one of claims 1 to 25, wherein the subject has had at least one uterine surgery.

27. 1. A method for preventing or reducing tearing of a uterine incision in a subject in need thereof, comprising applying a mesenchymal stem cell (MSC) sheet to the uterus of the subject, the MSC sheet comprising one or more layers of aggregated, confluent mesenchymal stem cells (MSCs), wherein application of the MSC sheet to the uterus prevents or reduces tearing of the uterine incision compared to a uterine incision to which the MSC sheet is not applied.

28. 28. The method of any one of claims 1 to 27, wherein the MSCs are human umbilical cord mesenchymal stem cells (hUC-MSCs).

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