Human umbilical cord mesenchymal stem cell sheets and methods for their production

Abstract

To provide therapeutic uses of human umbilical cord MSCs (hUC-MSCs), a promising cell source for stem cell therapy.SOLUTION: The disclosure provides a human umbilical cord mesenchymal stem cell sheet comprising one or more layers of confluent human umbilical cord mesenchymal stem cells (hUC-MSCs). The disclosure also provides a method for producing hUC-MSC sheets comprising: culturing hUC- MSCs in a culture solution on a temperature-responsive polymer which has been coated onto a substrate surface of a cell culture support, the temperature-responsive polymer having a lower critical solution temperature in water of 0-80°C; adjusting a temperature of the culture solution to below the lower critical solution temperature, whereby the substrate surface is made hydrophilic and adhesion of the cell sheet to the surface is weakened; and detaching the cell sheet from the culture support.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 793,199, filed January 16, 2019, the contents of which are incorporated herein in their entirety. [Background technology]

[0002] Mesenchymal stem cells (MSCs) are multipotent somatic stem cells that can differentiate into osteoblasts, chondrocytes, neurons, skeletal muscle cells, vascular endothelial cells, and cardiac myocytes (Reyes et al., 2002, J. Clin. Invest. 109; 337-346; Toma et al., 2002, Circulation 105, 93-98; Wang et al., 2000, J. Thorac. Cardiovasc. Surg. 120, 999-1005; Jiang et al., 2002, Nature 41S, 41-49). The therapeutic properties of MSCs have been proposed to stem from their intrinsic ability to 1) differentiate into diverse and distinct cell lineages, 2) produce numerous soluble bioactive factors important for cell maintenance, survival and proliferation, 3) modulate the host immune response, and 4) migrate following recruitment to the site of injury to reduce injury and promote healing (Squillaro et al., 2016, Cell Transplant, 25(5), 829-848). In particular, among the various MSC types, human umbilical cord MSCs (hUC-MSCs) are a promising cell source for stem cell therapy, which has seen increasing clinical findings (Bartolucci et al., 2017, Circ Res, 121(10), 1192-1204; Ichim et al., 2010, Int Arch Med, 3, 30; Riordan et al., 2018, J Transl Med, 16(1), 57; Tuma et al., 2016, Cell Transplant, 25(9), 1713-1721). Thus, hUC-MSCs have great potential for various therapeutic uses. Summary of the Invention

[0003] In certain aspects, the present disclosure relates to a human umbilical cord mesenchymal stem cell sheet comprising one or more layers of confluent human umbilical cord mesenchymal stem cells (hUC-MSCs). In certain embodiments, the cell sheet consists essentially of hUC-MSCs. In certain embodiments, at least 50% of the cells in the cell sheet are hUC-MSCs. In certain embodiments, the cell sheet comprises an extracellular matrix. In certain embodiments, the extracellular matrix comprises one or more proteins selected from the group consisting of fibronectin, laminin, and collagen. In certain embodiments, the cell sheet comprises cell adhesion proteins and cell-cell junction proteins. In certain 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 certain embodiments, the hUC-MSCs are isolated from the subepithelial layer of human umbilical cord tissue. In certain embodiments, the hUC-MSCs express a protein selected from CD44, CD73, CD105, and CD90. In certain embodiments, the hUC-MSCs express one or more cytokines selected from the group consisting of human hematopoietic growth factor (HGF), vascular endothelial growth factor (VEGF), and interleukin-10 (IL-10). In certain embodiments, expression of the one or more cytokines in the cell sheet is increased compared to a hUC-MSC suspension containing a comparable number of cells. In certain embodiments, the cell sheet secretes tumor necrosis factor-α (TNF-α) into culture medium at a rate of less than 50 pg per mL of culture medium in 24 hours. In certain embodiments, the cell sheet expresses the one or more cytokines for at least 10 days after implantation into a tissue of a host organism. In certain 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 certain embodiments, the extracellular matrix proteins are selected from the group consisting of fibronectin, laminin, and collagen. In certain 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.In a particular embodiment, the initial cell density of the hUC-MSCs seeded in the cell culture support used to prepare the cell sheet is 0.5×10. 4 / cm 2 From 9×10 5 / cm 2 In certain embodiments, the hUC-MSCs do not express one or more of CD31, CD45, human leukocyte antigen-DR isotype (HLA-DR), human leukocyte antigen-DP isotype (HLA-DP), or human leukocyte antigen-DQ isotype (HLA-DQ). In certain embodiments, the hUC-MSCs comprise microvilli and filopodia. In certain embodiments, the cell sheet remains attached to the tissue of the host organism for at least 10 days after transplantation into the tissue.

[0004] In certain aspects, the present disclosure relates to compositions comprising the cell sheets described herein and polymer-coated culture supports that are removable from said cell sheets.

[0005] In certain aspects, the present disclosure relates to a method for generating a human umbilical cord mesenchymal stem cell sheet comprising one or more layers of confluent human umbilical cord-derived mesenchymal stem cells (hUC-MSCs), the method comprising: (a) culturing hUC-MSCs in a culture medium on a temperature-responsive polymer coated on a 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, thereby rendering the substrate surface hydrophilic and weakening adhesion of the cell sheet to the surface; and (c) detaching the cell sheet from the culture support.

[0006] In certain embodiments, the method further comprises culturing the hUC-MSCs through multiple subcultures prior to the culturing step (a). In certain embodiments, 2-10 subcultures of the hUC-MSCs are performed prior to the culturing step (a). In certain embodiments, the culture medium is a xeno-free culture medium. In certain embodiments, the culture medium comprises human platelet lysate (hPL). In certain embodiments, the culture medium comprises fetal bovine serum (FBS). In certain embodiments, the culture medium comprises ascorbic acid. In certain embodiments, the conditioning step (b) is performed when the hUC-MSCs are confluent. In certain embodiments, the culture step (a) comprises culturing the hUC-MSCs at 0.5×10 4 / cm 2 From 9×10 5 / cm 2 and adding the hUC-MSCs to the culture medium at an initial cell seeding density of 100-200 μg / ml. In certain embodiments, the hUC-MSCs are cultured in the culture medium on the temperature-responsive polymer for at least 24 hours prior to the conditioning step (b). In certain aspects, the present disclosure relates to cell sheets produced by the methods described herein.

[0007] In certain aspects, the present disclosure relates to a method of transplanting a cell sheet into a subject, comprising applying a cell sheet described herein to a tissue of the subject. In certain embodiments of the methods and cell sheets described herein, the hUC-MSCs in the cell sheet are allogeneic to the subject. In certain embodiments of the methods and cell sheets described herein, the subject is a human. [Brief description of the drawings]

[0008] [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 1 h at room temperature (RT) without the use of proteolytic enzyme treatment, preserving functional structures such as 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 in the form of aggregates without TRCD temperature change on days 5, 6, and 7 for the 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] Figure 5A-5E show the cell-cell structure analysis using immunohistochemistry (IHC) and transmission electron microscopy (TEM). 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 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, red arrows = ECM; yellow arrows = cell junctions. Scale bars indicate 100 μm in (a-c). 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 hu-MSC cell sheet was clearly identified in the subcutaneous tissue implantation site compared with (a) the normal subcutaneous tissue. Moreover, (e) abundant vascular structures were observed in 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 by temperature shift, B) cells were treated with enzyme (trypsin) to generate single cell suspensions and cell clumps, and C) cells were harvested using a cell scraper. [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 cell sheet was performed by H&E staining. The scale bar indicates 200 μm in A and D and 10 mm in C. [Figure 11A-H] Figure 11A-H show morphological observations of hUC-MSCs and hUC-MSC sheets. (A) The morphology of the cell surface was observed using scanning electron microscopy (SEM). (B) The microstructure of hUC-MSC sheets and hUC-MSCs was analyzed using transmission electron microscopy (TEM). The white dashed arrow in (E) points to the ECM, and the dark grey arrow in (H) points to the endoplasmic reticulum. Scale bars in SEM and TEM = 5 μm. [Figure 12A-C] Figures 12A-C show cell dynamics-related protein expression analysis using Western blot and immunohistochemistry. (A) Western blot of F-actin, vinculin and GAPDH in whole cell lysates (10 mg protein / lane). (B) Immunostaining of F-actin, (C) vinculin and DAPI (blue). Scale bar = 10 μm. [Figure 13A-C] Figures 13A-C show ECM protein expression analysis using Western blot and immunohistochemistry. (A) Western blot of fibronectin, laminin and GAPDH in whole cell lysates (10 mg protein / lane). (B) Fibronectin, (C) Laminin immunostaining and DAPI (blue). Scale bar = 10 μm. [Figure 14A-C]Figures 14A-C show cell-ECM and cell-cell junction protein expression analysis using Western blot and immunohistochemistry. (A) Western blot of integrin β-1, connexin 43 and GAPDH in whole cell lysates (10 mg protein / lane). (B) Immunostaining of integrin β-1, (C) connexin 43 and DAPI (blue). Scale bars = 10 μm. [Figure 15A-C] 15A-B show live and dead cell assays: (A) Live and dead staining of cell sheets and cell suspensions. Cells were stained with calcein and ethidium homodimer-1 immediately after cell detachment. Scale bar = 100 μm. [Figure 16] Figure 16 shows mechanosensor expression analysis using Western blot. 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 in vivo HGF expression in a hUC-MSC sheet implanted into 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 HGF expression, the samples were stained with a 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-20B show HLA DR, DP, and DQ expression in hUC-MSC single cell suspension cultures (A) and cell sheets (B). HLA expression was measured in single cell suspension cultures from passage 4 to passage 12 (A). The percentages in (A) represent the percentages of cells expressing HLA. HLA-DR gene expression was undetectable in hUC-MSC sheets, whereas cell sheets 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 (B). [Fig. 21A-E] Figures 21A-21E show the culture and doubling time of hUC-MSCs affected by FBS or hPL medium. MSCs were cultured with FBS (A,C) or hPL medium (B,D) at P6 (A,B) or P12 (C,D). The cell morphology was observed (A-D) and the cell number was counted during cell culture for all passages to calculate the cell doubling time (E). [Fig. 22A-D] Figures 22A-D show the differentiation potential of hUC-MSCs as affected by FBS or hPL medium. P6 MSCs were cultured in FBS (A, C) or hPL (B, D) medium before inducing differentiation. Osteogenic differentiation marked by Alizarin Red staining (A, B) and adipogenic differentiation marked by Oil Red O staining (C, D). [Fig. 23A-L] Figures 23A-L show the phenotype of hUC-MSCs influenced by FBS or hPL medium. P6 hUC-MSCs showed positive expression of CD73, CD105, and CD90, and negative expression of MHC II, CD45, and CD31 in FBS (A-F) and hPL (G-L) medium cultures. [Fig. 24A-H]Figures 24A-O show a comparison of hUC-MSC sheets in FBS and hPL media. When cells reached confluence on the TRCD (A, C, E, and G), the MSC sheets were detached by changing the culture temperature from 37°C to room temperature. Cells cultured beyond confluence (B, D, F, and H) produced cell sheets that were successfully detached in the form of sheets in FBS media (B and F), whereas cell sheets prepared in hPL media spontaneously detached from the TRCD at 37°C without any temperature change (D and H). Scale bars represent 100 μm (A-D, J, and K), 1 cm (E-H), and 50 μm (M and N). *p<0.05 (N=3-4). [Fig. 24I-O] Figures 24A-O show the comparison of hUC-MSC sheets in FBS and hPL media. Cell sheets prepared in hPL media were detached in sheet shape faster than those in FBS media after temperature reduction to room temperature (I). H&E staining is performed to observe the structural changes of MSC sheets in FBS (J) and hPL (K) media. The cytoskeleton structure of MSCs cultured in FBS (M) or hPL (N) media was imaged by phalloidin staining. The gene expression levels of β-actin and ITGB1 in FBS or hPL groups were investigated by q-PCR analysis (L, O). Scale bars represent 100 μm (A-D, J, and K), 1 cm (E-H), and 50 μm (M and N). *p<0.05 (N=3-4). [Fig. 25A-K] Figures 25A-K show cell sheet specific structure and characteristics analysis using IHC and q-PCR. Fibronectin (Fb) (B, F), β-catenin (β-CTNN) (C, G) and MHC II (D, H) positive areas in MSC sheets prepared in FBS (A-D) or hPL (E-H) medium were imaged and compared with the negative control group (A, E). Gene expression levels of Fb (I), β-CTNN (J), MHC II (K) in FBS and hPL groups were investigated. Non-significant (NS) differences in gene expression levels were observed for Fb (I), β-CTNN (J) and MHC II (K) (N=3-4). Scale bar represents 200 μm. [Figure 26A-C]Figures 26A-C show cytokine secretion of human HGF from hUC-MSC sheets over 24 hours (A). Immediately after harvesting the supernatant, the cells were counted (B). The amount of hHGF was normalized to the cell number of each group (C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] This disclosure describes the preparation and characterization of human umbilical cord mesenchymal stem cell (hUC-MSC) sheets to improve MSC engraftment efficiency and retention at target tissue sites in MSC therapy. Currently, injected MSC cell suspensions are harvested using enzymes, which impair MSC function and engraftment capacity, resulting in poor tissue retention and survival, and suboptimal therapeutic properties. For example, as described herein, cell sheets made without enzymes as living sheets with extracellular matrix (ECM) and intact cell receptors can be physically placed at tissue sites with highly improved retention and engraftment efficiency.

[0010] Human umbilical cord mesenchymal stem cells (hUC-MSCs) were used to prepare cell sheets in vitro on temperature-responsive cell culture dishes (TRCDs) coated with a temperature-responsive polymer. Confluent cell sheets were formed 4–6 days after seeding and were detached from the TRCD by cooling the culture to room temperature. Various culture conditions were identified that allowed for the successful generation of robust and uniform hUC-MSC sheets containing a monolayer 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 fetal bovine serum (FBS) or human platelet lysate (hPL) to the cell culture medium, and the culture time in the TRCD before detachment from the temperature-responsive polymer. The hUC-MSC sheets generated by these methods showed several beneficial properties compared to current injectable cell suspensions for improving allogeneic MSC cell therapy. These beneficial properties include sustained secretion of cytokines, low HLA expression profile, in vivo retention of intact hUC-MSC sheets on the implanted target tissue site for 10 days, and neovascular recruitment to the sheets on the target tissue. Furthermore, the harvested monolayer hUC-MSC sheets retained tissue-like architecture, extracellular matrix (ECM), cell-cell junctions, and cell-ECM junctions, and had higher cell viability compared to conventional chemical disruption methods such as trypsinization. With reliable deployment to local tissue sites, high engraftment efficiency, and long-term retention and survival in vivo, the hUC-MSC sheets generated by the methods described herein have the potential to greatly improve the therapeutic value of allogeneic cell therapy compared to currently used injectable mesenchymal stem cell suspensions.

[0011] In some embodiments, other mesenchymal stem cells (MSCs) can be used to prepare cell sheets having one or more of the beneficial characteristics of hUC-MSC sheets described herein, including, but not limited to, sustained secretion of cytokines, low HLA expression profile, in vivo retention of an intact MSC sheet above the implantation target tissue site for 10 days, and neovascular recruitment to the sheet above the target tissue.

[0012] I. Human umbilical cord MSCs (hUC-MSCs) As used herein, the term "human umbilical cord mesenchymal stem cells" or "hUC-MSCs" refers to mesenchymal stem cells isolated from human umbilical cord.

[0013] 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).

[0014] 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).

[0015] 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 a monolayer 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 from harvested MSC suspensions in several ways. Suspensions of hUC-MSCs contain single cells that do not have ECM or intercellular junctions, as the adhesive proteins in their intercellular junctions must be removed (e.g., by trypsinization) to harvest the cells from the culture surface for preparation of cell suspension cultures. In contrast to single cell suspensions of hUC-MSCs, the hUC-MSC sheets described herein contain ECM and intercellular junctions between the hUC-MSCs that are generated during the formation of the cell sheet. The intact ECM and cell-cell junctions facilitate the attachment of hUC-MSC sheets to target tissues during transplantation into the host organism.

[0016] II. Cell sheets generated from human umbilical cord MSCs (hUC-MSCs) In certain aspects, the present disclosure relates to a human umbilical cord mesenchymal stem cell sheet comprising one or more layers of confluent human umbilical cord mesenchymal stem cells (hUC-MSCs). As used herein, the term "human umbilical cord mesenchymal stem cell sheet" or "hUC-MSC sheet" refers to a cell sheet obtained by growing human umbilical cord mesenchymal stem cells on a cell culture support in vitro. The one or more layers of hUC-MSC sheets described herein are harvested as sheets at temperature shift using a temperature-responsive culture dish (TRCD) without any enzymatic treatment. The hUC-MSC sheets maintain their sheet 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 related to cell therapy. Thus, the cell sheets described herein can include structural features that improve cell survival and cell function, including extracellular matrix, cell adhesion proteins, and cell junction proteins. Thus, the hUC-MSC sheets prepared by the methods described herein have several beneficial characteristics compared to MSCs generated by other methods. For example, chemical destruction (proteolytic enzyme treatment) is widely used on cells harvested for stem cell therapy. However, because the enzyme treatment destroys extracellular and intracellular proteins (intercellular and cell-ECM junctions), chemical destruction methods cannot maintain the tissue-like structure and cell-cell communication of cells. Therefore, protein cleavage by enzymes reduces cell viability and cell functions relevant to cell therapy.

[0017] 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.

[0018] The hUC-MSCs in the cell sheet 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 function and signaling.

[0019] In some embodiments, the cell sheet consists of hUC-MSCs. In some embodiments, the cell sheet consists essentially of hUC-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 hUC-MSCs. In some embodiments, 100% of the cells in the cell sheet are hUC-MSCs.

[0020] To optimize the formation or characteristics of the cell sheet, hUC-MSCs can be added to the culture medium on the temperature-responsive polymer in the cell culture support at various cell densities. For example, by controlling the initial cell density of hUC-MSCs in the cell culture support (e.g., TRCD), the cytokine expression level in hUC-MSCs can be optimized. In some embodiments, increasing the initial cell density of hUC-MSCs in the cell culture support increases cytokine expression (e.g., HGF). In some embodiments, decreasing the initial cell density of hUC-MSCs in the cell culture support decreases cytokine expression. In some embodiments, the initial cell density of hUC-MSCs in the cell culture support used for the preparation of the cell sheet is 0.5×10 4 / cm 2 From 9×10 5 / cm 2 In some embodiments, the initial cell density of hUC-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, 7×10 4 , 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 hUC-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.

[0021] The hUC-MSC sheet described herein can be transplanted into a target tissue of a host organism (e.g., human) for therapeutic use. Transplantation of the hUC-MSC sheet into the target tissue can result in the formation of 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 an important function for sheet engraftment, cell viability, and tissue regeneration. Furthermore, this new blood vessel recruitment to the sheet on the target tissue suggests that the implanted hUC-MSC sheet continuously secretes paracrine factors to modulate engraftment.

[0022] In some embodiments, the hUC-MSC sheet expresses one or more cytokines, such as one or more anti-inflammatory cytokines or one or more inflammatory cytokines. In some embodiments, the anti-inflammatory cytokine is selected from human 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, the expression of a cytokine (e.g., an anti-inflammatory cytokine or a proinflammatory cytokine) in the cell sheet is increased compared to a hUC-MSC suspension containing a comparable number of cells. In some embodiments, the expression of a cytokine (e.g., an anti-inflammatory cytokine or a proinflammatory cytokine) is decreased compared to a hUC-MSC suspension containing a comparable number of cells. For some therapeutic uses, it may be beneficial to reduce the 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.

[0023] The hUC-MSC sheet described herein can continuously express cytokines after transplantation into a 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 a 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 a tissue of a host organism.

[0024] The hUC-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.

[0025] 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 hUC-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.

[0026] 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 (DP, DM, DO, DQ and DR) corresponding to MHC class II present antigens from the outside of the cell to T lymphocytes. These antigens are then transferred to 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. Thus, minimizing the expression of HLA would be beneficial in minimizing immune responses to the hUC-MSC sheet transplanted into the host organism. In some embodiments, the hUC-MSC sheets described herein do not express one or more of 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, 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).

[0027] III. Methods for generating human umbilical cord MSC (hUC-MSC) sheets in vitro In certain aspects, the present disclosure provides a method for generating a cell sheet comprising a monolayer of aggregated, confluent human umbilical cord mesenchymal stem cells (hUC-MSCs), comprising: a) culturing hUC-MSC in a culture solution of a temperature-responsive polymer coated 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 (e.g., by water penetration); and c) Detaching the cell sheet from the culture support The present invention relates to a method comprising the steps of:

[0028] Methods for isolating human umbilical cord mesenchymal stem cells (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 a 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. SLs can be placed on top of a substrate in a cell culture dish without additional pretreatment to cell culture-treated plastic, or 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 cultures can then be cultured under normoxic or hypoxic culture conditions for a time sufficient to establish a primary cell culture (e.g., 3-7 days). 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.

[0029] 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 by reference in its entirety.

[0030] 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.

[0031] 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.

[0032] The substrate to be coated with the polymer can be any type commonly used in cell culture, including glass, modified glass, polystyrene, poly(methyl methacrylate), polyester, and ceramic.

[0033] Methods for coating a support with a temperature-responsive polymer are known in the art and are described, for example, in JP 211865 / 1990. Specifically, such coating can be achieved by subjecting the substrate and the above-mentioned monomer or polymer to, for example, electron beam (EB) exposure, irradiation with gamma rays, irradiation with ultraviolet rays, plasma treatment, corona treatment or organic polymerization reaction. Other techniques can be used, such as physical adsorption, as achieved by application and kneading of the coating.

[0034] 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.

[0035] The cultured cells can be detached and collected from the cell culture support by adjusting the temperature of the support to a temperature at which the polymer on the support substrate can hydrate and thus detach the cells. 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 any other isotonic liquid, whichever is suitable. In some embodiments, after culturing the hUC-MSCs in the culture medium on the temperature-responsive polymer for at least 12 hours, at least 24 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days, the temperature of the culture medium is adjusted below the lower critical solution temperature for the release of the cell sheet from the support.

[0036] 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 dehydrates in water at a temperature above 31° C., and the polymer chains aggregate and cause turbidity. Conversely, at temperatures below 31° C., the polymer chains 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, for example, by chemical or physical grafting or tethering. Thus, at temperatures above 31° C., the polymer on the substrate surface also dehydrates, but the polymer chains cover the substrate surface and are immobilized thereon, so that the substrate surface becomes hydrophobic with the dehydration of the polymer. Conversely, at temperatures below 31° C., the polymer on the substrate surface is hydrated, but the polymer chains cover the substrate surface and are immobilized thereon, so that the substrate surface becomes hydrophilic with the dehydration of the polymer. 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.

[0037] 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 culture media comprises human platelet lysate (hPL). In some embodiments, the culture media comprises fetal bovine serum (FBS). In some embodiments, cell sheets grown in culture media containing hPL grow to high density more quickly, exhibit faster detachment at lower temperatures, weaker cell adhesion, and tend to form cell aggregates more readily upon release under poorly controlled sheet-forming conditions, even without temperature reduction, compared to cell sheets grown in culture media containing FBS. In some embodiments, cell sheets grown in culture media containing hPL secrete more human growth factor (HGF) compared to cell sheets grown in culture media containing FBS. In some embodiments, cell sheets grown in culture media containing hPL have a higher cell density per sheet compared to cell sheets grown in culture media containing FBS.

[0038] 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] The hUC-MSCs may 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, the 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, the hUC-MSCs are subcultured 2-10 times, 4-8 times, or 1-12 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] hUC-MSC sheets can be prepared in a range of different sizes depending on the application. In some embodiments, the hUC-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 the range of sizes of hUC-MSC sheets. For example, in some embodiments, the hUC-MSC sheets have a diameter of 1 to 20 cm, 1 to 10 cm, or 2 to 10 cm. In some embodiments, the hUC-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 hUC-MSC sheet. For example, in some embodiments, the hUC-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 for Transplanting Human Umbilical Cord MSC (hUC-MSC) Sheets into Subjects The cell sheet described herein can be transplanted into a subject by applying it to the tissue of the subject. For example, as disclosed in Example 1 below, when a hUC-MS sheet was prepared by the method described herein and implanted into the dorsal subcutaneous pocket of an immunodeficient mouse, the cell sheet was stably engrafted into the subcutaneous tissue 10 days after implantation. In addition, on the 10th day after implantation, capillaries were formed in the tissue in which the cell sheet was implanted (angiogenesis), whereas only a few thin blood vessels were observed in the subcutaneous tissue in which the cell sheet was not implanted. Furthermore, in animals in which the cell sheet was implanted, numerous vascular structures were observed between the implanted cell sheet and the host tissue. These results indicate that the cell sheet of the present invention can be implanted, can be engrafted, and the cell sheet structure can be preserved in vivo for 10 days, and can induce capillary angiogenesis, which is an important ability for engraftment, viability, and tissue regeneration.

[0042] Thus, in some aspects, the present disclosure relates to a method of transplanting a cell sheet into a subject, comprising applying the cell sheet described herein to a tissue of the subject. In certain embodiments, the subject is a human. One advantage of the hUC-MSC sheet described herein is that the extracellular matrix of the cell sheet acts as an adhesive to bind the cell sheet to the tissue of the subject, so that no stitches are required to attach the cell sheet to the tissue. A support membrane can be used to release the harvested hUC-MSC sheet from the culture surface and transfer it to the tissue of the subject. Support membranes for such transfer can be, for example, poly(vinylidene difluoride) (PVDF), cellulose acetate, and cellulose esters. 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 hUC-MSC sheet adheres to the target tissue within 5, 10, 15, 20, 25, or 30 minutes of contact with the tissue. Once the hUC-MSC sheet has adhered to the target tissue, the support membrane can be excised. In certain embodiments, the hUC-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 thus not genetically identical at one or more loci. In certain reported cases, MSCs appear to avoid allogeneic rejection in humans and animal models (Jiang et al., 2005, Blood, 105(10), 4120-4126). Thus, the hUC-MSC sheets described herein can be used in allogeneic cell therapy as an off-the-shelf product, avoiding the unfavorable costs and developmental disincentives associated with autologous stem cell therapy methods.

[0043] 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, as described above, the hUC-MSC cell sheet described herein is stably engrafted in the subcutaneous tissue 10 days after transplantation. Therefore, the hUC-MSC sheet described herein provides distinct advantages compared to mesenchymal stem cell suspensions.

[0044] [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.

[0045] 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.

[0046] 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 2 Cells 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.

[0047] 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 5Cells 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 × 10 5 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.

[0048] 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 2Passage 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).

[0049] 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).

[0050] 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).

[0051] 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.

[0052] 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, Pharmacia ...

[0053] 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.

[0054] 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 with TapMan universal PCR master mix using an Applied Biosystems Step One instrument (Applied Biosystems™, Foster City, USA). Gene expression levels were assessed for the following genes: 1) glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs02786624_g1) as housekeeping gene, 2) integrin-linked kinase (ILK, Hs00177914_m1), 3) N-cadherin (N-cad, Hs00983056_m1). All primers were manufactured by Applied Biosystems (the respective sequences are shown in Supplementary Data Table S1). 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.

[0055] result Preparation of hUC-MSC sheets using different initial cell numbers and passage numbers hUC-MSCs were cultured in flasks and subcultured using trypsin every 5 days from passage 4 to 12 (Table 1). Cells expanded 16-20 fold from the initial cell seeding number during passages 4 to 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 to 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.

[0056] [Table 1] Table 1. Proliferation rate of hUC-MSCs during passages 4–12

[0057] 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.

[0058] hUC-MSC surface marker characterization CD44 and CD90 are known to be expressed in hUC-MSC. CD44 and CD90 expression was measured in hUC-MSC suspension culture and hUC-MSC sheet in vitro. As shown in FIG. 4, hUC-MSC expressed CD44 and CD90 in suspension culture (FIG. 4A and FIG. 4B) and cell sheet (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 sheet maintained the hUC-MSC-specific phenotype. In particular, the results in FIG. 4C and FIG. 4D indicate that the cell sheet contained hUC-MSC and did not contain other cell types and differentiated cells derived from the umbilical cord.

[0059] 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.

[0060] 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.

[0061] 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).

[0062] 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.

[0063] 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.

[0064] 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).

[0065] 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 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.

[0066] 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.

[0067] Central to these results is the reliable ability to produce stable and robust monolayers of hUC-MSCs using commercially available TRCDs grafted with a temperature-responsive polymer coating that facilitates cell harvesting without destructive enzymes using reduced temperature (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).

[0068] 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.

[0069] 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, homogenous monolayer 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.

[0070] [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.

[0071] 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.

[0072] 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.

[0073] 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).

[0074] 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).

[0075] 2.6 Cell viability assay Cell viability was measured by live and dead viability / cytotoxicity assay (Thermo Fisher Scientific, MA). 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:

[0076]

number

[0077] The percentage of dead cells was calculated to compare cell viability in each sample, as shown in FIG. 15B.

[0078] 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.

[0079] 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).

[0080] 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.

[0081] 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 TRCDs. 5Cells were seeded at a density of 100 μg / ml 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 proliferation and morphology of the cells. 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 a monolayer and maintained cell-binding proteins like the native structure (Figure 10d).

[0082] Morphological observation of hUC-MSC sheets The surface and intercellular structures of hUC-MSC sheets were observed by scanning electron microscopy (SEM) (Fig. 11A-D) and transmission electron microscopy (TEM) (Fig. 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 native structures formed when they were cultured on cell culture dishes. Native cell membrane structures are composed of cell surface proteins and membrane proteins, which are related to cell adhesion and function. This finding suggests that hUC-MSC sheets can retain cell surface proteins and membrane proteins and improve cell adhesion and cell function (Albuschies et al., 2013, Sci Rep 3: 1658). In contrast, hUC-MSCs treated with 0.05% trypsin showed single-cell shape without connected tissue (Fig. 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.

[0083] 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 filopodia on their cell surface and had an unclear shape 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 disruption) causes damage to cell and tissue structures (i.e., junction proteins, ECM, nuclei, and endoplasmic reticulum).

[0084] 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 disrupts actin in the cytoplasm. To observe the cytoskeleton structure, hUC-MSCs were stained with actin. When cells are attached to the cultureware surface, actin forms stress fiber structures that play 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 structures. 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.

[0085] Vinculin is a membrane cytoskeleton protein that forms focal adhesions by linking the integrin receptor 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.

[0086] 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 fibronectin expression. 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.

[0087] To observe the structure of ECM proteins, 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 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 detachment of cells from the cell culture dish.

[0088] hUC-MSC sheets maintain cell junction proteins associated with cell communication Integrin β-1 is a major member of the integrin family, which are transmembrane proteins that form cell-ECM junctions. Integrins are linked to the actin cytoskeleton through adaptor proteins (e.g., vinculin, talin) and are known to be involved in cell 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 constitutes gap junctions and facilitates cell-cell communication. Connexin 43 plays an essential role in maintaining cell and tissue homeostasis and function through the exchange of biological information (Ribeiro-Rodrigues, 2017, J Cell Sci 130(21): 3619-3630). Connexin 43 was expressed in the cell sheet, 0.05% trypsin treatment (5, 20, 60 min) and 0.5% trypsin treatment (5 min) groups. However, 0.50% trypsin treatment for 20 and 60 min showed no expression of connexin 43. This suggests that connexin 43 protein was cleaved by 0.50% trypsin when treated for 20 and 60 min.

[0089] 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 throughout 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 expression of connexin 43 throughout the area. This demonstrates that the cell sheet has a connected tissue structure and maintains cell-junction proteins. In contrast, trypsin treatment (chemical disruption) cleaved the junction proteins.

[0090] 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 a result, the ratio of dead cells to live cells in the 0.05% trypsin treatment groups for 5 and 20 min was similar. Notably, the ratio of dead cells to live cells in the 0.05% trypsin treatment group for 60 min was significantly increased compared to cells treated with 0.05% trypsin for 5 and 20 min (FIG. 15B). This result suggests that cell death was induced by trypsin treatment (chemical destruction).

[0091] Apoptotic cell death is activated by chemical destruction Mechanosensors control cell homeostasis by transducing extracellular physical stimuli into intracellular chemical stimuli (Humphrey, 2014, Nat Rev Mol Cell Biol 15(12): 802-12). Yes-associated protein (YAP) is one of the major mechanosensor proteins 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 apoptosis. When cells lose cell-ECM junctions, apoptotic cell death, i.e., anoikis, is induced following YAP phosphorylation (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 the cellular response towards apoptosis.

[0092] Consideration Chemical disruption method is 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 method (trypsinized cells) had insufficient ECM, which is necessary to adhere to the target tissue, and insufficient cell junctions to maintain their cell function through graft-host communication (Figures 13 and 14). On the other hand, hUC-MSC sheets harvested by cell sheet technique using TRCD maintained tissue-like structures such as smooth surface, microvilli, ECM and cell junctions of connected cells (Figures 10, 13 and 14).

[0093] 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 treated with 0.05% trypsin for 20 min, and cell nuclei were disassembled after 0.05% trypsin treatment for 60 min. Furthermore, endoplasmic reticulum associated with cell death was observed after 0.05% trypsin treatment for 60 min (Figure 11). Integrins are key proteins involved in the interaction between cell membranes and ECM, and also form focal adhesions by linking with the cytoskeleton (actin) (Kim et al., 2011, J Endocrinol 209(2): 139-51). Chemical disruption induced cleavage of integrin β-1, as well as the 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 the chemical disruption method (trypsinization) could result in a harsh environment for cell survival due to enzymatic proteolytic destruction.

[0094] 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 occurs in the chemically disrupted group (Figures 11, 15 and 16). In contrast, hUC-MSC sheets maintain integrin β-1 and have lower expression of pYAP (Figures 14 and 16), which shows 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. The hUC-MSC sheets showed cytoskeletal fiber structures of F-actin, indicating active actin polymerization, even after cell detachment from the cell culture dish (Figure 12). This suggests that the hUC-MSC sheets retain integrin β-1 (cell-ECM junctions) and F-actin structures, allowing the cell sheets to maintain higher cell viability compared to trypsin treatment (a traditional chemical disruption method). These findings suggest that cell-ECM junctions and actin fiber structures are important factors for cell survival, and that it is difficult to maintain high cell viability when using chemical disruption methods.

[0095] This study demonstrated that ECM, cell-cell junction and cell-ECM junction proteins are important to maintain higher cell viability. In conventional stem cell therapy using chemical disruption method for harvesting, low engraftment rate and low cell viability cannot be avoided because chemical disruption (e.g. trypsinization) cuts cell-cell junction, cell-ECM junction and cell adhesion proteins. The cell sheet technology made it possible to harvest cells in the form of a sheet without any structural disruption. Furthermore, the cell sheet technology maintained the important structures of cells (ECM, cell-ECM junction, cell-cell junction, cytoskeleton and mechanosensor) related to cell viability, engraftment rate and various cell functions. As a result, the cell viability in hUC-MSC sheet was significantly higher than that of cells harvested by chemical disruption method.

[0096] conclusion We demonstrated that tissue-like structures such as ECM cell-cell junctions and cell-ECM junctions are associated with cell viability of transplanted cells. The cell sheet technology allows cells to be cultured and harvested in the form of sheets without using any enzymes (chemical disruption). The harvested hUC-MSC monolayer sheets, which retained the tissue-like structures, ECM, cell-cell junctions and cell-ECM junctions, had higher cell viability compared to the traditional chemical disruption method (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 of functioning cells in regenerative medicine research.

[0097] [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.

[0098] 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.

[0099] 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-dependent cell proliferation was enhanced by increasing the initial cell density from hUC-MSCs in TRCD. As shown in FIG. 19, increasing the initial cell density increased HGF expression in a concentration-dependent manner. For example, at an initial cell density of 10×10 4 Cells / cm 2 The cell sheet generated in 4 , 4×10 4 , 6×10 4 , 8×10 4 Or 10 x 10 4 Cells / cm2 The hUC-MSC sheets had higher HGF gene expression than the cell sheets produced with the 100% 15 ...

[0100] 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 reduced immune responses to cell sheets transplanted into a host organism. 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.

[0101] [Example 4] Effect of culture medium on the fabrication of allogeneic human umbilical cord mesenchymal stem cell (hUC-MSC) sheets In this study, fetal bovine serum (FBS) and human platelet lysate (hPL), the most common media used for MSC culture, were compared with respect to MSC differentiation capacity, MSC-specific phenotype, cell sheet preparation, cell sheet-specific traits and paracrine secretion.

[0102] Materials and Methods Human umbilical cord mesenchymal stem cell (hUC-MSC) culture hUC-MSCs (Jadi Cell LLC, Miami, USA) stored in a bank (Patel et al., 2013, Cell Transplant 22:513-519) were cultured in cell culture medium with Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, USA) supplemented with either 10% hPL (Jadi Cell LLC) or 10% FBS (Life Technologies), 1% Glutamax (Life Technologies), 1% MEM NEAA (Life Technologies), and 1% penicillin streptomycin (Life Technologies) at 37°C in a humidified atmosphere containing 5% CO2 from the second passage (P2). A working cell bank was established at P4. The cell culture medium was changed every 2 days. Cell morphology was observed at P6 and P12 using an AX10 microscope (Carl Zeiss Microimaging, Germany) equipped with AxioVision software (Carl Zeiss Microimaging) prior to heat-induced detachment of the cell sheet from the culture device.

[0103] hUC-MSC proliferation rate Cells were seeded on 6-well plates at a cell density of 2.3 × 104 / cm2. Cell numbers in FBS or hPL cultures were counted 8, 24, 33, 48, and 72 hours after seeding on P4-12. Cell doubling times were calculated by polynomial approximation using Origin Pro 2017 (Origin Lab, Massachusetts, USA).

[0104] MSC differentiation ability To investigate osteogenic or adipogenic differentiation at passage 6, cells were seeded in 6-well plates. For osteogenic differentiation, cells were seeded at 5 × 10 3 Cells / cm 2At 60% confluence, cells were induced with osteogenic differentiation medium containing αMEM, 10 nM dexamethasone, 82 μg / mL ascorbic acid 2-phosphate, and 10 mM β-glycerol phosphate (Sigma-Aldrich). Cells were cultured in osteogenic medium at 37°C for 21 days, with medium changed every 3 days. Cells were fixed with 4% cold paraformaldehyde (PFA) for 12 min and stained with Alizarin Red S-(Sigma-Aldrich) using standard protocols. For adipogenic differentiation, cells were cultured at 1 × 10 4 Cells / cm 2 At 80% confluence, 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, with medium changed every 3 days. Cells were fixed with 4% cold paraformaldehyde for 12 min and stained with Oil Red O (Sigma-Aldrich) using standard protocols.

[0105] Stem cell surface phenotyping assay hUC-MSCs were cultured in cell growth medium on tissue culture flasks (Genesee Scientific, CA, USA) for 5 days and then detached using TrypLE (Gibco, Waltham, MA, USA) for MSC phenotyping assays. After harvesting, the cell suspension was incubated with 2% w / v bovine serum albumin (BSA) in PBS for 30 min and then diluted with 3–5 × 10 5 The 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: CD73, CD105, CD90, MHC II, CD45 and CD31 (Biolegend, San Diego, USA). Primary antibodies were added to each aliquot to achieve a cell to antibody ratio of approximately 20:1 in buffer. Approximately 3–5 × 10 5Cells were stained with saturating concentrations of (fluorophore)-conjugated primary antibodies. Cells were incubated for 30 min on ice in the dark. After incubation, cells were washed three times, then resuspended in 1×PBS and immediately analyzed by flow cytometry (Becton Dickinson FACS Canto, BD Biosciences, Sparks, USA). Unstained cells were used for calibration of the flow cytometer. Cells were gated by forward vs. side scatter to exclude doublets. A minimum of 10,000 events were counted for each analysis.

[0106] hUC-MSC sheet preparation hUC-MSC sheets were prepared using P6 cells, which were cultured at 2.3 × 10 4 / cm 2 The cells were seeded at a density of 100 μg / ml and cultured in cell culture medium with DMEM supplemented with either 20% FBS or 20% hPL at 37°C in a humidified atmosphere of 5% CO2 for 4 (FBS) or 3 (hPL) days, respectively. One day after seeding, ascorbic acid (16.4 μg / ml) was added. To analyze cell sheet properties, confluent cells were detached 3 or 4 days after seeding by changing the culture temperature from 37°C to room temperature (RT) within 1 h. The spontaneous surface detachment time of the sheet was determined when the cells TRCD were transferred from 37°C incubation to room temperature without any additional manipulation such as pipetting, scraping, or medium exchange.

[0107] Histological analysis For H&E staining, cell sheets were fixed with 4% PFA for 15 min and then embedded in paraffin. Embedded specimens were sectioned in 4 μm slices. Specimens were treated with hematoxylin solution (Sigma-Aldrich) for 3 min and then with eosin solution (ThermoFisher Scientific, Waltham, USA) for 5 min. H&E stained specimens were dehydrated and mounted with Permount™ (ThermoFisher Scientific). H&E images were obtained using a BX 41 microscope (Olympus, Hamburg, Germany). For immunohistochemistry (IHC), when cells reached confluence, they were fixed using 4% PFA for 10 min. Cell membranes were permeabilized with 0.1% Triton X (Sigma-Aldrich, St. Louis, USA) for 15 min. Non-specific binding was blocked for 1 h at room temperature in PBS 1x containing 10% goat serum (Vector Laboratories, Burlingame, USA). Cells were stained with Alexa Fluor 488® phalloidin (ThermoFisher Scientific) to visualize the cytoskeleton or with primary antibodies (Abcam, Cambridge, USA) to image fibronectin (Fb, ab2413) (Abcam, Cambridge, USA), β-catenin (β-CTNN, ab16051) (Abcam), HLA DR, DP, DQ (MHC II, ab7856) (Abcam), and negative control rabbit IgG (NC, x0936) (DAKO, Santa Clara, CA). The specimens were treated with Alexa Fluor 594-conjugated secondary antibodies (Life Technologies) (1:200) for 1 h for Fb, β-CTNN, and MHC II staining. Stained cells were mounted with ProLong Gold Antifade reagent containing DAPI (Life Technologies). Immunofluorescence images were obtained using an AX 10 microscope (Carl Zeiss Microimaging) and analyzed with Axiovision software (Carl Zeiss Microimaging).

[0108] Gene expression analysis of cell sheets Total RNA from cell sheets was extracted using Trizol and PureLink RNA Mini Kit (Life Technologies, Carlsbad, USA) according to the manufacturer's protocol. cDNA was prepared from 1 μg of total RNA using High Performance cDNA Reverse Transcription Kit (Life Technologies). qPCR analysis was performed with TapMan Universal PCR Master Mix using an Applied Biosystems Step One instrument (Applied Biosystems™, Foster City, USA). Gene expression levels were investigated for the following genes: (1) glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905_m1) as a housekeeping gene, (2) β-actin (Hs999999903_m1), (3) integrin β-1 (ITGB1, Hs01127536_m1), (4) fibronectin (Fb, Hs01549976_m1), (5) β-catenin (Hs00355049_m1), and (6) major histocompatibility complex II (MHC II: HLA-DRB, Hs04192464_m1). All primers were manufactured by Applied Biosystems. Relative gene expression levels were quantified by the comparative CT method (Schmittgen et al., 2009, Nat Protoc 3:1101-1108). Gene expression levels were normalized to GAPDH expression levels. Gene expression levels are relative to the hPL group for β-actin and ITGB1 analysis and to the FBS group for Fb, β-CTNN and MHC-II analysis.

[0109] Hepatocyte growth factor (HGF) secretion assay The supernatant medium of adherent cells cultured for 24 h was collected immediately before cell sheet detachment from TRCD at room temperature. The amount of HGF secreted from the cell sheets was measured by human HGF Quantikine ELISA (R&D Systems, Minneapolis, USA) according to the manufacturer's protocol. To count the number of cells constituting each cell sheet, the cells were detached with TrypLE. The amount of HGF was normalized to the cell sheet or cell number.

[0110] 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 (ANOVA) using Origin 2017 software (OriginLab, Northampton, USA). Probability values ​​less than 0.05 (p < 0.05) were considered statistically significant.

[0111] result Changes in hUC-MSC morphology and proliferation rate MSCs were cultured in cell culture medium containing FBS or hPL and observed 1 and 4 days after seeding at P6 (Figures 21A and 21B) and P12 (Figures 21C and 21D), respectively. MSCs in hPL medium showed a more elongated spindle shape, whereas MSCs in FBS were more flat (Figures 21A and 21B). hUC-MSCs cultured in FBS medium proliferated in a homogeneous dispersion state, whereas those in hPL medium tended to proliferate in a cohesive dispersion state (Figures 21A-21D). The cohesive, dispersed proliferation of the hPL group was more obvious with increasing passage number, especially for P12 (Figure 21D). Cells in the hPL group showed significantly higher proliferation ability compared to those in the FBS group from P4 to P8. However, the doubling time of cells in the hPL group increased rapidly after P8 (Figure 21E).

[0112] Differentiation potential and MSC cell-specific phenotype To investigate how FBS and hPL media affect hUC-MSC differentiation capacity and stem cell-specific phenotype, differentiation capacity and stem cell surface markers were investigated after two passages of culture in FBS (Figures 22A and 22C) or hPL (Figures 22B and 22D) media. The differentiation capacity in osteogenesis and adipogenesis was not affected by either FBS or hPL media (Figures 2A-2D). Furthermore, cells cultured in FBS (Figures 23A-23F) or hPL (Figures 23G-23L) both displayed stem cell-specific surface markers; positive expression of CD73, CD105, CD90 (Figures 3A-3C and 3G-3I) and negative expression of MHC II, CD45, CD31 (Figures 23D-23F and 23J-23L).

[0113] Cell sheet production Cell sheets were prepared in cell culture medium containing 20% ​​FBS or hPL. Cells in the FBS or hPL group reached confluence on the TRCD 4 or 3 days after seeding, respectively, and were then harvested as detached sheets using a small temperature change from 37°C to room temperature without additional manipulation (Figures 24A-24H). Cell sheets cultured in FBS medium were harvested when the cells exceeded confluence (Figures 24B and 24F), whereas cell sheets cultured in hPL medium were detached spontaneously at 37°C as more cell aggregates (Figures 24D and 24H). Cell sheets in the FBS and hPL groups were harvested within 50 and 15 minutes at room temperature, respectively (Figure 24I). Cells in the hPL group showed a higher density of cell nuclei in the cell sheet compared to those in the FBS group (Figures 24J and 24K). The actin cytoskeleton (F-actin) of cells in the FBS group was aligned and ordered (Figure 24M). On the other hand, that of the hPL group showed random crisscrossing actin cytoskeleton patterns (Figure 24N). The gene expression levels of β-actin and ITGB1, which are related to stable cell adhesion, in the FBS group were significantly higher than those in the hPL group (Figures 24L and 24O). The data indicate that hPL cell sheets grow rapidly to high density, exhibit faster detachment at low temperature, weaker cell adhesion, and tend to easily form cell aggregates upon release under poorly controlled sheet-forming conditions even without reduction in temperature.

[0114] Phenotype of hUC-MSC cell sheets For cell sheets prepared in FBS and hPL media, the desired cell sheet-specific traits (e.g., retention of endogenous cell adhesion proteins) that correlate with therapeutic and engraftment efficacy were investigated by IHC analysis. Both cell sheets in the FBS and hPL groups showed positive expression of cell adhesion proteins (ECM: fibronectin (Fb) and cell junction protein: β-catenin (β-CTNN)) compared to the negative control (NC) (Figures 25A-25C and 25E-25G). Furthermore, gene expression levels of Fb and β-CTNN were investigated by q-PCR analysis and were all similar in both FBS and hPL groups (Figures 25I and 25J).

[0115] Histocompatibility markers for hUC-MSC sheets MHC II expression is an indicator of a possible host immune response when allogeneic cells are implanted in vivo (Zantvoort et al., 1996, Transplantation 61:841-844). In cell sheets prepared in FBS and hPL groups, positive expression of MHC II was barely detectable (Figures 25D and 25H). Gene expression levels related to MHC II were also similar in FBS and hPL groups (Figure 25K). These data demonstrate that neither FBS nor hPL medium culture conditions activate this immune response antigen.

[0116] Cell sheet paracrine factor secretion Human growth factor (HGF) secreted from each cell sheet condition at 24 hours was measured in the medium supernatant. hPL cell sheets secrete significantly more HGF compared to FBS cell sheets (Figure 26A). Because hPL cell sheets contain more cells than FBS cell sheets, when secreted HGF was normalized to the number of cells per cell sheet (Figure 26B), the amount of HGF in the FBS and hPL groups is similar (Figure 26C). This result indicates that the increased paracrine factor secretion from hPL cell sheets relative to FBS cell sheets is attributed to their higher proliferation capacity (Figure 26B) and the resulting higher cell density per sheet (Figure 26C).

[0117] Consideration Many MSC studies, including their current and diverse clinical applications, use media supplemented with FBS to promote cell attachment, proliferation, and provide essential nutrients during MSC culture and storage. To date, FBS culture has been accepted as a clinical-scale cell production method. However, xeno-derived media and additives have known risks of contamination from prions, viruses, and zoonotic pathogens, as well as the potential for stimulating host immune responses. Recently, hPL has been considered as an alternative to FBS to avoid potential immune cross-reactivity. Furthermore, some data indicate that hPL supports greater MSC proliferation capacity than FBS for large-scale MSC production. In this study, MSC culture in hPL medium showed greater proliferation rates (1.2-1.4 times higher) than MSC cultured in FBS medium at early passage numbers (i.e., between P4 and P8) (Figure 21E). However, MSC proliferation rates after P8 in hPL were similar to that in FBS medium. hPL medium induces a wide difference in MSC proliferation rate for different passage numbers (Figure 21E). Moreover, both FBS and hPL conditions maintain cell sheet differentiation capacity and phenotypes characteristic of MSCs (Figures 22 and 23). Therefore, cell culture medium containing hPL that promotes higher cell proliferation capacity without altering MSC-specific traits at early passage numbers would be valuable for large-scale MSC production. However, cell sheets in hPL medium show unexpected detachment from the TRCD without any temperature change when cells exceed confluence, possibly induced by the higher cell density in the cell sheet (Figure 24H). Therefore, cell sheet fabrication protocols using hPL medium should be standardized with special attention to the culture time to avoid unexpected cell sheet detachment during sheet fabrication.

[0118] In this study, although the exact mechanism is unclear, MSCs in hPL medium show randomly arranged multidirectional actin cytoskeleton patterns, whereas MSCs in FBS medium cytoskeleton structures are unidirectionally aligned (Figures 24M and 24N). Random crisscrossing actin cytoskeleton structures induce more contractile forces, because contractions of cells with randomly aligned cytoskeleton structures occur simultaneously from multiple directions, whereas cells with unidirectionally aligned cytoskeleton structures contract from only one direction (Takahashi et al., 2015, Adv Healthc Mater 4: 2388-240). Cytoskeleton structures are also well recognized to contribute to cell morphology. MSCs in hPL show elongated cell shapes with randomly arranged actin cytoskeleton structures that are crisscrossed. MSCs in FBS medium cover a larger area with flat cell shapes (Figures 21A-21B and 24M-24N). Furthermore, the cytoskeletal structure correlated with cell adhesion, as the binding of ITGB1 to the actin cytoskeleton is involved in cell adhesion (Fernandez-Rebollo et al., 2017, Sci Rep 7:5132). In this study, MSCs in FBS medium expressed more actin and ITGB1 markers compared to MSCs in hPL medium (Figures 24L and 24O). Some studies also demonstrate that MSCs in hPL medium result in a more spindle-shaped fibroblast-like morphology and weaker cell adhesion to tissue culture plastic compared to MSCs in FBS medium (Fernandez-Rebollo et al., 2017, Sci Rep 7:5132). These characteristics suggest that their aligned cytoskeletal structure in FBS medium provides stable cell morphology and adhesion. Cell sheet fabrication generates confluent cells that adhere under cell-cell tension on the TRCD and are harvested as a continuous sheet from these firm culture surfaces at RT. Spontaneous heat-induced cell sheet detachment from this taut, adherent state results in spontaneous cell sheet contraction through intrinsic cytoskeletal contraction after release from the TRCD. MSC sheets derived from hPL medium are rapidly detached from the TRCD within 15 min (Figure 24I); this is faster than cell sheets in FBS medium (detachment time: 49 min).These differences in MSC sheet detachment times from TRCDs in FBS medium versus hPL medium likely result from a combination of weaker MSC adhesion to the TRCD surface in hPL culture and rapid contraction in the MSC sheet due to random crisscrossing cytoskeleton structures. Overall, hPL medium rapidly generates MSC sheets through random actin cytoskeleton and weaker cell adhesion, but is accompanied by an unexpected strong intercellular contractile response after non-temperature-dependent cell sheet detachment.

[0119] Recently, cell-based therapies have increasingly focused on allogeneic stem cell sources for several advantages: (1) reducing the high production costs for autologous supply, (2) reducing donor variability (e.g., aging and disease pathophysiology) that affect stem cell quality, and (3) allowing cell bank sourcing of healthy donors for "off-the-shelf" products. However, currently, allogeneic cell therapy is also limited by host-patient immunocompetence and transplant compatibility (surveyed through major histocompatibility complex, MHC), as well as inconsistent cell delivery methods to effectively result in cell retention and therapeutic engraftment. This study uses hUC-MSCs, which have been shown to present a low antigenic / histocompatibility profile (MHC) (Patel et al., 2013, Cell Transplant 22:513-519). Consistent with this, MSC sheets prepared with both FBS and hPL media expressed low MHC class II antigens (Figure 23) and maintained low MHC class II antigen expression during cell sheet preparation (Figures 25D and 25H). Cell sheets prepared with FBS and hPL also retained critical cell adhesion proteins related to therapeutic and engraftment efficacy (Figures 25D, 25C, 25F and 25G). No significant difference in the expression levels of genes for critical cell adhesion protein production was found between the FBS and hPL groups (Figures 25I and 25J). Overall, FBS and hPL media retained MSC phenotypes and structural organization required for cell sheets that are believed to be important for host immune tissue compatibility and optimal therapeutic and transplantation processes. These findings suggest that cell culture media supplemented with either FBS or hPL can be used in standard production methods to maintain cell sheet specific traits.

[0120] HGF is a cytokine important for mechanisms that suppress fibrosis and promote tissue repair in vivo (Inoue et al., 2003, FASEB J 17:268-270). 4At 1000 cells / well, MSC sheets prepared in hPL medium secreted higher HGF levels than those prepared in FBS medium (Figure 26A). On TRCD, the hPL group produced 2.5-fold higher final cell numbers in MSC sheets compared with the FBS group (Figure 26B). The amount of HGF normalized to the final cell sheet number in each medium was similar in the FBS and hPL groups (Figure 26C), indicating that the increased HGF secretion from hPL-produced cell sheets was due to the higher cell proliferation capacity in the hPL-cultured sheets and the resulting difference in cell sheet numbers. Based on this, cytokine secretion of MSC cell sheets may be controlled through cell density and proliferation capacity during culture. Future studies will investigate the effect of MSC cell density in cell sheets on paracrine production and signaling.

[0121] We have demonstrated the influence of cell culture media on certain MSC sheet properties that are important for the generation, standardization and quality control of the final cell sheet. To determine and establish these MSC-specific sheet processes, MSC-specific biomarkers, secreted products, phenotypes and immunogenic antigen / histocompatibility profiles become essential. Both FBS and hPL supplemented media maintain MSC phenotypes and cell sheet specific structures (e.g. cell adhesion proteins) that have been shown to be important for their in vivo therapeutic and engraftment efficacy (Sekine et al., 2011, Tissue Eng Part A 17:2973-2980). However, the consistency of the final cell sheet product prepared in hPL media was lower than that prepared in FBS media. Cell sheets cultured in hPL media spontaneously detached from the TRCD without the desired applied temperature release trigger and often yielded MSC aggregates instead of stable MSC sheets when they were released. This is argued to be due to weaker cell adhesion and higher contractile forces within the sheets due to changes in the cytoskeleton structure. Due to the increased final cell sheet number in hPL culture, HGF cytokine secretion is increased in MSC sheets prepared in hPL medium versus FBS medium.

[0122] conclusion MSCs cultured with FBS and hPL exhibit different cytoskeleton structures. This feature affects cell adhesion to the TRCD surface and cell sheet contraction force, which are important aspects of the cell sheet fabrication process. FBS medium MSC culture shows advantages in consistent MSC cell sheet generation, showing stable cell adhesion to the TRCD, phenotypic stability, and controlled contraction force within the released and harvested MSC sheets. hPL medium MSC culture allows for more rapid cell sheet generation and enhanced HGF cytokine secretion. Both FBS and hPL media retain the desired MSC cell sheet specific traits that are essential for their ultimate therapeutic goals and efficacy. These findings provide an initial foundation for establishing a standardized MSC sheet fabrication method that is essential for large-scale MSC sheet production for eventual clinical studies.

Claims

1. A human umbilical cord mesenchymal stem cell sheet comprising one or more layers of confluent human umbilical cord mesenchymal stem cells (hUC-MSCs).

2. The cell sheet of claim 1 , wherein the cell sheet essentially consists of hUC-MSCs.

3. The cell sheet of claim 1, wherein at least 50% of the cells in the cell sheet are hUC-MSCs.

4. The cell sheet according to claim 1 , wherein the cell sheet comprises an extracellular matrix.

5. The cell sheet according to claim 4, wherein the extracellular matrix comprises one or more proteins selected from the group consisting of fibronectin, laminin and collagen.

6. The cell sheet according to claim 1 , wherein the cell sheet comprises a cell adhesion protein and an intercellular junction protein.

7. The cell sheet of claim 6, 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.

8. The cell sheet of any one of claims 1 to 7, wherein the hUC-MSCs are isolated from the subepithelial layer of human umbilical cord tissue.

9. The cell sheet of claim 1 , wherein the hUC-MSCs express a protein selected from CD44, CD73, CD105 and CD90.

10. The cell sheet of any one of claims 1 to 9, wherein the hUC-MSCs express one or more cytokines selected from the group consisting of human growth factor (HGF), vascular endothelial growth factor (VEGF), and interleukin-10 (IL-10).

11. The cell sheet of claim 10, wherein expression of said one or more cytokines in said cell sheet is increased compared to a hUC-MSC suspension containing a comparable number of cells.

12. 12. The cell sheet of claim 1, wherein the cell sheet secretes tumor necrosis factor-α (TNF-α) into a culture medium at a rate of less than 50 pg per mL of culture medium over 24 hours.

13. The cell sheet of any one of claims 10 to 12, wherein the cell sheet expresses the one or more cytokines for at least 10 days after transplantation into a tissue of a host organism.

14. 14. The cell sheet of claim 1, 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.

15. The cell sheet of claim 14, wherein the extracellular matrix protein is selected from the group consisting of fibronectin, laminin and collagen.

16. The cell sheet of claim 14, 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.

17. The initial cell density of the hUC-MSCs seeded in the cell culture support used to prepare the cell sheet was 0.5×10 4 / cm 2 From 9×10 5 / cm 2 The cell sheet according to any one of claims 1 to 16,

18. The cell sheet of any one of claims 1 to 17, wherein the hUC-MSCs do not express one or more of CD31, CD45, human leukocyte antigen-DR isotype (HLA-DR), human leukocyte antigen-DP isotype (HLA-DP), or human leukocyte antigen-DQ isotype (HLA-DQ).

19. The cell sheet of any one of claims 1 to 18, wherein the hUC-MSCs comprise microvilli and filopodia.

20. 20. The cell sheet of any one of claims 1 to 19, wherein the cell sheet remains attached to the tissue of the host organism for at least 10 days following transplantation into the tissue.

21. 21. A composition comprising the cell sheet of any one of claims 1 to 20 and a polymer-coated culture support that is removable from the cell sheet.

22. 1. A method for generating a human umbilical cord mesenchymal stem cell sheet comprising one or more layers of confluent human umbilical cord-derived mesenchymal stem cells (hUC-MSCs), comprising: a) culturing hUC-MSCs in a culture medium on a temperature-responsive polymer coated on a substrate surface of a cell culture support, the temperature-responsive polymer having a lower critical solution temperature in water of 0-80°C; b) adjusting the temperature of the culture medium below the lower critical solution temperature, thereby rendering the substrate surface hydrophilic and weakening the adhesion of the cell sheet to the surface; c) detaching the cell sheet from the culture support; The method includes:

23. 23. The method of claim 22, further comprising culturing the hUC-MSCs through multiple subcultures prior to the culturing step (a).

24. The method of claim 23, wherein 2 to 10 passages of the hUC-MSCs are carried out prior to the culturing step (a).

25. 25. The method of any one of claims 22 to 24, wherein the culture medium is a xeno-free culture medium.

26. 26. The method of any one of claims 22 to 25, wherein the culture medium comprises human platelet lysate (hPL).

27. 27. The method of any one of claims 22 to 26, wherein the culture medium comprises fetal bovine serum (FBS).

28. 28. The method of any one of claims 22 to 27, wherein the culture medium comprises ascorbic acid.

29. 29. The method of any one of claims 22 to 28, wherein the conditioning step (b) is carried out when the hUC-MSCs are confluent.

30. The culturing step (a) comprises culturing the hUC-MSCs at 0.5×10 4 / cm 2 From 9×10 5 / cm 2 30. The method of any one of claims 22 to 29, comprising adding to the culture medium at an initial cell seeding density of

31. 31. The method of any one of claims 22 to 30, wherein the hUC-MSCs are cultured in the culture medium on the temperature-responsive polymer for at least 24 hours prior to the conditioning step (b).

32. 32. A cell sheet produced by the method of any one of claims 22 to 31.

33. A method for transplanting a cell sheet into a subject, the method comprising applying the cell sheet of any one of claims 1 to 20 or 32 to a tissue of the subject.

34. 34. The method of claim 33, wherein the hUC-MSCs in the cell sheet are allogeneic to the subject.

35. 35. The method of claim 33 or 34, wherein the subject is a human.

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