New site for transplantation

Abstract

The present invention provides a new site for transplantation. Specifically, the present invention relates to a method for transplanting cells or tissues, particularly pancreatic islets, into the sub-rectus sheath site. The present invention also relates to a method for treating diabetes by transplanting pancreatic islets, particularly hPSC pancreatic islets, into a sub-rectus sheath position.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of PCT International Application No. PCT / CN2022 / 095086, filed on May 25, 2022, the entire content of which is incorporated herein by reference.

[0002] The present invention relates to the field of transplantation of cells, tissues or organs. Specifically, the present disclosure provides a method for transplanting cells or tissues into the sub-rectus sheath site. After transplantation, the cells or tissues are grafted under the rectus sheath, and preferably, most of the cells or tissues are grafted into the space between the rectus sheath and the rectus muscle. The new transplantation site of the present invention is particularly suitable for ectopic transplantation of, for example, hormone-secreting cells or hormone-secreting tissues. Also, the new transplantation site provides a desirable environment for grafts that are not fully differentiated or mature at the time of transplantation.

Background Art

[0003] Human pluripotent stem cells have shown great potential in cell replacement therapies for the treatment of diseases including diabetes. In our previous studies with non-human primates, it has been shown that human pluripotent stem cell-derived islets (hPSC islets) can be transplanted for the treatment of type 1 diabetes.

[0004] However, there are still challenges in the clinical application of hPSC-derived cells and tissues such as hPSC islets, one of which is the lack of transplantation sites that can effectively support the functional maturation and long-term survival of grafts.

[0005] Taking hPSC islets as an example, portal vein injection is the most commonly used in clinical islet transplantation, but it is not satisfactory due to reasons such as substantial loss of islets in the early stage of transplantation, low revascularization rate of transplanted islets, and progressive loss of graft function in the long term. To maintain the therapeutic effect level of functional islets, an excessive amount of cells for transplantation is required to compensate for the loss. However, such an excessive amount may lead to hypoglycemia. Furthermore, liver transplantation results in direct exposure of hPSC islets to high concentrations of immunosuppressive drugs as well as the coagulation and complement systems, which will further hinder their functional maturation and long-term maintenance. Previous experience in mouse models has shown that the subrenal capsule is ideal for the transplantation of various cells and tissues, including both primary human islets and hPSC islets (Du, Y. et al., Human pluripotent stem-cell-derived islets ameliorate diabetes in non-human primates. Nat Med 28, 272-282, doi:10.1038 / s41591-021-01645-7 (2022); Pagliuca, F. W. et al., Generation of functional human pancreatic beta cells in vitro, Cell 159, 428-439, doi:10.1016 / j.cell.2014.09.040 (2014); Rezania, A. et al., Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32, 1121-1133, doi:10.1038 / nbt.3033 (2014)). However, this site is not suitable for clinical use because of limited space for high transplant mass and invasive surgical procedures.

[0006] Considerable efforts have been made to investigate alternative sites for islet transplantation in animal models. Although successful in rodent models, most transplantation sites have been non-transplantable in large animal models.

[0007] There is still a need in the art for a transplantation site that can provide a permissive microenvironment for the cell survival, growth, and maintenance of grafts, particularly hPSC-derived grafts.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0009] The inventors have discovered that when transplanted under the rectus sheath, hPSC islets gradually matured and exhibited excellent graft function, which led to an overall improvement in blood glucose control in diabetic non-human primates. Notably, C-peptide secretion increased dramatically, responding to a meal challenge from 6 weeks post-transplantation (wpt), and the stimulation index was comparable to that of native islets. The mean postprandial C-peptide level was maintained at approximately 2.0 ng / ml from 8 wpt, which was five-fold that of intraportal injection. Thus, the mean HbA1c decreased by more than 44% at 12 wpt. Furthermore, recipient macaques recovered rapidly from cell transplantation and showed a lower risk of hypoglycemia within the first 24 hours. Collectively, the data demonstrate that the sub-rectus sheath is an optimal transplantation site for hPSC islets that can be extended to other derivatives of hPSCs. This simple and non-invasive transplantation strategy combined with hPSC derivatives may introduce a new paradigm for cell replacement therapy and thus complete the present invention. -1 was maintained, which was five-fold that of intraportal injection. Thus, the mean HbA1c decreased by more than 44% at 12 wpt. Furthermore, recipient macaques recovered rapidly from cell transplantation and showed a lower risk of hypoglycemia within the first 24 hours. Collectively, the data demonstrate that the sub-rectus sheath is an optimal transplantation site for hPSC islets that can be extended to other derivatives of hPSCs. This simple and non-invasive transplantation strategy combined with hPSC derivatives may introduce a new paradigm for cell replacement therapy and thus complete the present invention.

Means for Solving the Problems

[0010] Accordingly, in a first aspect, the present application provides a method for transplanting one or more cells or tissues, or to a subject in need thereof, the method comprising introducing the cells or tissues into a site under the rectus sheath of the subject (sub-rectus sheath site), preferably the site between the rectus sheath and the rectus muscle. In some embodiments, the cells or tissues to be transplanted can be cells or tissues derived from hPSCs. In some aspects, the cells or tissues to be transplanted are suitable for ectopic transplantation, such as secretory cells such as endocrine glands. In certain embodiments, the cells or tissues to be transplanted are hPSC-derived islets. In another aspect, the method enables one or more of the cells or tissues to be transplanted to be grafted to a site under the rectus sheath, preferably the site between the rectus sheath and the rectus muscle, after transplantation.

[0011] In a second aspect, the present application provides a method of treating a disease or condition caused by or associated with insulin deficiency in a subject in need thereof, the method comprising transplanting pancreatic islets into the subject, wherein the pancreatic islets are introduced into a site under the rectus sheath (sub-rectus sheath site). In some embodiments, the pancreatic islets are derived from hPSCs. In some embodiments, the disease is diabetes or a complication thereof.

[0012] In a third aspect, the present application provides a method for reducing the need for exogenous insulin in a subject having diabetes, the method comprising transplanting pancreatic islets into the subject, wherein the pancreatic islets are introduced into a site under the rectus sheath (sub-rectus sheath site). In some embodiments, the pancreatic islets are derived from hPSCs.

[0013] In a fourth aspect, the present application provides the use of pancreatic islets in treating a disease or condition caused by or associated with insulin deficiency, wherein the hPSC-derived pancreatic islets are introduced into a site under the anterior rectus sheath (sub-anterior rectus sheath site). In some embodiments, the disease is diabetes or a complication thereof.

[0014] In a fifth aspect, the present application provides an apparatus for injecting pancreatic islets, such as hPSC-derived pancreatic islets, into a site under the rectus sheath (sub-rectus sheath site), preferably into the space between the anterior rectus sheath and the rectus muscle. In some embodiments, the apparatus includes a needle and a cartridge containing a suspension of hPSC-derived pancreatic islets. In preferred embodiments, the apparatus comprises a trocar needle. In more preferred embodiments, the trocar needle has one or more needle tracks, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more needle tracks.

[0015] The new transplant site of the present invention offers at least the following advantages.

[0016] (1) Sub-rectus sheath transplantation of hPSC pancreatic islets achieves a desirable therapeutic effect showing functionality comparable to that of primary human pancreatic islets in healthy adults.

[0017] (2) The rectus abdominis muscle has abundant blood vessels and is surrounded by the rectus sheath, providing a desirable microenvironment for the survival, growth, and maintenance of the graft after sub-rectus sheath transplantation.

[0018] (3) As an extraperitoneal site, the sub-rectus sheath transplantation of hPSC islets can be completed by a simple and non-invasive method such as bedside injection. Such a simple procedure allows repeated injection and rapid recovery after the transplantation procedure.

[0019] (4) Sub-rectus sheath transplantation allows removal of the graft when the recipient no longer benefits from the transplanted tissue. For example, since the long-term safety of hPSC-derived tissues is still under investigation, it may be desirable to keep an open window for removing hPSC islets.

[0020] (5) Intraportal transplantation of islets induces an immediate blood-mediated inflammatory reaction (IBMIR) and activates coagulation. Therefore, the application of anticoagulants is continuously required during intraportal transplantation. Inadequate control of coagulation can lead to dangerous or even lethal hypercoagulability (thrombosis) or hypocoagulability (bleeding). IBMIR has been reported to result in dramatic β-cell death and, consequently, a decrease in blood glucose. Transplantation to the new site of the present invention suggests that hypoglycemia due to β-cell death was not observed and the problems caused by IBMIR were solved.

[0021] (6) The new transplantation site under the rectus sheath can also be used for the transplantation of various cells or tissues. Particularly preferred grafts are hPSC-derived cells or tissues, such as hPSC islets.

Brief Description of the Drawings

[0022]

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Modes for Carrying Out the Invention

[0023] Unless specifically defined otherwise elsewhere in this specification, all technical and scientific terms used in this specification have the meanings commonly understood by those of ordinary skill in the technical field to which this invention belongs.

[0024] As used in this specification, including the appended claims, the singular forms of words such as "a", "an", and "the" include their corresponding plural references unless the context clearly indicates otherwise.

[0025] In the context of the present disclosure, unless otherwise indicated, the term "comprising", as well as variations thereof such as "comprises" and "including", are understood to mean that the recited elements, such as amino acid sequences, nucleotide sequences, characteristics, steps, or groups thereof, are included, but do not exclude any other elements, such as amino acid sequences, nucleotide sequences, characteristics, and steps. As used herein, the term "comprising" or any variation thereof can be replaced with the term "containing", "including", or in some cases, "having" or an equivalent variation thereof. In certain embodiments, the expression "comprise" includes the scenario of "consisting of".

[0026] "Sub-rectus sheath transplantation" refers to introducing a graft or transplant into a position under and within the rectus sheath, preferably into the space between the rectus sheath and the rectus abdominis muscle. "Sub- anterior rectus sheath transplantation" specifically refers to administering a graft or transplant to a position under the anterior side of the rectus sheath, preferably into the space between the anterior rectus sheath and the anterior side of the rectus abdominis muscle. As shown in FIGS. 1a and 1b, it reaches a position under the anterior rectus sheath.

[0027] The terms "rectus abdominis", "abdominis rectus", "musculus rectus abdominis", or "abdominal muscle" can be used interchangeably in the context of the present invention and refer to two parallel and flat muscles that are on both sides of the linea alba and extend along the entire length of the anterior surface of the abdomen.

[0028] The term "subject" refers to an animal, preferably a mammal, such as a non-human primate or preferably a human. In some cases, the subject is the "recipient" of the graft.

[0029] The term "secretory cell" refers to any cell that has the function of producing a substance and secreting the substance into the extracellular space.

[0030] As used herein, the term "islet" refers to a cluster of cells containing insulin-producing cells capable of regulating blood glucose levels.

[0031] The term "islet equivalent" or "IEQ" is a standardized measure for representing a transplanted mass of islets. For the determination of IEQ, reference can be made to, for example, Lembert N et al. (Lembert N et al., Areal density measurement is a convenient method for the determination of porcine islet equivalents without counting and sizing individual islets. Cell Transplant. 2003;12(1):33-41).

[0032] As used herein, the term "pluripotent stem cell (PSC)" refers to an undifferentiated cell defined by its ability to self-renew and differentiate at the single-cell level. Stem cells can give rise to progeny cells, including self-renewing progenitor cells, non-replicating progenitor cells, and terminally differentiated cells. Stem cells can be characterized by their ability to differentiate into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm, and ectoderm). The abbreviation "hPSC" refers to human pluripotent stem cells. Preferably, the hPSCs in the present method are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) obtained from embryos that have not developed in vivo and are within 14 days after fertilization.

[0033] The term "hPSC-derived" cells or tissues refers to cells or tissues generated or differentiated from human pluripotent stem cells.

[0034] The term "hPSC islet" refers to an islet containing insulin-producing cells derived from human pluripotent stem cells.

[0035] Transplantation site The advantage of the present application lies in the discovery of a new implantation site under and within the rectus sheath, preferably within or near the space between the rectus sheath and the rectus abdominis muscle. The "implantation site" in the present invention refers to the site where one or more cells or tissues are delivered and introduced. The expression "a cell or tissue is grafted to a site" means that the transplanted cell or tissue remains at the site and grows after transplantation. In some cases, since a part of the transplanted cells may move to some extent, the site where the cell or tissue is grafted may not be exactly the same as or 100% overlapping with the implantation site.

[0036] For ease of understanding, with reference to FIGS. 1a to 1c, the new implantation site of the present application will be described. As shown in FIGS. 1a and 1c, the graft or implant is delivered to a location between the rectus sheath and the surface of the rectus abdominis muscle. However, due to the accuracy of the operator's manipulation, the exact injection site may vary and may sometimes be slightly deeper up to the superficial rectus muscle. In another embodiment, the graft or implant is delivered into the rectus abdominis muscle, preferably in a position close to the rectus sheath.

[0037] As shown in FIG. 1a, the two rightmost photos show a cross-sectional view of the human abdomen and illustrate exemplary positions where a graft or implant can be introduced in the present invention. The graft is delivered under a thin layer of the rectus sheath (specifically, the anterior rectus sheath in FIG. 1a) and in front of the rectus abdominis muscle. After delivery, the graft can spread along the rectus abdominis muscle and form a layer of the graft under the rectus sheath. It should be understood that the needle can penetrate any part of the skin at any angle and to any depth as long as it can reach the desired implantation site.

[0038] The implantation site of the present application can be easily reached by routine administration means such as injection with a device containing a needle. Therefore, the cells or tissues to be transplanted can be delivered by a less invasive method such as injection or infusion. The implantation site of the present application also allows for the removal of the graft if any undesirable side effects occur after transplantation or if the graft is no longer needed.

[0039] After the graft or transplant has been delivered to the desired site, most of the transplanted cells can remain in place, but some of the transplanted cells can also migrate within the rectus sheath to locations away from the original transplant site. As long as cells derived from the graft or transplant remain within the rectus sheath, they are within the scope of this application.

[0040] The transplant site of this application also provides an optimal environment for the maintenance of the graft or transplant. Within the area surrounded by the rectus sheath, sufficient space is provided for the graft or transplant to grow, and a rich vascular network delivers sufficient oxygen and nutrients to the graft or transplant to support their survival, growth, and maintenance. In some cases, vascular infiltration into the graft or transplant can be observed several weeks after transplantation.

[0041] The transplant site of this application is particularly suitable for "immature" grafts or transplants that may require an in vivo maturation process after transplantation. For example, the graft may contain certain portions of cells that are not fully developed or differentiated. Such a transplant environment that allows for functional maturation is particularly important for undifferentiated or not fully differentiated cells, especially cells or tissues derived from stem cells, endodermal stem cell lines, mesenchymal stem cells, progenitor cells, or precursor cells. In some embodiments, the graft or transplant is a cell or tissue derived from undifferentiated or not fully differentiated cells, such as cells differentiated from hPSCs, which are referred to as hPSC cells or hPSC tissue in the context of this application. For example, cells or tissues derived from human pluripotent stem cells (hPSCs) can be derived from embryonic stem cells or induced pluripotent stem cells (iPSCs). In certain embodiments, for the transplantation of hPSC-derived islets, post-transplant maturation enables improvement of the insulin-secreting ability and glucose challenge responsiveness of the hPSC islets, which are important for the therapeutic effect of the hPSC islets.

[0042] Transplantation process The present invention is not intended to limit the means for introducing the graft into the area under the rectus sheath. The means or device used for transplantation may depend on the type of graft, the amount of graft, and the like.

[0043] In some embodiments, the transplantation is performed by injection. FIG. 1b illustrates the injection of the graft under the rectus sheath. As shown in FIG. 1b, the puncture needle enters the body, first penetrates the skin, and then penetrates the rectus sheath and is manipulated to reach a position under the rectus sheath, preferably a position between the surface of the rectus muscle and the (anterior) rectus sheath. Then, the needle can penetrate deeper while continuing to move along the space between the (anterior) rectus sheath and the rectus muscle. After the needle enters the desired location, the injection of cells is initiated. While injecting the cells, the needle is slowly withdrawn along the track so that the injected cells remain along the needle track between the rectus sheath and the rectus muscle. The present invention does not require that all grafts be injected or seeded into the space between the rectus sheath and the rectus muscle. It will be understood that some of the transplanted cells can be seeded into the rectus muscle. As long as a part of the graft is within the rectus sheath, it is within the scope of the present invention.

[0044] For example, transplantation by injection can be performed on both sides of the body, left or right, i.e., under the rectus sheath surrounding the left or right rectus muscle, or on both sides.

[0045] For example, transplantation by injection can be performed with a syringe such as a syringe. In the case of injection, the injection device can include a needle such as a puncture needle, or any suitable needle known in the art. The needle should be configured to be sharp and rigid enough to penetrate the skin and the rectus sheath, and long enough to reach the desired position. The length of the needle can be any length between about 1 and 25 cm. The size of the needle can be selected according to a plurality of factors including the type, amount and formulation of the graft, and the needle insertion site.

[0046] For example, the needle can have an inner diameter of about 0.15 mm to 3 mm, such as 0.15 mm, 0.2 mm, 0.25 mm, 0.3 mm, 0.35 mm, 0.4 mm, 0.45 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3 mm. When injecting pancreatic islets, the needle can have an inner diameter of about 0.25 mm to 3 mm. Those skilled in the art will be able to appropriately determine the gauge of the needle. For example, the needle can be of any size from 12 gauge to 30 gauge. For example, when injecting pancreatic islets, the needle can be of any size from 12 gauge to 26 gauge.

[0047] For injection, the cells to be transplanted can be provided as a suspension, such as a suspension of cells in saline. When the total volume of the cell suspension is large, the injection can be performed by a device having a plurality of needle tracks, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more needle tracks, and each track has a part of the total volume of the cell suspension.

[0048] The number of cells or tissues transplanted at the subcostal position of the rectus sheath can range from 1 to about 1×10 11 cells. For example, the cells or tissues can be about 1 cell, 10 cells, 1×10 2 cells, 1×10 3 cells, 1×10 4 cells, 1×10 5 cells, 1×10 6 cells, 1×10 7 cells, 1×10 8 cells, 1×10 9 cells, 1×10 10 cells, or 1×10 11 cells. For example, the cells or tissues contained in the cartridge of the syringe to be transplanted can be about 10 cells, 1×10 2 cells, 1×10 3cells, 1×10 4 cells, 1×10 5 cells, 1×10 6 cells, 1×10 7 cells, 1×10 8 cells, 1×10 9 cells, 1×10 10 cells, or 1×10 11 cells may be included. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% of the transplanted cells are viable cells.

[0049] For example, in the case of transplanting pancreatic islets, the total amount of pancreatic islets can range from 100 to 200,000 IEQ per kg of the recipient's body weight, preferably from 500 to 150,000 IEQ per kg of the recipient's body weight, more preferably from 5,000 to 80,000 IEQ per kg of the recipient's body weight, or even more preferably from 10,000 to 50,000 IEQ per kg of the recipient's body weight.

[0050] To facilitate the accurate deposition of the graft, imaging techniques such as ultrasound can be used to guide the transplantation. In one embodiment, ultrasound imaging is used to facilitate the transplantation.

[0051] Type of graft The new transplantation site of the present application is suitable for the transplantation of a wide variety of grafts or transplants. The grafts of the present invention are preferably cells, tissues or organoids.

[0052] In the broadest scope of the present application, it is not intended to limit the type or source of the graft. The graft can be cells, tissues or organoids provided by an individual, e.g., a donor, and / or prepared by in vitro methods including induction, differentiation, maturation, etc.

[0053] In some embodiments, the graft is delivered to a subject for therapeutic purposes. In this case, the graft can be cells or tissues having the desired function.

[0054] In some embodiments, the graft is delivered to the subject for non-therapeutic purposes. For example, a harmful graft can be introduced into an animal to establish a disease model. In one example of this embodiment, the graft can be tumor-derived cells or tissues.

[0055] This application is particularly suitable for xenotransplantation. In this case, the graft is a xenograft. The success of xenotransplantation depends on the ability of the xenograft or graft to survive and function properly at a new site that is different from the location where they can naturally be found. In some embodiments, the graft comprises or consists of tissues or cells of a secretory gland such as an endocrine gland, for example, the thyroid, parathyroid, hypothalamus, pituitary gland, adrenal gland, pineal gland. In some embodiments, the graft or graft comprises or consists of pancreatic islets, pancreatic cells, hepatocytes, kidney cells, thymocytes, lung cells.

[0056] In some embodiments, the graft or graft of the present invention is autologous, allogeneic, xenogeneic or syngeneic to the subject or recipient.

[0057] The graft or graft of the present invention can be derived from any species of animal, preferably a mammal, more preferably a primate, and most preferably a human.

[0058] The graft of the present invention can include immature cells or tissues that are not fully differentiated or developed. For example, if the graft is prepared from pluripotent, multipotent cells, progenitor cells or precursor cells, the graft can include certain portions of such "immature" cells. In one embodiment, the graft can be cells derived from an embryo, such as cells derived from embryonic stem cells or embryonic stem cells (ESCs), such as human embryonic stem cells. In one embodiment, the human embryonic stem cells have not undergone in vivo development and are within 14 days after fertilization. In some aspects, the graft includes fully differentiated cells, developed cells or immature cells, and these cells are capable of undergoing differentiation and / or maturation in the recipient after transplantation.

[0059] In some embodiments, the graft can be obtained from pluripotent cells, such as ESCs or induced pluripotent stem cells (iPSCs), or multipotent cells, such as precursor cells or progenitor cells differentiated from precursor cells. Examples of precursor cells include endodermal precursor cells such as endodermal precursor cells that have the potential to differentiate into the pancreas or liver.

[0060] In some specific embodiments, the graft is an islet. In the context of this application, the term "islet" is understood to broadly include any cell or cell aggregate that secretes insulin. The islets of this application include islet organoids. Islets can be harvested from a donor, including an individual, such as an allogeneic donor, autologous donor, or xenogeneic donor. Islets can be derived from stem cells including ESCs and iPSCs, or islet promoting cells. Islets can include healthy islet cells or dysfunctional islet cells. In one specific embodiment, the islet is an hPSC islet differentiated from human iPSCs. Differentiation of stem cells into islets can be completed in vitro prior to transplantation. Alternatively, stem cells are differentiated into immature islets in vitro and then matured in vivo after transplantation.

[0061] The cells to be transplanted can be prepared as a cell suspension. In some embodiments, the cells can be dissociated by an enzyme prior to transplantation. Enzymes for dissociating cells can include, but are not limited to, Accutase, TrypLE, Versene, CTS TM TrypLE and CTS TM Versene.

[0062] Therapeutic uses The new transplantation site of the present application enables minimally invasive procedures and a supportive environment for the graft, thus having great potential in therapeutic use. Depending on the type of graft, the methods of the present application can treat various diseases and conditions. The method may have potential applications in a wide range of tissue types for the purpose of complementing or reconstructing organ function. Particularly suitable organ tissues transplanted by the method may include endocrine gland tissues such as the adrenal gland. Also of interest are organs that are not essential for orthotopic transplantation. For example, the method can be used for heterotopic transplantation.

[0063] For example, the method is particularly suitable for heterotopic transplantation of hormone-producing secretory cells. In this case, the method can be used to treat diseases or conditions associated with or caused by the absence or deficiency of a hormone.

[0064] In certain embodiments, the method includes the transplantation of islets, such as hPSC-derived islets, under the rectus sheath for treating conditions resulting from the absence or deficiency of insulin, including various types of diabetes that may be affected by genetic, metabolic, environmental, and / or immune factors. Conditions may include hyperglycemia, type 1 diabetes (T1D), type 2 diabetes (T2D), T1D- or T2D-related complications, and other rare types of diabetes.

[0065] Transplantation of islets under the rectus sheath can result in increased secretion of C-peptide. Transplantation of islets under the rectus sheath can improve long-term and short-term blood glucose control. Blood glucose control can be measured by HbA1c, fasting blood glucose, postprandial blood glucose, and blood glucose variability. Since the need for exogenous insulin can be reduced or eliminated, the subject can benefit from the transplantation of the present invention.

[0066] When transplanting islets, the transplantation of the present invention enables the desirable maintenance and maturation of the graft. In some embodiments, the grafted islets are positive for mature β-cell markers such as MAFA and UCN3 about 2, 3, or 4 weeks after transplantation.

[0067] In one embodiment, the method includes transplantation of hepatocytes under the rectus sheath.

[0068] The transplantation under the rectus sheath of the present application can be used to replace the current transplantation into the portal vein.

Example

[0069] For a more complete understanding and application of the present invention, the present invention will be described in detail below with reference to examples and drawings. Those skilled in the art will understand that the following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The scope of the present invention should be defined by the appended claims.

[0070] Method Cell culture One chemically induced pluripotent human stem cell line, hPSC8#, was used in this study. hPSC8# cells were induced from human adult adipose-derived cells using a chemical reprogramming strategy without gene transfer (Guan, J. et al., Chemical reprogramming of human somatic cells to pluripotent stem cells. Nature 605, 325 - 331, doi:10.1038 / s41586 - 022 - 04593 - 5 (2022)). hPSCs were cultured in mTeSR1 (STEMCELL Technologies, 85850) on 5% CO2, 37 °C, 1:40 diluted Matrigel-coated (BD BioSciences, 356231) 6-well plates (Corning, 353046) or 500 cm 2 Square TC-treated culture dishes (Corning, 431110). The medium was changed daily, and the cells were passaged at a split ratio of 1:10 - 1:15 every 5 - 6 days with ReleSR (STEMCELL Technologies, 05872). On the other hand, a mycoplasma negative test of the cells was performed using the MycoSEQ Mycoplasma Detection Kit (Thermo Fisher Scientific, 4460626).

[0071] Differentiation protocol for generating hPSC pancreatic islets. hPSCs were differentiated using a six-step protocol as previously described by the inventors (Du, Y. et al., Human pluripotent stem-cell-derived islets ameliorate diabetes in non-human primates. Nat Med 28, 272-282, doi:10.1038 / s41591-021-01645-7 (2022)). Briefly, hPSCs were dispersed into single cells using Accutase (EMD Millipore, SCR005) and seeded at approximately 1.35×10 TM EasyFill TM -2 Cell Factory TM cells / cm 5 in a Matrigel-coated Nunc 2 system (Thermo Fisher Scientific, 169171). Twenty-four hours after seeding, differentiation was initiated by replacing the medium with a medium suitable for the protocol supplemented with small molecules or cytokines (Tables 1 and 2). At the end of stage 3, the cells were dispersed with Accutase. After rinsing with DMEM basal medium, the cells were seeded at 5×10 6 cells / well in 6-well AggreWell Microwell Plates (STEMCELL Technologies, 27940) containing stage 4 medium and spun down at 300 g for 5 minutes. After incubating at 37 °C in 5% CO2 for 24 hours, the clusters were transferred to an ultra-low attachment 6-well plate (Beaver Bio, 40406) containing stage 4 medium. The suspended aggregates were cultured in an incubator shaker (INFORS HT, Multitron) at 37 °C, 5% CO2 and 85% humidity with a rotational speed of 90 r.p.m. Detailed information on small molecules, cytokines, media and additives is listed in Table 3.

[0072]

Table 1

[0073]

Table 2

[0074]

Table 3

[0075] Cryopreservation and recovery of hPSC islets. Cryopreservation and recovery were performed as described above (Du, Y. et al., 2022, supra).

[0076] Cryopreservation. hPSC islets were dissociated using Accutase and rinsed with DMEM-basic. After counting with a Countess II Automated Cell Counter (Invitrogen, AMQAX1000), the cells were cryopreserved using a cryopreservation medium consisting of 35% FBS, 5% DMSO (Sigma-Aldrich, D2650), 60% stage 6 medium, and 10 μM Y27632 at a concentration of 1×10 7 cells / mL. The vials were then immediately transferred to a Thermo Fisher Scientific Mr.Frosty (5100-0001) and frozen at -80°C for 24 hours in a freezer. Subsequently, the vials were transferred to liquid nitrogen for long-term storage.

[0077] Recovery. The cryopreserved vials were thawed in a 37°C water bath. Next, each cell suspension was transferred to a 15 mL centrifuge tube containing 10 mL of DMEM basic medium and then centrifuged at 350 g for 3 minutes. The cells were resuspended in DMEM basic medium supplemented with 1% B27 and 10 μM Y27632. After confirming the viability and yield, the cells were seeded at 5×10 in 6-well AggreWell Microwell Plates 6Cells were seeded in wells and spun down at 300 g for 5 minutes in a microwell. After incubation at 37 °C for 24 hours in 5% CO2, the clusters were transferred to an ultra-low attachment 6-well plate containing DMEM-basic supplemented with 1% B27. The suspended aggregates were cultured in an incubator shaker at 37 °C, 5% CO2 and 85% humidity at a rotational speed of 90 r.p.m. for 24 hours and then used for transplantation.

[0078] Flow cytometry The clusters were dissociated into single cells using Accutase in a 37 °C water bath for 5 - 10 minutes and then stained for intracellular markers as described above (Du, Y. et al., 2022, supra). Briefly, individual cells were fixed and permeabilized with a fixation / permeabilization solution (BD BioSciences, 554714) at 4 °C for 20 minutes. The cells were then washed twice with Perm / Wash buffer (BD BioSciences, 554714) and incubated overnight at 4 °C with the primary antibody in Perm / Wash buffer. The cells were washed three times with Perm / Wash buffer, incubated with the secondary antibody in Perm / Wash buffer at 4 °C for 1 hour, then washed three times with Perm / Wash buffer and analyzed using BD CellQuest Pro. FlowJo version 10 software was used for flow cytometry analysis. The antibodies used are listed in Table 4.

[0079]

Table 4

[0080] Immunohistochemistry and immunofluorescence staining. For frozen tissue sections. The clusters or tissue samples were washed with PBS and fixed with 4% PFA at 4°C for 2 hours (hPSC pancreatic islet clusters) or 24 hours (tissue samples). The samples were washed three times with PBS and dehydrated overnight at 4°C in a 30% sucrose solution. The dehydrated samples were covered with OCT (Sakura, 4583), frozen in liquid nitrogen, and stored at -80°C. 10-μm frozen sections were cut, placed on slides, washed with PBS, and permeabilized with a PBST solution (PBS + 0.2% Triton X-100 + 5% donkey serum) at room temperature for 1 hour. The slides were then incubated overnight at 4°C with a primary antibody diluted in the PBST solution. After washing three times with PBS, the slides were incubated at a 1:1,000 dilution in the PBST solution with a secondary antibody conjugated to Alexa Fluor 488, 555, or 647 (Life Technologies) for 1 hour and stained with DAPI for 5 minutes at room temperature. Images were captured using a Leica TCS SP8 confocal microscope and a Zeiss LM710 confocal microscope.

[0081] For paraffin sections. The samples were fixed in a 10% formalin solution at room temperature for 7 days, embedded in paraffin, and sectioned. The sections were deparaffinized, rehydrated, and washed with PBS. After rehydration, hematoxylin and eosin (H&E) staining was performed. For immunohistochemistry, the slides were immersed in a preheated antigen retrieval solution and irradiated with microwaves until boiling for at least 15 minutes, then cooled to room temperature. The slides were washed with PBS for 5 minutes, immersed in a blocking reagent at room temperature for 1 hour in a humidified light-tight chamber, and washed three times with PBS. Next, the sections were stained with a primary antibody and a secondary antibody, followed by a color reaction and hematoxylin staining. All antibodies used are listed in Table 4.

[0082] Quantitative evaluation of graft volume. OCT was overlaid on tissue samples from each transplantation site and serially sectioned into 10-μm-thick slides using a cryomicrotome. The sections were stained with the human cell marker Stem121 and imaged using a KF-Pro-005 digital slide scanner. Stem121-positive human grafts were labeled using K-Viewer to measure the total area of the grafts. The volume of the grafts was calculated by multiplying the total area by the thickness of the sections.

[0083] qRT-PCR Total RNA was extracted from the clusters using the RNeasy Micro Kit (Qiagen, 74004) according to the manufacturer's instructions. cDNA was synthesized using the Transcript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, AT311-03). The KAPA SYBR FAST Universal qPCR Mix (KAPA Biosystems, KK4601) was used for qRT-PCR analysis, which was performed on a 7500 Real Time PCR System. The relative expression levels were normalized to the housekeeping gene GAPDH, and the results were analyzed using the ΔΔCt method. The primer sequences are listed in Table 5.

[0084]

Table 5

[0085] Glucose-stimulated insulin secretion (GSIS) Krebs buffer was prepared by dissolving 129 mM NaCl, 2.5 mM CaCl2, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1 mM Na2HPO4, 5 mM NaHCO3, 10 mM HEPES and 0.1% BSA in deionized and sterile filtered water. Separate batches of Krebs buffer containing 2.8 mM glucose, 16.7 mM glucose and 30 mM KCl were prepared and warmed to 37 °C. hPSC islets (20 - 50 clusters) were harvested and washed twice with Krebs buffer in a 24-well plate. The cells were then successively incubated at 37 °C for 1 h in Krebs buffer, Krebs buffer containing 2.8 mM glucose, Krebs buffer containing 16.7 mM glucose and Krebs buffer containing 30 mM KCl. The supernatants were collected and the cells were washed with fresh Krebs buffer after each incubation. The supernatant samples were frozen at -80 °C for C-peptide assay. After the assay, the cells were dispersed into individual cells using Accutase TM and the cells were counted using a Countess II Automated Cell Counter.

[0086] Non-human primate transplantation. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Institute of Medical Biology, Chinese Academy of Medical Science (Ethical number: DWLL201908013). Seven rhesus monkeys (Macaca mulatta) were used for hPSC islet transplantation. Three healthy monkeys were used to evaluate the feasibility of transplanting hPSC islets under the rectus sheath, in the subcutaneous space and intramuscularly in the brachioradialis muscle, and four diabetic monkeys were used to verify the functionality of hPSC islets when transplanted under the rectus sheath.

[0087] Induction of diabetes. Diabetes was induced by a single intravenous administration dose of STZ (AdooQ, A10868) injection as described above (Du, Y. et al., 2022, supra). Briefly, STZ (90 mg / kg) was diluted with 0.1 M citrate buffer (pH 4.3 - 4.5) and immediately (within 5 minutes) intravenously injected into rhesus monkeys that had been fasted overnight. After hydration, physiological saline (50 mL) was administered to each monkey. Omeprazole (0.5 mg / kg, Losec®, Astrazeneca AB) was intravenously injected after hydration to prevent nausea and vomiting. Blood glucose was monitored hourly for the first 12 hours after STZ injection and then four times a day. Exogenous insulin injection was initiated 3 days after STZ treatment. Short-acting insulin (Humalog®, Eli Lilly Italia S.p.A.) and long-acting insulin (Lantus®, Sanofi-Aventis Deutschland GmbH) were subcutaneously injected. Short-acting insulin was administered according to the dosing chart in Table 6. The levels of blood glucose, exogenous insulin, C-peptide, and HbA1c were recorded before hPSC islet transplantation.

[0088]

Table 6

[0089] Immunosuppression. The immunosuppressive regimen was initiated 9 days before transplantation (day 0) as described above (Du, Y. et al., 2022, supra). Detailed information is listed in Table 7. Induction therapy: Rituximab was injected on day -9. ATG was infused on days -5 and -3. Basiliximab was administered on day 0 of transplantation and day 2 after transplantation. Methylprednisolone, chlorpheniramine maleate tablets, and diphenhydramine were administered 10 minutes before rituximab and ATG treatment to reduce allergic reactions. Maintenance therapy: Belatacept was administered on day 0 of transplantation, day 4 after transplantation, and day 14 after transplantation, and then injected every other week. Sirolimus and tacrolimus were administered daily. The dosages of sirolimus and tacrolimus were adjusted according to trough blood levels (tacrolimus: 4 - 10 ng / mL, sirolimus: 4 - 10 ng / mL). The blood concentration of the drugs was tested using Viva-E (Vital Scientific N.V.).

[0090]

Table 7

[0091] Prophylactic and preemptive treatment. To prevent CMV infection, valganciclovir was administered daily from day -4 onwards. To reduce the inflammatory response, cobra venom factor (CVF) was administered on day -1 after transplantation, and etanercept was administered on day 0 (5 mg / kg, i.v.) and on days 3, 7, and 10 (2.5 mg / kg, i.h.).

[0092] Anesthesia. Anesthesia was initiated with propofol (0.5 mL / kg, Petsun Therapeutics) and maintained with isoflurane and oxygen. Heart rate, body temperature, blood oxygenation, and blood pressure were monitored in real time during hPSC islet transplantation. The overall quality of the hPSC islet preparation is described in Figure 1 and Tables 8 and 9.

[0093] Subcutaneous transplantation of hPSC islets. hPSC islets (1×10 4The islets (IEQ) were dispersed in IVM as reported (Yu, M. et al., Islet transplantation in the subcutaneous space achieves long-term euglycaemia in preclinical models of type 1 diabetes. Nat Metab 2, 1013-1020, doi:10.1038 / s42255-020-0269-7 (2020)) and filled into a syringe with a puncture needle (0.7×80 TWLB). After sterilizing the scalp preparation, the syringe containing hPSC islets was inserted into the subcutaneous space under ultrasound guidance. The total volume of the inoculation material was slowly injected into the subcutaneous space, and then the needle tip was wiped with iodine.

[0094] Radial forearm muscle hPSC islet transplantation. hPSC islets (1×10 4 The islets (IEQ) were dispersed in saline as reported (Bertuzzi, F., Colussi, G., Lauterio, A. & De Carlis, L., Intramuscular islet allotransplantation in type 1 diabetes mellitus. European review for medical and pharmacological sciences 22, 1731-1736, doi:10.26355 / eurrev_201803_14588 (2018)) and filled into a syringe with a puncture needle (0.7×80 TWLB). After sterilizing the forearm preparation, the syringe containing hPSC islets was inserted distally in the fiber direction of the radial forearm muscle and moved proximally through the muscle by ultrasound guidance. While slowly moving the needle from the proximal part to the distal part of the muscle, the total volume of the inoculation material was injected into the space between the fibers of the radial forearm muscle to obtain a bead-like distribution on the string of hPSC islets in the muscle, and then the needle tip was wiped with iodine.

[0095] hPSC pancreatic islet transplantation under the anterior sheath of the rectus abdominis muscle. hPSC pancreatic islets were dispersed in physiological saline and then filled into a syringe with a puncture needle (0.7×80TWLB). Under the guidance of ultrasonic images, the hPSC pancreatic islet suspension was injected into the space between the anterior sheath of the rectus abdominis muscle and the rectus abdominis muscle. For each monkey, 8 injections were applied in 4 injections on each side. Specifically, a 10MHz ultrasonic probe placed around the umbilicus was used to show the short-axis cross-section of the rectus abdominis muscle. When transplanting under the left lower side of the anterior sheath of the rectus abdominis muscle, the puncture needle entered from the confluence of the external oblique abdominal muscle, internal oblique abdominal muscle, and transversus abdominis aponeurosis. When transplanting under the right lower side of the anterior sheath of the rectus abdominis muscle, the puncture needle entered from the right edge of the white line in the left abdomen. When the needle tip punctured the anterior sheath of the rectus abdominis muscle and reached the edge of the rectus abdominis muscle, the puncture needle was inserted into the space between the anterior sheath of the rectus abdominis muscle and the rectus abdominis muscle, and the puncture needle was brought very close to the anterior layer of the rectus abdominis sheath. After reaching the target position, hPSC pancreatic islets were injected while removing the puncture needle. As a result, hPSC pancreatic islets were dispersed in and around the needle track.

[0096] Daily examinations. C-peptide secretion, HbA1c, complete blood count, serum creatinine, and liver function analysis were performed daily. The complete blood count was performed using a Sysmex XT-200i. HbA1c, serum creatinine, and liver function analysis were evaluated using a Mindray BS-2000.

[0097] Intravenous glucose tolerance test (IVGTT). After an overnight fast, 0.75 g / kg body weight of 50% glucose was intravenously injected into the monkeys within 1 minute. Blood samples were collected at 0 minutes, 5 minutes, 15 minutes, 30 minutes, 60 minutes, and 90 minutes after injection. Blood glucose was measured using a handheld glucometer, and C-peptide levels were measured by ELISA.

[0098] Arginine stimulation test. The C-peptide secretion response to intravenous arginine stimulation was measured at two different plasma glucose levels (Ref). Briefly, after an overnight fast, a dose of 70 mg / kg of 10% arginine hydrochloride (Sigma, catalog number A5006) was administered over 30 seconds, and its start was designated as 0 minutes. Blood samples were collected at 0 minutes, 2 minutes, 4 minutes, and 10 minutes after the first pulse of arginine. Then, 50% glucose was injected to raise the plasma glucose level to 20 mM and maintained. 50 minutes after the first pulse of arginine, a second arginine pulse (70 mg / kg) was injected. Blood samples were collected at 0 minutes, 2 minutes, 4 minutes, and 10 minutes after the second pulse of arginine. C-peptide levels were measured by ELISA.

[0099] ELISA The human C-peptide ELISA kit (ALPCO, 80-CPTHU-E10) was used according to the manufacturer's instructions to detect C-peptide. ELISA was performed in three technical replicates for all samples.

[0100] Autopsy and histological analysis Complete necropsy of all monkeys was performed by experienced primate pathologists. Tissue specimens of major organs were fixed with 4% PFA and 10% formalin for frozen sections and paraffin sections, respectively.

[0101] Statistical analysis. Data analysis was performed using GraphPad Prism software. Statistical significance was evaluated by t-test. Throughout the manuscript, n represents the number of biological replicates unless otherwise specified. P-values are presented as follows: *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.00005.

[0102] Example 1. Comparison of subcutaneous transplantation of islets under the rectus sheath with two other extraperitoneal transplantation strategies To evaluate the feasibility of transplanting hPSC islets into the rectus sheath, the rectus sheath approach was first compared with two previously reported extraperitoneal islet transplantation strategies, namely intramuscular transplantation and subcutaneous transplantation (Figure 1a) (Yu, M. et al., 2020, supra; Bertuzzi, F. et al., 2020, supra; Rafael, E. et al., Intramuscular autotransplantation of pancreatic islets in a 7-year-old child: a 2-year follow-up. Am J Transplant 8, 458-462, doi:10.1111 / j.1600-6143.2007.02060.x (2008); Sakata, N. et al., Strategy for clinical setting in intramuscular and subcutaneous islet transplantation. Diabetes Metab Res Rev 30, 1-10, doi:10.1002 / dmrr.2463 (2014)).

[0103] Figure 1a shows the three transplantation sites. In this example, the methods used for transplantation into the brachioradialis muscle and subcutaneous space were based on previously reported methods (Yu, M. et al., 2020, supra; Bertuzzi, F. et al., 2020, supra). For transplantation under the rectus sheath, the space between the anterior rectus sheath and the rectus muscle was selected as the graft site to keep it away from the peritoneum and avoid potential surgical damage to the blood vessels and nerve network surrounding the posterior rectus sheath (Figure 1b) (Sevensma, K.E., Leavitt, L. & Pihl, K.D., Anatomy, Abdomen and Pelvis, Rectus Sheath. StatPearls (book) (2022)).

[0104] Three healthy adult rhesus monkeys (Macaca mulatta) (Monkeys 1-3) were used (Table 8). As described in the section entitled "Methods" above, hPSC islets were differentiated from hPSCs and cryopreserved in single cell suspension. Two days before transplantation, hPSC islets were recovered, re-aggregated, and their characteristics are shown in Figure 2. Immunosuppressive therapy was applied as described in the section entitled "Methods" above. Under ultrasonic guidance, hPSC islets were transplanted into each of the three monkeys selected at a dose of approximately 8×10 4 islet equivalents (IEQ) per site (Figure 1a-b and Table 8).

[0105]

Table 8

[0106]

Table 9

[0107] Cell viability at the early stage after transplantation Monkey 1 was sacrificed at 1 week post-transplantation (wpt) to evaluate the cell survival and gene expression patterns at the early stage of the hPSC islet graft. Immunostaining of the human cell marker Stem121 detected hPSC islet grafts in all three sites (Figure 3a).

[0108] As shown in Figure 4a, the volumes of human grafts in the brachioradialis muscle and subcutaneous space were dramatically smaller than those under the rectus sheath, respectively. Furthermore, the proportion of C-peptide positive β-cells was significantly lower in intramuscular and subcutaneous grafts, and there was also a decrease in the expression of pancreatic transcription factors (NKX6.1, PDX1, and NKX2.2) compared to the proportion or expression level of the graft before transplantation (Figure 4b-d and Figure 3b-c). In contrast, the hPSC islet graft under the rectus sheath maintained its structural integrity with robust expression of islet hormones and transcription factors (Figure 4c-d and Figure 3b-3c).

[0109] In summary, these results suggest that hPSC islets showed better initial survival and maintenance when transplanted under the rectus abdominis anterior sheath.

[0110] Cell survival rate at the late stage after transplantation At 4 wpt, monkeys 2 and 3 were sacrificed. By serial sectioning of the 4-week grafts and staining with Stem121, human cells could be detected only in the grafts grown under the rectus abdominis anterior sheath, while the hPSC islets injected into the brachioradialis muscle and the subcutaneous space were observed to be completely excluded. The findings from both monkeys were consistent (Figures 5a and 6a).

[0111] Notably, most of the hPSC islets were grafted between the surface of the rectus abdominis muscle and the rectus abdominis anterior sheath, while a small proportion was found to be seeded within the rectus abdominis muscle (Figures 5a and 6a).

[0112] Immunofluorescence staining of 4-week human grafts under the rectus abdominis anterior sheath Further analysis of 4-week human grafts under the rectus abdominis fascia was performed by immunofluorescence staining. The results of immunofluorescence staining showed that the human grafts expressed essential pancreatic hormones and transcription factors (Figs. 5b–c and 6b–c). Notably, the mature β-cell markers MAFA and UCN3 were detectable (Figs. 5c and 6c) (Blum, B. et al., Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3. Nat Biotechnol 30, 261–264, doi:10.1038 / nbt.2141 (2012); Kaneto, H. et al., PDX-1 and MafA play a crucial role in pancreatic beta-cell differentiation and maintenance of mature beta-cell function. Endocrine journal 55, 235–252, doi:10.1507 / endocrj.k07e-041 (2008)). In addition, abundant blood vessels infiltrating the human grafts were observed (Figs. 5d and 6d). Immunopathological analysis showed the presence of rare CD3+ T cells, CD20+ B cells, and CD68+ macrophages within or around the grafts (Figs. 5e and 6e). Collectively, these results suggested that the rectus abdominis fascia provides an environment that promotes the functional maturation and maintenance of hPSC islets.

[0113] Example 2. Functional study of hPSC islets transplanted under the rectus abdominis fascia in diabetic monkeys In this example, the function of hPSC islets in improving the diabetic state when transplanted under the rectus abdominis fascia in diabetic monkeys was examined.

[0114] Four adult monkeys (Monkeys 4 - 7) were injected with a single high dose of streptozotocin (STZ) to induce a diabetic state as reported (Table 9) (Du, Y. et al., 2022, supra; Zhu, H., Yu, L., He, Y. & Wang, B. Nonhuman primate models of type 1 diabetes mellitus for islet transplantation. J Diabetes Res 2014, 785948, doi:10.1155 / 2014 / 785948 (2014)). After STZ injection, all macaques showed significantly increased blood glucose levels and extremely low C - peptide concentrations (fasting C - peptide: 0.08 ± 0.07 ng / ml, post - prandial C - peptide: 0.08 ± 0.07 ng / ml) (Figures 7a - d, 8a - d and 9a - d). Exogenous insulin was administered 3 days after STZ injection according to blood glucose levels, and its daily dose was 1.5 - 3.0 IU / kg, consistent with previous reports in the same model system. -1within the range of the day (Fig. 7i-l) (Du, Y. et al., 2022, supra; Zhu, H. et al., 2014, supra; Shin, J. S. et al., Long-term control of diabetes in immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets. Am J Transplant 15, 2837-2850, doi:10.1111 / ajt.13345 (2015)). HbA1c, a parameter commonly used to evaluate long-term average blood glucose concentration (Sherwani, S. I., Khan, H. A., Ekhzaimy, A., Masood, A. & Sakharkar, M. K. Significance of HbA1c Test in Diagnosis and Prognosis of Diabetic Patients. Biomark Insights 11, 95-104, doi:10.4137 / BMI.S38440 (2016); American Diabetes, A. 2. Classification and Diagnosis of Diabetes: Standards of Medical Care in Diabetes-2021. Diabetes Care 44, S15-S33, doi:10.2337 / dc21-S002 (2021)), dramatically increased from 3.6 ± 0.3% before STZ treatment to 7.7 ± 2.2% on the day of hPSC islet transplantation (Figs. 7e-h).

[0115] The dose of hPSC islets for transplantation under the rectus sheath was designed based on the dose used in the portal vein injection, ranging from 40,000 IEQ to 48,000 IEQ per kilogram of body weight (IEQ / kg), with an average of 45,000 IEQ / kg (Table 9) (Du, Y. et al., 2022, supra). The hPSC islets were injected in a tubular fashion into the space between the rectus sheath and the rectus muscle using a puncture needle, and the cell clusters were dispersed within and around the needle track (Figure 1b). Considering that high-density hPSC islets in a single needle track may impair cell survival, the hPSC islets were administered by eight injections in four parallel needle tracks on both sides of the rectus muscle (Figure 1c). Considering that heterologous immune attack-mediated clearance of hPSC islet grafts was observed 4 mpt under a human-monkey xenotransplantation setting as previously reported (Du, Y. et al., 2022, supra), to evaluate the persistence of the functional effects of hPSC islets, diabetic recipients were tested by continuously monitoring blood glucose and C-peptide secretion for 12 weeks after transplantation.

[0116] Improvement in glycemic control After injecting hPSC islets under the rectus sheath, all diabetic recipients showed a significant improvement in overall glycemic control (Figure 7). Blood glucose levels gradually decreased, and this change was accompanied by a reduction in exogenous insulin requirements (Figure 7a-d and Figure 7i-l). In all recipients, fasting blood glucose was stably maintained below 180 mg / dL -1 (10 mM) from 6 wpt, and blood glucose 2 hours after a meal decreased to levels below 144 mg / dL -1 (8.0 mM) from 5 wpt (Figure 7a-d and Figure 8a-d). The fluctuations in blood glucose levels also significantly attenuated (Figure 7a-d). As a result, the mean Hb1Ac decreased from 7.7 ± 2.2% at baseline to 4.3 ± 1.3% at 12 wpt (Figure 7e-h).

[0117] Notably, severe hypoglycemic events occurring during the perioperative period after portal vein injection were not detected in recipient macaques after transplantation under the anterior rectus sheath. In our previous study, when hPSC islets were injected into the hepatic portal vein, immediate blood-mediated inflammatory reaction (IBMIR)-mediated β-cell death often resulted in blood glucose levels below 54 mg / dl -1 (3.0 mM) in recipient macaques within the first 12 hours after transplantation, necessitating intravenous injection of high-concentration dextrose (Du, Y. et al., 2022, supra; Rickels, M. R. & Robertson, R. P. Pancreatic Islet Transplantation in Humans: Recent Progress and Future Directions. Endocr Rev 40, 631-668, doi:10.1210 / er.2018-00154 (2019); Shin, J. S. et al., 2015, supra; Faradji, R. N. et al., C-peptide and glucose values in the peritransplant period after intraportal islet infusions in type 1 diabetes. Transplant Proc 37, 3433-3434, doi:10.1016 / j.transproceed.2005.09.090 (2005)). These clear observations suggest an improvement in the initial survival rate of hPSC islets after transplantation under the anterior rectus sheath.

[0118] Reduced exogenous insulin requirement The exogenous insulin requirement decreased in all recipients of hPSC islet transplantation under the anterior rectus sheath (Fig. 7i-l). A rapid decrease in the exogenous insulin requirement was observed immediately after hPSC islet injection, followed by a continuous decreasing trend corresponding to the functional maturation of hPSC islets (Fig. 7i-l). At 12 wpt, the average exogenous insulin requirement decreased from 2.3 ± 0.5 IU / kg -1 / day to 1.3 ± 0.4 IU / kg -1 / day (Fig. 7i-l).

[0119] C-peptide secretion C-peptide secretion was also monitored in all monkeys. Both fasting and postprandial C-peptide significantly increased at the first 6 wpt, with mean levels of approximately 0.6 ng ml -1 and 2.0 ng ml -1 being maintained from 8 wpt (Figures 9a–d). The stimulation index after 6 wpt was equivalent to that of native islets (Figures 9a–d).

[0120] Furthermore, intravenous glucose tolerance tests (IVGTTs) showed that glucose clearance ability was gradually improved along with enhanced glucose-responsive insulin secretion (Figures 9e–l).

[0121] Furthermore, arginine-stimulated insulin secretion tests provided further evidence of the C-peptide secretory capacity of hPSC islet grafts (Figures 8e–h).

[0122] Collectively, these data revealed that hPSC islets transplanted under the rectus sheath effectively improved overall glycemic control in diabetic macaques through restored insulin secretion.

[0123] Histological analysis To confirm that the improvement in blood glucose levels was due to hPSC islet transplantation rather than recovery of endogenous β-cells, all recipient monkeys were sacrificed at 13 wpt for evaluation. Histological analysis of the native pancreas revealed that endogenous islets were severely damaged and C-peptide + cells were undetectable (Figures 10a–d). These results were consistent with the extremely low concentrations of endogenous C-peptide observed before transplantation (Figures 9–d and Figures 9i–l). Furthermore, CK19 + proinsulin +Cells were not observed, and this population has been reported to contribute to the recovery of endogenous β-cells in the pancreas after STZ treatment (Figure 10e) (Bottino, R. et al., Recovery of endogenous beta-cell function in nonhuman primates after chemical diabetes induction and islet transplantation. Diabetes 58, 442 - 447, doi:10.2337 / db08-1127 (2009)).

[0124] In contrast, a high proportion of C-peptide + β-cells were present in the human grafts under the rectus sheath, and strong expression of MAFA and UCN3 was detected (Figure 11c). Quantitative analysis showed that the proportion of β-cells in the transplanted hPSC islets was similar to that measured before transplantation, and more than half of the β-cells were co-stained with MAFA (Figure 11d - f). These observations were consistent in four diabetic monkeys (Figure 11d - f and Figure 12a - f).

[0125] Collectively, these results provided evidence that the improvement in glycemic control was due to the hPSC islet grafts under the rectus sheath.

[0126] Whole-body ultrasound examination Furthermore, whole-body ultrasound examination was performed on all four recipient monkeys at 13 wpt before sacrifice, and no evidence of teratoma formation was found (Figure 12). Complete necropsy further confirmed the absence of tumor formation and abnormalities in the major organs examined (Figure 12).

Claims

**Claim 1** A method for transplanting one or more cells or one or more tissues into a subject in need thereof, the method comprising introducing the cells or tissues into a site under the rectus sheath. **Claim 2** The method according to claim 1, wherein the cells or tissues are introduced into a site between the rectus sheath and the rectus abdominis muscle. **Claim 3** A method for transplanting one or more cells or one or more tissues into a subject in need thereof, the method enabling one or more of the transplanted cells or tissues to be grafted into a site under the rectus sheath after transplantation. **Claim 4** The method according to claim 3, enabling one or more of the transplanted cells or tissues to be grafted into a site between the rectus sheath and the rectus abdominis muscle. **Claim 5** The method according to claim 3 or 4, wherein one or more of the transplanted cells are grafted into the rectus abdominis muscle. **Claim 6** The method according to any one of claims 1 to 5, wherein the rectus sheath is the rectus sheath anterior or the rectus sheath posterior, preferably the rectus sheath anterior. **Claim 7** The method according to any one of claims 1 to 6, wherein the cells or tissues are cells or tissues generated in vitro or isolated from natural organs. **Claim 8** The method according to claim 7, wherein the cells or tissues are generated in vitro from one or more of pluripotent cells, multipotent cells, progenitor cells or precursor cells. **Claim 9** The method according to claim 8, wherein the cells or tissues are cells or tissues differentiated from human pluripotent stem cells (hPSCs). **Claim 10** The method according to claim 9, wherein the cells are hormone-secreting cells differentiated from hPSCs. **Claim 11** The method according to claim 10, wherein the cells are islets (hPSC islets) differentiated from hPSCs. **Claim 12** The method according to any one of claims 9 to 11, wherein the hPSCs are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). **Claim 13** The method according to any one of claims 8 to 12, wherein at least a portion of the transplanted cells or tissues undergoes maturation after the transplantation. **Claim 14** The method according to any one of claims 1 to 13, wherein the cells are introduced into the site by injection. **Claim 15** A method for treating a disease or condition caused by or associated with insulin deficiency in a subject in need thereof, comprising transplanting one or more pancreatic islets into the subject, wherein the one or more pancreatic islets are introduced into a site under the rectus sheath.

16. A method for reducing the need for exogenous insulin in a subject having a disease or condition caused by or associated with insulin deficiency, comprising transplanting one or more pancreatic islets into the subject, wherein the one or more pancreatic islets are introduced into a site under the rectus sheath.

17. The method according to claim 15 or claim 16, wherein the disease is diabetes or a complication thereof.

18. The method according to any one of claims 15 to 17, wherein the pancreatic islets are introduced into a site between the rectus sheath and the rectus abdominis muscle.

19. The method according to any one of claims 15 to 18, wherein one or more of the transplanted pancreatic islets are enabled to be grafted into a site under the rectus sheath after transplantation.

20. The method according to claim 19, wherein one or more of the transplanted pancreatic islets are enabled to be grafted into a site between the rectus sheath and the rectus abdominis muscle.

21. The method according to claim 19 or claim 20, wherein one or more of the transplanted pancreatic islets are grafted into the rectus abdominis muscle.

22. The method according to any one of claims 15 to 21, wherein the rectus sheath is the anterior rectus sheath or the posterior rectus sheath, preferably the anterior rectus sheath.

23. The method according to any one of claims 15 to 22, wherein the pancreatic islets are generated in vitro or isolated from the pancreas.

24. The method according to any one of claims 15 to 23, wherein the pancreatic islets are generated in vitro from one or more of pluripotent cells, multipotent cells, progenitor cells or precursor cells derived from hPSCs.

25. The method according to claim 24, wherein the pancreatic islets are differentiated from hPSCs (hPSC islets).

26. The method according to claim 25, wherein the hPSCs are ESCs or iPSCs.

27. The method according to any one of claims 15 to 26, wherein at least a part of the transplanted pancreatic islets undergoes maturation after the transplantation.

28. The method according to any one of claims 15 to 27, wherein the cells are introduced into the site by injection.

29. The method according to any one of claims 15 to 28, wherein the islets are transplanted in an amount of 100 to 200,000 islet equivalents (IEQ).

30. The method according to any one of claims 15 to 29, wherein the islets are transplanted into both of the left and right rectus abdominis muscles at a site under the corresponding rectus sheath.

31. The method according to any one of claims 15 to 30, comprising transplanting islets into the subject two or more times.

32. Use of hPSC islets for treating a disease or condition caused by or associated with insulin deficiency, wherein the hPSC islets are transplanted at a site under the rectus sheath anterior.

33. Use of hPSC islets for reducing the need for exogenous insulin, wherein the hPSC islets are transplanted at a site under the rectus sheath anterior.

34. The use according to claim 32 or claim 33, wherein the hPSC islets are transplanted at a site between the rectus sheath anterior and the rectus abdominis muscle.

35. An apparatus for injecting islets into a site within the rectus sheath, preferably a site between the rectus sheath and the rectus abdominis muscle.

36. The apparatus according to claim 35, wherein the islets are hPSC-derived islets.

37. The apparatus according to claim 35 or claim 36, comprising a needle.

38. The apparatus according to claim 35 or claim 36, comprising a plurality of needle tracks.

39. The apparatus according to claim 38, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more needle tracks.

40. The apparatus according to any one of claims 35 to 39, comprising a cartridge.

41. The apparatus according to claim 40, wherein the cartridge contains islets in an amount of 100 to 200,000 islet equivalents (IEQ) per kg of the recipient's body weight.

42. The cartridge is 1×10 ２ ~2×10 ７ 41. The device of claim 40, comprising an amount of pancreatic islets of IEQ.

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