New transplantation site

Abstract

The invention provides a new transplantation site. In particular, the present invention relates to methods of transplanting cells or tissues, in particular pancreas islets, into a site under the rectus abdominis sheath. The invention also relates to a method of treating diabetes by transplanting the pancreatic islet, in particular the hPSC-pancreatic islet, to the position under the rectus abdominis sheath.

Description

[0001] Cross-references

[0002] This application claims the benefit of PCT International Application No. PCT / CN2022 / 095086 filed on May 25, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of cell, tissue or organ transplantation. Specifically, the present disclosure provides a method for transplanting cells or tissues to a site below the rectus sheath. After transplantation, the cells or tissues are colonized below the rectus sheath, and preferably most of the cells or tissues are colonized in the space between the rectus sheath and the rectus abdominis. The new transplantation site of the present invention is particularly suitable for ectopic transplantation, such as ectopic transplantation of hormone-secreting cells or tissues. In addition, the new transplantation site provides a desired environment for transplants that are not fully differentiated or mature at the time of transplantation. Background Art

[0004] Human pluripotent stem cells have shown great potential in cell replacement therapy for diseases such as diabetes. Our previous studies on non-human primates have demonstrated the feasibility of transplanting human pluripotent stem cell-derived pancreatic islets (hPSC-islets) to treat type 1 diabetes.

[0005] However, challenges remain in the clinical application of hPSC-derived cells or tissues (including hPSC islets), one of which is the lack of transplantation sites that effectively support the functional maturation and long-term survival of the grafts.

[0006] Taking hPSC-islets as an example, clinical islet transplantation most often uses portal vein infusion; however, it is far from satisfactory due to the large loss of islets in the early transplantation stage, the low vascular regeneration of transplanted islets, and the long-term progressive loss of graft function. In order to maintain the level of therapeutic effect of functional islets, transplantation requires an excess of cells to compensate for the loss. However, this excess may lead to hypoglycemia. In addition, liver implantation causes hPSC islets to be directly exposed to high concentrations of immunosuppressive drugs and coagulation and complement systems, which will further hinder their functional maturation and long-term maintenance. Previous experience in mouse models has shown that the infrarenal capsule is desirable for a variety of cell and tissue transplants, including both primary human islets and hPSC-islets (Du, Y. et al. Human pluripotent stem-cell-derived islets ameliorated iabetes 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 invitro 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 due to the limited space provided by the high graft volume and the invasive surgical procedure.

[0007] Many efforts have been made to study alternative sites for islet transplantation in animal models. Success has been achieved in rodent models, but few transplantation sites have been transferable to larger animal models.

[0008] There remains a need in the art for transplantation sites that can provide a microenvironment that allows for cell survival, growth, and maintenance of transplanted cells, particularly hPSC-derived transplants. Summary of the invention

[0009] The present inventors have found that when transplanted under the anterior rectus sheath, hPSC-islets gradually mature and show excellent colonization function, which leads to an overall improvement in glycemic control in diabetic non-human primates. Notably, C-peptide secretion increased significantly from 6 weeks post-transplantation (wpt) and responded to meal challenge, with a stimulation index comparable to that of native islets. The average postprandial C-peptide level was maintained at about 2.0 ng ml starting from 8 wpt. -1 , 5 times higher than intraportal infusion. As a result, the average HbA1c reduction was more than 44% at 12wpt. In addition, the recipient macaques recovered quickly from the cell transplantation and showed a lower risk of hypoglycemia within the first 24 hours. In summary, the data indicate that the anterior rectus sheath is the optimal transplantation site for hPSC-islets, which can be extended to other derivatives of hPSCs. This simple, non-invasive transplantation strategy combined with hPSC derivatives can introduce a new paradigm for cell replacement therapy, thereby completing the present invention.

[0010] Therefore, in the first aspect, the present application provides a method for transplanting one or more cells or tissues to a subject in need thereof, comprising introducing the cells or tissues into a site (subrectus sheath site) below the rectus sheath of the subject, preferably a site between the rectus sheath and the rectus abdominis. In some embodiments, the cells or tissues to be transplanted may be cells or tissues derived from hPSC. In some embodiments, the cells or tissues to be transplanted are those suitable for ectopic transplantation, such as secretory cells, such as endocrine glands. In a specific embodiment, the cells or tissues to be transplanted are pancreatic islets derived from hPSC. In another aspect, the method allows one or more transplanted cells or tissues to be implanted in a site below the rectus sheath after transplantation, preferably a site between the rectus sheath and the rectus abdominis.

[0011] In a second aspect, the present application provides a method for treating a disease or condition caused by or associated with insulin deficiency in a subject in need thereof, comprising transplanting islets to the subject, wherein the islets are introduced into a site below the rectus sheath (subrectus sheath site). In some embodiments, the islets are derived from hPSCs. In some embodiments, the disease is diabetes or its complications.

[0012] In a third aspect, the present application provides a method of reducing the need for exogenous insulin in a subject suffering from a disease or condition caused by or associated with insulin deficiency, comprising transplanting one or more pancreatic islets to the subject, wherein the one or more pancreatic islets are introduced into a site below the rectus sheath (subrectus sheath site). In some embodiments, the pancreatic islets are derived from hPSCs.

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

[0014] In a fifth aspect, the present application provides a device for injecting islets (e.g., hPSC-derived islets) into a site below the rectus sheath (subrectus sheath site), preferably between the anterior rectus sheath and the rectus abdominis. In some embodiments, the device comprises a needle and a cartridge containing a suspension of hPSC-derived islets. In a preferred embodiment, the device comprises a puncture needle. In a more preferred embodiment, the puncture needle has a single or multiple needle tracks, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more needle tracks.

[0015] The novel transplantation site of the present invention provides at least the following advantages.

[0016] (1) Transplantation of hPSC islets under the rectus sheath has achieved the desired therapeutic effect, showing comparable function to primary human islets from healthy adults.

[0017] (2) The rectus abdominis muscle is rich in blood vessels and is surrounded by the rectus sheath, which provides the microenvironment required for the implant to survive, grow, and maintain after transplantation under the rectus sheath.

[0018] (3) As an extraperitoneal site, the subrectus sheath transplantation of hPSC islets can be accomplished in a simple and non-invasive manner, such as by infusion at bedside. This convenient procedure allows for repeated infusions and rapid recovery after the transplantation procedure.

[0019] (4) Subrectus sheath transplantation allows for removal of the graft in any situation where the recipient no longer benefits from the transplanted tissue. For example, since the long-term safety of hPSC-derived tissue is still under investigation, it may be necessary to leave a window open to remove hPSC islets.

[0020] (5) Intraportal transplantation of pancreatic islets triggers an immediate blood-mediated inflammatory reaction (IBMIR) and activates coagulation. Therefore, anticoagulants need to be continuously used during intraportal transplantation. Improper control of coagulation may lead to dangerous or even fatal hypercoagulability (thrombosis) or hypocoagulability (hemorrhage). IBMIR has been reported to cause significant β-cell death, which in turn leads to a decrease in blood sugar. By transplanting at the new site of the present invention, hypoglycemia due to β-cell death was not observed, indicating that the problem caused by IBMIR has been solved.

[0021] (6) The new transplantation site under the rectus sheath can also be used for transplantation of many types of cells or tissues. Particularly preferred transplants are hPSC-derived cells or tissues, such as hPSC-islets. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 ac|Schematic diagram of the hPSC-islet transplantation strategy. a. hPSC-islets were transplanted into three extraperitoneal sites for comparison. b. hPSC-islets were transplanted under the anterior sheath using a puncture needle under the guidance of ultrasound imaging. c. hPSC-islets were delivered via eight injections in four parallel needle tracks on both sides of the rectus abdominis muscle.

[0023] Figure 2 af | Characterization of hPSC-islets for transplantation. a. Representative bright field images of hPSC-islets. Scale bar, 200 μm. b. Representative flow cytometry data showing the expression of islet hormones and transcription factors in hPSC-islets. c. Representative immunofluorescence staining of islet hormones in hPSC islet sections. Scale bar, 25 μm. d. Representative immunostaining of key β-cell transcription factors and C-peptide in hPSC islet sections. Scale bar, 50 μm. e. C-peptide secretion in static glucose stimulation assays of hPSC-islets (n=3) under low glucose (2.8 mM), high glucose (16.7 mM), and 30 mM KCl depolarization. The glucose stimulation index is shown above the bar graph. f. qRT-PCR analysis of key islet genes in hPSC-islets (n=4) and human islets (n=3).

[0024] Figure 3 ac| Immunostaining of hPSC-islet colonies at three extraperitoneal transplantation sites in monkey 1 1 week after transplantation. a. Representative immunohistological staining of the human cell marker Stem121. Scale bar, 400 μm; magnification (indicated by green border) scale bar, 50 μm. (bc). Representative immunofluorescence staining of C-peptide and β-cell-specific transcription factors PDX1 (b) or NKX2.2 (c) in human colonies. Scale bar, 50 μm.

[0025] Figure 4 ad|Evaluation of hPSC-islet colonies at three extraperitoneal transplantation sites in Monkey 1 1 week after transplantation. a. Quantitative assessment of the volume of hPSC-islet colonies at the three designated sites. b. Proportion of C-peptide expressing cells in human colonies at the three designated sites (n=12). c. Representative immunofluorescence staining of islet hormones in hPSC-islet colonies. d. Representative immunofluorescence staining of C-peptide and β-cell-specific transcription factor NKX6.1 in human colonies. Scale bar, 50 μm. Data are shown as mean ± SEM.

[0026] Figure 5 ae | Evaluation of hPSC-islet colonies in monkey 2 4 weeks after transplantation. a. Representative immunohistological staining images of the human cell marker Stem121 at the three indicated sites. Scale bar, 400 μm; magnification (indicated by green border) scale bar, 50 μm. (bc). Representative immunofluorescence staining of islet hormones, pancreatic transcription factors, and UCN3 in colonies under the anterior rectus sheath. Scale bar, 50 μm. d. Representative immunohistological staining of the endothelial marker CD31 in colonies under the anterior rectus sheath. Scale bar, 100 μm; magnification (indicated by green border) scale bar, 25 μm. e. Representative immunohistological staining of the T cell marker CD3, the B cell marker CD20, and the macrophage marker CD68 in colonies under the anterior rectus sheath. Scale bar, 100 μm; magnification (indicated by green border) scale bar, 25 μm.

[0027] Figure 6 ae | Evaluation of hPSC-islet colonies in monkey 3 4 weeks after transplantation. a. Representative immunohistological staining of the human cell marker Stem121 in colonies under the anterior rectus sheath. Scale bar, 400 μm; magnification (indicated by green border) scale bar, 50 μm. (bc). Representative immunofluorescence staining of islet hormones, pancreatic transcription factors, and UCN3 in colonies under the anterior rectus sheath. Scale bar, 50 μm. d. Representative immunohistological staining of the endothelial marker CD31 in colonies under the anterior rectus sheath. Scale bar, 100 μm; magnification (indicated by green border) scale bar, 25 μm. e. Representative immunohistological staining of the T cell marker CD3, the B cell marker CD20, and the macrophage marker CD68 in colonies under the anterior rectus sheath. Scale bar, 100 μm; magnification (indicated by green border) scale bar, 25 μm.

[0028] Figure 7a-1 | hPSC-islet transplantation under the anterior rectus sheath leads to improved glycemic control in immunosuppressed diabetic macaques. ad. Fasting, pre-prandial, and postprandial blood glucose were continuously followed before and after hPSC-islet transplantation under the anterior rectus sheath in STZ-treated monkeys. Each data point represents a weekly value based on the mean blood glucose (n=7 independent values), except for monkey 5 at -5wpt (n=5 independent values), monkey 6 at -5wpt (n=6 independent values), and monkey 7 at -5wpt (n=6 independent values). eh. Glycated hemoglobin (HbA1c) levels were continuously followed in diabetic macaque recipients before STZ treatment (grey), before hPSC-islet transplantation (0wpt, brown), and after hPSC-islet transplantation (black). il. Exogenous insulin requirements of diabetic macaque recipients were continuously followed before and after hPSC-islet transplantation. Each data point represents a weekly value based on the mean daily exogenous insulin dose (n=7 independent dose values). Exogenous insulin doses in the last week before transplantation and at 12-wpt are indicated in brown. Data are shown as mean ± SEM.

[0029] Figure 8 ah | Evaluation of hPSC-islet colonization in monkey 3 4 weeks after transplantation. ad. Daily fasting blood glucose levels before (grey), before (brown) and after (black) STZ treatment, hPSC-islet transplantation under the anterior rectus sheath (infusion procedure was performed on day 0). eh. C-peptide secretion in response to glucose-enhanced arginine (Arg) stimulation, performed at 12 wpt (n=3, technical replicates) and before transplantation.

[0030] Fig. 9 al | C-peptide secretion in diabetic rhesus macaques with hPSC-islet transplantation under the anterior rectus sheath. ad. Fasting (grey) and postprandial (red) C-peptide secretion in diabetic macaque recipients was measured before STZ treatment, before hPSC-islet transplantation (0 wpt), and after hPSC-islet transplantation (n=3 technical replicates). The stimulation index is shown above the bar graph. el. Changes in blood glucose levels (eh) and C-peptide levels (il) in diabetic macaque recipients after intravenous glucose tolerance test (IVGTT) before transplantation (Pre-Tx) and 4, 8, and 12 wpt (technical replicates; n=2 (eh); n=3 (il)).

[0031] Fig.10ad|Gross anatomy, histology, and immunological analysis of native pancreas from STZ-treated recipient monkeys. a. Gross anatomy and H&E staining of pancreatic sections from recipient monkeys, with islet structures outlined in green. Scale bar, 100 μm. b. C-peptide staining of pancreatic sections from STZ-treated recipient monkeys. Scale bar, 200 μm; magnification (indicated by green border) scale bar, 50 μm. ce. Representative immunofluorescence staining of pancreatic sections from STZ-treated recipient monkeys for CHGA (c), islet hormones (d), and CK19 and proinsulin (e). Scale bar, 50 μm.

[0032] Fig.11 ag|Characterization of hPSC-islet colonies in monkey 4 at 13wpt. a. Representative immunohistological staining of the human cell marker Stem121 in colonies under the anterior rectus sheath. Scale bar, 400 μm; magnified image (indicated by green border) scale bar, 100 μm. (bc). Representative immunofluorescent staining of islet hormones, pancreatic transcription factors, and UCN3 in colonies under the anterior rectus sheath. Scale bar, 50 μm. d. Proportion of human colonies expressing C-peptide (n=10). e. Proportion of C-peptide expression in PDX1 or NKX6.1-positive cells in hPSC-islet colonies (n=4). f. Proportion of MAFA expression in C-peptide-positive cells in hPSC-islet colonies (n=4). g. Representative immunohistological staining of the endothelial marker CD31 in colonies under the anterior rectus sheath. Scale bar, 100 μm; magnified image (indicated by green border) scale bar, 25 μm.

[0033] Fig.11 ag | Characterization of hPSC-islet colonies in monkeys 5-7 at 13wpt. (af). Representative immunofluorescence staining of islet hormones, pancreatic transcription factors, and UCN3 in colonies under the anterior rectus sheath. Scale bar, 50μm. g. Representative immunohistological staining of endothelial marker CD31 in colonies under the anterior rectus sheath. Scale bar, 100μm; magnification (indicated by green border) scale bar, 25μm.

[0034] Fig.12 al|Ultrasound examination and autopsy of major organs of transplanted diabetic macaques. Gross anatomy (a, d, g, and j), H&E staining (b, e, h, and k), and ultrasound examination (c, f, i, and l) of major organs of monkeys 4-7. Scale bar, 200 μm. DETAILED DESCRIPTION

[0035] Unless specifically defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0036] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their plural counterparts unless the context clearly dictates otherwise.

[0037] In the context of the present invention, unless otherwise stated, "comprising" and its variants such as "including" and "containing" are understood to mean including the elements, such as amino acid sequences, nucleotide sequences, properties, steps or a group thereof, but not excluding any other elements, such as amino acid sequences, nucleotide sequences, properties and steps. When used herein, the term "comprising" or any variant thereof can be replaced with "containing", "including" or "having" or synonymous variants. In certain embodiments, the word "comprising" also includes the situation of "consisting of".

[0038] "Transplantation under the rectus sheath" refers to the introduction of a graft or implant into a position below and within the rectus sheath, preferably into the space between the rectus sheath and the rectus abdominis. "Transplantation under the anterior rectus sheath" specifically refers to the application of a graft or implant into a position below the anterior side of the rectus sheath, preferably into the space between the anterior rectus sheath and the anterior side of the rectus abdominis, and to a position below the anterior rectus sheath, such as Figure 1 As shown in a and 1b.

[0039] The terms "rectus abdominis / abdominis rectus", "musculus rectus abdominis" or "abdominal muscle" are used interchangeably in the context of the present invention and refer to the two rows of parallel flat muscles located on either side of the linea alba and extending along the entire length of the anterior abdomen.

[0040] 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 a transplant.

[0041] The term "secretory cell" refers to any cell that has the function of producing a substance and secreting that substance into the extracellular space.

[0042] The term "pancreatic islets" as used herein refers to clusters of cells containing insulin-producing cells, which are able to regulate blood sugar levels.

[0043] The term "islet equivalents" or "IEQ" is a standardized measure that describes the mass of pancreatic islets to be transplanted. Reference may be made, for example, to Lembert N et al. for measuring IEQ (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).

[0044] The term "pluripotent stem cell (PSC)" herein refers to an undifferentiated cell, which is defined as having the ability to self-renew and differentiate at the single cell level. Stem cells can produce daughter cells, including self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells. Stem cells may be characterized by their ability to differentiate into various cell lineages from multiple germ layers (endoderm, mesoderm, and ectoderm). The abbreviation "hPSC" refers to human pluripotent stem cells. Preferably, the hPSC in the method of the present invention is an induced pluripotent stem cell (iPSC), or an embryonic stem cell (ESC) obtained from an embryo that has not yet developed in vivo and within 14 days after fertilization.

[0045] The term "hPSC-derived" cells or tissues refers to those cells or tissues generated or differentiated from human pluripotent stem cells.

[0046] The term "hPSC-islets" refers to pancreatic islets comprising insulin-producing cells, which are derived from human pluripotent stem cells.

[0047] Transplantation site

[0048] The advantage of the present application is that a new transplantation site is found below and within the rectus sheath, preferably within or near the space between the rectus sheath and the rectus abdominis. In the present invention, "transplantation site" refers to a site where one or more cells or tissues are delivered and introduced. The expression "cells or tissues are implanted at a certain site" refers to the residence and growth of transplanted cells or tissues at the site after transplantation. In some cases, the site where cells or tissues are implanted is not completely consistent with the transplantation site or 100% overlapped, because some transplanted cells may migrate to a certain extent.

[0049] For easier understanding, refer to Figure 1 ac describes the new transplantation site of the present application. Figure 1As shown in Figs. 1a and 1c, the implant or implant is delivered to a position between the rectus sheath and the surface of the rectus abdominis muscle. However, due to the accuracy of the physician's operation, the exact injection site may vary and may sometimes be slightly deeper into the superficial rectus abdominis muscle. In another embodiment, the implant or implant is delivered to a position in the rectus abdominis muscle, preferably near the rectus sheath.

[0050] like Figure 1 a, the two rightmost images show cross-sectional views of the human abdomen, showing exemplary locations where a graft or implant may be introduced into the present invention. The graft is delivered to the thin layer of the rectus sheath (specifically, Figure 1 The position of the abdominis anterior sheath of the rectus abdominis in a) and the anterior side of the rectus abdominis. After delivery, the graft can spread along the rectus abdominis and form a graft layer under the rectus abdominis sheath. It should be understood that the needle can penetrate any part of the skin at any angle to any depth, as long as it can reach the desired transplant site.

[0051] The transplantation site of the present application can be easily reached by conventional administration, for example, by injecting with a device comprising a needle. Therefore, cells or tissues to be transplanted can be delivered by less invasive methods such as injection or infusion. The transplantation site of the present application also allows the removal of colonization in the event of any undesirable side effects or no longer needing a transplant after transplantation.

[0052] After the graft or colonization is delivered to the desired site, most of the transplanted cells can reside in situ, while some transplanted cells can also migrate to a position away from the original transplantation site in the rectus sheath. As long as the cells derived from the graft or colonization remain in the rectus sheath, it is within the scope of the present application.

[0053] The transplantation site of the present application also provides the best environment for the maintenance of graft or plant.In the scope surrounded by the rectus sheath, enough space can be provided for graft or plant to grow, and abundant vascular network provides enough oxygen and nutrients to support their survival, growth and maintenance for graft or plant.In some cases, can observe the vascular infiltration in graft or plant after transplanting for several weeks.

[0054] The transplantation site of the present application is particularly suitable for "pre-maturity" transplantation or planting that may need a post-transplantation in vivo maturation process. For example, the planting plant may include a part of an incompletely developed or differentiated cell. This transplantation environment that allows functional maturation is particularly important for cells or tissues derived from undifferentiated cells or incompletely differentiated cells (especially stem cells, endoderm stem cell lines, mesenchymal stem cells, progenitor cells or precursor cells). In some embodiments, the transplant or planting plant is a cell or tissue derived from undifferentiated cells or incompletely differentiated cells, such as those differentiated from hPSC, which are referred to as hPSC-cells or hPSC-tissues in the context of the present application. For example, cells or tissues derived from human pluripotent stem cells (hPSC) can be those derived from embryonic stem cells or induced pluripotent stem cells (iPSC). In a specific embodiment, for the transplantation of hPSC-derived islets, post-transplantation maturation allows the improvement of the insulin secretion ability of hPSC-islets and the sensitivity to glucose challenges, which is critical for the therapeutic effect of hPSC-islets.

[0055] Transplantation process

[0056] The present invention is not intended to be limited to methods of introducing a implant into a site beneath the rectus sheath. The means or device used to perform the implant may depend on the type of implant, the amount of implant, and the like.

[0057] In some embodiments, transplantation is performed by injection. Figure 1 b illustrates the subrectus sheath injection of the graft. Figure 1 As shown in FIG. 1 , the puncture needle is operated to enter the body, pierce through the skin and the rectus sheath, and reach a position below the rectus sheath, preferably between the surface of the rectus abdominis and the (front) rectus sheath. Then, while keeping moving along the space between the (front) rectus sheath and the rectus abdominis, the needle is allowed to penetrate deeper. After the needle is in the desired position, cell injection is started. When injecting cells, the needle is slowly withdrawn along the needle track so that the injected cells remain along the track between the rectus sheath and the rectus abdominis. The present invention does not require that all grafts are injected or inoculated into the space between the rectus sheath and the rectus abdominis. It is understood that a portion of the transplanted cells can be inoculated into the rectus abdominis. As long as a portion of the graft is in the rectus sheath, it is within the scope of the present invention.

[0058] The implant, for example by injection, can be placed on either side of the body (left or right), ie, beneath the rectus sheath surrounding the left or right rectus abdominis muscle, or on both sides.

[0059] Transplantation, for example, by injection, can be carried out by syringe such as syringe.In the case of injection, injection device can include needle, for example puncture needle or any suitable needle known in the art.The needle should be configured to be sharp enough and hard to penetrate skin and rectus sheath and long enough to reach the desired position.The length of needle can be any length between about 1-25cm.The size of needle can be selected according to various factors (including implant type, amount and preparation, and needle insertion site).

[0060] For example, needle can have an inner diameter of about 0.15mm to 3mm, such as 0.15mm, 0.2mm, 0.25mm, 0.3mm, 0.35mm, 0.4mm, 0.45mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3mm. In the case of injecting islets, needle can have an inner diameter of about 0.25mm to 3mm. Those skilled in the art can determine the specification of needle accordingly. For example, needle can be any size between No. 12 and No. 30. For example, in the case of injection of pancreatic islets, the needle can be any size between 12 gauge and 26 gauge.

[0061] 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 multiple needle tracks (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more needle tracks), each track having a portion of the total volume of the cell suspension.

[0062] The total number of cells or tissues to be transplanted under the rectus sheath can be 1×10 11 For example, the cells or tissues may include 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 For example, the cells or tissues to be transplanted contained in the cartridge of the injection device may include about 10 cells, 1×10 2 cells, 1×103 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. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% of the cells to be transplanted are viable cells.

[0063] For example, in the case of transplanting pancreatic islets, the total amount of islets may be 100 to 200,000 IEQ / kg recipient body weight, preferably 500 to 150,000 IEQ / kg recipient body weight, more preferably 5,000 to 80,000 IEQ / kg recipient body weight, or even more preferably 10,000 to 50,000 IEQ / kg recipient body weight.

[0064] To facilitate precise placement of the implant, imaging techniques such as ultrasound can be used to guide the implant. In one embodiment, ultrasound imaging is used to facilitate the implant.

[0065] Transplant type

[0066] The novel transplantation site of the present application is suitable for transplantation of various transplants or implants. The transplant of the present invention is preferably a cell, tissue or organoid.

[0067] In the broadest scope of the present application, it is not intended to limit the type or source of the transplant. The transplant can be cells, tissues or organoids provided by an individual (eg, a donor) and / or prepared by in vitro methods including induction, differentiation, maturation, etc.

[0068] In some embodiments, the transplant is delivered to a subject for therapeutic purposes. In this case, the transplant can be a cell or tissue with a desired function.

[0069] In some embodiments, the implant is delivered to a subject with a non-therapeutic purpose. For example, a harmful implant can be placed in an animal to establish a disease model. In an example of this embodiment, the implant can be a cell or tissue derived from a tumor.

[0070] The application is particularly suitable for heterotopic transplantation. In this case, the transplant is an ectopic transplant. The success of ectopic transplantation depends on the ability of ectopic transplants or implants to survive and function normally in new sites that are different from the positions that can be naturally found by them. In some embodiments, the transplant comprises tissue or cells of secretory glands (e.g., endocrine glands, such as thyroid, parathyroid, hypothalamus, pituitary, adrenal gland, pineal gland) or consists of them. In some embodiments, the transplant or implant comprises islets of Langerhans, pancreatic cells, hepatocytes, kidney cells, thymocytes, pneumocytes or consists of them.

[0071] In some embodiments, the transplant or colonization of the invention is autologous, allogeneic, xenogeneic, or syngeneic to the subject or recipient.

[0072] The graft or implant of the present invention may be derived from any species of animal, preferably mammals, more preferably primates, and most preferably humans.

[0073] Graft of the present invention can include pre-mature cells or tissues of incomplete differentiation or development.For example, when graft is prepared by pluripotent cells, multipotent cells, progenitor cells or precursor cells, graft can include a certain part of such "immature" cells.In one embodiment, graft can be a cell derived from embryo, for example embryonic stem cell or a cell derived from embryonic stem cell (embryonic stem cell, ESC), for example human embryonic stem cell.In one embodiment, human embryonic stem cell does not experience in vivo development and in 14 days after fertilization.In some embodiments, graft includes fully differentiated, developing or immature cells, which allow to experience differentiation and / or maturation in recipient after transplantation.

[0074] In some embodiments, the transplant can be obtained by differentiation of multipotent cells (e.g., ESC or induced pluripotent stem cells (induced pluripotent stem cell, iPSC)) or pluripotent cells (e.g., precursor cells or progenitor cells). Examples of precursor cells include endodermal progenitor cells, such as endodermal progenitor cells with the potential to differentiate into pancreas or liver.

[0075] In some specific embodiments, the transplant is an islet. In the context of the present application, the term "islet" is broadly understood to include any cell or cell aggregate that secretes insulin. The islet of the present application includes islet organoids. Islets can be taken from an individual, such as a donor, including an allogeneic donor, an autologous donor, or a xenogeneic donor. Islets can be derived from stem cells (including ESC and iPSC) or islet progenitor cells. Islets can include healthy islet cells or dysfunctional islet cells. In a specific embodiment, islets are hPSC-islets differentiated from human iPSC. Differentiation from stem cells to islets can be completed in vitro before transplantation. Alternatively, stem cells differentiate into immature islets in vitro and then mature in vivo after transplantation.

[0076] The cells to be transplanted can be prepared as a cell suspension. In some embodiments, the cells can be enzymatically dissociated prior to transplantation. The enzyme used to dissociate the cells can include, but is not limited to, Accutase, TrypLE, Versene, CTS TM TrypLE and CTS TM Versene.

[0077] Therapeutic Uses

[0078] The new transplantation site of the present application has great potential in therapeutic use because it allows less invasive processes and a supportive environment for colonization. According to the type of the transplant, the method of the present application can treat a variety of diseases and illnesses. The method of the present invention has potential use in the transplantation of a wide range of tissue types intended to supplement or rebuild organ function. Particularly suitable organ tissues transplanted by the present method may include endocrine gland organ tissues, such as adrenal glands. Also interested in those organs that are not necessarily transplanted in situ. For example, the present method can be used for ectopic transplantation.

[0079] For example, the method is particularly suitable for ectopic transplantation of secretory cells that produce hormones. In this case, the method can be used to treat diseases or conditions related to or caused by hormone deficiency or insufficiency.

[0080] In a specific embodiment, the method comprises the sub-rectus sheath transplantation of pancreatic islets (e.g., hPSC-derived islets) to treat conditions caused by insulin deficiency or insufficiency, including various types of diabetes that may be affected by genetic, metabolic, environmental and / or immune factors. The conditions may include hyperglycemia, type 1 diabetes (T1D), type 2 diabetes (T2D), T1D or T2D-related complications, and other rare types of diabetes.

[0081] Transplantation of islets under the rectus sheath can result in increased C-peptide secretion. Transplantation of islets under the rectus sheath can improve long-term and short-term glycemic control. Glycemic control can be measured by HbA1c, fasting blood glucose, postprandial blood glucose, and blood glucose fluctuations. Subjects can benefit from the transplantation of the present invention because the need for exogenous insulin can be reduced or eliminated.

[0082] In the case of transplanted islets, the transplantation of the invention allows for the desired maintenance and maturation of the transplant. In some embodiments, the colonized islets are positive for mature beta cell markers such as MAFA and UCN3 after about 2, 3, or 4 weeks post-transplantation.

[0083] In one embodiment, the method comprises transplantation of hepatocytes beneath the rectus sheath.

[0084] The subrectus sheath transplantation of the present application can be used to replace the current intraportal vein transplantation.

[0085] Example

[0086] In order to more completely understand and apply the present invention, the present invention will be described in detail below with reference to Examples and accompanying drawings. It will be appreciated by those skilled in the art 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 limited by the appended claims.

[0087] method

[0088] Cell culture

[0089] This study used a chemically induced pluripotent human stem cell line, hPSC-8#. hPSC-8# cells were induced from human adult adipocytes 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 placed in 1:40 diluted matrix Matrigel-coated (BD BioSciences, 356231) six-well plates (Corning, 353046) or 500 cm at 5% CO2 and 37°C in mTeSR1 (STEMCELL Technologies, 85850). 2The cells were cultured on a TC-treated culture dish (Corning, 431110). The culture medium was changed every day, and the cells were passaged every 5-6 days using ReleSR (STEMCELL Technologies, 05872) at a split ratio of 1:10-1:15. At the same time, the cells were verified to be mycoplasma-free using the MycoSEQ Mycoplasma Detection Kit (ThermoFisher Scientific, 4460626).

[0090] Differentiation protocol for generating hPSC-islets.

[0091] hPSCs were differentiated using a six-stage protocol previously described by the present 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 (EMDMillipore, SCR005) and cultured at ∼1.35 × 10 5 Cells per cm 2 Matrigel-coated Nunc in mTESR1 supplied with 10 μM Y27632 TM EasyFill TM -2Cell Factory TM System (ThermoFisher Scientific, 169171). Differentiation was initiated 24 hours after seeding by replacing the medium with protocol-appropriate medium supplemented with small molecules or cytokines (Tables 1 and 2). At the end of stage 3, cells were dispersed with Accutase. After rinsing with DMEM basal medium, cells were plated at 5×10 6Cells / wells were seeded in stage 4 medium supplemented with 10 μM Y27632 in six-well AggreWell microplates (STEMCELL Technologies, 27940) and decelerated at 300 g for 5 minutes. After incubation for 24 hours at 37 ° C in 5% CO2, the clusters were transferred to ultra-low attachment six-well plates (Beaver Bio, 40406) with stage 4 medium. The suspended aggregates were cultured in a shaking incubator (INFIRSHT, Multitron) at a rotation rate of 90 rpm at 37 ° C, 5% CO2 and 85% humidity. The details of small molecules, cytokines, culture media and supplements are listed in Table 3.

[0092] Table 1. Base medium recipe

[0093]

[0094] Table 2. Description of differentiation protocol

[0095]

[0096] Table 3. Small molecules, cytokines, basal media, and supplements used in the differentiation protocol

[0097]

[0098]

[0099] Cryopreservation and retrieval of hPSC-islets

[0100] Cryopreservation and retrieval were performed as described previously (Du, Y. et al., 2022, supra).

[0101] Keep frozen. hPSC-islets were separated using Accutase and washed with DMEM basal medium. After counting with a CountessII automated cell counter (Invitrogen, AMQAX1000), the cells were cryopreserved in a freezing medium consisting of 35% FBS, 5% DMSO (Sigma-Aldrich, D2650), 60% stage 6 medium, and 10 μM Y27632 at a rate of 1×10 7 The cells were cryopreserved at a concentration of 10 cells / mL. The vials were then immediately transferred to a Thermo Fisher Scientific Mr. Frosty (5100-0001) and frozen in a -80°C freezer for 24 hours. Subsequently, the vials were transferred to liquid nitrogen for long-term storage.

[0102] recover.The cryopreserved vials were thawed in a 37°C water bath. Each cell suspension was then transferred to a 15 mL centrifuge tube containing 10 mL of DMEM basal medium, followed by centrifugation at 350°C for 3 minutes. The cells were resuspended in DMEM basal medium supplemented with 1% B27 and 10 μM Y27632. After verifying viability and yield, the cells were plated at 5 × 10 6 Cells / well were seeded in 6-well AggreWell microplates and spun down at 350g for 5 minutes into the microwells. After incubation at 37°C in 5% CO2 for 24 hours, the clusters were transferred to ultra-low attachment six-well plates in DMEM basal medium supplemented with 1% B27. The suspended aggregates were cultured in a shaking incubator at a rotation rate of 90 rpm at 37°C, 5% CO2 and 85% humidity for 24 hours before being used for transplantation.

[0103] Flow cytometry

[0104] The clusters were dissociated into single cells using Accutase in a 37°C water bath for 5-10 minutes, and then intracellular markers were stained as previously described (Du, Y. et al., 2022, supra). In brief, single 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 in Perm / Wash buffer (BD BioSciences, 554714) and incubated with the primary antibody overnight in Perm / Wash buffer at 4°C. The cells were washed three times in Perm / Wash buffer and incubated with the secondary antibody overnight in Perm / Wash buffer at 4°C, then washed three times in Perm / Wash buffer, and flow cytometry analysis was performed using BD CellQuest Pro.FlowJo version 10 software. The antibodies used are listed in Table 4.

[0105] Table 4. Flow cytometry and immunohistochemistry antibodies

[0106]

[0107] Immunohistochemistry and immunofluorescence staining.

[0108] For frozen tissue sections. The clusters or tissue samples were washed with PBS and fixed with 4% PFA for 2 hours (hPSC-islet clusters) or 24 hours (tissue samples) at 4°C. The samples were washed three times with PBS and dehydrated overnight in a 30% sucrose solution at 4°C. The dehydrated samples were covered with OCT (Sakura, 4583), frozen in liquid nitrogen and stored at -80°C. 10 μm frozen sections were cut and placed on slides, washed with PBS, and permeabilized with PBST solution (PBS + 0.2% TritonX-100 + 5% donkey serum) at room temperature for 1 hour. The slides were then incubated with diluted primary antibodies in PBST solution overnight at 4°C. After washing three times with PBS, the slides were incubated with secondary antibodies conjugated to AlexaFluor488, 555 or 647 (Life Technologies) at 1:1,000 in PBST solution for 1 hour, and then 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.

[0109] For paraffin sections . The samples were fixed with 10% formalin solution at room temperature for 7 days, embedded in paraffin and sectioned. The sections were dewaxed, rehydrated and washed in PBS. Hematoxylin and eosin (H&E) staining was performed after rehydration. For immunohistochemistry, the slides were immersed in preheated antigen retrieval solution, heated to boiling in a microwave for at least 15 minutes, and then cooled to room temperature. The slides were washed with PBS for 5 minutes, immersed in blocking reagent for 1 hour at room temperature in a humid, light-proof chamber, and washed three times with PBS. Next, the sections were stained with primary and secondary antibodies, followed by color development and hematoxylin staining. All antibodies used are listed in Table 4.

[0110] Quantitative evaluation of colonization volume . Tissue samples from each transplant site were covered with OCT and serially sectioned into 10 μm thick slides using a cryostat. The sections were stained with the human cell marker Stem121 and imaged with a KF-Pro-005 digital slide scanner. Stem121-positive human colonization was marked using a K viewer to measure the total area of ​​the colonization. The volume of the colonization was calculated by multiplying the total area by the section thickness.

[0111] qRT-PCR

[0112] Total RNA was extracted from the cluster using RNeasy Micro Kit (Qiagen, 74004) according to the manufacturer's instructions. cDNA was synthesized using transcript one-step gDNA removal and cDNA synthesis (TransGen Biotech, AT311-03). qRT-PCR analysis was performed using KAPASYBR FAST Universal qPCR Mix (KAPA Biosystems, KK4601), which was performed on a 7500 real-time PCR system. Relative expression levels were standardized to housekeeping gene GAPDH, and the results were analyzed using the ΔΔCt method. Primer sequences are listed in Table 5.

[0113] Table 5. Primers used for qRT-PCR

[0114]

[0115] Glucose stimulated insulin secretion (GSIS)

[0116] Krebs buffer was prepared by dissolving 129mM NaCl, 2.5mM CaCl2, 4.8mM KCl, 1.2mM KH2PO4, 1.2mM MgSO4, 1mMNa2HPO4, 5mM NaHCO3, 10mM HEPES and 0.1% BSA in deionized and sterile filtered water. Separate batches of Krebs buffer containing 2.8mM glucose, 16.7mM glucose and 30mM KCl were prepared and warmed to 37°C. hPSC-islets (20-50 clusters) were collected and washed twice with Krebs buffer in a 24-well plate. The cells were then incubated in Krebs buffer, Krebs buffer containing 2.8mM glucose, Krebs buffer containing 16.7mM glucose and Krebs buffer containing 30mM KCl at 37°C for 1 hour. The supernatant was collected after each incubation and the cells were washed with fresh Krebs buffer. The supernatant samples were frozen at -80°C until C-peptide assay. After the assay, cells were dispersed into single cells using Accutase and Countess TM II automated cell counter for counting.

[0117] Nonhuman primate transplantation.

[0118] All experimental procedures were approved by the Animal Care and Use Committee of the Institute of Medical Biology, Chinese Academy of Medical Sciences (Ethics Number: DWLL201908013). Seven rhesus monkeys (Macaca mulatta) were used for hPSC-islet transplantation. Three healthy monkeys were used to evaluate the feasibility of hPSC-islet transplantation under the anterior rectus sheath (in the subcutaneous space and brachioradialis muscle), and four diabetic monkeys were used to verify the functionality of hPSC-islet transplantation under the anterior rectus sheath.

[0119] Diabetes induction . Diabetes was induced using a single intravenous injection of STZ (AdooQ, A10868) at a dose as previously described (Du, Y. et al., 2022, supra). Briefly, STZ (90 mg / kg) was diluted in 0.1 M citrate buffer (pH 4.3-4.5) and immediately (within 5 minutes) intravenously injected into overnight fasted rhesus monkeys. Saline (50 mL) was administered to each monkey after hydration. To prevent nausea and vomiting, omeprazole (0.5 mg / kg, Astrazeneca AB). Monitor blood glucose every hour for the first 12 hours after STZ injection and then monitor 4 times a day. Start exogenous insulin injection 3 days after STZ treatment. Subcutaneous injection of short-acting insulin ( Eli LillyItalia SpA) and long-acting insulin ( Sanofi-Aventis Deutschland GmbH). Short-acting insulin was administered according to the dosing schedule in Table 6. Blood glucose, exogenous insulin, C-peptide and HbA1c levels were recorded before hPSC-islet transplantation.

[0120] Table 6. Dose adjustment table for short-acting insulin based on blood glucose levels

[0121]

[0122] Immunosuppression. As previously described (Du, Y. et al., 2022, supra), the immunosuppressive regimen was started 9 days before transplantation (day 0). See Table 7 for details. Induction therapy: Rituximab was injected on the first 9 days. ATG was infused on the first 5 days and the first 3 days. Basiliximab was given on days 0 and 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 injected on days 0, 4, and 14, and once every two weeks thereafter; rapamycin and tacrolimus were injected once a day. The doses of rapamycin and tacrolimus were adjusted according to the trough blood levels (tacrolimus: 4-10 ng / mL; rapamycin: 4-10 ng / mL). The blood concentrations of the drugs were tested using Viva-E (Vital Scientific NV).

[0123] Table 7. Drugs used for immunosuppression and preventive therapy

[0124]

[0125] Preventive and preemptive treatment To prevent CMV infection, valganciclovir was injected daily from day -4. To alleviate the inflammatory response, cobra venom factor (CVF) was injected on day 1, and etanercept was injected on day 0 (5 mg / kg intravenously) and on days 3, 7, and 10 after transplantation (2.5 mg / kg intravenously).

[0126] anaesthetization Anesthesia was initiated with propofol (0.5 mL / kg, Petsun Therapeutics) and maintained with isoflurane and oxygen. Heart rate, temperature, blood oxygenation, and blood pressure were monitored in real time during hPSC-islet transplantation. The overall quality of hPSC-islet preparations is recorded in Figure 1 and in Tables 8 and 9.

[0127] Subcutaneous transplantation of hPSC-islets As reported (Yu, M. et al. Islet transplantation in the subcutaneous space achieves long-term euglycaemia in preclinical models if type 1 diabetes. Nat Metab 2, 1013-1020, doi: 10.1038 / s42255-020-0269-7(2020)), hPSC-islets (1×10 4IEQ) was dispersed in IVM and loaded into a syringe using a puncture needle (0.7×80TWLB). After aseptic treatment of the scalp, the syringe containing hPSC-islets was inserted into the subcutaneous tissue under ultrasound guidance, the entire inoculum was slowly injected into the subcutaneous tissue, and the needle tip was wiped with iodine.

[0128] Brachioradialis muscle hPSC-islet transplantation 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_20180314588(2018)), hPSC-islets (1×10 4 IEQ) was dispersed in saline and loaded into a syringe using a puncture needle (0.7×80TWLB). After sterilizing the forearm, the syringe containing hPSC-islets was inserted distally along the brachioradialis muscle fibers and moved through the muscle to the proximal end under ultrasound guidance. The total volume of the inoculum was infused into the space between the brachioradialis muscle fibers while slowly moving the needle from the proximal part of the muscle to the distal part to obtain a beaded distribution of hPSC islets in the muscle, and then the needle tip was wiped with iodine tincture.

[0129] hPSC-islet transplantation under the anterior rectus sheath . hPSC-islets were dispersed in saline and loaded into a syringe using a puncture needle (0.7×80TWLB). Under ultrasound guidance, the hPSC-islet suspension was injected into the space between the anterior rectus sheath and the rectus abdominis. For each monkey, 8 injections were applied, 4 injections on each side. Specifically, a 10MHz ultrasound probe was used, which was placed around the umbilicus to display the short-axis section of the rectus abdominis. When transplanted to the left side of the anterior rectus sheath, the puncture needle entered from the confluence of the external oblique muscle, the internal oblique muscle, and the transverse abdominal aponeurosis. When transplanted to the right side of the anterior rectus sheath, the puncture needle entered from the right edge of the left abdominal white line. When the needle tip pierced the anterior rectus sheath and reached the edge of the rectus abdominis, the puncture needle was inserted into the space between the anterior rectus sheath and the rectus abdominis, and the puncture needle was very close to the anterior layer of the rectus sheath. After reaching the target position, the hPSC-islets were injected and the puncture needle was withdrawn at the same time. Therefore, the hPSC-islets are dispersed in and around the needle track.

[0130] Routine testC-peptide secretion, HbA1c, complete blood count, serum creatinine, and liver function analysis were routinely performed. Complete blood count was performed using Sysmex XT-200i. HbA1c, serum creatinine, and liver function analysis were evaluated using Mindray BS-2000.

[0131] Intravenous glucose tolerance test (IVGTT) After an overnight fast, 50% glucose was infused intravenously into the monkeys at 0.75 g / kg body weight. Blood samples were collected at 0, 5, 15, 30, 60, and 90 minutes after injection. Blood glucose was measured using a handheld glucometer, and C-peptide levels were measured using ELISA.

[0132] Arginine stimulation test . The C-peptide secretion response stimulated by intravenous arginine was measured at two different blood glucose levels (reference). Briefly, after an overnight fast, 10% arginine hydrochloride (Sigma, Cat#A5006) was administered at 70 mg / kg within 30 seconds, with the start time designated as 0 minutes. Blood samples were collected at 0, 2, 4, and 10 minutes after the first arginine pulse. 50% glucose was then injected to increase and further maintain plasma glucose levels at 20 mM. 50 minutes after the first arginine pulse, a second arginine pulse (70 mg / kg) was injected. Blood samples were collected at 0, 2, 4, and 10 minutes after the second arginine pulse. C-peptide levels were measured by ELISA.

[0133] ELISA

[0134] C-peptide was detected using a human C-peptide ELISA kit (ALPCO, 80-CPTHU-E10) according to the manufacturer's instructions. ELISA was performed in triplicate for all samples.

[0135] Necropsy and histological analysis

[0136] Complete necropsies of all monkeys were performed by an experienced primate pathologist. Tissue specimens of major organs were fixed with 4% PFA and 10% formalin for frozen and paraffin sections, respectively.

[0137] Statistical analysis

[0138] Data analysis was performed using GraphPad Prism software. Statistical significance was assessed by t-test. Throughout the specification, unless otherwise indicated, n represents the number of biological replicates. P values ​​are indicated as follows: *P<0.05; **P<0.005; ***P<0.0005; ****P<0.00005.

[0139] Example 1. Comparison of pancreatic islet transplantation under the rectus sheath and two other extraperitoneal transplantation strategies

[0140] To evaluate the feasibility of hPSC-islet transplantation into the rectus sheath, we first compared the sub-rectus sheath strategy with two previously reported strategies for extraperitoneal islet transplantation: intramuscular transplantation and subcutaneous transplantation ( Figure 1 a) (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-yearfollow-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)).

[0141] Figure 1 a shows three transplantation sites. In this example, the method used for brachioradialis muscle and subcutaneous space transplantation was based on previous reports (Yu, M. et al. 2020, as described above; Bertuzzi, F. et al., 2020, as described above). For transplantation under the rectus sheath, the space between the anterior rectus sheath and the rectus abdominis muscle was selected as the implantation site to stay away from the peritoneum and avoid potential surgical damage to the blood vessels and neural network around the posterior rectus sheath ( Figure 1 b) (Sevensma, KE, Leavitt, L & Pihl, KD Anatomy, Abdomen and Pelvis, Rectus Sheath. StatPearls (book) (2022)).

[0142] Three healthy adult rhesus monkeys (Macaca mulatta) (monkeys 1-3) (Table 8) were used. hPSC-islets were differentiated from hPSCs and cryopreserved as single cell suspensions as described in the section entitled "Methods" above. Two days before transplantation, hPSC-islets were recovered and reaggregated, and their characteristics were as follows Figure 2 Immunosuppressive therapy was applied as described above in the section entitled “Methods”. Under ultrasound guidance, hPSC-islets were transplanted at a rate of approximately 8 × 10 per site. 4The islet equivalent (IEQ) dose was transplanted into each of the three selected monkeys ( Figure 1 ab and Table 8).

[0143]

[0144] Cell survival in the early stages after transplantation .

[0145] Monkey 1 was sacrificed 1 week after transplantation (wpt) to assess cell survival and gene expression patterns in the early stages of hPSC-islet colonization. hPSC-islet engraftment was detected at all three sites by immunostaining for the human cell marker Stem121 ( Figure 3 a).

[0146] like Figure 4 As shown in a, the volume of human implants in the brachioradialis muscle and subcutaneous space was significantly smaller than that under the anterior rectus sheath. In addition, the proportion of C-peptide-positive β cells in intramuscular and subcutaneous implants was significantly lower than that in the pre-implantation implants, which also indicated a decreased expression of pancreatic transcription factors (NKX6.1, PDX1, and NKX2.2) ( Figure 4 bd and Figure 3 bc). In contrast, hPSC-islet colonies beneath the anterior rectus sheath maintained structural integrity, with robust expression of islet hormones and transcription factors ( Figure 4 cd and Figure 3 bc).

[0147] Taken together, these results indicate that hPSC-islets show better early survival and maintenance when transplanted under the anterior rectus sheath.

[0148] Cell survival at late stages after transplantation .

[0149] Monkeys 2 and 3 were sacrificed at 4 wpt. By serial sectioning and Stem121 staining of the 4-week colonies, human cells were detected only in the colonies growing under the anterior sheath of the rectus abdominis muscle, while hPSC-islets injected into the brachioradialis muscle and subcutaneous space were completely absent. The findings in the two monkeys were consistent ( Figure 5 a and Figure 6 a).

[0150] Notably, the majority of hPSC-islets were found to colonize between the surface of the rectus abdominis muscle and the anterior rectus sheath, whereas a small portion was seeded into the rectus abdominis muscle ( Figure 5 a and Figure 6 a).

[0151] Immunofluorescence staining of 4-week-old human implants under the anterior rectus sheath .

[0152] The 4-week-old implants under the anterior rectus sheath were further analyzed by immunofluorescence staining. The results of immunofluorescence staining showed that the implants expressed essential pancreatic hormones and transcription factors ( Figure 5 bc and Figure 6 bc). Notably, mature β-cell markers MAFA and UCN3 were detectable ( Figure 5 c and Figure 6 c)(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, a large number of vascular infiltrations into human colonies were observed ( Figure 5 d and Figure 6 d). Immunopathological analysis showed rare CD3+T cells, CD20+B cells, and CD68+ macrophages in or around the colonies ( Figure 5 e and Figure 6 e) Taken together, these results suggest that the anterior rectus sheath provides an environment that promotes functional maturation and maintenance of hPSC islets.

[0153] Example 2. Functional study of hPSC islets transplanted under the anterior sheath of the rectus abdominis in diabetic monkeys

[0154] In this example, the function of hPSC-islets in improving diabetic conditions when transplanted under the anterior rectus sheath of diabetic monkeys was investigated.

[0155] As reported (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)), four adult monkeys (monkey 4-7) (monkey 4-7) were injected with a single high dose of streptozotocin (STZ) to induce diabetic fatty liver (Table 9). After STZ injection, all rhesus monkeys showed significantly elevated blood glucose levels and extremely low C-peptide concentrations (fasting C-peptide: 0.08 ± 0.07 ng / ml; postprandial C-peptide: 0.08 ± 0.07 ng / ml) ( Figure 7 Three days after STZ-injection, exogenous insulin was given according to blood glucose levels, with daily doses ranging from 1.5 to 3.0 IU kg / day. -1 , consistent with previous reports in the same model system ( Figure 7i-1) (Du, Y. et al., 2022, supra; Zhu, H., et al., 2014, supra; Shin, JS 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 assess long-term average blood glucose concentration (Sherwani, SI, Khan, HA, Ekhzaimy, A., Masood, A. & Sakharkar, MK 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)) was significantly increased from 3.6±0.3% before STZ treatment to 7.7±2.2% on the day of hPSC-islet transplantation ( Figure 7 eh).

[0156] The dose of hPSC islets for transplantation into the anterior inferior rectus sheath was designed based on the dose used in intraportal infusion, ranging from 40,000 to 48,000 IEQ / kg 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 manner into the gap between the anterior rectus sheath and the rectus abdominis using a puncture needle, with cell clusters dispersed in and around the needle track ( Figure 1 b). Considering that a high density of hPSC-islets in a single needle track may be detrimental to cell survival, hPSC-islets were administered by 8 injections in 4 parallel needle tracks on each side of the rectus abdominis muscle ( Figure 1c). Diabetic recipients were studied by continuously monitoring blood glucose and C-peptide secretion during 12 weeks after transplantation to assess the durability of the functional effects of hPSC-islets, since, as previously reported, xenogeneic immune attack-mediated hPSC-islet colonization clearance was observed after 4 mpt in the setting of human-monkey xenotransplantation (Du, Y. et al., 2022, supra).

[0157] Improved blood sugar control

[0158] After hPSC-islet injection under the anterior rectus sheath, all diabetic recipients showed significant improvement in overall glycemic control ( Figure 7 ). Blood glucose levels gradually decrease, and this change is accompanied by a decrease in the need for exogenous insulin ( Figure 7 In all recipients, fasting blood glucose was stably maintained at 180 mg dl from 6 wpt -1 (10mM) or less, blood sugar dropped from 5wpt to less than 144mg dl two hours after meal -1 (8.0mM) Figure 7 ad and Figure 8 ad). The fluctuation of blood sugar level is also greatly reduced ( Figure 7 ad). Thus, the mean Hb1ac changed from 7.7±2.2% at baseline to 4.3±1.3% at 12wpt ( Figure 7 eh).

[0159] It is noteworthy that no severe hypoglycemic events were detected in recipient rhesus monkeys after transplantation under the anterior rectus sheath (which occurred perioperatively after intraportal infusion). In previous studies by the inventors, when hPSC-islets were infused into the hepatic portal vein, β-cell death mediated by the immediate blood-mediated inflammatory reaction (IBMIR) in recipient rhesus monkeys often resulted in blood glucose levels below 54 mg dl-1 within the first 12 hours after transplantation. -1(3.0 mM) and requires intravenous injection of high concentrations of glucose (Du, Y. et al., 2022, supra; Rickels, MR & Robertson, RP Pancreatic Islet Transplantation in Humans: Recent Progress and Future Directions. Endocr Rev 40, 631-668, doi: 10.1210 / er.2018-00154 (2019); Shin, J Set al., 2015, supra; Faradji, R Net 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 striking observations suggest that early-stage survival of hPSC islets is improved following transplantation under the anterior rectus sheath.

[0160] Reduced exogenous insulin requirements

[0161] In all recipients of hPSC-islet transplantation under the anterior rectus sheath, the requirement for exogenous insulin was reduced ( Figure 7 i-1). A sharp reduction in exogenous insulin requirement was observed immediately after hPSC-islet injection, followed by a continuous gradual reduction, a trend that corresponds to the functional maturation of hPSC-islets ( Figure 7 i-1). At 12 wpt, the mean exogenous insulin requirement increased from 2.3 ± 0.5 IU kg / day to -1 Reduced to 1.3 ± 0.4 IU kg / day -1 ( Figure 7 i-1).

[0162] C-peptide secretion

[0163] C-peptide secretion was also monitored in all monkeys. Fasting C-peptide and postprandial C-peptide increased significantly in the first 6 wpt, with mean levels of approximately 0.6 ng ml -1 and 2.0 ng ml -1 , starting from 8wpt ( Fig. 9 ad). The stimulation index after 6 weeks is equivalent to that of natural islets ( Fig. 9 ad).

[0164] In addition, the intravenous glucose tolerance test (IVGTT) showed that glucose clearance capacity gradually increased with the enhancement of glucose-responsive insulin secretion ( Fig. 9 el).

[0165] In addition, arginine-stimulated insulin secretion assays provided further evidence of the C-peptide secretion capacity of hPSC-islet colonies ( Figure 8 eh).

[0166] Together, these data demonstrate that hPSC-islets transplanted under the anterior rectus sheath effectively improve overall glycemic control in diabetic rhesus monkeys through restoration of insulin secretion.

[0167] Histological analysis

[0168] To confirm that the improvement in blood glucose levels was caused by hPSC-islet transplantation rather than restoration of endogenous β cells, all recipient monkeys were sacrificed at 13 wpt for evaluation. Histological analysis of native pancreas showed that endogenous islets were severely destroyed and C-peptide + Cells cannot be detected ( Fig.10 ad). These results are consistent with the very low concentrations of endogenous C-peptide observed before transplantation ( Fig. 9 ad and 9i-1). In addition, no CK19 was observed + Proinsulin + cells, a population reported to contribute to endogenous β-cell recovery in the pancreas after STZ treatment ( Fig.10 e) (Bottino, R. et al. Recovery of endogenous beta-cell function in nonhuman primates after chemical diabetes induction and islettransplantation. Diabetes 58, 442-447, doi: 10.2337 / db08-1127 (2009)).

[0169] In contrast, a high proportion of C-peptide was present in the human implant beneath the anterior sheath of the rectus abdominis muscle. + β cells, strong expression of MAFA and UCN3 was detected ( Fig.11 c). 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 ( Fig.11 df). These observations were consistent in four diabetic monkeys ( Fig.11 df and Fig.12 af).

[0170] Together, these results suggest that improved glycemic control is attributable to hPSC islet colonization under the anterior rectus sheath.

[0171] Whole body ultrasound

[0172] In addition, whole-body ultrasound examinations of all four recipient monkeys at 13 wpt before sacrifice revealed no evidence of teratoma formation ( Fig.12 A full autopsy further confirmed the absence of tumorigenesis and abnormalities in the major organs examined ( Fig.12 ).

Claims

1. A method of transplanting one or more cells, or one or more tissues, into a subject in need thereof, comprising introducing the cells or tissues into a site beneath the rectus sheath.

2. The method of claim 1, wherein the cells or tissue are introduced into a site between the rectus sheath and the rectus abdominis muscle.

3. A method of transplanting one or more cells, or one or more tissues, into a subject in need thereof, wherein the method allows one or more of the transplanted cells or tissues to colonize a site beneath the rectus sheath after transplantation.

4. The method of claim 3, wherein the method allows one or more of the transplanted cells or tissues to colonize a site between the rectus sheath and the rectus abdominis muscle after transplantation.

5. The method of claim 3 or 4, wherein one or more of the transplanted cells colonizes the rectus abdominis muscle.

6. The method according to any one of claims 1 to 5, wherein the rectus abdominis sheath is the anterior rectus abdominis sheath or the posterior rectus abdominis sheath, preferably the anterior rectus abdominis sheath.

7. The method according to any one of claims 1 to 6, wherein the cells or tissues are cells or tissues produced in vitro or isolated from natural organs.

8. The method of claim 7, wherein the cells or tissues are produced in vitro from one or more of pluripotent cells, multipotent cells, progenitor cells, or precursor cells.

9. The method of claim 8, wherein the cells or tissues are cells or tissues differentiated from human pluripotent stem cells (hPSCs).

10. The method of claim 9, wherein the cells are hormone-secreting cells differentiated from hPSCs. The method according to claim 10 , wherein the cells are pancreatic islets differentiated from hPSCs (hPSC-islets).

12. The method according to any one of claims 9 to 11, wherein the hPSC is an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC).

13. The method of any one of claims 8 to 12, wherein at least a portion of the transplanted cells or tissue undergoes maturation after transplantation.

14. The method according to any one of claims 1 to 13, wherein the cells are introduced at the site by injection.

15. A method of 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 beneath the rectus abdominis sheath.

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

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

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

19. The method of any one of claims 15 to 18, wherein the method allows one or more of the transplanted islets to colonize a site beneath the rectus sheath following transplantation.

20. The method of claim 19, wherein the method allows one or more of the transplanted islets to colonize a site between the rectus sheath and the rectus abdominis muscle.

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

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

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

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

25. The method of claim 24, wherein the islets are differentiated from hPSCs (hPSC-islets).

26. The method of claim 25, wherein the hPSC is an ESC or an iPSC.

27. The method of any one of claims 15 to 26, wherein at least a portion of the transplanted islets undergo maturation after transplantation.

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

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

30. The method of any one of claims 15 to 29, wherein the pancreatic islets are transplanted into sites beneath the corresponding rectus sheaths of the left and right rectus abdominis muscles.

31. The method of any one of claims 15 to 30, comprising transplanting pancreatic islets into the subject more than once.

32. Use of hPSC-islets for treating a disease or condition caused by or associated with insulin deficiency, wherein the hPSC-islets are transplanted to a site beneath the anterior sheath of the rectus abdominis muscle.

33. Use of hPSC-islets for reducing the requirement for exogenous insulin, wherein the hPSC-islets are transplanted to a site beneath the anterior sheath of the rectus abdominis muscle.

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

35. A device for injecting pancreatic islets into the rectus sheath, and preferably at a site between the rectus sheath and the rectus abdominis muscle.

36. The device of claim 35, wherein the islets are hPSC-derived islets.

37. A device according to claim 35 or claim 36, wherein the device comprises a needle.

38. A device according to claim 35 or claim 36, wherein the device comprises a plurality of needle tracks.

39. The device of claim 38, wherein the device comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more needle tracks.

40. A device according to any one of claims 35 to 39, wherein the device comprises a cartridge.

41. The device of claim 40, wherein the cartridge comprises islets in an amount of 100 to 200,000 islet equivalents (IEQ) per kilogram of recipient body weight.

42. The device of claim 40, wherein the cartridge comprises 1×10 2 Up to 2×10 7 IEQ islets.

Images