Fat-related lymphatic clusters as sites for transplantation, tissue regeneration, organogenesis and function of multiple tissues

Abstract

The present disclosure relates to implantation or proliferation of cells in fat-related lymphatic clusters ("FALC" or "milk spots"), which can be used to generate functional ectopic tissues. The present disclosure also provides methods and compositions for transplanting and proliferating cells in FALC by activating lymphotoxin beta receptor (LTbeta R) and / or NF-[kappa] B-induced kinase (NIK) signal transduction pathways. The disclosure also provides methods and compositions for establishing ectopic liver tissue in FALC (breast spot) and using such ectopic liver tissue for obtaining therapeutic benefits, and methods and compositions for generating ectopic kidney tissue in FALC, which can be used for obtaining therapeutic benefits in a subject.

Description

[0001] Priority information

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 460,267 filed on February 17, 2017 and U.S. Provisional Patent Application Serial No. 62 / 574,119 filed on October 18, 2017, the contents of each of which are incorporated herein by reference in their entirety and claim their respective priority.

[0003] Funding Information

[0004] none.

[0005] introduction

[0006] The present disclosure relates to the engraftment and proliferation of organ cells in Fat-Associated Lymphoid Clusters ("FALCs", also known as "milky spots") to generate ectopic tissue that can be used to supplement or replace organ function in a subject. Background Art

[0007] There is a lack of effective treatment for patients with liver disease. Liver disease causes more than 31,000 deaths in the United States each year. Orthotopic liver transplantation (OLT) is essentially a last resort and is currently the only treatment for severe disease (1, 2). In addition, although it is estimated that 100,000 patients need a new liver, only more than 6,000 patients receive liver transplants each year. The shortage of available donors is one of the major challenges faced by patients affected by end-stage liver disease. Patients with comorbidities and advanced age are either not considered candidates for OLT or are expected to have low survival rates after transplantation (3-5). In addition, transplant operations are costly in terms of spending and health care resources. For these reasons, it has been proposed that cell-based transplantation be used as a treatment alternative or bridge to OLT while patients wait for available organs (6).

[0008] To date, the functional utility of hepatocyte transplantation has been demonstrated in animal models. From transgenic urokinase (7-9) to inducible tyrosinemic mice (10-15), hepatocyte transplantation has successfully established its therapeutic potential to completely regenerate the liver. Despite these encouraging results, human hepatocyte transplantation remains in the experimental stage of clinical exploration (6, 16) in the hope that the positive results of animal studies can be translated into therapeutic applications for human disease.

[0009] However, for patients with advanced liver disease, there are additional challenges: most envisioned cell therapies target cell implantation in the diseased liver itself. Transplanted hepatocytes are typically injected via the spleen (e.g., via the splenic artery in human patients or via the splenic parenchyma in rodents) or via the portal vein. Hepatocytes migrate rapidly, either actively or passively, to the diseased liver, where liver regeneration by the transplanted hepatocytes is expected to occur. This approach limits and likely precludes the efficacy of cell therapy in the vast majority of patients with severe liver disease due to the presence of cirrhosis and fibrosis (17), common pathological features in diseased livers. Therefore, native liver regeneration in these patients, those who are most in need of treatment, is a major challenge.

[0010] Therefore, patients with advanced liver disease need new therapies. An option that has been developed is an engineered auxiliary liver (18-20). Typically, this consists of a healthy liver graft that is implanted in an ectopic or orthotopic location, while retaining all or part of the original native liver intact. This method is not only likely to avoid OLT in patients of a particular type, but also includes the possibility of spontaneously regenerating the natural liver and ultimately avoiding immunosuppressive drugs (21-24). Although problems occurred in early trials (18-20, 25-27), recent favorable results have been reported, which are encouraging in the case of acute liver failure (25), metabolic disorders (28-31) and even cirrhosis (26, 27). So far, the cellular and molecular mechanisms necessary for maintaining long-term stability and vitality of hepatocytes and auxiliary livers have not been understood.

[0011] Many researchers have transplanted hepatocytes at a variety of extrahepatic sites (34-40). Most hepatocyte implantations at extrahepatic sites are associated with variable outcomes. It has been previously demonstrated that hepatocytes transplanted into lymph nodes can generate functional auxiliary livers capable of restoring liver function long-term (greater than 6 months) in mice (see, e.g., U.S. Pat. No. 9,125,891). These results lay the foundation for a new paradigm of tissue regeneration by using lymph nodes as in vivo bioreactors for the growth of tissue or organ replacements (32-36).

[0012] In addition to liver disease, kidney disease is prevalent in the population and causes more than 47,000 deaths in the United States each year. Known treatments for treating kidney disease include rest, dietary changes, drug therapy, hemodialysis therapy and kidney transplantation, and depend on the severity of the disease. When renal failure is reached, dialysis therapy such as hemodialysis or peritoneal dialysis or kidney transplantation is required. Although hemodialysis therapy can eliminate waste accumulated in the body, the function of the damaged kidneys cannot be restored. In the case of renal failure, unless the patient receives a kidney transplant, the patient must continue dialysis therapy throughout his or her lifetime. However, there are many difficulties associated with kidney transplantation, including donor shortages, tissue compatibility difficulties and avoiding rejection reactions. Therefore, new therapies for patients with low renal function are needed.

[0013] Lymph nodes and fat-associated lymphoid clusters ("FALCs", also referred to herein as "milky spots") are secondary lymphoid organs of the lymphatic system and are well vascularized. FALCs occur in many anatomical locations, including the omentum (see Figure 1 ). They play an important role in the immune system, allowing for the massive expansion of white blood cells under various conditions, such as during bacterial or viral infections. Interestingly, lymph nodes and milky spots are also of clinical significance in cancer, as they are the sites of early metastatic events (i.e., tumor invasion and initial metastatic growth). Tumor metastasis in lymph nodes is often used for staging and prognosis of cancer. SUMMARY OF THE INVENTION

[0015] The present disclosure relates to the implantation and proliferation of cells in adipose-associated lymphoid clusters ("FALCs" or "milky spots"), which can be used to generate functional ectopic tissue for transplantation into a host subject. This is based (at least in part) on the discovery that hepatocytes transplanted intraperitoneally into mice with hepatic insufficiency localize and proliferate in the FALCs (milky spots) of the omentum, as well as the FALCs of the mesentery, spleen, portal, and / or gonadal fat to produce ectopic liver tissue that can advantageously enhance liver function in mice. This is also based (in part) on the discovery that fetal kidney cells proliferate in the omental FALCs to produce ectopic kidney tissue. This is further based (in part) on the discovery that LTβR signaling in stromal cells promotes successful vascularization / angiogenesis of transplanted cells.

[0016] In certain embodiments, the present disclosure provides methods and compositions for establishing ectopic liver tissue in the FALC (milky spots) of the omentum and the FALC of the mesentery, spleen, hilum and / or gonadal fat and using such ectopic liver tissue for therapeutic benefit. In certain embodiments, the present disclosure also provides methods and compositions for generating ectopic kidney tissue in the FALC, which can be used to obtain therapeutic benefit in a subject.

[0017] In certain embodiments, the present disclosure also provides methods and compositions for transplanting and proliferating cells (e.g., hepatocytes or kidney cells) in FALC or lymph nodes by activating lymphotoxin beta receptor (LTβR) and / or NF-κB-inducing kinase (NIK) signaling pathways.

[0018] The present disclosure provides a method for developing ectopic tissue in a subject. In certain embodiments, the method includes introducing one or more cells into a fat-associated lymph cluster or lymph node of a subject and providing one or more reagents that promote proliferation and / or angiogenesis of the one or more cells to form ectopic tissue. In certain embodiments, the one or more cells are hepatocytes, and the ectopic tissue is ectopic liver tissue. In certain embodiments, the one or more cells are kidney cells and / or kidney tissue fragments, and the ectopic tissue is ectopic kidney tissue. In certain embodiments, the fat-associated lymph cluster is located in adipose tissue of the pleura and / or pericardium and / or peritoneal cavity. In the peritoneal cavity, FALC can be located in the omentum and / or mesentery and / or spleen and / or liver hilum and / or gonadal fat of the subject.

[0019] In some embodiments, the agent that promotes ectopic tissue formation comprises one or more bone marrow-derived cells and / or stromal cells. In some embodiments, one or more agents comprise stromal cells, e.g., fibroblasts. In some embodiments, the stromal cells express one or more of podoplanin, NIK, and / or LTβR. In some embodiments, the stromal cells are treated with an activator of the LTβR and / or NIK signal transduction pathway.

[0020] In certain embodiments, the agent that promotes the formation of ectopic tissue comprises an activator of the LTβR and / or NIK signal transduction pathway, for example, an activator of the LTβR and / or NIK. In certain embodiments, the agent that promotes the formation of ectopic tissue comprises an activator of the non-classical NF-κB signal transduction pathway. In certain embodiments, the activation of LTβR and / or NIK and / or the activation of the non-classical NF-κB signal transduction pathway promotes the proliferation and / or angiogenesis of one or more cells to form ectopic tissue.

[0021] In certain embodiments, the agent that promotes ectopic tissue formation comprises an agent that promotes inflammation.In certain embodiments, inflammation is induced prior to introducing one or more cells into an adipose-associated lymph cluster or lymph node of a subject.

[0022] The present disclosure further provides a method for treating an object in need of enhanced liver function. In certain embodiments, the method includes administering a therapeutically effective amount of hepatocytes to the object, and promoting the proliferation of hepatocytes in the adipose-associated lymph nodes or lymph nodes of the object to form ectopic liver tissue. In certain embodiments, the formation of ectopic liver tissue in the adipose-associated lymph nodes is promoted by locally administering hepatocytes to the anatomical region of the adipose-associated lymph nodes. In certain embodiments, the adipose-associated lymph nodes are located in the omentum and / or mesentery and / or spleen and / or liver hilum and / or gonadal fat.

[0023] In certain embodiments, the formation of ectopic liver tissue in adipose-associated lymph nodes or lymph nodes is promoted by co-administering one or more bone marrow-derived cells or stromal cells. For example, but not limited to, co-administering stromal cells. In certain embodiments, the stromal cells are fibroblasts. In certain embodiments, the stromal cells express one or more of podoplanin, NIK, and / or LTβR.

[0024] In certain embodiments, the formation of ectopic liver tissue in adipose-associated lymph nodes or lymph clusters is promoted by co-administering an activator of the LTβR and / or NIK signaling pathway. In certain embodiments, the formation of ectopic liver tissue in adipose-associated lymph nodes or lymph nodes is promoted by co-administering an activator of the non-classical NF-κB signaling pathway.

[0025] In certain embodiments, the formation of ectopic liver tissue in an adipose-associated lymph node or lymph cluster is promoted by inducing inflammation in a subject. In certain embodiments, inflammation is induced by administering to the subject an agent that promotes inflammation. In certain embodiments, inflammation is induced prior to introducing one or more cells into an adipose-associated lymph node or lymph cluster of a subject.

[0026] The present disclosure further provides a method for generating an ectopic liver, wherein the method includes introducing one or more hepatocytes into adipose-associated lymph nodes or lymph nodes, and providing at least one reagent that promotes the formation of ectopic liver tissue. In certain embodiments, the method is implemented in vivo or in vitro. In certain embodiments, the reagent that promotes the formation of ectopic liver tissue comprises one or more bone marrow-derived cells and / or stromal cells. In certain embodiments, the stromal cells are fibroblasts. In certain embodiments, the stromal cells express one or more of podoplanin, NIK and / or LTβR. In certain embodiments, the stromal cells are treated with an activator of the LTβR and / or NIK signal transduction pathway. In certain embodiments, the reagent that promotes the formation of ectopic tissue comprises an activator of the LTβR and / or NIK signal transduction pathway. In certain embodiments, the reagent that promotes the formation of ectopic tissue comprises an activator of the LTβR and / or NIK signal transduction pathway. In certain embodiments, the reagent that promotes the formation of ectopic tissue comprises an agent that promotes inflammation.

[0027] In certain embodiments, a method for generating ectopic liver tissue in a subject comprises introducing cells comprising one or more hepatocytes and one or more stromal cells into an adipose-associated lymph cluster or lymph node of the subject, and providing an activator of the LTβR and / or NIK signaling pathway, wherein activation of the LTβR and / or NIK signaling pathway in the one or more stromal cells promotes proliferation and / or angiogenesis of the one or more hepatocytes to form ectopic liver cells.

[0028] In certain embodiments, a method for generating ectopic liver tissue in a subject comprises introducing cells comprising one or more hepatocytes and one or more stromal cells into an adipose-associated lymph node or lymph node of the subject, and providing an activator of LTβR and / or NIK, wherein activation of LTβR and / or NIK in the one or more stromal cells promotes proliferation and / or angiogenesis of the one or more hepatocytes to form ectopic liver cells. In certain embodiments, the kidney cells comprise cells isolated from embryonic kidneys, metanephroi, cells isolated from kidney organoids formed in vitro, or any combination thereof.

[0029] The present disclosure further provides a method for treating an object requiring enhanced renal function. In certain embodiments, the method includes administering a therapeutically effective amount of renal cells (or renal tissue fragments) to the object, and promoting the proliferation of renal cells in the adipose-associated lymph nodes or lymph nodes of the object to form ectopic renal tissue. In certain embodiments, the formation of ectopic renal tissue in the adipose-associated lymph nodes is promoted by locally administering hepatocytes to the anatomical region of the adipose-associated lymph nodes. In certain embodiments, the adipose-associated lymph nodes are located in the omentum and / or mesentery and / or spleen and / or liver hilum and / or gonadal fat.

[0030] In certain embodiments, the formation of ectopic renal tissue in adipose-associated lymph nodes or lymph nodes is promoted by co-administering one or more bone marrow-derived cells or stromal cells. For example, but not limited to, co-administering stromal cells. In certain embodiments, the stromal cells are fibroblasts. In certain embodiments, the stromal cells express one or more of podoplanin, NIK, and / or LTβR.

[0031] In certain embodiments, the formation of ectopic renal tissue in adipose-associated lymph nodes or lymph clusters is promoted by co-administering an activator of the LTβR and / or NIK signaling pathway. In certain embodiments, the formation of ectopic renal tissue in adipose-associated lymph nodes or lymph nodes is promoted by co-administering an activator of the non-classical NF-κB signaling pathway.

[0032] In certain embodiments, the formation of ectopic renal tissue in an adipose-associated lymph node or lymph node is promoted by inducing inflammation in a subject. In certain embodiments, inflammation is induced by administering to a subject an agent that promotes inflammation. In certain embodiments, inflammation is induced prior to introducing one or more renal cells into an adipose-associated lymph node or lymph node of a subject.

[0033] The present disclosure further provides a method for generating an ectopic kidney, wherein the method includes introducing one or more kidney cells (or one or more kidney tissue fragments) into adipose-associated lymph nodes or lymph nodes, and providing at least one reagent that promotes the formation of ectopic kidney tissue. In certain embodiments, the method is implemented in vivo or in vitro. In certain embodiments, the reagent that promotes the formation of ectopic kidney tissue comprises one or more bone marrow-derived cells and / or stromal cells. In certain embodiments, the stromal cells are fibroblasts. In certain embodiments, the stromal cells express one or more of podoplanin, NIK and / or LTβR. In certain embodiments, the stromal cells are treated with an activator of the LTβR and / or NIK signal transduction pathway. In certain embodiments, the reagent that promotes the formation of ectopic kidney tissue comprises an activator of the LTβR and / or NIK signal transduction pathway. In certain embodiments, the reagent that promotes the formation of ectopic kidney tissue comprises an activator of the LTβR and / or NIK signal transduction pathway. In certain embodiments, the reagent that promotes the formation of ectopic kidney tissue comprises an agent that promotes inflammation.

[0034] In certain embodiments, a method for generating ectopic renal tissue in a subject comprises introducing cells comprising one or more renal cells and one or more stromal cells into an adipose-associated lymph cluster or lymph node of the subject, and providing an activator of the LTβR and / or NIK signaling pathway, wherein activation of the LTβR and / or NIK signaling pathway in the one or more stromal cells promotes proliferation and / or angiogenesis of the one or more renal cells to form ectopic renal cells.

[0035] In certain embodiments, a method for generating ectopic renal tissue in a subject comprises introducing cells comprising one or more renal cells and one or more stromal cells into an adipose-associated lymph cluster or lymph node of the subject, and providing an activator of LTβR and / or NIK, wherein activation of LTβR and / or NIK in the one or more stromal cells promotes proliferation and / or angiogenesis of the one or more renal cells to form ectopic liver cells.

[0036] The present disclosure further includes compositions for generating ectopic tissue. In certain embodiments, the compositions comprise a plurality of cells and one or more agents that promote the formation of ectopic tissue. In certain embodiments, the compositions for generating ectopic liver tissue comprise a plurality of hepatocytes and one or more agents that promote the formation of ectopic liver. In certain embodiments, the compositions for generating ectopic kidney tissue comprise a plurality of kidney cells and one or more agents that promote the formation of ectopic kidney.

[0037] In certain embodiments, the composition of the present disclosure includes one or more agents comprising a plurality of bone marrow-derived cells and / or a plurality of stromal cells. In certain embodiments, the stromal cells express one or more of podoplanin, NIK and / or LTβR. In certain embodiments, the one or more agents are activators of the LTβR and / or NIK signal transduction pathways. In certain embodiments, the one or more agents are agents that promote inflammation. In certain embodiments, the one or more agents include two or more of the following: (a) a plurality of bone marrow-derived cells and / or a plurality of stromal cells; (b) an activator of the LTβR and / or NIK signal transduction pathway; and (c) an agent that promotes inflammation. In certain embodiments, the composition further comprises a synthetic culture medium.

[0038] In certain embodiments, a method for treating a subject in need of enhanced liver function comprises administering to the subject a therapeutically effective amount of hepatocytes and providing one or more agents that promote the formation of ectopic liver tissue in the subject's lymph nodes, wherein the one or more agents comprise two or more of the following: (a) a plurality of bone marrow-derived cells, a plurality of stromal cells, or a combination thereof; (b) an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof; or (c) an agent that promotes inflammation.

[0039] In certain embodiments, the present disclosure further provides a method for treating a subject in need of enhanced renal function, the method comprising administering to the subject a therapeutically effective amount of renal cells, renal tissue fragments, or a combination thereof, and providing one or more agents that promote the formation of ectopic renal tissue in the subject's lymph nodes, wherein the one or more agents comprise two or more of the following: (a) a plurality of bone marrow-derived cells, a plurality of stromal cells, or a combination thereof; (b) an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof; or (c) an agent that promotes inflammation.

[0040] The present disclosure further provides kits comprising one or more compositions disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : Fat-associated lymphoid clusters; schematic diagram (upper panel) and distribution (lower panel).

[0043] Figure 2A -D: Generation of ectopic liver in secondary lymphoid tissue. (A) 12 weeks after IP transplantation of hepatocytes, Fah - / - Macroscopic images of mice. Yellow circles highlight multiple ectopic liver nodules (milky spots and lymph nodes (LNs)) generated in the lymphoid tissue. (B) Mesenteric porcine lymph node 2 months after portal vein shunt, partial hepatectomy and hepatocyte transplantation. Left, OCT block and cryosections from the block stained with CK18 for hepatocytes and PNad for high endothelial cells. 51.5% of the LN mass was identified as CK18+ hepatocytes. Right, cryosections of control liver and ectopic liver stained with CK18 (hepatocytes) and ER-TR7 (fibroblasts). H&E shows normal microangiogenesis present in porcine ectopic liver (C) Top image, macroscopic view of the mouse peritoneal space showing the larger omentum (O) covering the stomach (S) and intestine (I), adjacent to the liver (L) and located on the pancreas (not shown). Middle image, mouse omentum under a dissecting microscope, showing a large number of blood vessels. Bottom image, schematic diagram of the omentum showing the vascular tree supplying the milky spots. (D) IP transplantation of GFP+ hepatocytes. Ectopic nodules were generated in the papillary plaques of Fah- / - mice 12 weeks after hepatocyte implantation.

[0044] Figure 3 :Fah 3-4 weeks after IP transplantation of hepatocytes - / - Mouse; dilation of capillary spaces and sprouting of blood vessels in milky spots.

[0045] Figure 4 : Omental milky spots in mice.

[0046] Figure 5 : Engraftment of transplanted hepatocytes in the omental milky spots. Hepatocytes are labeled with GFP and are present as bright areas of green fluorescence. ER-TR7 is an antigen present in the extracellular matrix of lymphoid tissues and is fluorescent blue in this image (indicated by arrows).

[0047] Figure 6 :FRGN(Fah - / - Rag2 / γc - / - Immunofluorescence staining of wild-type (left) and FRGN omentum, with milky spots ("MS") marked.

[0048] Figure 7 : Engraftment of hepatocytes in the omentum of FRGN mice.

[0049] Figure 8 : No growth of hepatocytes implanted in the omentum was observed in FRGN mice during 12 weeks.

[0050] Fig. 9: Restoration of milky spots in the omentum of FRGN mice by bone marrow transplantation.

[0051] Fig.10 : Restoration of milky spots in the omentum of FRGN mice by bone marrow transplantation. The arrows in the right image show the implanted hepatocytes in the milky spots.

[0052] Fig.11 : Ectopic liver formation in FRGN mice after bone marrow transplantation.

[0053] Fig.12 : Heterotopic liver formation in FRGN mice after bone marrow transplantation. The left panel shows the results without bone marrow transplantation-hepatocyte aggregation, but no ectopic liver formation was observed. The right panel shows the results of bone marrow transplantation, generating an ectopic liver.

[0054] Fig.13 : Stromal cell / lymphoid tissue inducer interactions (left) and NIK signaling pathway (right).

[0055] Fig.14 NIK-deficient (aly / aly) and control Fah cells were injected intraperitoneally (IP) into the spleen (SP). - / - Schematic representation of a study in mice testing the effect of NIK function on rescue of liver function. Ten 6 Kaplan Meier survival curves of Fah- / - mice and aly / aly Fah- / - mice with 100 hepatocytes.

[0056] Fig.15 NIK function is rescued by IP injection of Fah - / - Mice required. 6 GFP+ hepatocytes were inoculated into Fah - / - Neutralization of alymphoplasia in mice - / - Mouse (aly / alyFah - / - ). Whole-mount images of retinal milky spots were taken at various time points. Shown is a bright-field image merged with the fluorescence of donor hepatocytes (green). Top image, Fah - / - Mouse; bottom image, aly / aly Fah - / - Mouse.

[0057] Fig.16 : Schematic diagram of the results observed after IP hepatocyte transplantation.

[0058] Fig.17A-D: (A) Distribution of podoplanin (PDPLN) and CD31 in fibroblastic reticular cells ("FRC"), lymphatic endothelial cells (LEC), and brain endothelial cells (BEC), and flow cytometric analysis of these markers in lymph nodes compared to milky spots. (B) In milky spots, hepatocytes establish themselves in proximity to ER-TR7 stromal cells. (C) Isolated stromal cells, transplanted IP, migrate back into omental milky spots. (D) Milky spots with transplanted hepatocytes.

[0059] Fig.18 : Immunofluorescence studies demonstrated that hepatocytes were positive for lymphotoxin α and β, whereas stromal cells were positive for NIK and lymphotoxin β receptors.

[0060] Fig.19 Wild-type GFP+ (green fluorescent protein) hepatocytes were transplanted intraperitoneally into Fah - / - or Fah / LTbR - / - This image shows the implantation in the omentum of these animals.

[0061] Fig. 20A-G: (A) Top panel, images from left to right are human fetal kidney, jejunal LN (after kidney fragment transplantation), and LN (3 weeks after transplantation). Middle left, H&E stained sections of paraffin-embedded donor human fetal kidney. Middle right, hematoxylin-stained frozen sections of LN 3 weeks after kidney transplantation. The area surrounded by the orange dashed line indicates the area of ​​non-implanted LN. Bottom panel, these insets show immature (left) or mature (right) glomeruli. (B) Representative immunofluorescence staining of 3-week transplant sections for NCAM, WT-1, PDPLN, α-SMA, Megalin, AQ1, LTL, NKCC2, BRN1 / DBA, LTL / DBA, K8 / AQ2, or EPO. (C) Representative 3D reconstruction of 8-week transplants. Left, images of implanted glomeruli and tubules in LNs. Right, images of glomeruli and tubules only. (D) 10,000 kDa MW Texas Red dextran accumulation in 11-week-old mice with grafts. (E) Representative fluorescent immunostaining of 1, 3, and 8-week graft sections for mouse and human CD31. (F) Left, representative immunofluorescence staining for NCAM, SIX2, WT-1, or E-CADH in mouse NP organoids before transplantation. Right, representative immunofluorescence staining for PDPLN / CD31, PDPLN / CD105, or PDPLN / Ly-76 shows glomerular-like structures of vascularization in LNs 8 weeks after NP organoid transplantation. (G) Left, representative whole-mount immunofluorescence imaging of iPSC-derived kidney organoids for DBA / BRN1 and E-CADH / LTL. Bottom panel shows serial sections of two different nephron-like structures near LTL / Megalin-responsive proximal-like tubules, each of which includes a glomerulus-like structure containing putative podocytes (PDPLN, SYNPO, PODXL), glomerular mesangial cells (CIV), and endothelial cells (CD31) 6 weeks after organoid transplantation into LN. Nuclei were counterstained with Hoechst (blue).

[0062] Fig.21 : Schematic representation of the canonical and non-canonical NF-κB signaling pathways.

[0063] Fig.22A-G: (A) Left, schematic diagram of the experimental protocol. Mice were injected IP with 100 μg LTβR-Fc or control Ig 2 days before receiving transplanted embryonic GFP+ kidney fragments into their LNs (day 1). Mice were treated again on days 8 and 15. LNs were collected on day 22. Right, LNs isolated from whole mounts of kidneys from treated or untreated mice. (B) Representative immunofluorescence staining of serial sections of kidney transplants as described in A for PDPLN, LTL, CD31, CD105, Ly-76. (C) Flow cytometric profiles of fibroblast reticular cells (FRC), lymphatic endothelial cells (LEC), and blood endothelial cells (BEC) in LN or omental stromal cell populations (CD45-). (D) Representative immunofluorescence staining of PDPLN and LTβR for omental stromal cells. (E) Whole mount renal omentum from wild type and / or LTβR- / - mice after transplantation of embryonic GFP+ kidneys, 6 weeks after transplantation (left is a merged fluorescence / bright field image, and the right is a dissecting scope image). Neo-kidney is shown compared to adult mouse kidneys. (F) Representative immunofluorescence staining of serial sections of intraomental kidney transplants as described in E for CD31, CD105 and Ly-76. (G) Representative immunofluorescence staining of ERTR-7, LTα, LTβ, LTβR, NIK, NIK / CD34 on sections of embryonic GFP+ (left and middle) or wild type (right) kidney LN transplants. Nuclei were counterstained with Hoechst (blue).

[0064] Fig.23 : Peritoneal inflammation induces dramatic changes in the omentum. Top: Schematic diagram of the experiment. Zymosan was injected intraperitoneally (IP) on day 0. 3 days later, 1 million hepatocytes were injected IP. Samples were collected 7 days after hepatocyte injection. Bottom left: Macroscopic images of the omentum from zymosan-induced and control animals. Bottom right: The weight of the omentum was significantly increased after zymosan-induced inflammation when compared to controls.

[0065] Fig.24 : FALC and liver growth. Omental and mesenteric fat of C57bl / 6 mice one week after GFP+ hepatocyte transplantation and after zymosan or PBS injection. Zymosan inflammation induced a significant increase in FALC number and size that allowed engraftment of GFP+ hepatocytes. Omental and mesenteric fat were compared with GFP+ hepatocytes identified under fluorescence microscopy.

[0066] Fig.25 Peritoneal inflammation increases survival of tyrosinemic mice after hepatocyte transplantation. Fah with or without prior induction of inflammation - / -C57bl / 6 mice were transplanted with wild-type hepatocytes via IP, and then two rounds of selection (NTBC removal) were performed. Animals without inflammation (left figure) continued to lose weight after the second selection, and animals H146 and H148 died about 8 weeks after transplantation. Animals with previously induced inflammation (right figure) gained weight during the second selection, indicating rescue for liver disease with restoration of functional liver quality.

[0067] Fig.26 : Peritoneal inflammation increases liver mass present in lipid-containing FALCs. Macroscopic images of peritoneal fat containing liver tissue from Fah- / - C57bl / 6 mice without (H146) and with (H150) previously induced inflammation. Inflammation supports ectopic liver growth in FALCs. DETAILED DESCRIPTION OF THE INVENTION

[0069] For purposes of clarity and not limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections:

[0070] (i) definitions;

[0071] (ii) treatment methods;

[0072] a. Treatment of liver diseases and disorders;

[0073] b. Treatment of kidney diseases and disorders; and

[0074] (iii) Composition.

[0075] 5.1 Definitions

[0076] Unless defined otherwise, all technical and scientific terms used in this application have the same meanings as commonly understood by one of ordinary skill in the art.

[0077] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend on how the value is measured or determined, i.e., the limits of the measurement system. For example, "about" may mean within 3 standard deviations or greater than 3 standard deviations as practiced in the art. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within a certain order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0078] As used herein, fat-associated lymphoid clusters ("FALCs", also referred to herein as "milky spots") are structures of the non-lymphatic lymphatic system. FALCs are present in many anatomical locations, including but not limited to the mesentery, mediastinum, pericardium, and subcutaneous tissue (see Figure 1). FALC is located in the adipose tissue of the pleura, pericardium, and peritoneal cavity. In the peritoneal cavity, FALC is located in the omentum and mesentery, or spleen, hilum, and gonadal fat of a subject. In certain non-limiting embodiments, FALC for ectopic tissue growth, such as liver or kidney tissue growth, is located in the omentum. In certain non-limiting embodiments, FALC is located in the mediastinum or pericardium.

[0079] As used herein, "lymph node" or "LN" refers to any lymph node, such as, but not limited to, abdominal lymph nodes, celiac lymph nodes, para-aortic lymph nodes, splenic hilar lymph nodes, hepatic hilar lymph nodes, gastric lymph nodes (left and right), gastroepiploic (omental) lymph nodes (left and right), retroperitoneal lymph nodes, pyloric lymph nodes (suprapyloric, subpyloric, retropyloric), pancreatic lymph nodes (superior pancreatic, inferior pancreatic, splenic line lymph nodes), hepatic lymph nodes (cystic, containing foramina of Winslow), pancreaticoduodenal lymph nodes (superior pancreaticoduodenal, inferior pancreatic duodenal), inguinale), superior mesenteric, ileocolic, prececal, retrocecal, appendiceal, mesocolonic (pericolic, left colic, middle colic, right colic, inferior mesenteric, sigmoid, superior rectal), common iliac (medial common ilium, middle common ilium, lateral common ilium, subaortic common ilium, anterior promontory common ilium) and external iliac (medial external ilium, middle external ilium, lateral external ilium, medial lacunar-femoral, middle lacunar-femoral, lateral lacunar-femoral, interiliac external ilium, obturator-obturator), jejunal, popliteal, and axillary nodes.

[0080] The cells of an organ may include one or more cell types. In some embodiments, the cells include multiple cell types present in the original organ, but do not necessarily need to include all cell types present in the organ. A non-limiting example, "liver cells" for implantation (engraftment) according to the present disclosure include hepatocytes and may also include one or more of liver progenitor cells, biliary cells, endothelial cells and stem cells. Another non-limiting example, "kidney cells" for implantation according to the present disclosure include renal parenchymal cells and may also include one or more of kidney progenitor cells, glomerular cells, endothelial cells and stem cells. In some embodiments, the cells are dissociated before the introduction for implantation. In some embodiments, the cells are included in aggregates before introduction. In some embodiments, the cells include organoids expanded in vitro. In some embodiments, the cells include iPS or other stem cells expanded in vitro. In some embodiments, the cells include tissue fragments obtained from a complete organ, but the fragment composition does not exceed about 5% of the organ, or does not exceed about 10%, or does not exceed about 25% or does not exceed about 50%.

[0081] As used herein, "activator" refers to a compound or molecule (e.g., a small molecule, a peptide, a peptide mimetic, a natural compound, or an antibody) that activates (e.g., increases, promotes, or enhances) the activity, function, expression, and / or production of a protein or pathway. An activator can be any compound or molecule that promotes any activity of a specified protein (molecule, any molecule related to a specified molecule or a specified related molecule) such as LTβR or NIK, or promotes the interaction of a specified protein (e.g., LTβR or NIK) with a signal transduction partner or binding partner. Activators may also include molecules that indirectly regulate the biological activity of a specified protein (e.g., LTβR or NIK) by intercepting upstream signal transduction molecules. In certain embodiments, an activator may include a molecule that promotes, increases, and / or enhances the production and / or production of a specified protein such as LTβR or NIK by increasing the activity and / or function of an enzyme that generates and / or produces LTβR or NIK.

[0082] As used herein, "treatment" (and grammatical variations thereof) refers to an attempt to alter the natural course of the individual being treated, and may be used for prevention or clinical intervention in the course of clinical pathology. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, alleviating or relieving the disease state, and alleviating or improving prognosis. In certain embodiments, "treatment" may refer to reducing the severity of complications, symptoms, and / or exacerbations. For example, but not limited to, reduction may be, for example, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% reduction in the severity of complications, symptoms, and / or exacerbations relative to similar control subjects not receiving treatment. In certain embodiments, "treatment" may also refer to an extension of survival relative to the expected survival period without treatment.

[0083] As used herein, "therapeutically effective amount" or "effective amount" refers to an amount that can achieve one or more of the following: alleviate symptoms, reduce the rate of disease progression, prolong survival, slow or alleviate the disease state and / or improve prognosis.

[0084] "Subject" or "individual" are used interchangeably herein and may refer to a human or non-human subject. Non-limiting examples of non-human subjects include non-human primates, dogs, cats, horses, mice, rats, hamsters, rabbits, pigs, etc.

[0085] The terms or phrases "transplantation," "cell replacement," or "implantation" are used interchangeably herein to refer to the introduction of cells into a target tissue, such as FALC or a tissue comprising FALC.

[0086] As used herein, "in combination with..." can mean that one or more cells to be implanted or a composition thereof and an agent (e.g., an activator of the LTβR and / or NIK signaling pathway) can be administered to a subject as part of a treatment regimen or plan. In certain embodiments, the combined use does not require that the one or more cells and the one or more agents be physically combined prior to administration or be administered within the same time frame. For example, but not limited to, the one or more cells to be implanted and the one or more agents can be administered to the subject being treated at the same time, or can be administered at the same time or sequentially in any order or at different time points. In certain embodiments, the methods of the present disclosure may include administering one or more agents prior to administering one or more cells to be implanted, such as hepatocytes or kidney cells.

[0087] 5.2 Treatment methods

[0088] In certain embodiments, the present disclosure relates to the transplantation or proliferation of cells in adipose-associated lymphoid clusters ("FALC" or "milky spots") to generate ectopic tissue.

[0089] The present disclosure provides methods for generating ectopic tissue in or around a FALC. In certain embodiments, the method for generating ectopic tissue may include introducing cells, such as hepatocytes or kidney cells, into the FALC of a subject to form ectopic tissue, for example, ectopic liver tissue or ectopic kidney tissue. In certain embodiments, the method for generating ectopic tissue may include introducing cells into the lymph nodes of a subject. In certain embodiments, the methods of the present disclosure may be used to treat medical conditions and / or conditions (e.g., pathologies, disease syndromes) that may benefit from the generation of ectopic tissue. For example, but not limited to, a subject may suffer from a disease or condition, such as, but not limited to, liver disease and / or failure or kidney disease and / or failure.

[0090] In certain non-limiting embodiments, the subject can be a human or a non-human. Non-limiting examples of non-humans include non-human primates, dogs, cats, horses, mice, rats, hamsters, rabbits, pigs, etc. In certain embodiments, the subject is a human.

[0091] In certain embodiments, the cells to be implanted into the FALC of a subject can be human cells, non-human cells, or both. In some embodiments, the cells are human cells. In certain embodiments, human cells can be implanted into the FALC of a non-human subject (e.g., a mouse), or a region comprising the FALC.

[0092] Cells for transplantation can be obtained from an object undergoing transplantation. In certain embodiments, cells can be obtained from a source other than the object undergoing transplantation. In certain embodiments, cells can be obtained from a fresh or frozen cell population. In certain embodiments, cells can be isolated from tissues and grown in vitro under various culture conditions before transplantation. In certain embodiments, cells can be obtained from embryonic, fetal, child or adult tissues. In certain embodiments, cells can be progenitor cells or precursor cells. In certain embodiments, progenitor cells or precursor cells can differentiate into cell types to be transferred.

[0093] Cells for use according to the present disclosure can be prepared by any method known in the art. In certain embodiments, cell solutions, such as compositions disclosed herein, are used for injection. Non-limiting examples of compositions for the disclosed methods are described below in Section 5.3. In certain embodiments, cells can be prepared according to the method of Li et al. J Tissue Culture Methods 14: 139-146 (1992). In short, separation includes collagenase perfusion with only a sample of a limited size (<50g) on ​​the surface of an incision. In certain embodiments, a cell population enriched in specificity can be used, such as those disclosed in U.S. Patent No. 7,211,404.

[0094] The cells may be administered to the subject by any method known in the art. In certain embodiments, the cells may be injected, including but not limited to, intraperitoneal, intravenous or intraarterial injection or by local perfusion. In certain embodiments, the cells may be surgically transplanted, for example, by conventional or endoscopic surgical techniques. In certain embodiments, the cells may be administered locally. For example, but not limited to, the cells may be introduced into the vicinity of an anatomical location containing the FALC, such as, but not limited to, the gonads, omentum, mesentery, mediastinum, pericardium, and subcutaneous tissue. In certain embodiments, the anatomical region is the omentum. In certain embodiments, for example, hepatocytes or kidney cells (or fragments of kidney tissue) may be introduced into the vicinity of the omentum (where the FALC resides), for example, by targeted intraperitoneal injection or by conventional or endoscopic surgical techniques. In certain embodiments, the cells to be transplanted may be initially expanded in a culture medium and then introduced into the FALC or lymph nodes in vivo.

[0095] In certain embodiments, the cells can be suspended in any suitable injection buffer. Non-limiting examples of such buffers include saline, phosphate-buffered saline, Hank's salt, and Ringer's solution, among others.

[0096] In certain embodiments, methods for generating ectopic tissue in or near a FALC or lymph node include providing an agent that promotes the formation of ectopic tissue. For example, but not limited to, the method may include administering a combination of one or more cells to be implanted and one or more agents that promote the formation of ectopic tissue. In certain embodiments, one or more agents may be included in a composition containing one or more cells to be implanted. Alternatively and / or in addition, one or more agents may be administered before, after, or at the same time as the implantation of one or more cells. In certain embodiments, the agent promotes the proliferation and / or vascularization of one or more cells to form ectopic tissue. Similar methods may be used to generate ectopic tissue in a lymph node.

[0097] In certain embodiments, the reagent that promotes the formation of ectopic tissue can be a cell type different from the cell (e.g., kidney cell or liver cell) that forms the ectopic tissue. In certain embodiments, the reagent includes a variety of bone marrow-derived cells and / or matrix. In certain embodiments, the reagent includes stromal cells, for example, fibroblasts, fibroblast reticular cells (FRC), foliated dendritic cells (FDC), lymphatic endothelial cells (LEC), blood endothelial cells (BEC), α-7 integrin pericytes (AIP) and double negative cells (DNC). In certain embodiments, the fibroblasts can be human foreskin fibroblasts, human embryonic fibroblasts, mouse embryonic fibroblasts, skin fibroblasts, vascular fibroblasts, myofibroblasts, smooth muscle cells, mesenchymal stem cells (MSC)-derived fibroblasts or a combination thereof. For example, but not limited to, a method for generating ectopic tissue may include (a) introducing one or more cells, such as hepatocytes or kidney cells, into the FALC (or an anatomical region comprising the FALC) or lymph nodes (or an anatomical region comprising lymph nodes) of a subject to form ectopic tissue, for example, ectopic liver tissue or ectopic kidney tissue, and (b) introducing one or more bone marrow-derived cells and / or stromal cells to promote the formation of ectopic tissue, for example, by promoting proliferation and / or vascularization of one or more cells to form ectopic tissue. In some embodiments, one or more cells to be implanted and one or more bone marrow-derived cells and / or stromal cells may be included in the same composition.

[0098] In certain non-limiting embodiments, stromal cells, such as stromal endothelial cells or fibroblasts, that promote ectopic tissue formation may express detectable levels of one or more of podoplanin, lymphotoxin beta receptor ("LTβR"), NIK, or a combination thereof. Alternatively and / or in addition, stromal cells may be treated with an activator of the LTβR and / or NIK signaling pathway, e.g., an activator of LTβR or NIK or its downstream targets, before, during, or after administration to a subject. In certain embodiments, stromal cells, such as stromal endothelial cells or fibroblasts, may be treated with an activator of NIK. In certain embodiments, stromal cells may be genetically modified to exogenously express LTβR and / or NIK and / or overexpress LTβR and / or NIK.

[0099] In certain embodiments, an agent that promotes the formation of ectopic tissue, for example, by promoting proliferation and / or angiogenesis of one or more cells to form ectopic tissue, can be an activator of the LTβR and / or NIK signal transduction pathway. For example, but not limited to, a method of generating ectopic tissue in or near the FALC or lymph node can include introducing cells, such as hepatocytes or kidney cells, into the FALC (or an anatomical region containing the FALC) or lymph node of a subject and providing at least one activator of the LTβR and / or NIK signal transduction pathway, wherein activation of the LTβR and / or NIK or its downstream targets promotes the formation of ectopic tissue, for example, ectopic liver tissue or ectopic kidney tissue. In certain embodiments, the activator of the LTβR and / or NIK signal transduction pathway is an activator of the LTβR and / or NIK or an activator of the non-classical NF-κB signal transduction pathway. In certain embodiments, one or more cells to be implanted and the activator of the LTβR and / or NIK signal transduction pathway can be included in the same composition. Alternatively, the activator can be administered before, during, or after the one or more cells to be implanted are introduced into the subject.

[0100] In certain embodiments, an activator of the LTβR and / or NIK signaling pathway can increase the expression of LTβR or NIK in cells (e.g., stromal cells or stromal endothelial cells) present in a subject undergoing implantation. For example, but not limited to, an activator of the LTβR and / or NIK signaling pathway activates a signaling pathway in stromal cells present in the subject's FALC.

[0101] Alternatively and / or additionally, the cells to be implanted may be a heterogeneous cell population, for example, a composition comprising a heterogeneous cell population comprising stromal cells or stromal endothelial cells, and an activator of the LTβR and / or NIK signaling pathway may increase the expression of LTβR or NIK in the stromal cells or stromal endothelial cells present in the composition to be implanted. For example, but not limited to, a method for generating ectopic tissue may include (a) introducing one or more cells, such as hepatocytes or kidney cells, into the FALC or lymph nodes of a subject to form ectopic tissue, for example, ectopic liver tissue or ectopic kidney tissue, (b) introducing one or more bone marrow-derived cells and / or stromal cells to promote proliferation and / or angiogenesis of the one or more cells to form ectopic tissue, and (c) administering an activator of the LTβR and / or NIK signaling pathway. In certain embodiments, an activator of the LTβR and / or NIK signaling pathway is present in a composition comprising one or more cells to be implanted and one or more bone marrow-derived cells and / or stromal cells, or an activator of the LTβR and / or NIK signaling pathway is present in a composition comprising one or more bone marrow-derived cells and / or stromal cells.

[0102] In certain embodiments, the activator of the LTβR and / or NIK signal transduction pathway may be an activator of LTβR or a downstream target of LTβR. In certain embodiments, the activator may be an activator of NIK or a downstream target of NIK, for example, an activator of the non-classical NF-κB signal transduction pathway. In certain embodiments, the activator of the LTβR and / or NIK signal transduction pathway may be an agonist antibody (or an antibody fragment thereof) or a single-chain antibody that specifically binds to LTβR or NIK. Non-limiting examples of anti-LTβR agonist antibodies include: monoclonal antibodies produced by Adipogen Life Sciences catalog number AG-20B-0008, anti-LTβR agonist antibody CBE11 (see, e.g., Lukashev et al., Cancer Res. 66(19):9617-9624 (2006)), anti-LTβR agonist antibody BS-1 (see, e.g., Hu et al., Carcinogenesis 34(5):1105-1114 (2013)), and anti-LTβR agonist antibody 4H8 (see, e.g., Scarzello et al., Gut 65:1765-1775 (2016)). Other non-limiting examples of anti-LTβR agonist antibodies are disclosed in U.S. Patent Application No. 2002 / 0090366, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, an activator of the LTβR and / or NIK signaling pathway may enhance the function and / or activity of LTβR or NIK or its downstream targets.

[0103] In certain embodiments, the present disclosure provides a method for generating ectopic liver tissue in a subject, comprising introducing one or more cells into the FALC or lymph nodes of the subject, and providing an activator of LTβR and / or NIK, wherein the activation of LTβR and / or NIK promotes proliferation and / or angiogenesis of one or more cells to generate ectopic cells. In certain embodiments, the one or more cells comprise hepatocytes, and the ectopic tissue comprises ectopic liver tissue. In certain embodiments, the one or more cells comprise kidney cells, and the ectopic tissue comprises ectopic kidney tissue. In certain embodiments, the one or more cells further comprise one or more stromal cells, and the activation of LTβR and / or NIK results in activation of LTβR and / or NIK signal transduction pathways in stromal cells.

[0104] In certain embodiments, the agent that promotes the formation of ectopic tissue can be an agent that promotes inflammation in a subject. For example, but not limited to, the method for generating ectopic tissue in or near a FALC or lymph node can also include inducing inflammation in a subject, for example, by administering an agent that promotes inflammation in a subject. Non-limiting examples of such agents include cytokines, vasodilators, histamine, serotonin, bradykinin, and zymosan. Non-limiting examples of cytokines include: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, IFN-α, IFN-α2, IFN-β or IFN-γ, TNF-α and TNF-β, TGF-α and TGF-β, lymphotoxin-α and lymphotoxin-β. Non-limiting examples of chemokines include: CCL18, CCL19, CCL21, CXCL8, CCL2, CCL3, CCL4, CCL5, CCL7, CXCL12 and CXCL13. In certain embodiments, the agent comprises a compound and / or molecule that activates the complement system. In certain embodiments, the agent comprises zymosan. In certain embodiments, the agent is administered in and / or near the FALC in which the cells have been implanted or the cells will be implanted. In certain embodiments, inflammation is induced locally, for example, in or near the anatomical region of the FALC or lymph nodes in which the cells have been implanted or the cells will be implanted. In addition and / or alternatively, inflammation is induced systemically.

[0105] In certain embodiments, the inflammation is temporary. For example, but not limited to, the inflammation lasts no more than about 10 weeks, no more than about 9 weeks, no more than about 8 weeks, no more than about 7 weeks, no more than about 6 weeks, no more than about 5 weeks, no more than about 4 weeks, no more than about 3 weeks, no more than about 2 weeks, no more than about 1 week, no more than about 6 days, no more than about 5 days, no more than about 4 days, or no more than about 3 days.

[0106] In certain embodiments, inflammation is induced prior to the introduction of one or more cells to be transplanted. For example, but not limited to, inflammation is induced at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, 1 day before, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days before the introduction of one or more cells to be implanted.

[0107] Additionally and / or alternatively, inflammation is induced after the one or more cells to be transplanted are introduced. For example, but not limited to, inflammation is induced at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days after the one or more cells to be implanted are introduced.

[0108] In certain non-limiting embodiments, cells such as liver cells or kidney cells transplant recipients can be additionally administered with immunosuppressants to minimize the immune rejection of implanted cells (e.g., liver cells or kidney cells). By inhibiting or suppressing any innate immune system activity, including but not limited to T cell and / or B cell activity, immunosuppressants can reduce or prevent adverse immune responses to foreign or transplanted tissues in recipient mammals. The administration of immunosuppressants may include, but is not limited to, administration of radiotherapy and / or administration of immunosuppressive drugs. Examples of suitable immunosuppressive drugs include, but are not limited to, steroids (such as corticosteroids, dexamethasone and prednisone), Cox-1 and Cox-2 inhibitors, macrolide antibiotics (such as rapamycin and tacrolimus) and other substances that limit, reduce or inhibit B cells, T cells and / or other innate immune activity. In certain embodiments, immunosuppressants particularly suitable for use in the present disclosure include those known for liver and / or kidney transplantation, including but not limited to, steroids, cyclosporin, rapamycin, azathioprine, prednisone and OKT3. In certain embodiments, the hepatocyte transplant recipient is administered the immunosuppressant tacrolimus, also known as FK506, a calcineurin inhibitor that in turn may inhibit IL-2 production and downstream B lymphocyte activation.

[0109] In certain non-limiting embodiments, the subject is not administered an immunosuppressive agent, such as, for example, the agents listed in the above paragraphs (for example, but not limited to, where autologous liver or kidney cells are transferred).

[0110] 5.2.1 Treatment of liver diseases and disorders; The present disclosure provides methods for treating subjects in need of enhanced liver function. In certain embodiments, the present disclosure provides methods for propagating hepatocytes in FALC or lymph nodes to produce ectopic liver tissue, which can be used to obtain therapeutic benefit in a subject, for example, in a subject with reduced liver function. In certain embodiments, the FALC is located in the omentum.

[0111] The subject who needs to enhance liver function is a subject who lacks sufficient functional liver to maintain a healthy state, including but not limited to such a subject, the subject has a fibrotic and / or cirrhotic liver, or a liver damaged by disease, trauma or toxic effects. For example, a healthy state is demonstrated by one or more liver function parameters falling within the normal range of the subject. Non-limiting examples of liver function parameters include albumin levels, alanine aminotransferase ("ALT") levels, aspartate aminotransferase ("AST") levels, creatinine levels, total bilirubin levels, direct bilirubin levels, phenylalanine levels, alanine levels, glycine levels, valine levels, glutamate levels, prothrombin time, lactate dehydrogenase and alkaline phosphatase; the normal ranges of these levels for various subjects including human subjects are known in the art. In certain non-limiting embodiments, one or more, or two or more, or three or more of the parameters increase by at least about 25% or at least about 50% relative to normal levels, indicating that liver function needs to be enhanced. Achieving an increase in liver function in a subject means moving at least one liver function parameter toward a normal range, for example, but not limited to, an improvement of at least about 10% or at least about 20% or at least about 30% or at least about 40% or at least about 50%.

[0112] In certain non-limiting embodiments, enhanced liver function is indicated by prolonged survival of the subject, such as by at least about 20% or at least about 30%.

[0113] In certain embodiments, the subject in need of enhancing liver function may suffer from liver disease, liver disorder, liver failure and / or reduced liver function. Non-limiting examples of liver diseases and / or disorders that can be treated by the disclosed methods include: metabolic disorders, Crigler-Najjar syndrome type I, acute liver failure, cirrhosis, hemochromatosis, hyperoxaluria, oxalosis, Wilson disease, alpha-1 antitrypsin deficiency, liver cancer, hepatitis (alcohol and autoimmune), fatty liver disease and non-alcoholic fatty liver disease. Non-limiting examples of diseases and / or disorders associated with the bile system (and indirectly associated with the liver) that can be treated by the disclosed methods include primary biliary cirrhosis and primary sclerosing cholangitis.

[0114] In certain non-limiting embodiments, the present disclosure provides methods for treating a subject in need of enhanced liver function, comprising administering to the subject a therapeutically effective amount of hepatocytes, wherein at least one of the administered hepatocytes proliferates in or near the FALC or lymph nodes and generates ectopic liver tissue, thereby enhancing the subject's liver function.

[0115] In certain embodiments, the hepatocytes transplanted into the subject can be human hepatocytes, non-human hepatocytes, or both. The hepatocytes can be homologous or allogeneic to the intended recipient. In non-limiting embodiments, the hepatocytes are autologous to the intended recipient (e.g., harvested and amplified in culture medium before transplantation, or generated by stem cells or precursor cells of the intended recipient). In certain non-limiting embodiments, the hepatocytes of one species can be transplanted into another species, such as, but not limited to, transplanting human hepatocytes into non-human (e.g., mouse) hosts. For example, but not limited to, non-human hepatocytes can be implanted into the FALC of a human recipient.

[0116] An effective amount or therapeutically effective amount of liver cells to be implanted comprises about 10 4 -10 11 Hepatocytes or about 10 5 -10 10 In some embodiments, the effective amount of hepatocytes may include one or more hepatocytes. In some embodiments, about 10 5 to about 10 11 , 10 5 to about 10 9 , 10 5 to about 10 8 , 10 5 to about 10 7 , 10 5 to about 10 6 , 10 6 to about 10 11 , 10 6 to about 10 10 , 10 6 to about 10 9 , 10 6 to about 10 8 , 10 6 to about 10 7 , 10 7 to about 10 11 , 10 7 to about 10 10 , 10 7 to about 10 9 The amount of liver cells.

[0117] In certain embodiments, the hepatocytes can be administered to a subject more than once to achieve the desired ectopic liver tissue. For example, but not limited to, the hepatocytes can be administered to a subject at least twice, at least three times, or at least four times. Alternatively or in addition, the hepatocytes can be implanted into two or more different anatomical regions, including FALC or lymph nodes, to obtain two or more ectopic liver tissues.

[0118] In certain non-limiting embodiments, the present disclosure provides a method for treating an object in need of enhanced liver function, comprising administering a therapeutically effective amount of hepatocytes to the object, and promoting the proliferation of at least one hepatocyte in the FALC of the object. In certain embodiments, such proliferation can be promoted, for example but not limited to, by locally administering hepatocytes to such an anatomical region, the anatomical region comprising a FALC targeted for ectopic liver tissue formation, and / or by providing at least one agent that promotes ectopic liver tissue formation, as described above. In certain embodiments, the agent can be, for example but not limited to, a variety of bone marrow-derived cells and / or a variety of stromal cells. In certain embodiments, the agent can be an activator of the LTβR and / or NIK signal transduction pathway, for example, an activator of LTβR or NIK or its downstream targets. In certain embodiments, the agent can be a bone marrow-derived cell, a stromal cell, an activator of the LTβR and / or NIK signal transduction pathway, or a combination thereof. Similar methods can be used to generate ectopic tissue in lymph nodes to enhance liver function.

[0119] In certain non-limiting embodiments, the present disclosure provides a method for generating ectopic liver tissue, comprising introducing hepatocytes into FALC and providing at least one agent that promotes the formation of ectopic liver tissue, for example, as described above. For example, but not limited to, a method for treating a subject in need of enhanced liver function, comprising (a) administering a therapeutically effective amount of hepatocytes to the subject's FALC to form ectopic liver tissue, and (b) administering one or more bone marrow-derived cells and / or stromal cells to the subject to promote the formation of ectopic liver, for example, by promoting proliferation and / or angiogenesis of one or more cells to form ectopic liver tissue. Similar methods can be used to generate ectopic tissue in lymph nodes.

[0120] In certain non-limiting embodiments, methods for treating a subject in need of enhanced liver function may include (a) administering to the subject a therapeutically effective amount of hepatocytes to the subject's FALC to form ectopic liver cells, (b) providing the subject with an activator of the LTβR and / or NIK signaling pathway to promote the formation of ectopic liver tissue. Similar methods can be used to generate ectopic tissue in lymph nodes to enhance liver function.

[0121] In certain non-limiting embodiments, methods for treating a subject in need of enhanced liver function may include (a) administering a therapeutically effective amount of hepatocytes to the subject into the FALC of the subject to form ectopic liver tissue, (b) administering one or more bone marrow-derived cells and / or stromal cells to the subject to promote the formation of ectopic liver, for example, by promoting proliferation and / or angiogenesis of one or more cells to form ectopic liver tissue, and (c) administering an activator of the LTβR and / or NIK signaling pathway. Similar methods can be used to generate ectopic tissue in lymph nodes to enhance liver function.

[0122] In certain embodiments, the method for treating a subject in need of enhanced liver function may include inducing inflammation in the subject. For example, but not limited to, inflammation may be induced by administering to the subject an agent that promotes inflammation, as disclosed above. In certain embodiments, inflammation is induced prior to introducing one or more hepatocytes to be transplanted, as described above.

[0123] In certain embodiments, the disclosed methods are performed in vivo, as described above. In certain embodiments, they are performed in vitro / ex vivo, for example, as cultured tissue explants. In certain non-limiting embodiments, the disclosed subject matter provides liver tissue produced with such in vitro / ex vivo methods, for example, for subsequent transplantation into a subject in need of enhanced liver function.

[0124] 5.2.2 Treatment of kidney diseases and conditions;

[0125] The present disclosure provides methods for treating subjects in need of enhanced renal function. In certain embodiments, the present disclosure provides methods for propagating renal cells or fragments of renal tissue in FALC or lymph nodes to produce ectopic renal tissue, which can be used to obtain therapeutic benefit in a subject, for example, in a subject with reduced renal function. In certain embodiments, the FALC is located in the omentum.

[0126] A subject in need of enhanced renal function is a subject that lacks sufficient functional kidneys to maintain a healthy state, including but not limited to such subjects, the subject has renal failure, or a kidney damaged by disease, trauma or toxic effects. For example, but not limited to, a healthy state is demonstrated by one or more renal function parameters falling within the normal range of the subject. Non-limiting examples of renal function production include creatinine levels, protein levels, and albumin levels; the normal ranges of these levels for various subjects, including human subjects, are known in the art. In certain non-limiting embodiments, one or more, or two or more, or three or more of the parameters are increased by at least about 25% or at least about 50% relative to normal levels, indicating a need for enhanced renal function. Achieving enhanced renal function in a subject means moving at least one renal function parameter toward the normal range, for example, but not limited to, improving by at least about 10% or at least about 20% or at least about 30% or at least about 40% or at least about 50%. In certain embodiments, a decrease in the glomerular filtration rate (GFR) of a subject indicates decreased renal function. For example, but not limited to, a decrease in the GFR of a subject by about 10%, about 20%, about 30%, about 40%, or about 40% indicates decreased renal function. In certain embodiments, the implanted cells are capable of producing concentrated urine.

[0127] In certain non-limiting embodiments, improved renal function is indicated by prolonged survival of the subject, such as by at least about 20% or at least about 30%.

[0128] In certain embodiments, the subject in need of enhancing kidney function may suffer from kidney disease, kidney disorder, kidney failure and / or reduced kidney function. Non-limiting examples of kidney diseases and / or disorders that can be treated by the methods of the present disclosure include acute renal failure, chronic kidney disease, glomerulonephritis, lupus, polycystic kidney disease, nephropathy, nephrosis, kidney malformations and renal cancer. Other non-limiting examples associated with kidney disease include reduced erythropoiesis, lack of activated vitamin D and atypical non-thyroid disease.

[0129] In certain non-limiting embodiments, the present disclosure provides methods of treating a subject in need of enhanced renal function, comprising administering to the subject a therapeutically effective amount of renal cells, wherein at least one administered renal cell proliferates in or near the FALC or lymph nodes and generates ectopic liver tissue, thereby enhancing the subject's renal function.

[0130] In some embodiments, the fragment of kidney cell or kidney tissue is implanted in or near FALC or lymph node to generate ectopic kidney tissue. In some embodiments, the fragment of kidney cell or kidney tissue transplanted into the object can be human or non-human cell. The non-limiting example of non-human kidney cell includes non-human primate kidney cell and kidney cell from various non-human animals such as dog, cat, horse, mouse, rat, hamster, rabbit, pig, etc. In some embodiments, the fragment of kidney cell or kidney tissue can be homologous or allogeneic for the expected receptor. In non-limiting embodiments, kidney cell and / or kidney tissue fragment is autologous for the expected receptor (for example, harvested and amplified in culture medium before transplantation, or generated by stem cells or precursor cells of the expected receptor). In some embodiments, kidney cell can be obtained from embryonic kidney cell or tissue, for example, metanephroi. In some embodiments, kidney cell is not metanephroi, for example, complete metanephroi. In some embodiments, kidney cell can be renal progenitor cell. In certain non-limiting embodiments, kidney cells and / or kidney tissue fragments from one species can be transplanted into another species, such as, but not limited to, transplanting human kidney cells into a mouse host. For example, but not limited to, non-human kidney cells and / or kidney tissue fragments can be implanted into the FALC of a human recipient.

[0131] In certain embodiments, the effective amount of kidney cells comprises about 10 4 -10 11 kidney cells or about 10 5 -10 10 In some embodiments, the effective amount of renal cells may include one or more renal cells. In some embodiments, about 10 5 to about 10 11 , 10 5 to about 10 9 , 10 5 to about 10 8 , 10 5 to about 10 7 , 10 5 to about 10 6 , 10 6 to about 10 11 , 10 6 to about 10 10 , 10 6 to about 10 9 , 10 6 to about 10 8 , 10 6 to about 10 7 , 10 7to about 10 11 , 10 7 to about 10 10 , 10 7 to about 10 9 The amount of kidney cells.

[0132] In certain embodiments, the renal cells and / or renal tissue fragments can be administered to a subject more than once to achieve the desired ectopic renal tissue. For example, but not limited to, the renal cells and / or renal tissue fragments can be administered to a subject at least twice, at least three times, or at least four times. Alternatively or in addition, the renal cells and / or renal tissue fragments can be implanted into two or more different anatomical regions, including FALC or lymph nodes, to obtain two or more ectopic renal tissues.

[0133] In certain embodiments, the present disclosure also provides a method for treating a subject in need of enhanced renal function, comprising administering to the subject a therapeutically effective amount of renal cells (or a fragment of renal tissue), and promoting the proliferation of renal cells in the adipose-associated lymph nodes or lymph nodes of the subject. In certain embodiments, such proliferation can be promoted, for example but not limited to, by locally administering renal cells to such an anatomical region that contains a FALC targeted for ectopic renal tissue formation, and / or by providing at least one agent that promotes ectopic renal tissue formation, as described above.

[0134] In certain embodiments, the agent can be, for example, but not limited to, a plurality of bone marrow-derived cells and / or a plurality of stromal cells or stromal endothelial cells. Additionally and / or alternatively, the agent can be an activator of the LTβR and / or NIK signaling pathway, for example, an activator of LTβR or NIK or its downstream targets. In certain embodiments, the agent can be an activator of the non-classical NF-κB signaling pathway. In certain embodiments, the agent can be a bone marrow-derived cell, a stromal cell, an activator of the LTβR and / or NIK signaling pathway, an activator of the non-classical NF-κB signaling pathway, or a combination thereof.

[0135] In certain non-limiting embodiments, the present disclosure provides a method for generating ectopic renal tissue, comprising introducing a composition comprising renal cells into the FALC, and providing at least one agent that promotes the formation of ectopic renal tissue, for example, as described above. For example, but not limited to, a method for treating a subject in need of enhanced renal function, comprising (a) administering to the subject a therapeutically effective amount of renal cells or renal tissue fragments into the subject's FALC to form ectopic renal tissue, and (b) administering to the subject one or more bone marrow-derived cells and / or stromal cells to promote the formation of an ectopic kidney, for example, by promoting proliferation and / or angiogenesis of one or more cells to form ectopic renal tissue. Similar methods can be used to generate ectopic tissue in lymph nodes.

[0136] In certain non-limiting embodiments, a method for treating an object in need of enhanced renal function comprises (a) administering to the object a therapeutically effective amount of renal cells or renal tissue fragments to the object's FALC or lymph nodes to form ectopic renal tissue, and (b) administering to the object one or more bone marrow-derived cells and / or stromal cells to promote the formation of ectopic kidneys, for example, by promoting proliferation and / or angiogenesis of one or more cells to form ectopic renal tissue. Similar methods can be used to generate ectopic tissue in lymph nodes to enhance renal function.

[0137] In certain non-limiting embodiments, methods for treating a subject in need of enhanced renal function may include (a) administering to the subject a therapeutically effective amount of renal cells or renal tissue cells to the subject's FALC or lymph nodes to form ectopic renal cells, (b) providing the subject with an activator of the LTβR and / or NIK signaling pathway to promote the formation of ectopic renal tissue. Similar methods can be used to generate ectopic tissue in lymph nodes to enhance renal function.

[0138] In certain embodiments, the method for treating a subject in need of enhanced renal function may include inducing inflammation in the subject. For example, but not limited to, inflammation may be induced by administering to the subject an agent that promotes inflammation, as disclosed above. In certain embodiments, inflammation is induced prior to introducing one or more hepatocytes to be transplanted, as described above.

[0139] In certain embodiments, the disclosed methods are performed in vivo, as described above. In certain embodiments, they are performed in vitro / ex vivo, for example, as cultured tissue explants. In certain non-limiting embodiments, the disclosed subject matter provides kidney tissue produced with such in vitro / ex vivo methods, for example, for subsequent transplantation into a subject in need of enhanced kidney function.

[0140] 5.3 Compositions and kits

[0141] The presently disclosed subject matter provides compositions for generating ectopic tissue. The presently disclosed subject matter provides compositions for generating ectopic tissue, comprising one or more pericytes and one or more agents that can be used to promote the formation of ectopic tissue from the one or more cells.

[0142] In certain embodiments, the one or more cells present in the composition include hepatocytes. In certain embodiments, the one or more cells present in the composition include kidney cells. In certain embodiments, hepatocytes or kidney cells can be prepared by transplantation, and / or the transplantation process can be performed using the methods described in U.S. Patent No. 9,125,891, incorporated herein by reference.

[0143] Cells suitable for use in the present disclosure may be derived from any suitable source. For example, but not limited to, cells may be derived from autologous sources. In certain embodiments, cells may be derived from the object into which the cells are to be implanted. In certain embodiments, cells may be derived from a heterologous source. In certain embodiments, cells may be derived from an individual different from the object into which the cells are to be implanted. In certain embodiments, cells may also be generated from stem cells derived from various sources that will then differentiate into relevant cell types, such as hepatocytes or kidney cells. In certain embodiments, cells may be cultured under various conditions for a period of time to induce certain phenotypes prior to use in the compositions and / or methods of the present disclosure.

[0144] In certain embodiments, the number of cells included in the compositions disclosed herein can vary. In certain embodiments, the composition can include at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 50, about 100, about 150, about 200, about 300, about 400, about 500, about 1000, about 10,000, about 100,000, or about 1,000,000 cells, such as liver cells or kidney cells. In certain embodiments, the composition can include about 10 4 - Approx. 10 11 Cells, for example, liver cells or kidney cells.

[0145] In certain embodiments, the present disclosure provides compositions for generating ectopic livers or treating objects that need to enhance liver function. In certain embodiments, the compositions include a variety of hepatocytes and one or more of the following agents that promote the formation of ectopic livers. In certain embodiments, the agent may be one or more cells that are not hepatocytes. For example, but not limited to, one or more agents may include a variety of bone marrow-derived cells and / or a variety of stromal cells. In certain embodiments, bone marrow-derived cells may be hematopoietic stem cells. In certain embodiments, stromal cells may be fibroblasts or fibroblast-like cells, such as fibroblast reticular cells (FRC), lobed dendritic cells (FDC), lymphatic endothelial cells (LEC), blood endothelial cells (BEC), α-7 integrin pericytes (AIP) and double negative cells (DNC).

[0146] In certain embodiments, the present disclosure provides compositions for generating ectopic kidneys or treating objects that need to enhance renal function. In certain non-limiting embodiments, the present disclosure provides compositions for generating ectopic renal tissues, which include a variety of renal cells (or fragments of renal tissues) and one or more reagents that promote the formation of ectopic kidneys. In certain embodiments, reagents can be one or more cells that are not renal cells. For example, but not limited to, one or more reagents can include a variety of bone marrow-derived cells and a variety of stromal cells. In certain embodiments, bone marrow-derived cells can be hematopoietic stem cells. In certain embodiments, stromal cells can be fibroblasts or fibroblast-like cells, such as fibroblast reticular cells (FRC), lobed dendritic cells (FDC), lymphatic endothelial cells (LEC), blood endothelial cells (BEC), α-7 integrin pericytes (AIP) and double negative cells (DNC).

[0147] In certain embodiments, the composition may further comprise an activator of the LTβR and / or NIK signal transduction pathway. Non-limiting examples of activators of the LTβR and / or NIK signal transduction pathway are as described above. In certain embodiments, the composition may further comprise an activator of a non-classical NF-κB signal transduction pathway. For example, but not limited to, the composition of the present disclosure may comprise a variety of cells, for example, kidney cells (or fragments of kidney tissue) or hepatocytes, and a therapeutically effective amount of an activator of the LTβR and / or NIK signal transduction pathway.

[0148] In certain embodiments, the present disclosure provides compositions that may include (a) a plurality of cells, e.g., kidney cells (or fragments of kidney tissue) or liver cells, (b) a plurality of bone marrow-derived cells and a plurality of stromal cells and (c) a therapeutically effective amount of an activator of the LTβR and / or NIK signaling pathway.

[0149] In some embodiments, the composition may optionally include a synthetic culture medium and / or a scaffold. In some embodiments, the culture medium and / or the scaffold may include, but are not limited to, peptides, proteins, carbohydrates, matrigel, hyaluronic acid, collagen, fibrin, fibrinogen, fibronectin, polyorthoesters, polyvinyl alcohol, polyamide, polycarbonate, agarose, alginate, poly(ethylene) glycol, polylactic acid, polyglycolic acid, polycaprolactone, polyvinyl pyrrolidone, marine adhesive proteins, cyanoacrylates, polymer hydrogels, analogs or combinations thereof.

[0150] In certain embodiments, the composition may optionally include an antibiotic. Non-limiting examples of antibiotics include, but are not limited to, penicillins, such as penicillin and amoxicillin; cephalosporins, such as cephalexin; sulfonamides, such as trimethoprim and trimethoprim; macrolides, such as erythromycin, clarithromycin and azithromycin; fluoroquinolones, such as ciprofloxacin, levofloxacin and ofloxacin; tetracyclines, such as tetracycline and doxycycline; and aminoglycosides, such as gentamicin and tobramycin.

[0151] The present disclosure further provides compositions comprising ectopic tissue produced by the methods disclosed herein. For example, but not limited to, the compositions of the present disclosure may comprise human liver tissue produced in a non-human host, as disclosed above. In certain embodiments, the compositions may optionally comprise synthetic culture media and / or scaffolds. In certain embodiments, the compositions may optionally comprise antibiotics.

[0152] The present disclosure further provides a kit comprising one or more compositions disclosed herein. In certain embodiments, if the kit comprises one or more compositions, each composition can be provided in its own container in the kit. In certain embodiments, the kit of the present disclosure further provides instructions for using the kit, for example, instructions for administering the compositions present in the kit. In certain embodiments, the kit herein may also include one or more components such as those described below: such as instructions for use, devices, and other reagents and components, such as tubes, containers, or syringes for implementing the aforementioned methods.

[0153] The following examples are provided to more fully illustrate the present disclosure but should not be construed as limiting the scope thereof.

[0154] 6. Example 1: Bioengineering the Liver in Secondary Lymphoid Tissue

[0155] 6.1. Generation of helper livers in lymph nodes (LN) and milky spots (FALC)

[0156] In an effort to identify novel sites for hepatocyte transplantation as an alternative to whole organ transplantation, we made the unusual observation that hepatocytes remained intact after intraperitoneal (IP) injection into the Fah- / After removing 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (“NTBC”) from the drinking water in mice deficient in the tyrosine catabolic enzyme fumarylacetoacetate hydrolase (Fah) (an induced liver failure model) (32), these hepatocytes were able to form ectopic nodules de novo. Initially, normal hepatocytes were observed to migrate to the lymphatic system to colonize the Fah - / - Mouse lymph nodes ( Figure 2A), and ectopically generated more than 70% of the native liver mass. This is a surprising result, especially considering that lymph nodes are a common site of initial metastasis for many cancers. Figure 3 Describes Fah 3-4 weeks after IP transplantation of hepatocytes - / - Mouse; dilation of capillary spaces and sprouting of blood vessels in milky spots.

[0157] This has led to the hypothesis that lymph nodes could be directly targeted as sites for heterotopic transplantation of multiple cell types to restore tissue and organ function. - / - Sufficient functional ectopic liver mass is generated in mouse lymph nodes (axillary, popliteal, or mesenteric) to rescue the survival of these mutant mice affected by a lethal metabolic disease (33).

[0158] Recently, it was demonstrated that this concept of generating ectopic livers can be translated to large animals after induction of liver disease ( Figure 2B Here, a porcine model of liver disease and ectopic liver tissue growth in the lymph nodes was generated by performing a portal vein shunt followed by transplantation of hepatocytes in the mesenteric lymph nodes. Six pigs were transplanted, with increasing additional liver damage, using partial hepatectomy. The results of this experiment demonstrated hepatocyte engraftment in the lymph nodes and generation of liver tissue in all transplanted animals ( Figure 2B ), with an increased amount of ectopic liver mass. Portal shunt surgery is the main surgical procedure for patients with severe liver disease, but is now less commonly used. It relieves debilitating portal hypertension by creating a new connection between the blood vessels (portal vein to vena cava) to reduce blood flow to the liver by 75%. In addition, this significant reduction in blood flow to the liver is known to induce hepatotrophic factors and hepatocyte proliferation (41).

[0159] Currently, a similar but preferred minimally invasive procedure is the transjugular intrahepatic portosystemic shunt (TIPS) to open a new connection between the portal and hepatic veins in cirrhotic patients with critical portal hypertension. Given these promising results in the porcine model and the potential translational results for patients after TIPS, it remains to be determined how and why the lymphatic microenvironment is such a good host for transplanted cells.

[0160] Hepatocytes were also implanted in the milky spots. - / - When hepatocytes were implanted into the site in mice, it was found that in addition to the lymph nodes, hepatocytes also migrated to the papillary spots of the omentum to form liver nodules ( Figure 2C -D, Figure 3-5 ). Note that if Figure 3As shown in Figure 1, dilation of the capillary spaces and the sprouting of blood vessels in milky spots (e.g., lower right). Milky spots are small "milky" areas of lymphoid tissue (fat-associated lymphoid clusters or "FALCs") found primarily in the greater omentum. Figure 2C ). These aggregates of hematopoietic cells (lymphocytes and macrophages) and stromal cells are called secondary lymphoid organs (42, 43), analogous to lymph nodes. Not surprisingly, milky spots have also been identified as early targets in peritoneal carcinomatosis, particularly metastatic ovarian cancer.

[0161] To confirm that ectopic liver formation requires a lymphoid tissue environment, we further isolated Fah cells carrying mutations in the recombinase activating gene-2 ​​(RAG2) and the common cytokine receptor gamma chain (γc). - / - IP transplantation was performed in NOD mice ("FRGN" mice); such mice are completely alymphoid (44). Figure 6 As shown, omental milky spots and FALC are absent in FRGN mice. IP transplantation of hepatocytes resulted in poor engraftment in the omentum of FRGN mice relative to wild-type controls ( Figure 7 ), and no growth of implanted cells was observed ( Figure 8 Interestingly, the ability to form milky spots and FALCs was restored in FRGN mice by bone marrow transplantation ( Fig. 9 and 10 ), and those milky spots and FALC can support ectopic liver formation ( Fig.11 and 12 ).

[0162] 6.2. Molecular mechanisms of ectopic liver development

[0163] Hepatocyte engraftment and Fah - / - Are lymph nodes required for survival? It is reasonable to assume that hepatocytes are colonizing milky spots and lymph nodes, and it is desirable to know whether Fah cells lacking functional lymph nodes - / - To test this hypothesis, we compared lymphoblastic mice (aly / aly) with mice that underwent induced liver failure (Fah - / - ) mice. Due to point mutations in NF-κB-inducing kinase (NIK) that disrupt non-canonical NF-κB signaling in cells, aly / aly mice lack lymph nodes, Peyer's patches, and have a disorganized spleen ( Fig.13 ) In the absence of non-canonical NF-κB signaling, lymph node organogenesis does not occur normally during development (no lymph nodes), although lymphatic vessels and important milky spots are present and functional in these mice (43).

[0164] We first tested whether IP-transplanted hepatocytes would engraft in aly / aly-Fah - / - In mice ( Fig.14 The results showed that most of the hepatocytes migrated to the milky spots in the omentum and confirmed that the hepatocytes engrafted into the aly / aly-Fah - / - Mouse, with Fah - / - The situation is similar in mice ( Fig.15 ), but failed to form large nodules during the 6-week period, and after 12 weeks (the time required to rescue control Fah- / - mice), none of the transplanted mice were rescued (n=28), regardless of the initial omental implantation ( Fig.14 , with Kaplan-Meier survival plot on the far right). At autopsy, compared to Fah - / - Hepatocytes were also transplanted into aly / aly-Fah - / - mice livers (injected via spleen) and found that the mice were rescued from liver failure ( Fig.14 , with the Kaplan-Meier survival plot on the far left). This result is important because it shows that aly / aly-Fah - / - The lack of hepatocyte peritoneal outgrowth in mice is specific to this site and is a consequence of disruption of the NIK pathway due to aly(NIK) mutation ( Fig.16 ). Hepatocytes injected into the liver (splenic injection) may replace dying hepatocytes within the normal liver structure without the need for the NIK pathway.

[0165] Next, studies were performed to characterize the lymphoid environment that could serve as a host for ectopic liver formation. Fig.17A Shown is the distribution of the lymphoid marker podoplanin and the endothelial marker CD31 in lymph nodes compared to milky spots. Fig. 17B As shown, transplanted hepatocytes formed tight associations with stromal cells carrying ER-TR7. Interestingly, when omental stromal cells were transplanted by intraperitoneal injection instead of hepatocytes, they also migrated back into the milky spots in the omentum ( Fig. 17C ).like Fig.18 As shown, stromal cells were found to express NIK and lymphotoxin beta receptors; in turn, hepatocytes were positive for lymphotoxin alpha (also known as tumor necrosis factor beta) and beta (also known as tumor necrosis factor C).

[0166] Given the observation that hepatocytes express lymphotoxin beta ("LTb") and stromal cells express lymphotoxin beta receptor ("LTbR"), it was of interest to see whether hepatocytes would express lymphotoxin beta in Fah cells lacking LTbR. - / - The results are shown in Fig.19 , where Fah / LTbR was observed - / -Growth of hepatocytes in mice is less than that in Fah - / - ,LTbR + Growth of hepatocytes in mice.

[0167] In summary, IP-injected hepatocytes undergo organogenesis, complete neovascularization to support Fah - / -ectopic liver mass in mice and that the NIK pathway is a unique pathway required for this process to be completed.

[0168] In Dr. Stephen Paget's (1889) classic seed and soil theory of cancer, the seed (metastatic cancer) requires soil (fertile environment) to succeed. The results described herein demonstrate that the seed (hepatocyte) requires soil (secondary lymphoid organs) to transplant and generate liver tissue at an ectopic site. Without being limited by any particular theory, the data support a mechanism in which hepatocytes accompanied by stromal cells activate the lymphotoxin / NIK pathway to generate auxiliary livers in milky spots. The data further suggest that the regeneration of liver tissue by transplanting hepatocytes to the liver (spleen pathway) or milky spots (IP pathway) is mechanistically different. Aly / aly point mutations in NF-κB-inducing kinase (NIK) disrupt ectopic liver development but do not disrupt native liver repair. NIK has a central function in the development of secondary lymphoid organs in non-classical NF-κB signaling. NIK is a member of the mitogen-activated protein 3 (MAP3) kinase, which accumulates to detectable levels after receptor ligation. This enables NIK to phosphorylate and activate inhibitor of κB kinase α (IKKα), thereby initiating IKKα-mediated phosphorylation of p100 (see Fig.13 ).

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[0217] 7. Example 2: Bioengineering the Kidney in Secondary Lymphoid Tissue

[0218] Mouse lymph nodes (LNs), a secondary lymphoid organ (SLO), can support the maturation of mouse metanephros into nephrons with glomerular and tubular function, and LNs can also contribute to the maturation of transplanted human fetal kidneys and renal organoids generated from mouse nephron progenitor cells (NPs) or human induced pluripotent stem cells (hiPSCs). To test whether LTβR signaling, a key signaling mediator in LN development, supports ectopic organogenesis, mice bearing LN kidney transplants were treated with LTβR-Fc fusion protein. LTβR-Fc fusion protein antagonizes LTβR-mediated effects by engaging LTβR ligands (LTα and LTβ) (see Fig.21 ).like Fig.22A As shown in Figure 2B, LTβR-Fc treatment significantly reduced the size of the grafts and their vascularization.

[0219] In addition to engaging LTβR ligands, LTβR-Fc also engages the non-TNF family member LIGHT, which not only interacts with LTβR, but can also interact with HVEM and Dcr3 receptors (see Fig.21 ). To exclude the possibility that impaired graft vascularization / angiogenesis due to LTβR-Fc interferes with signal transduction pathways other than LTβR-mediated signaling pathways, the results of kidney transplantation in an LTβR-deficient environment, such as those provided by LTβR- / - mice, were studied (see Figure 22). This approach also allows to exclude the possibility that the observed effects may be due to inhibition of donor renal cells or other cell types present in the donor tissue at the time of transplantation, including stromal cells and hematopoietic cells, by LTβR / HVEM / Dcr3.

[0220] Because LTβR- / - mice have no LNs, the larger omentum was used as a surrogate SLO for transplantation. The omentum contains lymphoid aggregates, called milky spots, which promote immunity to peritoneal antigens (see Figure 1 ). As with LN, a reticular network of fibroblastic reticular cells (FRC) supports leukocytes in milky spots. When the steady-state matrix composition of the omentum was investigated in comparison to LN, a similar stromal cell flow cytometric profile was found ( Fig. 22C In addition, PDPLN+ / LTβR+ and PDPLN- / LTβR+ cell subpopulations were identified in omental cell suspensions, suggesting the presence of rare LT-responsive FRC and brain endothelial cell (BEC) populations in the omentum ( Fig.22D Growth of embryonic kidney fragments was significantly affected in the LTβR- / - omentum; not only were the grafts smaller, but they were also less vascularized and displayed abnormal morphology when compared to their controls, establishing the importance of LTβR signaling in host stromal cells for successful vascularization / angiogenesis of heterotopic kidney grafts ( Fig.22EIn untreated LN renal allografts, NIK, a downstream target of LTβR and a central component of the non-canonical NF-κB pathway, was restricted to glomerular endothelial cells ( Figure 22G ). In contrast, LTβR-Fc treatment abolished NIK expression in these cells. Similarly, NIK expression was undetectable in grafts grown in LTβR- / - omentum. Without being bound by any particular theory or mechanism, LTβR may use NIK to propagate non-canonical NF-κB signaling and promote vascularization / angiogenesis of transplanted tissues. Furthermore, without being bound by any particular theory, stromal cells residing within secondary lymphoid organs appear to use LTβR signaling to support organogenesis, and stromal endothelial cell-restricted NIK activation may promote graph angiogenesis.

[0221] 8. Example 3: Inflammation enhances the growth of transplanted hepatocytes

[0222] Peritoneal inflammation induces an increase in the number and size of fat-associated lymphoid clusters (FALCs) (Benezech, C. et al. Inflammation-induced formation of fat-associated lymphoid clusters. Nature immunology 16, 819-828 (2015)). This effect is dependent on TNF expression by myeloid cells and TNFR signaling on stromal cells (Benezech et al. (2015)). To determine whether inflammation also leads to enhanced hepatocyte engraftment, sterile peritoneal inflammation driven by zymosan (a yeast-derived Toll-like receptor 2 ligand) was initiated in wild-type C57bl / 6 and control animals (wild-type C57bl / 6 injected with PBS) (see Fig.23 ). GFP+ hepatocytes were transplanted into the two groups of animals 3 days later and the animals were killed 1 week later.

[0223] like Fig.24 As shown, zymosan-induced inflammation significantly increased the presence of GFP+ cells, as can be seen in the tissue and by quantification of GFP+ hepatocytes in omental and mesenteric fat. Importantly, engraftment of hepatocytes in the FALC following IP injection was observed in wild-type mice, indicating that engraftment in the FALC is not dependent on liver injury. It remains to be investigated how FALC formation is related to the onset or resolution of inflammation and whether hepatocyte engraftment will persist during the resolution phase.

[0224] Next, in Fah - / - This preconditioning protocol was tested in C57bl / 6 mice to determine whether it would affect the survival of tyrosinemic mice after induction of liver disease (NTBC depletion). Fig.23 The same method as described in - / - C57bl / 6 mice were transplanted with wild-type hepatocytes and then induced to have liver disease (de-NTBC). Fig.25 As shown, animals without inflammation on the left panel were not rescued after two selections (weight loss = liver disease), whereas animals with inflammation were rescued after 8 weeks.

[0225] Necropsy of animals without induced inflammation revealed low / limited liver engraftment and liver mass in FALC present in the omentum, spleen, liver hilum, gonads, and mesenteric fat ( Fig.26 ). In contrast, animals with induced inflammation had large amounts of liver tissue in these locations.

[0226] The contents of various references, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims

1. A method for generating ectopic tissue in a subject, the method comprising introducing a cell or tissue fragment into an adipose-associated lymphoid cluster of the subject, thereby promoting the formation of ectopic tissue in the adipose-associated lymphoid cluster.

2. The method according to claim 1, in, The cells or tissue fragments comprise hepatocytes, and the ectopic tissue is ectopic liver tissue.

3. The method of claim 1, wherein the subject has decreased liver function.

4. The method of claim 1, wherein the subject has Crigler-Najjar syndrome type I, acute liver failure, cirrhosis, hemochromatosis, hyperoxaluria, oxalosis, Wilson's disease, alpha-1 antitrypsin deficiency, liver cancer, hepatitis, alcoholic hepatitis, autoimmune hepatitis, fatty liver disease, and nonalcoholic fatty liver disease.

5. The method of claim 1, wherein the cells or tissue fragments comprise kidney cells and the ectopic tissue is ectopic kidney tissue.

6. The method of claim 1, wherein the cells or tissue fragments comprise cells isolated from embryonic kidney, metanephros, cells isolated from renal organoids formed in vitro, or any combination thereof.

7. The method of claim 1, wherein the subject is a human.

8. The method of claim 1, wherein the fat-associated lymphoid cluster is located in the pleural cavity of the subject.

9. The method of claim 1, wherein the cells or tissue fragments are allogeneic to the subject.

10. The method of claim 1, wherein the cells or tissue fragments are autologous to the subject.

11. The method of claim 1, wherein the fat-associated lymphoid cluster is located in the pericardial cavity of the subject.

12. The method of claim 1, wherein the fat-associated lymphoid cluster is located in the peritoneal cavity of the subject.

13. The method of claim 1, wherein the fat-associated lymph nodes are located in the omentum, mesentery, spleen, hilum and / or gonadal fat of the subject.

14. The method of claim 1, wherein the fat-associated lymphoid cluster is located in the omentum of the subject.

15. The method of claim 1, wherein the cells or tissue fragments are administered to the subject by intraperitoneal injection.

16. The method of claim 1, wherein the cells or tissue fragments are administered topically to the subject.

17. The method of claim 1, wherein the method is capable of extending the survival of the subject by at least 20%.

18. A method of generating ectopic tissue in a subject, the method comprising introducing cells into a lymph node of the subject and administering to the subject an agent that promotes the formation of the ectopic tissue.

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