Fat-associated lymphoid clusters as sites for transplantation, tissue regeneration, organ formation, and multiple tissue functions

Abstract

The present disclosure relates to the engraftment and proliferation of cells in adipose-associated lymphoid cell collections ("FALCs" or "milts"), which can be used to generate functional ectopic tissue. The present disclosure further provides methods and compositions for grafting and expanding cells in FALCs. The present disclosure also provides methods and compositions for establishing ectopic liver tissue in FALCs and using such ectopic liver tissue for therapeutic benefit, and methods and compositions for generating ectopic kidney tissue in FALCs, which can be used in a subject for therapeutic benefit. [Solution] A method for generating ectopic tissue in a subject, the method comprising introducing one or more cells into an adipose-associated lymphocyte accumulation in the subject and providing one or more agents that promote the formation of ectopic tissue.

Description

[Technical Field]

[0001] Priority information This application claims priority to U.S. Provisional Patent Application No. 62 / 460,267, filed February 17, 2017, and U.S. Provisional Patent Application No. 62 / 574,119, filed October 18, 2017, the contents of each of which are incorporated herein by reference in their entirety and to which priority is claimed.

[0002] Grant Information Not applicable.

[0003] 1. Introduction The present disclosure relates to the engraftment and proliferation of organ cells in adipose-associated lymphocyte collections ("FALCs," also known as "milky spots") to generate ectopic tissue that can be used to supplement or replace organ function in a subject. [Background technology]

[0004] 2. Background of the Invention There is a lack of effective treatments for patients with liver disease. Liver disease accounts for more than 31,000 deaths annually in the United States. Orthotopic liver transplantation (OLT) is very often a last resort and currently the only curative treatment for severe disease (1, 2). Furthermore, an estimated 100,000 patients require a new liver, yet barely more than 6,000 patients undergo liver transplantation each year. The lack of available donors is one of the major challenges faced by patients with end-stage liver disease. Elderly patients with comorbidities are not considered candidates for OLT or are predicted to have reduced post-transplant survival rates (3-5). Furthermore, transplant procedures are costly both financially and in terms of healthcare resources. For these reasons, cell-based transplantation has been proposed as an alternative treatment option or bridge to OLT as patients await available organs (6).

[0005] To date, hepatocyte transplantation has demonstrated its functional utility in animal models. From transgenic urokinase (7-9) to inducible tyrosinemia mice (10-15), hepatocyte transplantation has successfully established its therapeutic potential through complete liver regeneration. Despite these promising results, human hepatocyte transplantation remains in the experimental phase of clinical investigation (6, 16), raising hopes that positive results from animal studies can be translated into therapeutic utility for human disease.

[0006] However, for patients with end-stage liver disease, most potential cell therapies pose an additional challenge: they target cell engraftment in the diseased liver itself. Transplanted hepatocytes are typically infused via the spleen (e.g., through the splenic artery in human patients or into the splenic parenchyma in rodents) or the portal vein. The hepatocytes are expected to rapidly migrate, either actively or passively, to the diseased liver, where liver regeneration by the transplanted hepatocytes occurs. This approach may limit or even eliminate the efficacy of cell therapy in the majority of patients with severe liver disease due to the presence of cirrhosis and fibrosis, common pathological features of diseased livers (17). As a result, regeneration of the native liver remains a major challenge for these patients, i.e., most patients who require treatment.

[0007] Therefore, new therapies for patients with advanced liver disease are needed. One option that has been investigated is the engineering of an auxiliary liver (18-20). Generally, this involves implanting a heterotopically or orthotopically placed healthy liver graft while leaving all or part of the native liver intact. This approach not only has the potential to avoid OLT for certain categories of patients, but also encompasses the potential for temporary regeneration of the native liver and eventual withdrawal of immunosuppressive medications (21-24). Although early trials have shown problems (18-20, 25-27), recent favorable results have been reported and are promising in cases of acute liver failure (25), metabolic disorders (28-31), and even cirrhotic livers (26, 27). To date, the cellular and molecular mechanisms required to maintain hepatocytes and auxiliary livers stable and viable for long periods are not understood.

[0008] Several researchers have transplanted hepatocytes into various extrahepatic sites (37-40). Engraftment of hepatocytes at most extrahepatic sites has been met with variable results. It has previously been demonstrated that hepatocytes transplanted into lymph nodes in mice generated functional auxiliary livers capable of restoring liver function for long periods (>6 months) (see, e.g., U.S. Patent No. 9,125,891). These results formed the basis for a new paradigm of tissue regeneration by using lymphatic sites as in vivo bioreactors to grow tissue or organ replacements (32-36).

[0009] In addition to liver disease, kidney disease is widespread in the population, resulting in more than 47,000 deaths annually in the United States. Known therapeutic methods for treating kidney disease include rest, dietary changes, drug therapy, hemodialysis, and kidney transplantation, depending on the severity of the disease. When kidney failure is reached, dialysis treatment, such as hemodialysis, peritoneal dialysis, or kidney transplantation, is required. Hemodialysis treatment allows the body to eliminate waste products that accumulate in the body, but does not restore the function of damaged kidneys. In the case of kidney failure, patients will continue to undergo dialysis treatment for the rest of their lives unless they receive a kidney transplant. However, there are many challenges associated with kidney transplantation, including a shortage of donors, difficulties with tissue compatibility, and avoiding rejection. Therefore, new therapies for patients with impaired kidney function are needed.

[0010] Lymph nodes and adipose-associated lymphoid clusters ("FALCs," also referred to herein as "lacunae") are secondary lymphoid organs of the lymphatic system and are well vascularized. FALCs occur in several anatomical locations, including the omentum (see Figure 1). FALCs play an important role in the immune system, allowing for the extensive proliferation of white blood cells during various conditions, such as bacterial or viral infections. Interestingly, lymph nodes and lacunae also have clinical significance in cancer, as they are present at the sites of early metastatic events, i.e., tumor invasion and initial metastatic growth. Tumor metastasis in lymph nodes is commonly used for cancer staging and prognosis. Summary of the Invention

[0011] 3. Summary of the Invention The present disclosure relates to the engraftment and proliferation of cells in adipose-associated lymphoid cell collections ("FALCs" or "mammary 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 impaired liver function localize and proliferate in the FALCs (mammary spots) of the omentum, as well as in the FALCs of the mesenteric, splenic, portal, and / or gonadal fat, resulting in ectopic liver tissue that can beneficially enhance the liver function of the mice. It is also based, at least in part, on the discovery that fetal kidney cells proliferate in the FALCs of the omentum and produce ectopic kidney tissue. Furthermore, it is based, in part, on the discovery that LTβR signaling in stromal cells successfully promotes angiogenesis / angiogenesis of the grafted cells.

[0012] In certain embodiments, the present disclosure provides methods and compositions for establishing ectopic liver tissue in the FALC (fatty lining) of the omentum, and the FALC of the mesenteric, splenic, portal vein, and / or gonadal fat, and using such ectopic liver tissue for therapeutic benefit. In certain embodiments, the present disclosure further provides methods and compositions for generating ectopic liver tissue in the FALC, which can be used in a subject for therapeutic benefit.

[0013] In certain embodiments, the present disclosure further provides methods and compositions for grafting and expanding cells, e.g., hepatocytes or kidney cells, in FALC or lymph nodes by activating the lymphotoxin beta receptor (LTβR) and / or NF-κB-inducing kinase (NIK) signaling pathways.

[0014] 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 an adipose-associated lymphocyte accumulation or lymph node of a subject and providing one or more agents 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 adipose-associated lymphocyte accumulation is located in the adipose tissue of the pleural cavity and / or pericardial cavity and / or peritoneal cavity. In the peritoneal cavity, FALCs can be located in the fat of the omentum and / or mesentery and / or spleen and / or portal vein and / or gonadal glands of the subject.

[0015] In certain embodiments, the agent that promotes ectopic tissue formation comprises one or more bone marrow-derived cells and / or stromal cells. In certain embodiments, the one or more agents comprise stromal cells, e.g., 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 signaling pathway.

[0016] In certain embodiments, the agent that promotes the formation of ectopic tissue comprises an activator of the LTβR and / or NIK signaling pathway, e.g., an activator of LTβR and / or NIK. In certain embodiments, the agent that promotes the formation of ectopic tissue comprises an activator of the non-canonical NF-κB signaling pathway. In certain embodiments, activation of LTβR and / or NIK and / or activation of the non-canonical NF-κB signaling pathway promotes the proliferation of one or more cells and / or angiogenesis, resulting in the formation of ectopic tissue.

[0017] In certain embodiments, the agent that promotes ectopic tissue formation is an agent that promotes inflammation, hi certain embodiments, inflammation is induced prior to introducing one or more cells into an adipose-associated lymphoid accumulation or lymph node of a subject.

[0018] The present disclosure further provides a method for treating a subject in need of renal function enhancement. In certain embodiments, the method comprises administering a therapeutically effective amount of hepatocytes to the subject and promoting hepatocyte proliferation in the subject's adipose-associated lymphocyte accumulation or lymph node to form ectopic liver tissue. In certain embodiments, the formation of ectopic liver tissue in adipose-associated lymphocyte accumulation is promoted by locally administering hepatocytes to the anatomical region of adipose-associated lymphocyte accumulation. In certain embodiments, the adipose-associated lymphocyte accumulation is located in the fat of the omentum and / or mesenteric and / or spleen and / or portal vein and / or gonadal fat.

[0019] In certain embodiments, the formation of adipose-associated lymphoid accumulation or ectopic liver tissue in lymph nodes is promoted by co-administration of one or more bone marrow-derived cells or stromal cells. For example, but not limited to, stromal cells are co-administered. In certain embodiments, the stromal cells are fibroblasts. In certain embodiments, the stromal cells express one or more of podoplanin, NIK, and / or LTβR.

[0020] In certain embodiments, adipose-associated lymphocyte accumulation or the formation of ectopic liver tissue in lymph nodes is promoted by co-administration of an activator of the LTβR and / or NIK signaling pathway. In certain embodiments, adipose-associated lymphocyte accumulation or the formation of ectopic liver tissue in lymph nodes is promoted by co-administration of an activator of the non-canonical NF-κB signaling pathway.

[0021] In certain embodiments, the formation of ectopic liver tissue in adipose-associated lymphocyte accumulation or lymph node is promoted by inducing inflammation in the subject.In certain embodiments, inflammation is induced by administering the agent that promotes inflammation in the subject.In certain embodiments, inflammation is induced before introducing one or more cells into the adipose-associated lymphocyte accumulation or lymph node of the subject.

[0022] The present disclosure further provides a method for generating an ectopic liver, the method comprising introducing one or more hepatocytes into an adipose-associated lymphoid accumulation or lymph node and providing at least one agent that promotes the formation of ectopic liver tissue. In certain embodiments, the method is performed in vivo or in vitro. In certain embodiments, the agent 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 signaling pathway. In certain embodiments, the agent that promotes the formation of ectopic tissue comprises an activator of the LTβR and / or NIK signaling pathway. In certain embodiments, the agent that promotes the formation of ectopic tissue comprises an agent that promotes inflammation.

[0023] In certain embodiments, a method for generating ectopic liver tissue in a subject includes introducing cells comprising one or more hepatocytes and one or more stromal cells into an adipose-associated lymphocyte accumulation 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 one or more hepatocytes to form ectopic liver tissue.

[0024] In certain embodiments, the method for generating ectopic liver tissue in object comprises: introducing the cell of object's adipose-associated lymphoid accumulation or lymph node, comprising one or more hepatocytes and one or more stromal cells, and providing LTβR and / or NIK activator, wherein the activation of LTβR and / or NIK in one or more stromal cells promotes the proliferation and / or angiogenesis of one or more hepatocytes, thereby forming ectopic liver tissue.In certain embodiments, renal cell comprises the cell that is separated from embryonic kidney, metanephroi, the cell that is separated from the kidney organoid that forms in vitro, or any combination thereof.

[0025] The present disclosure further provides a method for treating a subject in need of enhanced renal function. In certain embodiments, the method comprises administering a therapeutically effective amount of renal cells (or renal tissue fragments) to the subject and promoting proliferation of renal cells in the subject's adipose-associated lymphocyte accumulation or lymph nodes to form ectopic kidney tissue. In certain embodiments, the formation of ectopic kidney tissue in adipose-associated lymphocyte accumulation is promoted by administering hepatocytes locally to the anatomical region of adipose-associated lymphocyte accumulation. In certain embodiments, the adipose-associated lymphocyte accumulation is located in the fat of the omentum and / or mesenteric and / or spleen and / or portal vein and / or gonadal fat.

[0026] In certain embodiments, adipose-associated lymphoid accumulation or formation of ectopic kidney tissue in lymph nodes is promoted by co-administration of one or more bone marrow-derived cells or stromal cells. For example, but not limited to, stromal cells are co-administered. In certain embodiments, the stromal cells are fibroblasts. In certain embodiments, the stromal cells express one or more of podoplanin, NIK, and / or LTβR.

[0027] In certain embodiments, adipose-associated lymphocyte accumulation or the formation of ectopic kidney tissue in lymph nodes is promoted by co-administration of an activator of the LTβR and / or NIK signaling pathway. In certain embodiments, adipose-associated lymphocyte accumulation or the formation of ectopic kidney tissue in lymph nodes is promoted by co-administration of an activator of the non-canonical NF-κB signaling pathway.

[0028] In certain embodiments, the formation of ectopic kidney tissue in adipose-associated lymphocyte accumulation or lymph nodes is promoted by inducing inflammation in a subject. In certain embodiments, inflammation is induced by administering an agent that promotes inflammation to the subject. In certain embodiments, inflammation is induced before introducing one or more kidney cells into the adipose-associated lymphocyte accumulation or lymph nodes of the subject.

[0029] The present disclosure further provides a method for generating an ectopic kidney, the method comprising introducing one or more renal cells (or one or more renal tissue fragments) into an adipose-associated lymphoid depot or lymph node and providing at least one agent that promotes the formation of ectopic renal tissue. In certain embodiments, the method is performed in vivo or in vitro. In certain embodiments, the agent that promotes the formation of ectopic renal 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 signaling pathway. In certain embodiments, the agent that promotes the formation of ectopic renal tissue comprises an activator of the LTβR and / or NIK signaling pathway. In certain embodiments, the agent that promotes the formation of ectopic renal tissue comprises an agent that promotes inflammation.

[0030] In certain embodiments, a method for generating ectopic renal tissue in a subject includes introducing cells comprising one or more renal cells and one or more stromal cells into an adipose-associated lymphoid depot 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 one or more renal cells to form ectopic renal tissue.

[0031] In certain embodiments, a method for generating ectopic renal tissue in a subject includes introducing cells comprising one or more renal cells and one or more stromal cells into an adipose-associated lymphoid depot 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 one or more renal cells to form ectopic renal tissue.

[0032] The present disclosure further provides a composition for generating ectopic tissue. In certain embodiments, the composition comprises a plurality of cells and one or more agents that promote the formation of ectopic tissue. In certain embodiments, the composition for generating ectopic liver tissue comprises a plurality of hepatocytes and one or more agents that promote the formation of ectopic liver. In certain embodiments, the composition for generating ectopic kidney tissue comprises a plurality of kidney cells and one or more agents that promote the formation of ectopic kidney.

[0033] In certain embodiments, the one or more agents included in the composition of the presently disclosed subject matter include 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 signaling pathway. 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: (a) a plurality of bone marrow-derived cells and / or a plurality of stromal cells, (b) an activator of the LTβR and / or NIK signaling pathway, and (c) an agent that promotes inflammation. In certain embodiments, the composition further comprises a synthetic culture medium.

[0034] In certain embodiments, a method of treating a subject in need of enhanced liver function comprises administering a therapeutically effective amount of hepatocytes to the subject 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: (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.

[0035] In certain embodiments, the present disclosure further provides a method of treating a subject in need of enhanced renal function, 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: (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.

[0036] The present disclosure further provides kits comprising one or more of the compositions disclosed herein.

[0037] 4. Brief description of the drawings [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 shows a diagram of adipose-associated lymphocyte accumulation; a schematic diagram (upper panel) and distribution (lower panel). [Figure 2-1] Figure 2A-D: Ectopic liver formation in secondary lymphoid tissue. (A) Microscopic image of a Fah- / - mouse 12 weeks after IP transplantation of hepatocytes. Yellow circles highlight the numerous ectopic liver nodules (milks and lymph nodes (LNs)) that formed in the lymphoid tissue. (B) Mesenteric lymph node of a pig 2 months after portocaval shunting, partial hepatectomy, and hepatocyte transplantation. Left panel: OCT block and frozen section from this block stained with CK18 for hepatocytes and PNAd for high endothelial venules in the LN. 51.5% of the LN mass was identified as CK18+ hepatocytes. Right panel: Frozen sections of a control liver and an ectopic liver stained with CK18 (hepatocytes) and ER-TR7 (fibroblasts). H&E shows the normal microvasculature present in the ectopic pig liver (C). Top panel: Macroscopic view of the mouse peritoneal cavity showing the larger omentum (O) (not shown) adjacent to the liver (L) and covering the stomach (S) and small intestine (I) above the pancreas. Middle panel: Mouse omentum under an operating microscope showing abundant vasculature. Bottom panel: Schematic diagram of the omentum showing the vascular tree supplying the macula. (D) IP transplantation of GFP+ hepatocytes. Ectopic nodules developed in the macula of Fah- / - mice 12 weeks after hepatocyte engraftment. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3] Photographs show enlargement of capillary spaces and sprouting of blood vessels in the mammary glands in Fah − / − mice 3 to 4 weeks after IP transplantation of hepatocytes. [Figure 4] FIG. 1 shows the mammary gland in mice. [Figure 5]This photograph shows the engraftment of transplanted hepatocytes in the mammary gland of the omentum. The hepatocytes are GFP-labeled and appear as bright areas of green fluorescence. ER-TR7 is an antigen found in the extracellular matrix of lymphoid tissue and is fluorescent blue in this image (indicated by the arrow). [Figure 6] Photographs showing that omentum mammary spots are absent in FRGN (Fah- / -Rag2 / γc- / -Nod) mice. Immunofluorescent staining of wild-type (left) and FRGN omentum, mammary spots labeled ("MS"). [Figure 7] 10 shows photographs showing engraftment of hepatocytes in the omentum of FRGN mice. [Figure 8] Photographs showing that engrafted hepatocytes in the omentum were not observed to grow in FRGN mice over a 12-week period. [Figure 9] Photographs showing that mammary glands are restored in the omentum of FRGN mice by bone marrow transplantation. [Figure 10] Photographs showing that mammary glands are restored in the omentum of FRGN mice by bone marrow transplantation. Note that the arrows in the right panel indicate engrafted hepatocytes in the mammary glands. [Figure 11] FIG. 1 shows the formation of ectopic liver in FRGN mice after bone marrow transplantation. [Figure 12] Photographs showing the formation of ectopic livers in FRGN mice after bone marrow transplantation. The left panel shows the results without bone marrow transplantation - no ectopic liver formation, but hepatocyte aggregation is visible. The right panel shows the results with bone marrow transplantation - ectopic liver formation. [Figure 13] FIG. 1 shows stromal cell / lymphoid tissue inducer interactions (left) and the NIK signaling pathway (right). [Figure 14]Schematic of a study to test the impact of NIK function in rescuing liver function by intraperitoneal (IP) versus spleen (SP) injection in NIK-deficient (aly / aly) and control Fah- / - mice. Kaplan-Meier survival curves for Fah- / - and aly / aly Fah- / - mice transplanted with 106 hepatocytes by spleen (SP) injection (left) or IP injection (right). [Figure 15] Photographs showing that NIK function is required to rescue Fah- / - mice by IP injection. 106 GFP+ hepatocytes were inoculated into Fah- / - mice and Fah- / - mice with lymphoid dysplasia (aly / aly Fah- / -) by IP injection, respectively. Whole-mount images of the omental milky spot at various time points. Bright-field images merged with the fluorescence (green) of donor hepatocytes are shown. Upper panel, Fah- / - mouse; lower panel, aly / aly Fah- / - mouse. [Figure 16] FIG. 1 is a schematic diagram of the results observed after IP hepatocyte transplantation. [Figure 17-1] Figure 17A-D: (A) Distribution of podoplanin (PDPLN) and CD31 in reticular fibroblasts (FRCs), lymphatic endothelial cells (LECs), and brain endothelial cells (BECs), and flow cytometry analysis of these markers in lymph nodes compared to mammary glands. (B) Photographs showing that hepatocytes establish themselves adjacent to ER-TR7 stromal cells in mammary glands. (C) Photographs showing that isolated stromal cells, transplanted IPs, will migrate back into the mammary glands of the omentum. (D) Photographs showing mammary glands containing transplanted hepatocytes. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18] 1 is a photograph showing immunofluorescence studies demonstrating that hepatocytes are lymphotoxin alpha and beta positive and interstitial cells are NIK and lymphotoxin beta receptor positive. [Figure 19] Photographs showing wild-type GFP+ (green fluorescent protein) hepatocytes transplanted intraperitoneally into either Fah- / - or Fah / LTbR- / - mice after 2, 4, and 6 weeks, demonstrating engraftment in the omentum of these animals. [Figure 20-1] Figure 20A-G: (A) Top, from left to right, photographs show images of a human fetal kidney, a jejunal LN after kidney fragment transplantation, and an LN 3 weeks after transplantation. Middle left, H&E-stained section of a paraffin-embedded donor human fetal kidney. Middle right, hematoxylin-stained frozen section of LN 3 3 weeks after kidney transplantation. The orange dashed box indicates the non-engrafted LN area. Bottom, insets show immature (left) or mature (right) glomeruli. (B) Photographs show representative immunofluorescence staining of 3-week-old graft 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 an 8-week-old graft. Left, images of engrafted glomeruli and tubules within the LN. (Right) Images of glomeruli and tubules only. (D) Photograph showing accumulation of 10,000 kDa MW Texas Red Dextran in 11-week-old transplant-bearing mice. (E) Photograph showing representative immunofluorescent staining of mouse and human CD31 sections from 1-, 3-, and 8-week-old transplants. (F) Left) Photograph showing representative immunofluorescent staining of NCAM, SIX2, WT-1, or E-CADH in NP organoids from mice before transplantation. Right) Photograph showing representative immunofluorescent staining of PDPLN / CD31, PDPLN / CD105, or PDPLN / Ly-76 showing angiogenic glomerular-like structures in LNs 8 weeks after NP organoid transplantation. (G) Left) Photograph showing representative whole-mount immunofluorescent staining of iPSC-derived kidney organoids for DBA / BRN1 and E-CADH / LTL. Bottom: Serial sections showing two distinct nephron-like structures, each composed of a glomerular-like structure containing putative glomerular epithelial cells (PDPLN, SYNPO, PODXL), mesangial cells (CIV), and endothelial cells (CD31), adjacent to LTL / megalin-reactive proximal-like tubules, 6 weeks after organoid transplantation into the LN. Nuclei were counterstained with Hoechst (blue). [Figure 20-2] This is a continuation of Figure 20-1. [Figure 20-3] This is a continuation of Figure 20-2. [Figure 20-4] This is a continuation of Figure 20-3. [Figure 21] FIG. 1 is a schematic diagram of the canonical and non-canonical NF-κB signaling pathways. [Figure 22-1] Figure 22A-G: (A) Left, schematic of the experimental design. Mice were IP injected with 100 μg of LTβR-Fc or control Ig 2 days before receiving transplants of GFP+ embryonic kidney fragments into their LNs (day 1). Mice were treated again on days 8 and 15. LNs were harvested on day 22. Right, whole tissue specimens of kidneys bearing LNs isolated from treated or untreated mice. (B) Representative immunofluorescent staining of serial sections of the kidney graft in A for PDPLN, LTL, CD31, CD105, and Ly-76. (C) Flow cytometry profiles of reticular fibroblasts (FRCs), lymphatic endothelial cells (LECs), and vascular endothelial cells (BECs) in LNs or the omental stromal cell population (CD45-). (D) Representative immunofluorescent staining of omental stromal cells for PDPLN and LTβR. (E) Photographs showing whole tissue preparations of omentum-bearing kidneys from wild-type or LTβR- / - mice 6 weeks after transplantation of a GFP+ embryonic kidney (merged fluorescent / bright-field image on the left and surgical scope image on the right). The new kidney is shown in comparison to an adult mouse kidney. (F) Photographs showing representative immunofluorescent staining for CD31, CD105, and Ly-76 of a contiguous section of the interomental kidney graft from E. (G) Photographs showing representative immunofluorescent staining for ERTR-7, LTα, LTβ, LTβR, NIK, and NIK / CD34 of a section of an LN graft from a GFP+ (left and middle) or wild-type (right) embryonic kidney. Nuclei were counterstained using Hoechst (blue). [Figure 22-2] This is a continuation of Figure 22-1. [Figure 22-3] This is a continuation of Figure 22-2. [Figure 22-4] This is a continuation of Figure 22-3. [Figure 22-5] This is a continuation of Figure 22-4. [Figure 23]Figure 1 shows that peritoneal inflammation induces dramatic changes in the omentum. Top panel: Schematic of the experiment. On day 0, zymosan is injected intraperitoneally (IP). Three days later, one million hepatocytes are injected IP. Samples are collected 7 days after hepatocyte injection. Bottom left panel: Macroscopic view of omentum from zymosan-induced and control animals. Bottom right panel: Dramatic increase in omentum weight after zymosan-induced inflammation compared to control. [Figure 24] Figure 1 shows FALC and liver growth. Omental and mesenteric fat in C57BL / 6 mice after zymosan or PBS injection one week after GFP+ hepatocyte transplantation. The number and size of FALC after zymosan-induced inflammation dramatically increased GFP+ hepatocyte engraftment. Omental and mesenteric fat were compared, and GFP+ hepatocytes were identified under a fluorescent microscope. [Figure 25] Figure 1 shows that peritoneal inflammation increases the survival rate of tyrosinemic mice after hepatocyte transplantation. Wild-type hepatocytes were IP transplanted into Fah- / - C57bl / 6 mice with or without previous inflammation induction, followed by two rounds of sorting (without NTBC). Animals without inflammation (left panel) continued to lose weight after the second round of sorting, and animals H146 and H148 died around / 8 weeks after transplantation. Animals with previous inflammation induction (right panel) gained weight during the second round of sorting, demonstrating rescue of liver disease with restoration of functional liver mass. [Figure 26] Photographs showing that peritoneal inflammation increases liver mass present in the fat containing FALC. Macroscopic images of intraperitoneal fat containing liver tissue from Fah- / - C57bl / 6 mice without (H146) and with (H150) previously induced inflammation. Inflammation supports ectopic liver growth in FALC. DETAILED DESCRIPTION OF THE INVENTION

[0039] 5. Detailed Description of the Invention For purposes of clarity, and not by way of limitation, the detailed description of the subject matter of the present disclosure is divided into the following subsections: (i) Definition; (ii) treatment methods; a. Treatment of liver disease and liver damage; b. Treatment of kidney disease and disorders; and (iii) Composition.

[0040] 5.1 Definition Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art.

[0041] 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 varies depending on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more standard deviations, as is common in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within the same order of magnitude of a value, preferably within 5-fold, and more preferably within 2-fold.

[0042] Adipose-associated lymphocyte collections ("FALCs," or alternatively referred to herein as "milk spots"), as used herein, are lymphoid structures that are not lymph nodes. FALCs are present in several anatomical locations, including, but not limited to, the mesentery, mediastinum, pericardium, and subcutaneous tissue (see FIG. 1). FALCs are located in the adipose tissue of the pleural, pericardial, and peritoneal cavities. In the peritoneal cavity, FALCs are located in the fat of the omentum and mesentery, spleen, portal vein, and gonads of a subject. In certain non-limiting embodiments, FALCs used for ectopic tissue growth, e.g., liver or kidney tissue growth, are located in the omentum. In certain non-limiting embodiments, FALCs are located in the mediastinum or pericardium.

[0043] "Lymph node" or "LN," as used herein, refers to any lymph node, including, 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), gastric omentum (omentum) lymph nodes (left and right), retroperitoneal lymph nodes, pyloric lymph nodes (suprapyloric, infrapyloric, retropyloric), pancreatic lymph nodes (superior pancreatic, inferior pancreatic, splenic linear lymph nodes), hepatic lymph nodes (gallbladder, foramen including foramen of Winslow), pancreaticoduodenal lymph nodes (superior pancreaticoduodenal, inferior pancreaticoduodenal), superior pancreaticoduodenal lymph nodes, inferior pancreaticoduodenal lymph nodes, superior pancreaticoduodenal lymph nodes, hepatic lymph nodes (gallbladder, foramen including foramen of Winslow), duodenal pancreatic lymph nodes (superior duodenal, inferior duodenal), superior pancreaticoduodenal lymph nodes, inferior pancreaticoduodenal lymph nodes, ... This refers to the mesenteric lymph nodes, ileocolic lymph nodes, prececal lymph nodes, retrocecal lymph nodes, appendicular lymph nodes, mesocolic lymph nodes (paracolic, left colon, mesocolic, right colon, inferior mesenteric lymph nodes, sigmoid, superior rectal), common iliac lymph nodes (promontory medial common iliac, middle common iliac, lateral common iliac, aorticized common iliac, common iliac lymph nodes), and external iliac lymph nodes (medial external iliac, middle external iliac, lateral external iliac, medial fossa-femoral, middle fossa-femoral, lateral fossa-femoral, interiliac external iliac, obturator external iliac), jejunal, popliteal, and axillary lymph nodes.

[0044] Cells of an organ may be composed of one or more cell types. In certain embodiments, the cells are composed of multiple cell types found in the organ of origin, but do not necessarily include all cell types found in that organ. As a non-limiting example, "liver cells" for engraftment according to the present disclosure include hepatocytes and may further include one or more bile cells, endothelial cells, stem cells, and progenitor cells of the liver. As another non-limiting example, "renal cells" for engraftment according to the present disclosure include renal parenchymal cells and may further include one or more glomerular cells, endothelial cells, stem cells, and progenitor cells of the kidney. In certain embodiments, the cells are dissociated before introduction for engraftment. In certain embodiments, the cells are contained in aggregates before introduction. In certain embodiments, the cells are composed of organoids grown in vitro. In certain embodiments, the cells are composed of iPS or other stem cells grown in vitro. In certain embodiments, the cells are contained in a tissue fragment obtained from an intact organ, wherein the fragment comprises less than about 5%, or less than about 10%, or less than about 25%, or less than about 50% of the organ.

[0045] "Activator," as used herein, refers to a compound or molecule (e.g., a small molecule, peptide, peptidomimetic, natural compound, or 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 named protein (molecule, any molecule associated with the named molecule, or a named related molecule), such as LTβR or NIK, or promotes the interaction of the named protein, e.g., LTβR or NIK, with a signaling or binding partner. Activators can also include molecules that indirectly regulate the biological activity of a named protein, e.g., LTβR or NIK, by interfering with an upstream signaling molecule. In certain embodiments, activators can include molecules that promote, increase, and / or enhance the production and / or generation of a named protein, such as LTβR or NIK, for example, by increasing the activity and / or function of an enzyme that produces and / or produces LTβR or NIK.

[0046] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the treated individual and can be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or palliating the disease state and remission or improving prognosis. In certain embodiments, "treatment" may refer to a reduction in the severity of complications, symptoms, and / or exacerbations. For example, and without limitation, a reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% reduction in the severity of complications, symptoms, and / or exacerbations, e.g., relative to a control subject not receiving treatment. In certain embodiments, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0047] A "therapeutically effective amount" or "effective amount," as used herein, refers to an amount that can achieve one or more of the following: alleviation of symptoms, slowing the rate of disease progression, prolonging survival, improving or alleviating the disease state, and / or improving prognosis.

[0048] "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.

[0049] The terms or phrases "transplantation," "cell replacement," or "grafting" are used interchangeably herein and refer to the introduction of cells into a target tissue, e.g., FALC or a tissue containing FALC.

[0050] "In combination with," as used herein, can mean that one or more cells or compositions thereof to be grafted and an agent, e.g., an activator of the LTβR and / or NIK signaling pathway, are administered to a subject as part of a treatment regimen or plan. In certain embodiments, used in combination does not require that the one or more cells and the one or more agents be physically combined prior to administration or that they be administered over the same time frame. For example, and without limitation, the one or more cells to be grafted and the one or more agents may be administered to a subject to be treated simultaneously, or may be administered sequentially at the same time or in any order or at different times. In certain embodiments, the methods of the present disclosure can include administration of one or more agents prior to administration of one or more cells to be grafted, e.g., hepatic or renal cells.

[0051] 5.2 Treatment Methods In certain embodiments, the present disclosure relates to the engraftment and proliferation of cells in adipose-associated lymphoid cell collections ("FALCs" or "milt areas") to generate ectopic tissue.

[0052] The present disclosure provides methods for generating ectopic tissue within or around a FALC. In certain embodiments, the methods for generating ectopic tissue may include introducing cells, such as hepatocytes or renal cells, into a subject's FALC to form ectopic tissue, such as ectopic liver tissue or ectopic kidney tissue. In certain embodiments, the methods for generating ectopic tissue may include introducing cells into a lymph node of the subject. In certain embodiments, the methods of the present disclosure can be used to treat medical conditions and / or disorders (e.g., pathologies, diseases, symptoms) in which the generation of ectopic tissue may be beneficial. For example, but not limited to, the subject may be suffering from a disease or disorder, such as, but not limited to, liver disease and / or liver failure or kidney disease and / or kidney failure.

[0053] In certain embodiments, the subject may 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.

[0054] In certain embodiments, the cells grafted into the FALC of a subject may be human cells, non-human cells, or both. In certain embodiments, the cells are human cells. In certain embodiments, human cells may be grafted into the FALC, or a region containing FALC, of ​​a non-human subject, e.g., a mouse.

[0055] Cells for engraftment can be obtained from the subject receiving the graft. In certain embodiments, cells can be obtained from a source other than the subject receiving the graft. In certain embodiments, cells can be obtained from fresh or frozen cell populations. In certain embodiments, cells can be isolated from tissue and grown in vitro under various culture conditions prior to transplantation. In certain embodiments, cells can be obtained from embryonic, fetal, pediatric, or adult tissue. In certain embodiments, cells can be progenitor or precursor cells. In certain embodiments, progenitor or precursor cells can be differentiated into the cell type to be transplanted.

[0056] Cells for use according to the present disclosure can be prepared by any means known in the art. In certain embodiments, cell solutions, such as compositions disclosed herein, are used for injection or other purposes. Non-limiting examples of compositions for use in the methods of the present disclosure are described in Section 5.3 below. In certain embodiments, cells can be prepared according to the method of Li et al., J Tissue Culture Methods 14:139-146 (1992). Briefly, isolation involves collagenase perfusion of a sample of limited size (less than 50 g) with only one cut surface. In certain embodiments, enriched cell populations, such as those disclosed in U.S. Pat. No. 7,211,404, can be used.

[0057] Cells can be administered to a subject by any method known in the art. In certain embodiments, cells can be injected by, but not limited to, intraperitoneal, intravenous, or intra-arterial injection or by local instillation. In certain embodiments, cells can be surgically engrafted, for example, by conventional or endoscopic surgical techniques. In certain embodiments, cells can be administered locally. For example, but not limited to, cells can be introduced near the FALC, for example, in anatomical locations including, but not limited to, the gonads, omentum, mesentery, mediastinum, pericardial, 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) can be introduced near the omentum (and the FALC present therein), for example, by targeted intraperitoneal injection or by conventional or endoscopic surgical techniques. In certain embodiments, the engrafted cells can first be grown in culture before being introduced into the FALC or lymph nodes in vivo.

[0058] In certain embodiments, the cells can be suspended in any suitable buffer for injection, non-limiting examples of which include saline, phosphate-buffered saline, Hank's salts, and Ringer's solution, among others.

[0059] In certain embodiments, the method for generating ectopic tissue in or around the FALC or lymph nodes may further include providing an agent that promotes the formation of ectopic tissue. For example, without limitation, the method may include administering one or more cells to be grafted in combination with one or more agents that promote the formation of ectopic tissue. In certain embodiments, the one or more agents may be included in a composition containing the one or more cells to be grafted. Alternatively and / or additionally, the one or more agents may be administered before, after, or simultaneously with grafting the one or more cells. In certain embodiments, the agent promotes proliferation and / or angiogenesis of one or more cells to form ectopic tissue. Similar methods may be used to generate ectopic tissue in lymph nodes.

[0060] In certain embodiments, the agent that promotes the formation of ectopic tissue may be a cell type different from the cell type of the cells that form the ectopic tissue, e.g., kidney cells or hepatocytes. In certain embodiments, the agent comprises one or more bone marrow-derived cells and / or stromal cells. In certain embodiments, the agent comprises stromal cells, e.g., fibroblasts, reticular fibroblasts (FRCs), follicular dendritic cells (FDCs), lymphatic endothelial cells (LECs), vascular endothelial cells (BECs), alpha 7 integrin pericytes (AIPs), and double-negative cells (DNCs). In certain embodiments, the fibroblasts may be human foreskin fibroblasts, human embryonic fibroblasts, mouse embryonic fibroblasts, skin fibroblasts, vascular fibroblasts, myofibroblasts, smooth muscle cells, mesenchymal stem cell (MSC)-derived fibroblasts, or combinations thereof. For example, without limitation, a method for generating ectopic tissue may include: (a) introducing one or more cells, e.g., hepatocytes or kidney cells, into a subject's FALC (or an anatomical region containing FALC) or lymph node (or an anatomical region containing lymph node) to form ectopic tissue, e.g., 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 the ectopic tissue, e.g., by promoting proliferation and / or angiogenesis of the one or more cells to form the ectopic tissue. In certain embodiments, the one or more cells to be grafted and the one or more bone marrow-derived cells and / or stromal cells may be included in the same composition.

[0061] In certain non-limiting embodiments, stromal cells, e.g., stromal endothelial cells or fibroblasts, that promote the formation of ectopic tissue can express detectable levels of one or more of podoplanin, lymphotoxin beta receptor ("LTβR"), NIK, or a combination thereof. Alternatively and / or additionally, stromal cells can be treated with an activator of the LTβR and / or NIK signaling pathway, e.g., an activator of LTβR or NIK or a downstream target thereof, before, during, or after administration to a subject. In certain embodiments, stromal cells, e.g., stromal endothelial cells or fibroblasts, can be treated with an activator of NIK. In certain embodiments, stromal cells generally can be modified to exogenously express LTβR and / or NIK and / or overexpress LTβR and / or NIK.

[0062] In certain embodiments, an agent that promotes the formation of ectopic tissue, for example, by promoting the proliferation and / or angiogenesis of one or more cells to form ectopic tissue, may be an activator of the LTβR and / or NIK signaling pathway. For example, but not limited to, a method for generating ectopic tissue in or around FALC or lymph nodes may include introducing cells, such as hepatocytes or kidney cells, into a subject's FALC (or an anatomical region containing FALC) or lymph node, and providing at least one activator of the LTβR and / or NIK signaling pathway, wherein activation of LTβR and / or NIK or their 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 signaling pathway is an activator of LTβR and / or NIK or an activator of the non-canonical NF-κB signaling pathway. In certain embodiments, the one or more cells to be grafted and the activator of the LTβR and / or NIK signaling pathway may be included in the same composition, or the activator is administered before, during, or after the introduction of the one or more cells to be grafted into the subject.

[0063] In certain embodiments, activators of the LTβR and / or NIK signaling pathway present in a subject receiving a transplant can increase expression of LTβR or NIK in cells, such as stromal cells or stromal endothelial cells. For example, but not limited to, activators of the LTβR and / or NIK signaling pathway activate signaling pathways in stromal cells present in the subject's FALC.

[0064] Alternatively and / or additionally, the cells to be grafted may be a heterogeneous cell population comprising stromal cells or stromal endothelial cells, e.g., a composition comprising a heterogeneous cell population, 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 transplanted composition. For example, without limitation, a method for generating ectopic tissue may include (a) introducing one or more cells, e.g., hepatocytes or renal cells, into a subject's FALC or lymph node to form ectopic tissue, e.g., 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 grafted 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.

[0065] In certain embodiments, the activator of the LTβR and / or NIK signaling 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, such as an activator of the non-canonical NF-κB signaling pathway. In certain embodiments, the activator of the LTβR and / or NIK signaling pathway may be an agonistic antibody (or antibody fragment thereof) or single-chain antibody that specifically binds to LTβR or NIK. Non-limiting examples of anti-LTβR agonist antibodies include the monoclonal antibody manufactured 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)). Further non-limiting examples of anti-LTβR agonist antibodies are disclosed in U.S. Patent Application Publication No. 2002 / 0090366, the contents of which are incorporated herein in their entirety. In certain embodiments, an activator of the LTβR and / or NIK signaling pathway may be a small molecule that increases the function and / or activity of LTβR or NIK or its downstream targets.

[0066] In certain embodiments, the present disclosure provides a method for generating ectopic tissue in a subject, the method comprising introducing one or more cells into the subject's FALC or lymph node and providing an activator of LTβR and / or NIK, wherein activation of LTβR and / or NIK promotes proliferation and / or angiogenesis of the one or more cells to generate ectopic tissue. 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 LTβR and / or NIK signaling pathways in the stromal cells are activated by the activator of LTβR and / or NIK.

[0067] In certain embodiments, the agent that promotes the formation of ectopic tissue may be an agent that promotes inflammation in a subject. For example, without limitation, the method of generating ectopic tissue in or around a FALC or lymph node may further include inducing inflammation in a subject, e.g., by administering an agent that promotes inflammation in the 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-alpha, IFN-alpha2, IFN-beta, or IFN-gamma, TNF-alpha and TNF-beta, TGF-alpha and TGF-beta, lymphotoxin-alpha and lymphotoxin-beta. 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 to and / or around the FALC into which the cells have been or will be grafted. In certain embodiments, inflammation is induced locally, for example, in or near the anatomical region containing the FALC or lymph node into which the cells have been or will be transplanted. Alternatively and / or additionally, inflammation is induced systemically.

[0068] In certain embodiments, the inflammation is temporary, for example, but not limited to, the inflammation lasts for about 10 weeks or less, about 9 weeks or less, about 8 weeks or less, about 7 weeks or less, about 6 weeks or less, about 5 weeks or less, about 4 weeks or less, about 3 weeks or less, about 2 weeks or less, about 1 week or less, about 6 days or less, about 5 days or less, about 4 days or less, or about 3 days or less.

[0069] In certain embodiments, inflammation is induced prior to the introduction of the one or more cells to be grafted. For example, and without limitation, 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, 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 prior to the introduction of the one or more cells to be grafted.

[0070] Alternatively and / or additionally, inflammation is induced after introduction of the one or more grafted cells. For example, and without limitation, 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 introduction of the one or more grafted cells.

[0071] In certain non-limiting embodiments, a recipient of a cell, e.g., hepatic or renal cell, transplant may further be administered an immunosuppressant to minimize immune rejection of the grafted cells, e.g., hepatic or renal cells. The immunosuppressant can reduce or prevent an adverse immune response in the recipient mammal against the foreign or grafted tissue by inhibiting or suppressing any innate immune system activity, including, but not limited to, T cell and / or B cell activity. Administration of an immunosuppressant includes, but is not limited to, administration of radiation therapy and / or administration of immunosuppressive drugs. Examples of suitable immunosuppressive drugs include, but are not limited to, steroids (e.g., corticosteroids, dexamethasone, and prednisone), Cox-1 and Cox-2 inhibitors, macrolide antibiotics (e.g., rapamycin and tacrolimus), and other substances that limit, reduce, or suppress B cell, T cell, and / or other innate immune activity. In certain embodiments, immunosuppressants particularly suitable for use in connection with the present disclosure include immunosuppressants known for use in connection with liver and / or kidney transplantation, including, but not limited to, steroids, cyclosporine, rapamycin, azathioprine, prednisone, and OKT3. In certain embodiments, hepatocyte transplant recipients may be administered the immunosuppressant tacrolimus, also known as FK506, which inhibits IL-2 and downstream B lymphocyte activity.

[0072] In certain non-limiting embodiments, the subject is not administered an immunosuppressant, such as an agent described above in this paragraph (for example, but not limited to, when autologous liver or kidney cells are transplanted).

[0073] 5.2.1 Treatment of Liver Disease and Liver Injury The present disclosure provides a method for treating a subject who needs to enhance liver function. In certain embodiments, the present disclosure provides a method for propagating hepatocytes to FALC or lymph nodes to produce ectopic liver tissue, which can be used for therapeutic benefit in a subject, for example, in a subject with reduced liver function. In certain embodiments, FALC is located in the omentum.

[0074] A subject in need of enhanced liver function is one who lacks a functional liver sufficient to maintain a healthy state, including, but not limited to, a subject with fibrotic liver and / or cirrhosis, or a liver damaged by disease, trauma, or toxic effects. For example, a healthy state is evidenced by one or more liver function parameters within the normal range for the subject. Non-limiting examples of liver function parameters include albumin level, alanine transaminase ("ALT") level, aspartate transaminase ("AST") level, creatinine level, total bilirubin level, direct bilirubin level, phenylalanine level, alanine level, glycine level, valine level, glutamic acid level, prothrombin time, lactate dehydrogenase, and alkaline phosphatase, the normal ranges of which are known in the art for various subjects, including human subjects. In certain non-limiting embodiments, an increase of at least about 25% or at least about 50% relative to normal levels of one or more, two or more, or three or more of the above parameters indicates the need for enhanced liver function. Achieving enhanced liver function in a subject means shifting at least one liver function parameter into the normal range, for example, but not limited to, an improvement of at least about 10 percent, or at least about 20 percent, or at least about 30 percent, or at least about 40 percent, or at least about 50 percent.

[0075] In certain non-limiting embodiments, enhanced liver function is indicated by an increase in subject survival, for example, by at least about 20% or at least about 30%.

[0076] In certain embodiments, the subject who needs to enhance liver function may suffer from liver disease, liver damage, liver failure and / or decreased liver function.Non-limiting examples of liver diseases and / or liver damage that can be treated by the method of the present disclosure include metabolic disorders, Criglan-Nager syndrome type I, acute liver failure, cirrhosis, hemochromatosis, hyperoxaluria, oxalosis, Wilson's disease, alpha-1 antitrypsin deficiency, liver cancer, hepatitis (alcoholic and autoimmune), fatty liver and non-alcoholic fatty liver.Non-limiting examples of diseases and / or disorders related to the biliary system (and indirectly to the liver) that can be treated by the method of the present disclosure include primary biliary cirrhosis and primary sclerosing cholangitis.

[0077] In certain non-limiting embodiments, the present disclosure provides a method of 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 around the FALC or lymph nodes, producing ectopic liver tissue, thereby enhancing the subject's liver function.

[0078] In certain embodiments, the hepatocytes transplanted into a subject may be human hepatocytes, non-human hepatocytes, or both. The hepatocytes may be syngeneic or allogeneic to the intended recipient. In non-limiting embodiments, the hepatocytes are autologous to the intended recipient (e.g., harvested and expanded in culture prior to transplantation, or generated from the intended recipient's progenitor cells or hepatocytes). In certain non-limiting embodiments, hepatocytes of one species may be transplanted into another species, for example, but not limited to, human hepatocytes may be transplanted into a non-human (e.g., mouse) host. For example, but not limited to, non-human hepatocytes can be grafted into the FALCs of a human recipient.

[0079] The effective or therapeutically effective amount of hepatocytes to be grafted is about 10 4 From 10 11 Between 10 and 20 hepatocytes, or approximately 10 5 From 10 10In certain embodiments, the effective amount of hepatocytes may comprise one or more hepatocytes. In certain embodiments, the effective amount of hepatocytes may comprise between about 10 and 20 hepatocytes. 5 from about 10 11 pieces, about 10 5 from about 10 9 pieces, about 10 5 from about 10 8 pieces, about 10 5 from about 10 7 pieces, about 10 5 from about 10 6 pieces, about 10 6 from about 10 11 pieces, about 10 6 from about 10 10 pieces, about 10 6 from about 10 9 pieces, about 10 6 from about 10 8 pieces, about 10 6 from about 10 7 pieces, about 10 7 from about 10 11 pieces, about 10 7 from about 10 10 pieces, about 10 7 from about 10 9 Pieces or about 10 7 from about 10 8 Hepatocytes can be administered.

[0080] In certain embodiments, hepatocytes can be administered to a subject more than once to achieve the desired ectopic liver tissue. For example, but not limited to, hepatocytes can be administered to a subject at least twice, at least three times, or at least four times. Alternatively, or in addition, hepatocytes can be grafted into two or more different anatomical regions containing FALC or lymph nodes to obtain two or more ectopic liver tissues.

[0081] In certain non-limiting embodiments, the present disclosure provides a method of treating a subject in need of enhanced liver function, comprising administering a therapeutically effective amount of hepatocytes to the subject and promoting proliferation of at least one of the hepatocytes in the subject's FALC. In certain embodiments, such proliferation can be promoted, for example, but not limited to, by locally administering hepatocytes to an anatomical region containing the FALC targeted for ectopic liver tissue formation, and / or by providing at least one agent that promotes ectopic liver tissue formation, as disclosed above. In certain embodiments, the agent may be, for example, but not limited to, a plurality of bone marrow-derived cells and / or a plurality of stromal cells. In certain embodiments, the agent may be an activator of the LTβR and / or NIK signaling pathway, as described above, for example, an activator of LTβR or NIK or its downstream target. In certain embodiments, the agent may be bone marrow-derived cells, stromal cells, an activator of the LTβR and / or NIK signaling pathway, or a combination thereof. Similar methods may be used to generate ectopic tissue in lymph nodes to enhance liver function.

[0082] In certain non-limiting embodiments, the present disclosure provides a method for generating ectopic liver tissue, the method comprising introducing hepatocytes into a FALC and providing at least one agent that promotes the formation of ectopic liver tissue, e.g., as described above. For example, without limitation, a method for treating a subject in need of enhanced liver function comprises (a) administering to the subject a therapeutically effective amount of hepatocytes into the subject's FALC to form ectopic liver tissue, and (b) administering to the subject one or more bone marrow-derived cells and / or stromal cells to promote the formation of ectopic liver, e.g., by promoting the proliferation and / or angiogenesis of one or more cells to form ectopic liver tissue. Similar methods may be used to generate ectopic tissue in lymph nodes.

[0083] In certain non-limiting embodiments, a method for treating a subject in need of enhanced liver function can include (a) administering to the subject a therapeutically effective amount of hepatocytes into the subject's FALC to form ectopic liver tissue; (b) administering to the subject an activator of the LTβR and / or NIK signaling pathway to promote the formation of ectopic liver tissue. Similar methods can also be used to generate ectopic tissue in lymph nodes to enhance liver function.

[0084] In certain non-limiting embodiments, a method for treating a subject in need of enhanced liver function can include: (a) administering to the subject a therapeutically effective amount of hepatocytes into the subject's FALC to form ectopic liver tissue; (b) administering to the subject one or more bone marrow-derived cells and / or stromal cells to promote the formation of ectopic liver, for example, by promoting the 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 also be used to generate ectopic tissue in lymph nodes to enhance liver function.

[0085] In certain embodiments, the method for treating the subject who needs to enhance liver function can further comprise inducing inflammation in the subject.For example, but not limited to, as disclosed above, inflammation can be induced by administering to the subject an agent that promotes inflammation.In certain embodiments, inflammation is induced before introducing one or more hepatocytes to be transplanted, as described above.

[0086] In certain embodiments, the method of the present disclosure is carried out in vivo, as described above.In certain embodiments, the method of the present disclosure is carried out in vitro / ex vivo, for example, as a cultured tissue explant.In certain non-limiting embodiments, the subject of the present disclosure provides liver tissue produced by such in vitro / ex vivo method, for example, for subsequent transplantation into a subject in need of enhanced liver function.

[0087] 5.2.2 Treatment of Renal Disease and Impairment The present disclosure provides a method for treating a subject in need of enhanced renal function. In certain embodiments, the present disclosure provides a method for propagating renal cells or renal tissue fragments to FALC or lymph nodes to produce ectopic renal tissue that can be used for therapeutic benefit in a subject, for example, in a subject with reduced renal function. In certain embodiments, the FALC is located in the omentum.

[0088] A subject in need of enhanced renal function is one who lacks sufficient functional kidneys to maintain a healthy state, including, but not limited to, a subject with renal failure or kidneys damaged by disease, trauma, or toxic effects. For example, and without limitation, the healthy state is evidenced by one or more renal function parameters within the normal range for the subject. Non-limiting examples of renal function parameters include creatinine levels, protein levels, and albumin levels, and the normal ranges for these levels are known in the art for various subjects, including human subjects. In certain non-limiting embodiments, an increase of at least about 25% or at least about 50% relative to normal levels in one or more, two or more, or three or more of the above parameters indicates the need for enhanced renal function. Achieving enhanced renal function in a subject refers to the transition of at least one renal function parameter into the normal range, for example, but not limited to, an improvement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In certain embodiments, the subject's glomerular filtration rate (GFR) is an indicator of declining renal function. For example, but not by way of limitation, a decrease in a subject's GFR of about 10%, about 20%, about 30%, about 40%, or about 50% is indicative of decreased renal function. In certain embodiments, the engrafted cells are capable of producing concentrated urine.

[0089] In certain non-limiting embodiments, enhanced renal function is indicated by an increase in subject survival, for example, by at least about 20% or at least about 30%.

[0090] In certain embodiments, a subject in need of enhanced renal function may suffer from renal disease, renal impairment, renal failure, and / or reduced renal function. Non-limiting examples of renal diseases and / or renal impairments that can be treated by the methods of the present disclosure include acute renal failure, chronic renal disease, glomerulonephritis, lupus, polycystic kidney disease, nephropathy, nephrosis, renal malformation, and renal cancer. Other non-limiting examples related to renal disease include decreased erythropoiesis, active vitamin D deficiency, and abnormal non-thyroid diseases.

[0091] In certain non-limiting embodiments, the present disclosure provides a method 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 of the administered renal cells proliferates in or around the FALC or lymph nodes, producing ectopic renal tissue, thereby enhancing renal function in the subject.

[0092] In certain embodiments, renal cells or renal tissue fragments are grafted into or around the FALC or lymph nodes to generate ectopic kidney tissue. In certain embodiments, the renal cells or renal tissue fragments transplanted into a subject may be human or non-human. Non-limiting examples of non-human renal cells include non-human primate renal cells and renal cells from various non-human animals, such as dogs, cats, horses, mice, rats, hamsters, rabbits, and pigs. In certain embodiments, the renal cells and / or renal tissue fragments may be syngeneic or allogeneic to the intended recipient. In non-limiting embodiments, the renal cells and / or renal tissue fragments are autologous to the intended recipient (e.g., harvested and expanded in culture prior to transplantation or generated from the intended recipient's progenitor or stem cells). In certain embodiments, the renal cells are fetal kidney cells or tissue, e.g., metanephros. In certain embodiments, the renal cells are not metanephros, e.g., intact metanephros. In certain embodiments, the renal cells may be renal progenitor cells. In certain non-limiting embodiments, kidney cells and / or kidney tissue fragments of one species may be transplanted into another species, for example, but not limited to, human kidney cells may be transplanted into a mouse host. For example, but not limited to, non-human kidney cells and / or kidney tissue fragments can be grafted into FALCs of a human recipient.

[0093] In certain embodiments, the effective amount of kidney cells is about 10 4 and 10 11 between 10 and 10 kidney cells, or approximately 10 5 From 10 10 The amount of renal cells or FALCs may comprise an amount found to be effective to generate at least one site of ectopic kidney tissue in a subject between about 10 and 20% of the total kidney tissue. In certain embodiments, the effective amount of renal cells may comprise one or more renal cells. In certain embodiments, the effective amount of renal cells may comprise about 10 or more renal cells. 5 from about 10 11 pieces, about 10 5 from about 10 9 pieces, about 10 5 from about 10 8 pieces, about 10 5 from about 107 pieces, about 10 5 from about 10 6 pieces, about 10 6 from about 10 11 pieces, about 10 6 from about 10 10 pieces, about 10 6 from about 10 9 pieces, about 10 6 from about 10 8 pieces, about 10 6 from about 10 7 pieces, about 10 7 from about 10 11 pieces, about 10 7 from about 10 10 pieces, about 10 7 from about 10 9 Pieces or about 10 7 from about 10 8 A quantity of kidney cells can be administered.

[0094] In certain embodiments, renal cells and / or renal tissue fragments can be administered to a subject more than once to achieve the desired ectopic kidney tissue. For example, without limitation, 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, renal cells and / or renal tissue fragments can be grafted into two or more different anatomical regions containing FALCs or lymph nodes to obtain two or more ectopic kidney tissues.

[0095] In certain embodiments, the present disclosure further provides a method of treating a subject in need of enhanced renal function, comprising administering to the subject a therapeutically effective amount of renal cells (or fragments of renal tissue) and promoting proliferation of renal cells in the subject's adipose-associated lymphocyte accumulation or lymph nodes. In certain embodiments, such proliferation can be promoted, for example, but not limited to, by administering renal cells locally to an anatomical region containing the adipose-associated lymphocyte accumulation or lymph nodes targeted for formation of ectopic renal tissue, and / or by providing at least one agent that promotes the formation of ectopic renal tissue.

[0096] In certain embodiments, the agent may 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. Alternatively and / or in addition, the agent may be an activator of the LTβR and / or NIK signaling pathway, e.g., an activator of LTβR or NIK or a downstream target thereof. In certain embodiments, the agent may be an activator of the non-canonical NF-κB signaling pathway. In certain embodiments, the agent may be bone marrow-derived cells, stromal cells, an activator of the LTβR and / or NIK signaling pathway, an activator of the non-canonical NF-κB signaling pathway, or a combination thereof.

[0097] In certain non-limiting embodiments, the present disclosure provides methods for generating ectopic renal tissue, the methods comprising introducing a composition comprising renal cells into a FALC and providing at least one agent that promotes the formation of ectopic renal tissue, e.g., as described above. For example, without limitation, a method for treating a subject in need of enhanced renal function comprises (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, e.g., by promoting the proliferation and / or angiogenesis of one or more cells to form the ectopic renal tissue. Similar methods may be used to generate ectopic tissue in lymph nodes.

[0098] In certain non-limiting embodiments, a method of treating a subject in need of enhanced renal function includes (a) administering to the subject a therapeutically effective amount of renal cells or renal tissue fragments into the subject's FALC or lymph nodes 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, e.g., by promoting proliferation and / or angiogenesis of one or more cells to form the ectopic renal tissue. Similar methods may be used to generate ectopic tissue in lymph nodes to enhance renal function.

[0099] In certain non-limiting embodiments, a method of treating a subject in need of enhanced renal function can include (a) administering to the subject a therapeutically effective amount of renal cells or renal tissue fragments into the subject's FALC or lymph nodes to form ectopic renal tissue, and (b) administering to the subject an activator of the LTβR and / or NIK signaling pathway to promote the formation of ectopic renal tissue. Similar methods can also be used to generate ectopic tissue in lymph nodes to enhance renal function.

[0100] In certain embodiments, the method for treating a subject who needs to enhance renal function can further comprise inducing inflammation in the subject.For example, but not limited to, as disclosed above, inflammation can be induced by administering to the subject an agent that promotes inflammation.In certain embodiments, inflammation is induced before introducing one or more hepatocytes to be transplanted, as described above.

[0101] In certain embodiments, the methods of the present disclosure are performed in vivo, as described above. In certain embodiments, the methods of the present disclosure are performed in vitro / ex vivo, for example, as cultured tissue explants. In certain non-limiting embodiments, the presently disclosed subject matter provides kidney tissue produced by such in vitro / ex vivo methods, for example, for subsequent transplantation into a subject in need of enhanced kidney function.

[0102] 5.3 Compositions and Kits The presently disclosed subject matter provides compositions for generating ectopic tissue, including one or more cells and one or more agents that can be used to promote the formation of ectopic tissue from the one or more cells.

[0103] In certain embodiments, the one or more cells present in the composition comprise hepatocytes. In certain embodiments, the one or more cells present in the composition comprise kidney cells. In certain embodiments, the hepatocytes or kidney cells can be prepared for transplantation and / or the transplantation procedure can be performed using the methodology described in U.S. Patent No. 9,125,891, incorporated herein by reference.

[0104] Cells suitable for use in the present disclosure may be derived from any suitable source. For example, without limitation, cells may be derived from an autologous source. In certain embodiments, cells may be derived from the subject into which the cells are implanted. In certain embodiments, cells may be derived from a xenogeneic source. In certain embodiments, cells may be derived from an individual different from the subject into which the cells are implanted. In certain embodiments, cells, such as hepatocytes or kidney cells, may also be generated from stem cells derived from various sources and then differentiated into the relevant cell type. In certain embodiments, cells may be cultured under various conditions for a period of time to induce a particular phenotype before use in the compositions and / or methods of the present disclosure.

[0105] In certain embodiments, the number of cells contained in the compositions disclosed herein may vary. In certain embodiments, the composition may comprise 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 may comprise at least about 10 4 from about 10 11 The cells may include individual cells, for example, liver cells or kidney cells.

[0106] In certain embodiments, the present disclosure provides a composition for generating an ectopic liver or treating a subject in need of enhanced liver function. In certain embodiments, the composition comprises a plurality of hepatocytes and one or more of the following agents that promote the formation of an ectopic liver. In certain embodiments, the agent may be one or more cells that are not hepatocytes. For example, but not limited to, the one or more agents may include a plurality of bone marrow-derived cells and / or a plurality of stromal cells. In certain embodiments, the bone marrow-derived cells may be hematopoietic stem cells. In certain embodiments, the stromal cells may be fibroblasts or fibroblast-like cells, such as reticular fibroblasts (FRCs), follicular dendritic cells (FDCs), lymphatic endothelial cells (LECs), vascular endothelial cells (BECs), alpha-7 integrin pericytes (AIPs), and double-negative cells (DNCs).

[0107] In certain embodiments, the present disclosure provides compositions for generating an ectopic kidney or treating a subject in need of enhanced renal function. In certain non-limiting embodiments, the present disclosure provides compositions for generating an ectopic kidney, comprising a plurality of renal cells (or fragments of renal tissue) and one or more agents that promote the formation of an ectopic kidney. In certain embodiments, the agents may be one or more cells that are not renal cells. For example, without limitation, the one or more agents may include a plurality of bone marrow-derived cells and a plurality of stromal cells. In certain embodiments, the bone marrow-derived cells may be hematopoietic stem cells. In certain embodiments, the stromal cells may be fibroblasts or fibroblast-like cells, such as reticular fibroblasts (FRCs), follicular dendritic cells (FDCs), lymphatic endothelial cells (LECs), vascular endothelial cells (BECs), alpha-7 integrin pericytes (AIPs), and double-negative cells (DNCs).

[0108] In certain embodiments, the composition may further comprise an activator of the LTβR and / or NIK signaling pathway. Non-limiting examples of activators of the LTβR and / or NIK signaling pathway are discussed above. In certain embodiments, the composition may further comprise an activator of the non-canonical NF-κB signaling pathway. For example, but not limited to, a composition of the present disclosure may comprise a plurality of cells, such as kidney cells (or kidney tissue fragments) or liver cells, and a therapeutically effective amount of an activator of the LTβR and / or NIK signaling pathway.

[0109] 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 hepatocytes, (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.

[0110] In certain embodiments, the composition may optionally include a synthetic culture medium and / or scaffold, which may include, but is not limited to, peptides, proteins, carbohydrates, Matrigel, hyaluronic acid, collagen, fibrin, fibrinogen, fibronectin, polyorthoesters, polyvinyl alcohol, polyamides, polycarbonates, agarose, alginic acid, poly(ethylene) glycol, polylactic acid, polyglycolic acid, polycaprolactone, polyvinylpyrrolidone, marine adhesive proteins, cyanoacrylates, polymeric hydrogels, analogs or combinations thereof.

[0111] In certain embodiments, the composition may optionally include an antibiotic, non-limiting examples of which include, but are not limited to, penicillins such as penicillin and amoxicillin; cephalosporins such as cephalexin; sulfonamides such as cotrimoxazole and trimethoprim; macrolides such as erythromycin, carithromycin, and azithromycin; fluoroquinolones such as ciprofloxacin, levofloxacin, and ofloxacin; tetracyclines such as tetracycline and doxycycline; and aminoglycosides such as gentamicin and tobramycin.

[0112] The present disclosure further provides a composition for inducing ectopic tissue produced by the method disclosed above. For example, but not limited to, the composition of the present disclosure may include human liver tissue produced in a non-human host as disclosed above. In certain embodiments, the composition may optionally include a synthetic culture medium and / or a scaffold. In certain embodiments, the composition may optionally include an antibiotic.

[0113] The present disclosure further provides a kit comprising one or more compositions disclosed herein.In certain embodiments, when a 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 of this disclosure can further include instructions for use, one or more components such as devices and additional reagents, and components such as tubes, containers and syringes for carrying out the above-disclosed methods.

[0114] The following examples are provided to more fully illustrate the present disclosure, but should not be construed as limiting its scope. [Example]

[0115] 6. Example 1: Liver Bioengineering in Secondary Lymphoid Tissue 6.1. Generation of auxiliary livers in lymph nodes (LN) and femoral foci (FALC) To identify new sites for hepatocyte transplantation as an alternative to whole organ transplantation, we developed a hepatocyte-induced liver failure model, Fah. - / - We made the unusual observation that mice lacking the tyrosine-degrading enzyme fumarylacetoacetate hydrolase (Fah) formed ectopic nodules de novo and subsequently were able to remove 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione ("NTBC") from their drinking water after intraperitoneal (IP) injection (32). Initially, normal hepatocytes migrated to the lymphatic system and transported Fah - / - We observed that hepatocytes colonized the lymph nodes of mice (Figure 2A) and ectopically generated over 70% of the native liver mass. This was a surprising result, especially considering that lymph nodes are a common site of early metastasis for several cancers. Figure 3 shows the Fah 3 to 4 weeks after IP transplantation of hepatocytes. - / - Mice showing enlargement of capillary spaces and sprouting of blood vessels in the mammary plaque.

[0116] This leads to the hypothesis that lymph nodes can be directly targeted as sites for ectopic transplantation of multiple cell types to restore tissue and organ function. - / - It was established that direct injection into mouse lymph nodes (axillary, popliteal, or mesenteric) generated ectopic liver masses that were sufficiently functional to rescue survival in these mutant mice suffering from a lethal metabolic disease ( 33 ).

[0117] More recently, it was demonstrated that the concept of generating ectopic livers can be translated to larger animals after inducing liver disease (Figure 2B). Here, a porcine model of liver disease was created, and a portocaval shunt was performed to grow ectopic liver tissue in the lymph nodes, followed by hepatocyte transplantation into the mesenteric lymph nodes. A partial hepatectomy was used to further increase the size of the liver injury, and six pigs were transplanted. The results of this experiment demonstrated the generation of liver tissue with hepatocyte engraftment in the lymph nodes and increased ectopic liver mass in all transplanted animals (Figure 2B). Portocaval shunts are a primary surgical procedure for patients with severe liver disease, but are no longer commonly used. Portocaval shunts reduce the liver's blood flow by 75% by creating a new connection between the blood vessels (from the portal vein to the vena cava) to alleviate the debilitating effects of portal hypertension. Furthermore, this dramatic reduction in blood flow to the liver is known to induce hepatotrophic factors and hepatocyte proliferation (41).

[0118] Today, a similar but preferred minimally invasive procedure for initiating a new connection between the portal vein and the hepatic vein in cirrhotic patients with significant portal hypertension is the transjugular intrahepatic portosystemic shunt (TIPS). With these promising results in a porcine model and successful patient conversion after TIPS, it was necessary to determine how and why the lymphatic microenvironment is such a good host for transplanted cells.

[0119] Hepatocytes also engraft in the milk spots. - / -Careful analysis of hepatocyte engraftment sites in mice revealed that, in addition to lymph nodes, hepatocytes also migrate to the lacteal areas of the omentum, which generate hepatic lymph nodes (Figures 2C-D, 3-5). As shown in Figure 3, note the expansion of capillary spaces and vascular sprouting in the lacteal areas (e.g., bottom right panel). Lacteal areas are small, "milky" areas of lymphoid tissue (adipose-associated lymphocyte clusters, or "FALCs") most commonly found in the larger omentum (Figure 2C). These aggregates of hematopoietic stem cells (lymphocytes and macrophages) and stromal cells are known as secondary lymphoid organs, similar to lymph nodes (42, 43). Not surprisingly, lacteal areas have also been identified as early targets of peritoneal carcinomatosis, particularly in metastatic ovarian cancer.

[0120] To confirm that the formation of ectopic liver required a lymphoid tissue environment, IP transplantation was performed using Fah mice, which additionally carry mutations in the recombination activating gene 2 (RAG2) and common cytokine receptor gamma chain (gamma c). - / - This study was performed in NOD mice ("FRGN" mice); these mice are completely lymphocyte-free (44). As shown in Figure 6, the mammary glands in the omentum and FALC are absent in FRGN mice. IP transplantation of hepatocytes resulted in poor engraftment in the omentum of FRGN mice compared with wild-type controls (Figure 7), and no growth of engrafted cells was observed (Figure 8). Interestingly, the ability to form mammary glands and FALC was restored in FRGN mice by bone marrow transplantation (Figures 9 and 10), and these mammary glands and FALC were able to support the formation of ectopic livers (Figures 11 and 12).

[0121] 6.2. Molecular mechanisms underlying ectopic liver development The lymph nodes were then examined for hepatocyte engraftment and Fah - / - Hepatocytes are thought to colonize mammary glands and lymph nodes, and are essential for the survival of Fah, which lack functional lymph nodes. - / -We sought to determine whether mice could develop an ectopic liver mass in the mammary gland sufficient to survive. To test this hypothesis, lymphoblastic mice (aly / aly) were bred into mice subjected to inducible renal failure (Fah). - / - ) were crossed with aly / aly mice. The aly / aly mice lack lymph nodes, Peyer's patches, and have disorganized spleens due to a point mutation in NF-κB-inducing kinase (NIK), which disrupts non-canonical NF-κB signaling in the cells (Figure 13). In the absence of non-canonical NF-κB signaling, lymph node organogenesis naturally does not occur during development (no lymph nodes), but lymphatic vessels and, importantly, mammary glands are present and functional in these mice (43).

[0122] IP transplanted hepatocytes were aly / aly-Fah - / - We first tested whether the hepatocytes could be engrafted in mice (Fig. 14). It was found that the majority of hepatocytes migrated to the milky spots of the omentum, and the hepatocytes were expressed in the Fah - / - aly / aly-Fah, as in mice - / - Although the cells engrafted in the omentum of mice (Fig. 15), they failed to form large nodules even after 6 weeks, confirming that transplanted mice (n=28) were not rescued after 12 weeks (the time required to rescue control Fah- / - mice) despite initial engraftment in the omentum (Fig. 14, rightmost Kaplan-Meier survival plot). - / - Limited angiogenesis was observed compared to the ectopic nodules generated in mice. Hepatocytes also expressed aly / aly-Fah - / - When transplanted into the liver (via spleen injection) of mice, the mice were found to be rescued from liver failure (Figure 14, Kaplan-Meier survival plot on the far left). - / - This is important because the lack of intraperitoneal growth of hepatocytes in mice is specific to this site and is the result of disruption of the NIK pathway by the aly(NIK) mutation (Figure 16). Hepatocytes injected into the liver (spleen injection) do not require the NIK pathway and likely replace dying hepatocytes within the normal liver structure.

[0123] Next, we conducted studies to characterize the lymphocyte environment that may serve as a host for the formation of ectopic liver. Figure 17A shows the distribution of the lymphocyte marker podoplanin and the endothelial marker CD31 in lymph nodes compared with the mammary plaque. As shown in Figure 17B, transplanted hepatocytes formed close associations with ER-TR7-bearing stromal cells. Interestingly, when omental stromal cells, but not hepatocytes, were transplanted by intraperitoneal injection, they also migrated back into the intraomental mammary plaque (Figure 17C). As shown in Figure 18, stromal cells were found to express both NIK and lymphotoxin beta receptors, and hepatocytes were, in turn, positive for lymphotoxin alpha (also known as tumor necrosis factor beta) and lymphotoxin beta (also known as tumor necrosis factor C).

[0124] Given the observation that hepatocytes express lymphotoxin beta ("LTb") and interstitial cells express lymphotoxin beta receptor ("LTbR"), it is possible that hepatocytes express Fah receptors that lack LTbR. - / - It will be of interest to see whether the Fah / LTbR cells can engraft and grow in the mammary glands of mice. The results are shown in Figure 19. - / - Hepatocyte growth in mice is associated with Fah - / - , LTbR + Lower hepatocyte growth was observed in mice.

[0125] In conclusion, IP-injected hepatocytes undergo organogenesis and - / - New blood vessel formation supporting ectopic liver mass in mice is complete, and the NIK pathway is a unique pathway required for this process to be completed.

[0126] In the classic seed-and-field hypothesis of cancer proposed by Dr. Stephen Paget (1889), the seed (metastatic cancer) requires a field (a fertile environment) to thrive. The results described herein demonstrate that the seed (hepatocytes) requires a field (a secondary lymphoid organ) to engraft and generate liver tissue at ectopic sites. Without being bound by any particular theory, this data supports a mechanism in which hepatocytes with stromal cells activate the lymphotoxin / NIK pathway to generate an auxiliary liver in the mammary gland. Furthermore, this data demonstrates that liver tissue regeneration by transplantation of hepatocytes in the liver (spleen route) or mammary gland (IP route) is mechanistically distinct. An aly / aly point mutation in NF-κB-inducing kinase (NIK) disrupted ectopic liver development beyond native liver repair. NIK plays a central role in noncanonical NF-κB signaling for secondary lymphoid organ development. NIK is a member of the mitogen-activated protein 3 (MAP3) kinases, which accumulate to detectable levels after receptor ligation, thereby phosphorylating NIK and activating inhibitor of κB kinase α (IKKα), thus initiating IKKα-mediated phosphorylation of p100 (see Figure 13).

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Can hepatocytes proliferate when transplanted into the spleen? Demonstration by autohistoradiography in the rat. European surgical research. Europaische chirurgische Forschung. Recherches chirurgicales europeennes 19, 381-387 (1987). 42. Shah, S. et al. Cellular basis of tissue regeneration by omentum. PloS one 7, e38368 (2012). 43. Rangel-Moreno, J. et al. Omental milky spots develop in the absence of lymphoid tissue-inducer cells and support B and T cell responses to peritoneal antigens. Immunity 30, 731-743 (2009). 44. Mazurier F1, Fontanellas A, Salesse S, Taine L, Landriau S, Moreau-Gaudry F, Reiffers J, Peault B, Di Santo JP, de Verneuil H., A novel immunodeficient mouse model--RAG2 x common cytokine receptor gamma chain double mutants--requiring exogenous cytokine administration for human hematopoietic stem cell engraftment. J Interferon Cytokine Res. 1999 May;19(5):533-41. 45. Fabregat I, Moreno-Caceres J, Sanchez A, Dooley S, Dewidar B, Giannelli G, Ten Dijke P; IT-LIVER Consortium., TGF-β signalling and liver disease. FEBS J. 2016 Jun;283(12):2219-32. 46. Rogers S., Lowell J., Hammerman N., and Hammerman M., Transplantation of developing metanephroi into adult rats. Kidney International 1998; 54:27-37.

[0128] 7. Example 2: Kidney Bioengineering in Secondary Lymphoid Tissue Mouse lymph nodes (LNs), secondary lymphoid organs (SLOs), can support the maturation of mouse metanephros glomeruli and tubules into functional nephrons. LNs can also promote the maturation of transplanted human fetal kidneys, as well as renal organoid cultures derived from mouse nephron progenitor cells (NPs) or human induced pluripotent stem cells (hiPSCs) (Figure 20). To test whether LTβR signaling, a key signaling mediator of LN origin, supported ectopic organ formation, mice bearing LN kidney grafts were treated with LTβR-Fc fusion protein. LTβR-Fc fusion protein antagonizes LTβR-mediated effects by binding to LTβR ligands (LTα and LTβ) (see Figure 21). As shown in Figures 22A-B, LTβR-Fc treatment significantly reduced graft size and graft angiogenesis.

[0129] In addition to binding to LTβR ligands, LTβR-Fc also binds LIGHT, a non-TNF family member that can interact not only with LTβR but also with HVEM and Dcr3 receptors (see Figure 21). To exclude the possibility that the impaired graft angiogenesis / angiogenesis resulted from LTβR-Fc interference with signaling pathways other than those mediated by LTβR, we investigated the outcome of kidney transplantation in an LTβR-deficient environment, such as that provided by LTβR- / - mice (see Figure 22). This approach also allowed us to exclude the possibility that the observed effects could be due to LTβR / HVEM / Dcr3 inhibition in donor kidney cells or other cell types present in the donor tissue at the time of transplantation, including stromal and hematopoietic cells.

[0130] Because LTβR- / - mice lack LNs, we used the larger omentum as an alternative SLO for transplantation. The omentum contains lymphocyte aggregates called mammary plaques, which promote immunity to antigens in the peritoneal cavity (see Figure 1). As in LNs, a reticular network of reticular fibroblasts (FRCs) supports leukocytes in the mammary plaques. When we examined the steady-state stroma composition of the omentum compared with LNs, we found similar flow cytometry profiles of stromal cells (Figure 22C). Furthermore, subsets of PDPLN+ / LTβR+ and PDPLN- / LTβR+ cells were identified in omentum cell suspensions, indicating the presence of rare populations of LT-reactive FRCs and brain endothelial cells (BECs) in the omentum (Figure 22D). Growth of embryonic kidney fragments was significantly affected in LTβR- / - grafts; grafts were not only smaller but also poorly vascularized and displayed abnormal morphology compared with their control counterparts, pointing to the importance of LTβR signaling in host stromal cells for successful angiogenesis / neovascularization of heterotopic kidney grafts (Figure 22E-F). In untreated LN kidney grafts, NIK, a downstream target of LTβR and a central component of the noncanonical NF-κB pathway, was restricted to glomerular endothelial cells (Figure 22G). Conversely, LTβR-Fc treatment abrogated NIK expression in these cells. Similarly, NIK expression was undetectable in grafts grown in LTβR- / - grafts. Without being bound by any particular theory, LTβR may use NIK to propagate noncanonical NF-κB signaling and promote angiogenesis / neovascularization of the graft tissue. Furthermore, without being bound by any particular theory, it appears that stromal cells present within secondary lymphoid organs may use LTβR signaling to support organogenesis, and NIK activation restricted to stromal endothelial cells may promote vascularization of the graft.

[0131] 8. Example 3: Inflammation Enhances the Growth of Grafted Hepatocytes Peritoneal inflammation induces an increase in the number and size of adipose-associated lymphoid cell clusters (FALCs) (Benezech, C. et al., Inflammation-induced formation of fat-associated lymphoid clusters. Nature Immunology 16, 819-828 (2015)). This effect depends on TNF expression by myeloid cells and TNFR signaling to stromal cells (Benezech et al., 2015). To determine whether inflammation also leads to increased hepatocyte engraftment, sterile peritoneal inflammation driven by zymosan (a yeast-derived ligand for Toll-like receptor 2) was initiated in wild-type C57BL / 6 mice and control animals (wild-type C57BL / 6 mice injected with PBS) (see Figure 23). After 3 days, GFP+ hepatocytes were transplanted into both groups of animals, which were then sacrificed one week later.

[0132] As shown in Figure 24, zymosan-induced inflammation dramatically increased the presence of GFP+ cells, as visualized by quantification of GFP+ hepatocytes in tissues and in the omental and mesenteric fat. Importantly, hepatocyte engraftment in FALCs after IP injection was observed in wild-type mice, indicating that engraftment in FALCs is independent of liver injury. How to time FALC formation with the onset or resolution of inflammation and whether hepatocyte engraftment is maintained during the resolution phase remains to be investigated.

[0133] This preconditioning regimen then - / - This was tested in C57bl / 6 mice to determine whether it affected the survival rate of tyrosinemic mice after induction of liver disease (without NTBC). Using the same procedures as described in Figure 23, Fah was administered with or without induction of inflammation. - / - C57bl / 6 mice were transplanted with wild-type hepatocytes and subsequently liver disease was induced (no NTBC). As shown in Figure 25, animals without inflammation in the left panel were not rescued after two rounds of sorting (weight loss = liver disease), whereas animals with inflammation were rescued after 8 weeks.

[0134] Necropsy of animals in which inflammation was not induced revealed reduced / limited liver engraftment and liver mass in FALCs located in the omentum, spleen, portal vein, gonadal, and mesenteric fat (Figure 26). In contrast, animals in which inflammation was induced had a larger mass of liver tissue in these locations.

[0135] Various references, patents and patent applications are cited herein, the contents of which are incorporated herein by reference in their entireties. The present invention encompasses, for example, the following embodiments: [Embodiment 1] A method for generating ectopic tissue in a subject, the method comprising introducing one or more cells into an adipose-associated lymphocyte accumulation in the subject, and providing one or more agents that promote the formation of ectopic tissue. [Embodiment 2] The method of embodiment 1, wherein the one or more agents that promote the formation of ectopic tissue include bone marrow-derived cells, stromal cells, or both. [Embodiment 3] The method of embodiment 1 or 2, wherein the one or more agents include stromal cells. [Embodiment 4] The method of embodiment 2 or 3, wherein the stromal cells comprise fibroblasts. [Embodiment 5] The method of any one of embodiments 2, 3, or 4, wherein the stromal cells express podoplanin, NIK, LTβR, or a combination thereof. [Embodiment 6] The method of any one of embodiments 2 to 5, wherein the stromal cells are treated with an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof. [Embodiment 7] The method of embodiment 6, wherein the activator is an activator of LTβR, NIK, or a combination thereof. [Embodiment 8] The method of embodiment 6, wherein the activator is an activator of the non-canonical NF-κB signaling pathway. [Embodiment 9] The method of any of embodiments 6, 7, or 8, wherein activation of the LTβR signaling pathway, the NIK signaling pathway, the non-canonical NF-κB signaling pathway, or a combination thereof promotes proliferation of one or more cells, angiogenesis of ectopic tissue, or a combination thereof. [Embodiment 10] The method of any one of embodiments 1 to 9, wherein the one or more agents that promote the formation of ectopic tissue include an agent that promotes inflammation. [Embodiment 11] The method of any one of embodiments 1 to 9, further comprising inducing inflammation in the subject. [Embodiment 12] The method of embodiment 11, wherein the inflammation is induced by administering to the subject an agent that promotes inflammation. [Embodiment 13] The method of any one of embodiments 10 to 12, wherein inflammation is induced prior to introducing one or more cells into the adipose-associated lymphocyte accumulation of the subject. [Embodiment 14] The method of any one of embodiments 1 to 13, wherein the one or more cells comprise hepatocytes and the ectopic tissue comprises ectopic liver tissue. [Embodiment 15] The method of any one of embodiments 1 to 13, wherein the one or more cells comprise renal cells, one or more renal tissue fragments, or a combination thereof, and the ectopic tissue comprises ectopic renal tissue. [Embodiment 16] A method according to any one of embodiments 1 to 15, wherein the adipose-associated lymphocyte accumulation is located in the adipose tissue of the pleural cavity, pericardial cavity and / or peritoneal cavity, such as the fat of the omentum, mesenteric, splenic, portal vein and / or gonadal glands of the subject. [Embodiment 17] A method for treating a subject in need of enhanced liver function, comprising administering a therapeutically effective amount of hepatocytes to the subject and promoting the formation of ectopic liver tissue in the subject's adipose-associated lymphocyte accumulation. [Embodiment 18] The method according to embodiment 17, wherein the formation of ectopic liver tissue in adipose-associated lymphocyte accumulation is promoted by locally administering hepatocytes to the anatomical region of adipose-associated lymphocyte accumulation. [Embodiment 19] The method described in embodiment 17 or 18, wherein the adipose-associated lymphocyte accumulation is located in the adipose tissue of the subject's omentum, mesenteric, splenic, portal vein and / or gonadal areas. [Embodiment 20] The method described in any one of embodiments 17 to 19, wherein the formation of ectopic liver tissue in adipose-associated lymphocyte accumulation is promoted by co-administration of one or more bone marrow-derived cells or stromal cells. [Embodiment 21] The method of embodiment 20, wherein stromal cells are co-administered. [Embodiment 22] The method of embodiment 20 or 21, wherein the stromal cells comprise fibroblasts. [Embodiment 23] The method described in any one of embodiments 20 to 22, wherein the stromal cells express podoplanin, NIK, LTβR, or a combination thereof. [Embodiment 24] The method according to any one of embodiments 17 to 23, wherein the formation of ectopic liver tissue in adipose-associated lymphocyte accumulation is promoted by simultaneous administration of an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof. [Embodiment 25] The method according to any one of embodiments 17 to 24, wherein the formation of ectopic liver tissue in adipose-associated lymphocyte accumulation is promoted by co-administration of an activator of the non-canonical NF-κB signaling pathway. [Embodiment 26] The method of any one of embodiments 17 to 24, further comprising inducing inflammation in the subject. [Embodiment 27] The method of embodiment 26, wherein the inflammation is induced by administering to the subject an agent that promotes inflammation. [Embodiment 28] The method of embodiment 26 or 27, wherein inflammation is induced before administering the hepatocytes. [Embodiment 29] The method described in any one of embodiments 17 to 25, wherein the formation of ectopic liver tissue in adipose-associated lymphocyte accumulation is promoted by administering to the subject an agent that promotes inflammation. [Embodiment 30] The method of embodiment 29, wherein a drug that promotes inflammation is administered to the subject before administering the hepatocytes. [Embodiment 31] A method for generating an ectopic liver, comprising introducing one or more hepatocytes into an adipose-associated lymphocyte accumulation and providing at least one agent that promotes the formation of ectopic liver tissue. [Embodiment 32] The method of embodiment 31, carried out in vivo. [Embodiment 33] The method of embodiment 31, carried out in vitro. [Embodiment 34] The method of any one of embodiments 31, 32, or 33, wherein the agent that promotes the formation of ectopic liver tissue comprises bone marrow-derived cells, stromal cells, or a combination thereof. [Embodiment 35] The method of embodiment 34, wherein the agent comprises stromal cells. [Embodiment 36] The method of embodiment 34 or 35, wherein the stromal cells comprise fibroblasts. [Embodiment 37] The method of any one of embodiments 34, 35, or 36, wherein the stromal cells express one of podoplanin, NIK, LTβR, or a combination thereof. [Embodiment 38] The method of any one of embodiments 34 to 37, wherein the stromal cells are treated with an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof. [Embodiment 39] The method of any one of embodiments 31 to 38, wherein the agent that promotes the formation of ectopic tissue includes an activator of the LTβR signaling pathway, an NIK signaling pathway, an agent that promotes inflammation, or a combination thereof. [Embodiment 40] The method of any one of embodiments 31 to 38, further comprising inducing inflammation. [Embodiment 41] The method of embodiment 40, wherein the inflammation is induced by administering an agent that promotes inflammation. [Embodiment 42] A method for treating a subject in need of enhanced renal function, comprising administering to the subject a therapeutically effective amount of renal cells, renal tissue fragments, or a combination thereof, and promoting the formation of ectopic renal tissue in the subject's adipose-associated lymphocyte accumulation. [Embodiment 43] The method of embodiment 42, wherein the formation of ectopic kidney tissue in adipose-associated lymphocyte accumulation is promoted by administering kidney cells locally to the anatomical region of adipose-associated lymphocyte accumulation. [Embodiment 44] The method described in embodiment 42 or 43, wherein the adipose-associated lymphocyte accumulation is located in the adipose tissue of the subject's omentum, mesenteric, splenic, portal vein, and gonadal areas. [Embodiment 45] The method described in any one of embodiments 42 to 44, wherein the formation of ectopic kidney tissue in adipose-associated lymphocyte accumulation is promoted by co-administration of one or more bone marrow-derived cells, stromal cells, or a combination thereof. [Embodiment 46] The method of embodiment 45, wherein stromal cells are co-administered. [Embodiment 47] The method of embodiment 45 or 46, wherein the stromal cells comprise fibroblasts. [Embodiment 48] The method described in any one of embodiments 45 to 47, wherein the stromal cells express podoplanin, NIK, LTβR, or a combination thereof. [Embodiment 49] The method described in any one of embodiments 42 to 48, wherein the formation of ectopic kidney tissue in adipose-associated lymphocyte accumulation is promoted by simultaneous administration of an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof. [Embodiment 50] The method of any one of embodiments 42 to 49, wherein the formation of ectopic kidney tissue in adipose-associated lymphocyte accumulation is promoted by co-administration of an activator of the non-canonical NF-κB signaling pathway. [Embodiment 51] The method of any one of embodiments 42 to 50, wherein the formation of ectopic kidney tissue in adipose-associated lymphocyte accumulation is promoted by administering an agent that promotes inflammation. [Embodiment 52] The method of embodiment 51, wherein the agent that promotes inflammation is administered before administering kidney cells, kidney tissue fragments, or a combination thereof. [Embodiment 53] The method of any one of embodiments 42 to 50, further comprising inducing inflammation in the subject. [Embodiment 54] The method of embodiment 53, wherein the inflammation is induced by administering to the subject an agent that promotes inflammation. [Embodiment 55] The method of any one of embodiments 51 to 54, wherein inflammation is induced prior to introducing one or more cells into the adipose-associated lymphocyte accumulation of the subject. [Embodiment 56] A method for generating ectopic kidney tissue, comprising introducing one or more kidney cells, kidney tissue fragments, or a combination thereof into an adipose-associated lymphocyte accumulation, and providing at least one agent that promotes the formation of ectopic kidney tissue. [Embodiment 57] The method of embodiment 56, performed in vivo. [Embodiment 58] The method of embodiment 56, carried out in vitro. [Embodiment 59] The method of any of embodiments 56, 57, or 58, wherein the agent that promotes the formation of ectopic liver tissue comprises bone marrow-derived cells, stromal cells, or a combination thereof. [Embodiment 60] The method of embodiment 59, wherein the agent comprises stromal cells. [Embodiment 61] The method of embodiment 59 or 60, wherein the stromal cells comprise fibroblasts. [Embodiment 62] The method of embodiment 59, 60, or 61, wherein the stromal cells express podoplanin, NIK, LTβR, or a combination thereof. [Embodiment 63] The method of any one of embodiments 59 to 62, wherein the stromal cells are treated with an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof. [Embodiment 64] The method of any of embodiments 56 to 63, wherein the agent that promotes the formation of ectopic tissue comprises an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, an agent that promotes inflammation, or a combination thereof. [Embodiment 65] The method of any one of embodiments 56 to 63, further comprising inducing inflammation in the subject. [Embodiment 66] The method of embodiment 65, wherein the inflammation is induced by administering to the subject an agent that promotes inflammation. [Embodiment 67] A method for generating ectopic renal tissue in a subject, comprising introducing cells comprising one or more renal cells and one or more stromal cells into an adipose-associated lymphocyte accumulation in the subject, and providing an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof, wherein activation of the LTβR signaling pathway, the NIK signaling pathway, or a combination thereof in the one or more stromal cells promotes the formation of ectopic renal tissue. [Embodiment 68] A method for generating ectopic liver tissue in a subject, comprising introducing cells comprising one or more hepatocytes and one or more stromal cells into an adipose-associated lymphocyte accumulation in the subject, and providing an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof, wherein activation of the LTβR signaling pathway, the NIK signaling pathway, or a combination thereof in the one or more stromal cells promotes the formation of ectopic liver tissue. [Embodiment 69] A composition for generating ectopic tissue, comprising a plurality of cells and one or more agents that promote the formation of ectopic tissue. [Embodiment 70] A composition for generating ectopic liver tissue, comprising a plurality of hepatocytes and one or more agents that promote the formation of an ectopic liver. [Embodiment 71] A composition for generating ectopic kidney tissue, comprising a plurality of kidney cells and one or more agents that promote the formation of an ectopic kidney. [Embodiment 72] A composition described in any of embodiments 69, 70, or 71, wherein the one or more agents include a plurality of bone marrow-derived cells, a plurality of stromal cells, or a combination thereof. [Embodiment 73] The composition described in embodiment 72, wherein the stromal cells express podoplanin, NIK, LTβR, or a combination thereof. [Embodiment 74] A composition described in any of embodiments 69, 70, or 71, wherein the one or more agents include an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof. [Embodiment 75] A composition described in any of embodiments 69, 70, or 71, wherein the one or more agents include an agent that promotes inflammation. [Embodiment 76] A composition described in any of embodiments 69, 70, or 71, wherein the one or more agents include two or more of: (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. [Embodiment 77] The composition of any one of embodiments 69 to 76, further comprising a synthetic culture medium. [Embodiment 78] A kit comprising a composition described in any one of embodiments 69 to 77. [Embodiment 79] A method for generating ectopic kidney tissue in a subject, the method comprising introducing an effective amount of kidney cells, kidney tissue fragments, or a combination thereof into the subject's adipose-associated lymphocyte accumulation. [Embodiment 80] The method of embodiment 79, wherein the renal cells include cells isolated from an embryonic kidney, a metanephroi, cells isolated from a renal organoid formed in vitro, or any combination thereof. [Embodiment 81] A method for treating a subject in need of enhanced liver function, comprising administering a therapeutically effective amount of hepatocytes to the subject 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 include two or more of: (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. [Embodiment 82] A method for treating a subject in need of enhanced renal function, 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 include two or more of: (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. [Embodiment 83] A method for generating ectopic tissue in a subject, the method comprising introducing one or more cells into a lymph node of the subject and providing one or more agents that promote the formation of ectopic tissue. [Embodiment 84] The method of embodiment 83, wherein the one or more agents that promote the formation of ectopic tissue include bone marrow-derived cells, stromal cells, or both. [Embodiment 85] The method of embodiment 83 or 84, wherein the one or more agents comprise stromal cells. [Embodiment 86] The method of any of embodiments 83, 84, or 85, wherein the stromal cells comprise fibroblasts. [Embodiment 87] The method of any one of embodiments 84, 85, or 86, wherein the stromal cells express podoplanin, NIK, LTβR, or a combination thereof. [Embodiment 88] The method of any of embodiments 84 to 87, wherein the stromal cells are treated with an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof. [Embodiment 89] The method of any of embodiments 83 to 88, wherein the one or more agents that promote the formation of ectopic tissue are activators of the LTβR signaling pathway, activators of the NIK signaling pathway, or a combination thereof. [Embodiment 90] The method of embodiment 88 or 89, wherein the activator is an activator of LTβR, NIK, or a combination thereof. [Embodiment 91] The method described in embodiment 88 or 89, wherein the activator is an activator of the non-canonical NF-κB signaling pathway. [Embodiment 92] A method described in any one of embodiments 88, 89, 90, or 91, wherein activation of the LTβR signaling pathway, activation of the NIK signaling pathway, activation of the non-canonical NF-κB signaling pathway, or a combination thereof promotes proliferation of one or more cells, angiogenesis of ectopic tissue, or a combination thereof. [Embodiment 93] The method of any one of embodiments 83 to 92, wherein the one or more agents that promote the formation of ectopic tissue include an agent that promotes inflammation. [Embodiment 94] The method of any one of embodiments 83 to 92, further comprising inducing inflammation in the subject. [Embodiment 95] The method of embodiment 94, wherein the inflammation is induced by administering to the subject an agent that promotes inflammation. [Embodiment 96] The method of any of embodiments 93 to 95, wherein inflammation is induced prior to introducing one or more cells into the subject's lymph nodes. [Embodiment 97] The method of any of embodiments 83 to 96, wherein the one or more cells comprise hepatocytes and the ectopic tissue comprises ectopic liver tissue. [Embodiment 98] The method of any of embodiments 83 to 96, wherein the one or more cells comprise renal cells, one or more renal tissue fragments, or a combination thereof, and the ectopic tissue comprises ectopic renal tissue. [Embodiment 99] A method for treating a subject in need of enhanced liver function, comprising administering a therapeutically effective amount of hepatocytes to the subject and promoting the formation of ectopic liver tissue in the subject's lymph nodes. [Embodiment 100] The method of embodiment 99, wherein the formation of ectopic liver tissue in the lymph node is promoted by administering hepatocytes locally to the anatomical region of the lymph node. [Embodiment 101] The method described in embodiment 99 or 100, wherein the formation of ectopic liver tissue in lymph nodes is promoted by co-administration of one or more bone marrow-derived cells or stromal cells. [Embodiment 102] The method of embodiment 101, wherein stromal cells are co-administered. [Embodiment 103] The method of embodiment 101 or 102, wherein the stromal cells comprise fibroblasts. [Embodiment 104] The method of any one of embodiments 101 to 103, wherein the stromal cells express podoplanin, NIK, LTβR, or a combination thereof. [Embodiment 105] The method described in any one of embodiments 99 to 104, wherein the formation of ectopic liver tissue in lymph nodes is promoted by simultaneous administration of an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof. [Embodiment 106] The method of any of embodiments 99 to 105, wherein the formation of ectopic liver tissue in the lymph node is promoted by co-administration of an activator of the non-canonical NF-κB signaling pathway. [Embodiment 107] The method of any one of embodiments 99 to 106, further comprising inducing inflammation in the subject. [Embodiment 108] The method of embodiment 107, wherein the inflammation is induced by administering to the subject an agent that promotes inflammation. [Embodiment 109] The method of embodiment 107 or 108, wherein inflammation is induced before administering the hepatocytes. [Embodiment 110] The method described in any one of embodiments 99 to 106, wherein the formation of ectopic liver tissue in the lymph nodes is promoted by administering to the subject an agent that promotes inflammation. [Embodiment 111] The method of embodiment 110, wherein a drug that promotes inflammation is administered to the subject before administering the hepatocytes. [Embodiment 112] A method for generating an ectopic liver, comprising introducing one or more hepatocytes into a lymph node and providing at least one agent that promotes the formation of ectopic liver tissue. [Embodiment 113] The method of embodiment 112, performed in vivo. [Embodiment 114] The method of embodiment 112, carried out in vitro. [Embodiment 115] The method of any one of embodiments 112, 113, or 114, wherein the agent that promotes the formation of ectopic liver tissue comprises bone marrow-derived cells, stromal cells, or a combination thereof. [Embodiment 116] The method of embodiment 115, wherein the agent comprises stromal cells. [Embodiment 117] The method of embodiment 115 or 116, wherein the stromal cells comprise fibroblasts. [Embodiment 118] The method of any one of embodiments 115, 116, or 117, wherein the stromal cells express one of podoplanin, NIK, LTβR, or a combination thereof. [Embodiment 119] The method of any of embodiments 115 to 118, wherein the stromal cells are treated with an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof. [Embodiment 120] The method described in any of embodiments 112 to 119, wherein the agent that promotes the formation of ectopic tissue includes an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, an agent that promotes inflammation, or a combination thereof. [Embodiment 121] The method of any one of embodiments 112 to 119, further comprising inducing inflammation. [Embodiment 122] The method of embodiment 121, wherein the inflammation is induced by administering an agent that promotes inflammation. [Embodiment 123] A method for treating a subject in need of enhanced renal function, comprising administering to the subject a therapeutically effective amount of renal cells, renal tissue fragments, or a combination thereof, and promoting the formation of ectopic renal tissue in the subject's lymph nodes. [Embodiment 124] The method of embodiment 123, wherein the formation of ectopic renal tissue in the lymph node is promoted by administering renal cells locally to the anatomical region of the lymph node. [Embodiment 125] The method of embodiment 123 or 124, wherein the formation of ectopic kidney tissue in the lymph node is promoted by co-administration of bone marrow-derived cells, stromal cells, or a combination thereof. [Embodiment 126] The method of embodiment 125, wherein stromal cells are co-administered. [Embodiment 127] The method of embodiment 125 or 126, wherein the stromal cells comprise fibroblasts. [Embodiment 128] The method of any one of embodiments 125 to 127, wherein the stromal cells express podoplanin, NIK, LTβR, or a combination thereof. [Embodiment 129] The method described in any one of embodiments 123 to 128, wherein the formation of ectopic renal tissue in the lymph node is promoted by simultaneous administration of an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof. [Embodiment 130] The method described in any one of embodiments 123 to 129, wherein the formation of ectopic kidney tissue in the lymph node is promoted by co-administration of an activator of the non-canonical NF-κB signaling pathway. [Embodiment 131] The method described in any one of embodiments 123 to 130, wherein the formation of ectopic renal tissue in the lymph nodes is promoted by administering an agent that promotes inflammation. [Embodiment 132] The method of embodiment 131, wherein an agent that promotes inflammation is administered before administering renal cells, renal tissue fragments, or a combination thereof. [Embodiment 133] The method of any one of embodiments 123 to 131, further comprising inducing inflammation in the subject. [Embodiment 134] The method of embodiment 133, wherein the inflammation is induced by administering to the subject an agent that promotes inflammation. [Embodiment 135] The method of any of embodiments 131 to 134, wherein inflammation is induced prior to introducing one or more cells into the subject's lymph nodes. [Embodiment 136] A method for generating ectopic renal tissue, comprising introducing one or more renal cells, renal tissue fragments, or a combination thereof into a lymph node, and providing at least one agent that promotes the formation of ectopic renal tissue. [Embodiment 137] The method described in embodiment 136, which is carried out in vivo. [Embodiment 138] The method of embodiment 136, carried out in vitro. [Embodiment 139] The method of any one of embodiments 136, 137, or 138, wherein the agent that promotes the formation of ectopic liver tissue comprises bone marrow-derived cells, stromal cells, or a combination thereof. [Embodiment 140] The method of embodiment 139, wherein the agent comprises stromal cells. [Embodiment 141] The method of embodiment 139 or 140, wherein the stromal cells comprise fibroblasts. [Embodiment 142] The method of any of embodiments 139, 140, or 141, wherein the stromal cells express podoplanin, NIK, LTβR, or a combination thereof. [Embodiment 143] The method of any of embodiments 139 to 142, wherein the stromal cells are treated with an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof. [Embodiment 144] The method of any of embodiments 136 to 143, wherein the agent that promotes the formation of ectopic tissue includes an activator of the LTβR signaling pathway, an NIK signaling pathway, an agent that promotes inflammation, or a combination thereof. [Embodiment 145] The method of any one of embodiments 136 to 142, further comprising inducing inflammation in the subject. [Embodiment 146] The method of embodiment 145, wherein the inflammation is induced by administering to the subject an agent that promotes inflammation. [Embodiment 147] A method for generating ectopic renal tissue in a subject, comprising introducing cells comprising one or more renal cells and one or more stromal cells into a lymph node of the subject, and providing an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof, wherein activation of the LTβR signaling pathway, activation of the NIK signaling pathway, or a combination thereof in the one or more stromal cells promotes the formation of ectopic renal tissue. [Embodiment 148] A method for generating ectopic liver tissue in a subject, comprising introducing cells comprising one or more hepatocytes and one or more stromal cells into a lymph node of the subject, and providing an activator of the LTβR signaling pathway, an activator of the NIK signaling pathway, or a combination thereof, wherein activation of the LTβR signaling pathway, activation of the NIK signaling pathway, or a combination thereof in one or more stromal cells promotes the formation of ectopic liver tissue.

Claims

1. 1. A composition for use in a method of generating ectopic tissue in an adipose-associated lymphoid accumulation in a subject, comprising cells or tissue fragments, The method includes introducing a composition into an adipose-associated lymphoid accumulation in a subject, thereby forming ectopic tissue; the cells or tissue fragments comprise hepatocytes and the ectopic tissue is ectopic liver tissue; or The cells or tissue fragments comprise kidney cells or kidney tissue fragments, and the ectopic tissue is ectopic kidney tissue. composition.

2. The composition of claim 1, wherein the subject has impaired liver function.

3. 10. The composition of claim 1, wherein the subject has Crill-Nager syndrome type 1, acute liver failure, cirrhosis, hemochromatosis, hyperoxaluria, oxalosis, Wilson's disease, alpha-1 antitrypsin deficiency, liver cancer, hepatitis, alcoholic hepatitis, autoimmune hepatitis, fatty liver disease, or non-alcoholic fatty liver disease.

4. The composition of claim 1, wherein the cells or tissue fragments comprise cells isolated from an embryonic kidney, a metanephroi, or cells isolated from a renal organoid formed in vitro.

5. The composition of claim 1, wherein the subject is a human.

6. The composition of claim 1, wherein the adipose-associated lymphocyte accumulation is located in the pleural cavity of the subject.

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

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

9. The composition of claim 1, wherein the adipose-associated lymphocyte accumulation is located in the pericardial space of the subject.

10. The composition of claim 1, wherein the adipose-associated lymphocyte accumulation is located in the peritoneal cavity of the subject.

11. 2. The composition of claim 1, wherein the adipose-associated lymphocyte accumulation is located in the omental, mesenteric, splenic, portal vein, or gonadal fat of the subject.

12. The composition of claim 1, wherein the adipose-associated lymphocyte accumulation is located in the omentum.

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

14. The composition of claim 1, wherein the cells or tissue fragments are administered to the subject by topical administration.

15. 10. The composition of claim 1, wherein the composition extends survival of the subject by at least 20%.

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