WO2014138486A1 - Ganglion lymphatique comme site de transplantation, organogenèse et fonctionnement pour de multiples tissus et organes - Google Patents

Ganglion lymphatique comme site de transplantation, organogenèse et fonctionnement pour de multiples tissus et organes Download PDF

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WO2014138486A1
WO2014138486A1 PCT/US2014/021420 US2014021420W WO2014138486A1 WO 2014138486 A1 WO2014138486 A1 WO 2014138486A1 US 2014021420 W US2014021420 W US 2014021420W WO 2014138486 A1 WO2014138486 A1 WO 2014138486A1
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cells
lymph node
cell
lymphoid
mice
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Eric Lagasse
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University Of Pittsburgh - Of The Commonwealth System Of Higher Education
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Priority to US14/810,064 priority Critical patent/US11191785B2/en
Priority to US17/514,136 priority patent/US20220288127A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • the present invention relates to methods and compositions for transplanting non-lymphoid tissues into lymphoid organs. It may be used to cultivate organ tissues for purposes including supplementing or reconstituting organ function. Tissues that may be propagated in this manner include but are not limited to lung, kidney, thyroid, intestine, and brain.
  • the lymph node is a key organ of the mammalian immune system that has evolved to mount an immediate and orchestrated response against invading pathogens.
  • the lymph node acts as a checkpoint where migrating T and B cells may encounter foreign antigens (8,9). If a foreign antigen is identified, T cells undergo rapid cell division and also signal for help and recruit additional T cells (8,9). To accommodate this sudden increase in cell number, lymphocytes need a special environment, which the lymph node provides.
  • lymph node is also one of the first clinically observed sites of most cancer metastasis. Selected cancer cells will often migrate away from a primary tumor and colonize the lymph node (10). Lymphatic vessels are designed to facilitate the uptake of surrounding fluid and cells, which are then transported to a nearby lymph node (10). Therefore, malignant tumor cells take advantage of this route normally traveled by immune cells. On arrival in the lymph node, tumor cells can survive, perhaps because the architecture of the lymph node provides direct access through the high endothelial venules to essential nutrients and growth factors found in the blood. The lymph node also contains fibroblastic reticular cells and other stromal cells that secrete chemokines to enhance cell recruitment and survival (8,9,1 1).
  • the present invention relates to the use of the lymph node environment to promote the survival and expansion of healthy cells and tissues. Healthy cell and tissue growth in lymph nodes would provide a new approach for cell therapies in regenerative medicine.
  • the present invention is based, at least in part, on the discoveries that (i) hepatocytes injected directly into a single jejunal, popliteal, axillary or periportal lymph node generate an ectopic hepatic mass and rescue mice from lethal liver failure; (ii) thymic tissue injected into single jejunal lymph nodes of athymic nude mice generates functional ectopic thymuses; (iii) pancreatic islets transplanted into single jejunal lymph nodes of streptozotocin-induced diabetic mice engraft and secrete insulin to normalize glucose concentrations; (iv) additional fetal tissues including brain, lung, intestine, kidney and thyroid could also be successful engrafted into lymph nodes, and in various cases produced a histologic resemblance to the native organ.
  • the present invention provides for methods of inducing tolerance in a subject to transplantation of allograft tissue.
  • the method of inducing tolerance comprises conditioning a transplant recipient with cells immune-matched to a donor of a subsequent allograft.
  • the immune-matched cells are thymic cells.
  • the thymic cells are transplanted into the lymph node of the recipient.
  • the method of inducing tolerance comprises increasing regulatory T cell (Treg) induction associated with cross-talk between donor thymus tissue and recipient thymus tissue.
  • Treg regulatory T cell
  • the Treg cells are CD4+, CD25+ and/or FoxP3+ Treg cells.
  • the present invention also provides a method of transplanting allograft tissue to a subject comprising (i) introducing non-lymphoid cells in a lymphoid tissue of the subject under conditions such that the cells are able to proliferate; and (ii) introducing allograft tissue to the subject after the non-lymphoid cells have been introduced into the lymphoid tissue of the subject.
  • the non-lymphoid cells are immune-matched to the allograft tissue.
  • the non-lymphoid cells are thymus cells.
  • FIGURE 1 Direct injection of hepatocytes into a single lymph node of a C57BL/6 wild-type mouse,
  • Jejunal lymph node LN, yellow dotted oval
  • primary hepatocytes which were mixed with 3% Evans blue dye and Matrigel before injection.
  • Scale bars 1 mm.
  • SP spleen
  • IP intraperitoneally
  • the bar graphs show the number of GFP+ hepatocytes and the percentage of BrdU+ hepatocytes per section after immunostaining at 2 weeks after transplantation in mice with or without PHx.*P ⁇ 0.05, **P ⁇ 0.0001.
  • Data (mean ⁇ s.e.m.) are representative of one experiment with three to five mice per group. The experiment was repeated twice. Scale bars, 100 ⁇ .
  • FIGURE 2 Direct injection of hepatocytes into a single lymph node of a Fah-/- mouse, (a) Macroscopic appearances of nontransplanted and transplanted (hepatized) lymph nodes (lymph nodes are marked by yellow dotted ovals). For nontransplanted axillary and popliteal lymph nodes, the presence of the lymph node is highlighted by injected
  • Glutamine synthetase (GS, red) expression shows unique zonal restriction surrounding terminal hepatic venules. Sections were counterstained with Hoechst 33342 (blue). Scale bars, left, 1 mm;
  • mice were injected with the immunosuppressive agents CTLA4-Ig and MR1 on days 0, 2, 4 and 6 after transplantation. The experiment was repeated twice. The images n the bottom show one representative LN-Tx mouse injected with CTLA4-Ig and MR1. Also shown are the macroscopic appearances of hepatized jejunal lymph nodes (yellow dotted ovals) and
  • FIGURE 3 Functional ectopic thymus in the jejunal lymph node
  • (a) Flow cytometric analysis of T cells in the peripheral blood.
  • WT wild-type C57BL/6
  • Nude BALB/c nude
  • BALB/c nude mice into which C57BL/6 GFP+ thymic cells were injected into a jejunal lymph node
  • LN-Tx nude The number values assigned to the gates and quadrants represent the percentage of total live cells within that gate or quadrant. All contour plots display 10% probability contours.
  • mice were WT 5 nude, KC ⁇ Tx nude (BALB/c nude mice transplanted under the kidney capsule) and LN-Tx nude.
  • the thin black line indicates the mean ⁇ s.e.m. No statistical significance (NS) was observed between kidney capsule and lymph node transplantation in each of the three groups, (b) Left, representative flow cytometric analysis of cells present in wild-type
  • C57BL/6 (WT) and LN-Tx nude mouse tissues The number values assigned to the gates and quadrants represent the percentage of total live cells within that gate or quadrant.
  • Bottom whole-mount jejunal lymph node of a nude mouse engrafted in the lymph node with GFP+ thymic cells (top left). The bright-field image was merged with the fluorescence.
  • Top right a frozen section with GFP+ donor thymic cells.
  • Bottom left WT native thymus stained for K5 (red) and 8 (green). Scale bars, 200 ⁇ .
  • Graphs show the data from individual mice, labeled as in panel c. The thin black line indicates the mean, (e) Top, Kaplan-Meier curves showing the survival of skin grafts from C57BL/6 or CBA/CaJ donor mice transplanted onto BALB/c nude recipients that had previously undergone
  • FIGURE 4 Ectopic pancreas generation in the jejunal lymph node after islet transplantation, (a) Top, whole-mount lymph node of a streptozotocin- treated diabetic C57BL/6 mouse engrafted with C57BL/6 GFP+ pancreatic islets. The bright-field image was merged with the fluorescence. Other images show
  • FIGURE 5 Neovascularization of ectopic tissue
  • (a) Vascular trees are shown in a native lymph node and native liver of a GFP transgenic mouse and in mice after hepatocyte transplantation into the lymph node
  • CD31 is a marker for blood vessels
  • CD 105 and Collagen IV are markers for neovascularization.
  • Vasculature markers are shown in red, and ectopic tissue is shown in green. All sections were counterstained with Hoechst 33342 in blue. All scale bars are 100 ⁇ .
  • FIGURE 6 Distribution of immune cells in the hepatized jejunal LN of a rescued C57BL/6 Fah-/- mice at 12 weeks after transplantation. Left to right panels, immunostaining of frozen LN serial sections with mAbs (red) against
  • CD4/CD8 CD4 and CDS T cells
  • Gr-1 granulocytes
  • F4/80 macrophages
  • FIGURE 7 Representative flow cytometry analysis of peripheral blood T cells. Analysis of CD4 and CD8 T cells from C57BL/6 GFP+ mice, wild type C57BL/6, BALB/c Nude, kidney capsule (KC) transplanted (Tx) BALB/c Nude, and lymph node (LN) transplanted BALB/c Nude mice.
  • the number values assigned to the gates and quadrants represent the percentage of total live cells within that gate or quadrant. All contour plots display 10% probability contours.
  • FIGURE 8 Presence of peripheral T cells 10 months after C57BL/6
  • FIGURE 10A-B A. C57BL/6 GFP+ thymic tissue engrafts in the subcapsular space of the jejunal BALB/c Nude LN. Frozen section of an ectopic thymus with GFP+ donor thymic cells and stained with cytokeratin 8 (K8) in red. Hoechst counterstain is shown in blue. Scale bar: 100mm.
  • FIGURE 11 Harvest of the uterine horns from a pregnant mouse, and removal of placenta and fetal membranes from an embryo. Sagittal and transversal paraffin sections of an embryo stained with Hematoxylin and Eosin. B, Scheme of the jejunal lymph node injection procedure using different mouse fetal tissues.
  • FIGURE 12 Each panel shows paraffin (A, B and D) or frozen (C) sections of donor C57BL/6 GFP+ tissues stained with Hematoxylin and Eosin or Hoechst, respectively, whole-mount jejunal lymph nodes of C57BL/6 mice 3 weeks after transplantation, and immunofluorescence staining of frozen lymph node serial sections with the presence of GFP+ cells.
  • FIGURE 13 (Upper) Dissection of mouse thyroid gland. (Bottom) Scheme of the jejunal lymph node injection procedure, whole-mount jejunal lymph node 3 weeks after transplantation, and immunofluorescence staining of a frozen lymph node section with the presence of GFP+ cells.
  • FIGURE 14A-C Problems associated with determining gestational age.
  • FIGURE 15 Table showing mice injected and lymph nodes repopulated for various organ types.
  • FIGURE 16 Transplantation of thyroid gland into lymph node.
  • FIGURE 17 Transplantation of liver into lymph node.
  • FIGURE 18 Transplantation of brain tissue into lymph node.
  • FIGURE 19 Transplantation of lung tissue into lymph node.
  • FIGURE 20 Transplantation of intestinal tissue into lymph node.
  • FIGURE 21A-C Transplantation of kidney tissue into lymph node.
  • FIGURE 22A-D Transplantation of kidney tissue into lymph node.
  • FIGURE 23A-D The lymph node is a permissive site for kidney organogenesis.
  • A Schematic view of kidney transplantation into the lymph node.
  • B Hematoxylin and Eosin (H&E) staining of a paraffin section of donor C57BL/6 GFP+ embryonic kidney showing S-shaped bodies (upper left); whole-mount jejunal lymph node of a C57BL/6 mouse 3 weeks after embryonic kidney transplantation (upper right), and picture of a frozen lymph node section stained for reticular fibroblasts and reticular fibers (ER-TR7), with the presence of GFP+ cells (lower). Nuclei were counterstained using Hoechst. (C).
  • FIGURE 24A-C Proliferative and urine-concentrating ability of 6- week ectopic grafts.
  • A GFP positivity (left) and H&E staining (right) of a frozen or paraffin section of a jejunal lymph node 6 weeks after transplantation.
  • B Picture of paraffin lymph node sections stained for GFP or BrdU (AEC, red color).
  • C C).
  • FIGURE 25 Host cells vascularize the developing tissue.
  • FIGURE 26A-F Renal cyst development in repopulated lymph nodes.
  • A Whole-mount jejunal lymph node of a C57BL/6 mouse 12 weeks after embryonic kidney transplantation showing GFP positivity (left), and hematoxylin and eosin (H&E) staining showing cysts (right).
  • B Whole-mount jejunal lymph node of a C57BL/6 mouse 12 weeks after embryonic kidney transplantation showing GFP positivity (left), and hematoxylin and eosin (H&E) staining showing cysts (right).
  • B Whole-mount jejunal lymph node of a C57BL/6 mouse 12 weeks after embryonic kidney transplantation showing GFP positivity (left), and hematoxylin and eosin (H&E) staining showing cysts (right).
  • cyst #1 epithelium stained with H&E periodic acidschiff (PAS), masson's trichrome (TRI), picro-sirius red (PSR), GFP, BrdU, aquaporin-1 (AQP1), and sodium-potassium-chloride transporter 2 (NKCC2) (left), and of cyst #2-3 epithelium stained with H&E, PAS, TRI, PSR, GFP, BrdU, AQP1 and 2 (right, yellow arrows indicates vacuoles).
  • C Details of proteinaceous material and fibers found inside cyst #1 (left) and of round globules found inside cyst #2 and 3 (right), after staining with H&E, PAS, TRI, and PSR.
  • D Details of proteinaceous material and fibers found inside cyst #1 (left) and of round globules found inside cyst #2 and 3 (right), after staining with H&E, PAS, TRI, and PSR.
  • FIGURE 27A-D Bone marrow-derived host cells contribute to mesangial cells and podocyte regeneration.
  • A Fluorescence intensity profiles of GFP expressing leukocytes in peripheral blood of bone marrow chimeric mice. Blood of a wild type and a GFP+ mouse were used as negative and positive control, respectively.
  • B Overview of experimental plan (IR, irradiation; EK, embryonic kidney; LN, lymph node).
  • C Representative ectopic glomerulus grown inside lymph nodes of bone marrow chimeric mice, showing bone marrow- derived cell contribution to glomerular mesangium. Sections were stained with collagen IV antibody and nuclei were counterstained using Hoechst.
  • D The following procedures were stained with collagen IV antibody and nuclei were counterstained using Hoechst.
  • FIGURE 28A-C Nephrectomy accelerates kidney organogenesis and degeneration.
  • A Overview of experimental plan (EK, embryonic kidney; Nx, nephrectomy; LN, lymph node; K, kidney; see Materials and Methods section for details).
  • B Renal cell proliferation shown by bromodeoxyuridine (BrdU)
  • FIGURE 29 Schematic view of transplantation of E14.5-15.5 or P3 kidney into the lymph node, and immunofluorescence staining for podoplanin of an adult kidney section isolated from a GFP+ mouse (lower).
  • FIGURE 30A-B The immunofluorescence staining for podoplanin of lymph node frozen sections 3 weeks after transplantation of embryonic (left) or new born (right) kidney. Nuclei were counterstained using Hoechst. FIGURE 30A-B.
  • A Gating strategy for fluorescence-activated cell sorting (FACS) analysis of peripheral blood of chimeric mice 6 weeks after bone marrow transplant. Blood cells were gated on live cells, singlets, leukocytes (upper). Both GFP+ and GFP- leukocyte subsets were analyzed for CD3, CD19/CD45R or CD1 lb/Ly6G-LY6C (middle). The CD3+ cell population was further analyzed for CD4/CD8 (lower). (B).
  • FACS fluorescence-activated cell sorting
  • Dot plot graph showing percentages of different GFP+ leukocyte populations from bone marrow chimeric mice.
  • C Stacked bar graph showing percentages of donor GFP+/Ly6G-LY6C+ versus host GFP-/Ly6G-LY6C+ cells. Cells were gated on live, Ly6G-LY6C, GFP. A representative histogram profile is shown. Blood from a wild type and a GFP+ mouse were used as control.
  • FIGURE 31 Representative ectopic glomerulus grown inside lymph nodes of bone marrow chimeric mice 10 weeks after transplantation showing nodular lesion. Sections were stained with claudin-2, WT-1, podoplanin, CD31, keratin-8, and vimentin. Nuclei were counterstained using Hoechst.
  • FIGURE 32A-C Astrogenesis in the developing ectopic brain.
  • A Schematic view of transplantation of multiple embryonic tissues into the lymph node (scale bar, 1mm).
  • B Table shows percentages of engraftment into the mouse lymph node for different tissues.
  • CI Hematoxylin and Eosin (H&E) staining of a paraffin section of donor embryonic brain (upper left); whole-mount jejunal lymph node 3 weeks after embryonic brain transplantation (GB3 LN, upper right), and pictures of frozen lymph node (GB3 and GB4 LN) sections with the presence of GFP+ cells (lower). Nuclei were counterstained using Hoechst.
  • C2 Mouse embryonic brain transversal section (upper), and pictures of GB3 and GB4 LN sections stained for GFAP6 with the presence of GFP+ cells (lower). Nuclei were counterstained using Hoechst,
  • FIGURE 33A-D Granulocyte/macrophage progenitor accumulation following embryonic thymus transplantation into the lymph node (LN), and host contribution in the generation of the ectopic thymic cortex.
  • A Gating strategy for FACS analysis of peripheral blood of mice (M1-M5) receiving thymus transplant into their lymph nodes. Blood cells were gated on live cells, leukocytes, singlets, granulocyes/myeloid cells or lymphocytes.
  • B Representative fluorescence intensity histograms of granulocyes/myeloid cells from M2 analyzed for Ly6G-Ly6C (upper) or CD1 lb (lower) at 0, 3, 6, 12, or 21 weeks after thymus transplant.
  • C Representative fluorescence intensity histograms of granulocyes/myeloid cells from M2 analyzed for Ly6G-Ly6C (upper) or CD1 lb (lower) at 0, 3, 6, 12, or 21 weeks after thymus transplant.
  • Each symbol represents one mouse, and the horizontal bars represent the median values.
  • FIGURE 34A-C Contribution of the host in the generation of the ectopic thymic cortex.
  • A Agarose gel electrophoresis of PCR products following semi-quantitative RT-PCR analysis for GM-CSF (expected amplicon size of 431 bp) in embryonic thymus (EmT), 6- (6wEcT) or 21 -week ectopic thymus (21wEcT), and adult thymus (AdT). Wild type lymph node (LN) was used as a negative control. Amplification of GAPDH was used as an internal control.
  • the densitometric scanning data from two experiments are shown as bar graphs of GM-CSF/GAPDH ratio on the right (6wEcTs were isolated from M4 and M5, while 21wEcTs were isolated from Ml and M3).
  • B Picture of thymus glands isolated from a C57BL/6 GFP+ embryo (upper left) and Ff&E staining of a paraffin section of embryonic thymus (EmT, upper right); whole-mount mouse jejunal lymph nodes 21 weeks after embryonic thymus transplantation, showing different engraftment (21wEcT, lower).
  • C Picture of thymus glands isolated from a C57BL/6 GFP+ embryo (upper left) and Ff&E staining of a paraffin section of embryonic thymus (EmT, upper right); whole-mount mouse jejunal lymph nodes 21 weeks after embryonic thymus transplantation, showing different engraftment (21wEcT, lower).
  • FIGURE 35A-C Presence of terminally differentiated, mucus- producing cells in ectopic lung, stomach and intestine tissues.
  • A-C Each panel shows H&E staining of a paraffin section of donor embryonic lung (ErnL), stomach (EmS) or intestine (Eml); whole-mount jejunal lymph node 3 weeks after
  • FIGURE 36 Ectopic liver, pancreas and thymus has been successfully grown in lymph node tissue.
  • FIGURE 37 Experimental plan for assessing the utility of transplanted thymus in mediating acceptance of allografts.
  • 129 Fah-/- mice were transplanted with neonatal (d2-4) Balb/c GFP thymus in the lymph node (LN).
  • An immunosuppression regiment consisting of MR-1 and rapamycin was started concomitantly.
  • 6 weeks after thymus transplant mice were either given skin grafts or hepatocyte transfers to assess ability of thymus to mediate acceptance of subsequent allogeneic grafts. Mice receiving just immunosuppression (IS controls) were used as controls.
  • FIGURE 38A-C Presence of donor-specific GFP+ cells in the lymph node (LN) of 129sv mice transplanted with Balb/c-GFP neonatal thymus in the lymph node.
  • LN lymph node
  • B GFP+ cells in thymus-transplanted LN were positive for CD1 lc and EpCam, markers of thymic dendritic cells (DCs) and epithelial cells, respectively.
  • C Thymus-transplanted LN showed presence of CD4/CD8 double-positive (DP) T cells (a marker of T cell development), similar to a regular thymus. No presence of DP T cells in a non-transplanted LN.
  • FIGURE 39A-D Long-term acceptance of allogeneic hepatocytes mediated by ectopic thymus in the lymph node.
  • A C57BL6/J and Balb/c skin grafts in 129.Fah-/- mice receiving Balb/c thymus
  • B Rescue of liver failure as evidenced by weight gain and survival in Balb/c thymus-transplanted 129.Fah-/- mice receiving Balb/c hepatocytes.
  • C Liver in Balb/c hepatocyte transferred mice showed normal liver morphology by hematoxylin and eosin (H&E) staining (i) and positivity for FAH by immunohistochemistry (IHC) (ii).
  • (iii) shows the presence of transplanted GFP+ Balb/c hepatocytes that are also positive for FAH by immunofluorescence (IF).
  • (D) Balb/c thymus-transplanted mice show normal levels of liver enzymes in the serum after Balb/c hepatocyte transfer.
  • FIGURE 40A-C Ectopic thymus induces donor-specific tolerance and increased incidence of Tregs in recipients.
  • A Splenocytes from Balb/c thymus- transplanted mice were labeled with CellTraceTM and co-cultured with naive 129.sv (top panel), C57BL6/J (middle panel) or Balb/c splenocytes (bottom panel). After 72 hours, cells were analyzed for CellTraceTM dilution by flow cytometry.
  • B IFNy levels from cell culture supematants in (A).
  • FIGURE 1A-C Characterization of cells migrating from ectopic to native thymus.
  • A Presence of GFP+ cells in the native thymus of mice transplanted with Balb/c mice (top panel, 4x magnification).
  • B GFP+ cells present in the native thymus were MHC-II + (middle panel) and CD1 lc+ (bottom panel).
  • C GFP+ cells in the native thymus of mice transplanted with Balb/c-GFP thymus in the LN are observed to be interacting with CD4+ cells (i, inset) and CD4+CD25+ cells (ii and iii, inset). Images in C (i) and C (ii, iii) are at lOx and 40x magnification, respectively.
  • FIGURE 42A-C Migrating DCs induce Tregs capable of suppressing T cell activation.
  • A CD1 lc+ GFP+ cells from native thymus were co-cultured with naive CD4+ thymocytes. After 72 hours, the culture was analyzed for proportion of CD4+CD25+ cells (Tregs) in culture.
  • B Summarized data for Treg percentages in cultures described in (A).
  • C Tregs generated in culture in (B) are efficient in suppressing CD4+ T cell proliferation, as determined by ⁇ levels. Results representative of one of three independent experiments.
  • FIGURE 43 Model depicting cross-talk between ectopic and native thymus in transplant recipient showing migration of antigen-presenting cells which induce Tregs.
  • the present invention provides for methods of propagating non- lymphoid cells in a lymphoid tissue for the purpose of producing a non-lymphoid tissue and or/providing a desirable biological function.
  • Suitable cells include but are not limited to embryonal cells, non- embryonal cells, progenitor cells, and reprogrammed somatic cells (47).
  • Cells may be human or non-human cells (eg non-human primate, dog, cat, pig, cow, horse, sheep, goat, mouse, rat, rabbit, etc.). Cells may be autologous, allogeneic, or xenogeneic.
  • Suitable cells include but are not limited to a kidney cell or a partially differentiated kidney cell or a kidney progenitor cell, a lung cell or a partially differentiated lung cell or a lung progenitor cell, a thyroid cell or a partially differentiated thyroid cell or a thyroid precursor cell, a brain cell or a partially differentiated brain cell or a brain progenitor cell, an intestinal cell or a partially differentiated intestinal ceil or an intestinal precursor cell; a thymus cell or a partially differentiated thymus cell or a thymic progenitor cell, a pancreas cell or a partially differentiated pancreas cell or a pancreas precursor cell (in the foregoing, including islet cells), a liver cell a partially differentiated liver cell and a liver precursor cell, a stomach cell or a partially differentiated stomach cell or a stomach precursor cell and so forth.
  • a kidney cell or a partially differentiated kidney cell or a kidney progenitor cell a lung cell or a partially differentiate
  • Cells can be implanted into a lymph node or multiple lymph nodes. Such methods are intended to encompass implantation into any lymph node, including, but not limited to: abdominal lymph nodes, celiac lymph nodes, paraaortic lymph nodes, splenic hilar lymph nodes, porta hepatis lymph nodes, gastric lymph nodes (left and right), gastroomental (gastroepiploic) lymph nodes (left and right), retroperitoneal lymph nodes, pyloric lymph nodes (suprapyloric, subpyloric, retropyloric), pancreatic lymph nodes (superior pancreatic, inferior pancreatic, splenic lineal lymph nodes), hepatic lymph nodes (cystic, foraminal— including foramen of Winslow), pancreaticoduodenal lymph nodes (superior pancreaticoduodenal, inferior pancreaticodoudenal), superior mesenteric lymph nodes, ileocolic lymph
  • the cells are injected into a recipient's lymph node from a donor's tissue.
  • the present invention provides for a method of propagating non-lymphoid cells in a lymphoid tissue for the purpose of producing a non-lymphoid tissue and or/providing or supplementing a desirable biological function, comprising introducing, into a lymph node in a host, non-lymphoid cells, under conditions such that the cells are able to proliferate.
  • the host may be a human or non-human (eg non-human primate, dog, cat, pig, cow, horse, sheep, goat, mouse, rat, rabbit, etc).
  • non-human primate dog, cat, pig, cow, horse, sheep, goat, mouse, rat, rabbit, etc.
  • the present invention provides for a method of providing or supplementing a biological function in a host comprising introducing, into a lymph node of a host, a non-lymphoid cell of an organ where the cell or organ normally provides said function; for example, but without limitation, introducing an islet cell to provide insulin production, or , introducing a thyroid cell to provide thyroxin, or introducing a kidney cell to provide kidney function (filtering, etc.), or introducing a thymus cell to provide immune function, etc.
  • the present invention provides for methods of inducing tolerance in a subject to transplantation of allograft tissue.
  • the method of inducing tolerance comprises conditioning a transplant recipient with cells immune-matched to a donor of a subsequent allograft.
  • the immune-matched cells are thymic cells.
  • the thymic cells are transplanted into the lymph node of the recipient.
  • the allograft tissue comprises a liver cell or tissue. In certain embodiments, the allograft tissue comprises a skin cell or skin tissue.
  • the method of inducing tolerance comprises increasing regulatory T cell (Treg) induction associated with cross-talk between donor thymus tissue and recipient thymus tissue.
  • Treg regulatory T cell
  • the Treg cells are CD4+, CD25+ and/or FoxP3+ Treg cells.
  • the present invention also provides a method of transplanting allograft tissue to a subject comprising (i) introducing non-lymphoid cells in a lymphoid tissue of the subject under conditions such that the cells are able to proliferate; and (ii) introducing allograft tissue to the subject after the non-lymphoid cells have been introduced into the lymphoid tissue of the subject.
  • the non-lymphoid cells are immune-matched to the allograft tissue.
  • the non-lymphoid cells are thymus cells.
  • donor hepatocytes were isolated from C57BL/6 GFP+ mice (GFP transgene under the control of the human ubiquitin C promoter, C57BL/6-Tg(UBC- GFP)30Scha/J, Jackson Laboratory) and transplanted into 129sv Fah-/- mice.
  • C57BL/6 GFP+ mice GFP transgene under the control of the human ubiquitin C promoter, C57BL/6-Tg(UBC- GFP)30Scha/J, Jackson Laboratory
  • 129sv Fah-/- mice were backcrossed for more than eight generations with C57BL/6 mice (Jackson
  • Luciferase C57BL/6 transgenic mice (Luc+) expressing firefly luciferase under the control of the broadly expressed ⁇ -actin promoter were kindly provided by S. Thorne (University of Pittsburgh).
  • Donor primary hepatocytes were isolated from adult (5- to 8-week-old) mice. Donors and recipients were not matched according to gender.
  • Newborn (1- to 3 -day-old) C57BL/6 GFP+ mice were used as donors of thymic cells.
  • Athymic BALB/c nude Foxnl u (Harlan) mice were used as recipients of thymic cells. Blood collection (100 ⁇ ) was performed using the submandibular bleeding technique.
  • hepatocytes were isolated using the two-step collagenase perfusion technique. The number and viability of the cells were determined by trypan blue exclusion. For each recipient, 100,000-500,000 viable cells were suspended in 20 ⁇ Matrigel (BD Biosciences) and kept on ice until transplantation. Recipient mice were anesthetized with 1-3% isoflurane and laparotomized. The jejunal lymph node was exposed, and cells were injected using a 28 G needle under a dissecting scope (Leica). Just after injection, the contact site was clipped for 5 min by micro clamp to prevent cell leakage. In the experiments with Fah-/- mice, the mice were kept on NTBC-containing drinking water at a concentration of 8 mg/1 until transplantation.
  • NTBC was discontinued just after surgery.
  • Thymuses were harvested from newborn GFP+ transgenic mice and cut into small fragments. The jejunal lymph nodes were exposed, and cells were injected with minced thymus tissue through a 20 G needle. Thymus tissue was also grafted beneath the kidney capsule as a positive control. For the kidney capsule experiments, an incision was made on the left side of the peritoneal cavity, and the kidney was exposed. A small hole was made in the capsule, and the thymus was inserted between the kidney capsule and the arenchyma. Light cauterization was used to seal the opening. The wound was closed with surgical sutures.
  • mice Primary hepatocytes from C57BL/6 GFP+ mice were transplanted into 11 mice, as described above. Recipient mice were given drinking water containing 0.8 mg/ml BrdU immediately after surgery. After 1 week, we euthanized three mice for analysis. Partial hepatectomy (PHx), in which two-thirds of the liver of a mouse is removed, was then performed on 5 of the 11 mice, and the remaining 3 mice were used for controls. One week later, all eight mice were euthanized for analysis. Using histological sections, we determined the amount of hepatocyte engraftment in the lymph nodes by counting GFP+ hepatocytes and determined the ratio of proliferating hepatocytes by counting BrdU+ hepatocytes.
  • mice Primary hepatocytes from C57BL/6 GFP+ mice were transplanted into ten 129sv Fah-7- mice by lymph node or splenic injection. Five out of ten mice from each group were intraperitoneally injected with the immunosuppressive drugs CTLA4-Ig (0.25 mg) and MR1 (0.25 mg), a kind gift from F. Lakkis (University of Pittsburgh), on days 0, 2, 4 and 6 after transplantation.
  • CTLA4-Ig 0.25 mg
  • MR1 MR1
  • Antibodies specific to the following antigens were purchased for immunom ' stochemistry: ER-TR7, LYVE1 , GFP, glutamine synthetase, CCR7, S1PR1 (EDG1), F4/80 (Abeam), PNAd, B220, CD4, CD8, CD31, CD105 and Gr-1 (BD Biosciences), BrdU (Santa Cruz Biotechnology), DPPIV (AbD Serotec), E-cadherin (Zymed), Collagen IV (SouthemBiotech), keratin 5 (Covance), keratin 8 (DSHB) and C-peptide and glucagon (Cell Signaling Technologies).
  • Antibodies specific to the following antigens were purchased for flow cytometric analysis: APC mouse CD3-s, APC-Cy7 mouse CD8-a, phycoerythrin (PE)-Cy7 mouse CD45 and PE mouse CD4, a mouse ⁇ TCR screening panel, a mouse naive and memory T cell panel (Pharmingen) and a mouse regulatory T (Treg) cell detection kit (Miltenyi).
  • Tissue was fixed in 4% paraformaldehyde for 4 h, stored in 30% sucrose for 12 h and then embedded in optimal cutting temperature (OCT) medium, frozen and stored at -80 °C.
  • Sections 5-10 (5-10 (m) were mounted on glass slides and fixed in cold acetone for 10 min.
  • For immunohistochemical staining sections were washed with PBS and blocked with 5% bovine serum albumin (BSA) or milk for 30 min. Sections were then incubated with primary antibody for 1 h and secondary antibody for 30 min. Sections were mounted with Hoechst mounting media. Images were captured with an Olympus FluoView 1000 Confocal Microscope or an Olympus 1X71 inverted microscope.
  • FACS fluorescence- activated cell sorting
  • Antibodies were added at a dilution of 1/10 in blood and mixed by gentle pipetting. Reactions were incubated in the dark in an ice slurry bath for 30 min. Three milliliters of Red Blood Cell Lysing Buffer (Sigma) was added to each tube, lightly vortexed and incubated for an additional 5 min. Two milliliters of flow buffer (2% FBS in HBSS) was added to the tubes, mixed and centrifuged at 500g for 5 min. The supernatant was aspirated, and secondary antibody was added at a dilution of 1/50 in flow buffer and mixed by gentle pipetting.
  • FACS fluorescence- activated cell sorting
  • FACSVantage Cell Sorter a BD Aria II Cell Sorter or a Miltenyi MACSQuant.
  • Tail skin graft Tail skin graft.
  • Recipient mice (BALB/c nude) containing an ectopic thymus
  • C57BL/6 in the lymph node were anesthetized, and the tail skin (5 mm ⁇ 5 mm), from CBA/CaJ or C57BL/6 mice was grafted on the left and right lateral sides of the superior dorsal region of the recipient mouse, respectively. A bandage was applied and removed 7 d after surgery. The grafts were observed daily for rejection. Tumor cell transplantation.
  • Tumor size was recorded once per week using a caliper and calculated as ( ⁇ /6) x (length (mm) ⁇ width2 (mm 2 )).
  • LPS (1 mg per kg of body weight, Sigma) was injected intraperitoneally into C57BL/6 wild-type or normoglycemic mice that previously received an islet transplant to the lymph node. Mice were bled to determine glucose concentrations or measure serum cytokine levels. Cytokine level was determined by ELISA assay for TNF-a, IL- ⁇ and IL-6 (eBioscience).
  • hepatocytes were distributed mainly in the subcapsular sinus of the lymph nodes but were not present in the lymph node follicles or germinal centers (Fig. lc). Additionally, the hepatocytes rapidly formed patches of hepatic tissue expressing E-cadherin (Fig. Id). This hepatocyte-to-hepatocyte attachment may help retain hepatocytes within the site of transplantation (14).
  • sphingosine-1 -phosphate receptor 1 S1PR1
  • CCR7 a molecule known to control the migration of memory T cells and/or tumor cells into the lymph nodes (17) was expressed by hepatocytes in the lymph nodes (Fig. Id).
  • PHx partial hepatectomy
  • the number of GFP-expressing hepatocytes and the percentage of BrdU+ hepatocytes in the lymph nodes of the mice subjected to PHx were significantly higher than those in the mice not subjected to PHx (Fig. le), indicating that hepatocytes in the lymph nodes respond to growth stimuli after PHx.
  • the 'hepatized' lymph nodes were composed mostly of newly formed liver tissue containing Fah+GFP+ hepatocytes but also included remnants of the lymphatic system, as revealed by surrounding LYVE1+ lymphatic endothelial cells (Fig. 2b).
  • the lymph nodes were transformed into hepatic organoids composed of characteristically cuboidal hepatocytes but with the absence of a biliary system (12).
  • hepatized lymph node microarchitecture To define the hepatized lymph node microarchitecture, we stained recipient lymph nodes for ER-TR7, a marker of fibroblastic reticular cells (FRCs), dipeptidyl peptidase-4 (DPP IV), a marker for brush borders of hepatocytes and bile canaliculi organization, and glutamine synthetase, a marker of zonality for hepatocytes surrounding the terminal hepatic venules 22 (Fig. 2b). GFP+ hepatocytes resided in cellular proximity with ER-TR7+ FRCs. FRCs are known to have a crucial role in establishing the reticular network, as well as in regulating immune function (1 1,23).
  • DPP IV was localized throughout the hepatized lymph nodes. Moreover, we identified glutamine synthetase-positive hepatocytes surrounding some of the hepatic veins in the newly formed liver tissues. Notably, we observed survival of the mice (Fig. 2c) and long- term persistence (over 25 weeks) of the graft after transplantation to the lymph nodes (Fig. 2a), and we found no immune responsiveness to the grafts, as indicated by the presence of very few lymphocytes and macrophages around the transplantation zones (Fig. 6).
  • lymph nodes perform a central function for allogeneic recognition (25), we next tested whether successful engraftment is possible in allogeneic lymph nodes.
  • 129sv and C57BL/6 mice share the major histocompatibility complex haplotype H2b but differ in their minor
  • thymic epithelia have been distinguished by their cytokeratin 5 (K5) and cytokeratin 8 (K8) phenotypes (30).
  • the ectopic thymuses were present in the subcapsular sinus of the lymph nodes (Fig. 10) and contained both 5- and K8-positive regions, which correspond with thymic medullary and cortical epithelia, respectively (Fig. 3b).
  • the ectopic thymuses were also analyzed for the presence of recipient double-positive CD4+CD8+ thymocytes, which represent immature T cells undergoing thymic selection (31).
  • the ectopic thymuses contained recipient double-positive thymocytes as well as single-positive CD4+ and CD8+ T cells, indicating a selective mechanism of T-cell commitment and maturation (Fig. 3b).
  • TCR T-cell receptor
  • Heterozygous BALB/c nude mouse splenocytes expressed 10 out of the 15 ⁇ segments, which is consistent with known partial or complete genetic deletions of certain ⁇ segments ( ⁇ 3, ⁇ 5.1,5.2, ⁇ 9, ⁇ 1 1 and ⁇ 12) in this strain.
  • splenocytes in LN-Tx nude recipients expressed a ⁇ profile similar to that of the heterozygous BALB/c nude mice (Fig. 3c).
  • splenic T cells in LN-Tx nude mice by flow cytometry to determine whether regulatory, naive, central memory and effector memory T cell subsets were present.
  • We detected regulatory T cells by analyzing FoxP3 expression in CD4+CD25+ T cells.
  • naive and memory T cells using differential expression of CD44 and CD62L (where naive cells are CD44-CD62L+, central memory cells are CD44+CD62L+, and effector memory cells are CD44+CD62L-).
  • naive and memory T cell subsets among splenocytes from C57BL/6, heterozygous nude and LN-Tx nude mice Fig. 3d.
  • the percentage of effector memory T cells was higher and the percentage of naive T cells was lower in the LN-Tx nude mice than in the other two groups, perhaps because these mice had been previously exposed to skin grafts (see below).
  • LN-Tx nude mice contained a range of peripheral single- positive CD4+ and CD8+ T cells, we asked whether the de novo immune system in these recipient mice could mount a T cell-mediated response against a skin allograft.
  • lymph node might also provide a suitable environment for pancreatic islet transplantation.
  • GFP+ islets within the subcapsular sinus of the lymph nodes adjacent to the densely packed lymphocytes that are indicative of a typical lymph node (Fig. 4a).
  • TNF-a tumor necrosis factor a
  • IL- ⁇ interleukin- ⁇
  • IL-6 IL-6
  • Lymph nodes injected with hepatocytes, thymic cells or islets contained many recipient-derived CD31+ endothelial cells pervading the areas of engraftment, suggesting that extensive bloodvessel remodeling took place during the ectopic tissue engraftment (Fig. 5b and Supplementary Fig. 5).
  • CD 105+ (Endoglin) cells and Collagen 1V+ cells which are markers of neovascular remodeling
  • lymph nodes there are over 500 lymph nodes in the human body, many of which are relatively easily accessible. Although a single lymph node structurally limits the number of donor cells that can be transplanted, it is technically feasible to transplant more than one lymph node to gain sufficient organ or tissue function from the transplanted cells. The potential loss of function in a few lymph nodes does not seem to compromise the overall function of the lymphatic system. In fact, lymphedema is the most common complication after lymphadenectomy in patients with cancer and only affects a limited number of these patients (37). Compression syndrome and lymphatic spread are two of the common issues in patients with cancer that have metastatic
  • Lymph node biopsies by fine-needle aspiration are a routine diagnostic procedure, with lymph nodes often being readily identified by palpation or ultrasound guidance.
  • the clinical application of lymph node injection has been validated, with patients rating the procedure as less painful than venous puncture (38).
  • ultrasound guidance can be used to successfully inject the visceral mesenteric lymph nodes (39).
  • the strong clinical precedent of ultrasound-guided lymph node injections may help make this technique readily adaptable to a clinical setting.
  • These minimally invasive techniques may also provide a potential therapy for patients who are ineligible for a more invasive therapy because of comorbidities.
  • liver function gradually deteriorates and transplantation of several lymph nodes with hepatocytes will create enough hepatic mass to stabilize the liver disease.
  • the hepatic mass generated in the lymph node may provide enough hepatic function to facilitate regeneration of the native liver.
  • transplantation of heterotopic liver has been discussed at length in the literature as a possible alternative to orthotopic liver transplantation (34,40,41).
  • Thymus transplants have been performed exclusively in the quadriceps of pediatric patients with complete DiGeorge syndrome (4). Unfortunately, children with DiGeorge syndrome often show poor growth, and transplantation is frequently postponed to allow for further development (42). Furthermore, a lack of
  • Transplanting thymic cells into the lymph node may represent an advantageous site to provide thymic function.
  • pancreatic islet transplantation to increase function, reduce necessary implantation mass and decrease immunogenicity is still under debate (6,44-46).
  • proximity to a good vascular supply is clearly essential for the survival of islet cells, as well as for that of hepatocytes and thymic epithelial cells.
  • lymph node One concern for cell transplant into the lymph node is the rapid immune response that can be initiated by the introduction of a foreign antigen into a site that is densely packed with lymphocytes.
  • lymphocytes One concern for cell transplant into the lymph node is the rapid immune response that can be initiated by the introduction of a foreign antigen into a site that is densely packed with lymphocytes.
  • Reprogramrning somatic cells provides an exciting potential source of donor cells for regenerative medicine (47).
  • these cells can be derived from autologous material and are capable of being recognized as 'self by the host immune system, they can potentially overcome immunologic barriers.
  • recent studies have suggested that these autologous cells may not be entirely protected from the immune system (48).
  • the lymph node may be an effective transplantation site for reprogrammed somatic cells that can be developed for organ regeneration purposes.
  • lymph node as a site for functional cellular transplant.
  • hepatocytes thymuses or pancreatic islets
  • This new approach of using the lymph node as an in vivo bioreactor in which to regenerate functional organs ma be beneficial to the field of regenerative medicine.
  • jejunal lymph nodes have been identified as alternative ectopic sites able to provide a permissive environment for liver, pancreas and thymus cells. We aimed at
  • Fig. 12a-d Our preliminary data suggests that jejunal lymph nodes can favor the engraftment/maturation of several tissues. Differentiation of lung tissue from a pseudoglandular to a mixed saccular/alveolar morphology was observed (Fig. 12b) . Crypt- like structures developed following injection of intestinal fragments into lymph nodes (Fig. 12c). Importantly, goblet-like cells were observed all along these structures, suggesting the presence of terminally differentiated intestinal cell types. Similarly, well-developed renal corpuscles were found in repopulated lymph nodes (12d).
  • lymph nodes carry out specific functions.
  • Our lab has previously shown that primary hepatocytes injected intraperitoneally into mice lacking fumarylacetoacetate hydrolase (a mouse model of tyrosinemia type I) migrate and colonize the host abdominal lymphatics and restore hepatic functions (1). More recently, hepatocytes and pancreatic islets injected directly into a single lymph node were shown to generate functional tissues, rescuing mice from lethal liver failure, and streptozotocin-induced diabetes, respectively (2). Similarly, de novo thymus function could be generated in athymic mice by injecting thymic tissues into lymph nodes (2).
  • the lymph node Beyond its capacity in supporting the maturation of fetal tissues, the lymph node also provided a suitable environment for adult thyroid gland regeneration (Fig. 13). Indeed, follicle-like structures were observed in the repopulated lymph node, suggesting that the thyroid function might be restored in human patients after total thyroidectomy, by simply using their lymph nodes as bioreactors, thus avoiding thyroid hormone replacement medication for the rest of their life.
  • Fig. 11 Fetal tissues were isolated for implantation as set forth in Fig, 11. There are a number of problems associated with determining gestational age in embryonic mice (see Figs 14A-C). Injections of various fetal tissues were performed and in many cases resulted in lymph node repopulation (Fig. 15).
  • Fig. 16 shows exemplary transplantation of thyroid gland tissue into lymph node. Transplantation of liver tissue in lymph node is shown in Fig.17.
  • Fig. 18 shows the results of transplanting brain tissue into lymph node; brain tissue was observed to grow well in lymph node.
  • transplantation of lung tissue into lymph node was observed to result in differentiation of lung tissue from a pseudoglandular to a mixed saccular/alveolar morphology.
  • Fig. 15 shows exemplary transplantation of thyroid gland tissue into lymph node. Transplantation of liver tissue in lymph node.
  • Fig. 18 shows the results of transplanting brain tissue into lymph node; brain tissue was observed to grow well in lymph node.
  • Figs 21 A-C show the results of kidney tissue transplant into lymph node showing the presence of renal-like histology and the presence of renal cell markers.
  • a well-developed renal corpuscle was found within GFP+ tissue inside the repopulated lymph node, and its mean volume was increased 3-fold with respect to parental tissue.
  • the glomerulus- like structure was not GFP+.
  • GK2, GK3, and GK4 lymph nodes were injected with kidneys isolated from embryos derived from the same mother, however, well- developed renal corpuscles were observed only in the GK3 lymph node. This could reflect variability among embryos or recipient mice. Further results relating to the transplant of kidney tissue into lymph node are shown in Figs 22A-D. These results, inter alia, indicate that the mouse lymph node can support the maturation of kidney. Morphogenesis of the S-shaped body to a structure that contains vascular loops of the glomerulus and Bowman's capsule was observed. REFERENCES (for Examples 1-3)
  • Terato carcinoma transplantation rejection loci an H-2-linked tumor rejection locus. Immunogenetics 9, 207-220 (1979).
  • lymph node permitted mouse metanephroi to engraft, mature and perform glomerular filtration. Host cells likely contributed to this process. Over time, production of waste fluid resulted in some cases in graft degeneration. Indeed, urinelike fluid-containing cysts and glomerular alterations were observed in several grafts after 12 weeks post transplantation. Importantly, the kidney graft adapted in response to a loss of host renal mass, speeding its development.
  • the lymph node provides a unique tool for studying the mechanisms of renal maturation or cell proliferation and fluid secretion. This innovative system can also be used to validate the lymph node.
  • kidneys were retrieved from timed pregnant GFP+ or wild- type C57BL/6 black mice under a dissecting microscope (embryos were considered 0.5 days old when the vaginal plug was detected in the morning).
  • kidneys were isolated from 3-day ⁇ old (P3) GFP+ C57BL/6 black mice. All kidneys were chopped in PBS and kept on ice until injection.
  • a ⁇ , threaded plunger syringe (Hamilton, 81341) with a removable needle (gauge 20) was used to slowly inject kidney fragments into a single lymph node (paired kidneys from an embryo per mouse were injected). Light cauterization was used to seal the opening. The wound was then closed with surgical sutures. Ketoprofen treatment (2 mg kg, IM) for postoperative pain relief was initiated right after surgery, and continued for 2 additional consecutive days. After 3, 6, 12 or 16 weeks from transplantation, mice were euthanized for analysis. Mice were bred and housed in the Division of Laboratory Animal Resources facility at the University of Pittsburgh Center for Biotechnology and Bioengineering. Experimental protocols followed US National Institutes of Health guidelines for animal care and were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.
  • H&E Hematoxylin and eosin
  • PAS Periodic acid-Schiff
  • TRI Masson's triclirome
  • PSR Picro-sirius red
  • Sections were also stained with antibodies against ER-TR7 (Abeam, ab51824), Collagen IV (SB, 1340-01), CD31 (BD, 550274), Podoplanin (Angiobio, 1 1- 033), Claudin-2 (Abeam, ab76032), Keratin-8 (DSHB, TROMA-1), Erythropoietin (SCBT, sc-7956), GFP (Abeam, ab6556), BrdU (SCBT, sc-32323), AQP1 (Abeam, abl5080), NKCC2 (SCBT, sc- 133823), AQP2 (Abeam, ab85876), CD45 (BD, 550539), CD106 (SCBT, sc-8304), CD3 (BD, 550275), CD4 (550280), CDS (BD, 553027), CD45R/B220 (BD, 550286), Ly6C/G (BD, 550291), F8/40 (Cal
  • BrdU staining sections were incubated in 2N HCl for 30 minutes in order to denature DNA. Five min incubation in 0.1 M borate buffer pH 8.0 was then carried out to neutralize the acid. Finally, BrdU antibody was added. Alexa Fluor 594 (Invitrogen) or biotinylated (Dako, LSAB2 System-HRP) secondary antibodies were used to detect primary antibodies. Biotin labeling was then revealed using streptavidin-HRP conjugate (Dako, LSAB2 System- HRP) and AEC substrate- chromogen (BioGenex, HK129-5K). Nuclei were counterstained using Hoechst or hematoxylin.
  • AAACTGAAGCTG ACACGGGAGA-3 ' Rev, 5 ' -GG AGCA AGTTCGTCGGTCC- 3'; GAPDH, Fwd, 5 '-GGCATCCTGGGCTACACTGA-3 ', Rev, 5'- GGAGTGGGTGTCGCTGTTG-3'.
  • anemia was induced by a rapid withdrawal of blood for 3 consecutive days before mouse sacrifice and lymph node collection.
  • BUN Blood urea nitrogen
  • Bone marrow cells were harvested from tibias and femurs of a GFP+
  • Sulfamethoxazole Sulfamethoxazole
  • Antibodies were added at a dilution of 1/50 in blood and mixed by gentle pipetting. Antibodies used were as follows: PerCP Cy5.5 CD45 (BD, 550994), APC CD3 (BD, 553066), PE CD4 (BD, 553730), APC Cy7 CD8 (BD, 557654), PE CD45R/B220 (BD, 553090), APC CD19 (BD, 550992), PE CDl lb (BD, 55331 1), and APC Ly6G-Ly6C (BD, 553129). Reactions were incubated in the dark in an ice slurry bath for one hour.
  • Red Blood Cell Lysing Buffer (Sigma) was added to each tube, lightly vortexed and incubated for an additional 15 min.
  • Two milliliters of flow buffer (2% FBS in HBSS) was added to the tubes, mixed and centrifuged at 500g for 5 min. The supernatant was aspirated. The red blood cell lysis and centrifuge were repeated as described. The final cell pellet was resuspended in 400 ⁇ of flow buffer with Sytox Blue dye. Cells were analyzed using a Miltenyi MACSQuant and FlowJo software (Tree Star).
  • mice received left nephrectomy (Nx), while the remaining 4 mice received a sham operation. All mice were given drinking water containing 0.8 mg/ml BrdU and 1% sucrose immediately after surgery.
  • BrdUcontaining drinking water was prepared fresh and replaced daily for 9 consecutive days, after which, it was replaced with regular water. After 5 additional days, all mice were euthanized for analysis. The number of BrdU-positive
  • proliferating cells in the recipient kidneys was determined per renal cross-section.
  • Repopulated jejunal lymph nodes were fixed 2 hours in 4% PFA, and embedded in OCT for further analysis. Sections were stained with antibodies against Podoplanin (Angiobio, 1 1-033), Claudin-2 (Abeam, ab76032), WT-1 (SCBT, sc-192), CD31 (BD, 550274), eratin-8 (DSHB, TROMA-1) or Vimentin (SCBT, sc-5565). Alexa Fluor 594 antibodies (Invitrogen) were used to detect primary antibodies.
  • the lymph node is a permissive site for kidney organogenesis
  • Renal tissues were harvested from C57BL/6 GFP+ transgenic embryos, isolated from ureteric buds, minced, and injected directly into a single jejunal lymph node of adult wild-type C57BL/6 mice (Fig. 23 a). Following 3 weeks, recipient mice were sacrificed, lymph nodes collected, and histologically examined. Morphogenesis of S-shaped bodies into more mature renal corpuscles was observed in ectopic grafts 3 weeks after embryonic kidney transplantation (Fig. 23b). Developing renal corpuscles expressed type IV collagen in their glomerular basement membranes (GBM) as well in mesangial areas (Fig.
  • GBM glomerular basement membranes
  • glomeruli with loose structure of the tuft, and capillary loops lined with typical flat epithelia were defined as mature glomeruli.
  • glomeruli with at least half of the circumference of capillary loops densely lined with cuboidal epithelial cells were defined as immature glomeruli.
  • Ectopic grafts also showed some S-shaped bodies, further indicating that kidney maturation was not completed at the time of lymph node collection.
  • ectopic glomeruli contained different cell types present in the adult glomerulus, including CD31 -positive endothelial cells, and podoplaninpositive podocytes (Fig. 23d).
  • Developing kidneys also comprised rudimental claudin ⁇ 2-and keratin-8-positive tubules (Fig. 23d).
  • ectopic grafts showed tubular erythropoietin (Epo) expression, indicating hormonal competence (Fig. 23d).
  • kidney organogenesis into the lymph node was critically dependent on the stage of renal development at the time of transplantation.
  • P3 kidneys show glomerular maturity, they failed to efficiently engraft into the lymph node (Fig. 29).
  • Embryonic day (E) 14.5 to 15.5 kidneys generated larger and thicker grafts as compared to P3 kidneys following 3 weeks from transplantation into the lymph node.
  • embryonic kidneys acquired more mature morphological characteristics into the lymph node
  • newborn kidneys failed to recapitulate their native morphology, resulting in an imperfect glomerulogenesis.
  • Even extending the growth of newborn kidney fragments into the lymph node up to 12 weeks did not result in a better engraftment and maturation, confirming the idea that fetal kidney harbors more regenerative potential than newborn kidney.
  • Fig. 24a Three weeks after transplantation, it was not possible to confirm the presence of mature, functional nephrons. However, some mature nephrons were distinguishable 6 weeks after transplantation (Fig. 24a). indeed, attached to the developed renal corpuscles, various segments of the renal tubule could be observed. Furthermore, the presence of erythrocytes inside the glomerulus capillary tuft of these elongated structures indicated a probable blood filtration capacity (Fig. 24a). Importantly, such structures were still proliferating at the time of lymph node collection, as indicated by bromodeoxyuridine (BrdU) incorporation, administered to the mouse 24 hours before its sacrifice (Fig. 24b).
  • bromodeoxyuridine (BrdU) incorporation administered to the mouse 24 hours before its sacrifice
  • 6-week ectopic grafts showed urine-concentrating ability, as indicated by RT-PCR analysis of different urea transporters (UT-A1, UT-A2, UT-A3, and UT-B) (Fig. 24c). It is not surprising that UT ⁇ B mRNA was detected in both control and repopulated lymph nodes, since this urea transporter is known to be expressed in non-renal tissues, as well as in erythrocytes [19]. Erythropoietin production was also confirmed in 6-week ectopic grafts by RT-PCR analysis of mRNA isolated from phlebotomized mice.
  • nephrons were mature by 12 weeks. These nephrons showed glomerular expression of podoplanin and CD31 (Fig. 25, left). CD31 staining also indicated that ectopic nephrons were vascularized by host arterioles (Fig. 25, right). Collagen IV was localized at GBM and tubules (Fig. 25, left), as well in the mesangial areas in the glomeruli (Fig. 25, right), and it likely had a hybrid origin, indeed, it did not always colocalize with GFP+ cells. Ectopic nephrons also showed keratin ⁇ 8- and erythropoietin-positive tubules (Fig. 25, left).
  • Renal cysts develop within repopulated lymph nodes as a result of ectopic kidney's ability to filter the blood and produce urine
  • Renal cystic disease has multiple etiologies. Renal cysts can result from defective differentiation of kidney tubules [20]. However, while proliferative activity of the renal tubular epithelium is an essential component of cyst formation, fluid secretion could have a commanding role in cyst development and expansion [21]. We believe in a scenario in which urine-like fluid is produced by ectopic kidneys as a result of their functional maturation into the lymph node.
  • ectopically produced bile juice is transported in the serum, and eventually processed in the native liver without affecting the host (our unpublished data).
  • fluids and wastes might be successfully drained into the lymphatic vessels, allowing the ectopic graft to better survive.
  • kidney products might accumulate inside the tubules, activating a positive loop of epithelial proliferation and vectorial fluid secretion, which eventually leads to cyst appearance.
  • cyst formation inside the repopulated lymph node could share some traits with multicystic dysplastic kidney (MCDK) and obstructive dysplasia (ORD), where urinary tract obstructive lesions cause urine retention in functioning nephrons and lead to cystogenesis [22].
  • MCDK multicystic dysplastic kidney
  • ORD obstructive dysplasia
  • Cyst #1 was lined by a simple squamous epithelium, showing an apical expression of the water channel aquaporin-1 (AQP1) and absence of sodium-potassium-chloride transporter 2 (NKCC2), indicating a possible origin from thin descending limbs of loop of Henle (Fig. 26b, left).
  • the epithelium was negative for BrdU indicating that cystic expansion had already ceased at the time of lymph node collection (Fig. 26b, left).
  • the loop of Henle plays a role in the transport of ions and water, allowing production of urine.
  • cyst #1 contained many oval to round, rhomboid, parallelepiped, and amorphous urinary crystals, with more or less sharply defined contours, some of them reaching ⁇ of length (Fig. 26d, left). Cysts #1 also contained eosinophilic, Periodic acid-Schiff (PAS) positive, acidfuchsinophilic with Masson's trichrome (TRI), and red with picro-sirius red (PSR) staining proteinaceous material, apart from amorphous fibers often containing a periodic banding pattern, and rarely TRI positive (Fig. 26c, left). The presence of urine in repopulated lymph nodes was confirmed by Blood Urea Nitrogen (BUN) test 16 weeks following kidney transplantation.
  • BUN Blood Urea Nitrogen
  • BUN levels were highly increased in lymph node fluid after kidney transplantation and cyst formation (Fig. 26d, right). However, BUN levels were not increased in repopulated lymph nodes where no macroscopic cysts could be observed, further indicating that the time window of ectopic kidney maturation and degeneration differs among mice. Approximately, S-shapes bodies take 6 weeks to be converted into mature nephrons, and these nephrons can degenerate by the 12th week, as well as be still healthy and functional at this stage,
  • cyst #2 was lined by a simple tall cuboidal epithelium, showing apical endocytic vacuoles and a PAS positive brush border, indicating an origin from proximal convoluted tubule (Fig. 26b, right). Accordingly, the epithelium stained positive for AQP1 and negative for aquaporin 2 (AQP2) (Fig. 26b, right). Moreover, it showed some positivity for the BrdU marker, indicating cystic expansion process was still active at the time of lymph node collection (Fig. 26b, right). The proximal convoluted tubule reabsorbs large molecules, such as proteins.
  • cyst #2 contained pale eosinophilic, PAS positive, intensely acid-fuchsinophilic with TRI, and yellowish with PSR staining round globules, ranging from 1 to 20 ⁇ diameter, thought to be protein globules (Fig. 26c, right). These structures likely are hyaline casts covered with fat droplets. The accumulation of hyaline droplets is the visible aspect of the damage to the glomerular capillary membrane, which leads to abnormal filtration and reabsorption of plasma proteins.
  • Structural glomerular alterations could be observed in the cyst- containing ectopic renal graft. Specifically, histological analyses often revealed compressed tuft, in the center of the glomerulus, surrounded by a circumferential cellular crescent (H&E) (Fig. 26f, upper). There was a clear space between tuft and the crescent. A mild focal thickening of glomerular basement membrane could be observed (PAS) (Fig. 26f, upper). Basement membrane thickening could be attributed to increased collagen accumulation (TRI and PSR) (Fig. 26f, upper). The cellular crescent contained some BrdU positive cells, indicating active proliferation (Fig. 26f, upper).
  • kidney regeneration inside the lymph node could not only be attributable to transplanted kidney stem/progenitor cells, but could also be attributable to the combination of transplanted kidney stem/progenitor cells and stem cells of host origin such as bone marrow.
  • Fig. 27b Following 8 weeks from bone marrow transplantation, all mice received injection of wild-type embryonic kidneys (Fig. 27b). Mice were sacrificed 6 or 10 weeks post kidney transplantation (Fig. 27b). Interestingly, bone marrow- derived collagen-producing cells were incorporated in developed renal corpuscles 6 weeks after transplantation (Fig. 27c). Ectopic grafts were also stained for several markers of cells of hematopoietic and nonhematopoietic origin, including CD45, CD 106 (VCAM-1), CD3, CD4, CD8, CD45R/B220, Ly6C/G, and F8/40.
  • PECs parietal epithelial cells
  • claudin-2+ cells shared the same location of WT-1+ podocytes (Fig. 31).
  • PECs lining the inner region of Bowman's capsule have been shown to migrate onto the glomerular tuft and differentiate into podocytes [27].
  • PECs might transdifferentiate into podocytes.
  • Cellular lesions also expressed the PEC marker keratin-8 and the podocyte marker vimentin (Fig. 31).
  • lesioned glomeruli showed a massive presence of BMDCs. It remains to understand whether BMDCs contribute to regeneration of damaged glomeruli or facilitate extracellular matrix deposition and as a consequence renal failure.
  • BMDCs did not contribute to vascularization of the ectopic graft, as no GFP+ cells were incorporated in CD31+ vessels (Fig. 27d). Similarly, BMDCs did not contribute to the formation of kidney tubular structures (Fig. 27d, bottom), confirming the idea that tubule regeneration mainly occurs through survival of dedifferentiated epithelial cells which proliferate and redifferentiate into mature functional epithelial cells [28],
  • nephrogenesis In contrast to lower vertebrates, in mammals, nephrogenesis is limited to gestation or early post-natal life. Although the adult kidney cannot make new nephrons, it can regenerate and recover in some circumstances. Indeed, tubular regenerative capacity widely changes going from acute kidney injury (AKI) to chronic kidney disease (CKD), as acute renal insults are handled with successful regeneration, while chronic injuries lead to ineffective or even more damaging cellular responses [29].
  • AKI acute kidney injury
  • CKD chronic kidney disease
  • nephron tubule epithelium is regenerated after AKI, while in the setting of CKD, tubular damage is not repaired, and this is accompanied by a sustained inflammatory response and activation of myofibroblasts, that eventually results in interstitial fibrosis, tubular atrophy, and nephron loss.
  • kidney repair Although many theories exist on kidney repair, the existence of an intratubular cell source fuelling nephron tubule epithelium regeneration after AKI is gaining consensus amongst researchers [30].
  • lymph node might be considered as a unique niche to grow several tissues.
  • embryonic kidneys were transplanted into the lymph node, blood vessels integrated into the glomeruli. Vascularization is likely attributable to migration and proliferation of resident endothelial cells, and does not involve BMDCs.
  • bone marrow hematopoietic and stromal cells were found in the ectopic kidney graft. These cells contributed to mesangial cells and podocyte regeneration.
  • lymph node furnish the developing tissue with host cells, but also provided it with growth and homeostatic signals, since a decrease in native renal mass could push maturation of the ectopic graft.
  • the present study provides evidence that ectopic kidney inside the lymph node can sense a stimulus and appropriately respond.
  • This system can also be used to validate in vivo the differentiation potential of candidate cells in regenerative nephrology, including ES or iPS.
  • Kidney tissue reconstruction by fetal kidney cell transplantation effect of gestation stage of fetal kidney cells. Stem Cells. 2007; 25(6):1393-1401.
  • Thony HC Luethy CM, Zimmermann A, Laux-End R, Oetliker OH and Bianchetti MG. Histological features of glomerular immaturity in infants and small children with normal or altered tubular function. European journal of pediatrics. 1995; 154(9 Suppl 4):S65-68.
  • Bone marrow is a reservoir of repopulating mesangial cells during glomerular remodeling. Journal of the American Society of Nephrology : JASN. 2001; 12(12):2625-2635.
  • Bone marrow contributes to renal parenchymal turnover and regeneration. The Journal of pathology. 2001 ; 195(2):229-235.
  • Kidney tubular epithelium is restored without replacement with bone marrow-derived cells during repair after ischemic injury. Kidney international. 2005; 68(5):1956-1961.
  • lymph node a lymph node that provides a unique tool to track and monitor stem cell behavior in vivo, in a location far from the native environment, but still responsive to physiologic and homeostatic signals.
  • Embryonic day (E) 14.5 to 15.5 tissues were retrieved from timed pregnant GFP+ C57BL/6 black mice under a dissecting microscope (embryos were considered 0.5 days old when the vaginal plug was detected in the morning). All tissue were chopped in PBS and kept on ice until injection.
  • a small incision was made in the abdomen to expose jejunal lymph nodes.
  • a ⁇ threaded plunger syringe Hamilton, 81341
  • a removable needle gauge 20
  • mice were bred and housed in the Division of Laboratory Animal Resources facility at the University of Pittsburgh Center for Biotechnology and Bioengineering. Experimental protocols followed US National Institutes of Health guidelines for animal care and were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.
  • Repopulated jejunal lymph nodes were fixed 2 hours in 4% PFA, and embedded in Optimal Cutting Temperature (OCT) following infiltration with 30% sucrose overnight. Sections were stained with antibodies against GFAP5 (Bioss, bs- 11016R), GFP (Abeam, ab6556), Keratin-8 (DSHB, TROMA-1), eratin-5 (Covance, PRB-160P), GFP (Abeam, ab6556), CD31 (BD, 550274), CD105 (BD, 550546), ER- TR7 (Abeam, ab51824), MUC5AC (Abeam, ab79082), ⁇ -catenin (CST, 8480), MUC2 (SCT, sc- 15334), or chromogranin A (SCT, sc- 18232).
  • GFAP5 Bioss, bs- 11016R
  • GFP Abeam, ab6556
  • Keratin-8 DSHB, TROMA-1
  • Alexa Fluor 594 (Invitrogen) secondary antibodies were then used. Nuclei were counterstained using Hoechst, Donor organs were embedded in paraffin and stained with hematoxylin and eosin (H&E) as described elsewhere.
  • GAPDH transcript levels served as the housekeeping control target. Sequences of primers were as follows: GM-CSF, Fwd, 5'- TTCCTGGGCATTGTGGTCT-3 Rev, 5 ' -TG ATTC AG AGC TGGCCTGG- 3 ' ; GAPDH, Fwd, 5 ' -GGCATCCTGGGCT AC ACTGA-3 ' , Rev, 5'- GGAGTGGGTGTCGCTGTTG-3 ' .
  • GM-CSF PCR mixtures were subjected to different numbers of amplification cycles; 25 cycles were eventually chosen to quantify gene expression.
  • the lymph node is a permissive site for tissue organogenesis.
  • lymph node was investigated the ability of lymph node to support engraftment of several mouse mid- embryonic tissues including brain, thyroid, thymus, lung, heart, stomach, intestine, liver, and adrenal gland. Tissues were harvested from
  • E14.5/E.15.5 GFP transgenic mice minced, and injected directly into a single jejunal lymph node of adult wild-type mice (Fig. 32A). Following 3 weeks, recipient mice were sacrificed, lymph nodes collected, and histologically examined. As shown in Figure 32B, transplants were variably prone to engraft into the lymph node. While the brain showed the highest ability of repopulating the lymph node, the thyroid was unable to engraft in. Although in some cases heart, liver and adrenal gland engrafted, their grafts were very small. We therefore focused our attention on brain, thymus, lung, stomach, and intestine.
  • glial fibrillary acid protein delta (GFAP5) starts being expressed at El 8. Accordingly, we found very low GFAP8 expression in E14.5 - E.15.5 mouse brain ( Figure 32C2, upper). Importantly, when transplanted into the LN, E14.5/15.5 brain engrafted ( Figure 32C1) and maturated ( Figure 32C2, lower), as indicated by presence of GFAP6 + cells with complex branching and extended processes, indicative of mature astrocytes.
  • Maturation of the thymic epithelium in an ectopic site contribution of the host in the generation of the thymic cortex
  • Embryonic lung fragments arranged in lobe-like structures into the lymph node, and showed sign of differentiation from a pseudoglandular to mixed saccular/alveolar morphology 3 weeks after transplantation.
  • ectopic lung comprised a glandular epithelium with MUCSAC-producing goblet cells 10 weeks after transplantation, indicating that it is possible to achieve postnatal stages of mouse lung development inside the lymph node ( Figure 35 A).
  • stem cells reside in a highly specialized three-dimensional (3D) structure, the socalled niche (17). Not only does the niche preserve the stem cell pool, but also promotes progenitor cell expansion and mobilization. Reproducing this dynamic and complex microenvironment in culture is challenging, either because the mechanisms that control stem cell fate in vivo have not yet been fully elucidated, or because of ethical and technical issues.
  • the current study indicates that the lymph node mimic the physiological environment of transplanred tissue and promotes the vascularization of the transplanted tissue. Lymph nodes have ready access to the bloodstream, and can therefore foster cell growth by providing nutrients as well as hormones and growth factors.
  • lymph node in supporting organogenesis of different tissues including brain, thymus, lung, stomach and intestine. Lymph node-grown tissues more closely recapitulate in vivo phenotypes under physiological conditions than any other culture system.
  • Allogeneic transplant rejection is one of the major problems plaguing the field of organ transplants today.
  • the allograft acceptance maybe mediated by the increased Treg induction associated with cross-talk between the two thymuses 11.1 Methods
  • mice with thymus transplants demonstrated long-term acceptance of allografts (skin grafts as well as hepatocyte transfers). Furthermore, as observed in the mixed lymphocyte reaction (MLR) assays, these mice were specifically tolerized to the Balb/c strain, but were reactive against the C57BL/6 strain ( Figure 40).
  • MLR mixed lymphocyte reaction
  • tDCs migration of cells from the ectopic to the native thymus
  • Thymus transplants induced acceptance of allogeneic hepatocytes and rescue of liver function in the 129.Fah-/- mouse model.
  • Migration of antigen-presenting cells to induce Tregs, and cross-talk between the two thymuses appears to be important for induction of central tolerance and allograft acceptance.

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Abstract

La présente invention concerne des procédés et des compositions pour transplanter des tissus non lymphoïdes dans des organes lymphoïdes. Elle peut être utilisée pour cultiver des tissus d'organes afin de compléter ou reconstituer le fonctionnement d'un organe. Les tissus qui peuvent se propager de cette manière comprennent, mais sans y être limités, le poumon, le rein, la tyroïde, l'intestin et le cerveau.
PCT/US2014/021420 2008-03-07 2014-03-06 Ganglion lymphatique comme site de transplantation, organogenèse et fonctionnement pour de multiples tissus et organes WO2014138486A1 (fr)

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CN109642210A (zh) * 2016-06-29 2019-04-16 横尾隆 一种肾脏的制造方法
CN110869032A (zh) * 2017-02-17 2020-03-06 匹兹堡大学联邦系统高等教育 脂肪相关淋巴簇作为多种组织的移植、组织再生、器官发生和功能的位点
EP3952895A4 (fr) * 2019-04-11 2022-12-28 University of Pittsburgh - Of the Commonwealth System of Higher Education Procédure de transplantation cellulaire à invasion minimale pour induire le développement d'une organogenèse in vivo
US11607425B2 (en) 2016-06-29 2023-03-21 Takashi Yokoo Kidney production method
EP4009990A4 (fr) * 2019-08-05 2023-08-09 University of Pittsburgh - Of the Commonwealth System of Higher Education Greffe de tissu thymique autologue

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109642210A (zh) * 2016-06-29 2019-04-16 横尾隆 一种肾脏的制造方法
US11559605B2 (en) 2016-06-29 2023-01-24 Takashi Yokoo Kidney production method
US11607425B2 (en) 2016-06-29 2023-03-21 Takashi Yokoo Kidney production method
CN110869032A (zh) * 2017-02-17 2020-03-06 匹兹堡大学联邦系统高等教育 脂肪相关淋巴簇作为多种组织的移植、组织再生、器官发生和功能的位点
JP2020508298A (ja) * 2017-02-17 2020-03-19 ユニバーシティ オブ ピッツバーグ −オブ ザ コモンウェルス システム オブ ハイヤー エデュケイション 移植、組織再生、器官形成及び複数の組織に対する機能のための部位としての脂肪関連リンパ球集積（ｆａｔ−ａｓｓｏｃｉａｔｅｄ ｌｙｍｐｈｏｉｄ ｃｌｕｓｔｅｒ）
EP3582794A4 (fr) * 2017-02-17 2020-12-09 University of Pittsburgh- Of the Commonwealth System of Higher Education Groupes lymphoïdes associés à la graisse comme sites de transplantation, de régénération tissulaire, d'organogenèse et de fonction pour de multiples tissus
IL268389B1 (en) * 2017-02-17 2024-02-01 Univ Pittsburgh Commonwealth Sys Higher Education Adipose-associated lymphoid clusters as a site for transplantation, tissue regeneration, organ generation, and a role for multiple tissues
IL268389B2 (en) * 2017-02-17 2024-06-01 Univ Pittsburgh Commonwealth Sys Higher Education Adipose-associated lymphoid aggregates as a site for transplantation, tissue regeneration, organ generation, and a role for multiple tissues
EP3952895A4 (fr) * 2019-04-11 2022-12-28 University of Pittsburgh - Of the Commonwealth System of Higher Education Procédure de transplantation cellulaire à invasion minimale pour induire le développement d'une organogenèse in vivo
GB2600256B (en) * 2019-04-11 2024-02-21 Univ Pittsburgh Commonwealth Sys Higher Education Minimally invasive cell transplant procedure to induce the development of in vivo organogenesis
EP4009990A4 (fr) * 2019-08-05 2023-08-09 University of Pittsburgh - Of the Commonwealth System of Higher Education Greffe de tissu thymique autologue

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