Enhancing tissue regeneration, growth, and ex VIVO propagation with a novel recombinant factor

Abstract

Methods and compositions for organoid, stem cell / progenitor growth and tissue regeneration, and ex vivo propagation, and methods of treatment using N-terminal domain of an FGFBP1 or FGFBP2 or FGFBP3 or fusion proteins thereof & related expression constructs.

Description

93597 / 7344 ENHANCING TISSUE REGENERATION, GROWTH, AND EX VIVO PROPAGATION WITH A NOVEL RECOMBINANT FACTOR CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 633,389, filedApril 12, 2024, and U.S. Provisional Application No.63 / 719,776, filed November 13, 2024, the contents of each of which are hereby incorporated by reference. STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under grant numbers DK128801,AG067014, and DK103155 awarded by the National Institutes of Health. The government has certain rights in the invention REFERENCE TO SEQUENCE LISTING

[0003] This application incorporates-by-reference nucleotide and / or amino acid sequenceswhich are present in the file named “250411_92326-A-PCT_93597- 7292_Sequence_Listing_AWG.xml”, which is 26,067 bytes in size, and which was created on April 10, 2025 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the XML file filed April 11, 2025 as part of this application. BACKGROUND

[0004] The goal of regenerative medicine is to maintain, produce, repair, replace, or regenerateimpaired human cells, tissues, and organs to restore or augment their function but without causing their neoplastic transformation. Viability and growth of organoids and transplant cells and tissues is an ongoing concern. New methods or treatments are needed to enhance viability and growth of organoids and transplant cells, tissues, and organs. New methods or treatments are needed to repair, replace, or regenerate damaged, injured, diseased, or aged cells, tissues, and organs. SUMMARY

[0005] A method of propagating and / or increasing viability of a culture of a mammalian tissuecomprising administering to the human tissue in culture an amount of a composition comprising a 1 4909-6074-3733v.193597 / 7344 domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of a mammalian tissue.

[0006] A method of propagating and / or increasing viability of a culture of stem or progenitorcells comprising administering to the stem or progenitor cells in culture an amount of a composition comprising a domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of stem or progenitor cells.

[0007] A method of maintaining or increasing the viability of a donor tissue or donor organintended for transplantation comprising administering to the donor tissue or donor organ an amount of a composition comprising a domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to maintain or increase viability of a donor tissue or donor organ.

[0008] A method of expanding organoids or growth of tissues in a culture comprisingadministering to the organoids or tissues in a culture an amount of a protein domain comprising heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to expand the organoids or growth of tissues.

[0009] In some embodiments, the organoids are being cultured for transplantation or are listedin Table 1.

[0010] A method of promoting healing of a wound in a tissue or organ in a subject comprisingadministering to the wound an amount of a composition comprising a domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote healing of a wound in a tissue or organ.

[0011] A method of promoting growth of an organoid, or of an organoid culture, comprisingadministering to the organoid or organoid culture an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3, or a fusion protein thereof, effective to promote growth of an organoid, or of an organoid culture.

[0012] A method of reducing the frequency of culture media changes required to maintainviability of organoids of an organoid culture comprising administering to the organoid culture an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote and / or maintain viability of the organoids culture thereby reducing the frequency of culture media changes required to maintain viability of the organoids. 2 4909-6074-3733v.193597 / 7344

[0013] A method of propagating and / or increasing viability of a culture of a mammalian tissuecomprising administering to the human tissue in culture an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of a mammalian tissue.

[0014] A method of propagating and / or increasing viability of a culture of stem or progenitorcells comprising administering to the stem or progenitor cells in culture an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of stem or progenitor cells.

[0015] A method of maintaining or increasing the viability of a donor tissue or donor organintended for transplantation comprising administering to the donor tissue or donor organ an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to maintain or increase viability of a donor tissue or donor organ.

[0016] A method of expanding organoids or growth of tissues in a culture comprisingadministering to the organoids or tissues in a culture an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to expand the organoids or growth of tissues.

[0017] A method of promoting healing of a wound in a tissue comprising administering to thewound an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote healing of a wound in a tissue.

[0018] A method of promoting growth of an organoid, or of an organoid culture, comprisingadministering to the organoid or organoid culture an amount of a protein domain comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or a fusion protein thereof, effective to promote growth of an organoid, or of an organoid culture.

[0019] A method of reducing the frequency of culture media changes required to maintainviability of organoids of an organoid culture comprising administering to the organoid culture an amount of a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote and / or maintain viability of the organoids culture thereby reducing the frequency of culture media changes required to maintain viability of the organoids.

[0020] A fusion protein comprising (i) a protein domain of an N-terminal portion of anFGFBP1 or FGFBP2 or FGFBP3 and (ii) a peptide or a protein not encoded by a Fgfbp1 gene or Fgfbp2 gene or Fgfbp3 gene. 3 4909-6074-3733v.193597 / 7344

[0021] An expression construct encoding a protein domain of an N-terminal portion of anFGFBP1 or FGFBP2, but not encoding a full length FGFBP1 or FGFBP2 or FGFBP3 protein.

[0022] An expression construct encoding an FGFBP1 domain having SEQ ID NO:8, but notencoding a full length FGFBP1 protein having SEQ ID NO:7 or encoding an FGFBP1 domain having SEQ ID NO:9, but not encoding a full length FGFBP1 protein having SEQ ID NO:17.

[0023] An expression construct encoding an FGFBP2 domain but not encoding a full lengthFGFBP2 protein having SEQ ID NO:1.

[0024] An expression construct encoding a fusion protein comprising (i) a protein domain ofan N-terminal portion of an FGFBP1 and (ii) a peptide or a protein not encoded by a fgfbp1 gene.

[0025] An expression construct encoding a fusion protein comprising (i) a protein domain ofan N-terminal portion of an FGFBP2 and (ii) a peptide or a protein not encoded by a fgfbp2 gene

[0026] A host cell transfected with an expression construct of described herein, or a progenyof such a host cell.

[0027] A method of producing a protein domain of an N-terminal portion of an FGFBP1 orFGFBP2, or fusion protein thereof, comprising culturing the host cell or progeny thereof as described herein in culture media, and isolating a secreted heparin-binding domain of an FGFBP1,or of an FGFBP2, or fusion protein thereof from a supernatant thereof.

[0028] An isolated protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 but notcomprising a full length FGFBP1 or FGFBP2 protein. In some embodiments, the FGFBP1 is human or mouse. In some embodiments, the FGFBP2 is human or mouse. In some embodiments, the FGFBP1 is human. In some embodiments, the FGFBP2 is human.

[0029] A domain of an N-terminal portion of an FGFBP1 is a domain of the protein whichdomain is primarily in the N-terminal half of the protein. For example, a domain of FGFBP1 whose primary sequence is mainly (>50%) in the N-terminal half of the protein’s primary sequence. A domain of an N-terminal portion of an FGFBP2 is a domain of the protein which domain is primarily in the N-terminal half of the protein. For example, a domain of FGFBP2 whose primary sequence is mainly (>50%) in the N-terminal half of the protein’s primary sequence. A domain does not include the whole sequence of the mature protein.

[0030] Similarly, a sequence of FGFBP1 amino acids which is located in an N-terminal portionof an FGFBP1 protein is a sequence which is primarily in the N-terminal half of the FGFBP1 protein. For example, an amino acid sequence of FGFBP1 whose entire sequence is mainly (>50%) 4 4909-6074-3733v.193597 / 7344 in the N-terminal half of the FGFBP1 protein’s primary sequence. Similarly, a sequence of FGFBP2 amino acids which is located in an N-terminal portion of an FGFBP1 protein is a sequence which is primarily in the N-terminal half of the FGFBP2 protein. For example, an amino acid sequence of FGFBP2 whose entire sequence is mainly (>50%) in the N-terminal half of the FGFBP2 protein’s primary sequence.

[0031] Examples of wild-type FGFBP1 sequences include, but are not limited to, NCBIReference Sequence NP_005121 (Homo sapiens) and NP_001258545 (Mus musculus).

[0032] Examples of wild-type FGFBP2 sequences include, but are not limited to, NCBIReference Sequence: NP_114156.1 (Homo sapiens).

[0033] In some embodiments of the methods and compositions and constructs and proteindomains disclosed herein, the protein domain of an N-terminal portion of an FGFBP1 comprises a heparin-binding domain of FGFBP1. In some embodiments of the methods and compositions and constructs and protein domains disclosed herein, the protein domain of an N-terminal portion of an FGFBP2 comprises a heparin-binding domain of FGFBP2.

[0034] An expression construct encoding a putative heparin-binding domain of a fibroblastgrowth factor binding protein 1 (FGFBP1), but not encoding a full length FGFBP1 protein or not encoding a mature FGFBP1.

[0035] An expression construct encoding a putative heparin-binding domain of a fibroblastgrowth factor binding protein 2 (FGFBP2), but not encoding a full length FGFBP2 protein or not encoding a mature FGFBP2.

[0036] An expression construct encoding an FGFBP1 domain having SEQ ID NO:8, but notencoding a full length FGFBP1 protein having SEQ ID NO:7 or encoding an FGFBP1 domain having SEQ ID NO:9, but not encoding a full length FGFBP1 protein having SEQ ID NO:17. An expression construct encoding an FGFBP1 domain having SEQ ID NO:8, but not encoding a mature FGFBP1 protein or encoding an FGFBP1 domain having SEQ ID NO:9, but not encoding mature FGFBP1 protein.

[0037] An expression construct encoding an FGFBP2 domain but not encoding a full lengthFGFBP2 protein having SEQ ID NO:1.

[0038] An expression construct encoding a fusion protein comprising (i) a putative heparin-binding domain of a fibroblast growth factor binding protein 1 (FGFBP1) and (ii) a peptide or a protein not encoded by a fgfbp1 gene. 5 4909-6074-3733v.193597 / 7344

[0039] An expression construct encoding a fusion protein comprising (i) a putative heparin-binding domain of a fibroblast growth factor binding protein 2 (FGFBP2) and (ii) a peptide or a protein not encoded by a fgfbp2 gene.

[0040] A protein domain comprising a putative heparin-binding domain of a fibroblastgrowth factor binding protein 1 (FGFBP1), but not comprising a full length FGFBP1 protein.

[0041] A fusion protein comprising (i) a putative heparin-binding domain of a fibroblastgrowth factor binding protein 1 (FGFBP1) and (ii) a peptide or a protein not encoded by a fgfbp1 gene.

[0042] A protein domain comprising a putative heparin-binding domain of a fibroblastgrowth factor binding protein 2 (FGFBP2), but not comprising a full length FGFBP2 protein.

[0043] A fusion protein comprising (i) a putative heparin-binding domain of a fibroblastgrowth factor binding protein 2 (FGFBP2) and (ii) a peptide or a protein not encoded by a fgfbp2 gene.

[0044] A method of promoting growth of an organoid, or of an organoid culture, comprisingadministering to the organoid or organoid culture an amount of a protein domain comprising a putative heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or a fusion protein thereof, effective to promote growth of an organoid, or of an organoid culture.

[0045] A host cell comprising an expression construct as described herein. A method ofproducing an isolated putative heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein thereof, comprising culturing the host cell or progeny thereof in culture media, and isolating a secreted putative heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 or fusion protein thereof from a supernatant thereof.

[0046] A method of reducing the frequency of culture media changes required to maintainviability of organoids of an organoid culture comprising administering to the organoid culture an amount of a protein domain comprising a putative heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote and / or maintain viability of the organoids culture thereby reducing the frequency of culture media changes required to maintain viability of the organoids.

[0047] A method of propagating and / or increasing viability of a culture of a mammalian tissuecomprising administering to the human tissue in culture an amount of a protein domain comprising 6 4909-6074-3733v.193597 / 7344 a putative heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of a mammalian tissue.

[0048] A method of propagating and / or increasing viability of a culture of stem or progenitorcells comprising administering to the stem or progenitor cells in culture an amount of a protein domain comprising a putative heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of stem or progenitor cells.

[0049] A method of maintaining or increasing the viability of a donor tissue or donor organintended for transplantation comprising administering to the donor tissue or donor organ an amount of a protein domain comprising a putative heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to maintain or increase viability of a donor tissue or donor organ.

[0050] A method of expanding or enhancing growth or enhancing survival of cells ororganoids or tissues in a culture comprising administering to the organoids or tissues in a culture an amount of a peptide comprising heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to expand or enhance cells, organoids, or tissues.

[0051] A method of promoting healing of a wound in a tissue comprising administering to thewound an amount of a protein domain comprising a putative heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote healing of a wound in a tissue.

[0052] A method of prophylaxis against a tissue injury, optionally from a chemotherapy orradiotherapy, comprising administering a putative heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to prophylactically protect a tissue injury.

[0053] An expression construct encoding an N-terminal domain of a fibroblast growth factorbinding protein 1 (FGFBP1), but not encoding a full length FGFBP1 protein. An expression construct encoding an N-terminal domain of a fibroblast growth factor binding protein 1 (FGFBP1), but not encoding a mature FGFBP1 protein.

[0054] An expression construct encoding an N-terminal domain of a fibroblast growth factorbinding protein 1 (FGFBP1), but not encoding an FGF-binding domain of an FGFBP1 is also provided. In some embodiments, the N-terminal domain of FGFBP1 is human. In some embodiments, the N-terminal domain of FGFBP1 comprises SEQ ID NO:8 or SEQ ID NO:9.

[0055] An expression construct encoding a fusion protein comprising (A) an N-terminaldomain of a fibroblast growth factor binding protein 1 (FGFBP1) linked to (B) a non-FGFBP 7 4909-6074-3733v.193597 / 7344 protein or peptide or polypeptide. A non-FGFBP protein or peptide or polypeptide is a protein or peptide or polypeptide sequence that does not occur in an FGFBP naturally.

[0056] A composition comprising an expression construct described herein is provided. Acomposition comprising a peptide described herein is provided. A composition comprising a fusion described herein is provided. A composition comprising a fragment of an FGFBP described herein is provided. A composition comprising an N-terminal protein domain described herein is provided.

[0057] A peptide comprising an N-terminal domain of a fibroblast growth factor bindingprotein 1 (FGFBP1), but not comprising a full length FGFBP1 protein.

[0058] A fusion protein comprising (A) an N-terminal domain of a fibroblast growth factorbinding protein 1 (FGFBP1), linked to (B) a non-FGFBP protein or peptide or polypeptide.

[0059] A method of propagating, promoting growth / proliferation of, and / or increasingviability of a stem cell or progenitor cell, comprising contacting the stem cell or progenitor cell with an amount of a composition comprising (i) an N-terminal domain of a fibroblast growth factor binding protein 1 (FGFBP1), (ii) an N-terminal domain of a fibroblast growth factor binding protein 2 (FGFBP2), or (iii) an N-terminal domain of a fibroblast growth factor binding protein 3 (FGFBP3), effective to propagate, promote growth / proliferation of, and / or increase viability of a stem cell or progenitor cell.

[0060] A method of propagating, promoting growth / proliferation of, and / or increasingviability of a stem cell or progenitor cell, comprising contacting the stem cell or progenitor cell with an amount of an expression construct encoding (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to propagate, promote growth / proliferation of, and / or increase viability of a stem cell or progenitor cell.

[0061] A method of propagating, promoting growth / proliferation of, and / or increasingviability of a tissue or organ, comprising contacting the tissue with an amount of a composition comprising (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to propagate, promote growth / proliferation of, and / or increase viability of a tissue or organ.

[0062] A method of propagating, promoting growth / proliferation of, and / or increasingviability of a tissue or organ, comprising contacting the tissue with an amount of an expression construct encoding (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an 8 4909-6074-3733v.193597 / 7344 FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to propagate, promote growth / proliferation of, and / or increase viability of a tissue or organ.

[0063] A method of treating a wound or a scar in a tissue or organ, comprising contacting thetissue or organ with an amount of a composition comprising, or an expression construct encoding, (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N- terminal domain of an FGFBP3, effective to treat a wound or a scar in a tissue or organ.

[0064] A method of treating a pathology associated with degeneration or aging of a tissue ororgan, comprising contacting the tissue or organ with an amount of a composition comprising, or an expression construct encoding, (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to treat a pathology associated with degeneration or aging of a tissue or organ.

[0065] A method of treating a pathology associated with acute or chronic inflammation of atissue or organ, comprising contacting the tissue or organ with an amount of a composition comprising, or an expression construct encoding, (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to treat a pathology associated with acute or chronic inflammation of a tissue or organ.

[0066] A method of promoting growth and / or viability of an organoid, or of an organoidculture, or expanding organoids in a culture, comprising administering to the organoid or organoid culture an amount of a composition comprising, or an expression construct encoding, (i) an N- terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to promote growth and / or viability of an organoid, or of an organoid culture, or expanding organoids in a culture.

[0067] A method of stimulating proliferation of stem cells comprising contacting the stem cellswith an amount of a composition comprising, or an expression construct encoding, (i) an N- terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to stimulate proliferation of stem cells.

[0068] A cell engineered to express an expression construct described herein is provided. Acell engineered to express a peptide described herein is provided. A cell engineered to express a fusion protein described herein is provided. A host cell comprising an expression construct described herein is provided. A host cell comprising a peptide described herein is provided. A host cell comprising a fusion protein described herein is provided. 9 4909-6074-3733v.193597 / 7344 BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIGS.1A-1K: Single-cell analysis identifies Fgfbp1 as a marker of a proliferative uppercrypt zone population that is transcriptionally and spatially distinct from Lgr5+ CBC cells. (A) Left, Co-embedded UMAP projection of 13,095 single cells, colored by inferred cell type, consisting of 11,218 FACS-sorted Lgr5–GFP+ (Lgr5-GFPHigh and Lgr5-GFPLow) and 1,877 Lgr5–GFP- cells (GSE92865). Right, UMAP projection of the 13,095 single cells highlighting in red either the Lgr5–GFP- control cells vs. Lgr5–GFP+ cells from all treatment conditions in aggregate. (B) Nebulosa density plots showing the expression of Lgr5, Ascl2 and putative “TA” cell marker Fgfbp1, as well as distribution of cells by predicted cell cycle phase. (C) Distribution and relative quantification of cell types in each treatment condition (cells belonging to a specified treatment group are highlighted in red and their relative proportions among the “CBC” and “TA” clusters overlaid, n=2 replicates). (D) DotPlot visualization of differentially expressed genes (DEGs) used for the cluster identity assignment shown in (A), highlighting Fgfbp1, Dmbt1, and Kcnn4 as putative “TA” signature genes. (E) Low magnification view of WT murine proximal small intestine following multiplex in situ hybridization (ISH) for Fgfbp1 and Lgr5 combined with Ki67 immunostaining. Representative data from duodenum, scale bar = 250 μm. (F) Higher magnification views of the multiplex ISH shown in (E), demonstrating the presence of proliferative, Lgr5- / Fgfbp1+ cells in the upper crypt zone along the WT murine small intestine length. The bottom panels represent higher magnification images of the crypts presented in the top panels (TA = upper crypt “transit-amplifying” (“TA”) zone, CBC = Crypt Base Columnar (CBC) zone). Scale bar = 50 μm. (G) Fgfbp1 ISH in Lgr5-DTR-GFP small intestine confirms spatial segregation of Fgfbp1 transcripts from Lgr5-GFP+ CBC cells, scale bar = 20 μm. (H) Quantitation of Fgfbp1 expression domain along the small intestine length as determined by Fgfbp1 ISH. Histogram distributions reflect the position of Fgfbp1+ cells along the intestine length relative to the center-most nuclei at the very crypt base (assigned as cell position “0”). (I) Projection of Yan et al., Nature, 2017 single-cell dataset (GSE92865) onto UMAP coordinates of Haber et al., Nature, 2017 (GSE92332) using Seurat unimodal UMAP projection, supporting capture of the Haber et al., “TA early” initial state by the Yan et al., “TA” signature and suggesting that these are similar populations. (J) RNA-Velocity analysis of the published Haber et al., Nature, 2017 mouse small intestine dataset. Velocity field arrows inferred using scVelo stochastic model and projected 10 4909-6074-3733v.193597 / 7344 onto the UMAP space indicate predicted lineage trajectories. (K) Schematic diagram for the spatial localization of Fgfbp1+ cells relative to Lgr5+ CBC cells at the crypt base (cell positions 0 to +3) in a prototypical jejunal crypt, based on ISH quantitation in panel H. Fgfbp1+ cells are centered around cell positions +4 to +13 in the upper crypt zone as determined by Fgfbp1 ISH.

[0070] FIGS. 2A-2H: Fgfbp1-TimeR allele reveals that the Fgfbp1+ upper crypt populationexhibits multi-potency and gives rise to the Lgr5+ CBC cells during homeostasis. (A) Targeting strategy and schema for the dual-fluorescence Fgfbp1-TimeR knock-in allele. Destabilized DsRedE2 and long-lived mTagBFP2 fluorophores are expressed stoichiometrically under control of the endogenous Fgfbp1 promoter. Allele preserves secreted endogenous Fgfbp1 gene product along with addition of FLAG epitope tag. Engineered LoxP sites enable conditional knock-out of Fgfbp1 (floxed allele). SI = synthetic intron. (B) In vivo validation of Fgfbp1-TimeR mice demonstrates spatial confinement of the labile DsRed signal to the upper crypt zone alongside distribution of long-lived mTagBFP2 along the crypt-villus axis in the intestine. Representative data from duodenum, scale bar = 50 μm. (C) Low magnification swiss-roll of Fgfbp1-TimeR mouse small intestine demonstrate robust DsRed / mTagBFP2 signal distribution across every single crypt-villus unit. High magnification views of crypts from the proximal and distal intestine highlight the DsRed signal is confined to the upper crypt zone, mirroring the Fgfbp1 mRNA distribution along the length of the intestine. In contrast, mTagBFP2 is observed along the crypt- villus axis. Scale bar for low magnification view = 1 mm, scale bar for high magnification images in selected boxed regions = 50 μm. (D) Co-localization of mTagBFP2 with enterocyte (FABP1), goblet (MUC2), enteroendocrine (CHGA), and Paneth cell (LYZ) markers demonstrates Fgfbp1+ upper crypt cells undergo multi-lineage differentiation during homeostasis. Scale bar = 25 μm. (E) Representative DsRed and mTagBFP2 IF in the Fgfbp1-TimeR; Lgr5-DTR-GFP small intestinal crypt. Overlapping expression of mTagBFP2 and Lgr5-GFP, but not DsRed and Lgr5-GFP, is observed. Representative image from jejunum, scale bar = 50 μm. (F) IF detection of mTagBFP2 and Lgr5-GFP across the length of the mouse small intestine demonstrates extensive overlap between long-lived mTagBFP2 signal and Lgr5-GFP, supporting a model in which Lgr5+ CBC cells are progeny of Fgfbp1+ upper crypt cells throughout the intestine. mTagBFP2 is pseudo- colored in red to visualize overlap with Lgr5-GFP (overlap is yellow), scale bar = 25 μm. (G) Left, FACS analysis of EPCAM+ / Lgr5-GFP+ cells from the proximal small intestine of Fgfbp1-TimeR; Lgr5-DTR-GFP mice reveals expression of mTagBFP2 within the Lgr5+ CBC population. Right, 11 4909-6074-3733v.193597 / 7344 mTagBFP2+ signal is observed in both Lgr5-GFPLow and Lgr5-GFPHigh epithelial cells at similar proportion by FACS analysis. Representative data from jejunum. (H) Left, Quantitation of mTagBFP2 and DsRed distribution among Lgr5-GFP+ cells in small intestine tissue sections highlighting the minimal overlap of Lgr5-GFP and DsRed (red bar) yet abundant overlap of Lgr5- GFP and mTagBFP (blue bar) along the length of the intestine. Top right, Relative position of Lgr5-GFP+ / DsRed- / mTagBFP2+ cells within the crypts of Fgfbp1-TimeR; Lgr5-DTR-GFP small intestine, suggesting the long-lived mTagBFP2 signal is transmitted down by progeny of upper crypt zone cells to the crypt base (0 to +3 cell positions, corresponding to the location of Lgr5+ CBC cells) and is not only restricted to the crypt base-upper crypt boundary (+4 cell position). Bottom right, Relative position of the rare Lgr5-GFP+ / DsRed+ / mTagBFP2+ cells found within the crypts of Fgfbp1-TimeR; Lgr5-DTR-GFP small intestine. While the vast majority of Lgr5- GFP+ cells are DsRed-, the rare cells that do co-express Lgr5-GFP and DsRed are primarily localized to the crypt base-upper crypt interface (at the +4 cell position). All data is represented as mean with S.D., n=3 mice.

[0071] FIGS. 3A-3I: Fgfbp1-CreERT2 lineage tracing reveals rapid regeneration of the Lgr5+CBC compartment from the upper crypt during homeostasis. (A) Targeting strategy and schema for Fgfbp1-CreERT2 knock-in allele. (B) Scattered tdTomato+ cells populate the upper crypt (UC), but not the crypt base (CBC, defined as 0 to +3 cell position) at 18h post-TAM administration in Fgfbp1-CreERT2; Rosa-tdTomato mice. Data from jejunum, scale bar = 50 μm. (C) Dot plot displaying relative positions of tdTomato+ cells along the crypt-villus axis over time. Data represents overall cell positions of tdTomato+ cells quantitated across >50 crypts per region throughout the small intestine, n=3 mice. (D) Fgfbp1-CreERT2 lineage tracing time-course in the mouse intestinal epithelium shows rapid regeneration of villus cells from the Fgfbp1+ upper crypt zone. Notably, tdTomato+ cells rise up the crypt-villus axis and reach the villus tip by D4 post- TAM. Representative data from jejunum, scale bar = 50 μm. (E) Fgfbp1-CreERT2 lineage tracing shows rapid Lgr5+ CBC cell replacement by the Fgfbp1+ upper crypt zone over the course of 14 days throughout the small intestine. At 18h and D2 timepoints, tdTomato+ labeled cells are localized to the upper crypt zone. Then tdTomato+ crypt base cells (see white arrowheads) are observed by D4, followed by an explosion of labeling events in CBC positions (0 to +3) by D7 and D14 post-TAM. Scale bar = 50 μm. (F) Low magnification full-length swiss-roll of Fgfbp1- CreERT2; Rosa26-tdTomato intestine, D4 and D7 post-TAM administration. White box notes 12 4909-6074-3733v.193597 / 7344 regions shown with higher magnification. The extent of regeneration at these timepoints is evident as observed by widespread tdTomato+ signal. Note greater abundance of tdTomato+ traces at the crypt base by D7 compared to D4. Scale bar for low, medium and high magnification views = 2.5 mm, 250 μm, and 50 μm, respectively. (G) Fgfbp1+ upper crypt cells give rise to Lgr5+ CBC cells within 7 days, as evidenced by the presence of tdTomato+ cells at cell positions 0 to +3 overlapping with Lgr5-GFP in Fgfbp1-CreERT2; Rosa-tdTomato; Lgr5-DTR-GFP mice (top) and OLFM4 expression (center), and interspersed between LYZ+ Paneth cells (bottom). Representative data from jejunum, D7 post-TAM. Scale bar = 50 μm. (H) Co-localization of tdTomato+ lineage traces with markers of secretory and absorptive cell types by IF validates the multi-lineage potential of the Fgfbp1+ upper crypt population. Data from Fgfbp1-CreERT2; Rosa26-tdTomato jejunum, D21 post-TAM, scale bar = 50 μm. (I) Clonal labeling in Fgfbp1- CreERT2; Rosa26-Confetti small intestine supports cellular multi-potency of Fgfbp1+ upper crypt cells, shown by mixed absorptive (ACE2+) and secretory (MUC2+) cells within a Confetti-RFP+ (Cf-RFP) clonal trace. Dashed lines represent outline of ACE2+ enterocyte, * represents MUC2+ goblet cell. Representative data from the jejunum, D10 post-TAM, scale bar = 25 μm.

[0072] FIGS.4A-4H: Fgfbp1+ cells persist to repopulate crypt bases upon targeted ablation ofLgr5+ CBC cells and exhibit differential response to Rspo modulation. (A) Top, Experimental schema for DT-mediated ablation of Lgr5-GFP+ cells in Fgfbp1-TimeR; Lgr5-DTR-GFP mice. Bottom, Confirmation of complete Lgr5-GFP+ cell ablation after DT administration by FACS. (B) Fgfbp1+ cells (DsRed+ / mTagBFP2+) and their progeny (mTagBFP2+) persist unperturbed in the epithelium despite DT-mediated ablation of Lgr5+ CBCs. Repopulation of crypt bases by Fgfbp1- derived mTagBFP2+ progeny (but not Fgfbp1+ cells themselves) is shown. Representative data from duodenum, scale bar = 50 μm. (C) Fgfbp1+ cells support regeneration throughout the crypt- villus axis in the absence of an Lgr5+ CBC cell compartment following DT-mediated ablation, as evidenced by mTagBFP2 signal all the way into the villus tips. Representative data from the duodenum. Scale bar for low magnification view = 250 μm, scale bar for higher magnification view = 50 μm. (D) Top, Experimental schema for adenoviral (Ad)-mediated Rspo signaling modulation in Fgfbp1-TimeR mice. Bottom, Confirmatory histological analysis by H&E and Lgr5 ISH for validation of adenoviral-mediated hepatic transduction and systemic overexpression of Lgr5-ECD (Ad Lgr5-ECD) and Rspo1 (Ad Rspo1) in mice, D4 post-treatment. H&E reveals ballooning and Paneth cell loss in Lgr5-ECD treated animals compared to control Ad Fc tissues, 13 4909-6074-3733v.193597 / 7344 as well as crypt enlargement following Rspo augmentation. Scale bar = 50 μm. (E) Response of crypt-residing Fgfpb1+ cells and their progeny to pharmacological Rspo inhibition and overexpression, showcasing the expansion of Fgfbp1+ upper crypt cells into the crypt bases following Rspo blockade by Lgr5-ECD and the expansion of the upper crypt Fgfbp1+ population upon Rspo augmentation. Scale bar = 50 μm. (F) Histograms demonstrate relative positions of DsRed+ cells along the crypt villus axis upon Rspo signaling modulation, highlighting changes in crypt occupancy and the mitogenic effect of R-spondin on the Fgfbp1+ cells. Representative data of duodenum, D4 post-treatment. (G) Fgfbp1+ cells support crypt-villus axis regeneration in the absence of a functioning Lgr5+ CBC cell compartment as evidenced by abundant mTagBFP2 signal in the crypts and villi following Lgr5-ECD-mediated Lgr5+ CBC cell depletion or Rspo- induced pharmacological “entrapment” of Lgr5+ CBC cells. Representative data from the duodenum, D4-post treatment. Scale bar = 50 μm. (H) FACS analysis demonstrating persistence of mTagBFP2+ populations in Fgfbp1-TimeR mice following treatment with Ad Lgr5-ECD, as well as expansion of mTagBFP2+ populations after Ad Rspo1 administration (black arrows), consistent with Fgfbp1+ upper crypt cells continued reconstitution of the epithelium under the context of both Rspo loss- and gain-of-function. Data from proximal jejunum, D4 post-treatment.

[0073] FIGS. 5A-5H: Fgfbp1 is essential for intestinal epithelial homeostasis. (A)Experimental schema for Villin-CreERT2-driven Fgfbp1 conditional knockout (cKO) in the adult murine intestinal epithelium over the course of 4 days. Note that high-dose tamoxifen treatment (9mg / 40g body weight) led to mortality of all mice by D3-4 so the tamoxifen dose was lowered to 6mg / 40g body weight in these experiments. (B) Vil-CreERT2-driven Fgfbp1 CKO results in gross reduction of intestine length, scale bar = 5 cm. (C) FLAG-FGFBP1 IF in control (Fgfbp1f / f) versus Fgfbp1 cKO (Vil-CreERT2;Fgfbp1f / f) animals, confirming successful Fgfbp1 deletion. Representative data from jejunum, scale bar = 50 μm. (D) H&E analysis of control versus Fgfbp1 cKO intestine demonstrates gross architectural abnormalities upon Fgfbp1 cKO. Villus blunting, crypt atrophy and crypt dropout are all morphological features of Fgfbp1 loss. Representative data from jejunum, scale bar = 50 μm. (E) Top, Cleaved caspase 3 (CC3) immunostaining highlights the accumulation of apoptotic cells in the villus tips. Bottom, LYZ immunostaining reveals the accumulation of LYZ+ Paneth cells in residual crypts without their normal interspersed CBC cells. Representative data from jejunum, scale bar = 50 μm. (F) Crypts from Fgfbp1 cKO mice fail to grow as organoids ex vivo, supporting a model for impaired regeneration upon Fgfbp1 deletion. 14 4909-6074-3733v.193597 / 7344 Data from jejunum-derived intestinal epithelial crypts, D4 in culture. Scale bar = 1 mm. (G) Representative H&E staining of intestinal crypt (cross-sectional view) reveal vast areas of crypt dropout and immune infiltration upon Fgfbp1 cKO. Representative data from jejunum, scale bar = 50 μm. (H) Multiplex ISH for Lgr5 and Fgfbp1 combined with Ki67 IF demonstrates decreased crypt proliferation as well as Fgfbp1+ cell and Lgr5+ CBC cell loss following Fgfbp1 cKO. Representative data from jejunum, scale bar = 50 μm.

[0074] FIGS. 6A-6D: Model of the upper crypt zone as the source of intestinal epithelialregeneration. (A) Fgfbp1+ ISCs in the upper crypt are multi-potent cells that give rise to all the mature lineages, including the Lgr5+ CBC cells, along the length of the small intestine during homeostasis. (B) Loss of Lgr5+ cells from selective ablation or pharmacological Rspo inhibition results in reconstitution of crypt bases by a pre-existing pool of Fgfbp1+ ISCs, highlighting their non-overlap with Lgr5+ CBC cells. (C) The Rspo signaling axis is a critical modulator in controlling the transition between Fgfbp1+ upper crypt cells and Lgr5+ CBC cells. Like Lgr5+ CBC cells, Fgfbp1+ upper crypt cells are Rspo-responsive and expand upon Rspo stimulation. However, unlike Lgr5+ CBC cells, Fgfbp1+ ISCs in the upper crypt do not require Rspo signaling (via LGR5) for their self-renewal. In the absence of Rspo, Lgr5+ CBC cells are depleted and Fgfbp1+ cells do not regenerate them. (D) FGFBP1 is essential for intestinal epithelial homeostasis. Fgfbp1 loss results in disrupted intestinal epithelial tissue architecture, including crypt dropout, impaired proliferation, and the loss of all the Lgr5+ CBC cells consistent with impaired regeneration.

[0075] FIG. 7: Mouse FGFBP1 protein structure and domains. AlphaFold prediction of 3Dstructures of murine and human FGFBP1 proteins, highlighting a signal peptide, N-terminus comprising a putative heparin Binding Domain and C-terminal FGF Binding Domain.

[0076] FIG.8: Experimental schema for rescue of Fgfbp1 genetic deletion organoid phenotypeby recombinant FGFBP1 Domain A ex vivo. (Peptide A and Domain A as used herein are exemplary embodiments of the N-terminal fragments or N-terminal domains (NTDs) as disclosed herein).

[0077] FIG. 9: Rescue of Fgfbp1 genetic deletion phenotype by recombinant FGFBP1Domain A ex vivo. Single cells from jejunum organoid of Vil-CreER; Fgfbp1fl / flmouse were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with standard WENR organoid culture medium supplemented with either vehicle (ethanol) control versus 4- 15 4909-6074-3733v.193597 / 7344Hydroxytamoxifen (4-OHT) to enable conditional knockout of Fgfbp1 ex vivo. Differentconcentration of FGFBP1 Domain A (N-terminal domain T34-R162) versus Domain B (C- terminal domain A208-C251 comprising FGF Binding Domain) versus stoichiometric Domain A plus Domain B were added medium containing 4-OHT for 5 days. Representative images of organoids were shown here.

[0078] FIG. 10: Fgfbp1 is essential for organoid growth and maintenance. Single cellsdissociated from established jejunum organoids of Vil-CreER; Fgfbp1fl / flmouse were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with standard WENR culture medium supplemented with either vehicle (ethanol) control versus 4-Hydroxytamoxifen (4-OHT) versus 4-OHT plus FGFBP1 Domain A for 5 days. Representative images of organoids are shown here. Organoid cell viability was measured by CellTiter Glo 3D following manufacturer’s protocol. P-value was calculated by Student’s t-test.

[0079] FIG. 11: Domain A increases WT mouse organoid number and promotes organoidgrowth. Single cells dissociated from established jejunum organoid of wide type (WT) mouse were resuspended with Matrigel and seeded in plates with indicated densities. The Matrigel dome was overlayed with standard WENR culture medium supplemented with either vehicle control or different concentrations of FGFBP1 Domain A for 5 days. Representative images of organoids are shown here.

[0080] FIG. 12: Domain A increases mouse organoid number and promotes organoid growth.Single cells from jejunum organoid of wide type (WT) mouse were resuspended with Matrigel and seeded in plates with indicated densities. Matrigel dome was overlayed with standard WENR culture medium supplemented with either vehicle control or different concentration of FGFBP1 domain A for 5 days. Organoid numbers were counted and fold change to control group was calculated. Organoid cell viability was measured by CellTiter Glo 3D following manufacturer’s protocol and fold change to control group was calculated. P-value was calculated by Student’s t- test.

[0081] FIG. 13: Domain A promotes human colon organoid growth, D5 and D6. Single cellsfrom human colon organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 Domain A for 5 and 6 days. Representative images of organoids are shown here. 16 4909-6074-3733v.193597 / 7344

[0082] FIG. 14: Domain A promotes human colon organoid growth, D7 and D8. Single cellsfrom human colon organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 Domain A for 7 and 8 days. Representative images of organoids are shown here.

[0083] FIG. 15: Domain A promotes human colon organoid growth, D9 and D10. Single cellsfrom human colon organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 Domain A for 9 and 10 days. Representative images of organoids are shown here.

[0084] FIG.16: Domain A promotes human colon organoid growth, D11 and D12. Single cellsfrom human colon organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 Domain A for 11 and 12 days. Representative images of organoids are shown here.

[0085] FIG. 17: Domain A promotes human colon organoid growth, D13 amd D14. Singlecells from human colon organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 Domain A for 13 and 14 days. Representative images of organoids are shown here.

[0086] FIG.18: Domain A promotes human colon organoid growth as determined by viabilityassay. Single cells from human colon organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 domain A for indicated time points. Organoid cell viability was measured by CellTiter Glo 3D following manufacturer’s protocol at each time point and fold change to control group was calculated. P- value was calculated by Student’s t-test.

[0087] FIG. 19: Domain A maintains viable long-term culture of human colon organoids withreduced need for culture medium change, D11. Single cells from human colon organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different 17 4909-6074-3733v.193597 / 7344 concentration of FGFBP1 Domain A for 11 days. Representative images of organoids are shown here.

[0088] FIG. 20: Domain A maintains viable long-term culture of human colon organoids withreduced need for culture medium change, D12. Single cells from human colon organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 Domain A for 12 days. Representative images of organoids are shown here.

[0089] FIG. 21: Domain A maintains viable long-term culture of human colon organoids withreduced need for culture medium change, D13. Single cells from human colon organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 Domain A for 13 days. Representative images of organoids are shown here.

[0090] FIG. 22: Domain A maintains viable long-term culture of human colon organoids withreduced need for culture medium change, D14. Single cells from human colon organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 Domain A for 14 days. Representative images of organoids are shown here.

[0091] FIG. 23: Human colon organoid before collecting for RNA isolation on D10. Singlecells from human colon organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 Domain A for 10 days. Representative images of organoids are shown here.

[0092] FIG. 24: Domain A promotes human duodenum organoid growth. Single cells fromhuman duodenum organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 Domain A for 6 and 9 days. Representative images of organoids are shown here. 18 4909-6074-3733v.193597 / 7344

[0093] FIG. 25: Domain A promotes human duodenum organoid growth as determined byviability assay. Single cells from human duodenum organoids were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentration of FGFBP1 Domain A for 6 and 9 days. Organoid cell viability was measured by CellTiter Glo 3D following manufacturer’s protocol and fold change to control group was calculated. P-value was calculated by Student’s t-test.

[0094] FIG.26: Fgfbp1 marks a population of upper crypt colon cells that are spatially distinctfrom the Lgr5+ CBC cells in both the proximal and distal colon. (A) Low magnification view of WT murine colon following multiplex in situ hybridization (ISH) for Fgfbp1 and Lgr5. (B) Higher magnification views of the multiplex ISH shown in (A), demonstrating the presence of Fgfbp1+ cells localized above the CBC zone along the WT murine colon.

[0095] FIG. 27: Fgfbp1+ cells exhibit multi-lineage differentiation and give rise to allepithelial cells in the colon including Lgr5+ CBC cells. (A) Fgfbp1-TimeR mouse colon reveals DsRed+ / Fgfbp1+ cells above the CBC compartment, yet their progeny give rise to CBC cells. Note that the Lgr5-GFP+ CBC cells also are mTagBFP2+ in Fgfbp1-TimeR; Lgr5-DTR-GFP mice. (B) Fgfbp1+ cells give rise to both absorptive (CA4+) and secretory lineage (MUC2+ and CHGA+) cells in the colon epithelium.

[0096] FIG. 28: Lineage tracing in Fgfbp1-CreER; Rosa26-tdTomato mice reveal long-livedlineage tracing originating from Fgfbp1+ the upper crypt (UC) above the CBC zone in the colon, consistent with stem cell activity. Yellow dashed lines denote boundary between the CBC and UC zones. Notably, tracing events originate in the UC and over time migrate down to the CBC zone and reconstitute the entire colon epithelium.

[0097] FIG. 29: Global genetic deletion of Fgfbp1 from all tissues in the adult mouse resultsin rapid impairment of colon epithelial homeostasis that is recapitulated by intestinal epithelium- specific deletion. (A) H&E staining of colon tissue shows marked disruption of colon architecture and loss of the abundant goblet cells in the colon following global knockout of Fgfbp1 in adult Rosa26-CreER; Fgfbp1f / fmice. (B) H&E staining of the colon tissue shows similar architecturalchanges upon intestinal epithelium-specific knockout of Fgfbp1 in adult Vil-CreER; Fgfbp1f / fmice. 19 4909-6074-3733v.193597 / 7344

[0098] FIG. 30: Global genetic deletion of Fgfbp1 from all tissues in the adult mouse resultsin marked apoptosis and loss of epithelial integrity, proliferation, and both absorptive and secretory lineage cells in the colon epithelium in Rosa26-CreER; Fgfbp1f / fmice.

[0099] FIG. 31: Intestinal epithelium-specific knockout of Fgfbp1 in the adult mouse resultsin marked apoptosis and loss of epithelial integrity, proliferation, and both absorptive and secretorylineage cells in the colon epithelium in Vil-CreER; Fgfbp1f / f mice, recapitulating the phenotype ofthe global knockout.

[0100] FIGS. 32A-32B: FGFBP1 NTD is sufficient to rescue regenerative impairment ofFgfbp1- / -intestinal organoids. A) Single cells dissociated from pre-established small intestinal organoids generated from Vil-CreER; Fgfbp1fl / flmouse jejunum were resuspended with Matrigel and seeded in plates. The cells embedded in Matrigel were then culture in standard WENR culture medium supplemented with either vehicle (ethanol) control vs. 4-Hydroxytamoxifen (4-OHT) with or without murine FGFBP1 NTD (1 µM) for 5 days. Representative images of organoids are shown here. Day 5 of culture. Scale bar: 200 µm. B) Organoid cell viability was measured by CellTiter Glo 3D viability assay following the manufacturer’s protocol. Percent viability was normalized to the control. P-value was calculated by unpaired Student’s t-test.

[0101] FIGS. 33A-33B: Commercially available human full length FGFBP1 produced in E.coli did not rescue regenerative impairment in Fgfbp1 cKO organoids. A) Single cells dissociated from pre-established small intestinal organoids generated from Vil-CreER; Fgfbp1fl / flmouse jejunum were resuspended with Matrigel and seeded in plates. The cells embedded in Matrigel were cultured in standard WENR culture medium supplemented with either vehicle (ethanol) control vs.4-Hydroxytamoxifen (4-OHT) with or without recombinant human full length FGFBP1 (Lys24 to Cys234 expressed in E. coli purchased from Peprotech / ThermoFisher, 8.4 nM) for 5 days. Representative images of organoid are shown here. Day 5 of culture. Scale bar: 400 µm. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value was calculated by unpaired Student’s t-test.

[0102] FIGS. 34A-34B: FGFBP1 NTD induces proliferation and growth of wild type murinesmall intestine and colon ex vivo. A) Single cells dissociated from pre-established small intestinal organoids generated from wild type (WT) mouse jejunum were resuspended with Matrigel and seeded in plates. The cells embedded in Matrigel were cultured in standard WENR culture medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 20 4909-6074-3733v.193597 / 7344 5 days. Representative images of organoids are shown here. Organoids cell viability was measured by CellTiter Glo 3D following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test. Scale bar: 400 µm. B) Single cells dissociated from pre-established colon organoids of WT mouse colon were resuspended with Matrigel and seeded in plates. The cells embedded in Matrigel were cultured in standard WENR culture medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 6 days. Representative images of organoids are shown here. Day 6 of culture. Scale bar: 400 µm. Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test.

[0103] FIGS. 35A-35B: FGFBP1 NTD induces proliferation and growth of primary humanintestine and colon ex vivo. A) Single cells dissociated from pre-established small intestinal organoids generated from primary adult human duodenum endoscopic biopsies were resuspended with Matrigel and seeded in plates. The cells embedded in Matrigel dome were cultured in Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 7 days. Representative images of organoids are shown here. Day 7 of culture. Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control (0 nM) was calculated by unpaired Student’s t-test. Scale bar: 400 µm. B) Single cells dissociated from pre- established colon organoids generated from primary adult human colon biopsies were resuspended with Matrigel and seeded in plates. The cells embedded in Matrigel dome were cultured in Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 13 days. Representative images of organoids are shown here. Day 13 of culture. Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control (0 nM) was calculated by unpaired Student’s t-test. Scale bar: 400 µm.

[0104] FIGS.36A-36B: Human FGFBP1 NTD exhibits similar bioactivity to murine FGFBP1NTD in expanding human intestinal organoids ex vivo. A) Single cells dissociated from pre- established small intestinal organoids generated from primary adult human duodenum were resuspended with Matrigel and seeded in plates. The cells embedded in Matrigel were cultured in Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or 21 4909-6074-3733v.193597 / 7344 different concentrations of human FGFBP1 NTD for 7 days. Representative images of organoids are shown here. Day 7 of culture. B) Organoid cell viability was measured by CellTiter Glo 3D following manufacturer’s protocol. RLU, relative light unit. P-value compared to control (0 nM) was calculated by unpaired Student’s t-test. Scale bar: 400 µm.

[0105] FIGS. 37A-37B: Murine FGFBP1 NTD promotes human stomach (gastric body)organoid growth. A) Single cells dissociated from pre-established gastric organoid generated from primary adult human stomach (gastric body) were resuspended with Matrigel and seeded in plates. The cells embedded in Matrigel were cultured in Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 7 days. Representative images of organoids are shown here. Day 7 of culture. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test. Scale bar: 400 µm.

[0106] FIGS. 38A-38B: FGFBP1 NTD promotes human fetal gut epithelial organoid growth.A) Single cells dissociated from pre-established gut organoids generated from primary human fetal gut (at 16 weeks gestation) were resuspended with Matrigel and seeded in plates. The cells embedded in Matrigel dome were cultured in Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 7 days. Representative images of organoids are shown here. Day 7 of culture. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test. Scale bar: 400 µm.

[0107] FIGS. 39A-39B: FGFBP1 NTD promotes aged mouse (29 month old) small intestinalorganoid growth and viability. A) Single cells dissociated from pre-established small intestinal organoids generated from an aged (29 month old) mouse jejunum were resuspended with Matrigel and seeded in plates. The cells embedded in Matrigel were cultured in standard WENR culture medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 5 days. Representative images of organoids are shown here. Day 5 of culture. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test. Scale bar: 400 µm. 22 4909-6074-3733v.193597 / 7344

[0108] FIGS. 40A-40B: FGFBP1 NTD promotes aged mouse (29 months) colon organoidgrowth and viability. A) Single cells from pre-established organoids generated from aged (29 month old) mouse colon were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with standard WENR culture medium supplemented with either vehicle control or different concentrations of mouse FGFBP1 NTD or peptide A for 6 days. Representative images of organoids are shown here. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test. Scale bar: 400 µm. (Peptide A and Domain A as used herein are exemplary embodiments of the N-terminal fragments or N-terminal domains (NTDs) as disclosed herein).

[0109] FIGS. 41A-41B: FGFBP1 NTD promotes celiac patient duodenum organoid growthand viability. A) Single cells from pre-established organoids generated from a celiac patient’s duodenal biopsies were resuspended with Matrigel and seeded in plates. The cells embedded in Matrigel were cultured with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 7 days. Representative images of organoids are shown here. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test. Scale bar: 400 µm.

[0110] FIGS. 42A-42B: FGFBP1 NTD promotes growth and viability of primary organoidsfrom inflamed Crohn’s disease ileum. A) Crypts isolated from normal vs adjacent inflamed ileum of patient with Crohn’s disease were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD or peptide A for 7 days. Representative images of organoids are shown here. Day 7 of culture. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test. Scale bar: 400 µm.

[0111] FIGS. 43A-43B: FGFBP1 NTD promotes growth and viability of primary organoidsfrom inflamed UC colon. A) Crypts isolated from biopsies of normal vs. adjacent inflamed colon of patient with ulcerative colitis were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with Human IntestiCultTMorganoid growth medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 7 days. Representative 23 4909-6074-3733v.193597 / 7344 images of organoids are shown here. Day 7 of culture. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test. Scale bar: 400 µm.

[0112] FIGS. 44A-44B: FGFBP1 NTD promotes growth and viability of human fetal liverorganoids. A) Single cells dissociated from organoids generated from primary human fetal liver were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with human fetal liver organoid growth medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 7 days. Representative images of organoids are shown here. Day 7 of culture. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test. Scale bar: 400 µm.

[0113] FIGS. 45A-45B: FGFBP1 NTD promotes growth and viability of organoids derivedfrom primary human fetal pancreas. A) Single cells dissociated from organoids generated from primary human fetal pancreas were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with human fetal pancreas organoid growth medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 7 days. Representative images of organoids are shown here. Day 7 of culture. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test. Scale bar: 400 µm.

[0114] FIGS. 46A-46B: FGFBP1 NTD promotes growth and viability of adult human liverorganoids. A) Single cells dissociated from organoids generated from primary human adult liver were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with human adult liver organoid growth medium supplemented with either vehicle control or different concentrations of murine FGFBP1 NTD for 7 days. Representative images of organoids are shown here. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpaired Student’s t-test. Scale bar: 400 µm.

[0115] FIGS. 47A-47B: FGFBP1 NTD promotes growth of human adult pancreas organoids.A) Single cells dissociated from organoids generated from primary human adult pancreas were resuspended with Matrigel and seeded in plates. Matrigel dome was overlayed with human adult pancreas organoid growth medium supplemented with either vehicle control or different 24 4909-6074-3733v.193597 / 7344 concentrations of murine FGFBP1 NTD for 7 days. Representative images of organoids are shown here. B) Organoid cell viability was measured by CellTiter Glo 3D assay following manufacturer’s protocol. RLU, relative light unit. P-value compared to control was calculated by unpair Student’s t-test. Scale bar: 400 µm.

[0116] FIG. 48: Systemic expression of murine FGFBP1 NTD-Fc in the circulation of micepost adenovirus intravenous injection and hepatic transduction. Time course of serum expression after a single intravenous injection of adenovirus into C57Bl / 6 mice. Anti-His western blot on serum at indicated time points after injection of Ad-FGFBP1 NTD-Fc (His-tagged).

[0117] FIGS. 49A-49B: FGFBP1 NTD protects gut epithelial against irradiation injury. A)Control adenovirus Ad Fc or Ad FGFBP1 NTD-Fc was injected intravenously into mice 24 hrs prior to 12 Gy whole body irradiation (WBI). Small intestine and colon were harvested 3 days post irradiation. B) Histological analysis shows control Ad Fc treated mice exhibit more severe epithelial damage with crypt loss and extensive immune infiltration after 12 Gy WBI consistent with irradiation injury. In contrast, Ad FGFBP1 NTD-Fc treated mice exhibited more intact and deeper crypt structures consistent with regenerating, proliferative crypts. H&E. Scale bars: 100 µm.

[0118] FIGS. 50A-50B: FGFBP1 NTD augments gut epithelial regeneration followingirradiation injury. A) Experiment schema. Control adenovirus Ad Fc or Ad FGFBP1 NTD-Fc was injected intravenously into mice 30 minutes post 12 Gy whole body irradiation (WBI). Small intestine and colon were harvested 3 days post irradiation. B) Histological analysis shows control Ad Fc treated mice demonstrated extensive crypt loss after 12Gy WBI consistent with loss of proliferative stem / progenitor cells. In contrast, Ad FGFBP1 NTD-Fc treated mice show preservation of crypts consistent with regeneration. H&E. Scale bars: 100 µm.

[0119] FIGS. 51A-51C: FGFBP1 NTD augments epithelial proliferation and repair followingirradiation injury. A) Experiment schema. Control adenovirus Ad Fc or Ad FGFBP1 NTD-Fc was injected intravenously into mice 30 minutes post 12 Gy whole body irradiation (WBI). Small intestine and colon were harvested 3 days post irradiation. B) Ad FGFBP1 NTD-Fc treated mice show larger proliferative (Ki67+) crypt structures in the small intestine (jejunum) compared to Ad Fc. Ki67 immunostaining. Top panels are lower magnification views. Bottom panels are higher magnification views. C) Ad FGFBP1 NTD-Fc treated mice how larger proliferative (Ki67+) crypt 25 4909-6074-3733v.193597 / 7344 structures in the colon compared to Ad Fc. Ki67 immunostaining. Top panels are lower magnification views. Bottom panels are higher magnification views. Scale bars: 50 µm.

[0120] FIGS. 52A-52B: FGFBP1 NTD augments epithelial proliferation and repair in a DSSinflammatory colitis mouse model. A) Experiment schema of DSS induced inflammatory colitis and adenovirus treatment.3% of DSS in drinking water was provided to mice for 11 days.7 days after initiation of DSS treatment, control adenovirus Ad Fc or Ad FGFBP1 NTD-Fc was injected intravenously into mice. Mouse colon was harvested 4 days post injection. B) Ad FGFBP1 NTD- Fc treated mice demonstrate a preserved epithelial barrier, lack of apoptosis and enhanced proliferation in the colon consistent with augmented proliferation and repair in the setting of DSS colitis. Ki67 (proliferation), CC3 (apoptosis), E-cadherin (epithelial barrier) immunostaining. Scale bars: 50 µm.

[0121] FIG.53: FGFBP1 NTD promotes proliferation in adult mouse liver in vivo. Control AdFc or Ad FGFBP1 NTD-Fc was injected intravenously into adult wild type C57Bl / 6 mice (8 weeks old) and liver was harvested 4 days post injection. Immunostaining of the liver demonstrates that Ad FGFBP1 NTD-Fc treatment results in high-level expression of the FLAG-tagged transgenic peptide in the liver that is accompanied by dramatic proliferation, which is not found in the control Ad Fc treated mice. Ki67 and anti-FLAG immunostaining. Scale bars: 50 µm.

[0122] FIG. 54: FGFBP1 NTD promotes hepatocyte proliferation in adult mouse liver in vivo.Control Ad Fc or Ad FGFBP1 NTD-Fc was injected intravenously into adult wild type C57Bl / 6 mice (8 weeks old) and liver was harvested 4 days post injection. Immunostaining of liver tissue sections demonstrates proliferating (Ki67+) hepatocytes (HNF4A+) following treatment with Ad FGFBP1 NTD-Fc but not control Ad Fc. Ki67 and HNF4A immunostaining. Scale bar: 50 µm.

[0123] FIG.55: FGFBP1 NTD promotes proliferation in the adult mouse lung in vivo. ControlAd Fc or Ad FGFBP1 NTD-Fc was injected intravenously into adult wild type C57Bl / 6 mice (8 weeks old) and lung was harvested 4 days post injection. Immunostaining of lung tissue sections from Ad FGFBP1 NTD-Fc treated mice reveal highly proliferative cells not observed in control Ad Fc treated mice. Immunostaining for Ki67 (proliferation), Nkx2.1 (airway, AT1, and AT2 cells) in the adult mouse lung. Scale bars: 100 µm.

[0124] FIG. 56: FGFBP1 NTD promotes massive proliferation throughout the adult mousekidney. Control Ad Fc or Ad FGFBP1 NTD-Fc was injected intravenously into adult wild type C57Bl / 6 mice (8 weeks old) and kidneys were harvested 4 days post injection. Immunostaining of 26 4909-6074-3733v.193597 / 7344 kidney tissue sections reveals massive proliferation throughout the kidney of Ad FGFBP1 NTD- Fc treated mice that was not seen in control Ad Fc treated mice. Immunostaining for Ki67.

[0125] FIG. 57: FGFBP1 NTD promotes proliferation in the adult mouse kidney in vivo.Control Ad Fc or Ad FGFBP1 NTD-Fc was injected intravenously into adult wild type C57Bl / 6 mice (8 weeks old) and kidneys were harvested 4 days post injection. Immunostaining of kidney tissue sections reveals proliferation of E-cadherin+ cells throughout the kidney of Ad FGFBP1 NTD-Fc treated mice that was not seen in Ad Fc treated control mice. Immunostaining for Ki67 and E-cadherin. Scale bars: 100 µm.

[0126] FIG. 58: FGFBP1 NTD promotes proliferation of multiple cell types in adult mousekidney in vivo. Control Ad Fc or Ad FGFBP1 NTD-Fc was injected intravenously into adult wild type C57Bl / 6 mice (8 weeks old) and kidneys were harvested 4 days post injection. Immunostaining of kidney tissue sections reveals proliferation of multiple cell types, including the Lrp2 / Megalin+ cells in the proximal tubules, in the kidney of Ad FGFBP1 NTD-Fc treated mice but not in the Ad Fc treated control mice. Co-immunostaining of Ki67, uromodulin, Lrp2 / Megalin (epithelial cells of proximal tubules).

[0127] FIG. 59: FGFBP1 NTD promotes gastric stem cell proliferation in the corpus of adultwild type mouse stomach in vivo. Control Ad Fc or Ad FGFBP1 NTD-Fc was injected intravenously into adult wild type C57Bl / 6 mice (8 weeks old) and their stomachs were harvested 4 days post injection. Immunostaining of stomach tissue sections reveals enhanced proliferation in the isthmus region of the gastric corpus, which is the zone known to harbor gastric stem cells, in Ad FGFBP1 NTD-Fc treated mice compared to Ad Fc treated controls mice. Co-immunostaining of Ki67 (red) and E-cadherin (green).

[0128] FIGS. 60A-60C: FGFBP1 NTD promotes healing of gastric ulcers. Control Ad Fc orAd FGFBP1 NTD-Fc was injected intravenously into adult wild type C57Bl / 6 mice (8 weeks old) 14 hrs prior to ulcer induction. Briefly, acute gastric ulcers were generated on the serosal surface of the gastric corpus using 100% acetic acid application with a glass capillary tube for 15 seconds model following laparotomy. The stomachs of these mice were harvested 12 days following adenovirus and ulcer induction. A) Gastric ulcer size is reduced with Ad FGFBP1 NTD-Fc treated mice compared to Ad Fc treated controls. Scale bars: 2 mm. B) Histological analysis of stomach tissue sections by H&E staining reveals reduced ulcer size and enhanced epithelial architecture in 27 4909-6074-3733v.193597 / 7344 Ad FGFBP1 NTD-Fc treated mice compared to Ad Fc treated controls. Scale bars: 200 µm C) Quantitation of gastric ulcer size from mice treated with Ad Fc vs Ad FGFBP1 NTD-Fc.

[0129] FIG. 61: FGFBP1 NTD expands white pulp in the adult mouse spleen. Control Ad Fcor Ad FGFBP1 NTD-Fc was injected intravenously into adult wild type C57Bl / 6 mice (8 weeks old) and they were sacrificed and spleen samples were harvested 4 days post injection. Histological analysis of spleen tissue sections by H&E staining reveals spleen white pulp is massively expanded in Ad FGFBP1 NTD-Fc treated mice compared to Ad Fc treated controls. Scale bars: 200 µm.

[0130] FIG. 62: FGFBP1 NTD increases bone marrow cellularity in adult mouse long boneupon adenovirus intravenous injection. Control Ad Fc or Ad FGFBP1 NTD-Fc was injected intravenously into adult wild type C57Bl / 6 mice (8 weeks old). Mice were sacrificed and long bone samples were harvested 4 days post injection. Histological analysis by H&E staining reveals that bone marrow cellularity is dramatically increased in Ad FGFBP1 NTD-Fc treated mice compared to Ad Fc treated controls. Day 4 post injection. Representative histology from n=3 mice (biological replicates) are shown. Scale bars: 60 µm.

[0131] FIG. 63: FGFBP1 NTD induces proliferation of islets, enlarges islets, and expandsinsulin+ beta cell number in adult wild type mouse pancreas in vivo. Control Ad Fc or Ad FGFBP1 NTD-Fc was injected intravenously into adult wild type C57Bl / 6 mice (8 weeks old). Mice were sacrificed and pancreas specimens were harvested 14 days post injection. Histological analysis by H&E and immunostaining revealed enlarged pancreatic islets with extensive proliferation, including proliferation of insulin+ beta cells in the pancreas of Ad FGFBP1 NTD-Fc treated mice compared to Ad Fc controls. Ad FGFBP1 NTD-Fc revealed enlarged islets with expanded number of insulin+ beta cells within those enlarged islets. Co-immunostaining with Ki67, glucagon, insulin. Scale bars: 100 µm.

[0132] FIG. 64: Systemic expression of human FGFBP2 NTD in the circulation of mice postadenovirus injection. Control Ad Fc or Ad FGFBP2 NTD-Fc was injected intravenously into adult mice. Anti-His Western blot of mouse serum on day 4 after intravenous injection of Ad FGFBP2 NTD-Fc (see Fig.71) shows that the His-tagged transgenic protein is indeed robustly secreted into the circulation. Western blot from n=2 mice per treatment condition.

[0133] FIGS. 65A-65C: Human FGFBP2 NTD augments epithelial proliferation and repairfollowing irradiation injury, similar to murine FGFBP1 NTD. A) Experiment schema of mouse whole body irradiation (WBI) and adenovirus treatment. Control Ad Fc or Ad FGFBP2 NTD-Fc 28 4909-6074-3733v.193597 / 7344 was injected intravenously into adult mice 30 minutes post 12 Gy whole WBI. Mice were harvested and the small intestine and colon were collected 4 days post irradiation and adenovirus injection. B-C) Histological analysis revealed that Ad FGFBP2 NTD-Fc treatment did not affect epithelial homeostasis of the small intestine (jejunum) or colon but augmented proliferation and epithelial repair in the context of irradiation injury, compared to Ad Fc treatment. Ki67 immunostaining. Day 4 post treatment. Scale bars: 50 µm.

[0134] FIG. 66: Human and Mouse full length FGFBP sequences including hFGFBP1 (SEQID NO:7), mFGFBP1 (SEQ ID NO:17), hFGFBP2 (SEQ ID NO:1), hFGFBP3 (SEQ ID NO:4) and mFGFBP3 (SEQ ID NO:16).

[0135] FIG. 67: Exemplary mammalian cell expression constructs including Human Nterminal FGFBP1 (K34-N158) Avitag-His (with signal peptide) (SEQ ID NO:10) and Mouse N terminal FGFBP1 (T34-G163) Avitag-FLAG-His (with signal peptide) (SEQ ID NO:18); Human N terminal FGFBP2 (G30-G146) Avitag-FLAG-His (with signal peptide) (SEQ ID NO:2); and fusion protein Human N terminal FGFBP2 (M1-G146) Human-IgGFc (using endogenous signal peptide) (SEQ ID NO:11). Note, Fc portion of fusion is also SEQ ID NO:12.

[0136] FIG. 68: Fragments including endogenous signal sequence and N terminal domain ofFGFBP3: Human (SEQ ID NO:19) and mouse (SEQ ID NO:20).

[0137] FIG. 69: Exemplary heparin binding domains or N-terminal domains of FGFBP:Human FGFBP1 (SEQ ID NO:8); mouse FGFBP1 (SEQ ID NO:9); Human FGFBP2 (SEQ ID NO:3); Human FGFBP3 (SEQ ID NO:5); mouse FGFBP3 (SEQ ID NO:6).

[0138] FIG. 70: Exemplary fragments containing NTD: mouse FGFBP1 fragment (SEQ IDNO:13); Human FGFBP1 fragment (SEQ ID NO:14); Human FGFBP2 fragment (SEQ ID NO:15).

[0139] FIG. 71: Exemplary Adenovirus constructs: Ad mFGFBP1 NTD-Fc (signal peptide,mFGFBP1 NTD T34-G163, murine Fc, 3xFLAG, 8xHis) (SEQ ID NO:21); and Ad hFGFBP2 NTD-Fc (signal peptide, hFGFBP2 NTD G30-G146, murine Fc, 3xFLAG, 8xHis (SEQ ID NO:22).

[0140] FIGS. 72A-72E: Annotated exemplary protein sequences of FGFBP1 (human 72A,mouse 72B), FGFBP2 (human 72C), and FGFBP3 (human 72D, mouse 72E). Signal peptides as predicted by SignalP6.06are annotated in grey. Putative heparin-binding domain (dark green) and fibroblast growth factor (FGF)-binding domain (light green) are annotated, as predicted by AlphaFold7. 29 4909-6074-3733v.193597 / 7344

[0141] FIGS. 73A-73D: Annotated protein sequences of expressed recombinant proteins.Signal peptides (grey), native protein sequences (yellow), putative heparin-binding domains (dark green) and C-terminal tags (blue) are annotated. 73A. Mouse FGFBP1 T34-G163. 73B. Human FGFBP1 K34-N158.73C. Human FGFBP2 G30-G146.73D. Human FGFBP2 M1-G146. 30 4909-6074-3733v.193597 / 7344 DETAILED DESCRIPTION

[0142] A novel fibroblast growth factor binding protein (FGFBP) fragments have beendiscovered that functions in physiological tissue regeneration. This factor is also important for generation and maintenance of in vitro or ex vivo organoids grown from small intestine and colon. A specific protein domain was identified within FGFBP1 (with similar domains in FGFBP2 and FGFBP3) enhances the viability, growth, and expansion of organoids in culture. The data indicate that this factor can also function to augment tissue repair processes in vivo. This factor can be used ex vivo for enhanced storage, maintenance, and propagation of human tissues for many biomedical applications, including for transplantation into human patients for the treatment of various diseases. The factor can augment the growth and enhance the utility of organoid technologies, which are currently used in a wide variety of applications including toxicology / drug screens, genome wide-screens, disease modeling, and potential use in transplantation into patients. The factor can be used to enhance tissue repair and regeneration in vivo in human patients to treat human diseases (i.e., as a therapeutic). Fragments, fusion protein, methods of use and expression constructs therefor are also provided, as well as host cells and methods of production.

[0143] An expression construct encoding (i) an N-terminal domain of a fibroblast growthfactor binding protein 1 (FGFBP1), but not encoding a full length FGFBP1 protein. An expression construct encoding (i) an N-terminal domain of a fibroblast growth factor binding protein 1 (FGFBP1), but not encoding a mature FGFBP1 protein.

[0144] In some embodiments, the N-terminal domain of FGFBP1 is human.

[0145] In some embodiments, the N-terminal domain of FGFBP1 comprises SEQ ID NO:8 orSEQ ID NO:9.

[0146] In some embodiments, the expression construct further encodes a signal sequenceand / or a promoter. In some embodiments, the promoter is not native to the polynucleotide encoding the N-terminal domain of the FGFBP. In some embodiments, the promoter is not a native signal sequence for FGFBP.

[0147] An expression construct encoding (i) an N-terminal domain of a fibroblast growthfactor binding protein 1 (FGFBP1), but not encoding an FGF-binding domain of an FGFBP1 is also provided. In some embodiments, the N-terminal domain of FGFBP1 is human. In some embodiments, the N-terminal domain of FGFBP1 comprises SEQ ID NO:8 or SEQ ID NO:9. 31 4909-6074-3733v.193597 / 7344

[0148] An expression construct encoding a fusion protein comprising (A) an N-terminaldomain of a fibroblast growth factor binding protein 1 (FGFBP1) linked to (B) a non-FGFBP protein or peptide or polypeptide. A non-FGFBP protein or peptide or polypeptide is a protein or peptide or polypeptide sequence that does not occur in an FGFBP naturally.

[0149] In some embodiments, (A) does not comprise a full length or mature FGFBP1. In someembodiments, (A) does comprise full length or mature FGFBP1. In some embodiments, the expression construct also encodes a signal sequence. In some embodiments, the expression construct also has a promoter sequence. In some embodiments, (A) does not comprise an FGF binding domain of an FGFBP1. In some embodiments, the promoter is not native to the polynucleotide encoding the N-terminal domain of the FGFBP. In some embodiments, the promoter is not a native signal sequence for FGFBP.

[0150] In some embodiments, (B) comprises (a) an immunoglobulin G Fc. In someembodiments, (a) comprises a human immunoglobulin G Fc. In some embodiments, (B) comprises (b) a serum albumin. In some embodiments, (b) comprises a human serum albumin.

[0151] The expression constructs comprise one or more polynucleotides. In someembodiments, the expression construct comprises DNA. In some embodiments, the DNA is a cDNA. In some embodiments, the expression construct comprises mRNA.

[0152] A composition comprising an expression construct described herein. In someembodiments, the composition is the expression construct. In some embodiments, the composition comprises the expression construct and one or more other ingredients.

[0153] A lipid nanoparticle composition is provided comprising an expression constructdescribed herein. In some embodiments, the liposomal nanoparticle comprises one or more of 306Oi10, tetrakis(8-methylnonyl) 3,3′,3″,3‴-(((methylazanediyl) bis(propane-3,1 diyl))bis (azanetriyl))tetrapropionate; 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate; A2-Iso5- 2DC18, ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H- imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; β- sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13- dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol; BAME-O16B, bis(2-(dodecyldisulfanyl)ethyl) 3,3′-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6- diazahexacosyl)azanediyl)dipropionate; BHEM-Cholesterol, 2-(((((3S,8S,9S,10R,13R,14S,17R)- 32 4909-6074-3733v.193597 / 7344 10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2- hydroxyethyl)-N-methylethan-1-aminium bromide; C12-200, 1,1′-((2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl) piperazin-1-yl)ethyl)azanediyl) bis(dodecan-2-ol); cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5- dione; DC-Cholesterol, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3- DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; DOTAP, 1,2- dioleoyl-3-trimethylammonium-propane; DOTMA, 1,2-di-O-octadecenyl-3- trimethylammonium-propane; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3-yl) 9,9′,9″,9‴,9″″,9‴″- ((((benzene-1,3,5- tricarbonyl)yris(azanediyl)) tris (propane-3,1-diyl)) tris(azanetriyl))hexanonanoate; Lipid H (SM- 102), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino) octanoate; OF- Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4, 1-diyl))bis(azanetriyl))tetrakis(ethane- 2,1-diyl) (9Z,9′Z,9″Z,9‴Z,12Z,12′Z,12″Z,12‴Z)-tetrakis (octadeca-9,12-dienoate); PEG2000- DMG, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000; TT3, N1,N3,N5-tris(3- (didodecylamino)propyl)benzene-1,3,5-tricarboxamide, and ionizable amphiphilic Janus dendrimers (IAJDs). In some embodiments, the composition comprises hybrid lipid-polymer nanoparticles comprising a PLGA core and a dipalmitoylphosphatidylcholine (DPPC) shell. In some embodiments, the lipid nanoparticles are GalNAc-lipid nanoparticles.

[0154] A peptide comprising an N-terminal domain of a fibroblast growth factor bindingprotein 1 (FGFBP1), but not comprising a full length FGFBP1 protein. A peptide comprising an N-terminal domain of a fibroblast growth factor binding protein 1 (FGFBP1), but not comprising a mature FGFBP1 protein. In some embodiments, the N-terminal domain of FGFBP1 comprises SEQ ID NO:8 or SEQ ID NO:9.

[0155] In some embodiments, the peptide further comprises a signal sequence. In someembodiments, the peptide does not comprise a signal sequence. In some embodiments, the peptide does not comprise an FGF-binding domain of an FGFBP1.

[0156] A fusion protein comprising (A) an N-terminal domain of a fibroblast growth factorbinding protein 1 (FGFBP1), linked to (B) a non-FGFBP protein or peptide or polypeptide. 33 4909-6074-3733v.193597 / 7344

[0157] In some embodiments, (A) does not comprise a full length FGFBP1. In someembodiments, (A) does not comprise a mature FGFBP1. In some embodiments, (A) does not comprise an FGF-binding domain of an FGFBP1.

[0158] In some embodiments, (A) comprises a full length FGFBP1. In some embodiments,(A) comprises a mature FGFBP1.

[0159] In some embodiments, (B) comprises (a) an immunoglobulin G Fc, optionally a humanimmunoglobulin G Fc. In some embodiments, (B) comprises (b) a serum albumin, optionally a human serum albumin.

[0160] A method of propagating, promoting growth / proliferation of, and / or increasingviability of a stem cell or progenitor cell, comprising contacting the stem cell or progenitor cell with an amount of a composition comprising (i) an N-terminal domain of a fibroblast growth factor binding protein 1 (FGFBP1), (ii) an N-terminal domain of a fibroblast growth factor binding protein 2 (FGFBP2), or (iii) an N-terminal domain of a fibroblast growth factor binding protein 3 (FGFBP3), effective to propagate, promote growth / proliferation of, and / or increase viability of a stem cell or progenitor cell.

[0161] In some embodiments of the methods disclosed herein relating to, the stem cells are exvivo. In some embodiments, the stem cells are in vivo or in situ. In some embodiments, the stem cells are in vitro. In some embodiments of the methods disclosed herein relating to, the progenitor cells are ex vivo. In some embodiments, the progenitor cells are in vivo or in situ. In some embodiments, the progenitor cells are in vitro. In some embodiments of the methods disclosed herein relating to, the stem cells or progenitor cells, the stem cells or progenitor cells are mammalian. In some embodiments, the stem cells or progenitor cells are human. In some embodiments of the methods disclosed herein, the cells, tissues, organs, or organoids are in vivo, in vitro, or ex vivo.

[0162] A method of propagating, promoting growth / proliferation of, and / or increasingviability of a stem cell or progenitor cell, comprising contacting the stem cell or progenitor cell with an amount of an expression construct encoding (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to propagate, promote growth / proliferation of, and / or increase viability of a stem cell or progenitor cell. 34 4909-6074-3733v.193597 / 7344

[0163] A method of propagating, promoting growth / proliferation of, and / or increasingviability of a tissue or organ, comprising contacting the tissue with an amount of a composition comprising (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to propagate, promote growth / proliferation of, and / or increase viability of a tissue or organ.

[0164] In some embodiments of the methods disclosed herein relating to tissues, the tissue isin a subject. In some embodiments, the tissue has been removed from a subject. In some embodiments, the tissue is a soft tissue. In some embodiments, the tissue is a connective tissue, epithelial tissue, muscle tissue, and nervous tissue. In some embodiments of the methods disclosed herein relating to organs, the organ is in a subject. In some embodiments, the organ has been removed from a subject. In some embodiments the tissue or organ is a skin, cutaneous, ligament, a muscle, a tendon, a tongue, a pharynx, an esophagus, a stomach, a duodenum, a jejunum, a ileum, a cecum, an ascending colon, a transverse colon, a descending colon, a sigmoid colon, a rectum, a liver, a gallbladder, a mesentery, a pancreas, a pharynx, a larynx, a trachea, a bronchi, a bronchiole, a lung, a kidney, a ureter, a bladder, a urethra, an ovary, a fallopian tube, a uterus, a cervix, a placenta, a testicle, a pancreas, a heart, an artery, a vein, a capillary, arteries, bone marrow, a thymus, a spleen, a neuronal organ or tissue. In some embodiments, the tissue or organ is adult. In some embodiments, the tissue or organ is pediatric. In some embodiments, the tissue or organ is fetal.

[0165] A method of propagating, promoting growth / proliferation of, and / or increasingviability of a tissue or organ, comprising contacting the tissue with an amount of an expression construct encoding (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to propagate, promote growth / proliferation of, and / or increase viability of a tissue or organ.

[0166] A method of treating a wound or a scar in a tissue or organ, comprising contacting thetissue or organ with an amount of a composition comprising, or an expression construct encoding, (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N- terminal domain of an FGFBP3, effective to treat a wound or a scar in a tissue or organ.

[0167] In some embodiments, the scar or wound is caused by trauma. In some embodiments,the scar or wound is caused by fibrosis. In some embodiments, the scar or wound is caused by a tear or rip. In some embodiments, the scar or wound is caused by fibrosis. In some embodiments, 35 4909-6074-3733v.193597 / 7344 the scar or wound is secondary to another pathology. In some embodiments, the wound is an ulcer. In some embodiments, the wound is a burn.

[0168] In some embodiments, of the methods herein pertaining to tissues or organs, the tissueor organ is in a subject that has been treated with a radiotherapy or subject to irradiation that has caused, or is associated with, a tissue or organ injury. For example, a GI tissue or organ injury associated with a radiation therapy or with irradiation. In some embodiments, of the methods herein pertaining to tissues or organs, the tissue or organ is in a subject that has been treated with a chemotherapy or subject to a chemotherapeutic that has that has caused, or is associated with, a tissue or organ injury. For example, a GI tissue or organ injury associated with a chemotherapy or subject to a chemotherapeutic.

[0169] A method of treating a pathology associated with degeneration or aging of a tissue ororgan, comprising contacting the tissue or organ with an amount of a composition comprising, or an expression construct encoding, (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to treat a pathology associated with degeneration or aging of a tissue or organ.

[0170] In some embodiments, the pathology associated with degeneration or aging of a tissueor organ is a neurodegenerative disease. In some embodiments, the pathology associated with degeneration or aging of a tissue or organ is a nephrological disease. In some embodiments, the pathology associated with degeneration or aging of a tissue or organ is a hepatic disease. In some embodiments, the pathology associated with degeneration or aging of a tissue or organ is a sensory perception disease. In some embodiments, the pathology associated with degeneration or aging of a tissue or organ is a cardiac disease. In some embodiments, the pathology associated with degeneration or aging of a tissue or organ is a muscular disease. In some embodiments, the pathology associated with degeneration or aging of a tissue or organ is a pulmonary disease. In some embodiments, the pathology associated with degeneration or aging of a tissue or organ is a gastrointestinal disease. In some embodiments, the pathology associated with degeneration or aging of a tissue or organ is a reproductive organ disease. In some embodiments, the pathology associated with degeneration or aging of a tissue or organ is an integumentary organ disease.

[0171] A method of treating a pathology associated with acute or chronic inflammation of atissue or organ, comprising contacting the tissue or organ with an amount of a composition comprising, or an expression construct encoding, (i) an N-terminal domain of an FGFBP1, (ii) an 36 4909-6074-3733v.193597 / 7344 N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to treat a pathology associated with acute or chronic inflammation of a tissue or organ. In some embodiments, the pathology associated with acute or chronic inflammation of a tissue or organ is a gastrointestinal disease. In some embodiments, the pathology associated with acute or chronic inflammation of a tissue or organ is ulcerative colitis, Crohn’s disease or celiac disease.

[0172] In some embodiments, the stem cell is an adult mammalian stem cell or an inducedpluripotent stem cell, or an embryonic stem cell.

[0173] In some embodiments, the tissue is a mammalian tissue.

[0174] In some embodiments, the organ is a mammalian organ.

[0175] In some embodiments, the tissue is a donor tissue or wherein the organ is a donor organ.

[0176] In some embodiments, the composition comprises or the expression construct encodesan N-terminal domain of an FGFBP1.

[0177] In some embodiments, the composition comprises or the expression construct encodesan N-terminal domain of an FGFBP2.

[0178] In some embodiments, the composition comprises or the expression construct encodesan N-terminal domain of an FGFBP3.

[0179] In some embodiments, the composition comprises or the expression construct encodesan N-terminal domain of an FGFBP1 but not a full length FGFBP1.

[0180] In some embodiments, the composition comprises or the expression construct encodesan N-terminal domain of an FGFBP2 but not a full length FGFBP2.

[0181] In some embodiments, the composition comprises or the expression construct encodesan N-terminal domain of an FGFBP3 but not a full length FGFBP3.

[0182] In some embodiments, the composition is a fusion protein or the expression constructencodes a fusion protein.

[0183] In some embodiments, the composition does not comprise a fibroblast growth factor(FGF) -binding domain of an FGFBP1, FGFBP2 or FGFBP3, or the expression construct does not encode an FGF-binding domain of an FGFBP1, FGFBP2 or FGFBP3.

[0184] In some embodiments, the composition comprises a heparin-binding domain of anFGFBP1, FGFBP2 or FGFBP3, or the expression construct encodes heparin-binding domain of an FGFBP1, FGFBP2 or FGFBP3. 37 4909-6074-3733v.193597 / 7344

[0185] A method of promoting growth and / or viability of an organoid, or of an organoidculture, or expanding organoids in a culture, comprising administering to the organoid or organoid culture an amount of a composition comprising, or an expression construct encoding, (i) an N- terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to promote growth and / or viability of an organoid, or of an organoid culture, or expanding organoids in a culture.

[0186] In some embodiments, the mammalian is human or the donor is human.

[0187] In some embodiments, the tissue is a soft tissue, optionally a gastrointestinal tissue,cardiac tissue, hepatic tissue, skin tissue, renal tissue, neuronal tissue, reproductive tissue, dental tissue, cutaneous tissue, pancreatic tissue, or hematopoietic tissue.

[0188] In some embodiments, the organ is a duodenum, ileum, stomach, colon, liver, kidney,skin, or ovary.

[0189] In some embodiments, the wound is a burn or an ulcer.

[0190] A method of stimulating proliferation of stem cells comprising contacting the stem cellswith an amount of a composition comprising, or an expression construct encoding, (i) an N- terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to stimulate proliferation of stem cells.

[0191] In some embodiments, the stem cells are self-renewing.

[0192] In some embodiments, the stem cells are human.

[0193] In some embodiments, the stem cells are adult stem cells.

[0194] A method of stimulating proliferation of progenitor cells comprising contacting theprogenitor cells with an amount of a composition comprising, or an expression construct encoding, (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N- terminal domain of an FGFBP3, effective to stimulate proliferation of progenitor cells.

[0195] A cell engineered to express an expression construct described herein. A cellengineered to express a peptide described herein. A cell engineered to express a fusion protein described herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell.

[0196] A method of propagating and / or increasing viability of a culture of a mammalian tissuecomprising administering to the human tissue in culture an amount of a composition comprising a 38 4909-6074-3733v.193597 / 7344 domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of a mammalian tissue.

[0197] In some embodiments, the tissue is gastrointestinal tissue, cardiac tissue, pancreatictissue, hepatic tissue, ovarian tissues, skin or cutaneous tissue, or hematopoietic tissue.

[0198] In some embodiments, the tissue is a human tissue.

[0199] In some embodiments, the tissue is for transplantation.

[0200] A method of propagating and / or increasing viability of a culture of stem or progenitorcells comprising administering to the stem or progenitor cells in culture an amount of a composition comprising a domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of stem or progenitor cells.

[0201] In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs) orembryonic stem cells (ESCs).

[0202] In some embodiments, the stem or progenitor cells are human.

[0203] A method of maintaining or increasing the viability of a donor tissue or donor organintended for transplantation comprising administering to the donor tissue or donor organ an amount of a composition comprising a domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to maintain or increase viability of a donor tissue or donor organ.

[0204] In some embodiments, the donor tissue or donor organ has been removed from thedonor and is being maintained prior to transplantation.

[0205] A method of expanding organoids or growth of tissues in a culture comprisingadministering to the organoids or tissues in a culture an amount of a protein domain comprising heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to expand the organoids or growth of tissues.

[0206] In some embodiments, the organoids are being cultured for transplantation or are listedin Table 1.

[0207] A method of promoting healing of a wound in a tissue in a subject comprisingadministering to the wound an amount of a composition comprising a domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote healing of a wound in a tissue.

[0208] In some embodiments, the tissue is a skin tissue or a gastrointestinal tissue.

[0209] In some embodiments, the skin has suffered a burn.39 4909-6074-3733v.193597 / 7344

[0210] In some embodiments, the subject has ulcerative colitis, Crohn’s disease or celiacdisease.

[0211] A method of treating a disease associated with pancreatic beta cell degradation in asubject comprising administering to the subject an amount of a peptide, composition, fusion protein or expression construct as described herein an in amount effective to treating a disease associated with pancreatic beta cell degradation. In some embodiments, the disease is type I diabetes. In some embodiments, the disease is type 2 diabetes. In some embodiments, the method effects an increase in beta cell in islets of Langerhans of the pancreas.

[0212] A method of treating a disease associated with nephrosis or nephropathy in a subjectcomprising administering to the subject an amount of a peptide, composition, fusion protein or expression construct as described herein an in amount effective to treating a disease associated with nephrosis or nephropathy. In some embodiments, the disease is acute kidney injury. In some embodiments, the disease is chronic kidney disease. In some embodiments, the disease is kidney failure. In some embodiments, the disease is nephrotic syndrome. In some embodiments, the method effects an increase in epithelial cells of the kidney. In some embodiments, the method effects an increase or regeneration in the proximal tubules of the kidney.

[0213] A method of treating a disease associated with hepatocyte loss or degradation a liver ofa subject comprising administering to the subject an amount of a peptide, composition, fusion protein or expression construct as described herein an in amount effective to treating a disease associated with hepatocyte loss or degradation a liver. In some embodiments, the disease is liver failure. In some embodiments, the method effects an increase in hepatocytes of the liver.

[0214] A method of treating a disease associated with colon epithelium degradation in asubject comprising administering to the subject an amount of a peptide, composition, fusion protein or expression construct as described herein an in amount effective to treating a disease associated with colon epithelium degradation. In some embodiments, the method effects an increase in epithelial cells of the colon.

[0215] A method of treating a disease associated with small intestine degradation in a subjectcomprising administering to the subject an amount of a peptide, composition, fusion protein or expression construct as described herein an in amount effective to treating a disease associated with small intestine degradation. In some embodiments, the method effects an increase in small intestine growth. 40 4909-6074-3733v.193597 / 7344

[0216] A method of treating a disease associated with lung cell or tissue degradation in asubject comprising administering to the subject an amount of a peptide, composition, fusion protein or expression construct as described herein an in amount effective to treating a disease associated with lung cell or tissue degradation. In some embodiments, the method effects an increase in airway cells in the subject.. In some embodiments, the method effects an increase in alveolar cells in the subject. In some embodiments, the method effects an increase in AT1 cells in the subject. In some embodiments, the method effects an increase in AT2 cells in the subject.

[0217] A method of treating a gastric ulcer in a subject comprising administering to the subjectan amount of a peptide, composition, fusion protein or expression construct as described herein an in amount effective to treating a gastric ulcer. In some embodiments, the method effects decrease in gastric ulcer size. In some embodiments, the method effects healing of other gastrointestinal ulcers, including the small intestine and colon.

[0218] A method of treating a radiation-induced epithelial injury in a subject comprisingadministering to the subject an amount of a peptide, composition, fusion protein or expression construct as described herein an in amount effective to treat radiation-induced epithelial injury. In some embodiments, the method effects epithelial proliferation. In some embodiments, the method effects epithelial proliferation in the small intestine or colon.

[0219] A method of increasing bone marrow cell proliferation in a subject comprisingadministering to the subject an amount of a peptide, composition, fusion protein or expression construct as described herein an in amount effective to increase bone marrow cell proliferation. In some embodiments, the method effects an increase in bone marrow cellularity.

[0220] A method of treating a disease associated with bone marrow suppression, thediminished capacity to produce blood cells, or the destruction of blood cells in a subject comprising administering to the subject an amount of a peptide, composition, fusion protein or expression construct as described herein an in amount effective to treating a disease associated with diminished blood cells. In some embodiments, the disease is chemotherapy-induced or radiation- induced bone marrow insufficiency, bone marrow suppression due to blood cancers or metastatic cancer, bone marrow suppression due to infection or medications, aplastic anemia, sickle cell anemia, myelofibrosis, or autoimmune disease. In some embodiments, the bone marrow cells of a subject could be expanded ex vivo or in vitro with an amount of a peptide, composition, fusion 41 4909-6074-3733v.193597 / 7344 protein or expression construct as described herein and transplanted back into the subject or another subject.

[0221] A method of treating an inflammatory colitis in a subject comprising administering tothe subject an amount of a peptide, composition, fusion protein or expression construct as described herein an in amount effective to treat an inflammatory colitis. In some embodiments, the method effects an increase in proliferation of epithelial cells of the GI tissue afflicted with colitis. In some embodiments, the method effects an increase in number of epithelial cells of the GI tissue afflicted with colitis.

[0222] In some embodiments of the methods herein regarding stem cells, the stem cell is astem cell or an organ or a tissue as described herein. In some embodiments of the methods herein regarding progenitor cells, the progenitor cell is a progenitor cell or an organ or a tissue as described herein.

[0223] In some embodiments of the methods herein regarding stem cells, the stem cell ismammalian. In some embodiments of the methods herein regarding progenitor cells, the progenitor cell is mammalian.

[0224] A method of promoting growth of an organoid, or of an organoid culture, comprisingadministering to the organoid or organoid culture an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3, or a fusion protein thereof, effective to promote growth of an organoid, or of an organoid culture.

[0225] A method of reducing the frequency of culture media changes required to maintainviability of organoids of an organoid culture comprising administering to the organoid culture an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote and / or maintain viability of the organoids culture thereby reducing the frequency of culture media changes required to maintain viability of the organoids.

[0226] A method of propagating and / or increasing viability of a culture of a mammalian tissuecomprising administering to the human tissue in culture an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of a mammalian tissue.

[0227] A method of propagating and / or increasing viability of a culture of stem or progenitorcells comprising administering to the stem or progenitor cells in culture an amount of an expression 42 4909-6074-3733v.193597 / 7344 construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of stem or progenitor cells.

[0228] A method of maintaining or increasing the viability of a donor tissue or donor organintended for transplantation comprising administering to the donor tissue or donor organ an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to maintain or increase viability of a donor tissue or donor organ.

[0229] A method of expanding organoids or growth of tissues in a culture comprisingadministering to the organoids or tissues in a culture an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to expand the organoids or growth of tissues.

[0230] In some embodiments, the organoid is a tumor organoid grown from cancer ormalignancy or metastasis.

[0231] A method of promoting growth of an organoid, or of an organoid culture, comprisingadministering to the organoid or organoid culture an amount of a protein domain comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or a fusion protein thereof, effective to promote growth of an organoid, or of an organoid culture.

[0232] In some embodiments, the method effects an increase in organoid number in theorganoid culture.

[0233] In some embodiments, the method effects an increased growth rate of the organoid.

[0234] In some embodiments, the method effects increased viability of the organoid.

[0235] In some embodiments, the method effects increased regenerative capacity of theorganoid.

[0236] In some embodiments, the organoid is a mammalian tissue organoid.

[0237] A method of reducing the frequency of culture media changes required to maintainviability of organoids of an organoid culture comprising administering to the organoid culture an amount of a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote and / or maintain viability of the organoids culture thereby reducing the frequency of culture media changes required to maintain viability of the organoids.

[0238] In some embodiments, the organoids are gastrointestinal tract organoids, sebaceousglands organoids, hepatic tissue organoids, renal tissue organoids, lung organoids, pancreatic organoids, bile duct organoids, or are listed in Table 1. 43 4909-6074-3733v.193597 / 7344

[0239] In some embodiments, the organoids are gastric, colonic, duodenal, jejunal or ilealorganoids.

[0240] A method of promoting healing of a wound in a tissue comprising administering to thewound an amount of an expression construct encoding a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote healing of a wound in a tissue.

[0241] In some embodiments, the tissue is a skin tissue or a gastrointestinal tissue.

[0242] In some embodiments, the skin has suffered a burn.

[0243] In some embodiments, the subject has ulcerative colitis.

[0244] A fusion protein comprising (i) a protein domain of an N-terminal portion of anFGFBP1 and (ii) a peptide, polypeptide, domain or a protein not encoded by a fgfbp1 gene.

[0245] In some embodiments, (ii) comprises an immunoglobulin Fc.

[0246] An expression construct encoding a protein domain of an N-terminal portion of anFGFBP1 or FGFBP2, but not encoding a full length FGFBP1 or FGFBP2 protein. In some embodiments, the expression construct encodes a portion of an FGFBP1. In some embodiments, the expression construct encodes a fusion protein comprising a protein domain of an N-terminal portion of an FGFBP1 or FGFBP2.

[0247] In some embodiments, the FGFBP1 or FGFBP2 is human or mouse. In someembodiments, the protein domain of an N-terminal portion of an FGFBP1 comprises SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the protein domain of an N-terminal portion of an FGFBP2 comprises SEQ ID NO:3.

[0248] An expression construct encoding an FGFBP1 domain having SEQ ID NO:8, but notencoding a full length FGFBP1 protein having SEQ ID NO:7 or encoding an FGFBP1 domain having SEQ ID NO:9, but not encoding a full length FGFBP1 protein having SEQ ID NO:17.

[0249] An expression construct encoding an FGFBP2 domain but not encoding a full lengthFGFBP2 protein having SEQ ID NO:1.

[0250] In some embodiments, the expression construct encodes a signal peptide that controlssecretion the protein domain of an N-terminal portion of an FGFBP1 or FGFBP2. In some embodiments, the signal peptide is not naturally found in a gene encoding the full length FGFBP1 or FGFBP2 protein.

[0251] An expression construct encoding a fusion protein comprising (i) a protein domain ofan N-terminal portion of an FGFBP1 or FGFBP2 and (ii) a peptide, polypeptide, domain or a 44 4909-6074-3733v.193597 / 7344 protein not encoded by a fgfbp1 gene or fgfbp2 gene. In some embodiments, (ii) comprises an immunoglobulin Fc.

[0252] A host cell transfected with an expression construct of described herein, or a progenyof such a host cell.

[0253] A method of producing a protein domain of an N-terminal portion of an FGFBP1 orFGFBP2, or fusion protein thereof, comprising culturing the host cell or progeny thereof as described herein in culture media, and isolating a secreted heparin-binding domain of an FGFBP1 or FGFBP2 or fusion protein thereof from a supernatant thereof.

[0254] A composition comprising a protein domain of an N-terminal portion of an FGFBP1but not comprising a full length FGFBP1 or FGFBP2 protein. In some embodiments, the FGFBP1 is human. In some embodiments, the FGFBP1 is mouse. In some embodiments, the FGFBP2 is human. In some embodiments, the FGFBP2 is mouse.

[0255] In some embodiments, the domain of the N-terminal portion of an FGFBP1 comprisesSEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the domain of the N-terminal portion of an FGFBP1 comprises SEQ ID NO:8. In some embodiments, the protein domain comprises SEQ ID NO:8, but does not comprise a full length FGFBP1 protein having SEQ ID NO:7 or the protein domain comprises SEQ ID NO:9, but does not comprise a full length FGFBP1 protein having SEQ ID NO:17.

[0256] In some embodiments, the domain of the N-terminal portion of FGFBP2 is not a fulllength FGFBP2 protein having SEQ ID NO:1. In some embodiments, the domain of the N-terminal portion of FGFBP2 has SEQ ID NO:3.

[0257] In some embodiments of the methods and compositions, a domain of an N-terminalportion of an FGFBP1 is a domain of the protein which is primarily in the N-terminal half of the protein. For example, a domain of FGFBP1 whose primary sequence is mainly (>50%) in the N- terminal half of the protein’s primary sequence. In some embodiments of the methods and compositions, the composition is a portion of an FGFBP1 lacking the C-terminal FGF binding domain. In some embodiments of the methods and compositions, the composition comprises a fragment of a full length FGFBP1 but lacks the C-terminal FGF binding domain. The protein domains, fragments, and portions can be produced recombinantly. Thus, In some embodiments, the protein domains, fragments, and portions are not actual physical parts of the full length proteins, but have sequence identity therewith. For example, a domain having the same sequence, 45 4909-6074-3733v.193597 / 7344 or a modified version of, a domain of the native full length protein sequence is readily producible by, e.g., recombinant means.

[0258] In some embodiments of the methods and compositions, a domain of an N-terminalportion of an FGFBP2 is a domain of the protein which is primarily in the N-terminal half of the protein. For example, a domain of FGFBP2 whose primary sequence is mainly (>50%) in the N- terminal half of the protein’s primary sequence. In some embodiments of the methods and compositions, the composition is a portion of an FGFBP2 lacking the C-terminal FGF binding domain. In some embodiments of the methods and compositions, the composition comprises a fragment of a full length FGFBP2 but lacks the C-terminal FGF binding domain. The protein domains, fragments, and portions can be produced recombinantly. Thus, In some embodiments, the protein domains, fragments, and portions are not actual physical parts of the full length proteins, but have sequence identity therewith. For example, a domain having the same sequence, or a modified version of, a domain of the native full length protein sequence is readily producible by, e.g., recombinant means.

[0259] In some embodiments of the methods and compositions, a domain of an N-terminalportion of an FGFBP3 is a domain of the protein which is primarily in the N-terminal half of the protein. For example, a domain of FGFBP3 whose primary sequence is mainly (>50%) in the N- terminal half of the protein’s primary sequence. In some embodiments of the methods and compositions, the composition is a portion of an FGFBP3 lacking the C-terminal FGF binding domain. In some embodiments of the methods and compositions, the composition comprises a fragment of a full length FGFBP3 but lacks the C-terminal FGF binding domain. The protein domains, fragments, and portions can be produced recombinantly. Thus, In some embodiments, the protein domains, fragments, and portions are not actual physical parts of the full length proteins, but have sequence identity therewith. For example, a domain having the same sequence, or a modified version of, a domain of the native full length protein sequence is readily producible by, e.g., recombinant means.

[0260] The domains, fragments and portions disclosed herein can be produced in a variety ofways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides. In one embodiment, a recombinant domains, fragments and portions is produced by culturing a cell capable of expressing the domains, fragments and portions under conditions effective to produce the domains, fragments 46 4909-6074-3733v.193597 / 7344 and portions. The recombinant domains, fragments and portions may subsequently be secreted from the cell and recovered, or extracted from the cell and recovered, and is preferably purified away from contaminating molecules. It may or may not be further modified chemically or enzymatically. A preferred cell to culture is a recombinant cell. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit domain, fragment or portion production. An effective medium refers to any medium in which a cell is cultured to produce a polypeptide defined herein. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells defined herein can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.

[0261] In some embodiments of the methods and compositions and constructs and proteindomains, the domain of an N-terminal portion of an FGFBP1 comprises a heparin-binding domain of FGFBP1. In some further embodiments of the methods and compositions and constructs and protein domains, the composition comprising a domain of an N-terminal portion of an FGFBP1 does not comprise an FGF-binding domain of FGFBP1.

[0262] In some embodiments of the methods and compositions and constructs and proteindomains, the domain of an N-terminal portion of an FGFBP2 comprises a heparin-binding domain of FGFBP2. In some further embodiments of the methods and compositions and constructs and protein domains, the composition comprising a domain of an N-terminal portion of an FGFBP2 does not comprise an FGF-binding domain of FGFBP2.

[0263] In some embodiments of the methods and compositions and constructs and proteindomains, the domain of an N-terminal portion of an FGFBP3 comprises a heparin-binding domain of FGFBP3. In some further embodiments of the methods and compositions and constructs and protein domains, the composition comprising a domain of an N-terminal portion of an FGFBP3 does not comprise an FGF-binding domain of FGFBP3.

[0264] In some embodiments, the composition consists essentially of the protein domain of anN-terminal portion of an FGFBP1 and contains no other active ingredients. In some embodiments, the composition comprises the protein domain of an N-terminal portion of an FGFBP1 and 47 4909-6074-3733v.193597 / 7344 contains a physiological or pharmaceutically acceptable carrier. In some embodiments, the composition comprises a carrier comprising saline or a culture medium. In some embodiments, the composition comprises a portion of an FGFBP1. In some embodiments, the composition comprises a fusion protein comprising a protein domain of an N-terminal portion of an FGFBP1.

[0265] In some embodiments, the composition consists essentially of the protein domain of anN-terminal portion of an FGFBP2 and contains no other active ingredients. In some embodiments, the composition comprises the protein domain of an N-terminal portion of an FGFBP2 and contains a physiological or pharmaceutically acceptable carrier. In some embodiments, the composition comprises a carrier comprising saline or a culture medium. In some embodiments, the composition comprises a portion of an FGFBP2. In some embodiments, the composition comprises a fusion protein comprising a protein domain of an N-terminal portion of an FGFBP2.

[0266] In some embodiments, the composition consists essentially of the protein domain of anN-terminal portion of an FGFBP3 and contains no other active ingredients. In some embodiments, the composition comprises the protein domain of an N-terminal portion of an FGFBP3 and contains a physiological or pharmaceutically acceptable carrier. In some embodiments, the composition comprises a carrier comprising saline or a culture medium. In some embodiments, the composition comprises a portion of an FGFBP3. In some embodiments, the composition comprises a fusion protein comprising a protein domain of an N-terminal portion of an FGFBP3.

[0267] An expression construct is provided encoding a heparin-binding domain of a fibroblastgrowth factor binding protein 1 (FGFBP1), but not encoding a full length FGFBP1 protein. In some embodiments, the expression construct does not encode an FGF binding domain.

[0268] In some embodiments, the FGFBP1 is human or mouse. In some embodiments, theputative heparin-binding domain comprises SEQ ID NO:8. In some embodiments, the putative heparin-binding domain comprises SEQ ID NO:9. In some embodiments, the full length FGFBP1 comprises SEQ ID NO:7. In some embodiments, the full length FGFBP1 comprises SEQ ID NO:17. In some embodiments, the construct encodes SEQ ID NO:10, 2, or 18. In some embodiments, the construct encodes SEQ ID NO:13, 14, or 15. In some embodiments, the construct encodes SEQ ID NO:1. In some embodiments, the construct encodes SEQ ID NO:7. In some embodiments, the expression construct encodes a signal peptide that controls secretion of the heparin-binding domain of an FGFBP1. In some embodiments, the expression construct encodes a signal peptide that controls secretion of the heparin-binding domain of an FGFBP1, 48 4909-6074-3733v.193597 / 7344 wherein the signal peptide is not naturally found in a gene encoding the full length FGFBP1 protein. Examples of signal peptides include a gp67 signal peptide. Other signal peptides include NSP4, PelB5, DsbAss, baculoviral gp64, modified human serum albumin (mSA), human azurocidin (AZ), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgkC) and modified human Ig kappa chain V III region VG (mIgkH).

[0269] An expression construct is provided encoding a heparin-binding domain of a fibroblastgrowth factor binding protein 2 (FGFBP2), but not encoding a full length FGFBP2 protein. In some embodiments, the expression construct does not encode an FGF binding domain. In some embodiments, the FGFBP2 is human or mouse. In some embodiments, the expression construct encodes a signal peptide that controls secretion of the heparin-binding domain of an FGFBP2. In some embodiments, the expression construct encodes a signal peptide that controls secretion of the heparin-binding domain of an FGFBP2, wherein the signal peptide is not naturally found in a gene encoding the full length FGFBP2 protein. Examples of signal peptides include a gp67 signal peptide. Other signal peptides include NSP4, PelB5, DsbAss, baculoviral gp64, modified human serum albumin (mSA), human azurocidin (AZ), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgkC) and modified human Ig kappa chain V III region VG (mIgkH).

[0270] An expression construct encoding a fusion protein comprising (i) a heparin-bindingdomain of a fibroblast growth factor binding protein 1 (FGFBP1) and (ii) a peptide or a protein not encoded by a fgfbp1 gene. An expression construct encoding a fusion protein comprising (i) a heparin-binding domain of a fibroblast growth factor binding protein 2 (FGFBP2) and (ii) a peptide or a protein not encoded by a fgfbp2 gene.

[0271] A protein domain comprising a heparin-binding domain of a fibroblast growth factorbinding protein 1 (FGFBP1), but not comprising a full length FGFBP1 protein. In some embodiments, the FGFBP1 is human or mouse. The heparin-binding domain is also referred to herein as a putative heparin binding domain.

[0272] In some embodiments, the heparin-binding domain comprises SEQ ID NO:8. In someembodiments, the heparin-binding domain comprises SEQ ID NO:9.

[0273] In some embodiments, the protein domain comprises SEQ ID NO:8, but not comprisinga full length FGFBP1 protein having SEQ ID NO:7. In some embodiments, the protein domain comprises SEQ ID NO:9, but not comprising a full length FGFBP1 protein having SEQ ID NO:17. 49 4909-6074-3733v.193597 / 7344

[0274] A protein domain comprising a heparin-binding domain of a fibroblast growth factorbinding protein 2 (FGFBP1), but not comprising a full length FGFBP2 protein. In some embodiments, the FGFBP2 is human or mouse. The heparin-binding domain is also referred to herein as a putative heparin binding domain.

[0275] A fusion protein comprising (i) a heparin-binding domain of a fibroblast growth factorbinding protein 1 (FGFBP1) and (ii) a peptide or a protein not encoded by a fgfbp1 gene. A fusion protein comprising (i) a heparin-binding domain of a fibroblast growth factor binding protein 2 (FGFBP2) and (ii) a peptide or a protein not encoded by a fgfbp2 gene.

[0276] In some embodiments, (ii) comprises an immunoglobulin Fc.

[0277] A fusion protein of the heparin-binding domain of an FGFBP1 described hereincomprises the domain joined either directly, or indirectly through a linker, via a peptide bond at its N- or C-terminus to a second peptide or protein or polypeptide or protein domain which is heterologous relative to the FGFBP1 (not encoded by the same gene). Examples of such second peptide, protein, polypeptide or protein domain include, e.g., an immunoglobulin Fc portion, human serum albumin (HSA), transferrin, ELP repeat sequence (Phase Bio), proline-alanine- serine polymer, PAS (XL-protein GmbH), homo-amino acid polymer, HAP (homopolymer of glycine resides), gelatin-like protein GLK, or a non-structured polypeptide such as XTEN, a self- assembling protein nanoparticle carrier, all either directly or through a linker peptide sequence. The first protein domain and second protein, polypeptide or protein domain may be directly linked (for example, the C-terminus of the first protein domain is linked to the N-terminus of the second protein by a peptide bond), or indirectly linked (for example, the C-terminus of the first protein domain is directly linked to the N-terminus of a peptide linker by a peptide bond, and the C- terminus of the peptide linker is directly linked to the N-terminus of the second protein by a peptide bond). The invention also encompasses the protein domain described herein joined to second moiety to extend serum half-life such as a self-assembling protein nanoparticle carrier by a non-peptide chemical linker (such as a Sulfo-SIAB linker) or PEGylation. In an embodiment of the fusion protein comprising an Fc, the fusion protein can comprise glycosylation at a residue on the Fc portion corresponding to Asn297.

[0278] A fusion protein of the heparin-binding domain of an FGFBP2 described hereincomprises the domain joined either directly, or indirectly through a linker, via a peptide bond at its N- or C-terminus to a second peptide or protein or polypeptide or protein domain which is 50 4909-6074-3733v.193597 / 7344 heterologous relative to the FGFBP1 (not encoded by the same gene). Examples of such second peptide, protein, polypeptide or protein domain include, e.g., an immunoglobulin Fc portion, human serum albumin (HSA), transferrin, ELP repeat sequence (Phase Bio), proline-alanine- serine polymer, PAS (XL-protein GmbH), homo-amino acid polymer, HAP (homopolymer of glycine resides), gelatin-like protein GLK, or a non-structured polypeptide such as XTEN, a self- assembling protein nanoparticle carrier, all either directly or through a linker peptide sequence. The first protein domain and second protein, polypeptide or protein domain may be directly linked (for example, the C-terminus of the first protein domain is linked to the N-terminus of the second protein by a peptide bond), or indirectly linked (for example, the C-terminus of the first protein domain is directly linked to the N-terminus of a peptide linker by a peptide bond, and the C- terminus of the peptide linker is directly linked to the N-terminus of the second protein by a peptide bond). The invention also encompasses the protein domain described herein joined to second moiety to extend serum half-life such as a self-assembling protein nanoparticle carrier by a non-peptide chemical linker (such as a Sulfo-SIAB linker) or PEGylation. In an embodiment of the fusion protein comprising an Fc, the fusion protein can comprise glycosylation at a residue on the Fc portion corresponding to Asn297.

[0279] A fusion protein of the heparin-binding domain of an FGFBP3 described hereincomprises the domain joined either directly, or indirectly through a linker, via a peptide bond at its N- or C-terminus to a second peptide or protein or polypeptide or protein domain which is heterologous relative to the FGFBP3 (not encoded by the same gene). Examples of such second peptide, protein, polypeptide or protein domain include, e.g., an immunoglobulin Fc portion, human serum albumin (HSA), transferrin, ELP repeat sequence (Phase Bio), proline-alanine- serine polymer, PAS (XL-protein GmbH), homo-amino acid polymer, HAP (homopolymer of glycine resides), gelatin-like protein GLK, or a non-structured polypeptide such as XTEN, a self- assembling protein nanoparticle carrier, all either directly or through a linker peptide sequence. The first protein domain and second protein, polypeptide or protein domain may be directly linked (for example, the C-terminus of the first protein domain is linked to the N-terminus of the second protein by a peptide bond), or indirectly linked (for example, the C-terminus of the first protein domain is directly linked to the N-terminus of a peptide linker by a peptide bond, and the C- terminus of the peptide linker is directly linked to the N-terminus of the second protein by a peptide bond). The invention also encompasses the protein domain described herein joined to 51 4909-6074-3733v.193597 / 7344 second moiety to extend serum half-life such as a self-assembling protein nanoparticle carrier by a non-peptide chemical linker (such as a Sulfo-SIAB linker) or PEGylation. In an embodiment of the fusion protein comprising an Fc, the fusion protein can comprise glycosylation at a residue on the Fc portion corresponding to Asn297.

[0280] In an embodiment, the fusion protein comprises, between the heparin-binding domainand the second peptide, protein, polypeptide or protein domain, a linker peptide from 5 to 30 amino acid residues long. In an embodiment, the linker peptide comprises one or more of glycine, serine and threonine residues. In some embodiments, the linker peptide is 10-25 amino acids in length.

[0281] The protein domains of the invention can, in some embodiments, be modified by oneor more of site-specific mutations, incorporation of unnatural amino acids (e.g., for conjugating multiple copies of the protein domains in close proximity to enhance avidity), PEGylated, attached to other non-PEG polymers. Fusion proteins of the invention also encompass the protein domain fused, e.g. via a peptide bond or via a peptide, to an antibody, a single-chain variable fragment (e.g., to make a bispecific drug), a nanobody, an Fc, a Human serum albumin, as a polypeptide fusion (e.g., XTEN, a superhydrophilic zwitterionic peptide, a tannic acid modified protein). In some embodiments, the protein domains can be glycoengineered. In some embodiments, the protein domains can lipidated or made into lipid conjugates.

[0282] The protein domains herein can be as set forth in the relevant SEQ ID NOS. or have1,2, 3, 4, 5, 6, 7, 8, 9, 10 additional amino acids at the N-terminal, C-terminal, or independently in length at the N-terminal and C-terminals thereof. In some embodiments, the additional amino acids are consecutive from the full length FGFBP1 or FGFBP2 sequence. In some embodiments, the peptides have up to 15 amino acids at one or more terminals thereof. In some embodiments, the peptides have up to 20 amino acids at one or more terminals thereof. In some embodiments, the peptides have up to 25 amino acids at one or more terminals thereof.

[0283] Unless the SEQ ID NO for a NTD or a heparin-binding domain of an FGFBP1 isspecifically set forth, a NTD or a heparin-binding domain of an FGFBP1 means a protein domain having the same sequence as a NTD or a heparin-binding domain of an FGFBP1 regardless of whether that sequence was produced from an fgfbp1 gene or was produced by some other means, e.g., recombinant expression, e.g., from a cDNA. Thus, a human heparin-binding domain of an FGFBP1 peptide will have the sequence of a heparin-binding domain of an FGFBP1 encoded by a human gene, but without necessarily having been produced from a human gene. Similarly, a 52 4909-6074-3733v.193597 / 7344 mouse heparin-binding domain of an FGFBP1 peptide has the sequence of a heparin-binding domain of an FGFBP1 encoded by a mouse gene, but not necessarily produced from a mouse gene.

[0284] Unless the SEQ ID NO for a NTD or a heparin-binding domain of an FGFBP2 isspecifically set forth, a NTD or a heparin-binding domain of an FGFBP2 means a protein domain having the same sequence as a NTD or a heparin-binding domain of an FGFBP2 regardless of whether that sequence was produced from an fgfbp1 gene or was produced by some other means, e.g., recombinant expression, e.g., from a cDNA. Thus, a human heparin-binding domain of an FGFBP2 peptide will have the sequence of a heparin-binding domain of an FGFBP2 encoded by a human gene, but without necessarily having been produced from a human gene. Similarly, a mouse heparin-binding domain of an FGFBP2 peptide has the sequence of a heparin-binding domain of an FGFBP2 encoded by a mouse gene, but not necessarily produced from a mouse gene.

[0285] Unless the SEQ ID NO for a NTD or a heparin-binding domain of an FGFBP3 isspecifically set forth, a NTD or a heparin-binding domain of an FGFBP3 means a protein domain having the same sequence as a NTD or a heparin-binding domain of an FGFBP3 regardless of whether that sequence was produced from an fgfbp1 gene or was produced by some other means, e.g., recombinant expression, e.g., from a cDNA. Thus, a human heparin-binding domain of an FGFBP3 peptide will have the sequence of a heparin-binding domain of an FGFBP3 encoded by a human gene, but without necessarily having been produced from a human gene. Similarly, a mouse heparin-binding domain of an FGFBP3 peptide has the sequence of a heparin-binding domain of an FGFBP3 encoded by a mouse gene, but not necessarily produced from a mouse gene.

[0286] A host cell transfected with an expression construct described herein, or progenythereof, is provided.

[0287] A method of producing an isolated heparin-binding domain of an FGFBP1 or FGFBP2,or fusion protein thereof, comprising culturing the host cell or progeny thereof described herein in culture media, and isolating a secreted heparin-binding domain of an FGFBP1 or FGFBP2 or fusion protein thereof, respectively, from a supernatant thereof.

[0288] A method of promoting growth of an organoid, or of an organoid culture, comprisingadministering to the organoid or organoid culture an amount of a protein domain comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or a fusion protein thereof, effective to promote growth of an organoid, or of an organoid culture. 53 4909-6074-3733v.193597 / 7344

[0289] In some embodiments, the method effects an increase in organoid number in theorganoid culture. In some embodiments, the method effects an increased growth rate of the organoid. In some embodiments, the method effects increased viability of the organoid. In some embodiments, the method effects increased regenerative capacity of the organoid. An increase in organoid number is achieved when the number of organoids in the culture in the presence of the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 is greater than the number that exists under otherwise identical culture conditions except for the absence of the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3. An increase in growth rate is achieved when the growth rate of organoids in the culture in the presence of the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 is greater than the growth rate of organoids seen under otherwise identical culture conditions expect for the absence of the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 in the culture. An increase in regenerative capacity is achieved when the regenerative capacity of organoids in the culture in the presence of the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 is greater than the regenerative capacity of organoids seen under otherwise identical culture conditions expect for the absence of the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 in the culture.

[0290] A method of reducing the frequency of culture media changes required to maintainviability of organoids of an organoid culture comprising administering to the organoid culture an amount of a protein domain comprising heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote and / or maintain viability of the organoids culture thereby reducing the frequency of culture media changes required to maintain viability of the organoids.

[0291] In some embodiments of methods involving organoids, the organoid is a mammaliantissue organoid. In some embodiments, the organoid is comprised of epithelium only or epithelium with stromal cells or other non-epithelial cells.

[0292] In some embodiments of methods involving organoids, the organoids aregastrointestinal tract organoids, sebaceous glands organoids, hepatic tissue organoids, renal tissue organoids, lung organoids, pancreatic organoids, bile duct organoids, cardiac organoids, hematopoietic organoids, skin tissue organoids, ovarian tissue organoids, brain tissue organoids, placental tissue organoids or are listed in Table 1.

[0293] Table 1

[0294] Status of organoid regeneration medicine development54 4909-6074-3733v.193597 / 7344 ORGANOID TYPE INDICATION Intestinal organoid Ulcerative enteritis Refractory Crohn’s disease

[0295] In some embodiments, the organoid is produced from neoplastic tissues, malignancy,cancers, or metastases. 55 4909-6074-3733v.193597 / 7344

[0296] In some embodiments of methods involving organoids, the organoids are gastric,colonic, duodenal, jejunal or ileal organoids.

[0297] A method of propagating and / or increasing viability of a culture of a mammalian tissuecomprising administering to the human tissue in culture an amount of a protein domain comprising heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of a mammalian tissue.

[0298] In some embodiments, the tissue is a gastrointestinal tissue, cardiac tissue, hepatictissue, biliary tissues, skin tissue, hematopoietic tissue, ovarian or testicular tissues, placental tissues, joint tissues, pancreatic, ovarian, cutaneous, or dental tissues.

[0299] A method of propagating and / or increasing viability of a culture of stem or progenitorcells comprising administering to the stem or progenitor cells in culture an amount of a protein domain comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of stem or progenitor cells.

[0300] In some embodiments, the tissue is a human tissue. In some embodiments, the tissue isfor transplantation. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs) or their downstream products from directed differentiation. In some embodiments, the stem cells are somatic stem cells. In some embodiments, the stem or progenitor cells are human.

[0301] A method of maintaining or increasing the viability of a donor tissue or donor organintended for transplantation comprising administering to the donor tissue or donor organ an amount of a protein domain comprising heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to maintain or increase viability of a donor tissue or donor organ.

[0302] In some embodiments, the donor tissue or donor organ has been removed from thedonor and is being maintained prior to transplantation. In some embodiments, the transplant organ is a kidney, liver, heart, lung, pancreas, skin or intestine. In some embodiments, the tissue or organ for transplantation is from an autologous, an allogeneic or a bioengineered source.

[0303] A method of expanding organoids or growth of tissues in a culture comprisingadministering to the organoids or tissues in a culture an amount of a protein domain comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to expand the organoids or growth of tissues.

[0304] In some embodiments, the organoids are being cultured for transplantation or are listedin Table 1 hereinabove. 56 4909-6074-3733v.193597 / 7344

[0305] A method of promoting healing of a wound in a tissue in a subject comprisingadministering to the wound an amount of a protein domain comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote healing of a wound in a tissue.

[0306] In some embodiments, the tissue is a skin tissue.

[0307] In some embodiments, the skin has suffered a burn. In some embodiments, the tissueis interfollicular dermis.

[0308] In some embodiments, the subject has an inflammatory disease. In some embodiments,the subject has ulcerative colitis, Crohn’s disease, Behcet’s, vasculitides, ischemic colitis, or infectious colitis. In some embodiments, the subject has ulceration or damaged tissue from irradiation including but not limited to radiation stomatitis, radiation colitis / proctitis, radiation enteritis. In some embodiments, the subject has ulceration or tissue damage from drugs or medications including but not limited to chemotherapeutic agents, immunomodulators, steroids, anti-inflammatory drugs, or non-steroidal anti-inflammatory drugs. In some embodiments, the subject has peptic ulcer disease. In some embodiments, the subject has necrotizing enterocolitis. In some embodiments, the subject has short gut syndrome.

[0309] A method of promoting growth of an organoid, or of an organoid culture, comprisingadministering to the organoid or organoid culture an amount of an expression construct encoding a protein domain comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote growth of an organoid, or of an organoid culture.

[0310] A method of reducing the frequency of culture media changes required to maintainviability of organoids of an organoid culture comprising administering to the organoid culture an amount of an expression construct encoding a protein domain comprising heparin-binding domain of an FGFBP1 or FGFBP2 effective to promote and / or maintain viability of the organoids culture thereby reducing the frequency of culture media changes required to maintain viability of the organoids.

[0311] A method of propagating and / or increasing viability of a culture of a mammalian tissuecomprising administering to the human tissue in culture an amount of an expression construct encoding a protein domain comprising heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of a mammalian tissue.

[0312] A method of propagating and / or increasing viability of a culture of stem or progenitorcells comprising administering to the stem or progenitor cells in culture an amount of an expression 57 4909-6074-3733v.193597 / 7344 construct encoding a protein domain comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of stem or progenitor cells.

[0313] A method of maintaining or increasing the viability of a donor tissue or donor organintended for transplantation comprising administering to the donor tissue or donor organ an amount of an expression construct encoding a protein domain comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to maintain or increase viability of a donor tissue or donor organ.

[0314] A method of expanding organoids or growth of tissues in a culture comprisingadministering to the organoids or tissues in a culture an amount of an expression construct encoding a protein domain comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to expand the organoids or growth of tissues.

[0315] A method of promoting healing of a wound in a tissue comprising administering to thewound an amount of an expression construct encoding a protein domain comprising a heparin- binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote healing of a wound in a tissue.

[0316] In some embodiments, the protein domains of the invention can be administered in“naked” form, or as fusion proteins or fusion molecules with moieties that aid in delivery, serum half life time and / or stability and resistance to degradation, or as any of these forms in a composition. Examples of delivery systems for “naked” forms of the peptides, or as fusion proteins or fusion molecules thereof, include lipid-based nanocarriers such as oil-in-water nanoemulsions, self-emulsifying drug delivery systems (SEDDS), solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), liposomes and micelles.

[0317] In some embodiments of the compositions comprising the protein domains of theinvention, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 described herein is encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 described herein is attached to liposomes. In some instances, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 described herein is conjugated to the surface of liposomes. In some instances, 58 4909-6074-3733v.193597 / 7344 the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 described herein is encapsulated within the shell of a liposome. In some instances, the liposome is a cationic liposome.

[0318] In regard to fusion proteins or fusion molecules comprising the heparin-binding domainof an FGFBP1 or FGFBP2 or FGFBP3 described herein, i.e., comprising a second moiety which is not the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 described herein, exemplary second moieties include permeation enhancers, assisted for example by enteric coatings and / or the presence of peptidase-inhibiting excipients or direct-acting inhibitors, medium chain fatty acids (sodium caprylate C8, sodium caprate, C10) and derivatives (the Eligen® candidate, salcaprozate sodium (SNAC)), EDTA, bile salts, acyl carnitines, and SNAC.

[0319] The isolated heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 describedherein, whether “naked” or not in fusion protein or fusion molecule form, can be unmodified or modified. Modifications can be made anywhere in the heparin-binding domain of an FGFBP1 or FGFBP2 described herein, including the peptide backbone, the amino acid side-chains thereof, and the amino or carboxyl termini thereof. Certain common peptide modifications that are useful for modification of heparin-binding domain of an FGFBP1 or FGFBP2 described herein include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, and ADP-ribosylation.

[0320] In some embodiments of the inventions described herein, the heparin-binding domainof an FGFBP1 or FGFBP2 or FGFBP3 is isolated. As used herein, the term "isolated " refers to a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 that by virtue of its origin or source of derivation has one, two, three, or four of the following: (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, and (4) does not occur in nature absent the hand of man.

[0321] In some embodiments, peptide or fusion protein described herein is recombinantlyproduced. In some embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such, is produced in a eukaryotic expression system. In some embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such, is produced in a prokaryotic expression system. 59 4909-6074-3733v.193597 / 7344

[0322] Compositions or pharmaceutical compositions comprising the heparin-binding domainof an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein or fusion molecule comprising such, or the described expression constructs, are provided. Compositions or pharmaceutical compositions comprising the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein or fusion molecule comprising such or the described expression constructs disclosed herein are preferably comprise stabilizers to prevent loss of activity or structural integrity of the protein due to the effects of denaturation, oxidation or aggregation over a period of time during storage and transportation prior to use. The compositions or pharmaceutical compositions can comprise one or more of any combination of salts, surfactants, pH and tonicity agents such as sugars can contribute to overcoming aggregation problems. Where a composition or pharmaceutical composition of the present invention is used as an injection, it is desirable to have a pH value in an approximately neutral pH range, it is also advantageous to minimize surfactant levels to avoid bubbles in the formulation which are detrimental for injection into subjects. In an embodiment, the composition or pharmaceutical composition is in liquid form and stably supports high concentrations of bioactive heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such in solution and is suitable for inhalational or parenteral administration. In an embodiment, the composition or pharmaceutical composition is suitable for intravenous, intramuscular, intraperitoneal, intradermal and / or subcutaneous injection. In an embodiment, the composition or pharmaceutical composition is in liquid form and has minimized risk of bubble formation and anaphylactoid side effects. In an embodiment, the composition or pharmaceutical composition is isotonic. In an embodiment, the composition or pharmaceutical composition has a pH or 6.8 to 7.4.

[0323] In an embodiment the heparin-binding domain of an FGFBP1 or FGFBP2, or FGFBP3or fusion protein comprising such disclosed herein are lyophilized and / or freeze dried and are reconstituted for use.

[0324] Examples of pharmaceutically acceptable carriers include, for the pharmaceuticalcompositions of the invention, but are not limited to, phosphate buffered saline solution, sterile water (including water for injection USP), emulsions such as oil / water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline, for example 0.9% sodium chloride solution, USP. Compositions comprising such carriers are formulated by well-known conventional methods (see, 60 4909-6074-3733v.193597 / 7344 for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000, the content of each of which is hereby incorporated in its entirety). In non-limiting examples, this can comprise one or more of dibasic sodium phosphate, potassium chloride, monobasic potassium phosphate, polysorbate 80 (e.g. 2-[2-[3,5-bis(2- hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl (E)-octadec-9-enoate), disodium edetate dehydrate, sucrose, monobasic sodium phosphate monohydrate, and dibasic sodium phosphate dihydrate.

[0325] The compositions, or pharmaceutical compositions described herein can also belyophilized or provided in any suitable forms including, but not limited to, injectable solutions or inhalable solutions, gel forms and tablet forms.

[0326] In some embodiments the composition or pharmaceutical composition comprising theheparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such, described herein is substantially pure with regard to the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such. A composition or pharmaceutical composition comprising the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such, described herein is "substantially pure" with regard to the heparin- binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such when at least 60% to 75% of a sample of the composition or pharmaceutical composition exhibits a single species of the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such. A substantially pure composition or pharmaceutical composition comprising the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such, described herein can comprise, in the portion thereof which is the heparin- binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such, 60%, 70%, 80% or 90% of the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such, of the single species, more usually about 95%, and preferably over 99%. Purity or homogeneity may be tested by a number of means well known in the art, such as polyacrylamide gel electrophoresis or HPLC.

[0327] Administration as referred to in the methods disclosed herein, to a subject or to an organif relevant, can be auricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, via hemodialysis, via catheter, via injection, 61 4909-6074-3733v.193597 / 7344 interstitial, intra-abdominal, intra-amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophagus, intragastric, intravaginal, intragingival, intraileal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intraepicardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intraventricular, intravesical, intravitreal, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, rectal, inhalationally, retrobulbar, subarachnoid, subconjuctival, sublingual, submucosal, topically, transdermal, transmucosal, transplacental, transtracheal, ureteral, uretheral, vaginal, endoscopically, mucosally or by spray.

[0328] In some embodiments, administration is local or localized. In some embodiments,administration is systemic. In some embodiments of administration to a subject, the heparin- binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such is administered at a dose of 0.5mg / kg to 100 mg / kg. In some embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such is administered at a dose of 101 mg / kg to 250 mg / kg. In some embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such is administered at a dose of 251 mg / kg to 500 mg / kg. In some embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2, or fusion protein comprising such is administered at a dose of 501 mg / kg to 1000 mg / kg. In some embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such is administered at a dose of 1001 mg / kg to 2000 mg / kg. In some embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such is administered at a dose of up to 25 mg twice per daily, daily, every other day, weekly, monthly or every three months. In some embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such is administered at a dose of 25 to 100 mg twice per daily, daily, every other day, weekly, monthly or every three months. In some embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such administered at a dose of 100 to 250 mg twice per daily, daily, every other 62 4909-6074-3733v.193597 / 7344 day, weekly, monthly or every three months. In some embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such is administered at a dose of 250 to 500 mg twice per daily, daily, every other day, weekly, monthly or every three months. In some embodiments, the heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such is administered at a dose of 500 to 1000 mg twice per daily, daily, every other day, weekly, monthly or every three months. In some embodiments, heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or fusion protein comprising such is administered at a dose of 1000 to 2000 mg twice per daily, daily, every other day, weekly, monthly or every three months.

[0329] A method of promoting growth of an organoid, or of an organoid culture, comprisingadministering to the organoid or organoid culture an amount of an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or a fusion protein thereof, effective to promote growth of an organoid, or of an organoid culture.

[0330] A method of reducing the frequency of culture media changes required to maintainviability of organoids of an organoid culture comprising administering to the organoid culture an amount of an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote and / or maintain viability of the organoids culture thereby reducing the frequency of culture media changes required to maintain viability of the organoids.

[0331] A method of propagating and / or increasing viability of a culture of stem or progenitorcells comprising administering to the stem or progenitor cells in culture an amount of an FGFBP1 or FGFBP2 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 effective to propagate and / or increase viability of a culture of stem or progenitor cells.

[0332] A method of maintaining or increasing the viability of a donor tissue or donor organintended for transplantation comprising administering to the donor tissue or donor organ an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to maintain or increase viability of a donor tissue or donor organ.

[0333] A method of expanding organoids or growth of tissues in a culture comprisingadministering to the organoids or tissues in a culture an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to expand the organoids or growth of tissues. 63 4909-6074-3733v.193597 / 7344

[0334] A method of promoting healing of a wound in a tissue in a subject comprisingadministering to the wound an amount of an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote healing of a wound in a tissue.

[0335] In embodiments of the methods herein pertaining to tissues, the tissue may be geriatric.In embodiments of the methods herein pertaining to tissues, the tissue may be adult. In embodiments of the methods herein pertaining to tissues, the tissue may be pediatric. In embodiments of the methods herein pertaining to tissues, the tissue may be fetal.

[0336] A method of promoting growth of an organoid, or of an organoid culture, comprisingadministering to the organoid or organoid culture an amount of an expression construct encoding an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3, or a fusion protein thereof, effective to promote growth of an organoid, or of an organoid culture.

[0337] A method of reducing the frequency of culture media changes required to maintainviability of organoids of an organoid culture comprising administering to the organoid culture an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote and / or maintain viability of the organoids culture thereby reducing the frequency of culture media changes required to maintain viability of the organoids.

[0338] A method of propagating and / or increasing viability of a culture of a mammalian tissuecomprising administering to the human tissue in culture an amount of an expression construct encoding an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of a mammalian tissue.

[0339] A method of propagating and / or increasing viability of a culture of stem or progenitorcells comprising administering to the stem or progenitor cells in culture an amount of an expression construct encoding an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to propagate and / or increase viability of a culture of stem or progenitor cells.

[0340] A method of maintaining or increasing the viability of a donor tissue or donor organintended for transplantation comprising administering to the donor tissue or donor organ an amount 64 4909-6074-3733v.193597 / 7344 of an expression construct encoding an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to maintain or increase viability of a donor tissue or donor organ.

[0341] A method of expanding organoids or growth of tissues in a culture comprisingadministering to the organoids or tissues in a culture an amount of an expression construct encoding an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to expand the organoids or growth of tissues.

[0342] A method of promoting healing of a wound in a tissue comprising administering to thewound an amount of an expression construct encoding an FGFBP1 or FGFBP2 or FGFBP3 protein comprising a heparin-binding domain of an FGFBP1 or FGFBP2 or FGFBP3 effective to promote healing of a wound in a tissue.

[0343] In some embodiments of the methods or compositions, the composition comprising adomain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 comprises a fusion protein which comprises the domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3.

[0344] In some embodiments of the methods or compositions, the composition comprising adomain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 does not comprise a fusion protein comprising the domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3.

[0345] In some embodiments of the methods or expression constructs, the expression constructencoding a domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 encodes a fusion protein which comprises the domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3.

[0346] In some embodiments of the methods or expression constructs, the expression constructencoding a domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 does not encode a fusion protein which comprises the domain of an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3.

[0347] The invention also provides a composition comprising a non-naturally occurringpolypeptide, the polypeptide comprising a sequence of FGFBP1 or FGFBP2 or FGFBP3 amino acids which is located in an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 protein.

[0348] In some embodiments, the polypeptide is a recombinant protein comprising a sequenceof FGFBP1 or FGFBP2 or FGFBP3 amino acids which is located in an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 protein. 65 4909-6074-3733v.193597 / 7344

[0349] In some embodiments, the polypeptide is a fusion protein.

[0350] In some embodiments, the polypeptide further comprises an additional amino acidsequence, preferably an additional amino acid sequence derived from a protein other than the FGFBP1 or FGFBP2 protein.

[0351] In some embodiments, the polypeptide further comprises a synthetic amino acidsequence.

[0352] In some embodiments, the polypeptide further comprises an amino acid tag or signalsequence.

[0353] In some embodiments, the polypeptide consists of a sequence of FGFBP1 or FGFBP2or FGFBP3 amino acids which is located in an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 protein.

[0354] In some embodiments, the composition isa) effective to propagate, promote growth and / or increase viability of a culture of amammalian tissue; b) effective to propagate, functionally augment and / or increase viability of stem orprogenitor cells; c) effective to maintain or increase viability of a donor tissue or donor organ;d) effective to promote growth of an organoid, or of an organoid culture, orexpanding organoids in a culture; e) effective to promote healing of a wound in a tissue; and / orf) effective to promote and / or maintain viability of the organoids culture therebyreducing the frequency of culture media changes required to maintain viability of the organoids.

[0355] Also provided is a method of propagating, promoting growth and / or increasing viabilityof a culture of a mammalian tissue comprising administering to the mammalian tissue in culture an amount of the composition that is effective to propagate, promote growth and / or increase viability of a culture of a mammalian tissue.

[0356] Also provided is a method of propagating and / or increasing viability of stem orprogenitor cells comprising administering to the stem or progenitor cells, optionally in a in culture, an amount of the composition effective to propagate and / or increase viability of stem or progenitor cells. 66 4909-6074-3733v.193597 / 7344

[0357] Also provided is a method of maintaining or increasing the viability of a donor tissueor donor organ intended for transplantation comprising administering to the donor tissue or donor organ an amount of the composition effective to maintain or increase viability of a donor tissue or donor organ.

[0358] Also provided is a method of promoting growth of an organoid, or of an organoidculture, or expanding organoids in a culture, comprising administering to the organoid or organoid culture an amount of the composition effective to promote growth of an organoid, or of an organoid culture, or expanding organoids in a culture.

[0359] Also provided is a method of promoting healing of a wound in a tissue in a subjectcomprising administering to the wound an amount of the composition effective to promote healing of a wound in a tissue.

[0360] Also provided is a method of reducing the frequency of culture media changes requiredto maintain viability of organoids of an organoid culture comprising administering to the organoid culture (a) an amount of an expression construct encoding the polypeptide, or (b) an amount of the composition, effective to promote and / or maintain viability of the organoids culture thereby reducing the frequency of culture media changes required to maintain viability of the organoids

[0361] Also provided is a method of propagating, promoting growth and / or increasing viabilityof a culture of a mammalian tissue comprising administering to the mammalian tissue in culture an amount of an expression construct encoding the polypeptide effective to propagate, promote growth and / or increase viability of a culture of a mammalian tissue.

[0362] Also provided is a method of propagating and / or increasing viability of stem orprogenitor cells comprising administering to the stem or progenitor cells, optionally in a in culture, an amount of an expression construct encoding the polypeptide effective to propagate and / or increase viability of stem or progenitor cells.

[0363] Also provided is a method of maintaining or increasing the viability of a donor tissueor donor organ intended for transplantation comprising administering to the donor tissue or donor organ an amount of an expression construct encoding the polypeptide effective to maintain or increase viability of a donor tissue or donor organ.

[0364] Also provided is a method of promoting growth of an organoid, or of an organoidculture, or expanding organoids in a culture, comprising administering to the organoid or organoid 67 4909-6074-3733v.193597 / 7344 culture an amount of an expression construct encoding the polypeptide of effective to promote growth of an organoid, or of an organoid culture, or expanding organoids in a culture.

[0365] Also provided is a method of promoting healing of a wound in a tissue in a subjectcomprising administering to the wound an amount of an expression construct encoding the polypeptide.

[0366] In some embodiments, the stem cells are iPSCs or ESCs.

[0367] In some embodiments, the stem cells are human.

[0368] In some embodiments, the tissue is gastrointestinal tissue, cardiac tissue, pancreatictissue, hepatic tissue, ovarian tissue, skin or cutaneous tissue, or hematopoietic tissue.

[0369] In some embodiments, the tissue is a human tissue.

[0370] In some embodiments, the tissue is for transplantation.

[0371] In some embodiments, the donor tissue or donor organ has been removed from thedonor and is being maintained prior to transplantation.

[0372] In some embodiments, the organoids are being cultured for transplantation and / or arelisted in Table 1.

[0373] In some embodiments, the tissue is a skin tissue or a gastrointestinal tissue.

[0374] In some embodiments, the skin has suffered a burn.

[0375] In some embodiments, the subject has ulcerative colitis.

[0376] In some embodiments, the organoid is a tumor organoid grown from cancer ormalignancy or metastasis.

[0377] In some embodiments, the method effects an increase in organoid number in theorganoid culture.

[0378] In some embodiments, the method effects an increased growth rate of the organoid.

[0379] In some embodiments, the method effects increased viability of the organoid.

[0380] In some embodiments, the organoid is a mammalian tissue organoid.

[0381] In some embodiments, the organoids are gastrointestinal tract organoids, sebaceousglands organoids, hepatic tissue organoids, renal tissue organoids, lung organoids, pancreatic organoids, bile duct organoids, or are listed in Table 1.

[0382] In some embodiments, the organoids are gastric, colonic, duodenal, jejunal or ilealorganoids. 68 4909-6074-3733v.193597 / 7344

[0383] In some embodiments, the composition comprising a domain of an N-terminal portionof an FGFBP1 comprises a fusion protein which comprises the domain of an N-terminal portion of an FGFBP1.

[0384] The invention also provides an expression construct encoding polypeptide, thepolypeptide comprising a sequence of FGFBP1 or FGFBP2 or FGFBP3 amino acids which is located in an N-terminal portion of an FGFBP1 or FGFBP2 or FGFBP3 protein.

[0385] In some embodiments of the methods and compositions herein disclosed, the FGFBP1or FGFBP2 or FGFBP3 is human. In some embodiments of the methods and compositions herein disclosed, the FGFBP1 or FGFBP2 or FGFBP3 is mouse.

[0386] Without wishing to be bound by theory, there is no necessary reason that thecomposition, fragments, domains and portions described herein need actually bind to a heparin to effect the various method outcomes.

[0387] In some embodiments, the compositions or methods employing an NTD of a humanFGFBP2, but not a full length human FGFBP2, comprise a fragment, domain, portion or sequence, or comprise use of a fragment, domain, portion or sequence, which comprises SEQ ID NO:3 but does not comprise SEQ ID NO:1, nor SEQ ID NO:1 without its native signal sequence.

[0388] In some embodiments, the compositions or methods employing an NTD of a humanFGFBP3, but not a full length human FGFBP3, comprise a fragment, domain, portion or sequence, or comprise use of a fragment, domain, portion or sequence, which comprises SEQ ID NO:5 but does not comprise SEQ ID NO:4, nor SEQ ID NO:4 without its native signal sequence. In some embodiments, a portion comprising an N-terminal domain of human FGFBP3 has the sequence of SEQ ID NO:15.

[0389] In some embodiments, the compositions or methods employing an NTD of a mouseFGFBP3, but not a full length mouse FGFBP3, comprise a fragment, domain, portion or sequence, or comprise use of a fragment, domain, portion or sequence, which comprises SEQ ID NO:6 but does not comprise a full length mouse FGFBP3 with or without its signal sequence.

[0390] In some embodiments, the compositions or methods employing an NTD of a humanFGFBP1, but not a full length human FGFBP1, comprise a fragment, domain, portion or sequence, or comprise use of a fragment, domain, portion or sequence, which comprises SEQ ID NO:8 but does not comprise SEQ ID NO:7, nor SEQ ID NO:7 without its native signal sequence. In some 69 4909-6074-3733v.193597 / 7344 embodiments, a portion comprising an N-terminal domain of human FGFBP1 has the sequence of SEQ ID NO:14.

[0391] In some embodiments, the compositions or methods employing an NTD of a mouseFGFBP1, but not a full length mouse FGFBP1, comprise a fragment, domain, portion or sequence, or comprise use of a fragment, domain, portion or sequence, which comprises SEQ ID NO:9 but does not comprise a full length mouse FGFBP1 with or without its signal sequence. In some embodiments, a portion comprising an N-terminal domain of Mouse FGFBP1 has the sequence of SEQ ID NO:13.

[0392] In some embodiments, an expression construct encoding a human FGFBP2 NTD canencode SEQ ID NO:2. In some embodiments, an expression construct encoding a human FGFBP1 NTD can encode SEQ ID NO:10. In some embodiments, an expression construct encoding a mouse FGFBP1 NTD can encode SEQ ID NO:18. In some embodiments, a fusion protein comprising a human FGFBP2 NTD can be SEQ ID NO:11. In some embodiments, the expression constructs are adenoviral expression constructs. In some embodiments, an adenoviral expression construct encoding a human FGFBP2 NTD can encode SEQ ID NO:22. In some embodiments, an adenoviral expression construct encoding a mouse FGFBP1 NTD can encode SEQ ID NO:21.

[0393] In some embodiments, an protein domain or peptide comprising an N-terminal domainof a human fibroblast growth factor binding protein 1 (FGFBP1), but not comprising a full length or mature FGFBP1 protein, can comprise at the N-terminal of, e.g., SEQ ID NO:8 or 9, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more contiguous amino acids of the sequence of the full length FGFBP1 sequence extending beyond the N-terminal of SEQ ID NO:7 or 17. The same applies to the NTD of FGFBP2, and FGFBP3, e.g., with regard to SEQ ID NO:3 versus SEQ D NO:4, or with regard to SEQ ID NOS: 5 or 6 versus SEQ ID NOS: 4 or 16, respectively. For example, in the case of FGFBP2, as set forth in Fig.72C, the heparin binding domain of human FGFBP2, which can be utilized as an protein domain or peptide comprising an N-terminal domain of a human fibroblast growth factor binding protein 2 (FGFBP2), has the sequence QAPRQKQGSTGEE (SEQ ID NO:23) (hFGFBP2 Q20-E32) in between the C-terminal of the signal sequence and the N-terminal of heparin-binding domain sequence of human FGFBP2. In some embodiments, a protein domain or peptide comprising an N-terminal domain of a human fibroblast growth factor binding protein 2 (FGFBP2), but not comprising a full length or mature FGFBP2 protein, can comprise QAPRQKQGSTGEE (SEQ ID 70 4909-6074-3733v.193597 / 7344 NO:23) (hFGFBP2 Q20-E32) or any shorter sequence thereof that is contiguous with the N- terminal of SEQ ID NO:3, whether it be one, two, three, four, five, etc. or more contiguous amino acids from the sequence. The same applies, mutatis mutandis, to the C-terminal end of, e.g., SEQ ID NO:3. Thus in some embodiments, a protein domain or peptide comprising an N-terminal domain of a human fibroblast growth factor binding protein 2 (FGFBP2), but not comprising a full length FGFBP2 protein, can comprise at the C-terminal of, e.g., SEQ ID NO:3, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more contiguous amino acids of the sequence of the full length FGFBP2 sequence extending beyond the C-terminal of SEQ ID NO:3. Thus, with refence to Fig.72C, the C-terminal can extend one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more contiguous amino acids of the sequence of the full length human FGFBP2 sequence extending beyond the N- terminal of SEQ ID NO:3, i.e., any contiguous portion of the sequence of SEQ ID NO:1 starting after the C-terminal of the heparin binding domain up to the start of the FGF-binding domains (i.e., any C-terminal contiguous portion of PNQQPEAGTPSLRPKATVKLTEATQLGKDSMEELGKAKPTTRPTAKPTQ (SEQ ID NO:24) (hFGFBP2 P139-Q187) whether one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen etc. or more contiguous amino acids thereof. In some embodiments, the same applies to peptide or protein domains comprising an N-terminal domain of a human fibroblast growth factor binding protein 1 (FGFBP1), but not comprising a full length or mature FGFBP1 protein, and also to peptide or protein domains comprising an N-terminal domain of a human fibroblast growth factor binding protein 3 (FGFBP3), but not comprising a full length or mature FGFBP3 protein.

[0394] In some embodiments, a protein domain comprising an N-terminal domain of a humanfibroblast growth comprises from one to twenty additional amino acids at the N-terminal and / or C-terminal thereof, where the amino acids are independent of the N-terminal and / or C-terminal amino acids of the native FGFBP sequence.

[0395] Fragments of full length FGFBP1, FGFBP2, or FGFBP3 can include the signalsequence and an NTD, but exclude the FGF-binding domain. Fragments of full length FGFBP1, FGFBP2, or FGFBP3 containing an NTD can also exclude the native the signal sequence, and exclude the FGF-binding domain. Examples of such fragments are: 71 4909-6074-3733v.193597 / 7344

[0396] Fragments of full length FGFBP1 protein sequence (Human) (SEQ ID NO:7)containing an N-terminal domain (NTD) (e.g., S52-S153) can include, at the N-terminal portion thereof, any contiguous residues K24-K51 (i.e., the entire or less than the entire sequence between end of Signal Peptide and beginning of Heparin Binding Domain), and / or, independently, at the C-terminal portion thereof any contiguous residues T154-D200 (i.e., the entire or less than the entire sequence between end of Heparin Binding Domain and start of FGF-binding domain).

[0397] Fragments of full length FGFBP1 protein sequence (mouse) (SEQ ID NO:17)containing an NTD (e.g., S58-R162) can include, at the N-terminal portion thereof, any contiguous residues E21-T57 (i.e., the entire or less than the entire sequence between end of Signal Peptide and beginning of Heparin Binding Domain), and / or, independently, at the C-terminal portion thereof any contiguous residues G163-D211 (i.e., the entire or less than the entire sequence between end of Heparin Binding Domain and start of FGF-binding domain).

[0398] Fragments of full length FGFBP2 protein sequence (Human) (SEQ ID NO:1)containing an NTD (F33-E138) can include, at the N-terminal portion thereof, any contiguous residues Q20-E32 (i.e., the entire or less than the entire sequence between end of Signal Peptide and beginning of Heparin Binding Domain), and / or, independently, at the C-terminal portion thereof any contiguous residues P139-Q163 (i.e., the entire or less than the entire sequence between end of Heparin Binding Domain and start of FGF-binding domain).

[0399] Fragments of full length FGFBP3 protein sequence (Human) (SEQ ID NO:4)containing an NTD (G42-A151) can include, at the N-terminal portion thereof, any contiguous residues A26-P41 (i.e., the entire or less than the entire sequence between end of Signal Peptide and beginning of Heparin Binding Domain), and / or, independently, at the C-terminal portion thereof any contiguous residues S152-L230 (i.e., the entire or less than the entire sequence between end of Heparin Binding Domain and start of FGF-binding domain).

[0400] Fragments of full length FGFBP3 protein sequence (mouse) (SEQ ID NO:16)containing an NTD (S49-P151) can include, at the N-terminal portion thereof, any contiguous residues K31-S48 (i.e., the entire or less than the entire sequence between end of Signal Peptide and beginning of Heparin Binding Domain), and / or, independently, at the C-terminal portion thereof any contiguous residues S152-N215 (i.e., the entire or less than the entire sequence between end of Heparin Binding Domain and start of FGF-binding domain). 72 4909-6074-3733v.193597 / 7344

[0401] The compositions, peptides, fusion proteins, expression constructs, or methods of theinvention can in some embodiments apply or embody or encode any of the fragments described herein. In some embodiments, the peptide is a fragment.

[0402] It will be understood that, for the particular FGFBP polypeptides described here,natural variations can exist. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions which do not essentially alter biological and immunological activities, have been described, e.g. by Neurath et al in "The Proteins" Academic Press New York (1979). Amino acid replacements between related amino 15 acids or replacements which have occurred frequently in evolution are, inter alia, Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, Ile / Val (see Dayhof, M. D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3). Other amino acid substitutions include Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Thr / Phe, Ala / Pro, Lys / Arg, Leu / Ile, Leu / Val and Ala / Glu. Based on this information, Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science (1985) 227:1435) and determining the functional similarity between homologous proteins. Such amino acid substitutions of the exemplary embodiments of this invention, as well as variations having deletions and / or insertions are within the scope of the invention as long as the resulting proteins retain at least some of the one or more activities described herein. In some embodiments of the methods and compositions and fusion proteins, the N-terminal domain of the FGFBP (or the heparin-binding domain of the FGFBP) comprises a variant having a sequence identity of 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with a complete N-terminal domain as set forth in one of SEQ ID NOS: 3, 5, 6, 8 or 9. In some embodiments of the methods and compositions and fusion proteins, the N-terminal domain of the FGFBP (or the heparin-binding domain of the FGFBP) comprises a variant having a sequence identity of 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more with a complete N-terminal domain as set forth in one of SEQ ID NOS: 3, 5, 6, 8 or 9. Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, and GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, 73 4909-6074-3733v.193597 / 7344 Wisconsin). To determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are, or are about, of the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps.

[0403] In some embodiments of the methods and compositions and fusion proteins, the N-terminal domain of the FGFBP (or the heparin-binding domain of the FGFBP) comprises the complete domain (e.g. as set forth in SEQ ID NOS: 3, 5, 6, 8 or 9). In further aspects of the methods and compositions and fusion proteins herein, an active fragment of the N-terminal domain of the FGFBP (or the heparin-binding domain of the FGFBP) is employed or as part of the composition or fusion protein, i.e., a fragment of the N-terminal domain of the FGFBP (or the heparin-binding domain of the FGFBP) which is less than the entire N-terminal domain of the FGFBP (or the heparin-binding domain of the FGFBP) but which still retains some activity, e.g., some ability to promote propagation of, function of, and / or increasing viability of, T cells or NK cells. Such fragments may be as few as 8 amino acids of the N-terminal domain of the FGFBP (or the heparin- binding domain of the FGFBP) and of any amino acid number in length up to one less amino acid than the entire length of the N-terminal domain of the FGFBP (or the heparin-binding domain of the FGFBP). For example, a fragment of SEQ ID NO:5 (117 amino acids in length) that is an 8 to 116 amino acids fragment of SEQ ID NO:5 is encompassed. For example, a fragment of SEQ ID NO:6 (103 amino acids in length) that is an 8 to 102 amino acids fragment of SEQ ID NO:6. For example, a fragment of SEQ ID NO:3 (106 amino acids in length) that is an 8 to 105 amino acids fragment of SEQ ID NO:3. For example, a fragment of SEQ ID NO:8 (102 amino acids in length) that is an 8 to 101 amino acids fragment of SEQ ID NO:8. For example, a fragment of SEQ ID NO:9 (102 amino acids in length) that is an 8 to 101 amino acids fragment of SEQ ID NO:9. Fragments may be of any length as long as they retain some activity as described herein, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 etc. amino acids in length. All individual integer fragment lengths between the stated ranges are encompassed by the invention. In addition, all fragment length sub-ranges within the stated ranges are encompassed by the invention, e.g.20-30, 30-40, 40-50 etc. amino acids in length. 74 4909-6074-3733v.193597 / 7344

[0404] Full length FGFBP1 sequences are set forth in SEQ ID NOS:7 and 17. The matureFGFBP1 does not comprise the signal sequence, e.g. as set forth in FIG.72. Full length FGFBP2 sequence (Human only) is set forth in SEQ ID NO:1. The mature FGFBP2 does not comprise the signal sequence, e.g. as set forth in FIG.72. Full length FGFBP3 sequences are set forth in SEQ ID NOS:4 and 16. The mature FGFBP3 does not comprise the signal sequence, e.g. as set forth in FIG.72.

[0405] In some embodiments, the peptides or fusion proteins containing the N-terminaldomain of the FGFBP are modified in their backbones. Backbone modification includes the substitution of one or more L-amino acids by D-amino acids, insertion of one or more methylamino acids, and the incorporation of one or more β-amino acids and one or more peptoids. In some embodiments, the peptides are not modified in their backbones.

[0406] In some embodiments, the peptides or fusion proteins containing the N-terminaldomain of the FGFBP comprise one or more side chain modifications achieved by replacing the natural amino acids with their analogues during peptide synthesis, to improve their binding affinity and target selectivity. Variants of natural amino acid analogues encompassed include homoarginine, benzyloxytyrosine, and β-phenylalanine. In some embodiments, the peptides containing the N-terminal domain do not comprise one or more side chain modifications.

[0407] In some embodiments, the peptides or fusion proteins containing the N-terminaldomain of the FGFBP are PEGylated. PEG is formed by repetitive units of ethylene oxide and is a non-biodegradable, non-toxic, low-immunogenic polymer. PEGylation can increase the effective molecular weight of proteins to reduce their renal clearance by kidneyfiltration. The PEG moiety can also shield the proteins from digestion by proteolytic enzymes via increased steric hindrance, and help increase absorption by increasing the target protein’s water solubility. These advantages make PEGylation a prevalent strategy for modifying therapeutic proteins, and PEGylation has been applied for optimizing protein therapeutics since the 1970s, with great success. Conventional PEGylation often occurs at Lys or Cys residues. PEGylation can also be effected via genetic code expansion when making the peptides, e.g., recombinantly. Azide (e.g., p-azido-phenylalanine) or acetyl (e.g., p-acetylphenylalanine) groups can be introduced into therapeutic peptides and proteins by genetic code expansion to allow downstream PEGylation modifications including, e.g. mono- PEGylation. Different sized PEG chains are encompassed, e.g., PEG4, PEG8, PEG12 or PEG24. 75 4909-6074-3733v.193597 / 7344

[0408] Along with serum albumin or immunoglobulin Fc fusions, plasma half life of thecompositions of, and used in, the invention herein can be increased by conjugations. For example, in some embodiments, the peptides or fusion proteins containing the N-terminal domain of the FGFBP are conjugated to C14 / 16 / 18 fatty acids which can increase plasma circulation times and decrease kidney elimination.

[0409] In some embodiments, proximity-enabled reactive therapeutic strategy can be usedsuch as incorporating a non-canonical amino acid into the peptides or fusion proteins containing the N-terminal domain of the FGFBP, e.g. afluorosulfate-L-tyrosine.

[0410] In some embodiments, compositions comprising the peptides or fusion proteinscontaining the N-terminal domain of the FGFBP are formulated with permeation enhancers such as fatty acids or, e.g., with sodium N-[8-(2-hydroxybenzoyl amino]caprylate (SNAC). In some embodiments, the compositions are formulated with hydrogels or enzyme inhibitors.

[0411] Protein domains comprising an N-terminal domain of a human fibroblast growth factorbinding protein can be referred to herein as peptides.

[0412] In some embodiments, subsequences of, or encoding for, FGFBP1, 2 or 3, are provided.“Subsequence” means, for a polynucleotide, a polynucleotide having one or more (e.g., several) nucleotides absent from the 5′ and / or 3′ end of a mature polypeptide coding sequence, wherein the subsequence encodes a fragment having at least some level of an immune cell activity described herein. In one aspect, a subsequence contains at least 85%, e.g., at least 90% or at least 95% of the contiguous nucleotides of the mature FGFBP coding sequence. “Subsequence” means, for a peptide / protein, a peptide / protein having one or more (e.g., several) amino acids absent from the N- and / or C- end of a mature polypeptide sequence, wherein the subsequence encodes a fragment having at least some level of an immune cell activity described herein. In one aspect, a subsequence contains at least 85%, e.g., at least 90% or at least 95% of the contiguous amino acids of the mature FGFBP sequence. In addition, all orthologs of FGFBP1, 2 and 3 (and their NTDs) are encompassed by the invention for use in the methods or compositions, fusion proteins, peptides and fragments described herein. For example, UniProt Q9QY10 rat FGFBP1, Q9MZ06 bovine FGFBP1, D3ZVI0 rat FGFBP3, G3S6Y9 gorilla FGFBP2, and H2RAA8 chimpanzee FGFBP2 are all encompassed, as are other species’ FGFBP1, FGFBP2 and FGFBP3 set forth in UniProt. The encompassed NTD or N-terminal fragments or peptides are readily identifiable, e.g., by standard alignment techniques. 76 4909-6074-3733v.193597 / 7344

[0413] Expression constructs herein include expression vectors such a linear or circularpolynucleotide molecule (e.g., DNA) that comprises a polynucleotide encoding the peptides or fusion proteins described herein operably linked to control sequences that provide for its expression. Expression constructs are genetic constructs that are capable of expressing a nucleic acid sequence in a host cell. Such constructs can include segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more (e.g., several) control sequences. In some embodiments, the constructs include one or more control sequences operably linked to the sequence to be expressed. The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence. Control sequences include promoter sequences, which are well known in the art. Such control sequences also include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, and transcription terminator. In some embodiments, the control sequences at least include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a protein. Promoters include non-native promoters for the FGFBP N- terminal domain encoding polynucleotide. Promoters can be chosen based on the cell type one wishes to express the construct in. Promoters per se direct the initiation of transcription (e.g., binding sites for transcription factors and for DNA-dependent RNA-polymerase, TATA box, CAAT sequences, and 5′-capping elements). As long as this functionality of promoting transcription initiation is retained or substantially retained (e.g., at least 70%, at least 80%, at least 90% or at least 95% of wild-type activity, that is, activity of a full-length sequence), any truncated, mutated or otherwise modified variants of a (naturally occurring) wild-type promoter sequence are also encompassed. The promoter sequence is operably linked to the transcriptional start site of the nucleic acid sequence to be expressed.

[0414] In some embodiments, a host cell containing an expression construct as describedherein is disclosed. Such host cells are not limited unless otherwise stated and can be, e.g., mammalian, insect, bacterial, yeast, fungal. In some embodiments, transformant cell transformed with an expression construct as described herein is disclosed. Such transformant cells are not limited unless otherwise stated and can be, e.g., mammalian, insect, bacterial, yeast, fungal. 77 4909-6074-3733v.193597 / 7344

[0415] One can construct an expression construct encoding the peptides / NTD of the inventiononce the NTD sequence is known. The degeneracy of the genetic code means that various sequences can be employed, but codon optimization can be used to select preferred sequences.

[0416] Expression constructs can be delivered as vectors, and known vectors for such deliveryare established in the art and encompassed herein, including adenoviral and lentiviral vectors as well as AAV and retroviral vectors.

[0417] Signal peptides of the NTD compositions or fusion proteins, or encoded by theexpression constructs therefor, can be native or can be exogenous (e.g., in a non-limiting example, human BSA signal peptide). Signal peptides may be selected by one skilled in the art to enhance secretion from a particular cell type over another cell type, as desired.

[0418] Peptides of the invention can be conjugated to a ligand, such as biotin (e.g., via acysteine or lysine residue), a lipid molecule (e.g., via a cysteine residue), or a carrier protein (e.g., serum albumin, immunoglobulin Fc domain, keyhole limpet hemocyanin (KLH) via e.g., a cysteine or lysine residue). Attachment to ligands, such as biotin, can be useful for associating the peptide with ligand receptors, such as avidin, streptavidin, polymeric streptavidin, or neutravidin. Avidin, streptavidin, polymeric streptavidin, or neutravidin, in turn, can be linked to a signaling moiety (e.g., an enzyme, such as horse radish peroxidase (HRP) or alkaline phosphatase (ALP), or other moiety that can be visualized, such as a metallic nanoparticle or nanoshell (e.g., colloidal gold) or a fluorescent moiety), or a solid substrate (e.g., nitrocellulose membrane). Alternatively, the peptides of the invention can be fused or linked to a ligand receptor, such as avidin, streptavidin, polymeric streptavidin, or neutravidin, thereby facilitating the association of the peptides with the corresponding ligand, such as biotin and any moiety (e.g., signaling moiety) or solid substrate attached thereto. Examples of other ligand-receptor pairs are well-known in the art and can similarly be used.

[0419] Peptides of the invention can be fused to a fusion partner (e.g., a peptide or othermoiety) that can be used to improve purification, to enhance expression of the peptide in a host cell, to aid in detection, and to stabilize the peptide. Examples of suitable compounds for fusion partners include carrier proteins (e.g., serum albumin, immunoglobulin Fc domain, KLH), and enzymes (e.g., horse radish peroxidase (HRP), beta-galactosidase, glutathione-S-transferase, alkaline phosphatase). The fusion can be achieved by means of a peptide bond. For example, 78 4909-6074-3733v.193597 / 7344 peptides of the invention and fusion partners can be fusion proteins and can be directly fused in- frame or can comprise a peptide linker.

[0420] An IgG portion of an IgG-containing fusion protein herein can be, e.g., any of an IgG1,IgG2, IgG2a, IgG2b, IgG3 or IgG4 or a portion thereof. In an embodiment, the portion is an Fc region. In an embodiment the fusion protein comprises a sequence identical to an Fc portion of a human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3 or human IgG4. In an embodiment the fusion protein comprises a sequence identical to an Fc portion of a human IgG1. The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine of the Fc region may be removed, for example, by recombinantly engineering the nucleic acid encoding the fusion protein.

[0421] In an embodiment, the fusion protein components (e.g., a NTD and an Fc) are bounddirectly by a peptide bond from the NTD to the Fc domain. In an embodiment, the fusion protein components (e.g., a NTD and an Fc) are linked through a linker. In an embodiment a peptide linker is used which permits flexibility. In an embodiment, the linker is rigid. In an embodiment the linker is cleavable. Non-limiting examples of flexible linkers within the scope of the invention are Gn, and GGGGS, and (GGGGS)n where n = 2, 3, 4 or 5. Non-limiting examples of rigid linkers within the scope of the invention are (EAAAK)n, (XP)n. Non-limiting examples of cleavable linkers within the scope of the invention include disulfide links and protease cleavable linkers. In a preferred embodiment, the linker is a peptide linker.

[0422] In an embodiment, the Fc domain has the same sequence or 95% or greater sequencesimilarity with a human IgG1 Fc domain. In an embodiment, the Fc domain has the same sequence or 95% or greater sequence similarity with a human IgG2 Fc domain. In an embodiment, the Fc domain has the same sequence or 95% or greater sequence similarity with a human IgG3 Fc domain. In an embodiment, the Fc domain has the same sequence or 95% or greater sequence similarity with a human IgG4 Fc domain. In an embodiment, the fusion protein described herein is recombinantly produced. In an embodiment, the fusion protein is produced in a eukaryotic expression system. In an embodiment, the fusion protein produced in the eukaryotic expression system comprises glycosylation at a residue on the Fc portion corresponding to Asn297. In an 79 4909-6074-3733v.193597 / 7344 embodiment, the fusion protein is a homodimer. In an embodiment, the fusion protein is monomeric. In an embodiment, the fusion protein is polymeric.

[0423] In addition, peptides of the invention may be modified to include any of a variety ofknown chemical groups or molecules. Such modifications include, but are not limited to glycosylation, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment to polyethylene glycol (e.g., PEGylation), covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, modifications with fatty acids, and transfer-RNA mediated addition of amino acids to proteins such as arginylation. Analogues of an amino acid (including unnatural amino acids) and peptides with substituted linkages are also included. Peptides of the invention that consist of any of the sequences discussed herein may be modified by any of the discussed modifications.

[0424] To produce a recombinant peptide of the invention, a nucleic acid encoding the peptidecan be inserted into a suitable expression system. Generally, a recombinant molecule or vector is constructed in which the polynucleotide sequence encoding the selected peptide is operably linked to an expression control sequence permitting expression of the peptide. Numerous types of appropriate expression vectors are known in the art, including, e.g., vectors containing bacterial, viral, yeast, fungal, insect or mammalian expression systems. Methods for obtaining and using such expression vectors are well-known. For guidance in this and other molecular biology techniques used for compositions or methods of the invention, see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, current edition, Cold Spring Harbor Laboratory, New York; Miller et al, Genetic Engineering, 8:277-298 (Plenum Press, current edition), Wu et al., Methods in Gene Biotechnology (CRC Press, New York, N.Y., current edition), Recombinant Gene Expression Protocols, in Methods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa, N.J., current edition), and Current Protocols in Molecular Biology, (Ausabel et al., Eds.,) John Wiley & Sons, NY (current edition), and references cited therein. 80 4909-6074-3733v.193597 / 7344

[0425] Compositions of the invention can comprise the naked peptide or the peptide and oneor more additional agents. For example, a pharmaceutically acceptable carrier or a physiologically acceptable carrier.

[0426] In another aspect, provided are compositions comprising two or more peptides of theinvention, e.g. comprising an NTD of an FGFBP2 and an NTD of an FGFBP3, or comprising an NTD of an FGFBP2 and an NTD of an FGFBP1, or comprising an NTD of an FGFBP3 and an NTD of an FGFBP1. In another aspect, provided are compositions comprising three different peptides of the invention, e.g., comprising an NTD of an FGFBP2 and an NTD of an FGFBP3 and an NTD of an FGFBP1.

[0427] In another aspect, provided are the methods disclosed herein but which employcompositions comprising two or more peptides of the invention, e.g. comprising an NTD of an FGFBP2 and an NTD of an FGFBP3, or comprising an NTD of an FGFBP2 and an NTD of an FGFBP1, or comprising an NTD of an FGFBP3 and an NTD of an FGFBP1. In another aspect, provided are the methods disclosed herein but which employ compositions comprising three different peptides of the invention, e.g., comprising an NTD of an FGFBP2 and an NTD of an FGFBP3 and an NTD of an FGFBP1.

[0428] A peptide is provided which consists of SEQ ID NO:8. A peptide is provided whichconsists of SEQ ID NO:9. A peptide is provided which comprises SEQ ID NO:8 but does not comprise SEQ ID NO:7. A peptide is provided which comprises SEQ ID NO:9 but does not comprise SEQ ID NO:17.

[0429] In some embodiments, a peptide is provided which is or which comprises an N-terminaldomain of an FGFBP1. In some embodiments, said peptide is an N-terminal domain of an FGFBP1. In some embodiments, said peptide comprises an N-terminal domain of an FGFBP1, but is less than the full length FGFBP1 or is less than the mature FGFBP1 (without native signal sequence). In some embodiments, said peptide does not comprise a fibroblast growth factor- binding domain (FGF binding domain) of an FGFBP1. The FGF-binding domain is a C-terminal domain of FGFBP1. In some embodiments, said peptide comprising an N-terminal domain of an FGFBP1, but which is less than the full length FGFBP1 or is less than the mature FGFBP1, comprises one amino acid or more than one contagious amino acids at either the N-terminal or C- terminal thereof, independently, or at both the N-terminal and C-terminal thereof, independently, which amino acid or more than one contagious amino acids, are similarly existent in a naturally 81 4909-6074-3733v.193597 / 7344 occurring FGFBP1 at the N-terminal and / or C-terminal, respectively, of the N-terminal domain of an FGFBP1. In some embodiments, said peptide has at the N-terminal residue of the N-terminal domain of an FGFBP1, one amino acid or two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or up to 30, or up to 35, or up to 40, contiguous amino acids which occur in the naturally occurring FGFBP1 (e.g. SEQ ID NO:7 or SEQ ID NO:17) N-terminal portion of FGFBP1 relative to the N-terminal residue of the N-terminal domain of an FGFBP1 (e.g., such as SEQ ID NO:8 or SEQ ID NO:9). Thus, with reference to Fig. 72A, a peptide comprising an N-terminal domain of an FGFBP1, but which is less than the full length FGFBP1 or is less than the mature FGFBP1, such as the heparin-binding domain peptide demarcated in the figure, can comprise prior to the first marked residue of the heparin-binding domain at the N- terminal end thereof from one up to the full number of contiguous amino acids shown between the predicted signal peptide and heparin-binding domain. Thus, also with reference to Fig. 72A, a peptide comprising an N-terminal domain of an FGFBP1, but which is less than the full length FGFBP1 or is less than the mature FGFBP1, such as the heparin-binding domain peptide demarcated in the figure, can additionally, or alternatively, comprise subsequent to the last marked residue of the heparin-binding domain at the C-terminal end thereof, from one up to the full number of contiguous amino acids shown between the C-terminal end residue thereof up to and including the last residue prior to the FGF-binding domain thereof. SEQ ID NO:13 and SEQ ID NO:14 are non-limiting examples of such peptides.

[0430] In some embodiments, a peptide is provided which is or which comprises an N-terminaldomain of an FGFBP1, and which is a fragment of FGFBP1 less than the full length FGFBP1 or less than the mature FGFBP1 (without native signal sequence). Said peptide can comprise additional amino acids at the N- and / or -C-terminal thereof, the sequence or sequences of which do not naturally occur in a full length or mature FGFBP1.

[0431] Also in regard to compositions, fragments, fusion proteins and methods describedherein, mutatis mutandis, a peptide is provided, or employed in the methods, which is a fragment of an N-terminal domain of an FGFBP1, and has an amino acid sequence length which is less than the full amino acid sequence length of an N-terminal domain of an FGFBP1 (e.g., a fragment (less than full length) of SEQ ID NO:8 or SEQ ID NO:9). In some embodiments, said fragment has one or more of the activities of the NTD as described herein, even if at a lower activity amount than an entire NTD. In some embodiments, the peptide which is a fragment of an N-terminal domain 82 4909-6074-3733v.193597 / 7344 of an FGFBP1 is one of from 10 to 20, from 20 to 30, from 30 to 40, from 40 to 50, from 50 to 60, from 60 to 70, from 70 to 80, from 80 to 90, from 90 to 100, or from 100 to 110 contiguous amino acids of the N-terminal domain of an FGFBP1 but less than the full length N-terminal domain of the FGFBP1. Expression constructs encoding such peptides are also provided. In embodiments of the peptides, fragments or fusion proteins disclosed herein, the peptides, fragments or fusion proteins are glycosylated. In embodiments of the peptides, fragments or fusion proteins disclosed herein, the peptides, fragments or fusion proteins are not glycosylated.

[0432] In some embodiments, peptides can be up to 100, up to 150, or up to 200 amino acidsin length. In some embodiments, peptides are 200-300 amino acids in length.

[0433] Peptide A, and Domain A, as used herein are exemplary embodiments of the N-terminalfragments or N-terminal domains (NTDs) as disclosed herein.

[0434] Where a numerical range is provided herein, it is understood that all numerical subsetsof that range, and all the individual integers contained therein, are provided as part of the invention.

[0435] All combinations of the various elements, or the various embodiments, described hereinare within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0436] “And / or” as used herein, for example, with option A and / or option B, encompasses theseparate embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.

[0437] All combinations of the various elements described herein are within the scope of theinvention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0438] Definitions

[0439] The terms used in this specification generally have their ordinary meanings in the artwithin the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of 83 4909-6074-3733v.193597 / 7344 the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.

[0440] The term “subject” as used in this application means a mammal. Mammals includecanines, felines, rodents, bovine, equines, porcines, ovines, and primates including humans. Thus, the invention can be used in human medicine or also in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical and cosmetic applications. In a preferred embodiment the subject is a human.

[0441] The term “about” or “approximately” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0442] EXPERIMENTAL RESULTS

[0443] Example I

[0444] In the prevailing model, Lgr5+ crypt base columnar (CBC) cells are the only dedicatedintestinal stem cells (ISCs) that sustain epithelial regeneration during homeostasis by upward migration of their progeny from the crypt base through an elusive transit-amplifying (TA) intermediate in the upper crypt1-4. Paradoxically, loss of Lgr5+ cells does not disrupt intestinal crypts, proliferation, or epithelial homeostasis5,6. This finding has been attributed to plasticity or reprogramming of post-mitotic cell types7-10versus activation of quiescent populations11-14that repopulate the epithelium in the absence of CBC cells rather than to a distinct pool of ISCs15,16. Here, we identify a proliferative population of upper crypt cells marked by expression of Fgfbp1 in the location of putative TA cells. Single-cell RNA-seq profiling reveals Fgfbp1+ cells are 84 4909-6074-3733v.193597 / 7344 transcriptionally distinct from Lgr5+ CBC cells and RNA-Velocity trajectory analysis raised the possibility that they give rise to all lineages, including the CBC cells themselves. Using a novel kinetic knock-in allele for time-resolved fate mapping, we establish that Fgfbp1+ cells are multi- potent and give rise to Lgr5+ cells during homeostasis, consistent with their function as stem rather than TA cells. Fgfbp1-CreERT2 lineage tracing confirms rapid yet long-lived epithelial reconstitution of both villus and CBC compartments from upper crypt stem cells that secrete FGFBP1, which we demonstrate is an essential factor for crypt proliferation and epithelial homeostasis. Moreover, Fgfbp1+ cells sustain epithelial homeostasis after toxin-mediated CBC cell ablation and are resilient to R-spondin signaling blockade that completely depletes CBC cells. Together, these findings support a model in which the cellular source of regeneration is in the upper crypt, rather than the crypt base, from where Fgfbp1+ cells generate progeny that migrate bi-directionally along the crypt-villus axis and serve as a source of the Lgr5+ cells at the base.

[0445] The intestinal epithelium exhibits rapid physiological renewal with continuousturnover every 3-5 days supported by intestinal stem cells (ISCs) in the crypts1,2. However, the precise identity and localization of the ISCs have been intensely debated for decades. Historically, ISCs have been postulated to be either actively cycling crypt-based columnar (CBC) cells or quiescent label-retaining cells (LRC) residing at approximately the “+4” cell position from the crypt base2-4. Seminal studies using lineage tracing defined Lgr5, a Wnt target gene that encodes an R-spondin (Rspo) receptor, as a molecular marker of CBC-type ISCs interspersed between Paneth cells at the bottoms of the crypts that are able to persist long-term, self-renew, and give rise to all mature intestinal epithelial lineages5. Additionally, ex vivo clonogenic culture of isolated Lgr5+ cells yields growth of intestinal “organoids” capable of both multi-lineage differentiation and sustained proliferation, demonstrating stem-cell activity6.

[0446] A model in which Lgr5+ cells serve as the only homeostatic stem cell supportingintestinal epithelial regeneration has gained widespread acceptance1,2. In this model, the CBC cells that occupy the very bottoms of the crypt base (0 to +3 cell positions) and exhibit the highest levels of Lgr5 expression (Lgr5High) have the most self-renewal potential. Accordingly, the Lgr5Highstem cells give rise to downstream Lgr5Lowprogenitors (+4 to +5 cell positions) that represent short- lived transit-amplifying (TA) cells with more limited self-renewal capacity. However, selective toxin-mediated ablation of Lgr5+ ISCs demonstrated that crypt proliferation and epithelial homeostasis are maintained in the absence of Lgr5+ cells7. While the prevailing model is that of 85 4909-6074-3733v.193597 / 7344 a single ISC population responsible for renewal of the intestinal epithelium, these data suggest that perhaps multiple ISC populations co-exist to regenerate the intestinal epithelium under different biological contexts. Accordingly, multiple reports of molecular markers for lineage-tracing of quiescent +4 populations have been described that appear to mark facultative stem cell populations that become mobilized to repopulate the intestinal epithelium upon damage and / or loss of Lgr5+ ISCs7-13. However, these markers have been called into question due to their relatively broad, promiscuous mRNA expression pattern by in situ hybridization (ISH), the fact that their respective CreER alleles do not seem to selectively label previously reported +4 LRCs14-16, and their relative scarcity that fails to account for the large degree of regeneration that is needed to repopulate the epithelium upon injury or Lgr5+ ISC loss17.

[0447] Lineage-committed, post-mitotic populations have been suggested to harborregenerative potential via mechanisms of cellular plasticity. For example, the Notch ligand- expressing Dll1+ crypt cells represent early progenitors that generate short-lived clones of mixed secretory types under homeostasis but are suggested to exhibit plasticity and the ability to regain stemness upon radiation damage18. Similarly, quiescent +4 LRCs are secretory progenitors that co-express Lgr5 as well as Paneth and enteroendocrine (EE) markers under homeostasis, but can also undergo injury-inducible albeit short-lived lineage tracing19. Similar findings have been described for EE lineage cells14,20. Alkaline phosphatase (Alpi)-expressing enterocytes were also demonstrated to repopulate crypts after Lgr5+ ISC ablation21, suggesting some plasticity within the absorptive lineage to support epithelial reconstitution in the absence of Lgr5+ ISCs. Studies of the epigenetic landscape of Lgr5+ ISCs compared to differentiated cell types revealed that a permissive chromatin state is maintained throughout differentiation to support plasticity22,23. Recently, there are also reports of fetal reprogramming or “revival” stem cells that adopt a regenerative function upon tissue injury that further modify the model for regeneration24,25. Still, the prevailing paradigm is that Lgr5+ ISCs at the crypt base serve in homeostatic regeneration by generating differentiated progeny via a TA cell intermediate, and that upon Lgr5+ cell loss or tissue injury, other quiescent or post-mitotic cell types undergo activation, reprogramming, or de- differentiation to gain regenerative potential17,26.

[0448] Systemic Rspo modulation has demonstrated the essential role of the Rspo signalingaxis in Lgr5+ cell maintenance. Similar to DT-mediated ablation studies, Rspo loss-of-function led to complete depletion of Lgr5+ cells throughout the intestine and yet tissue homeostasis, crypt 86 4909-6074-3733v.193597 / 7344 architecture and proliferation were maintained, consistent with a non-Lgr5+ population regenerating the tissue. Systemic Rspo overexpression studies revealed striking phenotypes of intestinal crypt enlargement, enhanced crypt proliferation, Lgr5+ cell expansion, and tissue growth10. Notably, Lgr5 lineage tracing in this context revealed that all these overexpression phenotypes are not fully explained by Lgr5+ cell-mediated regeneration since all Lgr5+ cells and their progeny remain “trapped” at the crypt bases and do not contribute to tissue maintenance or growth nor fully account for the degree of crypt expansion observed27. These studies pointed to the existence of another regenerative crypt population of unclear identity aside from Lgr5+ CBC cells. Here, we sought to identify these regenerative cells.

[0449] One of the main challenges in studying intestinal epithelial regeneration arises from thefact that the intestinal crypt is populated by highly dynamic and heterogeneous cell types undergoing rapid cell transitions. While the cellular identity and functions of the Lgr5+ CBC cells are well-studied, the cell types residing in the upper crypt have eluded molecular definition due to a lack of specific and validated markers for their prospective study and isolation1,28. We therefore sought to use an unbiased approach to first define the identity and then study the function of the cell populations in the upper crypt zone as well as understand their lineage relationships to the Lgr5+ cells. This location is presumed to harbor TA cells, a population proposed to be immediately downstream of the Lgr5+ cell and operationally defined by its multi-potency but limited self- renewal potential, hence distinguishing it from bona-fide stem cells1,2. We aimed to dissect the cellular heterogeneity in the crypt compartment using single-cell RNA-seq (scRNA-seq) in the context of pharmacological modulation of the Rspo signaling axis that regulates Lgr5+ cell fate27,29. This allowed us not only to characterize the cell types present in the crypts under homeostasis, but also to understand their dynamic interplay in response to stem cell niche cues. Here, we found the marker Fibroblast growth factor binding protein 1 (Fgfbp1) is enriched in an immature, proliferative population of upper crypt cells distinct from Lgr5+ CBC cells. We validated the conserved spatial distribution of these cells in the upper crypt zone along the entire murine small intestine length, and functionally characterized them using a novel knock-in allele (termed “Fgfbp1-TimeR”) for time-resolved fate mapping. Our Fgfbp1-TimeR allele allows for constitutive labelling of Fgfbp1+ cells and their progeny in the absence of recombination or tamoxifen, providing characterization of the upper crypt lineage landscape. Our Fgfbp1-TimeR allele revealed the Fgfbp1+ upper crypt population is multi-potent, giving rise to all intestinal 87 4909-6074-3733v.193597 / 7344 lineages. Surprisingly, Fgfbp1+ cells were also found to give rise to all Lgr5+ cells within the crypt base during homeostasis, consistent with their role as stem rather than TA cells. These results were further confirmed by kinetic analysis using Fgfbp1-CreERT2 lineage tracing that highlights the rapid replacement of Lgr5+ cells by the upper crypt. Finally, Fgfbp1 conditional knockout (cKO) in the intestinal epithelium revealed that FGFBP1, secreted by upper crypt cells, is an essential factor for intestinal epithelial homeostasis, crypt regeneration, and Lgr5+ cell maintenance.

[0450] RESULTS

[0451] Identification of Fgfbp1 as a marker of proliferative upper crypt zone cells distinct fromLgr5+ CBC cells.

[0452] Lgr5+ CBC cell self-renewal is dependent on the Wnt / Rspo signaling axis, which canbe pharmacologically modulated at the ligand level to promote vs inhibit their self-renewal27,30. Pharmacological stimulation by systemic Rspo overexpression (via intravenous adenovirus injection with resultant hepatic transduction and secretion into the circulation) induces crypt hyperplasia, Lgr5+ cell expansion, and villus lengthening in mice10,27. In contrast, systemic overexpression of the soluble Lgr5 ectodomain (Lgr5-ECD) for Rspo signaling inhibition results in complete loss and suppression of all Lgr5+ ISCs throughout the intestine27. Notably, tissue architecture, crypt architecture, and crypt proliferation are maintained despite Lgr5+ cell depletion via the Lgr5-ECD27, analogous to what has been observed following diphtheria toxin (DT)- mediated ablation of the Lgr5+ population7. We sought to understand the early transcriptional changes with high granularity in the Lgr5-GFP cells upon Rspo modulation. Single-cell RNA-seq (scRNA-seq) analysis of FACS sorted Lgr5-GFP+ (both Lgr5-GFPHighand Lgr5-GFPLow) and Lgr5-GFP- cells from Lgr5-GFP-IRES-CreERT231mouse intestine revealed that Rspo vs Lgr5- ECD treatment acutely resulted in dynamic alterations in the relative proportions of cells in each cluster (Figure 1A-C), consistent with Rspo-mediated modulation of crypt cellular composition in vivo. Clusters were identified by canonical markers for each of the major described cell types14,27,32. Lgr5+ CBC signature genes32were used to identify two CBC cell clusters in different phases of the cell cycle, which were further annotated as “cycling CBCs” vs “non-cycling CBCs” by their cell cycle stage. A third dominant cluster contained proliferative cells that lacked the Lgr5+ CBC signature genes and was tentatively annotated as the putative “TA” cluster (Figure 1A-B). Acute Rspo treatment led to expansion of the Lgr5+ CBC clusters with concomitant 88 4909-6074-3733v.193597 / 7344 depletion of the “TA” cluster, consistent with Rspo function in enforcing Lgr5+ cell states. Conversely, both Lgr5-ECD or soluble Fzd8 cysteine-rich domain (CRD)27(a soluble inhibitor of Wnt ligands) led to expansion of the “TA” cluster with concomitant depletion of the CBC clusters (Figure 1A-C), consistent with blockade of Lgr5+ CBC states. We identified Fgfbp1 as a highly cluster-specific gene expressed in the “TA” population, along with other enriched genes such as Dmbt1 and Kcnn4 (Figure 1A-B & D). Dmbt1 is also expressed in cells identified by gene expression to be early enterocytes or their progenitors (Figure 1D). We next sought to map the anatomic location of the “TA” subset within the tissue. Accordingly, in situ hybridization (ISH) revealed that the Fgfbp1 mRNA is localized in proliferative cells of the upper crypt zone above the CBC compartment in the adult small intestine, minimally overlapping with Lgr5Lowcells (previously proposed to represent TA cells) at the +4 position (Figure 1E-H), concordant with the presumed location of TA cells and distinct from the Lgr5HighCBC cells at the crypt base. Similarly, Dmbt1 and Kcnn4, both co-enriched within the “TA” cluster, localized to the upper crypt zone above the CBC cells, consistent with the existence of a specific and distinct transcriptional program in cells occupying the upper crypt. Interestingly, Dmbt1 ISH demonstrated a similar mRNA distribution in the upper crypt zone but extended into the crypt-villus junction, consistent with expression in both TA cells and early enterocytes as predicted by scRNA-seq (Figure 1A & D).

[0453] The identification of the “TA” cluster and its gene expression signature was fromscRNA-seq data collected from multiple mice which underwent different in vivo pharmacological perturbations and analyzed aggregately27. Therefore, we queried normal unperturbed crypts for our “TA” signature by using a reference scRNA-seq atlas dataset. We projected our single-cell clusters onto a large scRNA-seq dataset comprising crypt-enriched normal epithelium collected along various regions spanning the length of the small intestine33. Indeed, our Fgfbp1-enriched “TA” cluster mapped directly onto the putative “TA / progenitor” populations annotated in the reference dataset, which were distinct from the Lgr5+ stem cell clusters (Figure 1I-J). Congruent with our ISH data, this finding additionally confirmed that the Fgfbp1+ cell population is not an artifactual or induced regenerative state secondary to our pharmacological manipulations, but rather that it is also present in the normal, unperturbed crypt during homeostasis. To understand the relationship between the “TA / progenitor” population and the Lgr5+ CBC cells, we performed RNA-Velocity analysis, which uses the ratio of nascent unspliced to mature spliced mRNA of individual genes to infer the directionality of cell state transitions between scRNA-seq clusters34,35, 89 4909-6074-3733v.193597 / 7344 on the reference dataset. Unexpectedly, the directionality inferred by RNA-Velocity trajectory analysis raised the possibility that the Fgfbp1-enriched “TA early / progenitor” cluster(s), corresponding to the “TA” cluster identified from our pharmacological perturbation analysis, is the origin of the mature cell lineages and also of the Lgr5+ CBC cells (Figure 1J). This raised the possibility that the Fgfbp1+ upper crypt cells may potentially serve as stem cells upstream of the Lgr5+ CBC cells, in contrast to the prevailing model in which Lgr5+ CBC cells give rise to downstream TA / progenitor cells prior to multi-lineage differentiation. Together, these data show Fgfbp1 is enriched in a discrete population in the upper crypt zone that is both spatially and transcriptionally distinct from the Lgr5+ CBC cells (Figure 1K) and raise the possibility of a potentially novel cellular hierarchy.

[0454] Fgfbp1-TimeR is a kinetic reporter allele for time-resolved fate mapping of upper cryptzone cells.

[0455] To experimentally validate these findings, we generated a novel knock-in kineticreporter allele (which we have termed “Fgfbp1-TimeR”) to study Fgfbp1 and the in vivo function of Fgfbp1-expressing cells in the mouse intestinal epithelium. Fgfbp1 is located on chromosome 5 and encodes a secreted glycoprotein that has been proposed to serve as a chaperone for fibroblast growth factors (FGFs) stored on heparan sulfate proteoglycans in the extracellular matrix and increase their availability to cognate FGF receptors (FGFRs) to positively modulate FGF signaling36,37. More recently, it has been shown to promote blood-brain barrier development via effects on brain endothelial cell Wnt / beta-catenin signaling, suggesting a possible role in augmenting canonical Wnt signaling38. We targeted the murine Fgfbp1 locus using a knock-in strategy to insert a dual fluorescent reporter cassette, comprised of DsRedE2-P2A-mTagBFP2, for time-resolved labeling of Fgfbp1+ cells and their progeny under control of the endogenous Fgfbp1 promoter (Figure 2A). The fluorophores were chosen for their reported spectral and non-cytotoxic properties in addition to their differential stability and protein folding kinetics, allowing for time- resolved mapping of Fgfbp1 expression39,40. In order for DsRedE2 to faithfully reflect transcriptional activation of the Fgfbp1 gene, we destabilized DsRedE2 to shorten its half-life using an N-terminal 1xUbVR motif41and a C-terminal PEST sequence42, resulting in an estimated half-life on the order of minutes to hours. The destabilized DsRed was separated by a P2A sequence from mTagBFP2, which has an estimated half-life of ~3.5 days40approximately corresponding to one turnover of the intestinal epithelium. The P2A self-cleaving peptide encoded 90 4909-6074-3733v.193597 / 7344 in the Fgfbp1-TimeR allele ensures stoichiometric expression of the two fluorophores, thus allowing for time-resolved fate mapping of the Fgfbp1+ cell lineage by using the ratio and relative intensities of DsRed:mTagBFP2 to track the time elapsed since Fgfbp1 transcriptional activation. Importantly, this labelling strategy does not require the use of CreER or tamoxifen, both of which have been reported to damage gut epithelial cells and is also problematic for studying proliferating cells43. Our Fgfbp1-TimeR reporter allele was engineered to preserve expression and secretion of the endogenous Fgfbp1 product (sFgfbp1), hence avoiding potential issues associated with hemizygosity / haploinsufficiency. Finally, in addition to inclusion of an N-terminal FLAG for epitope tagging of sFgfbp1, two LoxP sites were engineered to allow for conditional deletion of Fgfbp1 (Figure 2A). Since Fgfbp1 does not contain introns within its CDS, the second LoxP site was encrypted within a synthetic intron engineered in the Fgfbp1 gene body. Both the FLAG-tag and LoxP-containing synthetic intron constructs were extensively validated prior to incorporation into the Fgfbp1-TimeR allele.

[0456] The Fgfbp1-TimeR mice appeared normal and were fertile. The adult mouse smallintestine was grossly normal in architecture and all epithelial lineages were present. Histological analysis of the intestine demonstrated faithful DsRed reporter expression in the upper crypt above the Lgr5+ CBC compartment in a distribution that mirrored Fgfbp1 mRNA expression by ISH (Figure 2B-C). Due to the relative dimness and instability of the fluorophore against the high autofluorescence background of intestinal epithelial cells along the red spectrum, endogenous DsRed was not readily detected by flow cytometry of dissociated cells, but could be readily visualized by indirect immunofluorescence (IF) in fixed tissues (Figure 2B-C). The expression domain was additionally examined by anti-FLAG IF detection of FGFBP1 protein, which revealed an upper crypt epithelial expression pattern that mirrored that of the DsRed signal and the Fgfbp1 mRNA, thus congruent with the fidelity of the Fgfbp1-TimeR allele in reporting transcriptional activity, and confirmed that the Fgfbp1 expression domain is distinct from the Lgr5+ CBC compartment (defined as cell positions 0 to +3). On the other hand, endogenous mTagBFP2 was easily detected by flow cytometry. Notably, whereas DsRed was confined to the upper crypt zone by IF, mTagBFP2 was seen in epithelial cells of all crypts and villi throughout the intestine, identifying these as progeny derived from the DsRed+ cells in the upper crypt (Figure 2B-C). Importantly, no mTagBFP2 or DsRed signals were seen in WT control mouse intestine. Consistent with its broad expression along the crypt-villus axis, mTagBFP2 IF revealed extensive co- 91 4909-6074-3733v.193597 / 7344 localization with markers of both absorptive and secretory lineages, suggesting multi-potency of the Fgfbp1+ upper crypt population in the small intestine (Figure 2D). mTagBFP2+ traces were also observed in all the crypt bases, with overlap noted in scattered LYZ+ Paneth cells (Figure 2D) that likely represent newly-derived Paneth cells due to fewer LYZ+ cytoplasmic granules. Yet, the vast majority of Paneth cells were mTagBFP2 negative, which is consistent with time- dependent fluorescent decay attributable to their long lifespan of >3-8 weeks44. Thus, the absence of mTagBFP2 signal in the majority of Paneth cells further validates the fidelity of the Fgfbp1- TimeR allele as a kinetic reporter. Importantly, these findings were congruent with our RNA- Velocity analysis demonstrating a lineage trajectory from the Fgfbp1+ subset to the mature lineages.

[0457] The mTagBFP2+ signal present in all crypt bases, yet notably lacking in most Panethcells, prompted us to investigate whether the Fgfbp1+ cells in the upper crypt zone also gave rise to Lgr5+ CBC cells, as suggested by RNA-Velocity analysis. IF analysis of Fgfbp1-TimeR; Lgr5- DTR-GFP mouse tissues revealed direct overlap between mTagBFP2 and Lgr5-GFP, indicating that the Fgfbp1+ upper crypt zone cells do indeed give rise to the Lgr5+ CBC cells in the crypt base throughout the length of the small intestine (Figure 2E-F). This was further confirmed by FACS analysis, which revealed mTagBFP2 co-expression in the entire population of Lgr5-GFP+ cells, including both Lgr5-GFPHighand Lgr5-GFPLow(Figure 2G). Based on their mTagBFP2 co- expression, IF analysis and quantitation demonstrated that all (or nearly all) Lgr5-GFP+ cells derive from Fgfbp1+ cells, despite the fact that there is minimal overlap between DsRed and Lgr5- GFP at the crypt base (defined by cell positions 0 to +3, Figure 2E-H). Notably, in the rare instances where there is overlap between Fgfbp1 expression (denoted by DsRed signal) and Lgr5- GFP, the overlap occurs in the +4 cell position rather than the crypt base (Figure 2H). Together, these findings suggest that the Fgfbp1+ upper crypt zone cells (at cell positions +4 to +13) give rise to the Lgr5+ CBC cells (at cell positions 0 to +3) during homeostasis.

[0458] Fgfbp1-CreERT2 lineage tracing demonstrates Lgr5+ CBC cell regenerationoriginating from the upper crypt zone.

[0459] To further investigate the kinetics of regeneration from the Fgfbp1+ upper crypt zone,we generated a new Fgfbp1-CreERT2 knock-in allele (Figure 3A) and performed tamoxifen (TAM)-induced lineage tracing in Fgfbp1-CreERT2; Rosa26-tdTomato mice. Strikingly, acute TAM treatment resulted in tdTomato+ labeling of upper crypt zone cells above the Lgr5+ CBC 92 4909-6074-3733v.193597 / 7344 compartment by 18h (Figure 3B-E). With this TAM dose, tdTomato+ cells were observed in essentially all intestinal crypts. However, crypt-based labeling at 0 to +3 positions was notably absent throughout the intestine, with only sporadic isolated tdTomato+ cells found in 3.8% + / - 0.93% (S.E.) of all crypt bases. At D2 post-TAM, there continued to be a notable absence of labeled tdTomato+ cells at the crypt bases, and only rare, isolated tdTomato+ crypt-base cells could be observed in 10.4% + / - 4.3% (S.E.) of all crypts (Figure 3C-E). Between D2 and D4, tdTomato+ lineage traces rose up and reached the villus tips, consistent with rapid villus cell turnover originating from the upper crypt zone (Figure 3C-E). This was also appreciated by 3D whole mount confocal reconstruction. At D2 post-TAM labeling, there was clear spatial segregation between Lgr5-GFP and tdTomato signals observed in Fgfbp1-CreERT2; Rosa26- tdTomato; Lgr5-DTR-GFP mice, confirming that Fgfbp1 and Lgr5 label distinct zones. However, by D4, consistent infiltration of crypt bases by tdTomato+ cells was observed, followed by widespread regeneration along the crypt-villus axis by D7 (Figure 3C-F). The tdTomato+ cells found at the crypt bases at D7 were confirmed to be Lgr5+ CBC cells by IF (Figure 3G). Strikingly, the majority of Lgr5+ CBC cells within each crypt were tdTomato+ by D7 to D14, suggesting the Lgr5+ CBC compartment is rapidly regenerated by Fgfbp1+ cells from the upper crypt zone within two weeks (Figure 3E-G). Notably, regeneration of the CBC compartment is slower than regeneration of the villi, which occurs within 4 days. Consistent with multi-potent stem cell function, the tdTomato traces contained all mature cell lineages (Figure 3H). Additionally, clonal labeling using Fgfbp1-CreERT2; Rosa26-Confetti confirmed multi-potency of the upper crypt clones (Figure 3I). Together, these findings corroborate that the Fgfbp1+ upper crypt cells serve as an origin of homeostatic tissue self-renewal, reconstituting both villus and crypt base compartments (including the Lgr5+ CBC cells) via a bidirectional front of regeneration, consistent with the conclusions from the Fgfbp1-TimeR allele. Furthermore, the persistence of confluent lineage traces spanning the entire crypt-villus axis over 7 months suggests the ability of this population to sustain long-term self-renewal.

[0460] Regeneration originates from upper crypt Fgfbp1+ cells upon loss or suppression ofLgr5+ CBC cells.

[0461] Next, we wondered whether the Fgfbp1+ upper crypt zone cells play a role inregeneration upon loss or perturbation of the Lgr5+ CBC cells. We used DT to selectively ablate the Lgr5+ cells in Fgfbp1-TimeR; Lgr5-DTR-GFP mice (Figure 4A-C). Upon complete ablation 93 4909-6074-3733v.193597 / 7344 of the Lgr5-GFP+ cells, confirmed by direct microscopy and FACS analysis, we found that crypt architecture, crypt proliferation, and intestinal epithelial homeostasis were not impaired (Figure 4A-C). Moreover, the Fgfbp1+ cells remained unperturbed in the proliferative upper crypt zone (as demonstrated by both DsRed IF and Fgfbp1 ISH) and still gave rise to mTagBFP2 traces into the crypt bases, mirroring their observed function during homeostasis (Figure 4B-C). The resilience of upper crypt cells to targeted Lgr5+ CBC cell ablation was further confirmed by ISH of the co-enriched gene Dmbt1 in the Fgfbp1+ cell transcriptional signature. Similar to DT- mediated ablation of Lgr5+ CBC cells, pharmacological blockade of Rspo signaling using the Lgr5-ECD results in complete depletion of Lgr5+ cells and subsequent Paneth cell loss throughout the small intestine27while crypt architecture and proliferation remain intact (Figure 4D). Under these conditions, the Fgfbp1+ cells in the upper crypt zone persist and actually expand downward to occupy the crypt bases (Figure 4D-F), suggesting that Rspo signaling through LGR5 is not essential for their self-renewal. Rspo quantitatively expands Lgr5+ CBC cells10, which require Rspo signaling for their maintenance27. However, Rspo similarly enhances proliferation and cell number in the upper crypt zone, yet lineage tracing of Lgr5+ cells using Lgr5-GFP-IRES- CreERT2; Rosa26-tdTomato under conditions of Rspo overexpression shows that the upper crypt expansion does not arise from Lgr5+ cells, thus suggesting the existence of an Rspo-responsive population other than the Lgr5+ CBC cells27. Here, both Fgfbp1+ upper crypt cells and Lgr5+ CBC cells are expanded in response to systemic Rspo augmentation by Ad Rspo1 hepatic transduction, demonstrating responsiveness of both populations to Rspo overexpression (Figure 4D-H). Notably, the crypt expansion and tissue growth in this Rspo overexpression context is attributable to Fgfbp1+ upper crypt cells, which give rise to the expanded crypt and villus compartments (Figure 4G-H). This may potentially be mediated by the expression of Wnt and Rspo receptors / co-receptors in the Fgfbp1+ population. Additionally, the low-level expression of transcripts associated with previously reported markers of plasticity / reserve stem populations found in the Fgfbp1+ population may help reconcile previous findings with the central role of the upper crypt in fueling Lgr5+ CBC cell repopulation. Taken together, these findings support a model in which Fgfbp1+ cells in the upper crypt zone are spatially and functionally distinct from the Lgr5+ CBC cells, as they are resilient to loss of niche signals essential for the Lgr5+ cells and support tissue maintenance in the absence of Lgr5+ cells, and exhibit differential sensitivities and dependencies to the regulatory signals that govern Lgr5+ cell behavior. 94 4909-6074-3733v.193597 / 7344

[0462] FGFBP1 produced by upper crypt zone cells is essential for epithelial homeostasis andregeneration.

[0463] Given the high expression of FGFBP1 specifically confined to the upper crypt zone,we wondered whether Fgfbp1 is functionally important for regeneration. Since the Fgfbp1-TimeR allele harbors two LoxP sites that enable its use as a floxed allele, we used Villin-CreERT2; Fgfbp1TimeR / TimeR mice (herein referred to as Vil-CreERT2; Fgfbp1f / f) to conditionally delete Fgfbp1 in the intestinal epithelium. As the Fgfbp1+ upper crypt zone cells are highly proliferative, we initially used a high-dose (9mg / 40g body weight) TAM serial injection strategy in order to circumvent potential reconstitution of the tissue by escapers. At this TAM dose, all Vil-CreERT2; Fgfbp1f / f mice died within 2-3 days with no effect found in Fgfbp1f / f controls. Thus, we reduced the TAM dose (to 6mg / 40g body weight) to allow the mice to maintain a larger fraction of non- recombined crypts, and most of the Vil-CreERT2; Fgfbp1f / f mice survived for tissue harvest and histological analysis by D4 (Figure 5A). Macroscopically, the intestine of Fgfbp1 cKO mice appeared shorter in length compared to controls (Figure 5B). Anti-FLAG IF confirmed successful conditional knock-out (cKO) of Fgfbp1 in the Vil-CreERT2; Fgfbp1f / f intestinal epithelium, whereas the FLAG-tagged protein product is localized to the upper crypt epithelial cells and absent in the Lgr5+ CBC compartment of the controls (Figure 5C). Notably, tissue architecture was extensively disrupted in the Fgfbp1 cKO intestine, which revealed atrophic, shrunken villi filled with apoptotic cells (Figure 5D-E). The Fgfbp1 cKO intestine also exhibited marked crypt disruption, with patchy areas of crypt dropout directly adjacent to residual remnant crypts harboring nests of long-lived Paneth cells without histological evidence for any intercalating Lgr5+ CBC cells (Figure 5D-E). Importantly, the Fgfbp1 cKO crypts (or unfractionated epithelium) failed to generate and maintain organoids in ex vivo culture (Figure 5F), consistent with the impaired regeneration phenotype observed in vivo. Cross-sectional analysis of the intestine revealed widespread crypt loss and immune infiltration (Figure 5G) which were accompanied by diminished proliferation and complete loss of Lgr5+ CBC cells (Figure 5H). These results, which were further recapitulated using a Rosa26-CreERT2 driver for global Fgfbp1 cKO in adult mice, demonstrate that upper crypt zone epithelial-derived FGFBP1 is essential for crypt maintenance and homeostatic regeneration of the intestinal epithelium.

[0464] DISCUSSION95 4909-6074-3733v.193597 / 7344

[0465] The intestinal epithelium is a rapidly regenerating tissue that relies on stem / progenitorcells residing in the crypt compartment for its renewal. While the precise identity and location of the intestinal stem cell (ISC) have been controversial for decades, the current prevailing model is that regeneration originates from Lgr5-expressing cells at the crypt base. Previous studies using an Lgr5-GFP-CreERT2 allele suggested that Lgr5+ cells are actively cycling stem cells that can lineage trace during homeostasis to reconstitute the epithelium31. It is currently accepted that the Lgr5+ CBC cells at the crypt bases are stem cells that generate progeny that migrate up the crypt- villus axis, transitioning via short-lived transit-amplifying (TA) cell intermediates prior to terminal differentiation1,2. Thus, the flux of cells originates from the crypt bottom and flows upward in this model. Indeed, intravital microscopy studies focused on the crypt base CBC compartment initially concluded that those Lgr5+ CBCs at the very base harbor the most stem potential, with the ones at positions bordering the upper crypt zone (cell positions +3 / +4) becoming passively displaced onto the TA compartment and being ultimately lost to multi-lineage differentiation45. Interestingly, these authors revisited this question and recently noted that downward “retrograde” cellular movement is observed from the compartment above the crypt base, thus revising this uni- directional flux model. However, they did not define the identity of those cells migrating downward nor their origin46. Importantly, the molecular identity and nature of cells spatially localized to the upper crypt or “TA” zone, have remained elusive due to the lack of specific markers for their prospective identification or isolation28.

[0466] Despite the widespread acceptance of Lgr5 as the best marker of homeostatic stemcells, paradoxically it has been shown that Lgr5+ cells are dispensable for the maintenance of crypt architecture, proliferation, or regeneration of the intestinal epithelium. Indeed, the intestinal epithelium remains intact even when Lgr5+ cells are selectively depleted, such as via toxin- mediated ablation7or pharmacological blockade of Rspo signaling27. Various mechanisms have been evoked to reconcile these findings and explain how the intestine regenerates in the absence of homeostatic stem cells17,26. These alternative models include cellular plasticity / reprogramming so that differentiated cells can regain stem cell activity14,18,19,21,24,47or rare “+4 cells” that are normally quiescent but become activated upon Lgr5+ CBC cell loss7-12,25. However, controversy exists in the field due to numerous putative “+4” markers that have been shown to be promiscuously expressed throughout the crypt, even in the Lgr5+ CBC cells themselves32, or CreER alleles that are mosaic or do not faithfully mark cells according to their mRNA expression14-96 4909-6074-3733v.193597 / 734416. As a consequence, there currently is a lack of a consensus view on how the intestinal epithelium regenerates following perturbation or injury when Lgr5+ CBC cells are known to be absent.

[0467] Here, we used scRNA-seq to identify Fgfbp1 as a marker for a proliferative populationof immature cells that we tentatively assigned as “TA” cells due to their lack of the canonical Lgr5+ transcriptional signature. We confirmed that this Fgfbp1+ “TA” population is localized to the upper crypt, directly above the Lgr5+ stem cell zone, throughout the length of the small intestine, and thus that it is spatially and transcriptionally distinct from the Lgr5+ CBC cells. To our knowledge, this is the first specific and validated marker ever found for the “TA” zone. In light of these findings, we generated a novel knock-in allele that allows us to map in both real time and space the fate of Fgfbp1+ cells using a dual-fluorescence kinetic reporter48,49. Importantly, this Fgfbp1-TimeR allele is a faithful reporter of Fgfbp1 expression that does not perturb homeostasis as it does not disrupt the expression of the endogenous gene and does not require CreER activation or tamoxifen43. Therefore, our Fgfbp1-TimeR allele overcomes many of the technical problems of previous in vivo models and provides a novel tool to map early cell fate decisions in real time. We demonstrate that Fgfbp1+ “TA” cells are multi-potent in the small intestine, giving rise to all mature lineages. Unexpectedly, we found that these cells also give rise to the Lgr5+ cells during homeostasis, consistent with the upper crypt zone serving as the cellular origin of the Lgr5+ cells at the crypt base. Strikingly, these cells exhibit properties of stem cells rather than of short-lived “TA” cells. These conclusions were then confirmed using an orthogonal lineage tracing approach with a novel Fgfbp1-CreERT2 allele, highlighting the rapid and continuous homeostatic reconstitution of the villi and Lgr5+ CBC compartments from the upper crypt zone cells. Together, our data support a novel paradigm whereby regeneration originates from stem cells in the upper crypt and propagates bi-directionally throughout the epithelium (both down and up), producing Lgr5+ cells along the way (Figure 6A).

[0468] We also provide evidence that the targeted loss of Lgr5+ cells results in the generationof replacement non-Lgr5 crypt base cells by pre-existing upper crypt Fgfbp1+ cells (Figure 6B). Considering the numerous published markers for alternative populations regaining stem potential upon Lgr5+ CBC cell loss, our findings raise questions of how our upper crypt zone ISC model differs from the widely accepted cellular plasticity or reserve stem cell models. Although our data cannot rule out de-differentiation events, these are likely not the major driver for Lgr5+ CBC cell reconstitution under the conditions of Lgr5+ cell loss presented here. Instead, our model suggests 97 4909-6074-3733v.193597 / 7344 that Fgfbp1+ cells in the upper crypt zone represent the actual workhorse stem cells responsible for epithelial homeostatic regeneration and the ones that immediately repopulate the Lgr5+ CBC compartment following its loss (Figure 6B). These do not constitute a “reserve” population because they are mitotically active during homeostasis. Nor are they de-differentiating or undergoing plasticity, as they are normally found in the unperturbed crypt. Accordingly, single- cell analysis of regenerating crypts supports a model in which surviving, pre-existing cells with regenerative potential (rather than new or emerging cell types) are capable of reconstituting the epithelium following irradiation (ref companion manuscript by Malagola et al., BioRx https: / / doi.org / 10.1101 / 2022.04.26.489611). Thus, we believe our data reconcile the prevailing Lgr5 stem cell model with the perplexing finding that Lgr5+ cells are dispensable for tissue maintenance and regeneration.

[0469] Although our data support Fgfbp1+ cells as a cellular origin of Lgr5+ CBC cells,both Fgfbp1+ cells described here and the Lgr5+ CBC cells that have extensively characterized previously share characteristics compatible with ISC identity31. Interconversion between putative ISC pools is a model previously raised in the intestine7,12, therefore, it is possible that these are two distinct yet interacting stem or progenitor cell pools, perhaps with some intrinsic lineage bias19. However, our lineage tracing and Fgfbp1 cKO data highlight a previously unappreciated hierarchical relationship in which the upper crypt zone cells give rise to the CBC cells. While, “TA” zone cells have been previously suggested to play a role in reconstituting the CBC compartment, these prior models hinge upon mechanisms of TA de-differentiation that occur in the context of CBC cell loss and disruption of homeostatic mechanisms of tissue renewal. On the other hand, there is substantial literature to support the longevity of Lgr5-GFP-IRES-CreERT2 lineage tracing and their stem cell function31that seemingly contradicts our upper crypt ISC model. The currently accepted model of CBC cell “neutral-drift” dynamics was originally developed to explain the apparent paradox of how multiple equi-potent Lgr5+ cells can operate as part of a large stem cell pool and yet crypts become clonal over time50,51. These findings can now be reconciled by a model in which upper crypt stem cells fuel the displacement and regeneration of CBC cells at the base, providing an additional or alternative explanation for how the majority of Lgr5+ clones are lost over time. Intriguingly, recent intravital microscopy studies revealed extensive positional heterogeneity within the Lgr5+ compartment and found a bi-directional “shuffling” of cells at the border between the upper crypt and CBC zones45,46. Together, this could be explained by the 98 4909-6074-3733v.193597 / 7344 existence of a stem cell pool at or above the +4 cell “border” position, defined by its ability to long-term self-renew and contribute to all lineages in both villus and crypt base compartments. This +4 cell position, which harbors Lgr5Low / Fgfbp1+ cells, is indeed computationally predicted to have the highest stem potential (ref companion manuscript by Malagola et al., BioRxiv https: / / doi.org / 10.1101 / 2022.04.26.489611) and may indeed account for the lineage tracing observed in the Lgr5-GFP-IRES-CreERT2 model, in which the degree and longevity of tracing are highly tamoxifen-dose dependent. Taken together, we believe these data can help reconcile many historical observations about the location and nature of the ISC. Similarly, the identification of the upper crypt zone as a source of Lgr5+ CBC cells raises intriguing new questions about the role of the stem cell niche in regulating cell fate in these cellular transitions.

[0470] The intestinal epithelium is a highly zonated tissue, which is a consequence ofsignaling gradients from the niche that orchestrate the processes of self-renewal and differentiation52,53. The high degree of zonation within the crypt suggests that the Fgfbp1+ upper crypt zone cells occupy a distinct niche from that of Lgr5+ CBC cells. Therefore it is likely, and our data support the idea, that these distinct pools of cells are differentially regulated (Figure 6C). Given their stem properties and multi-potency, we anticipate that understanding the regulation of the upper crypt Fgfbp1+ cells will enable their use for tissue regeneration applications. Studies of the ISC niche have focused heavily on Wnt / Rspo signaling and have most recently centered around the underlying pericryptal mesenchyme52rather than contributions from the epithelium itself54,55. Since Fgfbp1+ ISCs exhibit some degree of Rspo independence, alternative signaling axes may be in place to support them. A cardinal feature of the upper crypt zone is production of an essential factor for epithelial regeneration. Importantly, our identification of the essential role of FGFBP1 in epithelial regeneration thus allows us to move from simply using gene markers and the anatomic localization for identification of the ISCs toward a biochemical understanding of regeneration.

[0471] An essential stem cell niche signaling axis is regulated by the gene Fgfbp1 as loss ofFGFBP1 in the intestinal epithelium markedly impairs homeostatic regeneration, leading to tissue architectural defects, crypt disruption, and loss of Lgr5+ CBC cells (Figure 6D). These results are in stark contrast to loss of Lgr5 via genetic knockout56or loss of Lgr5+ cells via DT-mediated ablation7or pharmacological perturbation27. Fgfbp1 has been implicated in regulating the FGF / FGFR signaling axis but its molecular functions remain unclear. FGF signaling has been shown to play an important role in gut development and adult tissue homeostasis, with numerous 99 4909-6074-3733v.193597 / 7344 of its components exhibiting high and localized expression along the crypt-villus axis57. One of these is Fgfr3, which specifically localizes to the intestinal crypts of both small intestine and colon in neonatal and adult mice58,59. Fgfr3 null mice have decreased number of crypts and reduction in Paneth cell number (also seen with Fgf10 overexpression), suggesting a role in regulation of ISCs and Paneth cells during development60but paradoxically mediating cell growth arrest in the adult crypt58. Indeed, Fgf10 and Fgfr2b are important for GI tract development61. Fgf10 signaling has been implicated in duodenal development since Fgf10 null mouse embryos exhibit duodenal atresia62-64. Cecal atresia has also been associated with Fgf10 signaling defects65. Fgfr2b null mice have defects in repair and regeneration following injury66. Fgf10 / Fgfr2b mesenchymal- epithelial interactions are important for the glandular stomach development with mutants exhibiting reduced growth of the stomach and reduced epithelial proliferation66. In contrast, Fgf10 overexpression expands the epithelium with enlargement of both the crypts and villi in tissue engineered intestine comprised of organoids67. Fgfbp1 contributes to embryonic development, angiogenesis, tumor growth and malignant progression68. Fgfbp1 is upregulated in colon, pancreas, breast, and skin cancers, and its expression accelerates skin wound healing in conditional overexpression mice68. Thus, it is possible that Fgfbp1 functions as a soluble autocrine / paracrine factor mediating cross-talk between the epithelial stem / progenitor compartment and the niche, reinforcing Fgfbp1+ cell identity and self-renewal and facilitating the Fgfbp1+ to Lgr5+ CBC cell transition within the crypts, a process that may involve Wnt signaling38,46. Understanding the molecular functions of this essential protein and the niche that supports the upper crypt stem cell self-renewal will be important toward any potential regenerative and therapeutic applications. 100 4909-6074-3733v.193597 / 7344

[0473] METHODS

[0474] Key resources tableREAGENT or RESOURCE SOURCE IDENTIFIER Antibodies 3 56101 4909-6074-3733v.193597 / 7344 Rabbit monoclonal anti-Cleaved Cell Signaling Cat#9664; RRID: AB_2070042 Caspase-3 (Asp175) (5A1E) Technology4909-6074-3733v.193597 / 7344 B-27 Supplement (50X) Gibco Cat#17504-044 EGF PeproTech Cat#AF-100-154909-6074-3733v.193597 / 7344 100X Non-Essential Amino Acids MP Biomedicals, Cat#1681049 LLC104 4909-6074-3733v.193597 / 7344 Anti-Flag ® M2 Magnetic Beads Millipore Sigma Cat#M8823-1ML RNAscope ® Multiplex Fluorescent Advanced Cell Cat#323100105 4909-6074-3733v.193597 / 7344 Mouse: Rosa26-tdTomato: B6.Cg- The Jackson JAX: 007914; RRID: Gt(ROSA)26Sortm14(CAG-tdTomato)Hze / J Laboratory IMSR_JAX:005703 co en ml106 4909-6074-3733v.193597 / 7344 R v4.0.3 R Core Team https: / / www.r-project.org / ab 2107 4909-6074-3733v.193597 / 7344

[0475] EXPERIMENTAL DETAILS

[0476] Mouse strains

[0477] All experimental procedures involving mice were performed in accordance withapproved protocols by the Institutional Animal Care and Use Committee (IACUC) at Columbia University Irving Medical Center. Mice were housed in 12-hour light / dark cycles with constant temperature and ad libitum access to food and water. Strains were maintained on a mixed C57BL / 6 background and both male and female mice aged 4-12 weeks were used. All experiments with mice were performed with n=3 to 5 biological replicates per group. Lgr5-DTR-GFP mice (generously provided by Dr. Frederic de Sauvage) were previously described5. For Lgr5+ CBC cell ablation, Lgr5-DTR-GFP mice were administered two doses of Diphtheria Toxin (Sigma D0564-1MG) at 50ug / kg dose via intraperitoneal injection and control mice were given PBS intraperitoneal injections, and intestine was harvested for histological analysis and flow cytometry analysis. For Wnt / Rspo signaling modulation, mice were administered adenoviruses via intravenous injections with either Ad Fc vs Ad Rspo vs Ad Lgr5-ECD as previously described6. For Fgfbp1-CreERT2 lineage tracing, mice were administered one intraperitoneal dose of tamoxifen (TAM) at a 6mg / 40g body weight dose and harvested at different time-points as stated within the manuscript. For Fgfbp1 conditional knockout (cKO), daily intraperitoneal doses of 6mg / 40g body weight TAM (for Vil-CreERT2 pan-epithelial cKO) or 9mg / 40g body weight TAM (for Rosa26-CreERT2 global cKO) were given over the course of 4 days.

[0478] The Fgfbp1-TimeR allele was generated using the Herbert Irving ComprehensiveCancer Center (HICCC) Transgenic Core Facility at Columbia University. Fgfbp1-TimeR mice were generated by homologous recombination in embryonic stem cells targeting a DsRed- mTagBFP2-IRES-FLAG-LoxP cassette to the mouse Fgfbp1 START codon, preserving the expression and secretory properties of the endogenous Fgfbp1 gene as well as all 3’UTR regulatory sequences. The Fgfbp1-TimeR allele was iteratively built through Gibson assembly and restriction enzyme-based cloning methods into a pCS2 vector backbone, followed by the introduction of flanking 5’ 2kb and 3’ 5 kb homology arms for homologous recombination into the mouse Fgfbp1 locus. Our LoxP-carrying synthetic intron was used as a harbor to an FRT-flanked neomycin resistance cassette for positive selection. The targeting construct was then transferred into a pMCS- DT.A backbone (a generous gift from Dr. Kosuke Yusa of Osaka University, Japan) and extensively verified by sequencing.100μg targeting construct were linearized and electroporated 108 4909-6074-3733v.193597 / 7344 into KV1 (129S6_C57BL / 6N Hybrid) mouse embryonic stem cells. Positive selection for insertional events was carried out with neomycin, while negative selection for random insertion took place using the diphtheria toxin gene encoded within the pMCS-DT.A vector.192 surviving clones were individually picked into two 96-well plates in duplicate. One of the plates was cryopreserved while the other underwent lysis for gDNA extraction. A preliminary check for homologous recombination in the murine Fgfbp1 locus was carried out by PCR spanning the 5’ homology arm. About 10% of the isolated clones were recombinants. The recombinant clones were thawed and expanded prior to high-quality DNA extraction. Homologous recombination was then confirmed for both homology arms by PCR, and the positive clones were injected into C57BL / 6 blastocysts and transferred into pseudopregnant females as per standard procedures. Born male chimeras were mated to homozygous Actin-Flpe females (Jax), allowing for both confirmation of germline transmission and excision of the FRT-flanked intervening neomycin resistance cassette. Complete removal of the neomycin resistance cassette was confirmed by PCR in the F2 generation.

[0479] The Fgfbp1-CreERT2 allele was also generated using the HICCC Transgenic CoreFacility using a similar approach as above. Fgfbp1-CreERT2 mice were generated by homologous recombination in embryonic stem cells targeting an mTagBFP2-CreERT2 cassette to the mouse Fgfbp1 STOP codon, and similarly derived as above.

[0480] METHODS DETAILS

[0481] Cell Transfection

[0482] For in vitro cell culture work, HEK293T (RRID: CVCL_0063) cells were transfectedwith Lipofectamine 3000 (Invitrogen L3000001) using 1µg of the corresponding expression constructs. Media was changed 24 hours post-transfection and the culture was allowed to grow for a further 24 hours prior to downstream analysis.

[0483] FLAG-Fgfbp1 secretion assay and affinity purification

[0484] A stable HEK293T cell line expressing FLAG-Fgfbp1 was made via lentiviral-mediated transduction and positively selected with puromycin. Following 24-hour culture in serum-free secretion media (2 / 3 ADMEM, 1 / 3 IMDM, 1X Non-essential amino acids & 1X PSQ), media was collected and incubated with 50 μl of Anti-Flag M2 Affinity Gel beads (Millipore Sigma M8823-1ML) for 24 hours at 4°C. Beads were washed as per manufacturer’s instructions and purified proteins immunoblotted using 1:1000 anti-FLAG (Sigma-Aldrich F1804) and 1:1000 109 4909-6074-3733v.193597 / 7344 anti-beta-actin (Cell Signaling Technology 4967) antibodies. 1:5000 IRDye® 800CW Donkey anti-Mouse IgG (LI-COR 926-32212) and 1:5000 IRDye® 680CW Donkey anti-Rabbit IgG (LI- COR 926-68073) were used as secondary antibodies. Membrane was visualized using the Odyssey CLx2 imager.

[0485] Generation of protein lysates and western blot for FLAG-Fgfbp1

[0486] Cells were lysed in RIPA buffer supplemented with protease inhibitors and blottedusing 1:1000 anti-FLAG (Sigma-Aldrich F1804) and 1:1000 anti-beta-actin (Cell Signaling Technology 4967) antibodies. 1:5000 IRDye® 800CW Donkey anti-Mouse IgG (LI-COR 926- 32212) and 1:5000 IRDye® 680CW Donkey anti-Rabbit IgG (LI-COR 926-68073) were used as secondary antibodies. Membrane was visualized using Odyssey Clx2.

[0487] Immunofluorescence and microscopy

[0488] Intestinal tissue was collected and fixed in 4% paraformaldehyde for OCT-frozen or10% neutral buffered formalin for paraffin sections. 7-10 μm OCT frozen sections or 5 μm paraffin-embedded sections were immunostained using the following primary antibodies: anti- RFP (Rockland, 600-401-379), anti-mTagBFP2 (NanoTag Biotechnologies N0502-AF647-L), anti-GFP (Aveslabs GFP-1020), anti-Ki67 (Cell Signaling Technology 9129, Invitrogen 14-5698- 82), anti-CHGA (Santa Cruz Biotechnology sc-1488, Abcam ab15160), anti-lysozyme 1 (Agilent Dako A0099, Santa Cruz Biotechnology sc-27958), anti-FLAG M2 (Sigma Aldrich #F1804 used with the ReadyProbes Mouse-on-Mouse IgG Blocking Solution from Invitrogen #R37621), and anti-FABP1 (Novus Biologicals, NBP1-87695). Heat-based antigen retrieval using either Tris- EDTA (pH 9.0) or citrate buffer (pH 6.0) was performed on all FFPE slides. All primary antibodies were used at 1:100 to 1:400 dilutions. Conjugated AlexaFluor 488, 568, and 647 (Invitrogen and Jackson ImmunoResearch) antibodies were used as secondary antibodies at a 1:500 dilution.

[0489] In situ hybridization (ISH)

[0490] ISH was performed using RNAscope Multiplex Fluorescent Reagent Kit v2 (AdvancedCell Diagnostics 323100) as per manufacturer’s instructions. The following probes and primary antibodies were used: Mm Lgr5 (Advanced Cell Diagnostics 312171), Mm Fgfbp1 (Advanced Cell Diagnostics 508831-C4), Mm Dmbt1 (Advanced Cell Diagnostics 418561-C2), 1:400 anti- GFP (Aveslabs GFP-1020), and 1:400 anti-Ki67 (Cell Signaling Technology 9129). AlexaFluor 488 and 568 secondary antibodies (Invitrogen and Jackson ImmunoResearch) were used at 1:500 dilutions. Kccn4 ISH was performed using proximity ligation in situ hybridization (PLISH)69. 110 4909-6074-3733v.193597 / 7344

[0491] Flow cytometry

[0492] FACS experiments were performed using standardized fresh epithelial preparations on5-10 cm of the mouse proximal jejunum. The intestine was isolated and the epithelium extracted and dissociated into single cells using an EDTA-based extraction method as previously described70. Singlet discrimination was performed using plots for forward scatter (FSC-A vs FSC- H), and dead cells were excluded using scatter characteristics and viability stains. All FACS experiments were performed on either Novocyte Quanteon or BD FACSAria II flow cytometers at the Columbia University CSCI Flow Cytometry Core. All data were analyzed using FlowJo v10.8.1.

[0493] Organoid Culture

[0494] Organoids were established using standardized fresh epithelial preparations on 5-10cmof the mouse proximal jejunum. The intestine was isolated and a suspension of intestinal epithelial crypts was obtained using an EDTA-based extraction method as previously described70. An equal number of crypts derived from both Fgfbp1 cKO and control animals was plated into Matrigel domes and cultured under Wnt-rich conditions as originally described6. Organoid growth was monitored over the course of 4 to 7 days by brightfield microscopy.

[0495] Tissue clearing for whole mount confocal 3D reconstruction

[0496] 4% PFA fixed whole-mount intestine tissue was micro-dissected under a dissectionmicroscope into ~4 mm x 1 mm strips of consecutive crypt-villus units. Tissue was rinsed in PBS and cleared with 8% SDS at 42°C for 24h, then transferred onto PBS to wash SDS off, and stained with DAPI. Refractive index matching took place overnight in a solution of HistoDenz1.46 (Sigma-Aldrich D2158). Tissue was then reverse mounted onto a coverslip in a solution of phytagel (Sigma-Aldrich 71010-52-1) / HD1.46 and loaded onto a glass slide for imaging.

[0497] Image acquisition

[0498] All fluorescence images were acquired on either a Zeiss Confocal Microscope LSM710 or Leica DMi8 Stellaris confocal microscope. H&E staining and live organoids images were acquired on a Leica Dmi8 widefield microscope.

[0499] Single-cell RNA-sequencing analysis and RNA-Velocity

[0500] 13,247 single cells, consisting of 11,218 FACS-sorted Lgr5-GFP+ and 1,877 Ad Fc-treated Lgr5-GFP− cells were analyzed from Gene Expression Omnibus (GEO) database accession GSE92865. Quality control to remove low-quality cells, doublets and empty droplets was 111 4909-6074-3733v.193597 / 7344 performed as described27. Additionally, Cd4+ and Cd8a+ immune cells were removed from the analysis. Altogether, 13,095 cells remained for analysis using the Seurat R package. The gene expression counts matrix were log-normalized and scaled using the NormalizeData and ScaleData functions from the Seurat package with default parameters. The first 30 principal components were used for UMAP projection using RunPCA and RunUMAP Seurat functions, and 10 clusters of cells were detected by the Louvain algorithm (resolution=0.07) based on the UMAP embeddings shared nearest neighbor graph. Differential expression analysis was performed using the Seurat wrapped function FindAllMarkers. Cell types were assigned using canonical markers as previously described27. Downstream analyses were conducted using the Seurat, Nebulosa and ggplot2 R packages.

[0501] 7,216 individual cells were analyzed from GSE92332. Cells were split by atlasbatch (n=10) and integrated using reciprocal PCA with the Seurat R package. Cells were identified and labelled using previously described cell type assignments from Haber et al., Nature, 201733. PCA and UMAP dimensionality reduction methods were used to analyze data as described above.

[0502] For RNA-Velocity analysis, spliced and unspliced expression matrices of Haber etal., Nature, 2017 dataset were estimated using the Velocyto command line interface (CLI). RNA velocity analysis was performed using the scVelo Python package34. Inference of directional trajectories was computed using the scVelo stochastic model on the top 3,000 variable genes.

[0503] For analysis of human intestinal dataset, 14,537 individual cells were analyzed fromGSE125970. Cells were split by sample (n=6) and integrated using reciprocal PCA with the Seurat R package. Cells were identified and labelled using previously described cell type assignments from Wang et al., JEM, 202071. PCA and UMAP dimensionality reduction methods were used to analyze data as described above.

[0504] Example II

[0505] The effects of NTDs of FGFBP on multiple organoid systems, tissues, cells and organsare shown in FIGS. 32-65, and the descriptions thereof hereinabove. The results show augmentation and enhancement of proliferation, growth, viability and repair in multiple tissue, cell and organ types.

[0506] Further Exemplary Sequences112 4909-6074-3733v.193597 / 7344

[0507] Human FGFBP2 is known in the art, For example, UniProt Q9BYJ0. E.g.,NP_114156.1 (encoded by NM_031950.4). Chicken, monkey, chimpanzee, dog, hamster, horse, bat, pigeon, reptile, amphibian, avian, and other FGFBP2 orthologs are also encompassed.

[0508] A full-length FGFBP2 protein sequence (Human):MKFVPCLLLVTLSCLGTLGQAPRQKQGSTGEEFHFQTGGRDSCTMRPSSLGQGAGEVW LRVDCRNTDQTYWCEYRGQPSMCQAFAADPKPYWNQALQELRRLHHACQGAPVLRPS VCREAGPQAHMQQVTSSLKGSPEPNQQPEAGTPSLRPKATVKLTEATQLGKDSMEELG KAKPTTRPTAKPTQPGPRPGGNEEAKKKAWEHCWKPFQALCAFLISFFRG (SEQ ID NO:1)

[0509] An exemplary expressed tagged construct comprising an active N-terminal domain ofFGFBP2 (human) is (Human N terminal FGFBP2 (G30-G146) Avitag-FLAG-His): MKWVTFISLLFLFSSAYSGEEFHFQTGGRDSCTMRPSSLGQGAGEVWLRVDCRNTDQT YWCEYRGQPSMCQAFAADPKPYWNQALQELRRLHHACQGAPVLRPSVCREAGPQAH MQQVTSSLKGSPEPNQQPEAGRGLNDIFEAQKIEWHEDYKDDDDKHHHHHH (SEQ ID NO:2)

[0510] An exemplary active N-terminal domain of FGFBP2 (human) (F33-E138) is:FHFQTGGRDSCTMRPSSLGQGAGEVWLRVDCRNTDQTYWCEYRGQPSMCQAFAADPK PYWNQALQELRRLHHACQGAPVLRPSVCREAGPQAHMQQVTSSLKGSPE (SEQ ID NO:3)

[0511] The FGFBP2 protein is not expressed in mice. There are homologs in primates, birds,reptiles, amphibians, and other species, but not rodents. Other (non-human) homologs of FGFBP2 are within the scope of the invention.

[0512] Human FGFBP3 is known in the art, for example, NP_689642.3 (encoded byNM_152429.5). For example, UniProt Q8TAT2. Genes encoding FGFBP3 are known in the art, for example, Gene ID: 143282, as updated on 8-Feb-2025. Mouse FGFBP3 is also known in the art, e.g., Uniprot Q8CDW7, and NP_082539.2 (encoded by NM_028263.1). Rat, chicken, monkey, chimpanzee, dog, hamster, horse, bat, pigeon, reptile, amphibian, avian, and other FGFBP3 orthologs are also encompassed

[0513] An exemplary full-length FGFBP3 protein sequence (Human), including the signalsequence is: 113 4909-6074-3733v.193597 / 7344 MTPPKLRASLSPSLLLLLSGCLLAAARREKGAASNVAEPVPGPTGGSSGRFLSPEQHACS WQLLLPAPEAAAGSELALRCQSPDGARHQCAYRGHPERCAAYAARRAHFWKQVLGGL RKKRRPCHDPAPLQARLCAGKKGHGAELRLVPRASPPARPTVAGFAGESKPRARNRGR TRERASGPAAGTPPPQSAPPKENPSERKTNEGKRKAALVPNEERPMGTGPDPDGLDGNA ELTETYCAEKWHSLCNFFVNFWNG (SEQ ID NO:4)

[0514] An exemplary active N-terminal domain (e.g. a heparin binding domain (HBD)) ofFGFBP3 (Human) (G42-A151) is predicted to be:GPTGGSSGRFLSPEQHACSWQLLLPAPEAAAGSELALRCQSPDGARHQCAYRGHPERCA AYAARRAHFWKQVLGGLRKKRRPCHDPAPLQARLCAGKKGHGAELRLVPRA (SEQ ID NO:5)

[0515] An exemplary full-length FGFBP3 protein sequence (mouse), including the signalsequence is: MSPPRPRASLSPLTLLLLLGGCLLSAAGRDKGAAGREVTRASRPTVGSSGRFVSPEQHAC SWQLLVPAPGTPTGGELALRCQTPGGASLHCAYRGHPERCAATGARRAHYWRRLLGA LRRRPRPCLDPAPLPPRLCARKTAGSDLHSPAHPSLPARPSEPPRSRARSPARSRQSVRSP SSQPEKKPLLVKSNSGGRKAGSDPVPEPPAAAGFQPNGLDQNAELTETYCTEKWHSLCN FFVNFWNG (SEQ ID NO:16)

[0516] An exemplary active N-terminal domain (e.g., HBD) of FGFBP3 (mouse) (S54-P156)is predicted to be: SGRFVSPEQHACSWQLLVPAPGTPTGGELALRCQTPGGASLHCAYRGHPERCAATGAR RAHYWRRLLGALRRRPRPCLDPAPLPPRLCARKTAGSDLHSPAHP (SEQ ID NO:6)

[0517] FGFBP1 (human) is well known – e.g., Uniprot entry Q14512. E.g., NP_005121.1(encoded by NM_005130.5). FGFBP1 (mouse) is well known – e.g., Uniprot entry Q62399. E.g., NP_001258545.1 (encoded by NM_001271616.1). Rat, chicken, monkey, chimpanzee, dog, hamster, horse, bat, pigeon, reptile, amphibian, avian, and other FGFBP1 orthologs are also encompassed.

[0518] An exemplary full-length FGFBP1 protein sequence (Human), including the signalsequence is: MKICSLTLLSFLLLAAQVLLVEGKKKVKNGLHSKVVSEQKDTLGNTQIKQKSRPGNKG KFVTKDQANCRWAATEQEEGISLKVECTQLDHEFSCVFAGNPTSCLKLKDERVYWKQV ARNLRSQKDICRYSKTAVKTRVCRKDFPESSLKLVSSTLFGNTKPRKEKTEMSPREHIKG 114 4909-6074-3733v.193597 / 7344 KETTPSSLAVTQTMATKAPECVEDPDMANQRKTALEFCGETWSSLCTFFLSIVQDTSC (SEQ ID NO:7)

[0519] An exemplary active N-terminal domain of FGFBP1 (Human) (S52-S153) is:SRPGNKGKFVTKDQANCRWAATEQEEGISLKVECTQLDHEFSCVFAGNPTSCLKLKDE RVYWKQVARNLRSQKDICRYSKTAVKTRVCRKDFPESSLKLVSS (SEQ ID NO:8)

[0520] An exemplary full-length FGFBP1 protein sequence (mouse), including the signalsequence is: MRLHSLILLSFLLLATQAFSEKVRKRAKNAPHSTAEEGVEGSAPSLGKAQNKQRSRTSK SLTHGKFVTKDQATCRWAVTEEEQGISLKVQCTQADQEFSCVFAGDPTDCLKHDKDQI YWKQVARTLRKQKNICRNAKSVLKTRVCRKRFPESNLKLVNPNARGNTKPRKEKAEVS AREHNKVQEAVSTEPNRVKEDITLNPAATQTMAIRDPECLEDPDVLNQRKTALEFCGES WSSICTFFLNMLQATSC (SEQ ID NO:17)

[0521] An exemplary active N-terminal domain of FGFBP1 (mouse) (S58-R162) is:SKSLTHGKFVTKDQATCRWAVTEEEQGISLKVQCTQADQEFSCVFAGDPTDCLKHDKD QIYWKQVARTLRKQKNICRNAKSVLKTRVCRKRFPESNLKLVNPNAR (SEQ ID NO:9)

[0522] An exemplary FGFBP1 expression construct sequence encoded (K34-N158), includinga histidine tag (Human N terminal FGFBP1 (K34-N158) Avitag-His): MKWVTFISLLFLFSSAYSKVVSEQKDTLGNTQIKQKSRPGNKGKFVTKDQANCRWAAT EQEEGISLKVECTQLDHEFSCVFAGNPTSCLKLKDERVYWKQVARNLRSQKDICRYSKT AVKTRVCRKDFPESSLKLVSSTLFGNSGRGLNDIFEAQKIEWHEHHHHHH (SEQ ID NO:10)

[0523] An exemplary FGFBP1 expression construct sequence encoded (T34-G163), includinga histidine tag (mouse N terminal FGFBP1 (T34-G163) Avitag-His): MKWVTFISLLFLFSSAYSTAEEGVEGSAPSLGKAQNKQRSRTSKSLTHGKFVTKDQATC RWAVTEEEQGISLKVQCTQADQEFSCVFAGDPTDCLKHDKDQIYWKQVARTLRKQKNI CRNAKSVLKTRVCRKRFPESNLKLVNPNARGRGLNDIFEAQKIEWHEDYKDDDDKHHH HHH (SEQ ID NO:18)

[0524] An exemplary NTD-FGFBP2 – fusion protein comprising an IgG Fc (Human Nterminal FGFBP2 (M1-G146) Human-IgGFc) (using endogenous signal peptide): (Italics = a human FGFBP2 fragment containing NTD; underlined = human IgG Fc): 115 4909-6074-3733v.193597 / 7344 MKFVPCLLLVTLSCLGTLGQAPRQKQGSTGEEFHFQTGGRDSCTMRPSSLGQGAGEVWL RVDCRNTDQTYWCEYRGQPSMCQAFAADPKPYWNQALQELRRLHHACQGAPVLRPSVCRE AGPQAHMQQVTSSLKGSPEPNQQPEAGGENLYFQSGASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK (Entire fusion protein is SEQ ID NO:11; IgG Fc portion is SEQ ID NO:12)

[0525] Exemplary sequences of active fragments containing N-terminal domains:

[0526] Mouse FGFBP1 T34-G163:TAEEGVEGSAPSLGKAQNKQRSRTSKSLTHGKFVTKDQATCRWAVTEEEQGISLKVQC TQADQEFSCVFAGDPTDCLKHDKDQIYWKQVARTLRKQKNICRNAKSVLKTRVCRKRF PESNLKLVNPNARG (SEQ ID NO:13)

[0527] Human FGFBP1 K34-N158KVVSEQKDTLGNTQIKQKSRPGNKGKFVTKDQANCRWAATEQEEGISLKVECTQLDHE FSCVFAGNPTSCLKLKDERVYWKQVARNLRSQKDICRYSKTAVKTRVCRKDFPESSLKL VSSTLFGN (SEQ ID NO:14)

[0528] Human FGFBP2 G30-G146GEEFHFQTGGRDSCTMRPSSLGQGAGEVWLRVDCRNTDQTYWCEYRGQPSMCQAFAA DPKPYWNQALQELRRLHHACQGAPVLRPSVCREAGPQAHMQQVTSSLKGSPEPNQQPE AG (SEQ ID NO:15).

[0529] Human FGFBP3 fragment (M1-A155) including endogenous signal peptide:MTPPKLRASLSPSLLLLLSGCLLAAARREKGAASNVAEPVPGPTGGSSGRFLSPEQHACS WQLLLPAPEAAAGSELALRCQSPDGARHQCAYRGHPERCAAYAARRAHFWKQVLGGL RKKRRPCHDPAPLQARLCAGKKGHGAELRLVPRASPPA (SEQ ID NO:19)

[0530] Mouse FGFBP3 fragment (M1-A155) including endogenous signal peptide:MSPPRPRASLSPLTLLLLLGGCLLSAAGRDKGAAGREVTRASRPTVGSSGRFVSPEQHAC SWQLLVPAPGTPTGGELALRCQTPGGASLHCAYRGHPERCAATGARRAHYWRRLLGA LRRRPRPCLDPAPLPPRLCARKTAGSDLHSPAHPSLPA (SEQ ID NO:20) 116 4909-6074-3733v.193597 / 7344

[0531] Exemplary Adenovirus constructs:

[0532] Ad mFGFBP1 NTD-Fc (signal peptide, mFGFBP1 NTD T34-G163, murine Fc,3xFLAG, 8xHis). Italics = a fragment containing an mFGFBP1 NTD; Underlined = a mouse Fc METDTLLLWVLLLWVPGSTGDTAEEGVEGSAPSLGKAQNKQRSRTSKSLTHGKFVTKDQA TCRWAVTEEEQGISLKVQCTQADQEFSCVFAGDPTDCLKHDKDQIYWKQVARTLRKQKNIC RNAKSVLKTRVCRKRFPESNLKLVNPNARGGSGLEPRGPTIKPCPPCKCPAPNLLGGPSVFIF PPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRV VSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKK QVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWV ERNSYSCSVVHEGLHNHHTTKSFSRTPGKGSDYKDHDGDYKDHDIDYKDDDDKGSHH HHHHHH- (SEQ ID NO:21)

[0533] Ad hFGFBP2 NTD-Fc (signal peptide, hFGFBP2 NTD G30-G146, murine Fc,3xFLAG, 8xHis). Italics = a fragment containing a hFGFBP2 NTD; Underlined = a mouse Fc METDTLLLWVLLLWVPGSTGDGEEFHFQTGGRDSCTMRPSSLGQGAGEVWLRVDCRNTD QTYWCEYRGQPSMCQAFAADPKPYWNQALQELRRLHHACQGAPVLRPSVCREAGPQAHMQ QVTSSLKGSPEPNQQPEAGGGSGLEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLM ISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQD WMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVT DFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSV VHEGLHNHHTTKSFSRTPGKGSDYKDHDGDYKDHDIDYKDDDDKGSHHHHHHHH- (SEQ ID NO:22)

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Claims

93597 / 7344 CLAIMS 1. An expression construct encoding (i) an N-terminal domain of a fibroblast growth factorbinding protein 1 (FGFBP1), but not encoding a full length FGFBP1 protein and / or not encoding a mature FGFBP1 protein.

2. The expression construct of Claim 1, wherein the N-terminal domain of FGFBP1 is human.

3. The expression construct of Claim 1, wherein the N-terminal domain of FGFBP1comprises SEQ ID NO:8 or SEQ ID NO:

9.

4. The expression construct of Claim 1, 2 or 3, which further encodes a signal sequence and / ora promoter.

5. An expression construct encoding a fusion protein comprising (A) an N-terminal domainof a fibroblast growth factor binding protein 1 (FGFBP1) linked to (B) a non-FGFBP protein or peptide / polypeptide.

6. The expression construct of Claim 5, wherein (A) does not comprise a full length FGFBP1and / or not comprise a mature FGFBP1 protein.

7. The expression construct of Claim 5 or 6, wherein (A) does not comprise an FGF bindingdomain of an FGFBP1.

8. The expression construct of Claim 5, 6 or 7, wherein (B) comprises (a) an immunoglobulinG Fc, optionally a human immunoglobulin G Fc, or (b) a serum albumin, optionally a human serum albumin.

9. The expression construct of any of Claims 1 – 8, comprising DNA or comprising mRNA.

10. A composition comprising the expression construct of any of Claims 1-9.122 4909-6074-3733v.193597 / 7344 11. A peptide comprising an N-terminal domain of a fibroblast growth factor binding protein1 (FGFBP1), but not comprising a full length FGFBP1 protein and / or not comprising a mature FGFBP1 protein.

12. The peptide of Claim 11, wherein the N-terminal domain of FGFBP1 comprises SEQ IDNO:8 or SEQ ID NO:

9.

13. The peptide of Claim 11 or 12, further comprising a signal sequence.

14. The peptide of Claim 11 or 12, which does not comprise a signal sequence.

15. A fusion protein comprising (A) an N-terminal domain of a fibroblast growth factorbinding protein 1 (FGFBP1), linked to (B) a non-FGFBP protein or peptide or polypeptide.

16. The fusion protein of Claim 15, wherein (A) does not comprise a full length FGFBP1and / or mature FGFBP1.

17. The fusion protein of Claim 15, wherein (A) comprises a full length FGFBP1 or comprisesa mature FGFBP1.

18. The fusion protein of Claim 15, 16 or 17, wherein (B) comprises (a) an immunoglobulin GFc, optionally a human immunoglobulin G Fc, or (b) a serum albumin, optionally a human serum albumin.

19. A method of propagating, promoting growth / proliferation of, and / or increasing viability ofa stem cell or progenitor cell, comprising contacting the stem cell or progenitor cell with an amount of a composition comprising (i) an N-terminal domain of a fibroblast growth factor binding protein 1 (FGFBP1), (ii) an N-terminal domain of a fibroblast growth factor binding protein 2 (FGFBP2), or (iii) an N-terminal domain of a fibroblast growth factor binding protein 3 (FGFBP3), effective to propagate, promote growth / proliferation of, and / or increase viability of a stem cell or progenitor cell. 123 4909-6074-3733v.193597 / 7344 20. A method of propagating, promoting growth / proliferation of, and / or increasing viability ofa stem cell or progenitor cell, comprising contacting the stem cell or progenitor cell with an amount of an expression construct encoding (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to propagate, promote growth / proliferation of, and / or increase viability of a stem cell or progenitor cell.

21. A method of propagating, promoting growth / proliferation of, and / or increasing viability ofa tissue or organ, comprising contacting the tissue with an amount of a composition comprising (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to propagate, promote growth / proliferation of, and / or increase viability of a tissue or organ.

22. A method of propagating, promoting growth / proliferation of, and / or increasing viability ofa tissue or organ, comprising contacting the tissue with an amount of an expression construct encoding (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to propagate, promote growth / proliferation of, and / or increase viability of a tissue or organ.

23. A method of treating a wound or a scar in a tissue or organ, comprising contacting thetissue or organ with an amount of a composition comprising, or an expression construct encoding, (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to treat a wound or a scar in a tissue or organ.

24. A method of treating a pathology associated with degeneration or aging of a tissue or organ,comprising contacting the tissue or organ with an amount of a composition comprising, or an expression construct encoding, (i) an N-terminal domain of an FGFBP1, (ii) an N- terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to treat a pathology associated with degeneration or aging of a tissue or organ. 124 4909-6074-3733v.193597 / 7344 25. The method of Claim 19 or 20, wherein the stem cell is an adult mammalian stem cell oran induced pluripotent stem cell, or an embryonic stem cell.

26. The method of any of Claims 21-24, wherein the tissue is a mammalian tissue.

27. The method of any of Claims 21-24, wherein the organ is a mammalian organ.

28. The method of any of Claims 26 or 27, wherein the tissue is a donor tissue or wherein theorgan is a donor organ.

29. The method of any of Claims 19-28, wherein the composition comprises or the expressionconstruct encodes an N-terminal domain of an FGFBP1.

30. The method of any of Claims 19-29, wherein the composition comprises or the expressionconstruct encodes an N-terminal domain of an FGFBP2.

31. The method of any of Claims 19-30, wherein the composition comprises or the expressionconstruct encodes an N-terminal domain of an FGFBP3.

32. The method of any of Claims 19-31, wherein the composition comprises or the expressionconstruct encodes an N-terminal domain of an FGFBP1 but not a full length FGFBP1 and / or not a mature FGFBP1.

33. The method of any of Claims 19-32, wherein the composition comprises or the expressionconstruct encodes an N-terminal domain of an FGFBP2 but not a full length FGFBP2 and / or not a mature FGFBP2.

34. The method of any of Claims 19-33, wherein the composition comprises or the expressionconstruct encodes an N-terminal domain of an FGFBP3 but not a full length FGFBP3 and / or not a mature FGFBP3.

35. The method of any of Claims 19-34, wherein the composition is a fusion protein or theexpression construct encodes a fusion protein. 125 4909-6074-3733v.193597 / 7344 36. The method of any of Claims 19-35, wherein the composition does not comprise afibroblast growth factor (FGF) -binding domain of an FGFBP1, FGFBP2 or FGFBP3, or the expression construct does not encode an FGF-binding domain of an FGFBP1, FGFBP2 or FGFBP3.

37. The method of any of Claims 19-36, wherein the composition comprises a heparin-bindingdomain of an FGFBP1, FGFBP2 or FGFBP3, or the expression construct encodes heparin- binding domain of an FGFBP1, FGFBP2 or FGFBP3.

38. A method of promoting growth and / or viability of an organoid, or of an organoid culture,or expanding organoids in a culture, comprising administering to the organoid or organoid culture an amount of a composition comprising, or an expression construct encoding, (i) an N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to promote growth and / or viability of an organoid, or of an organoid culture, or expanding organoids in a culture.

39. The method of any of Claims 25-28, wherein the mammalian is human or the donor ishuman.

40. The method of any of Claims 21-24, 26 or 28, wherein the tissue is a soft tissue,optionally a gastrointestinal tissue, cardiac tissue, hepatic tissue, skin tissue, renal tissue, neuronal tissue, reproductive tissue, dental tissue, cutaneous tissue, pancreatic tissue, spleen tissue, or hematopoietic tissue.

41. The method of any of Claims 21-24, 27 or 28, wherein the organ is a duodenum, ileum,stomach, colon, liver, kidney, skin, or ovary.

42. The method of Claim 23, 26 or 27, wherein the wound is a burn or an ulcer.

43. A method of stimulating proliferation of stem cells comprising contacting the stem cellswith an amount of a composition comprising, or an expression construct encoding, (i) an 126 4909-6074-3733v.193597 / 7344 N-terminal domain of an FGFBP1, (ii) an N-terminal domain of an FGFBP2, or (iii) an N-terminal domain of an FGFBP3, effective to stimulate proliferation of stem cells.

44. The method of Claim 43, wherein the stem cells are self-renewing.

45. The method of Claim 43 or 44, wherein the stem cells are human.

46. The method of any of Claims 43-45, wherein the stem cells are adult stem cells.

47. A cell engineered to express an expression construct of any of Claims 1-9.

48. A cell engineered to express a peptide of any of Claims 11-14.

49. A cell engineered to express a fusion protein of any of Claims 15-18.127 4909-6074-3733v.1

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