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US-20260125649-A1 - MEDIUM COMPOSITION AND METHOD FOR CULTURING MESENCHYMAL STEM CELLS

US20260125649A1US 20260125649 A1US20260125649 A1US 20260125649A1US-20260125649-A1

Abstract

The present invention generally relates to a medium composition and method for culturing mesenchymal stem cells (MSCs), in which the medium comprises an epithelial cell adhesion molecule (EpCAM) peptide, particularly a truncated EpCAM polypeptide containing the extracellular domain (EpEX). It significantly enhances cell proliferation and multipotency of the MSCs.

Inventors

  • Han-Chung Wu
  • I-I KUAN
  • Chien-Hsu Chen

Assignees

  • ACADEMIA SINICA

Dates

Publication Date
20260507
Application Date
20260102

Claims (10)

  1. 1 . A method for culturing mesenchymal stem cells (MSCs), comprising culturing the MSCs under a condition in the presence of an isolated EpCAM polypeptide.
  2. 2 . The method of claim 1 , wherein the EpCAM polypeptide is an extracellular domain of EpCAM.
  3. 3 . The method of claim 1 , wherein the extracellular domain of EpCAM comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID No: 2.
  4. 4 . The method of claim 1 , wherein the EpCAM polypeptide is present in an amount effective in promoting expansion and/or multipotency of the MSCs.
  5. 5 . The method of claim 1 , wherein the MSCs are cultured in a medium composition where the EpCAM polypeptide is present in an amount of 1-50 μg/mL.
  6. 6 . The method of claim 1 , wherein the medium composition comprises (i) Dulbecco's modified Eagle's medium-low glucose (DMEM-LG) supplemented with 0.1-5 mM glutamine, 5% to 25% FBS, and 1-50 μg/mL EpCAM polypeptide, or (ii) Dulbecco's modified Eagle's medium-high glucose (DMEM-HG) supplemented with 5% to 25% FBS, 0.05-1 μM dexamethasone, 1-50 mM β-glycerophosphate, 0.01-0.1 mM ascorbic acid-phosphate and 1-50 μg/mL EpCAM polypeptide.
  7. 7 . The method of claim 1 , wherein the EpCAM polypeptide is present in an amount effective in promoting expansion and multipotency of the MSCs.
  8. 8 . A method for enhancing osteogenesis of mesenchymal stem cells (MSCs), comprising culturing the MSCs in an osteogenic induction medium which comprises one or more components for osteogenic induction selected from the group consisting of β-glycerophosphate, ascorbic acid, dexamethasone and any combination thereof, wherein the medium further comprises an isolated EpCAM polypeptide.
  9. 9 . The method of claim 8 , wherein the EpCAM polypeptide is present in an amount effective in promoting expansion and/or multipotency of the MSCs.
  10. 10 . The method of claim 8 , wherein the EpCAM polypeptide is present in an amount effective in promoting expansion and multipotency of the MSCs.

Description

RELATED APPLICATIONS This application is a Divisional of application Ser. No. 17/273,256, filed Mar. 3, 2021, which is the National Phase of PCT/US2019/049344, filed Sep. 3, 2019, and which claims the benefit of U.S. provisional application No. 62/726,586, filed Sep. 4, 2018, under 35 U.S.C. § 119. The entire contents of prior applications are hereby incorporated by reference in their entireties. REFERENCE TO ELECTRONIC SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on Jan. 2, 2026, is named “2026_01_02_Sequence_Listing_5992-0237PUS3.xml” and is 12,563 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. TECHNOLOGY FIELD The present invention generally relates to a medium composition and method for culturing mesenchymal stem cells (MSCs), in which the medium comprises an epithelial cell adhesion molecule (EpCAM) peptide, particularly a truncated EpCAM polypeptide containing the extracellular domain (EpEX). It significantly enhances cell proliferation and multipotency of the MSCs. Specifically, the present invention provides a method for enhancing osteogenesis of MSCs by culturing the MSCs under osteogenic conditions in the presence of the EpCAM polypeptide. BACKGROUND OF THE INVENTION MSCs are found in compact bone, tendon, adipose, placenta, and umbilical cord (Brighton and Hunt, 1997), where these cells have the potential to differentiate into multiple lineages, including bone, cartilage, and muscle (Brighton and Hunt, 1997; Valero et al., 2012). In damaged tissues or organs, MSCs secrete chemokines and growth factors to create a microenvironment that promotes repair and recovery, which is especially important in bone regeneration (Briggs and King, 1952). MSC cytokine secretion also modulates the immune system, and because of these varied actions, MSCs are considered to be promising therapeutic candidates with wide-ranging clinical applications. Aging of hMSCs is known to attenuate proliferation, while increasing oxidative damage and senescence (Stolzing et al., 2008; Zhou et al., 2008). Therefore, the use of aged MSCs for autologous cell-based therapies is especially challenging (Stenderup et al., 2003; Stolzing et al., 2008). In addition to reduced proliferation of the MSCs themselves, aging is associated with decreased proliferative capacity in MSC-derived osteo-progenitor cells, which leads to decreased osteoblast cell number and eventually hinders bone formation (Stenderup et al., 2003; Zhou et al., 2008). Because of the diminished proliferative capacity and poor survival, MSCs derived from adult patients currently have limited potential for clinical use. In order to address these obstacles, methods to improve stemness and differentiation of MSCs are now under intensive development. EpCAM is a type I transmembrane protein with 314 amino acids and a molecular weight of about 39-42 kDa (Litvinov et al., 1994). It contains an extracellular domain (EpEX, 265 amino acids), a single transmembrane domain, and a short intracellular domain (EpICD, 26 amino acids). EpCAM is a well-known tumor-associated antigen, which is enriched in various carcinomas and involved in homotypic cell-cell adhesion in normal epithelium. (Litvinov et al., 1994). Previous research demonstrated that active proliferation is associated with enhanced EpCAM expression in neoplastic tissues. Furthermore, EpCAM is known to be relatively stable within the membrane of normal epithelial tissue, but is prone to cleavage in cancer tissue (Maetzel et al., 2009). Maetzel et al. first shed light on the mechanisms of EpCAM activation, showing that it occurs via regulated intramembrane proteolysis (RIP). During this process, EpCAM is cleaved, generating two products (EpEX and EpICD), which then induce EpCAM-mediated proliferative signaling (Maetzel et al., 2009). After RIP of EpCAM, EpICD associates with FHL2, β-catenin and Lef-1 to form a nuclear complex that binds to DNA at Lef-1 consensus sites and regulates gene transcription, potentially contributing to carcinogenesis. In a recent study we reported that EpCAM is enriched in human embryonic stem cells (hESCs), where it not only serves as an important surface marker, but it also regulates the four Yamanaka factors (Lu et al., 2010). Similarly, EpCAM plays a critical role in regulating self-renewal, cancer initiating ability, and invasiveness in colon cancer cells (Lin et al., 2012). It is also interesting to note that overexpression of EpCAM or EpICD decreased the levels of p53 and p21, and increased the promoter activity of Oct4 during iPSC derivation (Huang et al., 2011). Based on these findings, we recently further discovered that EpCAM/EpEX, together with Oct4 or Klf4 expression, can generate induced pluripotent stem cells (iPSCs) (Kuan et al.