Development and characterization of Gal KO porcine bone marrow‐derived mesenchymal stem cells

Abstract

Background:We demonstrated that neonatal porcine bone marrow-derived mesenchymal stem cell (npBM-MSCs) could improve a critical ischemic limb disease in rat model more efficiently compared with human MSCs. However, since porcine MSC presents galactosyl-alpha 1,3-galactose antigen (Gal antigen), MSC could be eliminated by the xenogeneic rejection. Recently, we established Gal knockout (KO) pigs by a technique of the electroporation of the CRISPR/Cas9 system into vitro-fertilized zygotes.In this study, we hypothesized that MSC from the established Gal KO pigs could further improve the efficacy. Before examining the hypothesis, in this study, we have established and characterized bone marrow-derived MSC from the Gal KO adult pigs (apBM-MSCs). Methods: Mononuclear cells (MNCs) were isolated from bone marrow cells of bothGal KO adult pigs and wild-type (WT) adult pigs. MNCs were further manipulated to create Gal KO apBM-MSCs and WT apBM-MSCs. Both MSCs were assessed by their surface markers, the capability of differentiation into adipocytes, osteocytes and chondrocytes, grow speed and colony-forming assay. To assess the efficacy of Gal KO apBM-MSCs, angiogenesis-related genes and immunosuppression-related genes were assessed by cytokine stimulation.Results: Gal KO apBM-MSC showed no Gal antigen on their cell surfaces. Both Gal KO apBM-MSCs and WT apBM-MSCs, presented little or no negative surface markers of MSCs, while they presented positive surface markers of MSCs. Furthermore, Gal KO apBM-MSCs were able to differentiate into adipocytes, osteocytes, and chondrocytes as well as WT apBM-MSCs. There was no difference in doubling time between Gal KO apBM-MSCs and WT apBM-MSCs. Interestingly, the colony-forming efficiency of Gal KO apBM-MSCs was about half that of WT apBM-MSC. However, angiogenesis and

Development and characterization of islet‐derived mesenchymal stem cells from clinical grade neonatal porcine cryopreserved islets

“…50 Additionally, we have previously reported the establishment of MSCs from pigs lacking the α-Gal antigen. 51 In the future, we plan to establish MSCs from pigs that are controlled and genetically regulated in a medical environment. This will improve donor supply and ensure stable cell quality for medical applications.…”

“…Thus, pig medical applications are being investigated, including the knockout of multiple antigen genes 50 . Additionally, we have previously reported the establishment of MSCs from pigs lacking the α‐Gal antigen 51 . In the future, we plan to establish MSCs from pigs that are controlled and genetically regulated in a medical environment.…”

Xenotransplantation of neonatal porcine bone marrow–derived mesenchymal stem cells improves diabetic wound healing by promoting angiogenesis and lymphangiogenesis

“…It is promised that xenotransplanted cells/tissues/organs from these pigs can be engrafted for a long period. For example, we recently developed Gal‐knockout porcine bone marrow‐derived MSCs and showed no differences in characteristics comparing with wild‐type porcine MSCs 49 . Further, Estrada et al., created the N‐acetyllactosaminide alpha‐1,3‐galactosyltransferase (GGTA1: encodes α‐Gal)/Cytidine monophospho‐N‐acetylneuraminic acid hydroxylase (CMAH: encodes Neu5Gc)/β1,4 N‐acetylgalactosaminyl transferase (β4GalNT2: encodes Sda) knockout pig and elucidated that the reactivity to human and non‐human primate antibodies was diminished in the triple knockout pig 50 .…”

Avoiding aggregation of human bone marrow–derived mesenchymal stem cells stored in cell preservation solutions

“…Dextran, a component of LR-3T-5D, suppresses cell sedimentation and thereby helps maintain a stable concentration of cells in the infusion line (Fujita et al 2020 ). This solution was shown to improve the preservation of porcine embryos (Lin et al 2022a ; Lin et al 2022b ), and the addition of dimethyl sulfoxide or propylene glycol allows solutions to be used for cryopreservation (Fujita et al 2021 ) of, for example, porcine bone marrow–derived MSCs (Nishimura et al 2019 ; Kikuchi et al 2021 ).…”