Development of PET Imaging to Visualize Activated Macrophages Accumulated in the Transplanted iPSc-Derived Cardiac Myocytes of Allogeneic Origin for Detecting the Immune Rejection of Allogeneic Cell Transplants in Mice

Abstract

Allogeneic transplantation (Tx) of induced pluripotent stem cells (iPSCs) is a promising tissue regeneration therapy. However, this inevitably induces macrophage-mediated immune response against the graft, limiting its therapeutic efficacy. Monitoring the magnitude of the immune response using imaging tools would be useful for prolonging graft survival and increasing the therapy longevity. Minimally invasive quantitative detection of activated macrophages by medical imaging technologies such as positron emission tomography (PET) imaging targets translocator protein (TSPO), which is highly expressed on mitochondrial membrane, especially in activated macrophage. N,N-diethyl-2-[4-(2-fluoroethoxy) phenyl]-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-acetamide (DPA-714) is known as a TSPO ligand used in clinical settings. We herein hypothesized that immune rejection of the transplanted iPSC-derived cardiomyocytes (iPSC-CMs) of allogeneic origin may be quantitated using 18F-DPA-714-PET imaging study. iPSC-CM cell-sheets of C57BL/6 mice origin were transplanted on the surface of the left ventricle (LV) of C57BL/6 mice as a syngeneic cell-transplant model (syngeneic Tx group), or Balb/c mice as an allogeneic model (allogeneic Tx group). 18F-DPA-714-PET was used to determine the uptake ratio, calculated as the maximum standardized uptake value in the anterior and septal wall of the LV. The uptake ratio was significantly higher in the allogeneic Tx group than in the syngeneic group or the sham group at days 7 and day 10 after the cell transplantation. In addition, the immunochemistry showed significant presence of CD68 and CD3-positive cells at day 7 and 10 in the transplanted graft of the allogeneic Tx group. The expression of TSPO, CD68, IL-1 beta, and MCP-1 was significantly higher in the allogeneic Tx group than in the syngeneic Tx and the sham groups at day 7. The 18F-DPA-714-PET imaging study enabled quantitative visualization of the macrophages-mediated immune rejection of the allogeneic iPSC-cardiac. This imaging tool may enable the understanding and monitoring host-immune response of the host, allogeneic cell transplantation therapy.

Syngeneic Mesenchymal Stem Cells Reduce Immune Rejection After Induced Pluripotent Stem Cell-Derived Allogeneic Cardiomyocyte Transplantation

“…Luciferase-miPSCs (959A2-1-6) generated from C57BL/6 (B6) (CLEA) mouse embryonic fibroblasts were cultured in the absence of serum and feeder cells using ESGRO Complete PLUS Clonal Grade Medium (Millipore, Burlington, MA, USA). Cardiomyogenic differentiation of the iPSCs was performed as described, with modifications, followed by purification with glucose-free medium supplemented with lactic acid 22,23 ; iPSCs (3 × 10 3 ) were resuspended in 100-µl aliquots of differentiation medium [DM; Dulbecco's Modified Eagle's Medium (DMEM; Nacalai Tesque, Kyoto, Japan) containing 15% foetal bovine serum (FBS; Biofill, Melbourne, Victoria, Australia), 100 mmol/l non-essential amino acids (NEAA; Invitrogen, Carlsbad, CA, USA), 2 mmol/l L-glutamine (Invitrogen), and 0.1 mmol/l 2-mercaptoethanol (Invitrogen)] containing 0.2 mmol/l 6-bromoindirubin-3′-oxime (BIO; a glycogen synthase kinase-3β inhibitor to activate the Wnt-signalling pathway; Calbiochem, San Diego, CA, USA), and cultured in 96-well Corning Costar Ultra-Low attachment multiwell plates (MilliporeSigma, Burlington, MA) for 3 days. On day 3, an additional 100 µl of DM without BIO was added to each well.…”

“…OCT4, NANOG, and LIN28, and using a previously reported protocol, they were differentiated into cardiomyocytes that expressed cardiomyocyte marker genes such as ANP-1, MYH7B, 1STL1, NKX2.5, MYH6, and TNNT2, with decreased OCT4, NANOG, and LIN28 expression ( Fig. 1a,b) [22][23][24] . Nearly all embryoid bodies exhibited self-beating at day 16 and the cells were positive for troponin T and alpha-actinin, indicating that they were iPSC-CMs (Fig.…”

Inflammatory projections after focal brain injury trigger neuronal network disruption: An 18F-DPA714 PET study in mice

“…18 F-DPA-714 preparation was described in our previous publication ( Kashiyama et al, 2016 ). Accordingly, 18 F-fluoride in 18 O-H 2 O was transferred to a UG-M1 synthesiser (Universal Giken, Kanagawa, Japan) and passed through an Accell Light QMA cartridge.…”

Optimizing the Use of iPSC-CMs for Cardiac Regeneration in Animal Models

“…In addition, different strategies are adopted for cardiac regeneration using stem cell-derived CMs. Cells can then be injected as single cells, patches, or sheets [11,43,47,155,160,[197][198][199]. Almost all improved some metrics of cardiac function, suggesting paracrine factors excreted from grafts exert some regenerative capacity.…”

“…Regardless of their maturity, researchers are currently studying the potential of iPSC-CMs for cardiac regeneration. Animal models such as rodents, canines, pigs, and primates are used [11,16,[41][42][43][44][45][46][47][48][49][50][51]. So far, some studies have shown encouraging results after iPSC-CM engraftment like long-term cell survival, increased cell maturation and contractile improvement while others have found the opposite.…”