Retention of Human iPSC-Derived or Primary Cells Following Xenotransplantation into Rat Immune-Privileged Sites

Abstract

In regenerative medicine, experimental animal models are commonly used to study potential effects of human cells as therapeutic candidates. Although some studies describe certain cells, such as mesenchymal stromal cells (MSC) or human primary cells, as hypoimmunogenic and therefore unable to trigger strong inflammatory host responses, other studies report antibody formation and immune rejection following xenotransplantation. Accordingly, the goal of our study was to test the cellular retention and survival of human-induced pluripotent stem cell (iPSCs)-derived MSCs (iMSCs) and primary nucleus pulposus cells (NPCs) following their xenotransplantation into immune-privileged knee joints (14 days) and intervertebral discs (IVD; 7 days) of immunocompromised Nude and immunocompetent Sprague Dawley (SD) rats. At the end of both experiments, we could demonstrate that both rat types revealed comparably low levels of systemic IL-6 and IgM inflammation markers, as assessed via ELISA. Furthermore, the number of recovered cells was with no significant difference between both rat types. Conclusively, our results show that xenogeneic injection of human iMSC and NPC into immunoprivileged knee and IVD sites did not lead to an elevated inflammatory response in immunocompetent rats when compared to immunocompromised rats. Hence, immunocompetent rats represent suitable animals for xenotransplantation studies targeting immunoprivileged sites.

Keywords: PTOA; cell therapy; immune privilege; inflammation; intervertebral discs; joint; mesenchymal stem cells; nucleus pulposus cells; xenotransplantation.

Figure 1

Experimental outline. (A): Schematic overview of the PTOA model. Purchased iPSCs are differentiated into their mesenchymal lineage (iMSCs) and labeled with lipophilic DiI dye. Subsequently, 1 × 10⁶ iMSC^DiI+ were injected into both knees of Nude (n = 7) and SD (n = 7) rats that underwent a non-invasive rupture of their right ACL 2 weeks prior. After 2 weeks, animals were anesthetized, and knee synovium tissues were processed for flow cytometric analyses and ELISA as well as histology and immunofluorescent imaging. (B): Schematic overview of the IVD model. NPCs of human donors were cultured, transduced with GFP and labeled with lipophilic DiO dye. Subsequently, 5 × 10⁵ NPC^GFP+/DiO+ were injected into each L3/4 and L4/5 lumbar discs of Nude (n = 4) and SD (n = 5) rats. After 1 week, animals were anesthetized, and lumbar disc synovium tissues processed for flow cytometric analyses and ELISA as well as histology and immunofluorescent imaging.

Figure 2

Materials and methods. (A,B): Representative images of a SD rat during non-invasive ACL rupture. (C–F): Biomechanical parameters (ultimate load (C), maximum displacement (D), stiffness (E), and toughness (F)) during non-invasive ACL rupture of Nude and SD rats for the PTOA model. Means  ±  SD. (G,H): Representative images of iMSCs (G) and NPCs (H) in culture prior to the labeling with lipophilic dyes. Scale bars = 100 µm. (I,J): Representative images of iMSC^DiI+ (I) and NPC^GFP+/DiO+ (J) in culture shortly before their xenotransplantation. Scale bars = 100 µm. (K–M): Representative images of X-ray C-Arm-guided injection of cells into the knee joints (PTOA model; (K)) and lumbar discs (IVD model; (L,M)) of Nude and SD rats. Scale bars: K = 13 mm, L + M = 8 mm.

Figure 3

Flow cytometry. (A–C): Total number of recovered cells (A) as well as the percentage of CD8⁺ (B) and DiI⁺ (C) cells in extracted tissue of the PTOA model. (D–F): Total number of recovered cells (D) as well as the percentage of CD8⁺ (E) and DiI⁺ (F) cells in extracted tissue of the PTOA model. Means  ±  SD. For the IVD model, a comparable number of cells could be isolated from L3-L4 and L4-L5 lumbar discs with no significant differences between the rat types (D). Using this recovered cell population, flow cytometric assessment further revealed a comparable percentage of CD8⁺ T-cells within both L3-L4 and L4-L5 lumbar discs after injection (E). Lastly, the percentage of GFP⁺ cells within the recovered cell population was assessed to determine the survival rate of injected NPC^GFP+/DiO+ after 1 week. The percentage of GFP⁺ cells within L3-L4 and L4-L5 lumbar discs was comparable between both Nude and SD rats (F).

Figure 4

(ELISA). (A,B): Systemic IL-6 (A) and IgM (B) levels (pg/mg) of Nude and SD rats throughout the 14-days experimental run of the PTOA model. Means  ±  SD. ** p  <  0.001 vs. Nude. (C,D): Systemic IL-6 (C) and IgM (D) levels (pg/mg) of Nude and SD rats throughout the one-week experimental run of the IVD model. Means  ±  SD.

Figure 5

Histology and immunofluorescence. (A–D): Representative images of knee (A,B) and lumbar disc (C,D) tissue of Nude (A,C) and SD (B,D) rats at the end of the experimental run (F = Femur, T = Tibia, AF = Annulus Fibrosus, NP = Nucleus Pulposus). Scale bars: A + B = 1 mm, C + D = 300 µm. (E–G): Representative images of DiI⁺ cells (arrows) inside knee tissue at the end of the 2-week experimental run of the PTOA model. Scale bars = 15 µm. (H–J): Representative images of DiO⁺ cells (arrows) inside the tissue of lumbar discs at the end of the 1-week experimental run of the IVD model. Scale bars = 15 µm.