Costimulation-adhesion blockade is superior to cyclosporine A and prednisone immunosuppressive therapy for preventing rejection of differentiated human embryonic stem cells following transplantation

Abstract

Rationale: Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction.

Objective: To test the hypothesis that a short-course, dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents.

Methods and results: We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein, and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury, as assessed by magnetic resonance imaging. Mechanistically, costimulation-adhesion therapy is associated with systemic and intragraft upregulation of T-cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile.

Conclusions: Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment, costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism.

Keywords: Costimulation blockade; Embryonic stem cells; Endothelial cells; Immune tolerance; Immunosuppressive drugs; Myocardial infarction.

© AlphaMed Press.

Figures

Figure 1

Characterization of hESC-ECs. (A) Endothelial cell differentiation protocol. Undifferentiated hESCs were grown on Matrigel and subcultured in low attachment dishes with differentiation medium supplement. At day 14, EBs in collagen were collected and digested and CD31+ cells were isolated by FACS and then sub-cultured in EGM-2 medium to expand. (B) Expression of endothelial cell markers CD31, CD144, and Laminin by confocal microscopy. Cell nuclei stained with DAPI (blue). Scale bars = 20 μm. (C) Gene expression profile (RT-PCR) of fibroblasts, undifferentiated ESCs, and hESC-ECS showing upregulation of CD31 and CD144 in hESC-ECs compared to undifferentiated hESCs and fibroblasts. Upregulation of pluripotency markers Sox2, Oct4, and Nanog is seen in undifferentiated hESCs compared to differentiated hESC-ECs and fibroblasts. Values were normalized to GAPDH and expression values are relative to fibroblasts. (D) hESC-ECs also can uptake ac-DiI-LDL and (E) form tube-like structures on Matrigel. Experiments were performed in triplicates.

Figure 2

hESC-EC transplantation and survival in immunodeficient hosts. (A) Schematic of double fusion reporter gene construct with a constitutive human ubiquitin promoter driving expression of Fluc and eGFP, with a self-inactivating (SIN) lentiviral vector. (B, C) Correlation of cell count with Fluc signal (R2 = 0.99). (D) Strong cell engraftment after hindlimb transplantation in immunodeficient NOD scid gamma (NSG) and Nude mice. (E) BLI signal quantification of panel D.

Figure 3

Costimulation-adhesion therapy (Costim) is superior to cyclosporine (CsA) and prednisone therapy in promoting hESC-EC survival. (A) CTLA4-Ig competes with CD28 for CD80/86 binding; anti-LFA-1 inhibits LFA-1/CAM-1 interaction; and CsA blocks production of IL-2. (B) Cell survival is limited in immunocompetent animals and is not significantly improved by CsA/Pred therapy. (C) BLI signal quantification of panel B. (D) Costimulation-adhesion therapy significantly improves hESC-EC survival compared to no treatment. (E) BLI signal quantification of panel D (*p<0.05; **p<0.01).

Figure 4

Costimulation-adhesion therapy promotes survival and engraftment of hESC-ECs in a myocardial infarction model. (A) Superior hESC-EC engraftment in immunodeficient (NOD/SCID) compared to immunocompetent (FVB) animals. Survival of hESC-ECs in immunocompetent mice was limited by day 10 whereas hESC-ECs in immunodeficient SCID mice engrafted up to day 21. (B) BLI signal quantification of panel A (*p<0.05). (C) Costimulation-adhesion therapy (costim) permits the strong engraftment of hESC-ECs compared to mice receiving CsA/Pred therapy. (D) BLI signal quantification of panel C (*p<0.05 costim compared to immunocompetent control, and costim compared to CsA/Pred).

Figure 5

Animals treated with costimulation-adhesion therapy and hESC-ECs showed improved cardiac function following myocardial infarction. (A–C) MRI analyses with plots for end diastolic volume (EDV), end systolic volume (ESV), and ejection fraction (EF) of animals with either (1) hESC-ECs + costimulation-adhesion therapy (costim); (2) hESC-ECs + no treatment; (3) hESC-ECs + CsA/Pred; (4) no cells + costim; or (5) no cells + PBS at day 2 and day 28 after MI (n=6–9/group). EDV and ESV were significantly lower and EF was significantly higher in animals treated with hESC-ECs and costimulation-adhesion agents compared to all other groups (ANOVA, *p<0.05 hESC-ECs + costim compared to all other groups), indicative of reduced remodeling and increased cardiac function. (D, E) Representative short-axis images of infarcted hearts at 2 days and 28 days after surgery of animals with (1) hESC-ECs + costim; (2) hESC-ECs + no treatment; (3) hESC-ECs + CsA/Pred; (4) no cells + costim, or (5) no cells + PBS. Scale bars = 5 mm; width of individual cardiac images = 18 mm.

Figure 6

Improved hESC-EC engraftment by costimulation-adhesion therapy is associated with upregulation of TIM3 and downregulation of a pro-inflammatory cytokine profile. (A, B) Marked increase in TIM3+PD1+ cells in splenocytes of costimulation-adhesion treated animals compared to untreated controls (*p<0.05). (C, D) Significant upregulation of TIM3+ cells in hindlimb muscle tissue implanted with hESC-ECs harvested from costimulation-adhesion-treated animals compared to untreated controls (*p<0.05). (E, F) RT-PCR of lymph node cells from costimulation-adhesion-treated animals also reveals upregulation of TIM3 and PD1 compared to untreated control animals. (G–J) Downregulation of IL-2, IFN-γ, and MIP1-α, and upregulation of IL-4 in splenocytes of costimulation-adhesion-treated animals compared to controls (*p<0.05, only in IL-2).