hESC-derived neural progenitors prevent xenograft rejection through neonatal desensitisation

Andreas Heuer, Agnete Kirkeby, Ulrich Pfisterer, Marie E Jönsson, Malin Parmar, Andreas Heuer, Agnete Kirkeby, Ulrich Pfisterer, Marie E Jönsson, Malin Parmar

Abstract

Stem cell therapies for neurological disorders are rapidly moving towards use in clinical trials. Before initiation of clinical trials, extensive pre-clinical validation in appropriate animal models is essential. However, grafts of human cells into the rodent brain are rejected within weeks after transplantation and the standard methods of immune-suppression for the purpose of studying human xenografts are not always sufficient for the long-term studies needed for transplanted human neurons to maturate, integrate and provide functional benefits in the host brain. Neonatal injections in rat pups using human fetal brain cells have been shown to desensitise the host to accept human tissue grafts as adults, whilst not compromising their immune system. Here, we show that differentiated human embryonic stem cells (hESCs) can be used for desensitisation to achieve long-term graft survival of human stem cell-derived neurons in a xenograft setting, surpassing the time of conventional pharmacological immune-suppressive treatments. The use of hESCs for desensitisation opens up for a widespread use of the technique, which will be of great value when performing pre-clinical evaluation of stem cell-derived neurons in animal models.

Keywords: Cyclosporine; Desensitisation; Immune response; Rejection; Stem cell; Transplant; Xenograft; hESC.

Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Fig. 1
Fig. 1
Rats desensitised with hESC-derived cells as neonates accept xenografts at a level comparable to CsA treated hosts. (A–G) Characterisation of the survival and immune response in the brain of CsA treated rats (CsA H9; n = 7), (H–N) neonatal desensitised rats engrafted as adults with identical tissue (Des H9; n = 8) and (O–U, W) desensitised rats engrafted with hiNs (Des hfl-iN; n = 2). Graft survival (A–B, H–I, O–P) and fibre innervation (B, I) of transplants from each group were analysed using a specific marker for human NCAM. Inflammation and immune responses were assessed using the following markers respectively: microglia (Ox42: C, J, Q), T-helper cells (CD4: D, K, R), T-cells (E, L, S), MHC-class II (Ox6: F, M, T) and MHC-class I (Ox18: G, N, U). Graft sizes were not different between the CsA control group and the neonatal desensitised rats (V). Optical density analysis for Ox42 positive microglia staining revealed increased staining intensity in the rejection group (*** = p 

Fig. 2

H9 hESC-derived cells desensitise the…

Fig. 2

H9 hESC-derived cells desensitise the host to other hESC lines. Graft survival of…

Fig. 2
H9 hESC-derived cells desensitise the host to other hESC lines. Graft survival of (A) CsA immune-suppressed (CsA H9; n = 8 + 3), (B) identical (Des H9; n = 8 + 2, H9 to H9-desensitised) and (C) different (Des RC17; n = 8 + 4, RC17 to H9-desensitised) hESC donor – host combinations. (E) A subset of animals was sacrificed after 6 weeks. The percentage of survival was similar in all groups 18 weeks post-transplantation (E, n = 8 per group). Graft sizes were different between the three groups, with RC17 transplants being significantly larger, whilst the graft areas were similar for H9 grafts in CsA or desensitised hosts. Grafts of H9 hESC origin into naïve animals were readily rejected within 6 weeks post transplantation (D; see also Fig. 3). Scalebar = 100 μM. All data in E are presented as mean ± SEM.

Fig. 3

Graft rejection response. Overview of…

Fig. 3

Graft rejection response. Overview of characterisation of graft rejection of human to rat…

Fig. 3
Graft rejection response. Overview of characterisation of graft rejection of human to rat xenografts in fully immune-competent rat hosts over the time-course of 18 weeks post-engraftment. All rats were engrafted in parallel with the rats that were inoculated. Time-points for assessment of the immune and inflammation responses were 2 weeks (n = 3), 6 weeks (n = 2) and 18 weeks (n = 2) post transplantation. No graft was detected via staining for human NCAM (A–C) later than the 2 week time-point. Inflammation and immune responses were strongest at the early time-point (2 weeks) and somewhat dampened at the last time-point of assessment (18 weeks). The respective infiltration of immune cells were visualized using antibodies against microglia (Ox42, D–E), T-helper cells (G–I), T-cells (J–L), MHC-class I (M–O), and MHC-class II (P–R). Scalebar = 200 μM. The rejection seen in H9-hESC grafts in fully immune-competent hosts demonstrates the capability of these cells to elicit an immune response as well as the need and validity of the neonatal desensitisation approach.

Fig. 4

Characterisation of reopening the Blood…

Fig. 4

Characterisation of reopening the Blood Brain Barrier. Rats that were neonatally desensitised using…

Fig. 4
Characterisation of reopening the Blood Brain Barrier. Rats that were neonatally desensitised using predifferentiated H9-hESCs and subsequently engrafted in adulthood using either H9-hESCs (Des H9 AAV; n = 4; A–F‴) or Cre-recombinase expressing H9-hESCs (Des H9-Cre AAV; n = 6; G–L) were subjected to a third opening of the blood brain barrier (after lesion and transplantation) to assess whether this would trigger an immune response causing the rejection of the transplant (A). We either used a GFP labelled AAV-vector to infect the host tissue (B–F‴) or a CRE-activatable direction inverse orientation (DIO) AAV-vector with a cassette encoding for EYFP to selectively infect the Cre-recombinase expressing donor cells (G–L) to select the cell population of interest. Transplants of both groups do survive long-term (> 24 weeks) in the rodent brain (B, G) and display limited inflammation (microglia; Ox42: C, H) and T-cell responses (CD4: D, I; CD8: E, J). Immune responses do not cause a rejection of the transplant within the four weeks after the respective AAV-transduction. Infection of the host cells was visualized using immune-fluorescent antibodies against DAPI (F) human nuclei (HuNU: F′), and GFP (F″). The composite merge shows the selective expression of the reporter in the host tissue (E‴). Selective transgene expression in the donor cells is demonstrated using fluorescent antibodies against human NCAM (J), Cre-recombinase (K′) and eYFP (K″). The composite merge (L) shows a cell that selectively expressed the EYFP reporter, clearly demonstrating the expression of nuclear Cre-recombinase and expression of EYFP. Scalebars: B–E = 500 μM; insets 50 μM; F–F‴ = 250 μM; G = 1000 μM, inset 50 μM; H–J = 1000 μM, inset 25 μM, K–K″ = 1000 μM, inset = 20 μM; L = 100 μM.
Fig. 2
Fig. 2
H9 hESC-derived cells desensitise the host to other hESC lines. Graft survival of (A) CsA immune-suppressed (CsA H9; n = 8 + 3), (B) identical (Des H9; n = 8 + 2, H9 to H9-desensitised) and (C) different (Des RC17; n = 8 + 4, RC17 to H9-desensitised) hESC donor – host combinations. (E) A subset of animals was sacrificed after 6 weeks. The percentage of survival was similar in all groups 18 weeks post-transplantation (E, n = 8 per group). Graft sizes were different between the three groups, with RC17 transplants being significantly larger, whilst the graft areas were similar for H9 grafts in CsA or desensitised hosts. Grafts of H9 hESC origin into naïve animals were readily rejected within 6 weeks post transplantation (D; see also Fig. 3). Scalebar = 100 μM. All data in E are presented as mean ± SEM.
Fig. 3
Fig. 3
Graft rejection response. Overview of characterisation of graft rejection of human to rat xenografts in fully immune-competent rat hosts over the time-course of 18 weeks post-engraftment. All rats were engrafted in parallel with the rats that were inoculated. Time-points for assessment of the immune and inflammation responses were 2 weeks (n = 3), 6 weeks (n = 2) and 18 weeks (n = 2) post transplantation. No graft was detected via staining for human NCAM (A–C) later than the 2 week time-point. Inflammation and immune responses were strongest at the early time-point (2 weeks) and somewhat dampened at the last time-point of assessment (18 weeks). The respective infiltration of immune cells were visualized using antibodies against microglia (Ox42, D–E), T-helper cells (G–I), T-cells (J–L), MHC-class I (M–O), and MHC-class II (P–R). Scalebar = 200 μM. The rejection seen in H9-hESC grafts in fully immune-competent hosts demonstrates the capability of these cells to elicit an immune response as well as the need and validity of the neonatal desensitisation approach.
Fig. 4
Fig. 4
Characterisation of reopening the Blood Brain Barrier. Rats that were neonatally desensitised using predifferentiated H9-hESCs and subsequently engrafted in adulthood using either H9-hESCs (Des H9 AAV; n = 4; A–F‴) or Cre-recombinase expressing H9-hESCs (Des H9-Cre AAV; n = 6; G–L) were subjected to a third opening of the blood brain barrier (after lesion and transplantation) to assess whether this would trigger an immune response causing the rejection of the transplant (A). We either used a GFP labelled AAV-vector to infect the host tissue (B–F‴) or a CRE-activatable direction inverse orientation (DIO) AAV-vector with a cassette encoding for EYFP to selectively infect the Cre-recombinase expressing donor cells (G–L) to select the cell population of interest. Transplants of both groups do survive long-term (> 24 weeks) in the rodent brain (B, G) and display limited inflammation (microglia; Ox42: C, H) and T-cell responses (CD4: D, I; CD8: E, J). Immune responses do not cause a rejection of the transplant within the four weeks after the respective AAV-transduction. Infection of the host cells was visualized using immune-fluorescent antibodies against DAPI (F) human nuclei (HuNU: F′), and GFP (F″). The composite merge shows the selective expression of the reporter in the host tissue (E‴). Selective transgene expression in the donor cells is demonstrated using fluorescent antibodies against human NCAM (J), Cre-recombinase (K′) and eYFP (K″). The composite merge (L) shows a cell that selectively expressed the EYFP reporter, clearly demonstrating the expression of nuclear Cre-recombinase and expression of EYFP. Scalebars: B–E = 500 μM; insets 50 μM; F–F‴ = 250 μM; G = 1000 μM, inset 50 μM; H–J = 1000 μM, inset 25 μM, K–K″ = 1000 μM, inset = 20 μM; L = 100 μM.

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