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Development of Histocompatible Primate-Induced Pluripotent Stem Cells for Neural Transplantation

From the July Edition of Stem Cells
By Stuart P. Atkinson

The risk of immune rejection and tumorigenesis of cells derived from pluripotent stem cells are both major concerns for regenerative medicine. The generation of patient specific induced pluripotent stem cells (iPSCs) promises a way round the problem of immunotolerance in cell/tissue replacement therapy, but iPSC could also be useful in the generation of a human leukocyte antigen (HLA)-haplotyped bank of pluripotent stem cell lines which could be used for widespread clinical use. Mauritanian cynomolgus macaques (CMs) exhibit limited major histocompatibility complex (MHC) diversity and may be useful in modelling human diseases, including neurodegeneration and transplantation research in non human primates (NHP). For this reason, the group of Ole Isacson at the Harvard Medical School/McLean Hospital, Massachusetts, USA attempted to produce MHC-matched iPSCs which could be of great value for immunological research and preclinical validation of the use of iPSCs. Their findings are presented in the July edition of Stem Cells (Deleidi et al).

Primary skin cells were isolated by biopsies from specific CMs so that resultant CM-iPSCs had MHC haplotypes which would provide a full match for 20% of recipients (MHC identical), a beneficial match for 60% of recipients (shared haplotype) and MHC-I match for 28% of recipients (MHC-I identical). Human OCT4, SOX2, KLF4 and MYC (OSKM) transgenes were then introduced into the cells via retroviral transduction and 6 to 8 weeks post-transfection, ESC-like colonies were identified and picked giving an efficiency of around 0.005%. Colonies showed morphology and growth rates similar to human embryonic stem cells (hESCs) and were positive for NANOG, OCT4, SSEA-4, TRA-1-60, TRA-1-81 and also for alkaline phosphatase (AP) activity. The CM-iPSC lines were also karotypically stable, showing efficient silencing of transgenes, full reactivation of endogenous genes and maintaining HLA haplotypes of the parental fibroblasts. CM-iPSCs could also form teratomas compromising tissues representative of all embryonic germ layers and so CM-iPSCs were deemed to be fully pluripotent. CM-iPSCs and hESCs (H9, used as a control) expressed very low levels of MHC-I, while MHC-II and CD16 were not detected. Treatment with IFNg, an important pro-inflammatory cytokine involved in the innate and adaptive immune response, led to the expression of MHC-I and CD16 in hESCs, but a more muted MHC-I expression in CM-iPSCs with no evidence of CD16 or MHC-II expression. Upon differentiation of CM-iPSCs, IFNg treatment led to an increase in MHC-I expression in neural progenitors (NPs) and differentiated neurons, while MHC-II and CD16 were unaffected.

Next, four of the CM-iPSC lines were tested for their potential to differentiate into midbrain dopaminergic (DA)-neurons with a previously described protocol (Sanchez-Pernaute et al) using staining for tyrosine hydroxylase (TH) and b-III-Tubulin (b-TubIII) as markers for DA-neurons. The cells readily differentiated, although at varying levels between the different CM-iPSC lines, and further Q-PCR analysis of midbrain markers (FOXA2, VMAT, CALB1, ALDH, AADC and EN1) confirmed DA-neuron differentiation. These cells were then transplanted into the striatum of naïve rats as a short-term in vivo bioassay, as the group had previously undertaken with human iPSCs (Hargus et al), and demonstrated the ability to survive and project axons into the host striatum. There was some evidence of immune reactivity by the presence of Iba1 around the transplanted cells, which is expressed in microglia/macrophages and is known to have a role in chronic transplant rejection, and also by the presence of T-cell infiltration, as shown by CD3 immunostaining, although this was minimal. There was no evidence of tumour formation and Ki67 staining showed that only a minority of cells from the graft were proliferative at 4 weeks. Importantly, no Oct4 expression was observed, meaning that no residual undifferentiated iPSCs were present. The next stage was to perform transplantation into the striatum of 6-OHDA-lesioned mice, an animal model of Parkinson’s disease (PD). Grafted DA neurons behaved as they had done in the naïve rat brain but reinnervated the host striatum and induced partial behavioural recovery, as measure by amphetamine and apomorphine responses in the PD mouse model as compared to the control PD mice, without transplanted cells. Importantly, the grafts survived in the long term (6 months) and showed no signs of tumorigenesis or inflammatory reaction.

This research does suggest that histocompatible iPSCs can be useful in the treatment of neurodegenerative diseases such as Parkinson’s and would also allow for the preclinical validation of safety and efficacy of iPSCs. However recent high impact research has shown that patient-specific iPSCs may still elicit an immune response (Zhao et al), but the lack of immune response after 6 months in the present study is very encouraging.

 

References

Deleidi M, Hargus G, Hallett P, Osborn T, Isacson O.
Development of histocompatible primate-induced pluripotent stem cells for neural transplantation.
Stem Cells. 2011 Jul;29(7):1052-63. doi: 10.1002/stem.662.

Sanchez-Pernaute R, Lee H, Patterson M, Reske-Nielsen C, Yoshizaki T, Sonntag KC, Studer L, Isacson O.
Parthenogenetic dopamine neurons from primate embryonic stem cells restore function in experimental Parkinson's disease.
Brain. 2008 Aug;131(Pt 8):2127-39. Epub 2008 Jul 22.

Hargus G, Cooper O, Deleidi M, Levy A, Lee K, Marlow E, Yow A, Soldner F, Hockemeyer D, Hallett PJ, Osborn T, Jaenisch R, Isacson O.
Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats.
Proc Natl Acad Sci U S A. 2010 Sep 7;107(36):15921-6. Epub 2010 Aug 23

Zhao T, Zhang ZN, Rong Z, Xu Y.
Immunogenicity of induced pluripotent stem cells.
Nature. 2011 May 13;474(7350):212-5. doi: 10.1038/nature10135.