Improved Cell Therapy Protocols for Parkinson's Disease Based on Differentiation Efficiency and Safety of hESC-, hiPSC-, and Non-Human Primate iPSC-Derived Dopaminergic Neurons

Abstract

The main motor symptoms of Parkinson’s disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson’s disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA-neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for pre-clinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate-iPSC (PiPSC)-derived DA neurons. According to our results, NCAM+/CD29low sorting enriched VM DA-neurons from pluripotent stem cell-derived neural cell populations. NCAM+/CD29low DA-neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM+/CD29low DA-neurons were able to restore motor function of 6-OHDA lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future.

Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors

“…This finding is an important advance for clinical application of pluripotent stem cells in neural grafting procedures, where some form of cell selection will almost certainly be required to eliminate potentially dangerous cell types, for example those capable of uncontrolled growth after transplantation. It extends on previous work in this area using fluorescent transgenes (4-6) or targeting of neurodevelopmental stage-specific transmembrane proteins (37,38) to eliminate tumorigenic cells before transplantation.…”

Canonical and noncanonical Wnt signaling in neural stem/progenitor cells

“…To do so, hES cells, normally cultured as embryoid bodies [115,152], are placed in stringent serum-free culture conditions that selectively facilitate the survival and growth of neural cells [153]. CHIR99021 has been widely used in order to keep cells in an undifferentiated and proliferative state in order to initially increase neural progenitor cell number, and ultimately neuronal yield [16,[154][155][156]. When the cells enter the neural lineage, rosette-like structures appear.…”

“…For example, Wexler et al showed that baseline -catenin signaling represses neuronal differentiation in human NSCs [131], and another study reported that inhibition of Wnt/ -catenin signaling using Dkk-1 or the tankyrase inhibitor XAV939 promotes neural precursor specification [164]. Moreover, GSK-3 inhibition, which induces stem cell renewal and sustains the expression of pluripotency markers [16], is a widely used tool to expand neural progenitors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 [154][155][156], and bFGF treatment of hES and iPS cells, which maintains the undifferentiated state, activates canonical Wnt signaling through inhibition of GSK-3 [165].…”

Treatment of Parkinson's disease using cell transplantation

“…This is unlikely to occur if the technology is used to create a cell bank with human leukocyte antigen haplotype-matching iPS cells to be used in allografting experiments [51]. Extensive axonal outgrowth from the human iPS cellderived dopaminergic neurons implanted into the denervated rodent striatum has not been convincingly demonstrated [38, [46][47][48][49][50], and their capacity to improve behavioural deficits relevant for the clinical condition is incompletely known. A rich dopaminergic terminal network extending throughout the striatum will definitely be needed for clinical efficacy.…”