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Improved Differentiation Strategy for Parkinson’s Therapy

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"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"

From Stem Cells

The replacement of dopaminergic (DA) neurons in the ventral midbrain (VM) is seen as a viable and beneficial treatment in Parkinson's disease (PD), but is however hampered by the poor dopaminergic differentiation capacity of pluripotent cells; the optimal sources for the derivation of these specialized neurons (Kirkeby et al, Kriks et al and Xi et al).   Sundberg et al. from the laboratory of Ole Isacson at the Neuroregeneration Laboratories, Harvard Medical School/McLean Hospital, USA now report on their optimisation of the signaling parameters for VM DA neuron specification from pluripotent stem cells sources through the application of knowledge gleaned from developmental studies (Cooper et al, Hynes et al, Prakash et al, and Ye et al).

After initial testing of different combinations of small molecules on growth factor-reduced Matrigel, human embryonic stem cells (hESCs) were differentiated towards FOXA2/OTX2/βIII tubulin-positive VM DA neuronal precursors using a complex process utilising LDN, SB, SHH (C24-II), purmorphamine, FGF8a, and 3 μM CHIR for 9 days (see end for abbreviations and targets).   This protocol was highly dependent on the level of GSK-3β inhibition by CHIR and was used for the differentiation of hESCs and human induced pluripotent stem cells (hiPSCs), while a modified protocol using LDN, SB, noggin, SAG, wnt1, FGF8a, RA, 1 μM CHIR alongside an additional cell proliferation step for primate iPSCs (PiPSCs).   After 30 days, the pluripotency markers Tra1-81 and SSEA-4 were highly downregulated in hESC- and hiPSC-derived cells, although in PiPSCs, SSEA-4 positive cells were still at 30% of total cell number.   Neural markers CXCR4 and NCAM, p75 neurotrophin receptor, β-integrin, FOXA2 and TH all increased during the differentiation process to similar levels across the three cell types assayed.   Analysis of neural subpopulations found that a purified NCAM+ β-integrinLo population expressed a high level of DA neuron-specific genes (NURR1, GIRK2, PITX3, and TH) with no pluripotent cells or endothelial-type cells detected.

PiPSC-derived DA neurons were then used for transplantation studies and, at 16 weeks, the formation of a choroid plexus endothelium cell-type overgrowth was detected in the 6-OHDA lesioned rat striatum (PD model) which was confirmed to have originated from the transplanted PiPSCs.   No abnormal cell growth was observed at 16 weeks and the presence of nigral A9-type DA neurons and rare ventral tegmental A10-type DA neurons was confirmed in unsorted and NCAM+/CD29low PiPSC-derived DA neuron grafts.  Additionally, graft-derived DA neurons were found to innervate host tissues.   Cell grafts purified by sorting prior to transplantation resulted in fewer numbers of engrafted cells than unsorted grafts, but had higher densities of TH+ cells.   These grafts displayed good outgrowth into the host striatum and the hosts showed excellent results in their rotational asymmetry in amphetamine-induced rotation test and contralateral paw usage in the cylinder test compared to lesioned control animals.   However Parkinsonian motor scores and daytime activity measurements were not changed 12 months after transplantation of PiPSC-derived DA neurons into a male cynomolgus monkey model of Parkinson’s.   Cells expressing FOXA2/TH/β-III tubulin with TH+ outgrowths were evident at the graft site and no graft overgrowth or cyst formation was detected.

The authors propose that their modified protocol, based on previously published studies (Kirkeby et al, Kriks et al and Xi et al), is a significantly improved differentiation strategy which, when combined with a novel sorting strategy, allowed them to select specifically for nigral A9 neurons, which have the ability to re-innervate the degenerate striatum after transplantation (Schultzberg et al and Thompson et al). This protocol also worked upon primate iPSCs, with transplantation of sorted DA neurons eliminating the tumourigenic potential of grafts and increasing functional recovery in 6-OHDA-lesioned rats.   Further, the autologous transplantation of these cells allowed the long term assessment of engrafted cells, finding that while no functional improvement was observed, grafted cells had survived and did not cause any abnormal growth, thus giving an impetus for further iPSC-based research in non-human primates.

Abbreviations and Targets: OTX2 (Orthodenticle homeobox 2), LDN (inhibitor of bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3), SB (SB431542, inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors), SHH (Sonic Hedgehog), Purmorphamine (agonist of Smoothened, a 7-transmembrane receptor of the hedgehog signaling pathway), FGF (Fibroblast Growth Factor), CHIR (CHIR99021 is the most selective inhibitor of glycogen synthase kinase 3β (GSK-3β)), SAG (smoothened agonist) , RA (retinoic acid), CXCR (CXC chemokine receptor), NCAM (Neural cell adhesion molecule), NURR1 (Nuclear receptor related-1), GIRK2 (KCNJ6, potassium inwardly-rectifying channel, subfamily J, member 6), PITX3 (paired-like homeodomain 3), TH (tyrosine hydroxylase), OHDA (6-hydroxydopamine).

 

References

  • Cooper O  et al. Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid. Mol Cell Neurosci 2010; 45: 258–266
  • Hynes M et al. Induction of midbrain dopaminergic neurons by Sonic hedgehog. Neuron 1995; 15: 35–44
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  • Prakash N et al. A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo. Development 2006; 133: 89–98
  • Schultzberg M et al. Dopamine and cholecystokinin immunoreactive neurons in mesencephalic grafts reinnervating the neostriatum: Evidence for selective growth regulation. Neuroscience 1984; 12: 17–32
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  • Ye W et al. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 1998; 93: 755–766

From Stem Cells.

Stem Cell Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.