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Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease.

Grealish S, Diguet E, Kirkeby A, Mattsson B, Heuer A, Bramoulle Y, Van Camp N, Perrier AL, Hantraye P, Björklund A, Parmar M - Cell Stem Cell (2014)

Bottom Line: Considerable progress has been made in generating fully functional and transplantable dopamine neurons from human embryonic stem cells (hESCs).We show long-term survival and functionality using clinically relevant MRI and PET imaging techniques and demonstrate efficacy in restoration of motor function with a potency comparable to that seen with human fetal dopamine neurons.Furthermore, we show that hESC-derived dopamine neurons can project sufficiently long distances for use in humans, fully regenerate midbrain-to-forebrain projections, and innervate correct target structures.

View Article: PubMed Central - PubMed

Affiliation: Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden. Electronic address: shane.grealish@med.lu.se.

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Comparison of A9 and A10 Midbrain DA Neuron Subtypes in Long-Term Grafts of hESC-DA Neurons and Human Fetal VM(A) Upon close inspection, hESC-DA neurons gave rise to TH+ dopaminergic neurons with an A9-like neuron morphology, characterized by large angular somas (upper panel), as well as A10-like neurons with small, round somas (lower panel).(B) The presence of neurons coexpressing TH and GIRK2 (red and green) and TH and Calbindin (red and blue) indicates that both midbrain DA neuron subtypes, A9 and A10, were contained in grafts of hESC-derived neurons.(C and D) Intrastriatal grafts of human fetal VM contained TH+ dopaminergic neurons with A9 and A10 morphologies, coexpressing GIRK2 and/or Calbindin.(E and F) Homotopic placement of hESC-DA neurons in SN resulted in large numbers of TH+ dopaminergic neurons with mature A9 and A10 morphologies, confirmed by coexpression of TH with GIRK2 and Calbindin, respectively.(G and H) Fetal VM tissue grafted to the SN displayed the same ability to generate transplants with dopaminergic neurons of A9 and A10 morphologies and protein expression (H).(I and J) The number and proportion (J) of TH+ neurons, coexpressing GIRK2, Calbindin, or both markers, was quantified in all groups (n = 3 per group).Calb, Calbindin; GIRK2, G protein-regulated inward-rectifying potassium channel 2; TH, tyrosine hydroxylase. In (I) and (J), data are represented as mean ± SEM. In (A), (C), (E), and (G), scale bars represent 50 μm. In (B), (D), (F), and (H), scale bars represent 20 μm.
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fig3: Comparison of A9 and A10 Midbrain DA Neuron Subtypes in Long-Term Grafts of hESC-DA Neurons and Human Fetal VM(A) Upon close inspection, hESC-DA neurons gave rise to TH+ dopaminergic neurons with an A9-like neuron morphology, characterized by large angular somas (upper panel), as well as A10-like neurons with small, round somas (lower panel).(B) The presence of neurons coexpressing TH and GIRK2 (red and green) and TH and Calbindin (red and blue) indicates that both midbrain DA neuron subtypes, A9 and A10, were contained in grafts of hESC-derived neurons.(C and D) Intrastriatal grafts of human fetal VM contained TH+ dopaminergic neurons with A9 and A10 morphologies, coexpressing GIRK2 and/or Calbindin.(E and F) Homotopic placement of hESC-DA neurons in SN resulted in large numbers of TH+ dopaminergic neurons with mature A9 and A10 morphologies, confirmed by coexpression of TH with GIRK2 and Calbindin, respectively.(G and H) Fetal VM tissue grafted to the SN displayed the same ability to generate transplants with dopaminergic neurons of A9 and A10 morphologies and protein expression (H).(I and J) The number and proportion (J) of TH+ neurons, coexpressing GIRK2, Calbindin, or both markers, was quantified in all groups (n = 3 per group).Calb, Calbindin; GIRK2, G protein-regulated inward-rectifying potassium channel 2; TH, tyrosine hydroxylase. In (I) and (J), data are represented as mean ± SEM. In (A), (C), (E), and (G), scale bars represent 50 μm. In (B), (D), (F), and (H), scale bars represent 20 μm.

Mentions: We next performed a direct histological comparison between hESC-DA neurons and authentic human fetal DA neurons prepared according to Rath et al. (2013), grafted in parallel, in order to validate the morphology, maturation, and phenotypic properties of the transplanted DA neurons (Figures 2D and 2E). In this analysis, we observed the presence of TH+ neurons with two distinct morphologies in the hESC-grafted animals (Mendez et al., 2005; Thompson et al., 2005): A9-like nigral neurons characterized by large angular somata and A10-like VTA neurons with small, round somata (Figure 3A). The morphology of the hESC-derived neurons was identical to that observed with intrastriatal transplants of fetal VM when analyzed at 6 months posttransplantation (Figure 3C) and consistent with graft appearance from previous postmortem reports from transplanted PD patients (Mendez et al., 2005). When staining the cells for markers commonly used to distinguish between SN (A9) and VTA (A10) neurons, TH+ neurons expressing GIRK2 (A9), Calbindin (A10), or both markers were readily detected (Figures 3B and 3D). Quantifications showed that TH+/GIRK2+ neurons were the most abundant subtype in both hESC-derived and fetal grafts (Figure 3I) and that the proportion was similar in both hESC- and fetal-derived grafts (Figure 3J). Taken together, this analysis shows that hESC-DA neurons are indistinguishable from their fetal counterparts on the basis of graft appearance, morphology, and marker expression 6 months after grafting and that the hESC-derived grafts are rich in both A9-like and A10-like DA neurons.


Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease.

Grealish S, Diguet E, Kirkeby A, Mattsson B, Heuer A, Bramoulle Y, Van Camp N, Perrier AL, Hantraye P, Björklund A, Parmar M - Cell Stem Cell (2014)

Comparison of A9 and A10 Midbrain DA Neuron Subtypes in Long-Term Grafts of hESC-DA Neurons and Human Fetal VM(A) Upon close inspection, hESC-DA neurons gave rise to TH+ dopaminergic neurons with an A9-like neuron morphology, characterized by large angular somas (upper panel), as well as A10-like neurons with small, round somas (lower panel).(B) The presence of neurons coexpressing TH and GIRK2 (red and green) and TH and Calbindin (red and blue) indicates that both midbrain DA neuron subtypes, A9 and A10, were contained in grafts of hESC-derived neurons.(C and D) Intrastriatal grafts of human fetal VM contained TH+ dopaminergic neurons with A9 and A10 morphologies, coexpressing GIRK2 and/or Calbindin.(E and F) Homotopic placement of hESC-DA neurons in SN resulted in large numbers of TH+ dopaminergic neurons with mature A9 and A10 morphologies, confirmed by coexpression of TH with GIRK2 and Calbindin, respectively.(G and H) Fetal VM tissue grafted to the SN displayed the same ability to generate transplants with dopaminergic neurons of A9 and A10 morphologies and protein expression (H).(I and J) The number and proportion (J) of TH+ neurons, coexpressing GIRK2, Calbindin, or both markers, was quantified in all groups (n = 3 per group).Calb, Calbindin; GIRK2, G protein-regulated inward-rectifying potassium channel 2; TH, tyrosine hydroxylase. In (I) and (J), data are represented as mean ± SEM. In (A), (C), (E), and (G), scale bars represent 50 μm. In (B), (D), (F), and (H), scale bars represent 20 μm.
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fig3: Comparison of A9 and A10 Midbrain DA Neuron Subtypes in Long-Term Grafts of hESC-DA Neurons and Human Fetal VM(A) Upon close inspection, hESC-DA neurons gave rise to TH+ dopaminergic neurons with an A9-like neuron morphology, characterized by large angular somas (upper panel), as well as A10-like neurons with small, round somas (lower panel).(B) The presence of neurons coexpressing TH and GIRK2 (red and green) and TH and Calbindin (red and blue) indicates that both midbrain DA neuron subtypes, A9 and A10, were contained in grafts of hESC-derived neurons.(C and D) Intrastriatal grafts of human fetal VM contained TH+ dopaminergic neurons with A9 and A10 morphologies, coexpressing GIRK2 and/or Calbindin.(E and F) Homotopic placement of hESC-DA neurons in SN resulted in large numbers of TH+ dopaminergic neurons with mature A9 and A10 morphologies, confirmed by coexpression of TH with GIRK2 and Calbindin, respectively.(G and H) Fetal VM tissue grafted to the SN displayed the same ability to generate transplants with dopaminergic neurons of A9 and A10 morphologies and protein expression (H).(I and J) The number and proportion (J) of TH+ neurons, coexpressing GIRK2, Calbindin, or both markers, was quantified in all groups (n = 3 per group).Calb, Calbindin; GIRK2, G protein-regulated inward-rectifying potassium channel 2; TH, tyrosine hydroxylase. In (I) and (J), data are represented as mean ± SEM. In (A), (C), (E), and (G), scale bars represent 50 μm. In (B), (D), (F), and (H), scale bars represent 20 μm.
Mentions: We next performed a direct histological comparison between hESC-DA neurons and authentic human fetal DA neurons prepared according to Rath et al. (2013), grafted in parallel, in order to validate the morphology, maturation, and phenotypic properties of the transplanted DA neurons (Figures 2D and 2E). In this analysis, we observed the presence of TH+ neurons with two distinct morphologies in the hESC-grafted animals (Mendez et al., 2005; Thompson et al., 2005): A9-like nigral neurons characterized by large angular somata and A10-like VTA neurons with small, round somata (Figure 3A). The morphology of the hESC-derived neurons was identical to that observed with intrastriatal transplants of fetal VM when analyzed at 6 months posttransplantation (Figure 3C) and consistent with graft appearance from previous postmortem reports from transplanted PD patients (Mendez et al., 2005). When staining the cells for markers commonly used to distinguish between SN (A9) and VTA (A10) neurons, TH+ neurons expressing GIRK2 (A9), Calbindin (A10), or both markers were readily detected (Figures 3B and 3D). Quantifications showed that TH+/GIRK2+ neurons were the most abundant subtype in both hESC-derived and fetal grafts (Figure 3I) and that the proportion was similar in both hESC- and fetal-derived grafts (Figure 3J). Taken together, this analysis shows that hESC-DA neurons are indistinguishable from their fetal counterparts on the basis of graft appearance, morphology, and marker expression 6 months after grafting and that the hESC-derived grafts are rich in both A9-like and A10-like DA neurons.

Bottom Line: Considerable progress has been made in generating fully functional and transplantable dopamine neurons from human embryonic stem cells (hESCs).We show long-term survival and functionality using clinically relevant MRI and PET imaging techniques and demonstrate efficacy in restoration of motor function with a potency comparable to that seen with human fetal dopamine neurons.Furthermore, we show that hESC-derived dopamine neurons can project sufficiently long distances for use in humans, fully regenerate midbrain-to-forebrain projections, and innervate correct target structures.

View Article: PubMed Central - PubMed

Affiliation: Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, 22184 Lund, Sweden; Lund Stem Cell Center, Lund University, 22184 Lund, Sweden. Electronic address: shane.grealish@med.lu.se.

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Related in: MedlinePlus