Long-Term Survival of Dopamine Neurons Derived from Parthenogenetic Primate Embryonic Stem Cells (Cyno-1) After Transplantation
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《干细胞学杂志》
a McLean Hospital/Harvard University Udall Parkinson’s Disease Research Center of Excellence and
b Neuroregeneration Laboratories, McLean Hospital, Belmont, Massachusetts, USA;
c Laboratory of Stem Cell & Tumor Biology, Division of Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
Key Words. Parkinson’s disease ? Embryonic stem (ES) cell ? Transplantation ? Primate ? Differentiation
Correspondence: Rosario Sánchez-Pernaute, M.D., Ph.D., McLean Hospital/Harvard University Udall Parkinson’s Disease Research Center of Excellence and Neuroregeneration Laboratories, McLean Hospital, 115 Mill St., Belmont, Massachusetts 02478, USA. Telephone: 617-855-3568 ; Fax: 617-855-2522; e-mail: rosario_pernaute@hms.harvard.edu
ABSTRACT
Embryonic stem (ES) cells provide an unlimited source of cells that can be tailored to meet specific criteria for cell-replacement therapies. ES cell lines have been derived from human and nonhuman primate blastocysts, including parthenogenetic primate blastocysts, . Neuronal and glial cell lineages have been derived in vitro from these primate and human ES cell lines and examined in vivo in transplantation paradigms . However, a number of critical questions remain to be addressed to develop an ES cell–based therapy for neurodegenerative disorders, in particular for Parkinson’s disease (PD). These questions relate to survival and proliferation, stability of neuronal phenotypes induced in vitro, and capacity of synaptic integration and are therefore a prerequisite for investigation of functional potential.
The Cyno-1 cell line used in this study was derived from the inner cell mass of a parthenogenetic blastocyst and has similar characteristics to normal fertilization–derived primate ES cells . Dopamine (DA) neurons have been derived in vitro from this cell line . Neural precursors derived from mouse ES cells can be directed into defined neuronal phenotypes by the timed exposure to factors that control antero-posterior and dorso-ventral fate specification during normal development in vivo, including fibroblast growth factors (FGF) 4 and 8, retinoic acid, and sonic hedgehog (SHH) . To obtain DA neurons from primate ES cells in this study, we first used a stromal cell feeder coculture system for neural induction , followed by the sequential exposure to inductive signals, such as SHH and FGF8, that control DA specification during embryogenesis and factors that promote DA neuron differentiation and maturation. In this study, transplantation of in vitro differentiated Cyno-1 cells was performed into adult immunosuppressed rats and into the primate striatum (allograft) without immunosuppression.
MATERIALS AND METHODS
Transplantation of Differentiated Primate ES Cells into the Rat Striatum
In this protocol for in vitro neuronal differentiation (Fig. 1A), undifferentiated Cyno-1 cells (Fig. 1B) on MS5 or MS5-wnt1 feeder cells formed neural rosettes within 10–14 days (Fig. 1C). After replating on coated (feeder-free) dishes, neural rosettes were exposed to a second induction phase with SHH and FGF8 (Fig. 1A) which resulted in neuronal differentiation (Fig. 1D) and expression of Pax2 (day 28, not shown) followed by engrailed 1 (Fig. 1E) in 20% of the cells at the end of passage 2 expansion (day 35). Upon differentiation of passage 2 cells for 7 more days in the absence of SHH and FGF8 and in the presence of various trophic factors (Fig. 1A), 30%–60% of the cells expressed neuronal markers (TuJ-1), and up to 70% of these neurons coexpressed TH (Figs. 1F, 1G). These cells were briefly exposed to fresh mitomycin C immediately before being harvested and dissociated into a cell suspension for grafting. We previously determined the effect of mitomycin C on graft size and proliferation 3 weeks after transplantation (see below and supplemental online Fig. 2). Some cells were replated in differentiation media and fixed 3, 5, and 12 days later for control of mitomycin C and cell-preparation effect (supplemental online Fig. 1). The replated cells formed small clusters in which the majority of cells were nestin or TuJ1-positive; 2%–3% of the TuJ1-positive cells were TH, and 10% were Bf1-positive cells (not shown); these percentages were similar at 5 and 12 days. Although these may not correspond exactly to the com-position of the population in the cell suspension, due to the effect of culture conditions upon replating, such as cell density and media, these data suggest that TH-positive cells are particularly sensitive to dissociation and replating compared with the overall neuronal population within the grafted cell suspension.
We transplanted these differentiated ES cells into rats (n = 7) and into one primate (see below). Rat brains were examined 16 weeks post-transplantation; six animals had surviving grafts. Graft volumes were small (2.32 ± 0.6 mm3), and PCNA expression in the graft core was low (supplemental online Fig. 2C). TH-positive neurons were found in all the grafts close to the host-graft interface (Figs. 2B, 2C) in a typical peripheral distribution around the core of the graft, which was strongly immunoreactive for double-cortin (Dcx, not shown), a neurofilament associated protein highly expressed by young and migrating neuroblasts, which is expressed only in neurogenic regions in the adult host brain. The TH-positive neurons in the graft were HNA-positive (Fig. 2D), DBH-negative, and calbindin-negative (not shown); some cell bodies and fibers expressed AADC and the transporters DAT and VMAT-2 (Figs. 2E–2H). DAT expression was observed in TH fibers projecting into the host (Fig. 2F). TH cell numbers in the grafts (557 ± 117, n = 6) were directly correlated with the graft volume (R2 = 0.91, p < 0.01) and inversely correlated with the percentage of cells expressing the forebrain marker Bf1 (R2 = 0.89, p < 0.01). Most of the HNA-positive cells in the core of the grafts expressed NeuN (Fig. 3B) and were positive for the forebrain transcription factor Bf1 (83% ± 4%, not shown). 5HT neurons (not shown) were present in the grafts in lower numbers (65 ± 19, 1:5 TH-positive). At 16 weeks, many primate (HNA-positive) cells were found away from the core of the graft (Fig. 3), mainly in the ipsilateral striatum and also along the corpus callosum and the anterior commissure, reaching the contra-lateral hemisphere. Semiquantitative estimation of HNA-positive cell density (Fig. 3A) showed a gradient away from the graft. Many of these cells away from the core expressed Dcx (some organized in migratory chains, Fig. 3G) or, in white matter tracts, glial markers (GFAP and O4, Figs. 3D–3F), and few were NeuN-positive, which were located in close proximity to the graft core. There were some HNA-positive cells in the subventricular zone (SVZ) (Fig. 3H), which were GFAP-negative and either positive or negative for Dcx.
Figure 2. Grafted cells were identified by HNA expression (B–D) 4 months post-transplantation into the rat striatum.(A):Computer-generated graft contour and (B) virtual slice acquired on a representative coronal section double immunostained for TH and HNA. The boxed area is shown in (B). TH-positive neurons derived from primate ES cells were (B) located at the periphery of the grafts and (C) showed nuclear expression of the primate and human specific marker HNA, as shown in (D) in a confocal reconstruction. (E, F): TH neurons in the grafts expressed AADC (E) and some TH-positive neurites expressed DAT (E–G). In (G) DAT and TH coexpression is shown in a 3-D reconstruction of a confocal z-stack through a DA fiber. (H): VMAT-2 was expressed in TH-positive and -negative (presumably serotonergic) fibers. Scale bars = 2 mm (A), 250 μm (B), 100 μm (C), 30 μm (D–F, H), and 10 μm (G). Abbreviations: AADC, aromatic amino acid decarboxylase; ac, anterior commisure; cx, cortex; DA, dopamine; DAT, dopamine transporter; ES, embryonic stem; g, graft; HNA, human nuclear antigen; st, striatum; TH, tyrosine hydroxylase; VMAT, vesicular monoamine transporter.
Figure 3. Primate HNA-positive cells (HNA is shown in red in all panels) migrated away from the graft core 4 months post-transplantation into the rat striatum. (A): Semiquantitative estimation of HNA-positive cell density was performed on serial sections, and the average density for the 6 grafted animals is shown on representative coronal sections (see Materials and Methods). (B, C): In the graft core, most HNA-positive cells expressed (B) NeuN, and not (C) GFAP. (D, F): HNA-positive glial cells were found along white matter tracts: astro-cytes and oligodendrocytes were observed along the corpus callosum. Outside the core of the graft there were some Dcx-positive neuroblasts in chain formations. (H): Some HNA-positive cells were found in the SVZ ipsilateral to the graft. Nuclear counterstain (Hoechst) is shown in blue in (D), (E), and (H), and white in (F). Scale bars = 100 μm (B–E), 15 μm (F), 30 μm (G), and 75 μm (H). Abbreviations: GFAP, glial fibrillary acidic protein; HNA, human nuclear antigen; il, ipsilateral to the graft; LV, lateral ventricle; SVZ, subventricular zone.
Transplantation of Differentiated Primate ES Cells into the Primate Striatum
Cells from the same preparation were transplanted into the right postcommissural putamen of a cynomolgus monkey. In this case (allograft), the animal did not receive immunosuppression. Motor scores were evaluated weekly and did not show any asymmetry up to 7 months post-transplantation. Because in this model parkinsonian signs are bilateral and symmetric and the transplant was unilateral, the absence of motor asymmetry is consistent with neither positive nor adverse effects of the grafts. Macroscopic examination of the brain disclosed two grafts at the sites of the injections. The graft volumes were 14 mm3 for the anterior deposit and 5.4 mm3 for the posterior graft (Fig. 4A). The grafts were homogeneous and had a slightly higher cell density than the host (Figs. 4D, 4F, 4H). There were no regions of non-neural differentiation, and the grafts did not displace or compress the striatal host parenchyma. Numerous TH-positive cells were located predominantly in the periphery of the graft cores (Figs. 4A, 4B), in a similar arrangement and morphology seen for TH/HNA-positive cells found in the rodent brain (Figs. 2B, 2C). Stereological counts of TH-positive cells showed 2,440 positive cells in the anterior and 880 in the posterior graft (thus, TH cells in vivo were 0.1% of grafted cells). Many of these TH neurons coexpressed AADC (Fig. 4E). As shown in the rodent grafts (Figs. 2E, 2F), some TH fibers within and close to the graft coexpressed DAT (Fig. 4E), but DAT immunoreactivity was not observed in the cell bodies. The grafts contained many NeuN-positive neurons (Figs. 4D, 4F) that coexpressed Bf1 (Fig. 4F). As in the rodent grafts, few neurons in these grafts expressed 5HT (not shown). Clusters of calretinin-positive neurons and some calbindin-positive neurons were observed within the grafts (Fig. 4G). Dcx expression was not detected in the graft core, in contrast with the widespread immunoreactivity for this marker observed in the core in rodent grafts at 4 months. Nevertheless, some Dcx-positive neuroblasts were observed in the primate grafts forming chains (Fig. 4H) reminiscent of those found in the rodents away from the graft core (Fig. 3G). The blood vessels located in the proximity of the grafts showed some perivascular cuffing with strong PCNA positivity, and microglial (CD68-positive) cells were observed in these areas (Fig. 4I).
Figure 4. Transplantation of DA cells derived from Cyno-1 ES cells into the primate putamen. (A): Computer-generated outline of the graft in the postcommissural putamen and map of the distribution of TH-positive neurons on a representative coronal section. Each green dot represents a TH cell. (B): Low power microphotograph of a graft, the boxed area is shown in (C) and the asterisks mark the same blood vessel in both images. Clusters of TH-positive cells (the DAB product appears dark brown) were located close to the graft-host interface, where there was a higher nuclear density and higher density of TH-positive fibers (C, D) than in the graft core; (E) many TH-positive cells in the grafts coexpressed AADC but not all and coexpressed the DAT in neurites but not in the cell bodies. (F, G): The graft core was predominantly neuronal (NeuN-positive) with most neurons coexpressing Bf1 (coexpression with NeuN appears pink in (F). Clusters of neurons inside the graft expressed calretinin (G) and calbindin . Although the morphology of the neurons inside and outside the graft was the same, neurons within the graft were closer to each other than in the host striatum. (H): Dcx-positive neuroblasts in a chain-like formation (see also Fig. 3). (I): PCNA-positive cells within and close to the graft were mainly located in perivascular spaces and some corresponded to microglia (CD68-positive) and astroglia (GFAP-positive). Scale bars = 2.5 mm (A), 125 μm (B), 100 μm (C), 20 μm (D–G), 50 μm (H) and (I). Nuclear counterstain (Hoechst) is shown in blue in (D) and (H) and in white in (F). * represents blood vessel. Abbreviations: AADC, aromatic amino acid decarboxylase; cd, caudate nucleus; DA, dopamine; DAB, 3, 3'-diaminobenzidine; DAT, dopamine transporter; ES, embryonic stem; GFAP, glial fibrillary acidic protein; GP, globus pallidus; ic, internal capsule; PCNA, proliferating cell nuclear antigen; put, putamen; TH, tyrosine hydroxylase.
DISCUSSION
This work was supported by the following grants: NINDS NS-39793 and USAMRMC DAMD17-01-1-0762; N.E.R.P.R.C. Center Grant P51RR00168. Support from the Orchard, Anti-Aging, and Stern foundations is greatly appreciated. D.F. is a fellow of the Vita-Salute San Raffaele University Ph.D. program.
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b Neuroregeneration Laboratories, McLean Hospital, Belmont, Massachusetts, USA;
c Laboratory of Stem Cell & Tumor Biology, Division of Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
Key Words. Parkinson’s disease ? Embryonic stem (ES) cell ? Transplantation ? Primate ? Differentiation
Correspondence: Rosario Sánchez-Pernaute, M.D., Ph.D., McLean Hospital/Harvard University Udall Parkinson’s Disease Research Center of Excellence and Neuroregeneration Laboratories, McLean Hospital, 115 Mill St., Belmont, Massachusetts 02478, USA. Telephone: 617-855-3568 ; Fax: 617-855-2522; e-mail: rosario_pernaute@hms.harvard.edu
ABSTRACT
Embryonic stem (ES) cells provide an unlimited source of cells that can be tailored to meet specific criteria for cell-replacement therapies. ES cell lines have been derived from human and nonhuman primate blastocysts, including parthenogenetic primate blastocysts, . Neuronal and glial cell lineages have been derived in vitro from these primate and human ES cell lines and examined in vivo in transplantation paradigms . However, a number of critical questions remain to be addressed to develop an ES cell–based therapy for neurodegenerative disorders, in particular for Parkinson’s disease (PD). These questions relate to survival and proliferation, stability of neuronal phenotypes induced in vitro, and capacity of synaptic integration and are therefore a prerequisite for investigation of functional potential.
The Cyno-1 cell line used in this study was derived from the inner cell mass of a parthenogenetic blastocyst and has similar characteristics to normal fertilization–derived primate ES cells . Dopamine (DA) neurons have been derived in vitro from this cell line . Neural precursors derived from mouse ES cells can be directed into defined neuronal phenotypes by the timed exposure to factors that control antero-posterior and dorso-ventral fate specification during normal development in vivo, including fibroblast growth factors (FGF) 4 and 8, retinoic acid, and sonic hedgehog (SHH) . To obtain DA neurons from primate ES cells in this study, we first used a stromal cell feeder coculture system for neural induction , followed by the sequential exposure to inductive signals, such as SHH and FGF8, that control DA specification during embryogenesis and factors that promote DA neuron differentiation and maturation. In this study, transplantation of in vitro differentiated Cyno-1 cells was performed into adult immunosuppressed rats and into the primate striatum (allograft) without immunosuppression.
MATERIALS AND METHODS
Transplantation of Differentiated Primate ES Cells into the Rat Striatum
In this protocol for in vitro neuronal differentiation (Fig. 1A), undifferentiated Cyno-1 cells (Fig. 1B) on MS5 or MS5-wnt1 feeder cells formed neural rosettes within 10–14 days (Fig. 1C). After replating on coated (feeder-free) dishes, neural rosettes were exposed to a second induction phase with SHH and FGF8 (Fig. 1A) which resulted in neuronal differentiation (Fig. 1D) and expression of Pax2 (day 28, not shown) followed by engrailed 1 (Fig. 1E) in 20% of the cells at the end of passage 2 expansion (day 35). Upon differentiation of passage 2 cells for 7 more days in the absence of SHH and FGF8 and in the presence of various trophic factors (Fig. 1A), 30%–60% of the cells expressed neuronal markers (TuJ-1), and up to 70% of these neurons coexpressed TH (Figs. 1F, 1G). These cells were briefly exposed to fresh mitomycin C immediately before being harvested and dissociated into a cell suspension for grafting. We previously determined the effect of mitomycin C on graft size and proliferation 3 weeks after transplantation (see below and supplemental online Fig. 2). Some cells were replated in differentiation media and fixed 3, 5, and 12 days later for control of mitomycin C and cell-preparation effect (supplemental online Fig. 1). The replated cells formed small clusters in which the majority of cells were nestin or TuJ1-positive; 2%–3% of the TuJ1-positive cells were TH, and 10% were Bf1-positive cells (not shown); these percentages were similar at 5 and 12 days. Although these may not correspond exactly to the com-position of the population in the cell suspension, due to the effect of culture conditions upon replating, such as cell density and media, these data suggest that TH-positive cells are particularly sensitive to dissociation and replating compared with the overall neuronal population within the grafted cell suspension.
We transplanted these differentiated ES cells into rats (n = 7) and into one primate (see below). Rat brains were examined 16 weeks post-transplantation; six animals had surviving grafts. Graft volumes were small (2.32 ± 0.6 mm3), and PCNA expression in the graft core was low (supplemental online Fig. 2C). TH-positive neurons were found in all the grafts close to the host-graft interface (Figs. 2B, 2C) in a typical peripheral distribution around the core of the graft, which was strongly immunoreactive for double-cortin (Dcx, not shown), a neurofilament associated protein highly expressed by young and migrating neuroblasts, which is expressed only in neurogenic regions in the adult host brain. The TH-positive neurons in the graft were HNA-positive (Fig. 2D), DBH-negative, and calbindin-negative (not shown); some cell bodies and fibers expressed AADC and the transporters DAT and VMAT-2 (Figs. 2E–2H). DAT expression was observed in TH fibers projecting into the host (Fig. 2F). TH cell numbers in the grafts (557 ± 117, n = 6) were directly correlated with the graft volume (R2 = 0.91, p < 0.01) and inversely correlated with the percentage of cells expressing the forebrain marker Bf1 (R2 = 0.89, p < 0.01). Most of the HNA-positive cells in the core of the grafts expressed NeuN (Fig. 3B) and were positive for the forebrain transcription factor Bf1 (83% ± 4%, not shown). 5HT neurons (not shown) were present in the grafts in lower numbers (65 ± 19, 1:5 TH-positive). At 16 weeks, many primate (HNA-positive) cells were found away from the core of the graft (Fig. 3), mainly in the ipsilateral striatum and also along the corpus callosum and the anterior commissure, reaching the contra-lateral hemisphere. Semiquantitative estimation of HNA-positive cell density (Fig. 3A) showed a gradient away from the graft. Many of these cells away from the core expressed Dcx (some organized in migratory chains, Fig. 3G) or, in white matter tracts, glial markers (GFAP and O4, Figs. 3D–3F), and few were NeuN-positive, which were located in close proximity to the graft core. There were some HNA-positive cells in the subventricular zone (SVZ) (Fig. 3H), which were GFAP-negative and either positive or negative for Dcx.
Figure 2. Grafted cells were identified by HNA expression (B–D) 4 months post-transplantation into the rat striatum.(A):Computer-generated graft contour and (B) virtual slice acquired on a representative coronal section double immunostained for TH and HNA. The boxed area is shown in (B). TH-positive neurons derived from primate ES cells were (B) located at the periphery of the grafts and (C) showed nuclear expression of the primate and human specific marker HNA, as shown in (D) in a confocal reconstruction. (E, F): TH neurons in the grafts expressed AADC (E) and some TH-positive neurites expressed DAT (E–G). In (G) DAT and TH coexpression is shown in a 3-D reconstruction of a confocal z-stack through a DA fiber. (H): VMAT-2 was expressed in TH-positive and -negative (presumably serotonergic) fibers. Scale bars = 2 mm (A), 250 μm (B), 100 μm (C), 30 μm (D–F, H), and 10 μm (G). Abbreviations: AADC, aromatic amino acid decarboxylase; ac, anterior commisure; cx, cortex; DA, dopamine; DAT, dopamine transporter; ES, embryonic stem; g, graft; HNA, human nuclear antigen; st, striatum; TH, tyrosine hydroxylase; VMAT, vesicular monoamine transporter.
Figure 3. Primate HNA-positive cells (HNA is shown in red in all panels) migrated away from the graft core 4 months post-transplantation into the rat striatum. (A): Semiquantitative estimation of HNA-positive cell density was performed on serial sections, and the average density for the 6 grafted animals is shown on representative coronal sections (see Materials and Methods). (B, C): In the graft core, most HNA-positive cells expressed (B) NeuN, and not (C) GFAP. (D, F): HNA-positive glial cells were found along white matter tracts: astro-cytes and oligodendrocytes were observed along the corpus callosum. Outside the core of the graft there were some Dcx-positive neuroblasts in chain formations. (H): Some HNA-positive cells were found in the SVZ ipsilateral to the graft. Nuclear counterstain (Hoechst) is shown in blue in (D), (E), and (H), and white in (F). Scale bars = 100 μm (B–E), 15 μm (F), 30 μm (G), and 75 μm (H). Abbreviations: GFAP, glial fibrillary acidic protein; HNA, human nuclear antigen; il, ipsilateral to the graft; LV, lateral ventricle; SVZ, subventricular zone.
Transplantation of Differentiated Primate ES Cells into the Primate Striatum
Cells from the same preparation were transplanted into the right postcommissural putamen of a cynomolgus monkey. In this case (allograft), the animal did not receive immunosuppression. Motor scores were evaluated weekly and did not show any asymmetry up to 7 months post-transplantation. Because in this model parkinsonian signs are bilateral and symmetric and the transplant was unilateral, the absence of motor asymmetry is consistent with neither positive nor adverse effects of the grafts. Macroscopic examination of the brain disclosed two grafts at the sites of the injections. The graft volumes were 14 mm3 for the anterior deposit and 5.4 mm3 for the posterior graft (Fig. 4A). The grafts were homogeneous and had a slightly higher cell density than the host (Figs. 4D, 4F, 4H). There were no regions of non-neural differentiation, and the grafts did not displace or compress the striatal host parenchyma. Numerous TH-positive cells were located predominantly in the periphery of the graft cores (Figs. 4A, 4B), in a similar arrangement and morphology seen for TH/HNA-positive cells found in the rodent brain (Figs. 2B, 2C). Stereological counts of TH-positive cells showed 2,440 positive cells in the anterior and 880 in the posterior graft (thus, TH cells in vivo were 0.1% of grafted cells). Many of these TH neurons coexpressed AADC (Fig. 4E). As shown in the rodent grafts (Figs. 2E, 2F), some TH fibers within and close to the graft coexpressed DAT (Fig. 4E), but DAT immunoreactivity was not observed in the cell bodies. The grafts contained many NeuN-positive neurons (Figs. 4D, 4F) that coexpressed Bf1 (Fig. 4F). As in the rodent grafts, few neurons in these grafts expressed 5HT (not shown). Clusters of calretinin-positive neurons and some calbindin-positive neurons were observed within the grafts (Fig. 4G). Dcx expression was not detected in the graft core, in contrast with the widespread immunoreactivity for this marker observed in the core in rodent grafts at 4 months. Nevertheless, some Dcx-positive neuroblasts were observed in the primate grafts forming chains (Fig. 4H) reminiscent of those found in the rodents away from the graft core (Fig. 3G). The blood vessels located in the proximity of the grafts showed some perivascular cuffing with strong PCNA positivity, and microglial (CD68-positive) cells were observed in these areas (Fig. 4I).
Figure 4. Transplantation of DA cells derived from Cyno-1 ES cells into the primate putamen. (A): Computer-generated outline of the graft in the postcommissural putamen and map of the distribution of TH-positive neurons on a representative coronal section. Each green dot represents a TH cell. (B): Low power microphotograph of a graft, the boxed area is shown in (C) and the asterisks mark the same blood vessel in both images. Clusters of TH-positive cells (the DAB product appears dark brown) were located close to the graft-host interface, where there was a higher nuclear density and higher density of TH-positive fibers (C, D) than in the graft core; (E) many TH-positive cells in the grafts coexpressed AADC but not all and coexpressed the DAT in neurites but not in the cell bodies. (F, G): The graft core was predominantly neuronal (NeuN-positive) with most neurons coexpressing Bf1 (coexpression with NeuN appears pink in (F). Clusters of neurons inside the graft expressed calretinin (G) and calbindin . Although the morphology of the neurons inside and outside the graft was the same, neurons within the graft were closer to each other than in the host striatum. (H): Dcx-positive neuroblasts in a chain-like formation (see also Fig. 3). (I): PCNA-positive cells within and close to the graft were mainly located in perivascular spaces and some corresponded to microglia (CD68-positive) and astroglia (GFAP-positive). Scale bars = 2.5 mm (A), 125 μm (B), 100 μm (C), 20 μm (D–G), 50 μm (H) and (I). Nuclear counterstain (Hoechst) is shown in blue in (D) and (H) and in white in (F). * represents blood vessel. Abbreviations: AADC, aromatic amino acid decarboxylase; cd, caudate nucleus; DA, dopamine; DAB, 3, 3'-diaminobenzidine; DAT, dopamine transporter; ES, embryonic stem; GFAP, glial fibrillary acidic protein; GP, globus pallidus; ic, internal capsule; PCNA, proliferating cell nuclear antigen; put, putamen; TH, tyrosine hydroxylase.
DISCUSSION
This work was supported by the following grants: NINDS NS-39793 and USAMRMC DAMD17-01-1-0762; N.E.R.P.R.C. Center Grant P51RR00168. Support from the Orchard, Anti-Aging, and Stern foundations is greatly appreciated. D.F. is a fellow of the Vita-Salute San Raffaele University Ph.D. program.
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