Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson's disease

Abstract

We report the first post-mortem analysis of two patients with Parkinson's disease who received fetal midbrain transplants as a cell suspension in the striatum, and in one case also in the substantia nigra. These patients had a favourable clinical evolution and positive 18F-fluorodopa PET scans and did not develop motor complications. The surviving transplanted dopamine neurons were positively identified with phenotypic markers of normal control human substantia nigra (n = 3), such as tyrosine hydroxylase, G-protein-coupled inward rectifying current potassium channel type 2 (Girk2) and calbindin. The grafts restored the cell type that provides specific dopaminergic innervation to the most affected striatal regions in the parkinsonian brain. Such transplants were able to densely reinnervate the host putamen with new dopamine fibres. The patients received only 6 months of standard immune suppression, yet by post-mortem analysis 3-4 years after surgery the transplants appeared only mildly immunogenic to the host brain, by analysis of microglial CD45 and CD68 markers. This study demonstrates that, using these methods, dopamine neuronal replacement cell therapy can be beneficial for patients with advanced disease, and that changing technical approaches could have a favourable impact on efficacy and adverse events following neural transplantation.

Figures

Fig. 1

(A–C) MRI study performed 24 h after the first surgery (patient 1). The four parallel needle tracks through the right putamen are visible in the axial (C) and sagittal (A) views (compare with 3D reconstruction of the grafts in Fig. 2D). (D–G) Parametric maps of F-DOPA uptake (Ki) overlaid on the patient’s MRI. (D–E) A preoperative PET scan showed a marked, asymmetrical decrease in putaminal 18F-DOPA uptake in the first patient, consistent with the diagnosis of idiopathic Parkinson’s disease. (F–G) Twenty-eight months after transplantation the PETs show a significant increase in 18F-DOPA uptake, more pronounced in the right putamen (>300% compared with preoperative values) than on the left (100% increase). Ki values are included in Table 1A. R = right; L = left.

Fig. 2

Morphological and cytoarchitectonic features of the cell suspension grafts in the putamen in patient 1. (A) Low-power microphotographs of TH immunostaining of a coronal section through the anterior portion of the grafts, at the level of the postcommissural putamen. (B) In the right putamen, clusters of TH-positive cells at the tip of three tracks are visible. (C) In the left hemisphere the cell infusion tracks are parallel to the section plane, so only the most anterior track is visible. TH-positive cells are predominantly located at the periphery of the grafts. (D–E) The spatial orientation of the grafts is demonstrated in the computer-assisted 3D reconstructions. This view shows the location of the grafts in the postcommissural putamen. The surviving cell aggregates spanned approximately 8 mm on the anterior–posterior axis in both hemispheres and the trajectories are easily identified in the sagittal view. The four parallel grafts (1–4) are numbered in anterior–posterior order for stereological analyses (see text and Table 2). P = putamen; Cd = caudate nucleus; V = lateral ventricle; A = anterior; Po = posterior; M = medial; L = lateral. Scale bars: A, 1 cm; B, C, 1 mm.

Fig. 3

(A) Parametric maps of F-DOPA uptake (Ki) overlaid on the second patient’s MRI before and 3 years after transplantation. Note the marked increase in 18F-DOPA uptake in the right putamen (>200%) while the loss progressed during this time period on the left side (45% loss) (Ki values are shown in Table 1B). (B) Macroscopic aspect of TH immunoreactivity at the level of the anterior commissure in the second patient. The right putamen was completely reinnervated by the TH neurons distributed in the six tracks (see schematic reconstruction in C) at this anatomical level. No surviving TH neurons were found in the left putamen (which received only one deposit), corresponding to in vivo data; partial volume effect and resolution of the PET precludes direct quantitative comparison between the histological and imaging studies. (C) 3D reconstruction of the six tracks in the right putamen. Numbers 1 and 2 followed a tangential direction from caudate to putamen; 3–6 are parallel (numbered anterior to posterior) to the major axis of the putamen. P = putamen; Cd = caudate nucleus; V = lateral ventricle; A = anterior; Po = posterior; M = medial; L = lateral. The boxed areas in B are shown at higher magnification in Fig. 4A and 4D. Scale bar in B, 1 cm.

Fig. 4

TH immunostaining of one of the transplants located in the right putamen of patient 2 showing the dense neurite outgrowth into the host putamen (central boxed area in Fig. 3B). Within the graft (B, boxed in A), TH-positive neurites from grafted neurons were thick and scarcely branched, while around the graft (C, boxed area in A), and further away (D, area shown boxed in Fig. 3B) they formed a dense network of fine branches approaching normal innervation in some areas of the putamen. Compare the fibre density with the contralateral putamen (E), where there was no graft survival. (F,G) GFAP immunostainings showing a representative transplant located in the putamen of patient 2. (F–H) Astroglial density was not increased in the graft core but around the graft deposits there was a band of variable thickness (<1 mm) of fibrous hypertrophic astrocytes (G, boxed in F). Further away, the astrocytic density and morphology were similar to those of normal striatum (H; illustrates a similar area from the putamen of patient 1). Microglial cells were identified by immunoreactivity against CD45 (common leucocyte antigen, CLA) and CD68 (activated microglia, not shown). (I–J) Representative microphotographs of CD45 immunostaining showing a local circumscribed increase in microglial cell density around needle tracks (arrows), which was very similar for all the grafts located in the putamen in patient 1 (shown in I) and in both the midbrain and the striatal deposits in patient 2. At higher magnification (J) a few macrophages could be observed along the needle track (arrow), but most microglial cells showed a typical resting branched morphology (see detail in the inset in K) comparable to that observed in striatal regions at a distance (away) from the grafts (K). Scale bar: A, 500 μm; F, I, 400 μm; G,H,J,K, 200 μm; C–E and inset in K, 75 μm; B, 25 μm.

Fig. 5

Distribution of subpopulations of DA neurons in human control midbrain. Maps of TH-positive neurons and their coexpression of calbindin (A) and Girk2 (B), two markers differentially expressed by Ventral mesencephalic DA neurons. Maps were generated from transverse serial sections double-immunolabelled for TH and each marker. Each dot represents a cell. (A) Calbindin/TH neurons, which are relatively spared in Parkinson’s disease, are located in medial and dorsal regions and not found in ventral SNc. (B) Girk2/TH neurons are predominantly located in the ventral tier of the SNc, the most vulnerable region in Parkinson’s disease. A8 = retrorubral area; A9 = substantia nigra; A10 = ventral tegmental area; CP = cerebral peduncle; RN = red nucleus; SNc = substantia nigra pars compacta; VTA = ventral tegmental area.

Fig. 6

Maps of the dopamine subpopulations, TH/Girk2-positive neurons (red) and TH/Girk2-negative neurons (green) in the putaminal grafts. The maps were generated from representative transverse sections double-immunolabelled for TH and Girk2 using Neurolucida software. Each dot represents one cell. (A, patient 1, and B, patient 2) TH/Girk2-positive neurons were preferentially located in the outer layer of the grafts in the putaminal grafts. (C) Confocal images of triple immunofluorescence studies of TH (green), Girk2 (red) and calbindin (blue) within a putaminal graft (see Table 2 for quantification). Numbers identified the tracks as described in Fig. 3. TH = tyrosine hydroxylase; CB = calbindin; Cd = caudate; Put = putamen; V = lateral ventricle; ic = internal capsule. Scale bar in C, 50 μm.

Fig. 7

Midbrain graft. (A) Brightfield photograph of a representative unstained section showing the two nigral deposits identified in the right midbrain, at the border between the red nucleus and the rostral part of the SN. (B) Schematic representation of the stereological 3D reconstruction of the grafts in the right midbrain. At this level two parallel deposits were found between the red nucleus and the rostral SN, spanning 6.5 mm along the rostrocaudal axis. (C–F) TH immunostaining showed a typical disposition of transplanted neurons in the periphery of the graft. There were fewer TH neurons (Table 2) than in the striatal deposits and limited outgrowth. Higher magnification of three areas in this same deposit showing the small TH neurons within the graft where there is no lipofuscin (reddish deposits in the microphotograph). Arrows in D and E point to host neurons which are bigger and heavily melanized. (G–H) Confocal images of TH neurons in the graft. Some TH neurons coexpressed Girk2 and calbindin (colours as in Fig. 3) (Table 2). (I) Two dopamine subpopulations TH-positive/Girk2-positive (red) and TH-positive/Girk2-negative (green) were mapped in the mesencephalon of this patient at the level of the red nucleus (graft location), showing a high expression of Girk2 except in the midline populations. Around 50% of TH cells in the midbrain grafts coexpressed Girk2 (Table 2). In the mapped sections there was no preferential distribution of the TH/Girk2 (red) population in the margins of the deposits. Scale bar: C, 150 μm; D–H, 50 μm.