Birth dating of midbrain dopamine neurons identifies A9 enriched tissue for transplantation into parkinsonian mice.

Clinical trials have provided proof of principle that new dopamine neurons isolated from the developing ventral midbrain and transplanted into the denervated striatum can functionally integrate and alleviate symptoms in Parkinson's disease patients. However, extensive variability across patients has been observed, ranging from long-term motor improvement to the absence of symptomatic relief and development of dyskinesias. Heterogeneity of the donor tissue is likely to be a contributing factor in the variable outcomes. Dissections of ventral midbrain used for transplantation will variously contain progenitors for different dopamine neuron subtypes as well as different neurotransmitter phenotypes. The overall impact of the resulting graft will be determined by the functional contribution from these different cell types. The A9 substantia nigra pars compacta dopamine neurons, for example, are known to be particularly important for motor recovery in animal models. Serotonergic neurons, on the other hand, have been implicated in unwanted dyskinesias. Currently little knowledge exists on how variables such as donor age, which have not been controlled for in clinical trials, will impact on the final neuronal composition of fetal grafts. Here we performed a birth dating study to identify the time-course of neurogenesis within the various ventral midbrain dopamine subpopulations in an effort to identify A9-enriched donor tissue for transplantation. The results show that A9 neurons precede the birth of A10 ventral tegmental area dopamine neurons. Subsequent grafting of younger ventral midbrain donor tissue revealed significantly larger grafts containing more mitotic dopamine neuroblasts compared to older donor grafts. These grafts were enriched with A9 neurons and showed significantly greater innervation of the target dorso-lateral striatum and DA release. Younger donor grafts also contained significantly less serotonergic neurons. These findings demonstrate the importance of standardized methods to improve cell therapy for Parkinson's disease and have significant implications for the generation and selectivity of dopamine neurons from stem cell based sources.

Lewy body pathology in fetal grafts.

Although fetal nigral transplants have been shown to survive grafting into the striatum, increased [(18)F]6-fluroro-L-3,4-dihydroxyphenylalanine ((18)F-DOPA) uptake and improved motor function in open-label assessments have failed to establish any clinical benefits in double-blind, sham-controlled studies. To understand morphological and neurochemical alterations of grafted neurons, we performed postmortem analyses on six Parkinson's disease (PD) patients who had received fetal tissue transplantation 18-19 months, 4 years, and 14 years previously. These studies revealed robust neuronal survival with normal dopaminergic phenotypes in 18-month-old grafts and decreased dopamine transporter and increased cytoplasmic alpha-synuclein in 4-year-old grafts. We also found a decline of both dopamine transporter and tyrosine hydroxylase and the formation of Lewy body-like inclusions in 14-year-old grafts, which stained positive for alpha-synuclein and ubiquitin proteins. These pathological changes suggest that PD is an ongoing process that affects grafted cells in the striatum in a manner similar to how resident dopamine neurons are affected in the substantia nigra.

Histological findings on fetal striatal grafts in a Huntington's disease patient early after transplantation.

Cell transplantation is a promising therapeutic approach that has the potential to replace damaged host striatal neurons and, thereby, slow down or even reverse clinical signs and symptoms during the otherwise fatal course of Huntington's disease (HD). Open-labeled clinical trials with fetal neural transplantation for HD have demonstrated long-term clinical benefits for HD patients. Here we report a postmortem analysis of an individual with HD 6 months after cell transplantation and demonstrate that cells derived from grafted fetal striatal tissue had developed into graft-derived neurons expressing dopamine-receptor related phosphoprotein (32 kDa) (DARPP-32), neuronal nuclear antigen (NeuN), calretinin and somatostatin. However, a fully mature phenotype, considered by the expression of developmental markers, is not reached by engrafted neurons and not all types of interneurons are being replaced at 6 months, which is the earliest time point human fetal tissue being implanted in a human brain became available for histological analysis. Host-derived tyrosine hydroxylase (TH) fibers had already heavily innervated the transplants and formed synaptic contacts with graft-derived DARPP-32 positive striatal neurons. In parallel, the transplants contained a considerable number of immature neuroepithelial cells (doublecortin+, Sox2+, Prox-1+, ss3-tubulin+) that exhibited a pronounced migration into the surrounding host striatal tissue and considerable mitotic activity. Graft-derived astrocytes could also be found. Interestingly, the immunological host response in the grafted area showed localized increase of immunocompetent host cells within perivascular spaces without deleterious effects on engrafted cells under continuous triple immunosuppressive medication. Thus this study provides for a better understanding of the developmental processes of grafted human fetal striatal neurons in HD and, in addition, has implications for stem cell-based transplantation approaches in the CNS.

The effect of cerebellar transplantation and enforced physical activity on motor skills and spatial learning in adult Lurcher mutant mice.

Lurcher mutant mice represent a model of olivocerebellar degeneration. They are used to investigate cerebellar functions, consequences of cerebellar degeneration and methods of therapy influencing them. The aim of the work was to assess the effect of foetal cerebellar graft transplantation, repeated enforced physical activity and the combination of both these types of treatment on motor skills, spontaneous motor activity and spatial learning ability in adult B6CBA Lurcher mice. Foetal cerebellar grafts were applied into the cerebellum of Lurchers in the form of solid tissue pieces. Enforced motor activity was realised through rotarod training. Motor functions were examined using bar, ladder and rotarod tests. Spatial learning was tested in the Morris water maze. Spontaneous motor activity in the open field was observed. The presence of the graft was examined histologically. Enforced physical activity led to moderate improvement of some motor skills and to a significant amelioration of spatial learning ability in Lurchers. The transplantation of cerebellar tissue did not influence motor functions significantly but led to an improvement of spatial learning ability. Mutual advancement of the effects of both types of treatment was not observed. Spontaneous motor activity was influenced neither by physical activity nor by the transplantation. Physical activity did not influence the graft survival and development. Because nerve sprouting and cell migration from the graft to the host cerebellum was poor, the functional effects of the graft should be explained with regard to its trophic influence rather than with any involvement of the grafted cells into neural circuitries.

A patient with Huntington's disease and long-surviving fetal neural transplants that developed mass lesions.

Transplantation of human fetal neural tissue into adult neostriatum is an experimental therapy for Huntington's disease (HD). Here we describe a patient with HD who received ten intrastriatal human fetal neural transplants and, at one site, an autologous sural nerve co-graft. Although initially clinically stable, she developed worsening asymmetric upper motor neuron symptoms in addition to progression of HD, and ultimately died 121 months post transplantation. Eight neural transplants, up to 2.9 cm, and three ependymal cysts, up to 2.0 cm, were identified. The autologous sural nerve co-graft was found adjacent to the largest mass lesion, which, along with the ependymal cyst, exhibited pronounced mass effect on the internal capsules bilaterally. Grafts were composed of neurons and glia embedded in disorganized neuropil; robust Y chromosome labeling was present in a subset of grafts and cysts. The graft-host border was discrete, and there was no evidence of graft rejection or HD pathologic changes within donor neurons. This report, for the first time, highlights the potential for graft overgrowth in a patient receiving fetal neural transplantation.

Dopamine neurons implanted into people with Parkinson's disease survive without pathology for 14 years.

Postmortem analysis of five subjects with Parkinson's disease 9-14 years after transplantation of fetal midbrain cell suspensions revealed surviving grafts that included dopamine and serotonin neurons without pathology. These findings are important for the understanding of the etiopathogenesis of midbrain dopamine neuron degeneration and future use of cell replacement therapies.

Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease.

Fourteen years after transplantation into the striatum of an individual with Parkinson's disease, grafted nigral neurons were found to have Lewy body-like inclusions that stained positively for alpha-synuclein and ubiquitin and to have reduced immunostaining for dopamine transporter. These pathological changes suggest that Parkinson's disease is an ongoing process that can affect grafted cells in the striatum in a manner similar to host dopamine neurons in the substantia nigra. These findings have implications for cell-based therapies and for understanding the cause of Parkinson's disease.

Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation.

Two subjects with Parkinson's disease who had long-term survival of transplanted fetal mesencephalic dopaminergic neurons (11-16 years) developed alpha-synuclein-positive Lewy bodies in grafted neurons. Our observation has key implications for understanding Parkinson's pathogenesis by providing the first evidence, to our knowledge, that the disease can propagate from host to graft cells. However, available data suggest that the majority of grafted cells are functionally unimpaired after a decade, and recipients can still experience long-term symptomatic relief.

Differentiation of xenografted human fetal lung parenchyma.

The goal of this study was to characterize xenografted human fetal lung tissue with respect to developmental stage-specific cytodifferentiation. Human fetal lung tissue (pseudoglandular stage) was grafted either beneath the renal capsule or the skin of athymic mice (NCr-nu). Tissues were analyzed from 3 to 42 days post-engraftment for morphological alterations by light and electron microscopy (EM), and for surfactant protein mRNA and protein by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC), respectively. The changes observed resemble those seen in human lung development in utero in many respects, including the differentiation of epithelium to the saccular stage. Each stage occurred over approximately one week in the graft in contrast to the eight weeks of normal in utero development. At all time points examined, all four surfactant proteins (SP-A, SP-B, SP-C, and SP-D) were detected in the epithelium by ICC. Lamellar bodies were first identified by EM in 14 day xenografts. By day 21, a significant increase in lamellar body expression was observed. Cellular proliferation, as marked by PCNA ICC and elastic fiber deposition resembled those of canalicular and saccular in utero development. This model in which xenografted lung tissue in different stages of development is available may facilitate the study of human fetal lung development and the impact of various pharmacological agents on this process.

Murine fetal small-intestine grafts: morphometric and immunohistochemical evaluation.

We investigated histopathological changes following murine fetal intestinal transplantation. Fetal intestine, obtained from a pregnant C57BL/6 mouse, was transplanted into BALB/c and C57Bl/6 mice. Recipients were divided into three groups: isogeneic, and allogeneic treated with 3 mg/kg/day gangliosides (Allo-a) or 9 mg/kg/day (Allo-b). One week after transplant, all grafts showed good viability, confirmed by cellular mitosis in the mucosa and a well-defined propria muscular layer. Isogeneic grafts showed a thicker muscular layer than in the Allo-a (P = 0.02) and Allo-b (P = 0.004) groups. There was no difference in number of mitotic cells among groups. Goblet cells were significantly reduced in allografts treated with 3 mg gangliosides (P = 0.013) or 9 mg gangliosides (P = 0.002) compared to isografts. Villi height was similar in all studied groups. There was no difference in positivity of the enteric nervous system among groups. Atrophy was more common in the allogeneic groups, suggesting that isografts had better development than allografts treated with gangliosides. (

Human fetal islet transplantation in type 1 diabetic patients: comparison of metabolic effects between single and multiple implantation regimens.

Previous studies suggest that multiple transplantations might be equally efficient to a single regimen for human adult islets. The aim of this study was to compare metabolic parameters after each of the two regimens of human fetal islet (HFI) transplantation in type 1 diabetics. In group A (single transplant, n = 9), 180 +/- 20 x 1000 HFI equivalents (IEQs) were implanted by a single IM injection; in group B (multiple transplants, n = 8) islets were implanted as three consecutive injections (60 +/- 10 x 1000 IEQs) at 7-day intervals. We analyzed the metabolic parameters on days -1, 30, 60, 90, 120, 150, and 180 after the procedure. Among the metabolic parameters, we evaluated insulin secretion capacity-ISC (C peptide, RIA), metabolic control (HbA1c, chromatography), and insulin daily dose IDD. We found that C peptide levels increased, peaking on day 90 (A: 0.38 +/- 0.15; B: 0.34 +/- 0.19 nmol/L, P = NS) and then rapidly decreasing without differences, the HbA1c levels and IDD decreased in the same manner without differences between the groups. Our results demonstrate that multiple and single islet transplant regimens are equally efficient to temporarily restore a significant ISC with improvement of metabolic and clinical parameters. The results imply that the two regimens have an equal clinical value.

Functional improvement with low-dose dopaminergic grafts in hemiparkinsonian rats.

OBJECTIVE: The beneficial functional effects of neural transplantation in Parkinson's disease are often directly attributed to the number of surviving dopaminergic cells within a graft. However, recent clinical trials of fetal neural transplantation suggest that a high number of dopaminergic cells may induce serious side effects. In this study, we explored the ability of low-dose dopaminergic grafts to produce functional benefits in the 6-hydroxydopamine rodent model of Parkinson's disease over a long period of observation. METHODS: Twelve rats received either 50,000 or 400,000 fetal ventral mesencephalic cells implanted into the striatum. Rotational behavior was assessed after the lesion and at 3, 6, 9, and 12 weeks after transplantation. Twelve weeks after transplantation, animals were perfused, and microtome sections were stained for tyrosine hydroxylase, glial fibrillary acidic protein, heat-shock protein 27, and vimentin. RESULTS: The low-dose group had a three-fold increase in tyrosine hydroxylase-positive cell survival rate compared with the high-dose group rate. The low-dose group also had a mean cell diameter significantly higher than the high-dose group. There was no significant difference between groups in fiber density; however, a higher percentage of longer fibers was encountered in the low-dose group. The low-dose group had a lower degree of trauma in the striatum, as assessed by optical density scores from glial fibrillary acidic protein, heat-shock protein 27, and vimentin staining. There was significant improvement in rotational behavior in the high-dose group at 3 weeks after transplantation, whereas the rotational behavior normalized in the low-dose group at 6 weeks after grafting. There was no significant difference in rotational behavior scores between groups at 6 weeks after grafting. CONCLUSION: This study demonstrates that over time, a low-dose dopaminergic graft has the capability of eliciting the same functional effect as a high-dose graft. Furthermore, low-dose grafts may increase graft survival, fiber outgrowth, and dopamine production and decrease trauma to the brain.

Amelioration of hyperglycemia in streptozotocin-induced diabetic mice with fetal pancreatic allografts: prevention of rejection by donor specific transfusion in conjunction with CTLA4Ig.

INTRODUCTION: Fetal pancreas has been considered as an alternative donor source for islet transplantation since it has potent capacity for beta cell differentiation and proliferation. However, prevention of fetal pancreatic allograft rejection can be hardly achieved compared with adult islet allografts. AIMS: The aim of the study is to determine whether donor specific transfusion (DST) in conjunction with CTLA4Ig has any favorable effect on prevention of fetal pancreatic allograft rejection in mice. METHODS: BALB/c splenocytes (SPC, 1 x 10) were injected iv into C57BL/6 mice in conjunction with CTLA4Ig (ip, 50 microgram, day 0, 2, and 4). Fourteen days later, the mice were made diabetic with streptozotocin (STZ, iv) and donor specific or third party pancreatic allografts were transplanted beneath the kidney capsule. RESULTS: Morphologically, it was found that rejection of fetal pancreatic allografts can be prevented at 30 days after transplantation only when donor specific allografts were grafted into the mice treated with DST in conjunction with CTLA4Ig. Functionally, 3 out of 9 diabetic mice became normoglycemic by 120 days after transplantation of fetal pancreatic allografts. CONCLUSION: DST in conjunction with CTLA4Ig can have a favorable effect on prevention of fetal pancreatic allograft rejection resulting in amelioration of STZ-induced diabetes in mice.

NF-kappa B-inducing kinase establishes self-tolerance in a thymic stroma-dependent manner.

Physical contact between thymocytes and the thymic stroma is essential for T cell maturation and shapes the T cell repertoire in the periphery. Stromal elements that control these processes still remain elusive. We used a mouse strain with mutant NF-kappaB-inducing kinase (NIK) to examine the mechanisms underlying the breakdown of self-tolerance. This NIK-mutant strain manifests autoimmunity and disorganized thymic structure with abnormal expression of Rel proteins in the stroma. Production of immunoregulatory T cells that control autoreactive T cells was impaired in NIK-mutant mice. The autoimmune disease seen in NIK-mutant mice was reproduced in athymic nude mice by grafting embryonic thymus from NIK-mutant mice, and this was rescued by supply of exogenous immunoregulatory T cells. Impaired production of immunoregulatory T cells by thymic stroma without normal NIK was associated with altered expression of peripheral tissue-restricted Ags, suggesting an essential role of NIK in the thymic microenvironment in the establishment of central tolerance.

Most IL-4-producing gamma delta thymocytes of adult mice originate from fetal precursors.

Thy-1(dull) gammadelta T cells constitute a distinct adult gammadelta T cell subset characterized by the expression of a TCR composed of Vgamma1Cgamma4 and Vdelta6Cdelta chains with limited junctional sequence diversity. However, several features of the expressed Thy-1(dull) TCR-gammadelta genes, in particular the absence or minimal presence of N region diversity and the almost invariable Ddelta2-Jdelta1 junction, are typical of rearrangements often found in the fetal thymus. In this study, we have investigated the origin of these cells. Few Thy-1(dull) gammadelta thymocytes developed in syngeneic radiation adult chimeras, regardless of whether the recipient mice were given adult bone marrow or fetal liver cells as a source of hemopoietic precursors. In contrast, normal numbers of Thy-1(dull) gammadelta T cells developed in fetal thymi grafted into adult syngeneic recipients. Interestingly, the majority of Thy-1(dull) gammadelta thymocytes present in the grafts were of graft origin, even when most conventional gammadelta and alphabeta thymocytes in the grafted thymi originated from T cell precursors of recipient origin. Single-cell PCR analyses of the nonselected TCR-gamma rearrangements present in adult Thy-1(dull) gammadelta thymocytes revealed that more than one-half of these cells represent the progenies of a limited number of clones that greatly expanded possibly during the first weeks of life. Finally, the second TCR-delta allele of a large number of Thy-1(dull) gammadelta T cells contained incomplete TCR-delta rearrangements, thus providing an explanation for the adult-type rearrangements previously found among nonfunctional V(D)J rearrangements present in Thy-1(dull) gammadelta thymocytes.

Pathogenesis of autoimmunity after xenogeneic thymus transplantation.

Thymus transplantation is a promising strategy to induce xenotolerance, but may also induce an autoimmune syndrome (AIS). The pathogenesis of this AIS was explored using nude rats as recipients. Thymus grafts consisted of fetal hamster thymic tissue with or without mixing with fetal rat tissue such as thymus, thyroid, salivary gland, and heart. All hamster thymus recipients died of AIS within 2-3 mo. In most recipients of xenothymus mixed with rat tissues such as thymus, thyroid, and salivary gland, but not heart, AIS was prevented, indicating an insufficient presence of rat epithelial cell Ags within the xenothymus. AIS could be transferred to control nude rats by whole splenocytes or by splenocyte subpopulations such as CD3(+), CD3(-), and B lymphocytes, but not by non-T, non-B cells from AIS animals. This transfer could be suppressed by cotransferring either CD4(+) or CD8(+) lymphocytes from euthymic rats, but not by splenocytes from recipients of syngeneic or xenogeneic thymus mixed with rat tissue, indicating a defective generation of regulatory lymphocytes. As for CD4(+) regulatory cells this defect was probably qualitative, because the percentages of CD4(+)CD25(+) or CD4(+)CD45RC(low) populations were normal after xenothymus transplantation. As for the CD8(+) regulatory cells, the defect was quantitative, as CD8(+) cell levels always remained low. The latter was related to the nonvascularized nature of thymus grafts. In conclusion, AIS after xenothymus transplantation in nude rats is due to a combination of insufficient intrathymic presence of host-type epithelial cell Ags and a defective generation of regulatory T lymphocytes.

Mucosally-derived HPV-40 can infect both human genital foreskin and cutaneous hand skin tissues grafted into athymic mice.

HPV-40 is a rare HPV type that has been detected only in genital mucosal tissues. This HPV type is very closely related to HPV-7, which has a predominantly cutaneous tissue tropism. We have shown, previously, that an isolate of HPV-40 (described here as HPV-40(Hershey) or HPV-40(H)) productively infected genital tissues. In this study, HPV-40(H) was tested for productive infection of cutaneous tissue. Fetal hand skin fragments were incubated with infectious HPV-40(H) and implanted subrenally into athymic mice. After 120 days, xenografts showed morphological changes consistent with HPV-40(H) infection and were HPV-40 DNA in situ positive and capsid antigen positive. The results demonstrated that hand skin can support HPV-40(H) infection thereby indicating that this viral type has the capacity to infect both genital mucosal and cutaneous tissues.

A new murine model of islet xenograft rejection: graft destruction is dependent on a major histocompatibility-specific interaction between T-cells and macrophages.

A new murine model of porcine islet-like cell cluster (ICC) xenograft rejection, avoiding interference of unspecific inflammation, was introduced and used to investigate rejection mechanisms. Athymic (nu/nu) mice were transplanted with syngeneic, allogeneic, or xenogeneic islets under the kidney capsule. After the original transplantation, immune cells in porcine ICC xenografts undergoing rejection in native immunocompetent mice were transferred to the peritoneal cavity of the athymic mice. At defined time points after transfer, the primary grafts were evaluated by immunohistochemistry and real-time quantitative RT-PCR to estimate cytokine and chemokine mRNA expression. Transfer of immunocompetent cells enabled athymic (nu/nu) mice to reject a previously tolerated ICC xenograft only when donor and recipient were matched for major histocompatibility complex (MHC). In contrast, allogeneic and syngeneic islets were not rejected. The ICC xenograft rejection was mediated by transferred T-cells. The main effector cells, macrophages, were shown to be part of a specific immune response. By day 4 after transplantation, there was an upreglation of both Th1- and Th2-associated cytokine transcripts. The transferred T-cells were xenospecific and required MHC compatibility to induce rejection. Interaction between the TCR of transferred T-cells and MHC on host endothelial cells and/or macrophages seems necessary for inducing ICC xenograft rejection.

A role of CXC chemokine ligand 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its receptor CXCR4 in fetal and adult T cell development in vivo.

The functions of a chemokine CXC chemokine ligand (CXCL) 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its physiologic receptor CXCR4 in T cell development are controversial. In this study, we have genetically further characterized their roles in fetal and adult T cell development using mutant and chimeric mice. In CXCL12(-/-) or CXCR4(-/-) embryos on a C57BL/6 background, accumulation of T cell progenitors in the outer mesenchymal layer of the thymus anlage during initial colonization of the fetal thymus was comparable with that seen in wild-type embryos. However, the expansion of CD3(-)CD4(-)CD8(-) triple-negative T cell precursors at the CD44(-)CD25(+) and CD44(-)CD25(-) stages, and CD4(+)CD8(+) double-positive thymocytes was affected during embryogenesis in these mutants. In radiation chimeras competitively repopulated with CXCR4(-/-) fetal liver cells, the reduction in donor-derived thymocytes compared with wild-type chimeras was much more severe than the reduction in donor-derived myeloid lineage cells in bone marrow. Triple negative CD44(+)CD25(+) T cell precursors exhibited survival response to CXCL12 in the presence of stem cell factor as well as migratory response to CXCL12. Thus, it may be that CXCL12 and CXCR4 are involved in the expansion of T cell precursors in both fetal and adult thymus in vivo. Finally, enforced expression of bcl-2 did not rescue impaired T cell development in CXCR4(-/-) embryos or impaired reconstitution of CXCR4(-/-) thymocytes in competitively repopulated mice, suggesting that defects in T cell development caused by CXCR4 mutation are not caused by reduced expression of bcl-2.