Transplantation Of Fetal Tissue Research Articles

Role of Neural Stem Cells in Parkinsons Disease

Human fetal mesencephalic tissue can be successfully used in dopamine (DA) cell replacement therapy in Parkinsons disease (PD), nevertheless, further advances have been impeded due to limited tissue availability. Stem cell-based approaches have received much hype as potential treatments for neurodegenerative disorders, but have shown poor cell survival. The brains of three patients with Parkinsons disease who had undergone transplants of fetal tissue were examined postmortem and it was observed that some transplanted cells contained clumps of protein containing alpha-synuclein. Inducible pluripotent stem (iPS) cells derived from somatic cells and human embryonic stem cells, represent a novel renewable source of tissue precursors and therapeutic safety necessitate much more in-depth research before the initiation of human clinical trials. Keywords: Parkinson's disease, neural stem cells, replacement therapy, induction of pluripotent stem cells, Stem Cells, therapy in Parkinson's disease, neurodegenerative disorders, Inducible pluripotent stem (iPS) cells, neural transplantation, somatic cells and human embryonic stem cells, embryonic stem cells, Stem Cell, –, Based Regeneration, Human ES cell-derived dopaminergic neurons, cell therapy in neurodegenerative diseases, neural stem cells in Parkinson's disease, Gene therapy, Regeneration-based therapies, embryonic germ cells, epigenetic reprogramming, Stem Cell Research, stem cell-specific expertise, human embryonic pluripotent stem cells, adult stem cells, stem cell-based therapies, progenitor cell transplants, Transplanted dopaminergic

Dopamine cell transplantation in Parkinson's disease: challenge and perspective

Functional imaging provides a valuable adjunct to clinical evaluation for assessing the efficacy of cell-based restorative therapies in Parkinson's disease (PD). In this article, we review the latest advances on the use of positron emission tomography (PET) imaging in evaluating the surgical outcome of embryonic dopamine (DA) cell transplantation in PD patients. These studies suggest long-term cell survival and clinical benefit following striatal transplantation of fetal nigral tissue in PD patients and in models of experimental parkinsonism. Adverse events subsequent to transplantation have also been noted and attributed to a variety of causes. Optimal outcomes of DA cell transplantation therapies are dependent on tissue composition and phenotype of DA neurons in the graft. Given continued progress in DA neuron production from stem cells in recent years, transplantation of neural stem cells may be the next to enter clinical trials in patients. The existing data from studies of embryonic DA transplantation for advanced PD have provided valuable insights for the design of new cell-based therapies for the treatment of this and related neurodegenerative disorders.

Graft‐induced dyskinesias in Parkinson's disease: High striatal serotonin/dopamine transporter ratio

AbstractGraft‐induced dyskinesias are a serious complication after neural transplantation in Parkinson's disease. One patient with Parkinson's disease, treated with fetal grafts 14 years ago and deep brain stimulation 6 years ago, showed marked improvement of motor symptoms but continued to suffer from OFF‐medication graft‐induced dyskinesias. The patient received a series of clinical and imaging assessments. Positron emission tomography and single‐photon emission computed tomography 14 years posttransplantation revealed an elevated serotonin/dopamine transporter ratio in the grafted striatum compatible with serotonergic hyperinnervation. Inhibition of serotonin neuron activity by systemic administration of a 5‐HT1A agonist suppressed graft‐induced dyskinesias. Our data provide further evidence that serotonergic neurons mediate graft‐induced dyskinesias in Parkinson's disease. Achieving a normal striatal serotonin/dopamine transporter ratio following transplantation of fetal tissue or stem cells should be necessary to avoid the development of graft‐induced dyskinesias. © 2011 Movement Disorder Society

Importance of stoichiometry in cells science: iPSC, CNS leukocytes, and more

the hope of becoming the source and basis of sideeffect–free ‘‘medication’’ and repair of deficits in our body,stem cells have been, for a few decades, of interest in basic cellresearch, regenerative medicine, and public opinion. Cytome-try was from the beginning a guiding technology in thisendeavor. The substantial past and present scientific interest isreflected by the fact that publications on stem cells and theirunequivocal identification are the ‘‘best sellers’’ regarding cita-tion and download rates also in the cytometry-focused litera-ture (1). Nevertheless, stem cell therapy still plays a minor rolein clinical practice. The main stem cell therapy is allogenic orautologous bone marrow transplantation in cancer or insevere life-threatening autoimmune diseases such as rheuma-toid arthritis. This transplantation is either done by crudebone marrow allografting or by using purified (i.e., sorted orotherwise enriched) material.Another far less frequent application is transplantation offetal brain tissue in Parkinson’s disease, rendering (temporary)relief from the symptoms. Fetal brain transplant in Hunting-ton’s disease was far less successful as indicated by clinicaltrials enrolling a limited number of patients. In addition,transplantation of parts of the fetal brain is a continuing issueof ethical concern. One of the reasons for the limited applica-tion of stem cell therapy in regenerative medicine is the limita-tion of appropriate cell sources. Mesenchymal stem cells fromfatty tissue (2), as one example, can be relatively easily har-vested but may be limited in their regenerative abilities.Consequently, the discovery of embryonic pluripotentstem cells has led to both a great deal of activity and hype inresearch for novel cell-based therapies. As ethical issuesinvolved in using embryonic stem cells exist, only a limitednumber of countries equipped with appropriate high-endresearch knowledge and technology can embrace these cells fortherapy development.Therefore, the discovery of inducible pluripotent stemcells (iPSCs)—somatic cells (of in principle any type) that canbe reprogrammed by the transfection of a limited number oftranscription factors to become pluripotent—was an impor-tant breakthrough. By their creative and diligent work, thegroup of Shinya Yamanaka from Kyoto University in Japandiscovered that the transfection of only four genes was suffi-cient to reprogram somatic cells to become iPSCs and formany possible cell type of an organism (3).How to optimize the process of transfection to producemaximum numbers of reprogrammed cells and how to per-form appropriate quality control of yield and cell type remainsa central issue. Tiemann and coworkers from Hannover andM€unster in Germany (this issue, page 426) developed a poly-chromatic flow cytometry approach by attaching each of thefour transfected transcription factors (Oct4, c-Myc, Sox2, andKlf4) to four different fluorescent proteins. Quadruplet posi-tive cells contain the reprogrammed iPSCs that can then besorted and cloned. By testing different stoichiometric combi-nations of these transcription factor expression levels,the authors were able to optimize iPSC yield and quantitateoptimal transcription factor levels.The importance of polychromatic cytometry has beenapplied to identify unusual biological material in the pri-mate brain. Bischoff from the University of G€ottingen,Germany and colleagues (this issue, page 436) focusedtheir interest on cells of the immune system infiltratingthe brain. The quantity and composition of these cells canrender information on inflammation processes in neuro-logical diseases and also in stem cell transplantation,

Biologicals and fetal cell therapy for wound and scar management.

Few biopharmaceutical preparations developed from biologicals are available for tissue regeneration and scar management. When developing biological treatments with cellular therapy, selection of cell types and establishment of consistent cell banks are crucial steps in whole-cell bioprocessing. Various cell types have been used in treatment of wounds to reduce scar to date including autolog and allogenic skin cells, platelets, placenta, and amniotic extracts. Experience with fetal cells show that they may provide an interesting cell choice due to facility of outscaling and known properties for wound healing without scar. Differential gene profiling has helped to point to potential indicators of repair which include cell adhesion, extracellular matrix, cytokines, growth factors, and development. Safety has been evidenced in Phase I and II clinical fetal cell use for burn and wound treatments with different cell delivery systems. We present herein that fetal cells present technical and therapeutic advantages compared to other cell types for effective cell-based therapy for wound and scar management.

Optimizing functional imaging protocols for assessing the outcome of fetal cell transplantation in Parkinson's disease

Clinical trials aiming to assess the safety and efficacy of fetal cell transplantation in Parkinson's disease rely on the hypothesis that the grafted tissue will survive and grow, restore striatal dopaminergic neurotransmission, improve the connectivity between striatum, thalamus and cortex and, thereby, produce long-lasting clinical improvement while avoiding the development of adverse effects. Although transplantation of human fetal ventral mesencephalic tissue has been reported as one of the most effective reparative therapies in Parkinson's disease patients to date, different studies have shown inconsistent results causing a paucity of new trials over the last decade. However, during this period, functional imaging alongside other scientific developments from clinical observations and animal work has significantly aided in understanding the mechanisms responsible for the success or failure of grafting human fetal tissue. Recent advances in functional imaging including both positron emission tomography and functional magnetic resonance imaging could be proven useful in vivo tools for the development and assessment of new clinically competitive trials. In this commentary we discuss how an optimized functional imaging protocol could assist new clinical trials using fetal cell transplantation in Parkinson's disease.

Prospect of cell therapy for Parkinson's disease

The hallmark of Parkinson's disease is on-going degeneration of dopaminergic neurons in the substantia nigra, which may be due to various etiologies. Various approaches to alleviate symptoms are available, such as life-long pharmacological intervention, deep brain stimulation, and transplantation of dopaminergic neuron-containing fetal tissue. However, each of these approaches has a disadvantage. Several studies have shown that various kinds of stem cells, induced pluripotent stem cells, and other cells can differentiate into dopaminergic neurons and may be promising for treating Parkinson's disease in the future. Therefore, this review addresses those cells in terms of their prospects in cell therapy for Parkinson's disease. In addition, the need for safety and efficacy studies, various cell delivery modes and sites, and possible side effects will be discussed.

Delayed Results of Transplantation of Fetal Neurogenic Tissue in Patients with Consequences of Spinal Cord Trauma

We analyzed delayed effects of transplantation of nervous and hemopoietic fetal cells to patients with consequences of spinal trauma. A decrease in neurological deficit associated with pronounced improvement of functional independence was observed in 48.9% cases. The best results were observed in patients receiving cell transplantation within the first 2 years after trauma and in younger individuals. The pattern of morphological changes in the spinal cord at site of injury, severity of damage, and the method of transplantation had no appreciable effects on its delayed results.

Serotonergic Neurons Mediate Dyskinesia Side Effects in Parkinson’s Patients with Neural Transplants

Troublesome involuntary movements in the absence of dopaminergic medication, so-called off-medication dyskinesias, are a serious adverse effect of fetal neural grafts that hinders the development of cell-based therapies for Parkinson's disease. The mechanisms underlying these dyskinesias are not well understood, and it is not known whether they are the same as in the dyskinesias induced by l-dopa treatment. Using in vivo brain imaging, we show excessive serotonergic innervation in the grafted striatum of two patients with Parkinson's disease, who had exhibited major motor recovery after transplantation with dopamine-rich fetal mesencephalic tissue but had later developed off-medication dyskinesias. The dyskinesias were markedly attenuated by systemic administration of a serotonin [5-hydroxytryptamine (5-HT)] receptor (5-HT(1A)) agonist, which dampens transmitter release from serotonergic neurons, indicating that the dyskinesias were caused by the serotonergic hyperinnervation. Our observations suggest strategies for avoiding and treating graft-induced dyskinesias that result from cell therapies for Parkinson's disease with fetal tissue or stem cells.

Trasplante de tejido retiniano y de células progenitoras retinianas: una promesa terapéutica para pacientes con enfermedad de la retina

La retinopatía diabética, la degeneración macular relacionada con la edad y la retinitis pigmentosa son las enfermedades retinianas más frecuentes en todo el mundo. A pesar de no contar con suficientes estudios que demuestren resultados funcionales positivos en cuanto a recuperar la función visual, el uso de células madre y células progenitoras retinianas y el trasplante de retina fetal parecen bastante promisorios. Hasta el momento no se han podido obtener resultados positivos sobre la funcionalidad de las células trasplantadas, pero sí se ha demostrado que el procedimiento para transferir el tejido retiniano es seguro y confiable. Aún no se ha intentado en seres humanos el trasplante de células progenitoras retinianas, pero dicho trasplante ha dado resultados satisfactorios en modelos múridos. Los estudios con células progenitoras retinianas han logrado demostrar en modelos múridos que se activan y expresan los fotorreceptores. Existen algunas barreras de disponibilidad para el uso de células progenitoras retinianas, que se deben superar con el fin de adelantar estudios que permitan aumentar las posibilidades de integración y diferenciación de dichas células hacia fotorreceptores.

Distinct Functions of Period2 and Period3 in the Mouse Circadian System Revealed by In Vitro Analysis

The mammalian circadian system, which is composed of a master pacemaker in the suprachiasmatic nuclei (SCN) as well as other oscillators in the brain and peripheral tissues, controls daily rhythms of behavior and physiology. Lesions of the SCN abolish circadian rhythms of locomotor activity and transplants of fetal SCN tissue restore rhythmic behavior with the periodicity of the donor's genotype, suggesting that the SCN determines the period of the circadian behavioral rhythm. According to the model of timekeeping in the SCN, the Period (Per) genes are important elements of the transcriptional/translational feedback loops that generate the endogenous circadian rhythm. Previous studies have investigated the functions of the Per genes by examining locomotor activity in mice lacking functional PERIOD proteins. Variable behavioral phenotypes were observed depending on the line and genetic background of the mice. In the current study we assessed both wheel-running activity and Per1-promoter-driven luciferase expression (Per1-luc) in cultured SCN, pituitary, and lung explants from Per2−/− and Per3−/− mice congenic with the C57BL/6J strain. We found that the Per2−/− phenotype is enhanced in vitro compared to in vivo, such that the period of Per1-luc expression in Per2−/− SCN explants is 1.5 hours shorter than in Per2+/+ SCN, while the free-running period of wheel-running activity is only 11 minutes shorter in Per2−/− compared to Per2+/+ mice. In contrast, circadian rhythms in SCN explants from Per3−/− mice do not differ from Per3+/+ mice. Instead, the period and phase of Per1-luc expression are significantly altered in Per3−/− pituitary and lung explants compared to Per3+/+ mice. Taken together these data suggest that the function of each Per gene may differ between tissues. Per2 appears to be important for period determination in the SCN, while Per3 participates in timekeeping in the pituitary and lung.

Plasticity and therapeutic potential of human fetal tissue

Rebuilding of brain structures and nervous circuitries with grafting of fetal tissue is currently being explored as a strategy to repair the damaged nervous system (Handb Clin Neurol 2009). Experimental studies demonstrated that transplanted neuroblasts can survive and proliferate in the host. Migration of primary or expanded neural precursors following transplantation has also been well documented. Cell migration plays a pivotal role in the organogenesis and contributes to multiple processes in adulthood, such as tissue repair and immune response. Migrating neuroblasts are capable of anatomically re-establishing damaged neural circuitries in the adult animal brain. These results suggest that there might also be a real potential for neurotransplantation in humans in order to promote reconstruction after brain injury. The challenge of such an approach is that the primordium recapitulates its ontogenesis in the host, being deprived of the fetal brain environmental cues, while it is subject to extrinsic cues coming from the diseased adult brain. In animals, recapitulation of normal development was achieved “simply“ by implanting fetal neurons. Striatal fetal grafting as a strategy of cell replacement has been extensively investigated in Huntington’s disease (HD) animal models. There is evidence that primary grafts of human fetal striatal tissue contain proliferative cells capable of migrating, differentiating and establishing robust graft-host interconnectivity, significantly recovering lesion-induced damage, without graft overgrowth or neoplastic proliferation (Lancet Neurol 2006). Based on these studies and the fact that current therapies do not prevent the unrelenting clinical course of HD, a clinical trial of human fetal striatal tissue transplantation for the treatment of HD has been initiated at the University of Florence. Fifteen HD patients underwent bilateral transplantation. The current report provides the first “in vivo“ evidence that unmanipulated grafted neural precursors from human striatal anlagen developed within the diseased striatum of HD patients and moved into the adult brain, reaching shortand long-distance appropriate anatomical targets. The anlagen development resulted in the building of metabolically active new nervous structures displaying striatal-like imaging features (Exp Neurol 2008, 2010). These events were accompanied by beneficial effects on the disease clinical course without negative outcomes, such as grafted tissue overgrowth or cyst formation.

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.

Human striatal neuroblasts develop and build a striatal-like structure into the brain of Huntington's disease patients after transplantation

Rebuilding brain structure and neural circuitries by transplantation of fetal tissue is a strategy to repair the damaged nervous system and is currently being investigated using striatal primordium in Huntington's disease (HD) patients. Four HD patients underwent bilateral transplantation with human fetal striatal tissues (9-12 week gestation). Small blocks of whole ganglionic eminencies were processed to obtain cell suspension and then stereotactically grafted in the caudate head and in the putamen. Follow-up period ranged between 18 and 34 months (mean, 24.7 months). Surgery was uneventful. Starting from the fourth month after grafting, neo-generation of metabolically active tissue with striatal-like MRI features was observed in 6 out of 8 grafts. The increase in D2 receptor binding suggested striatal differentiation of the neo-generated tissue in 3 patients. New tissue, connecting the developing grafts with the frontal cortex and, in one case, with the ventral striatum, was also observed. The new tissue growth halted after the ninth month post transplantation. All patients showed stabilization or improvement in some neurological indices. No clinical and imaging signs, suggestive of graft uncontrolled growth, were seen. This study provides the first evidence in humans that neuroblasts of a striatal primordium can develop and move into the brain after neurotransplantation. Primordium development resulted in the building of a new structure with the same imaging features as the corresponding mature structure, combined with short- and long-distance targeted migration of neuroblasts. The results of this study support both the reconstructive potential of fetal tissue and the remarkably retained plasticity of adult brain. Further studies are necessary to assess the clinical efficacy of the human fetal striatal transplantation.

Function Recovery after Transplantation of Fetal Brain Tissue into Injured Spinal Cord in Experimental Rats

Rakei, S.M., Rahmanian, A., Saffarian, A., Shafeian, R. and Mehrabani, D. 2009. Function recovery after transplantation of fetal brain tissue into injured spinal cord in experimental rats. J. Appl. Anim. Res., 36: 303–306. To determine the effect of transplantation of fetal brain tissue on regeneration of injured spinal cord and the limb function recovery, forty 30-week old male Sprague-Dawley 350–450 g rats were randomly and equally divided into two groups. In 5 pregnant rats the fetuses were removed and the embryonic brain tissues were sectioned into 0.05 mm segments under microscope. The vertebral column was exposed and a 0.1 mm segment of spinal cord medulla at the T10-T11 level was completely dissected in all rats of both groups. A 0.05 mm segment of rat fetus brain was implanted into the injured section of spinal cord of all rats in the case group. No further intervention was done in the control group. After 24 h lower extremity paraplegia was confirmed in all rats. After six weeks, no locomotor improvement was seen in the control group (Basso-Beattie-Bresnahan locomotor rating scale, BBB=0). 15% of rats in the case group had some degrees of recovery from spinal cord injury. Our findings suggest that fetal neural transplants could potentially be an effective surgical intervention for treatment of SCI even though more investigations are warranted.

Endogenous Rhythms inPeriod1Mutant Suprachiasmatic NucleiIn VitroDo Not Represent Circadian Behavior

The mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) controls daily rhythms of behavior and physiology. Lesions of the SCN cause arrhythmicity of locomotor activity, and transplants of fetal SCN tissue restore rhythmic behavior that is consistent with the periodicity of the donor's genotype, suggesting that the SCN determines the period of the circadian behavioral rhythm. While several studies have demonstrated that the circadian characteristics of in vitro SCN rhythms represent circadian behavior, others have shown that the periods of explanted SCN are not always congruent with locomotor activity. We find that the aberrant rhythms of ex vivo SCN lacking functional Period1 (Per1(-/-)) do not represent the behavioral rhythms of the mutant animals. Surprisingly, in C57BL/6J Per1(-/-) mice, the real-time circadian gene promoter activity rhythm is weak or absent in adult SCN slices in vitro even though the free-running wheel-running activity rhythm is indistinguishable from wild-type (Per1(+/+)) mice. While some neurons in Per1(-/-) SCN explants exhibit robust circadian rhythms, others have irregular and/or low-amplitude rhythms. Together, these data suggest that either a small population of rhythmic neurons in the Per1(-/-) SCN is sufficient to control wheel-running activity or that in vivo physiological factors can compensate for the aberrant endogenous rhythms of Per1(-/-) SCN.

Partial compensation of sexual receptivity deficits in female rats with bilateral lesions of the hypothalamic ventromedial nucleus by transplants of fetal mediobasal hypothalamic tissue.

Bilateral aspiration as well as bilateral electrolytic lesions of the hypothalamic ventromedial nucleus (VMN) in ovariectomized, adult female rats treated with testosterone or estrogen resulted in a long-lasting decrease of sexual receptivity. Fetal mediobasal hypothalamic (MBH) tissue was grafted successfully to the site of VMN lesions. The following presuppositions for a sufficient rate of long-term survival of the grafts could be defined: (1) careful lesioning and minimalizing of damage to the surrounding tissues, (2) high vitality of the donor fetuses, (3) delay of transplantation after lesioning, and (4) testosterone treatment of the recipient animals at an early stage of graft development. In rats with surviving MBH grafts the receptivity deficits resulting from bilateral aspiration of the VMN were compensated partially.

Therapeutic Microinjection of Autologous Adult Human Neural Stem Cells and Differentiated Neurons for Parkinson's Disease: Five-Year Post-Operative Outcome

Object. Neural stem cell-derived neurons offer new cellular therapeutic alternatives for diseases of the central nervous system. Selective neural repair can be particularly valuable in progressive degenerative diseases with discrete cell loss, like Parkinson's disease. Some benefits were previously demonstrated following transplantation of fetal embryonic tissue. This approach, however, carries inherent risks of immunological reactions, infectious transmission, and intractable dyskinesias, in addition to serious ethical concerns. Methods. Cortical and subcortical tissue samples were obtained during neurosurgical procedures. Neural stem cells were isolated and expanded in vitro for several months. Safety, differentiation and functional studies were performed during the expansion phases. Nine months after harvesting, autologous cell suspensions containing differentiated dopaminergic and GABAergic neurons were microinjected unilaterally in a patient with advanced Parkinson's disease. 18F-dopa PET stud- ies and neurological evaluations were performed serially (pre/post-operatively). Results. Over the next 36 months, the overall Unified Parkinson's Disease Rating Scale (UPDRS) improved by 81% while on medication and 83% while off medication. At five-years post-operatively, clinical motor scores returned to base- line. At three and twelve months post-operatively, 18F-dopa PET studies showed a 55.6% and 33.2% increase in dopa- mine uptake in the implanted left putamen. Conclusions. Adult neural stem cells derived from a patient's cerebral tissue can become a source of differentiated neu- rons, useful for grafting in the treatment of Parkinson's disease. The combined GABAergic and dopaminergic cells pro- duced a long lasting motor improvement. This approach has the potential to make neural stem cell therapy acceptable and available to a large number of patients.

臨床薬理試験認定医をめぐる課題 ４．プラセボの効果―特にパーキンソン病における効果について―

The placebo effect can be observed in various medical conditions, and has been documented by randomized placebo-controlled drug studies. One of the best examples is the placebo effect in Parkinson's disease (PD). The placebo effect is observable not only in drug trials but also with surgical therapies, such as deep brain stimulation, fetal tissue transplantation, and infusion therapy of a neurotrophic factor. A recent study using positron emission tomography (PET) with raclopride demonstrated that the release of endogeneous dopamine in the dorsal striatum mediates the placebo effect in PD, suggesting that the placebo effectiveness is achieved by endogenous dopamine supplementation. Although the detailed pathophysiological mechanism underlying the placebo effect is unclear, the placebo effect has been explained by two mechanisms: the conditioning theory (pavlovian conditioning) and the cognitive or expectancy theory (expectation of clinical improvement). Speculatively, it may be that the placebo effect in PD is related more to cognitive mechanisms. Numerous studies have suggested a functional relationship between dopamine and the reward system. Understanding the placebo effect is important for designing clinical trials and interpreting their results.

Tissue culture before transplantation of frozen-thawed human fetal ovarian tissue into immunodeficient mice

To evaluate the effects of tissue culture on the viability and development of follicles in frozen-thawed human fetal ovarian tissue before transplantation into severe combined immunodeficient (SCID) mice and to determine the optimal duration of pretransplant tissue culture. Experimental prospective study. Animal center and reproductive laboratories in university hospitals. Frozen-thawed human fetal ovarian tissue samples from 20-week-old abortuses were randomly divided into four groups and cultured in vitro for 0, 3, 6, or 9 days before being xenografted into kidney capsules of bilaterally oophorectomized severe combined immunodeficient (SCID) mice. Grafts were removed 16 weeks after transplantation. Histological analysis and assessment of proliferative cell nuclear antigen (PCNA) expression levels were used to evaluate the survival and development of follicles. The proportion of growing follicles was significantly increased in groups cultured before transplantation as compared with the noncultured group. Sixteen weeks after transplantation, the number of follicles in the cultured grafts was higher than that in the noncultured grafts. Grafts cultured for 6 or 9 days showed higher proportions of preantral and antral follicles than grafts cultured for 0 or 3 days. PCNA immunohistochemical analysis indicated that follicle cells were in a proliferative state after culture and transplantation. The viability and development of human fetal follicles may be improved by pretransplant tissue culture. The optimal culture duration before transplantation of fetal ovarian tissue is 6 days.